GREEN. Diethanolamine (DEA) and Related DEA-Containing Ingredients

Transcription

1 GREEN Diethanolamine (DEA) and Related DEA-Containing Ingredients CIR EXPERT PANEL MEETING MARCH 3-4, 2011

2 Memorandum To: From: CIR Expert Panel Members and Liaisons Monice M. Fiume MMF Senior Scientific Analyst/Writer Date: February 10, 2011 Subject: Re-Review of Diethanolamine (DEA) and Related DEA-Containing Ingredients At the December Panel meeting, the Panel made the decision to reopen the safety assessment of Triethanolamine (TEA), Diethanolamine, and Monoethanolamine (MEA). That decision was based on the need to incorporate new data, but most importantly, on the benefit of separating the ethanolamines, and having each of these ingredients be in its own report with a family of related ingredients created for each. The re-review of DEA and 68 DEA-containing ingredients is being submitted for your review. In considering the potential safety issues with DEA-containing ingredients, it was reasoned that, were they to penetrate the skin, the toxicity of most concern would be the DEA moiety. The acid salt ingredients, DEA Myristate, for example, would be expected to dissociate into DEA and the corresponding acid. The covalent DEA ingredients, such as cocamide DEA, do not readily dissociate into DEA and the other component. However, in the case of these covalent ingredients, DEA may be of concern as an impurity and/or metabolite. Since this is the first time the groupings are being presented to the Panel, there is an opportunity to make a further determination whether this family of ingredients is appropriate as currently grouped. If it is not, the Panel can make changes. The safety of 8 of the ingredients included in this re-review, as currently grouped, has been reviewed previously by the CIR. Summary information from the existing safety assessments is included in the current re-review document. Additionally, many of the ingredients included in this re-review include a component that has been reviewed by the CIR. For example, DEA-Isostearate is the DEA salt of isostearic acid; isostearic acid has been reviewed by the CIR. Table 2 provides the conclusions from the CIR reports on all the component ingredients. Finally, many of the ingredients are lacking safety data. The Panel should consider any existing CIR reports that can be used to determine the safety of ingredients that dissociate. For those that do not

3 dissociate, the Panel should consider whether the impurity level of DEA can be used as a determining factor in considering safety. As a reminder, NTP studies have results indicating clear evidence of carcinogenicity in mice for DEA and some DEA fatty acid esters. The Panel determined that the mode of action of DEA carcinogenesis in mice was understood and the penetration was sufficiently well-characterized, such that the carcinogenicity findings in mice were considered to have no relevance to human health from the use of cosmetics containing DEA. This re-review is the first of the three ethanolamine reports being presented. The re-reviews on TEA and MEA will be presented at later meetings. Also included for your review are previous CIR reports about ingredients discussed in this report.

4 Panel Book Page 1

5 TEA, DEA, MEA HISTRY riginal Report: In 1983, the Expert Panel determined that these ingredients were safe for use in cosmetic formulations designed for discontinuous, brief use followed by thorough rinsing from the surface of the skin. In products intended for prolonged contact with the skin, the concentration of ethanolamines should not exceed 5%. Ethanolamine (MEA) should be used only in rinse-off products. Triethanolamine (TEA) and diethanolamine (DEA) should not be used in products containing N-nitrosating agents. June 1999: discussed NTP carcinogenicity results; presentations were made by Dr. Lehman- McKeeman and Dr. Stott June 2008: presentation was made by Dr. Stott; Acetamide MEA was discussed, with reference to the MEA, DEA, TEA report June 2009: discussed DEA carcinogenicity; the DEA report was not be reopened December 2010: formal rereview package was presented to the Panel; report was split into 3 separate documents DEA, TEA, and MEA, add additional ingredients will be added to each report March 2011: the RR of DEA was presented to the Panel, including the new ingredient subgroups Panel Book Page 2

6 DEA X X X X X X X X X X X X X DEA Family Data Profile* March 2011 Writer, Monice Fiume Previously Reviewed CIR Review on Component Reported Use Free DEA Content log P value Toxicokinetics Data Animal Tox Acute, Dermal Animal Tox Acute, ral Animal Tox, Acute, Inhalation Animal Tox Rptd Dose, Dermal Animal Tox, Rptd Dose, ral Animal Tox Rptd Dose, Inhalation Repro/Dev Tox Genotoxicity Carcinogenicity Dermal Irr/Sens cular Irritation Panel Book Page 3 Diethanolamine Bisulfate X DEA-Isostearate DEA- Isostearate X DEA-Lauraminopropionate DEA-Linoleate X DEA-Myristate X DEA Stearate X X DEA-C12-13 Alkyl Sulfate DEA-C12-13 Pareth-3 Sulfate X DEA-C12-15 Alkyl Sulfate DEA-Ceteareth-2 Phosphate X DEA-Cetyl Phosphate DEA-Cetyl Sulfate X DEA-Di(2-Hydroxypalmityl)Phosphate X DEA-Dodecylbenzenesulfonate X DEA-Hydrolyzed Lecithin X DEA-Laureth Sulfate X X DEA-Lauryl Sulfate X X DEA-Methyl Myristate Sulfonate X DEA-Myreth Sulfate X DEA-Myristyl Sulfate X DEA-leth-3 Phosphate X DEA-leth-5 Phosphate DEA-leth-10 Phosphate X DEA-leth-20 Phosphate Inorganic Acid Salt rganic Acid Salts rgano-substituted Inorganic Acid Salts Alkyl Substituted Diethanolamines Butyl Diethanolamine N-Lauryl Diethanolamine X Methyl Diethanolamine X X X X X X X X X X

7 DEA Family Data Profile* March 2011 Writer, Monice Fiume Previously Reviewed CIR Review on Component Reported Use Free DEA Content log P value Toxicokinetics Data Animal Tox Acute, Dermal Animal Tox Acute, ral Animal Tox, Acute, Inhalation Animal Tox Rptd Dose, Dermal Animal Tox, Rptd Dose, ral Animal Tox Rptd Dose, Inhalation Repro/Dev Tox Genotoxicity Carcinogenicity Dermal Irr/Sens cular Irritation Panel Book Page 4 Diethanolamides Almondamide DEA X Apricotamide DEA X Avocadamide DEA X Babassuamide DEA X Behenamide DEA X Capramide DEA X X Cocamide DEA X X X X X X X X X X Cocoyl Sarcosinamide DEA X Cornamide DEA X Cornamide/Cocamide DEA X DEA-Cocoamphodipropionate X Diethanolaminooleamide DEA Hydrogenated Tallowamide DEA X Isostearamide DEA X X X X Lactamide DEA X Lanolinamide DEA X Lauramide DEA X X X X X X X X X X X X X X Lauramide/Myristamide DEA X X Lecithinamide DEA X Linoleamide DEA X X X X X X X X Minkamide DEA X Myristamide DEA X X X X X leamide DEA X X X X X X X X X X X livamide DEA X Palm Kernelamide DEA X X Palmamide DEA X Palmitamide DEA X X PEG-2 Tallowamide DEA X PEG-3 Cocamide DEA X Ricebranamide DEA X Ricinoleamide DEA X X X Sesamide DEA X Shea Butteramide/Castoramide DEA X Soyamide DEA X X Stearamide DEA X X X X X X X X

8 DEA Family Data Profile* March 2011 Writer, Monice Fiume Previously Reviewed CIR Review on Component Reported Use Free DEA Content log P value Toxicokinetics Data Animal Tox Acute, Dermal Animal Tox Acute, ral Animal Tox, Acute, Inhalation Animal Tox Rptd Dose, Dermal Animal Tox, Rptd Dose, ral Animal Tox Rptd Dose, Inhalation Repro/Dev Tox Genotoxicity Carcinogenicity Dermal Irr/Sens cular Irritation Stearamide DEA-Distearate X Stearamidoethyl Diethanolamine Stearamidoethyl Diethanolamine HCl Tallamide DEA X Tallowamide DEA Undecylenamide DEA X X Wheat Germamide DEA X * X indicates that data were available in a category for the ingredient Panel Book Page 5

9 DEA Search Info # uses conc data Toxline- Pubmed NLM EU FDA ChemPortal Misc NLM NTIS Registry NTIS Merck EU SCCS ECE- TC SIDS IARC NTP EAFUS TC HPVIS date searched 1-7& / DEA x x II x x x DEA Bisulfate DEA-Myristate x II x II DEA Stearate no DEA-Isostearate II DEA-Linoleate x II DEA-Lauraminopropionate DEA-Lauryl Sulfate x II x II DEA-C12-13 Alkyl Sulfate II DEA-Myristyl Sulfate x II DEA-C12-15 Alkyl Sulfate II DEA-Cetyl Sulfate x II DEA-Laureth Sulfate x II DEA-C12-13 Pareth-3 Sulfate II DEA-Myreth Sulfate II DEA-DodecylbenzeneSulfonate x II DEA-Methyl Myristate Sulfonate DEA-Cetyl Phosphate x II DEA-Ceteareth-2 Phosphate II DEA-leth-3 Phosphate II DEA-leth-5 Phosphate DEA-leth-10 Phosphate DEA-leth-20 Phosphate DEA-Hydrolyzed Lecithin II DEA-Di(2-Hydroxypalmityl) - Phosphate[ no x II II II II IUCLID data set Panel Book Page 6

10 Methyl Diethanolamine [ ] Butyl Diethanolamine N-Lauryl Diethanolamine [ ] Capramide DEA Undecylenamide DEA ; Lauramide DEA Myristamide DEA # uses conc data Toxline- Pubmed NLM EU FDA ChemPortal Misc NLM NTIS Registry NTIS Merck EU SCCS ECE- TC SIDS IARC NTP EAFUS TC HPVIS x no x x X x III x III x III x III x x x III Lauramide/ Myristamide DEA III Palmitamide DEA x III Stearamide DEA Behenamide DEA x III x III Lactamide DEA III Isostearamide DEA x X leamide DEA ; Linoleamide DEA Almondamide DEA Apricotamide DEA Avocadamide DEA Babassuamide DEA Cocamide DEA x III x x x III x III x III x III x III Cornamide DEA III Cornamide/ Cocamide DEA III Hydrogenated Tallowamide DEA x III Lanolinamide DEA [ ] x III x x x x III Lecithinamide DEA III Minkamide DEA x III IUCLID data set Panel Book Page 7

11 livamide DEA Palm Kernelamide DEA # uses conc data Toxline- Pubmed NLM EU FDA ChemPortal Misc NLM NTIS Registry NTIS Merck EU SCCS x III x III Palmamide DEA III Ricebranamide DEA III Ricinoleamide DEA x III Sesamide DEA Shea Butteramide/Castoramide DEA Soyamide DEA Tallamide DEA Tallowamide DEA Wheat Germamide DEA x III X ECE- TC SIDS IARC NTP EAFUS TC HPVIS x III x x III x x III x III PEG-2 Tallowamide DEA X PEG-3 Cocamide DEA X Stearamidoethyl Diethanolamine X Stearamidoethyl Diethanolamine HCl DEA-Cocoamphodipropionate no Diethanolaminooleamide DEA X Stearamide DEA-Distearate X Cocoyl Sarcosinamide DEA x X X IUCLID data set References rdered Whit NTIS Panel Book Page 8

12 TXNET Search Statements DEA Family of Ingredients Jan 7, 2011 SS1 DEA R DIETHANLAMINE R (only search last 12 mos) 50 hits in toxline; 31 in DART (all years) SS2 ((CCAMIDE R ISSTEARAMIDE R MYRISTAMIDE R STEARAMIDE) AND (DEA R DIETHANLAMINE)) R R R R (only since 1990) 26 hits in toxline SS R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R hits in toxline; 2 hits in DART SS4 ((DIETHANLAMINE R DEA) AND (BISULFITE R ISSTEARATE R LAURAMINPRPINATE R LINLEATE R MYRISTATE R LAURATE R STEARATE R ((ALKYL R PARETH R CETYL R LAURETH R LAURYL R MYRETH R MYRISTYL) AND SULFATE) R ((CETEARETH R CETYL R LETH R HYDRXYPALMITYL) AND PHSPHATE) R DDECYLBENZENESULFNATE R (METHYL AND MYRISTATE AND SULFNATE) R (HYDRLYZED AND LECITHIN) R BUTYL R LAURYL R METHYL)) 3 hits in toxline SS5 ((DIETHANLAMINE R DEA) AND (ALMNDAMIDE R APRICTAMIDE R AVCADAMIDE R BABASSUAMIDE R BEHENAMIDE R CAPRAMIDE R CCAMPHDIPRPINATE R DIETHANLAMINLEAMIDE R (HYDRGENATED AND TALLWAMIDE) R LACTAMIDE R LANLINAMIDE R LAURAMIDE R (LAURAMIDE AND MYRISTAMIDE) R LECITHINAMIDE R MINKAMIDE R LEAMIDE R LIVAMIDE R (PALM AND KERNELAMIDE) R PALMAMIDE R PALMITAMIDE R (PEG AND (TALLWAMIDE R CCAMIDE)) R RICEBRANAMIDE R RICINLEAMIDE R SESAMIDE R (SHEA AND BUTTER AND CASTRAMIDE) R SYAMIDE R (STEARAMIDE AND DISTEARATE) R (STEARYAMIDETHYL AND (HCL R HYDRCHLRIDE)) R TALLAMIDE R TALLWAMIDE R UNDECYLENAMIDE R (WHEAT AND GERMAMIDE) R (CCYL AND SARCSINAMIDE) R CRNAMIDE R (CRNAMIDE AND CCAMIDE) R LINLEAMIDE)) 15 hits in toxline SS6 (Jan 12, 2011) R R R R R R R R R R R R R R R R R R R R R R R R hits in toxline; 2 hits in DART Panel Book Page 9

13 FULL PANEL - December 2010 DR. BERGFELD: Thank you. So, the motion's been made to reopen and it's been seconded. Any further discussion? DR. MARKS: And with the intent -- and we'll -- as Paul mentioned earlier, at least for our team the intent was to add methylene glycol, but as we work through the report, we'll decide whether or not we want to continue that. DR. BERGFELD: All right. Call for the question, all those in favor, please indicate by raising your hand? Thank you. Unanimous. Then moving on to the second to the last ingredient which is the MEA/DEA/TEA. Dr. Belsito? DR. BELSIT: Yes, this is a re-review of the document and it's gone through a number of iterations. The initial was 1983, and since that time there have been a number of discussions regarding DEA. However, it's really time that we look at the original report which contained all three. And when we -- when my team looked at the data we really felt that perhaps with the exception of opening it to reassess MEA and changing it to our current way of stating that we had limited it to rinse off products because of irritation, to the current way of stating, could be used in leave-ons if formulated not to be irritating, it was really no reason to open the document. However, the reason to open it would be that there are a number of MEA, DEA, and TEA compounds that could be tagged onto this quite easily that we haven't reviewed. So we are recommending that, A, the report be split into three different reports: An MEA, a DEA, and a TEA report; and that all of the related cosmetic MEAs, DEAs, and TEAs be included in each of those reports. And that's a motion. DR. BERGFELD: Motion to reopen and split it into three different ingredient groups has been made. DR. MARKS: Second. DR. BERGFELD: Second. Any further discussion about reopening? John? DR. BAILEY: Yeah, I agree, but I think that it's really important how these groups are going to be constituted. And I would like to see the proposed group as soon as possible and then we will refer that to our Science and Support Committee just to make sure that they're comfortable with the way the group is put together. You know, there was some, I wouldn't say concern, but some interest in making sure that these groups are as rational and logical as possible, so we would need to get those as soon as we can. DR. BERGFELD: Alan? DR. ANDERSEN: Yeah, we will most certainly get the potential add-ons out ASAP. I see a primary focus of the March meeting on receiving that input from industry, receiving the input from the panel as the panel gets the opportunity to look at those groupings, and negotiating what actually should be done as add-ons. So, I don't know that we're -- I mean, unless we hit the nail perfectly on the head, there's going to be some negotiating in March. DR. BERGFELD: Call for the question then to reopen, all those in favor please indicate by raising your hands? Thank you. It's reopened. And then moving to the last ingredient to be considered this morning, human umbilical extract, Dr. Marks? DR. MARKS: In 2002, the CIR published its final safety assessments in the ingredients derived from human and animal placentas and umbilical cords with a conclusion that the available data were insufficient to support the safety. We recently had correspondence from a company specifically concerning use of human umbilical extract in cosmetic products. They supplied some data, but when you look at our insufficient data needs from the original safety assessment, really those data needs were not met and so our team moves not to reopen this safety assessment. DR. BERGFELD: Second? Is that a second? Comment? Panel Book Page 10

14 BELSIT TEAM - December 2010 CIR Meeting day 1 of 2 (Breakout Session) Page: 14 1 the risk assessment pages that you're looking at 2 will substantially make up the summary that you're 3 going to see at the next meeting. 4 DR. BELSIT: kay, good. Anything else 5 on this? No? kay. So moving on to the next 6 one, it's the re- review of MEA, DEA, and TEA. 7 And Alan has essentially already stolen my 8 thunder, which is basically how many salts and 9 esters of these can we make into super families? 10 And so I'm thinking we should be reopen it not 11 only to add those in, but I think our conclusion 12 that MEA should not be used in leave-on products 13 is based upon irritation. And we've taken a 14 different step now to say "when formulated not to 15 be irritating," so that conclusion may not be 16 correct either as it stands. So I would say that 17 we reopen the documents and take Alan's, split 18 them into three and add the salts and esters of 19 the MEAs, DEAs, and TEAs so that we get everything 20 that's out there. 21 DR. ANDERSEN: I think with that 22 strategy what you could expect to see at the next Anderson Court Reporting CIR Meeting day 1 of 2 (Breakout Session) Page: 15 1 meeting would be three more, three separate and 2 more comprehensive documents that list the rganic 3 Acid Salts that could conceptually be included and 4 then examines the question of going on to, let's 5 see, MEAs, for example, the DEA list. There is 6 yet a second group that takes off on the fact that 7 we've already reviewed cocamide DEA, lauramide 8 DEA, which are not -- may not technically be 9 considered as salts, but we'll look at forming 10 those groups as well. Monice and Bart have 11 already done a great deal of homework on this, and 12 are kind of ready to package that, but we just 13 kind of finished it last week and it seemed 14 disingenuous to dump all of that on the Panel for 15 this meeting. So if for all sorts of reasons, it 16 seems appropriate to reopen these, then we can 17 take the next step at the next meeting. 18 DR. BELSIT: Is everyone in agreement 19 of splitting them into three separate documents 20 when we do that? 21 DR. SNYDER: Yes. 22 DR. LIEBLER: Fine with it. Anderson Court Reporting CIR Meeting day 1 of 2 (Breakout Session) Page: 16 1 DR. BELSIT: kay, any comments? 2 DR. LIEBLER: I guess I had 3 misinterpreted the cover memo, and I thought that 4 the main reason to discuss these was the 5 appearance of new data on carcinogenicity. So 6 really that's not the main issue here. 7 DR. SNYDER: No. 8 DR. LIEBLER: kay. 9 MS. FIUME: riginally DR. ANDERSEN: I think, in fact, it's an 11 old issue at this point in terms of DEA 12 carcinogenesis. At this point in time arguably 13 explained process of choline metabolism in mice, 14 and it's not hugely relevant. 15 DR. LIEBLER: Right, so based on all of 16 that, I said don't reopen these, but I agree with 17 the reason now to reopen. 18 DR. BELSIT: Any other comments? kay, 19 dicarboxylic acid. kay, so in August we issued a 20 tentative report for the twelve dicarboxylic 21 acids, 44 diesters, finding them safe in present 22 practice of use and concentration. There was one MARKS TEAM - December 2010 CIR Meeting day 1 of 2 ( Main Session) Page: HIV, it put together all sorts of stuff, and we 2 started to separate the two boilerplates out, and 3 some time next year, we will be bringing to the 4 panel all of the boilerplates for boilerplate 5 re-review so we can go through and make sure 6 they're currently up to date. We felt there were 7 more than enough agenda items on this meeting to 8 not do it starting with this meeting. 9 DR. MARKS: Thank you. kay, onto the 10 next ingredient or ingredients. We're in the MEA, 11 DEA, TEA re- review. There's quite a history of 12 these ingredients, and I think where we're at at 13 this point is do we reopen, do we separate it out 14 into three different reports, do we put them 15 together? And I'll open it up for discussion. 16 And then, also, we should talk about if we reopen, 17 do we reopen it to add salts and simple esters, 18 also? And to further comment, and, Tom, I'd asked 19 you about the nitrosamine formulation concern, and 20 Ron's, where DEA has been banned in the EU and 21 Canada, plus it's salts and MEA and TEA has had 22 restrictions. So, let's go ahead and decide Anderson Court Reporting Panel Book Page 11 Anderson Court Reporting

15 CIR Meeting day 1 of 2 ( Main Session) Page: whether we're going to reopen and then do we do 2 them together or separate and what do we add? 3 DR. SLAGA: After reading this and 4 trying to compare to three of them, the DEA and 5 TEA and the MEA, so to speak, and with the data 6 related to EU and Canada, it seems to me it would 7 be a good idea to reopen and separate them. 8 In terms of nitrosamines, they all have 9 capabilities, don't they? 10 DR. SHANK: Not MEA. 11 DR. SLAGA: I mean, TEA. 12 DR. SHANK: Yes, TEA. 13 DR. SLAGA: TEA and DEA. 14 DR. HILL: I'm not sure I understand why 15 TEA does actually. I'm a little confused about 16 that. 17 DR. SLAGA: Chemistry. 18 DR. MARKS: So, let's go back. So, I 19 saw nodding of heads, all team members endorse the 20 idea of reopening? 21 DR. SHANK: Yes. 22 DR. MARKS: And to separate into the Anderson Court Reporting CIR Meeting day 1 of 2 ( Main Session) Page: mono, di, and tri? 2 DR. SHANK: What do you mean by 3 "separate?" Three reports or three sections of 4 one report? 5 DR. MARKS: That's the question. 6 Because my sense was there was a possibility of 7 doing three separate reports, but we can do -- 8 MS. BRESLAWEC: We could do it 9 administratively anyway. We just noticed that, 10 over the years, keeping them in one report has led 11 to unnecessary confusion. So, we would like to 12 either keep them separately in the same report or 13 put them in three different reports with cross 14 references. 15 DR. HILL: I guess I'd endorse the idea 16 of putting them in three separate reports on the 17 basis that there doesn't seem to be any 18 significant biotransformation, for example, of TEA 19 to DEA. The only relationships I see are in the 20 choline depletion, the ones that have that 21 activity, and I'm wondering, I mean, 22 monoethanolmine is relatively abundant endogenous Anderson Court Reporting CIR Meeting day 1 of 2 ( Main Session) Page: molecules. 2 So, until you get to really, really 3 artificial dose levels, I'm not sure -- so, my 4 personal bias, but I hadn't thought about keeping 5 them in the same report and just considering them 6 separately. My personal bias was to separate them 7 out into three individual reports. That is just 8 my personal bias based on everything I saw there. 9 DR. MARKS: Tom and Ron Shank? Together 10 or as separate? I should together in one report, 11 but separated within that report? 12 DR. SLAGA: I don't know. It seems to 13 me it'd be better in separate reports, not 14 confusing them. 15 DR. SHANK: I don't feel strongly about 16 it. If it were strictly up to me, I'd have one 17 report with three sections. 18 DR. MARKS: kay. Well it sounds like 19 at least at this point we'll go with separate 20 reports, and we'll see what the Belsito Team's 21 feelings are. 22 Any further comments before we -- CIR Meeting day 1 of 2 ( Main Session) Page: DR. SHANK: Yes, that being the case, 2 then I think TEA and MEA would carry the same 3 conclusion that the report has now, in that the 4 major changes would be in the DEA report. 5 MS. FIUME: Could you clarify what you 6 mean, Dr. Shank? 7 DR. SHANK: Yes, we have a report 8 already with all three ingredients in it. The 9 conclusion for that report would still apply to 10 TEA and MEA, even though you're splitting those 11 reports. And then the major changes would be in 12 the new report on DEA, diethanolamine. 13 MS. BRESLAWEC: Are you suggesting that 14 you would not reopen TEA and MEA? 15 DR. SHANK: You have to reopen it 16 because it's now one report, and now you're going 17 to split it into three. So, I don't see how you 18 can do that without reopening it. And now if 19 you're going to add the other ingredients that 20 pertain to each of those ethanolamines, that's 21 your opportunity to do that. 22 MS. BRESLAWEC: But you can reopen them Anderson Court Reporting Panel Book Page 12 Anderson Court Reporting

16 CIR Meeting day 1 of 2 ( Main Session) Page: just to add ingredients, which makes it a little 2 more expedient. DEA, it seems you're suggesting 3 to reopen to reconsider the conclusion, perhaps? 4 DR. SHANK: Correct. 5 DR. SLAGA: Yes. 6 DR. SHANK: How do you reopen? You're 7 creating three new reports. So, you're not 8 reopening DEA, you're not reopening the current 9 report. You're splitting it. 10 DR. MARKS: Yes DR. SHANK: How do you do that 12 procedurally? What words you use MS. BRESLAWEC: I think it's something 14 that we would do administratively. 15 DR. MARKS: No, that's a good point, 16 Ron, because in 1983, these were grouped together. 17 So, you're reopening that report, but if we decide 18 to do three separate reports, we're not reopening 19 them in that; we're reopening to separate it. So, 20 I guess administratively, you have to make sure 21 that that's not a problem with the CIR guidelines. 22 But, if there are, it seems to me just as we've Anderson Court Reporting CIR Meeting day 1 of 2 ( Main Session) Page: done with other reports; we've had major sections 2 within the report. There will be. Well, the 3 conclusion will just deal with it. 4 DR. BAILEY: And couldn't this also -- 5 in splitting these, wouldn't it be logical to 6 include adding the other alkanolamines within that 7 group, like a diethanol. I mean, it would be 8 dialkonalamines because there are some in the 9 dictionary now. 10 MS. BRESLAWEC: We've actually prepared, 11 and, Bart, maybe you'd like to come up here, as 12 well, but we've started looking at possible 13 add-ons for all there, MEA, DEA, and TEA, and we 14 are approaching it very systematically. There are 15 groups that seem to us to be natural add-ons, like 16 organic acid salts, for example, and then there 17 are groups that are related, but may be a little 18 far out or groups that are related, but probably 19 should be considered on their own. We're not 20 ready to present those groups for discussion right 21 now, but we have started the process, and we have 22 quite a bit of information on it, but it's Anderson Court Reporting CIR Meeting day 1 of 2 ( Main Session) Page: something that warrants more preparation before 2 it's presented to you all for discussion. 3 So, yes, we would like to consider 4 reopening all three reports for the potential of 5 adding new ingredients. 6 DR. MARKS: Halyna, how much do you see 7 in having separate reports that you're now going 8 to have a lot of refer to the other report to 9 support that the safety of the other ingredients. 10 Like Ron says TEA and MEA, the same conclusions. 11 So, does that make sense to separate them out if 12 we're going to be using data from one to support 13 the other? And, I, again, am looking forward in 14 terms of if there's going to be a lot of data 15 that's shared in all three reports, and does it 16 make sense to have there separate reports? 17 DR. BYER: For each of the three 18 chemicals, there is a lot of chemical-specific 19 information. So, it doesn't need to be a lot of 20 cross-reference and so forth. And DEA actually 21 stands out when you look at that data and the 22 mechanistic information that's been published and CIR Meeting day 1 of 2 ( Main Session) Page: so forth. So, I think from that perspective, it 2 certainly makes sense to separate them. 3 DR. HILL: Excuse me. And in regards to 4 potentially expanding the groups, I would just say 5 that I strongly suspect that there's going to be, 6 particularly with DEA, there's some toxicology 7 issues that might pertain to it that might not 8 pertain to anything even related. Now, amides of 9 DEA at some point, but those are really widely, 10 heavily used for cosmetic ingredients, and I think 11 moving in that direction would be right now with 12 great caution in my estimation because I think 13 there might not be that much to worry about. 14 DR. BYER: Right. 15 DR. HILL: And, so, if you tag related 16 to something where there clearly is a problem well, I say "clearly is a problem," seems to be a 18 problem. Don't know in humans, but you might be 19 creating a problem where there wasn't one before. 20 DR. MARKS: I think, again, for the 21 stenographers, that was Dr. Boyer who was 22 commenting earlier, correct? Anderson Court Reporting Panel Book Page 13 Anderson Court Reporting

17 CIR Meeting day 1 of 2 ( Main Session) Page: DR. BYER: Yes. 2 DR. MARKS: As new member of the CIR 3 support staff. Thank you. 4 To kind of reinforce what you said, Ron 5 Hill, for TEA, there's now am I reading 6 this correctly, 4,015 products that it's used in? 7 DR. ANSELL: The group is potentially 8 enormous depending on where you start drawing your 9 lines. 10 DR. MARKS: Yes. Plus it looks like and, obviously, there are also baby products 12 there, but a huge number of products that contain 13 this ingredient. 14 kay, so, it looks like I think what 15 we'll find out what the other team moves tomorrow, 16 but, for us, it's to reopen separate reports and 17 to consider add-ons, and we'll see that, I 18 presume, some time in a future meeting. And then 19 we'll start, I suspect, on looking at the add-ons 20 to begin with and then go from there. 21 Does that sound reasonable, team 22 members? Anderson Court Reporting CIR Meeting day 1 of 2 ( Main Session) Page: TEAM MEMBERS: (Nodding) 2 DR. MARKS: Anything else we need to 3 discuss about these three at this point? And, Ron 4 Shank, you've given us an insight of where the 5 safety assessments are going to go in the TEA and 6 MEA. It sounds like the same conclusion or 7 similar, and DEA, that I will have some 8 significant changes in the conclusion. 9 MR. SHANK: kay, so, we're not going to 10 discuss this until we see it in three different 11 reports? Is that what you're saying? 12 DR. MARKS: Well, that's what I 13 suggested, but I guess in discussing it MR. SHANK: Do you want to discuss the 15 mouse carcinogenicity assay? 16 DR. MARKS: Sure. 17 MR. SHANK: r not? Wait? 18 MS. FIUME: That's fine, because that 19 would be one reason to reopen that portion of the 20 report to separate than just to add. 21 MR. SHANK: Yes. 22 MS. FIUME: So, if there's information Anderson Court Reporting CIR Meeting day 1 of 2 ( Main Session) Page: you want taken care of there, I'd like to discuss 2 that part now. 3 DR. MARKS: Go ahead, Tom. 4 DR. SLAGA: (ff mike) restriction, too. 5 DR. MARKS: This isn't -- 6 DR. SHANK: I think the reason -- 7 DR. MARKS: This is the choline. 8 DR. SHANK: The main reason this was 9 coming up for re-review was there was a cancer 10 bioassay in the mouse on DEA that produced tumors, 11 and I think we need to address that mouse 12 bioassay. But if you want to wait until the 13 reports are split, then we can do it at that time. 14 DR. MARKS: I think that's up to you. 15 DR. SHANK: (ff mike) for three 16 different reports. 17 DR. MARKS: Yes, for Ron and Tom and Ron 18 Hill, there is that, and, also, the nitrosamine 19 formulation, we could discus that, also, at this 20 point and give a nice idea of the direction we're 21 going. Yes. 22 DR. SHANK: That's pretty simple. MEA, CIR Meeting day 1 of 2 ( Main Session) Page: it's a primary amine, and that's not 2 nitro-satiable. DEA and TEA are, and the 3 nitrosation products are in the literature. So, 4 that's not an issue. The issue is how does one 5 interpret the mouse cancer bioassay? 6 MS. DAHLIN: Dr. Marks, Dr. Shank, the 7 report, although under one cover, is in three 8 separate sections, as you've noted. So, we are 9 certainly prepared to hear a discussion on one of 10 the reports to see if you want any additional 11 scientific or safety information incorporated and 12 considered before considering add-ons. 13 DR. SHANK: No, I don't think there's a 14 data need. It's just how do we interpret that 15 assay? 16 MS. FIUME: And, Dr. Shank, I think it 17 was probably after this report was packaged and 18 sent out. We did find some information from I 19 want to say 1999 or the last time it was reviewed 20 where it was discussed and the panel at that time 21 had decided that the problem was it was the 22 choline deficiency causing the problem. It wasn't Anderson Court Reporting Panel Book Page 14 Anderson Court Reporting

18 CIR Meeting day 1 of 2 ( Main Session) Page: the DEA, it was the choline, and there was a 2 discussion. So, I will capture that, as well, it 3 was just discovered after it came out. But if you 4 don't agree what may have been said at that time, 5 then I'll capture something differently or look 6 for different information. 7 DR. SHANK: kay, I'd have to read that, 8 but I was on the panel at that time, so, I 9 shouldn't make the same argument all over again. 10 We don't need to discuss that now, and we'll see 11 what we said 11 years ago. 12 DR. MARKS: Well, basically in 2008, the 13 panel agreed that the NTP findings of 14 carcinogenesis in the mouse for DEA and certain 15 DEA fatty acid esters was related to choline 16 (inaudible) and not relevant to human health. 17 Tom, is that your recollection? 18 DR. SLAGA: (Nodding) 19 DR. MARKS: I think that's how we dealt 20 with the mouse carcinogenicity. 21 DR. HILL: But I had a question based on 22 information that was in both presentations at the Anderson Court Reporting CIR Meeting day 1 of 2 ( Main Session) Page: meeting. f course, that was long before my 2 participation. Both Dr. Lehman-McKeeman, I don't 3 know if I'm saying her name right, and Dr. Stott 4 mentioned that DEA is incorporated in ceramides 5 and possibly sphingomyelins, and then in the 6 discussion of DEA, that whole possible mechanism 7 is dropped, and because I guess there's a 8 pharmacologist in our department who's working on 9 that and effects on cancer stem cells and 10 apoptosis, I want to know if that thread of 11 biology has continued or people have just ignored 12 those pieces of information which came from 13 industry source presentations. Whether there's 14 been any follow-up whatsoever on that biology. 15 And that's one of the reasons why I was looking to 16 see this split was because there may be an issue 17 with DEA biology that doesn't show up at all that 18 shouldn't be an issue with TEA, that shouldn't be 19 an issue with monoethanolmine, but it very well 20 might be a big issue with DEA and only DEA. 21 DR. BYER: Well, that mechanism seems 22 to certainly distinguish DEA from the other two. Anderson Court Reporting CIR Meeting day 1 of 2 ( Main Session) Page: As far as I know, there has been no significant 2 progress in terms of developing information to 3 interpret or to determine the importance of those 4 observations, the observation that DEA seems to be 5 incorporated into possible lipids. And there's a 6 lot of speculation about what could happen and how 7 that mechanism might explain some of the toxic 8 effects not necessarily the carcinogenicity. 9 DR. HILL: Well, ceramides have a strong 10 role to play in regulating apoptotic pathways, as 11 well as proliferative pathways, and these were 12 mentioned in two different presentations by two 13 independent labs. So, I guess I'm raising it now 14 so that in mining the literature, whatever might 15 be out there, you will be attuned to looking for 16 anything. 17 DR. BYER: Absolutely. 18 DR. HILL: (off mike) 19 DR. BYER: Right. 20 DR. HILL: And I'm not thinking that 21 this is at all relevant in any of the amides of 22 DEA because I doubt that DEA is significantly CIR Meeting day 1 of 2 ( Main Session) Page: generated from those amides. But I think it might 2 be something specific to DEA, which I guess is 3 really not used much at all at this point. I get 4 the sense. 5 DR. BYER: Right. 6 DR. HILL: But it would be clean if 7 those three were dealt with separately then in 8 going to -- because I can envision language in 9 something that's reviewed that's structurally 10 similar, like the kinds of ingredients you were 11 suggesting to expand to. The panel has previously 12 reviewed DEA. We note the structural similarity, 13 but the specific toxicological issues pertaining 14 to that compound don't pertain to any of these, 15 and here's why. 16 DR. BYER: Yes. 17 DR. HILL: And, so, it would be very 18 clean to be able to refer to that single report 19 and not give issue with the other two that I don't 20 think have any same issues at all. 21 DR. MARKS: Would you like to, since 22 there are three separate reports within this Anderson Court Reporting Panel Book Page 15 Anderson Court Reporting

19 CIR Meeting day 1 of 2 ( Main Session) Page: document, should we, again, sort of have a preview 2 of what's coming down the road, take a look at 3 them individually? I think that the conclusion in it's going to be a little interesting if 5 we keep the same wording. So, his conclusion that 6 TEA, DEA, and MEA are safe for use in cosmetic 7 formulations designed for discontinuous brief use 8 followed by thorough rinsing from the surface of 9 the skin. And products intended for prolonged 10 contact with the skin, the concentration 11 ethanolamines should not exceed 5 percent, MEA 12 should only be used in products that do not 13 contain nitrosating agents. 14 So, I know the TEA and the MEA, Ron, you 15 suggested this same conclusion or something 16 similar is going to be okay. The DEA, there's 17 going to be changes. 18 Do you want to go through these 19 individually now? We dealt with the mouse, I 20 think, where the choline metabolism not relevant 21 to the human. We disused the nitrosamine 22 formulation concern left to deal with the ban in Anderson Court Reporting CIR Meeting day 1 of 2 ( Main Session) Page: EU and Canada. r restricted. 2 DR. SLAGA: Wasn't it suggested to have 3 two reports instead of three? I mean, I thought 4 that's what you were thinking, too. No? 5 DR. SHANK: No, my suggestion was one 6 report with three sections. But we all decided 7 three individual reports. I think. 8 DR. SLAGA: I thought you meant that you 9 wanted to have TEA and MEA combined because they 10 all have the same conclusions. 11 DR. ANDERSEN: I wanted all three 12 combined. ne single report with three sections. 13 But that's a minority opinion. 14 DR. HILL: Well, if there's nothing to 15 ceramides and if there's nothing to more than 16 choline deficiency in that particular assay then 17 you could keep them combined. I guess in my mind, 18 it's somewhat dependent on the toxicology here. 19 MS. BRESLAWEC: We really would prefer 20 separating them out in one form or another because 21 it's caused a lot of confusion when we've looked 22 at derivatives or components that contain DEA or Anderson Court Reporting CIR Meeting day 1 of 2 ( Main Session) Page: MEA or TEA. Administratively, it's very difficult 2 to deal with them in the same report. So, whether 3 it's one report with three sections, we're fine 4 with that, or three separate reports, we're fine 5 with that, as long as each of the ingredients are 6 handled separately. 7 DR. MARKS: Well, certainly, if they 8 were all in the same report, we wouldn't be 9 dealing with taking a combined report in 1983 and 10 now re-reviewing it and creating three separate 11 reports. 12 Ron Hill and Tom, does it matter to you 13 whether they all be combined in the one report and 14 three sections or three separate reports? 15 DR. SLAGA: Really, it's the same thing. 16 DR. MARKS: Yes, except we have to know 17 which way we're going to go as we proceed. Should 18 we wait and see what the Belsito Team says? I can 19 see there's not a strong DR. SLAGA: -- (ff mike) six reports. 21 DR. MARKS: Yes, six. I can see there's 22 not a great strong feeling one way or another, as CIR Meeting day 1 of 2 ( Main Session) Page: long as it's separated. So, we'll just say 2 separated in either3 separate reports or within 3 one. 4 Anything more in terms of looking at 5 these individual ones before we come back to this 6 in a future meeting? If there anything else you 7 wanted, Monice, to get any directions? 8 MS. FIUME: I just wanted to make sure 9 so from my understanding, what we will bring back 10 at the next meeting is three reports with what we 11 feel were the proper add-on ingredients that you 12 are more than welcome to take out, but this way, 13 we'd at least have it prepared for you as what we 14 think the next iteration of the reports are. 15 Is that correct? 16 DR. MARKS: Yes. Are there any data 17 needs for these individual ones at this point, and 18 is there enough in this report in terms of the 19 data? Certainly from irritation and 20 sensitization, I thought it was fine. Is there 21 anything else in terms of data needs? 22 Ron, you had mentioned one concern you Anderson Court Reporting Panel Book Page 16 Anderson Court Reporting

20 CIR Meeting day 1 of 2 ( Main Session) Page: had, but -- 2 DR. HILL: It was just an information, 3 sort of see if there's anything out there request. 4 Not a data need. 5 DR. MARKS: So, it sounds like the main 6 thing we're going to do next time is clarify the 7 discussion concerning the mouse and concerning the 8 nitrosamine formation for each of these as 9 separate ingredients, and then decide on the 10 add-ons, but in terms of data, it seems like we're 11 okay at this point. Is that DR. BRESLAWEC: Just to clarify one 13 thing, they'll be draft amended reports that 14 you'll get next time. 15 MS. FIUME: And then the only other 16 thing I was going to say is in Wave 2, you should 17 have received what the original re-review summary 18 was for DEA. So, I will pull from that 19 information, as well, that includes some of your 20 decision-making or conclusion as to why it went 21 the way it did. 22 DR. MARKS: Anything else? Anderson Court Reporting Panel Book Page 17

21 June 2009 Belsito Team Then our next item is DEA carcinogenesis. This is I think essentially being prompted because of issues of neurotoxicity data and we have these two papers by Craciunescu and by Craciunescu again looking at reports of DEA altering neurogenesis and inducing apoptosis and in fetal mice hippocampus, and then the other about dose response effects of dermally applied DEA neurogenesis. In the dermal paper even the authors acknowledged that the dermal penetration in the detectable plasma levels are such that they are far below the concentrations associated with perturbed brain development in mice. Then of course we had the prior issues that we had dealt with in the report about species difference, absorption differences and effects on choline metabolism that aren't really pertinent to humans. It wasn't clear to me what we're supposed to be doing with this, whether this was just an update to decide to open or not reopen. DR. ANDERSEN: I think my intent, and let's see if it matches what anybody actually read, is we had focused on the question of DEA carcinogenesis. There are now ample data both mechanistic, et cetera, to demonstrate that the positive findings are indeed species specific and they relate to choline metabolism. The summary that you have puts all of that together and on being published would resolve that question. The Panel would be on record as saying we buy into the choline metabolism as being the mechanistic cause and confirming that these materials as used in cosmetics do not present a risk of carcinogenesis. That was the purpose of summarizing all of that history. What we had not talked about in any way, shape or form were these new data on neurotoxicity and the question that is on the table is what should we do about those data. Should it cause another round of review or are you comfortable enough saying that it doesn't need to be reopened? When we had round after round for example with thalates and another new set of data came in, we did briefly reopen to consider those data. It turned out there was no formal need to reopen, but at least briefly we considered it. Were those data not there, it would just be a re-review summary and that would be the end of it. What do you think makes sense to do now that we have two studies with neurotox endpoints? DR. BELSIT: I think we addressed the species difference and the choline metabolism in our last re-review of something with DEA. DR. ANDERSEN: Yes. DR. BELSIT: In this case you have the same author who reports the effects on mice coming back and saying when you dermally apply it which is the relevant thing in cosmetics, there is no effect on the mice. So it's not like we have to defend the cosmetic use. The same author who had made these reports I think has defended it for us. I'm not sure that we really need to do anything with it. That was my feeling. It's not like we have to say this author reported it in mice but now you have to look at dermal absorption and you have to look at choline metabolism. He's already done that for us. DR. LIEBLER: Particularly in view of the fact that the neurotox effect is rationalized in terms of the choline metabolism effect as well. DR. SNYDER: I think the only issue is that we're kind of stuck because we're in the middle of a re-review and I really don't like not having it appear as though we didn't consider it these reports even though we understand that the mechanism is already known and we've already addressed the mechanism but it may not be readily apparently clear to the reader how we addressed that. DR. BELSIT: Was it time for a DEA re-review or did this just get accelerated because of these reports? I thought we just did it. DR. ANDERSEN: We just did it. This is the summary of what we did last year. We haven't had time to preparing the summary. DR. BELSIT: Because I thought we had already issued that and now we're looking at it again because of new data. DR. ANDERSEN: No. DR. BERGFELD: Don't you think you'd just add this to the paragraph on page 2 as to the choline to just update the reference? DR. BELSIT: Yes, I would just do that. DR. BERGFELD: I think it's still an unusual summary because it also includes the safety assessments of the others, and there are several of them which we haven't done particularly in the past. This is a new entry, or maybe once before. While we're discussing those I wondered if we couldn't go back and look at the citation that appears at the end of each paragraph under those ingredients containing N-nitrosating agents is one statement, another one is in which N-nitroso compounds are formed containing nitrosating agents. It seems to me we're talking about the same thing there even though we've used different terminology. DR. ANDERSEN: That's correct. ver time there have been three different ways that we've phrased it. The intention in all three cases is the same. Yes, we could develop a single language. My intent in presenting Table 1 was to simply capture what's in the documents as opposed to unifying them. DR. BERGFELD: I thought that was a good idea actually. DR. ANDERSEN: But it certainly brings into great relief the fact that we've used different phraseology over time. There's no question about that. Panel Book Page 18

22 DR. BELSIT: I would agree with Wilma's comment. I was confused. I thought we had already re-reviewed it and passed on it. If this is just our final review of the document, I don't think it changes anything. Just put statements as to these papers and include the author's conclusion which I think exonerates us. n the paper though I just have one question. n the second page, the fifth line down sort of in the middle it says, "In studies with multiple (3)", I'm assuming that there were three studies or was it three body lotion doses, and I think by putting the number in parenthesis now that we're using numbers for references also could be confusing as to whether that was the reference. Was it three studies with different body lotion doses, was it one study with three different body lotions? That needs to just be clarified. DR. ANDERSEN: It was the latter I'm pretty sure, but I'll confirm that. And, yes, we have to be careful about how we do stuff now that we're presenting it differently. DR. BERGFELD: n page 1 if I could interrupt, the second paragraph, "In addition, work done at the FDA." Were they reviews? What kind of work was done? What is the work done? DR. ANDERSEN: That was work that Bob Bronaugh presented during the discussion on his data on DEA penetration. DR. BERGFELD: Is there another way of stating that other than "work done"? DR. ANDERSEN: Yes, there. DR. BELSIT: And "data presented by"? DR. ANDERSEN: Yes, and that should have a reference as well. DR. BERGFELD: Could I ask a question? Are we going to have a book with all these summary statements somewhere so we could reflect on a format and the changes in the format? DR. ANDERSEN: Soon. That is one of the tasks that Halyna Breslawec has taken on and she's busily working on it. She was almost ready to put it on the agenda for this meeting but not quite, so a discussion of our precedent files you can expect depending on whether September's agenda is really heavy or not, but I'd like to get it on the September agenda. DR. BAILEY: So are you talking about consistency for the nitrosamine statement or just consistency of the format? DR. BERGFELD: All of it. DR. ANDERSEN: Everything. We're looking at soup to nuts. DR. BERGFELD: Particularly in these. We are beginning to enlarge these re-review summaries when we don't reopen and they are developing into a fair amount of text. I'd just to take a look at what we've been doing. DR. ANDERSEN: Thank you. June 2009 Marks Team DR. MARKS: -- see, let's move on. Since Wilbur isn't here, we have a couple other things to do before the cyclomethicones. Next in the agenda I have is the re-review summary of DEA carcinogenesis and then we happened to get a couple papers on the issue of neurogenesis and neurotoxicity with DEA. So let's just start first with this -- DR. ANDERSEN: Not -- in addition of neurogenesis. DR. MARKS: Yes. Right. DR. ANDERSEN: I mean if it actually increased neurogenesis -- SPEAKER: You'd want some. SPEAKER: (off mike) DR. HILL: Well, not in utero, however. DR. MARKS: So -- any rate -- Alan asked us to look over this re-review and summary and how does it appear? Tom, you're the one I have -- how do you like the summary? r do you have any -- any suggestions? DR. SLAGA: Well, I think the summary is in good shape -- I mean other than a few typos and that type of thing. The papers -- on the one hand at higher concentrations, you could have some apoptosis as well as some inhibition of neurogenesis. But in a small human study they did at (off mike) -- that was -- it had no effect on the brain development in the mouse at the levels they looked at it. The mouse was a much higher level than you would find in any cosmetics -- much higher. So -- to me -- I don't think it's an issue. DR. ANDERSEN: Well, in the metabolic issues, mouse versus human are still there in terms of colene deficiency, etc. DR. SLAGA: Right. Yeah -- no -- that colene part is definitely in amounts that doesn't seem to be a relationship with human. DR. ANDERSEN: Yeah, I was just concerned that our entire focus has been on DEA and DEA fatty acid carcinogenesis and I think that issue was nicely resolved -- DR. SLAGA: Yeah -- resolved. DR. ANDERSEN: -- and then this hit of something we hadn't really talked about. DR. SLAGA: We probably emphasized it a little too much, but at the same time it's in the literature a lot and it's good to Panel Book Page 19

23 eliminate if there is a concern. DR. SHANK: The neurotoxicity should be added to the review (off mike). DR. SLAGA: Yeah, to the review. But not (off mike). DR. ANDERSEN: (off mike) DR. SHANK: Acknowledge it. That's all. DR. ANDERSEN: Just include a sentence that says it exists. DR. SHANK: kay. DR. MARKS: kay. And in -- DR. ANDERSEN: Thank you. DR. MARKS: -- with that you include the 2009 use of concentration table, correct? Alan, (off mike)? DR. ANDERSEN: Well, that -- I decided not to do that for this summary because the focus was on simply the question of carcinogenesis. DR. MARKS: kay. DR. ANDERSEN: It didn't focus on the question of use concentrations at all. You didn't -- we never talked about that. The issue was is this stuff carcinogenic -- I'm sorry. Does it present a carcinogenic risk to humans? And the answer was no. So we didn't go on to talk about use concentration. There was an absence of a hazard, so exposure wasn't so important. We have to go -- Carol would have to go resurvey to get valid data for use and I'm not sure that's worth it. DR. MARKS: kay. I just -- DR. ANDERSEN: I think if -- you know -- brining it up tomorrow and let's see if anybody else is concerned. If so, then we can -- it's just a matter of doing another survey. DR. SLAGA: Nothing to it. DR. MARKS: No -- yeah -- I was just putting it in the format of what we usually do with a re-review. DR. ANDERSEN: It's different -- (off mike) it's different on purpose. DR. MARKS: Yeah. kay. Next is the hair dye epidemiology. We heard the presentation this morning by Julie Skare. Skare? June 2009 Full Panel DR. BERGFELD: I do. All right, then we'll move on. The last ingredient is a re-review summary of the DEA carcinogenicity, and that is Dr. Belsito. DR. BELSIT: Yes. This was a re-review we did the last time, and in the interim there were two reports that surfaced, one on diethanolamine altering neurogenesis and inducing apoptosis in fetal mouse hippocampus and then by the same author is the dose response effective dermally applied diethanolamine on neurogenesis. And while we found these interesting, they're really not relevant for several reasons. ne, we know that these effects are due to choline and there's a difference between murine and human metabolism. But even the authors in the second paper applying a commercially available skin lotion noted that the concentrations that were absorbed were far, far below the concentrations that would exert any effect. So, while we felt it was important that we add these references to the document just to show that where everything is up to date in Washington that it didn't really change that re-review summary and to add them and go ahead with it, issue it as final. DR. BERGFELD: Was it my understanding that you were going to add that to the paragraph on 2, which describes the choline metabolism as well? DR. BELSIT: Yes. DR. BERGFELD: And so your motion is -- DR. BELSIT: To go ahead with this as a final re- review with this, the simple addition of those two references. DR. BERGFELD: And agree -- DR. MARKS: Yea, our team concurs with that. DR. BERGFELD: Any further discussion then? Seeing none, I'll call for the vote. All those in favor of proceeding? Thank you. Unanimous. Re-review Summary of DEA Carcinogenesis June 2009 Executive Summary The Expert Panel reviewed and approved the summary of its earlier re-review decision to not reopen the safety Panel Book Page 20

24 assessment of Diethanolamine (DEA), Cocamide DEA, Cocamide MEA, Isostearamide DEA & MEA, Linoleamide DEA, Myristamide DEA & MEA, leamide DEA, and Stearamide DEA & MEA. This re-review was unusual in that it focused on an endpoint, carcinogenesis. The CIR Expert Panel reviewed the large body of data developed since studies conducted by the NTP reported clear evidence that dermal exposure to DEA and Cocamide DEA were hepatocarcinogenic in male and female mice. The body of work now available has demonstrated that DEA affects mouse choline metabolism, which leads to cellular choline depletion, which leads to altered DNA methylation, which leads to altered gene expression (increased DNA synthesis and oncogene expression, reduced tumor suppressor gene expression and apotosis). At this meeting, the Expert Panel also considered two newer studies that reported effects on neurogenesis in mice, noting that the mechanism of action likely involves the effect of DEA on mouse choline metabolism. June 2008 Presentation by Dr. Stott Minutes summarizing presentation Dr. William Stott, representing the Alkanolamines Panel of the American Chemistry Council, reviewed the large body of data developed since the point 10 years ago when studies conducted by the NTP reported clear evidence that DEA and Cocamide DEA were carcinogenic in male and female mice. The work has demonstrated that DEA affects mouse choline metabolism, which leads to cellular choline depletion, which leads to altered DNA methylation, which leads to altered gene expression (increased DNA synthesis and oncogene expression, reduced tumor suppressor gene expression and apoptosis). In work done at FDA and elsewhere, dermal penetration of DEA in personal care product vehicles was found to be significantly higher through mouse skin than either rat or human skin. In addition, the activity of choline oxidase, which is hundreds of times higher in the mouse compared to humans, suggesting that humans are resistant to choline deficiency choline oxidase levels in the rat, however, are even higher than in the mouse. verall, the available data support that DEA carcinogenesis in the mouse is related to choline depression and the effect is reversible and threshold-based. Given the known resistance in humans to choline deficiency, these data do not suggest a human health risk from the use of DEA and DEA fatty acids in cosmetic products. June 2008 Acetamide MEA Belsito Team (Valerie s Notes) Dr. Belsito: Why was there a 7.5% limit? This is inconsistent with the MEA/TEA/DEA report. If reopened to add, the issues with MEA in leave on products versus rinse off products will come up Dr. Eisenmann: Would MEA have its own group instead? Dr. Belsito: MEA ingredients as a group? Dr. Andersen: The MEA report was not addressed in the Acetamide MEA report. Dr. Eisenmann: There is a 2-gen. study going on. Dr. Andersen: Can be tabled to wait for the results. Dr. Belsito: Table until the results come in. What ingredients should be added? Any data needs? Format as Sodium Cetearyl Sulfate afterwards. Create an Alkonolamide MEA family report. Marks Team (Valerie s Notes) Dr. Slaga/Shank: Do not reopen. Dr. Shank: No add-ons due to the limitation in the original conclusion and sensitization. Dr. Bergfeld: Agree. Dr. Shank: The add-ons may have different absorption and sensitization due to the addition of fatty acid esters, which can increase penetration. Dr. Bailey: MEA ingredients have few uses. Dr. Marks: These add-ons are not no-brainers. Do not reopen/add. Panel Book Page 21

25 June 2008 Full Panel Chairman s pening Remarks Dr. Bergfeld welcomed the attendees to the 107 th meeting of the CIR Expert Panel. She noted that the following three presentations were made on the preceding day: (1) The presentation by Dr. Bill Stott was on DEA carcinogenicity. The data presented assured the Panel that there is no human risk that is associated with having these chemicals in cosmetics. It was learned that the mouse model that the Panel was somewhat worried about, i.e., due to liver tumors, is not relevant to humans. Acetamide MEA Dr. Marks stated that a CIR Final Report with the following conclusion on this ingredient was published in 1993: n the basis of the data presented in this report, the CIR Expert Panel concludes that Acetamide MEA is safe as a cosmetic ingredient at concentrations not to exceed 7.5% in leave-on products and is safe in the present practices of use in rinse-off products. Cosmetic formulations containing Acetamide MEA should not contain nitrosating agents or significant amounts of free acetamide. Dr. Marks said that this conclusion was reaffirmed by his Team and, thus, this safety assessment should not be reopened. He added that his Team considered the list of chemically similar ingredients that was provided and determined that, in light of the 7.5% concentration limit, actual data on all of the chemically similar ingredients would be needed if a decision to reopen the Final Report were made. Dr. Belsito said that his Team determined that the Final Report should be reopened, taking into consideration the following information: The CIR Final Report on MEA states that this ingredient should not be used in leave-on products, and it was noted that Acetamide MEA breaks down to MEA. After further review of the Final Report on Acetamide MEA to determine the basis for the 7.5% concentration limit for leave-on products, it was determined that, in the absence of leave-on product uses, it was necessary to establish a concentration limit for Acetamide MEA in these products. The 7.5% concentration limit is actually the highest concentration that was tested in skin sensitization studies. Dr. Belsito stated that his Team determined that the Final Report on Acetamide MEA should be reopened and that MEA should be added to this document along with other MEA ingredients selected from the list that was provided. Dr. Shank noted that MEA is one of 3 ingredients included in the published CIR Final Report on TEA, DEA, and MEA. Dr. Belsito said that the plan is to incorporate all of the data on MEA from this Final Report into the reopened document on Acetamide MEA, creating an MEA ingredient family. Dr. Shank said that MEA belongs in the primary document on ethanolamines, and not in the document on a secondary ingredient such as Acetamide MEA. Dr. Belsito noted that an MEA ingredient family is being created, consisting of MEA (lead ingredient), Acetamide MEA, and selected MEA ingredients. The plan is to reopen MEA, but not the Final Report on Acetamide MEA, and add Acetamide MEA and MEA ingredients selected from the list that was provided. Dr. Bailey said that he believes that Acetamide MEA is a very stable compound and would not break down very easily. He added that the idea that consumers would be exposed to MEA that has been released from Acetamide MEA is probably not true. Dr. Bailey recommended that the CIR Final Report on MEA, DEA, and TEA remain as a separate document and not be amended. He questioned the need to reopen the Final Report on Acetamide MEA because this ingredient is a stable compound and noted that this report should remain as a separate document. He added that the notion of grouping is reasonable, because the smaller molecule is going to be different from the longer-chain MEA derivatives (also very stable compounds). Dr. Belsito wanted to know if Dr. Bailey also thinks that, because Acetamide MEA is a short-chain compound, the Panel should not recommend the addition of other ingredients to this safety assessment. Dr. Bailey agreed that this approach would be reasonable because the longer-chain MEA derivatives would be somewhat different, both chemically and biologically. Dr. Belsito said that, typically, greater toxicity is associated with the shorter chain compounds. Dr. Shank noted that the fatty acid ester component would increase dermal penetration, making compounds more lipidsoluble. Dr. Bronaugh agreed. Dr. Snyder wanted to know whether there are any issues that are related to ingredient use in new product categories. Dr. Belsito said that Acetamide MEA is being used in some sprays/aerosol fixatives, and that the Panel s boilerplate relating to particle size and inhalation exposure could be incorporated. He added that use concentrations have not increased. Dr. Snyder said that there would likely be a new concentration limit if the Panel receives new data on Acetamide MEA indicating negative sensitization at higher concentrations. Panel Book Page 22

26 Dr. Belsito said that a statement expressing the following points should be incorporated into the discussion: The Panel is aware of the fact that MEA may be used in rinse-off, but not leave-on, products, but agrees that Acetamide MEA would be stable on the skin. Therefore, there are no safety concerns, particularly in light of the sensitization data in the final safety assessment on MEA. Dr. Belsito said that the preceding statement is needed because, otherwise, it appears that the Panel has issued contradictory opinions. The CIR Final Report on MEA states that this ingredient should not be used in leave-on products, yet the Expert Panel established a concentration limit for Acetamide MEA in leave-on products. Dr. Marks added that the discussion should also contain a statement explaining why the safety assessment on Acetamide MEA was not expanded to include chemically similar ingredients. This statement could read as follows: The Panel considered chemically similar ingredients, but, because of skin sensitization and increased skin penetration issues, the decision not to expand this group was made. The Panel voted unanimously in favor of not reopening the CIR Final Report on Acetamide MEA. Dr. Belsito said that if the Panel is not going to reopen this document, then there is no need to reopen the CIR Final Report on TEA, DEA, and MEA. Miscellaneous from the Minutes Near adjournment Dr. Bergfeld stated that the presentations on diethanolamine carcinogenesis and hair dye epidemiology from the preceding day will be captured in the minutes. In response to Dr. Snyder s concern, Dr. Andersen stated that CIR will formally undertake a re-review summary, for the Panel s consideration, that will describe all of the DEA fatty acids that have been reviewed and all of the available data on DEA. Dr. Andersen added that the overall plan is to prepare a short re-review summary that could be published and made available on-line, as the final denouement of the Panel s consideration of DEA. Dr. Andersen noted that Jonathon Busch, with the American Chemistry Council, has agreed to provide his extensive bibliography and make sure that CIR has all of the relevant information that needs to be captured. This will be presented to the Panel with a summary that captures the Panel s questions that were addressed to Dr. Stott on the preceding day and the Panel s comments to the effect that DEA carcinogenicity may be a mouse phenomenon and not relevant to DEA use in cosmetics (i.e., human exposure). Panel Book Page 23

27 CSMETIC INGREDIENT REVIEW ANNUNCEMENTS Dr. William Stott, representing the Alkanolamines Panel of the American Chemistry Council, reviewed the large body of data developed since the point 10 years ago when studies conducted by the NTP reported clear evidence that DEA and Cocamide DEA were carcinogenic in male and female mice. The work has demonstrated that DEA affects mouse choline metabolism, which leads to cellular choline depletion, which leads to altered DNA methylation, which leads to altered gene expression (increased DNA synthesis and oncogene expression, reduced tumor suppressor gene expression and apotosis). In work done at FDA and elsewhere, dermal penetration of DEA in personal care product vehicles was found to be significantly higher through mouse skin than either rat or human skin. In addition, the activity of choline oxidase, which is hundreds of times higher in the mouse compared to humans, suggesting that humans are resistant to choline deficiency choline oxidase levels in the rat, however, are even higher than in the mouse. verall, the available data support that DEA carcinogenesis in the mouse is related to choline depression and the effect is reversible and threshold-based. Given the known resistance in humans to choline deficiency, these data do not suggest a human health risk from the use of DEA and DEA fatty acids in cosmetic products. Panel Book Page 24

28 June 1999 Meeting Industry Presentation Diethanolamine - Update on Research Dr. Loretz stated that industry has conducted research in an effort to understand the mechanism of action of DEA in the NTP carcinogenicity study that was completed. Specifically, she noted that the Ethanolamines Panel of the Chemical Manufacturers Association (CMA) and Procter and Gamble Company have been involved in this research program. Today s speakers are Dr. William Stott (with Dow Chemical Company) and Dr. Lois Lehman-McKeeman (with Proctor and Gamble). The slide presentations by both speakers are inserted at the end of the minutes. Dr. Lehman-McKeeman s presentation is included below. I will discuss the results of ongoing research regarding the toxic effects of DEA. Particularly, I am going to focus on the work that is being done in my laboratory at Procter and Gamble. DEA is a secondary amine that is widely used in commerce; particularly, it is used in the synthesis of long-chain fatty acids for a variety of consumer products. The issue that brings us all here to discuss this research is the fact that in a long-term bioassay, DEA has been shown to be carcinogenic, specifically, in mice. This was a two-year dermal study in which mice were treated with 40, 80, or 160 mg/kg/day. Essentially, the tumor incidence was 100%, regardless of the dose that was administered. In these livers, there was also an observed increase in the actual multiplicity of tumors. So, as opposed to a single tumor per liver, DEA-treated mice typically exhibited four to six tumors per liver. It should be noted that, in this particular study (B6C3F1 mouse strain), there was a very high spontaneous rate of tumor formation. The rate, in this particular study, in the control group was between 65 and %. DEA also produced some incidence of kidney tumors in mice, particularly in males. In direct contrast to the effects in mice, dermal application in rats produced absolutely no carcinogenic effects. The dosage applied to the rats was significantly lower than that observed in the mouse. Actually, rats respond somewhat differently to DEA. It has been shown that the absorption across the skin is far less in a rat, and DEA is much more irritating to the skin of rats. In combination with this carcinogenicity data, it has also been shown in a fairly comprehensive battery of tests (particularly those conducted by NTP) that DEA is in no way DNA reactive. So, it appears that this is some kind of non-genotoxic event, leading to tumor formation. For the purpose of this talk, I would really like to focus on the hypothesis that we developed and the results that we have obtained in testing this hypothesis to explain the formation of these liver tumors in mice. Based on the structural similarity of DEA to phospholipid precursors, including ethanolamine and choline, we postulated that DEA treatment may, in fact, be disrupting choline homeostasis in some way that produces a biochemical condition that is similar to choline deficiency. If this were the case, then, the DEA-induced liver tumors may be resulting from chronic choline deficiency. I think that we all recognize that choline is an important precursor in the synthesis of a variety of essential cellular components, including phosphatidylcholine (phospholipid) and acetylcholine (neurotransmitter). It is also incorporated into agents such as sphingomyelin and liposhingomyelin, both of which are functionally important in signal transduction mechanisms, as well as platelet activating factor. So, with respect to the latter, it can contribute to allergic and inflammatory reactions. The significance of a relationship with choline deficiency or the perturbation of choline homeostasis is that choline deficiency, in and of itself, is known to be carcinogenic in rodents. Importantly, this is the only single nutritional deficiently that is known to be cancer-causing in rodents. The mechanism of this carcinogenic effect is not fully characterized. But, given the involvement of choline in so many fundamental processes, it has been postulated and shown that a variety of cellular changes occur in the presence of deranged choline homeostasis (i.e., cell proliferation, changes in methylation patterns of genes, activation of protein kinase C, etc.). To test this hypothesis, we chose to first evaluate it in a rapidly proliferating cell type, specifically the Chinese hamster ovary (CH) cell. The CH cell was chosen for the following two reasons: (1) As a proliferating cell, we felt that it would be sensitive to these effects. (2) Phosphatidylcholine synthesis and choline homeostasis are very well characterized and understood in the CH cell. So, we felt that if these experiments demonstrated an effect in this area, it would argue that there was some plausibility to this hypothesis. If, on the other hand, the CH cell did not show an effect, this would tell us that this is not a credible hypothesis. So, we simply did experiments with standard media which often used a CH cell culture that contained 100 μm choline in the medium. We exposed these cells to DEA and then used 33P phosphate to label the phospholipid pool. The cells Panel Book Page 25

29 were extracted after a 48 h culture and subjected to a TLC separation that allowed us to isolate and separate the major phospholipids in the cells. Elution of the phophatidylcholine is indicated on the autoradiograph. We found that at test concentrations of 20 to 1,000 μg/ml, there was basically no cytotoxicity until we reached a concentration of 1,000 μg/ml. So, there was really no change in cell number or in the total phosphate incorporated into those cells. However, we found a concentration-dependent reduction in the utilization of 33P into the phosphatidylcholine band. So, this was indicative of the fact that DEA was indeed disrupting phosphatidylcholine homeostasis in these cells. This effect can occur in two ways, and this schematic (in slide presentation) simply summarizes the ways in which phosphatidylcholine synthesis is regulated in the CH cell. Free choline is taken up into the cell and ultimately incorporated into the phosphotidylcholine phospholipids. First of all, the uptake of choline can occur in one of two ways, either by an energydependent facilitated transport or by simple diffusion at higher concentrations. nce taken up, it is phosphorylated and phosphocholine is the intracellular storage pool of choline. There is another way that these cells can incorporate phosphatidylcholine, and that is to actually take it up from the serum that exists in the media, which would then require hydrolysis to liberate the free choline. So, DEA would disrupt the uptake of choline into the cell, and, secondarily, it could also be affecting the incorporation of choline into these precursors, yielding phosphatidylcholine. So, we looked at both the uptake and the utilization of choline in the presence of DEA. To look at the uptake of choline, we simply tracked tritiated choline that was placed in the media. This is the time course (see slide presentation) over which choline is taken up, and we are focusing on the energy dependent transport. You can see that it begins to plateau after approximately 20 min. So, we chose to look at a time point of 10 min. of exposure to DEA and choline, and those results are shown here. I think that it is obvious that what we found was that at concentrations of 50 μg/ml and higher, DEA was blocking more than 90% of the uptake of free choline into the CH cell. So, this argues that one of the mechanisms by which DEA is disrupting choline homeostasis is very specific in terms of how it is being taken up into the cell. The second thing that we looked at was the utilization of choline into the phospholipid pool. The way that we looked at the effects of DEA on that utilization was to simply determine whether DEA in and of itself was being incorporated into phospholipids as a functional head group. Those results are shown here (see slide presentation). In this case, we cultured the CH cells with a 14C-labeled DEA for 48 h. This is the standard 33P extraction that was done simultaneously. You can see that DEA, once again, was being incorporated into the phospholipid pool. In fact, we tested this at a concentration of 500 μg/ml, and found that 20% of the total DEA ended up in the phospholipid fraction. At the moment, we are trying to identify the phospholipid tails in this fraction. This clearly demonstrates that the cells are actually utilizing DEA in place of or in addition to the natural lipid head groups. Finally, to look at this mechanism, what we wanted to discern was whether this was a reversible or an irreversible effect. So, we did two separate experiments to look at reversibility of the effect. The first one, shown on the left (see slide presentation), is a time-dependent experiment. Again, I am looking at the percent of the total phospholipid synthesis that is phosphatidylcholine. This is our standard result with a concentration of DEA of 500 μg/ml. We can see that there is a marked reduction in the 48 h incubation experiment. If, however, we culture the cells in the presence of DEA for 48 h, then remove the DEA and allow the cells to continue to grow for an additional 24 h, we begin to see that they did, in fact, recover and that the phosphatidylcholine synthesis was returning to normal. The other experiment that we did was to determine whether an excess of choline would prevent the changes that were shown previously. Choline (30 mm) completely overcame the effect of DEA, and there was no change in phosphatidylcholine synthesis. So, these experiments indicate that this is, in fact, a reversible phenomenon. I mentioned at the outset that in a series of standard genetic toxicity tests, DEA was uniformly negative. However, there have been some data published with the Syrian hamster embryo (SHE) cell system in which morphological transformation is evaluated, indicating that DEA does cause morphological transformation of the SHE cells. This assay has been developed and validated against approximately 200 chemicals. It is considered that cell transformation in the SHE cell is indicative of the ultimate carcinogenic potential of a chemical. These data (see slide presentation) simply summarize the concordance, sensitivity, and specificity of this assay. The results obtained using this cell system are approximately 85% predictive of the NTP bioassay results. These are the results (see slide presentation) that were published in a paper that was done in the validation experiment in concert with the NTP. These experiments were done prior to the completion of the DEA bioassay and were used to predict the carcinogenic potential of this chemical. These data were published by a colleague of mine, Dr. Bob Ledet, and Procter and Panel Book Page 26

30 Gamble. MTF represents morphological transformation, and you can see that DEA increased the transformation frequency in these cells. The prediction was that DEA would be carcinogenic. Given all of the work that we have done in the CH cell and recognizing these data, we basically stepped back from them and said, does DEA affect the SHE cell in a manner that is consistent with what is seen in the CH cell? We are in the process of completing these experiments. These are results of an experiment in which we supplemented SHE cells with choline in the presence of 500 μg/ml DEA. Basically, we saw the same effect in the SHE cell that was seen in the CH cell. That is, DEA inhibited phosphatidylcholine synthesis by more than 50%. In the presence of exogenous choline, we again inhibited or prevented that reduction from taking place. With that as background, we postulated that if there were anything to this choline hypothesis (i.e., mechanism involving the disruption of choline homeostasis), then the supplementation of these SHE cells in a standard assay would prevent the transformation from taking place. So, we did that experiment. Due to some toxicity, we lowered the concentration a bit. In the presence of the normal medium, we once again reproduced the transformation potential of DEA in the SHE cell. When we supplemented with choline, you can see that we totally prevented this transformation from taking place (see slide presentation). We did not affect toxicity in these cells nor did we alter the transformation frequency of a positive control that is always used in this assay (benzo[a]pyrene). You will notice that these controls are somewhat different. The historical control transformation frequency is between 0 and 0.6%. So, they don t consider these to be any different. But, when you compare between the cholines-upplemented and control medium, you can see that there is clearly an elimination of the transformation potential. So, we take these results as being fundamentally important and definitely supportive of the fact that DEA is disrupting choline homeostasis. That clearly has an effect on cell transformation, and, likely, on the carcinogenic potential of this chemical. We are now relating what we have seen in these cultured cells to the in vivo experience and the in vivo effects of this chemical. At the moment, I have virtually no data to show you in this regard. However, based on work that has been done by Bill Spunger, we do know that DEA does produce biochemical changes that mimic choline deficiency in the mouse. ne of the things that we are focusing on in our laboratory is to specifically ask why DEA is not carcinogenic in rats. The question is, does DEA produce choline deficiency in the rat? If it does not produce choline deficiency or mimic those changes in the rat, then I think that we have the basis for a species-specific effect related to these biochemical perturbations. If DEA does affect choline homeostasis in the rat, then, we may be looking at the sensitivity of the B6C3F1 mouse in this liver in responding to chemicals like this. The second thing that we are going to look at a little more closely is the dose response relationship for these changes in the mouse, again, recognizing that there is no dose response relationship in the carcinogenicity bioassay. There is also the question of whether the mechanism that has been deduced from cell culture carries over into the whole animal. Specifically, can we show that DEA does inhibit choline uptake into cells, particularly hepatocytes? If these changes are competitive and reversible, then there should be a critical concentration that is required to cause these effects, fundamentally important to a risk assessment process. Lastly, I want to introduce the concept of species differences that may come into play here. This diagram (see slide presentation) summarizes the interaction between the choline and phosphatidylcholine synthesis pathway, and the one-carbon pool in the utilization of methionine and S-adenosyl methionine. Fundamentally, one of the points that I would like to make from this slide is that there are actually significant species differences in the way that many of these intermediates are utilized. In the rodent, there is a very high activity of choline oxidase enzyme that oxidizes choline to betaine. Fundamentally, in rats and mice, if you don t use it, you lose it. It immediately oxidizes this. There are data dating back to 1960 from Manny Farber s laboratory. More recently, the oxidase that catalyzes this reaction has been cloned. What is recognized is that humans are inherently very slow at this oxidation, such that it has been postulated (since the early 1960's) that there are species differences in sensitivity to developing choline deficiency. This ability to inactivate or remove choline from the pathway in the rodent is believed to be one of the factors that really drives them to be very sensitive to these changes. So, as we go through this program (I haven t mentioned humans today, but we recognize this fundamental biochemical difference) and generate data, the critical question that we will be addressing is the human relevance of these findings. [End of Dr. Lehman-McKeeman s presentation] Panel Book Page 27

31 Dr. Slaga asked if there is really any difference between mice and rats. Dr. Lehman-McKeeman said that there is no difference between the two. She said that this is why understanding why that rat doesn t show these changes is critical to really positioning how good this hypothesis is. Dr. Bailey wanted to know if Dr. Lehman-McKeeman said that choline deficiency in mice also leads to liver tumors. Dr. Lehman-McKeeman said that choline deficiency clearly leads to liver tumors in mice and rats. Dr. Belsito recalled that Dr. Lehman-McKeeman had said that rat skin is more easily irritated than mouse skin, but that the skin of the rat is less absorptive. He viewed this as a possible simple solution to the difference between mice and rats that was observed in the carcinogenicity bioassay. Dr. Lehman-McKeeman said that the possible simple solution is that this is a function of the dose. She also said that there are data that show that the penetration across the rat skin is far less when compared to the mouse. Dr. Belsito asked whether any in vitro studies have been done, such as those that have been done using Chinese and Syrian hamster cells, on rat cells to see whether the same effects on phosphatidylcholine in vitro were observed. Dr. Lehman-McKeeman said that this type of study has not been done. She also said that the other part to that is to really address what that difference is in terms of exposure and how significant that is. Dr. McEwen wanted to know if the difference between rat and mouse skin sensitivity that was mentioned is based on data from other experiments or on data from the experiment that she discussed. Dr. Lehman-McKeeman said that it is indicated in the NTP bioassay report that the changes in the rat skin were extreme during the 91-day and two-year studies. She also said that her laboratory just completed a two-week experiment in which irritation was observed in the rat. Dr. McEwen wanted to know whether this observation is due to the fact that mice can groom, but that rats cannot. Dr. Lehman-McKeeman said that she does not know whether it is due to the fact that the mouse licks the material off of its back or whether it is a function of the rapidity and completeness with which this chemical penetrates mouse skin. Dr. McEwen said that it is not known whether the same kinds of skin effects would have been observed in the mouse, had the animal been properly collared and rendered unable to groom. Dr. Lehman-McKeeman agreed with Dr. McEwen s comment. However, in light of the comment, she also said that she believes that DEA penetrates the skin quickly and effectively and, therefore, does not remain on the surface. Dr. Slaga agreed that DEA is not DNA-reactive, but also recalled that it gives rise to free radicals. Additionally, he said that choline deficiency will modify DNA bases and increase free radicals. Dr. Lehman-McKeeman said that she has not seen any data suggesting that DEA gives rise to free radicals. She said that there has been some emphasis on whether or not, as a secondary amine, it can be nitrosated to a nitrosamine. Dr. Slaga said that there have been several published studies on a series of compounds in which the mouse was compared with the rat. He said that, in general, many compounds that are not DNA-reactive will lead to free radical damage in the mouse, but will not in the rat. For example, dieldrin causes tumors in the mouse liver but not in the rat. Dr. Slaga said that so many different compounds fall into this category, and that this may be the case with DEA. Dr. Lehman-McKeeman said that Dr. Slaga s point is well taken, and that it is possible that her laboratory may be addressing this phenomenon. Panel Book Page 28

32 The presentation by Dr. William Stott, on behalf of the Chemical Manufacturers Association Ethanolamines Panel, is included below. Along with some funding from CTFA, I have been sponsoring several studies (work in progress). When we looked at the tumor data, we said that the data resemble the effects of a nitrosamine and wanted to know whether there is an alternate method that could be promoting these effects. ur research program is focused on the following three areas: (1) potential nitrosamine formation in vivo in mice, (2) potential choline deficiency scenario, alone or promoting the effect, and (3) once we had identified what we thought might be a mechanism suggesting a mode of action, we felt that it would be important to do interspecies comparisons. In the first experiment, B6C3F1 mice (strain used in NTP bioassay) received a carcinogenic dose of 100 mg/kg DEA for two weeks by three different means. A skin painting study was performed with the intention of mimicking the NTP study. This study was conducted at Battelle-Columbus, using their methodology, with ethanol as a vehicle. The dosages applied to the skin were in the range of 1.7 ml/kg. Next, a second skin painting administration identical to the first (except for the use of daisy collars on the animals) was performed. We collected blood and urine samples and looked for NDELA (N-nitrosodiethanolamine) detection limits. This experiment was followed up with a second experiment, which was the same as the preceding one, with the exception that nitrite intake (in drinking water) was evaluated. Blood samples were analyzed for DEA and NDELA. Livers were submitted for analysis of phospholipids, choline, phosphocholine, and a number of compounds. Blood levels were determined in animals from the following four dose groups: (1) dermal, with collar; (2) dermal, no collar (dermal + oral); and (3) oral gavage. A nice dose response was observed, a 30% increase in DEA in moving from use of the collar to no collar (see slide presentation). The next experiment was a double gavage experiment (see slide presentation). This is not nitrite in the drinking water. However, it also is not nitrite added in the same syringe with the DEA. There was a fair dose response. This represents my estimation of what the NTP animals would have received primarily by the drinking water (bolus and non-bolus administration). This is NDELA concentration in the blood (in ppb) and in the ingesta. This is the dose level that we chose to administer in sodium nitrite (40 mg/kg) via the animal s water (non-bolus dose). Results indicated that it was below detection levels for practically everything. The preceding results moved us to explore our second hypothesis, choline deficiency. It doesn t rule out NDELA, but it certainly takes it out of a category of here is your primary suspect. The choline-methionine-c1 pool is fairly well known. There is dietary intake of choline and it can also be manufactured from phosphatidylcholine (see slide presentation). Phosphatidylcholine is a product of the trimethylation of ethanolamine. Phosphocholine was observed to decrease (approximately 80%) within two weeks of administration of a choline-deficient diet. DEA administration resulted in decreased hepatic and renal phosphatidylcholine levels. The DEA ended up primarily in ceramides. Choline deficiency does a number of things in animals. Most of this work has been done using rats because they develop a fatty liver (which is typical of a human condition). Mice don t appear to develop fatty liver. Rats subjected to choline deficiency also develop tumors. Choline deficiency also has the following effects: promotes tumor formation following initiation by genotoxins, increased cell proliferation and lipid peroxidation, hypomethylation of DNA, and decreased hematopoiesis and phosphatidylcholine in erythrocyte membranes. At two weeks post-administration of DEA, phosphocholine (the primary storage pool for choline in rodents) and choline decreased in rats, indicating choline deficiency, and sphingomyelin (i.e., ceramide) increased (see slide presentation). DEA can be incorporated into ceramide. Minimal decreases in phosphatidylcholine and phosphatidylethanolamine were also observed in rats after DEA administration. Regarding work in progress, B6C3F1 mice are dosed with DEA via oral gavage for four weeks. In blood, we are looking for indicators of hepatotoxicity. In the liver, we are looking for levels of the following: phospholipids (phosphatidylcholine and phosphatidylethanolamine), ceramide, sphingomyelin, diacylglycerol, choline, and phosphocholine. At Dow, Inc., we will be doing a gap junction intercellular communication assay in vivo. We will focus on protein kinase C activity and concentrations. Specifically, we will focus on two isoforms that have been shown to be induced by choline deficiency. We will use a PCNA method (an ELISA technique) for the indication of cell proliferation. We are also collecting urine and doing cross stains by an ELISA. There is a planned further experiment in which B6C3F1 mice will be exposed similarly. Hepatocellular proliferation will Panel Book Page 29

33 be evaluated via BrdU immunohistochemical staining and morphometrics. The protein kinase C (PKC) phosphorylation cascade is significant (see slide presentation) in the biochemistry of the cell. Ceramide is believed to turn-off PKC by a number of interactions with enzymes controlling the DAG - phosphatidic acid pathway. Levels of the following chemicals in the phosphorylation cascade will be measured: 1,2- sn-dag, P-choline, phosphatidylcholine, PKC, and sphingomyelin (a ceramide). PKC activity is linked to cell proliferation and phosphorylation, and is basically a benchmark of phorbol ester-type promotion. Ceramides are the primary metabolic pool that DEA is going to end up in. In summary, the CMA - CTFA research has, we feel, demonstrated choline deficiency in mice in vivo. There is no consistent NDELA formation in vivo that we can detect using a fairly sensitive methodology (detection limit = ppb). We have observed a 30% higher blood DEA level when you allow access to that dosing site in the animal. This is the direction of our research plan: cell proliferation and morphometrics, gap junction work, lipid peroxidation work, PKC activity and concentration. I failed to mention that a liver section will be sent to Procter and Gamble for S-adenosyl methionine (SAM) measurements. Should we observe a decrease in SAM, we will quickly move into DNA methylation status work. Finally, once we have identified what we feel are some key markers, we want to begin some in vitro interspecies comparisons of hepatocyte effects of DEA. [End of Dr. Stott s presentation] Dr. Belsito said that he did not understand the significance of measuring urinary cross stains. Dr. Stott said that this is an early measure of oxidative stress of lipid oxidation in the membranes. Dr. Klaassen wanted to know the status of FDA s involvement with DEA. Dr. Bailey said that DEA remains a central issue at FDA and that FDA is moving to complete its risk assessment before the end of this calendar year. He also said that the issue of a secondary mechanism is an intriguing one; however, from a risk assessment point of view, the data that would be needed to convincingly establish a secondary mechanism would have to be very complete. Dr. Klaassen said that some very interesting hypotheses are being tested, and that he would hope that enough time is provided such that some drastic decisions don t have to be made before the results are made available. Dr. Bailey said that the same presentation was made last week before FDA s toxicologists. He added that the issue of DEA is a very important public health issue that FDA wants to resolve as quickly as possible. Dr. Klaassen wanted to know if concern about DEA relates only to its presence in cosmetics. Dr. Bailey said that virtually all products are covered (foods, drugs, and cosmetics). He said that there is not that much direct use of DEA, but that the real issue relates to its presence as a contaminant in the various conjugates (all of which were studied by NTP) as well as triethanolamine. DEA is a contaminant of triethanolamine. In response to Dr. Belsito s question, Dr. Bailey confirmed that FDA s risk assessment is based on the NTP data. Dr. Bailey also said that if additional data are received that could change this, they will be considered. He reiterated that the standard for demonstrating secondary mechanisms has to be very convincing. Panel Book Page 30

34 Draft Amended Report Diethanolamine (DEA) and Related DEA-Containing Ingredients as Used in Cosmetics March 4, 2010 The 2011 Cosmetic Ingredient Review Expert Panel members are: Chairman, Wilma F. Bergfeld, M.D., F.A.C.P.; Donald V. Belsito, M.D.; Curtis D. Klaassen, Ph.D.; Daniel C. Liebler, Ph.D.; Ronald A Hill, Ph.D. James G. Marks, Jr., M.D.; Ronald C. Shank, Ph.D.; Thomas J. Slaga, Ph.D.; and Paul W. Snyder, D.V.M., Ph.D. The CIR Director is F. Alan Andersen, Ph.D. This report was prepared by Monice Fiume, Senior Scientific Analyst/Writer. Cosmetic Ingredient Review th Street, NW, Suite 412 " Washington, DC " ph " fax " cirinfo@cir-safety.org Panel Book Page 31

35 Table of Contents Introduction... 1 Chemistry... 2 Definition and Structure... 2 Method of Manufacture... 4 Analytical Methods... 4 Impurities... 4 Use... 5 Cosmetic... 5 Non-Cosmetic... 6 Toxicokinetics... 6 Absorption, Distribution, Metabolism and Excretion... 6 In-Vitro... 7 Dermal... 9 Non-Human... 9 Human ral Non-Human Intravenous Non-Human N-Nitrosodiethanolamine (NDELA) Formation Toxicological Studies Acute (Single Dose) Toxicity Dermal ral Inhalation ther Repeated Dose Toxicity Dermal ral Inhalation Reproductive and Developmental Toxicity Dermal ral Inhalation Effect on Hippocampal Neurogenesis and Apoptosis Genotoxicity In Vitro Carcinogenicity Dermal Possible Mode of Action for Carcinogenic Effects Irritation and Sensitization Irritation Skin Non-Human Human Mucosal In Vitro Non-Human Sensitization Non-Human Human Co-Reactivity Provocative Testing Phototoxicity/Photosensitivity Human Case Studies Miscellaneous Studies Inhibition of Choline Uptake ccupational Exposure Summary Discussion Tables Table 1. Definitions and Structures Table 2. Conclusions of previously reviewed ingredients and components ii Panel Book Page 32

36 Table of Contents (continued) Table 3. Physical and chemical properties Table 4a. Historical and current frequency and concentration of use according to duration and type of exposure Table 4b. Frequency (2010) and concentration of use according to duration and type of exposure Table 4c. Ingredients not reported to be in use Table 5. Status for use in Europe according to the EC CosIng Database Table 6. Conclusions of NTP dermal carcinogenicity studies References iii Panel Book Page 33

37 INTRDUCTIN In 1983, the Cosmetic Ingredient Review (CIR) Expert Panel issued a report on the safety of Triethanolamine, Diethanolamine, and Monoethanolamine. In 2010, the Panel decided to reopen that safety assessment as three separate reports and to add additional related ingredients included in each of the new reviews. This assessment addresses diethanolamine (DEA) and related DEA-containing ingredients. In considering the potential safety issues with DEA-containing ingredients, it was reasoned that, were they to penetrate the skin, the toxicity of most concern would be the DEA moiety. The acid salt ingredients (as recited below) would be expected to dissociate into DEA and the corresponding acid, some of which have been reviewed separately. In most cases, this means that the composition of these salts is stoichiometrically half DEA (i.e. accessible DEA is a major component of these ingredients). The covalent DEA ingredients (see alkyl substituted diethanolamines and diethanolamides, below), however, do not readily dissociate into DEA. In the case of these covalent ingredients, DEA may be of concern as an impurity, but not as a major component. In the 1983 review, the Expert Panel concluded that DEA, an ingredient that functions in cosmetics as a ph adjuster, is safe for use in cosmetic formulations designed for discontinuous, brief use followed by thorough rinsing from the surface of the skin. In products intended for prolonged contact with the skin, the concentration of DEA should not exceed 5%. 1 DEA should not be used with products containing N-nitrosating agents. The following are the lists of ingredients, sorted by chemical class, that the CIR is proposing to include in the rereview of DEA. Those marked with an asterisk have been previously reviewed by the CIR. Inorganic Acid Salt Diethanolamine Bisulfate rganic Acid Salts DEA-Isostearate DEA-Lauraminopropionate DEA-Linoleate rgano-substituted Inorganic Acid Salts DEA-C12-13 Alkyl Sulfate DEA-C12-13 Pareth-3 Sulfate DEA-C12-15 Alkyl Sulfate DEA-Ceteareth-2 Phosphate DEA-Cetyl Phosphate DEA-Cetyl Sulfate DEA-Di(2-Hydroxypalmityl)Phosphate DEA-Dodecylbenzenesulfonate* DEA-Hydrolyzed Lecithin DEA-Myristate DEA Stearate DEA-Laureth Sulfate DEA-Lauryl Sulfate DEA-Methyl Myristate Sulfonate DEA-Myreth Sulfate DEA-Myristyl Sulfate DEA-leth-3 Phosphate DEA-leth-5 Phosphate DEA-leth-10 Phosphate DEA-leth-20 Phosphate Alkyl Substituted Diethanolamines Butyl Diethanolamine N-Lauryl Diethanolamine (JPN) Methyl Diethanolamine Diethanolamides Almondamide DEA Apricotamide DEA Avocadamide DEA Babassuamide DEA Behenamide DEA Capramide DEA Cocamide DEA* 1 Cocoyl Sarcosinamide DEA Cornamide DEA Cornamide/Cocamide DEA DEA-Cocoamphodipropionate Diethanolaminooleamide DEA Hydrogenated Tallowamide DEA Isostearamide DEA* Panel Book Page 34

38 Lactamide DEA Lanolinamide DEA Lauramide DEA* Lauramide/Myristamide DEA Lecithinamide DEA Linoleamide DEA* Minkamide DEA Myristamide DEA* leamide DEA livamide DEA* Palm Kernelamide DEA Palmamide DEA Palmitamide DEA PEG-2 Tallowamide DEA PEG-3 Cocamide DEA Ricebranamide DEA Ricinoleamide DEA Sesamide DEA Shea Butteramide/Castoramide DEA Soyamide DEA Stearamide DEA* Stearamide DEA-Distearate Stearamidoethyl Diethanolamine Stearamidoethyl Diethanolamine HCl Tallamide DEA Tallowamide DEA Undecylenamide DEA Wheat Germamide DEA Lauramide DEA, linoleamide DEA, and oleamide DEA have previously been reviewed by the Expert Panel. In 1986, the Panel concluded that these ingredients are safe as used, and that they should not be used in products containing nitrosating agents. 2 Cocamide DEA was also included in that 1986 assessment; an amended report on cocamide DEA was issued in In 1996, the Panel concluded cocamide DEA is safe as used in rinse-off products and safe at concentrations 10% in leave-on cosmetic products. Cocamide DEA should not be used as an ingredient in cosmetic products in which N- nitroso compounds are formed. In 1995, the Expert Panel concluded that isostearamide DEA, myristamide DEA, and stearamide DEA are safe for use in rinse-off products. 4 In leave-on products, these ingredients are safe for use at concentrations that will limit the release of free ethanolamines to 5%, with a maximum use concentration of 40%. In 2009, the Expert Panel concluded that DEA-dodecylbenzenesulfonate is safe as used when formulated to be non-irritating. 5 This family of 69 ingredients, which includes previously reviewed ingredients, has been created to provide a single comprehensive review of related DEA-containing ingredients. While the ingredients in each subgroup listed above were presented alphabetically, the order in the report will follow ingredient groupings and chain length, and is provided in Table 1. The ingredients now included in this review consist of DEA and one or more component. The safety of many of these components has been reviewed by the CIR. The conclusions of the previously reviewed ingredients, and of the components that have been reviewed, are provided in Table 2. CHEMISTRY Definition and Structure DEA is an amino alcohol. DEA is produced commercially by aminating ethylene oxide with ammonia. The replacement of two hydrogens of ammonia with ethanol groups produces DEA. DEA contains small amounts of triethanolamine (TEA) and ethanolamine (MEA). H NH Figure 1. DEA DEA is structurally similar to choline and ethanolamine. 6 DEA is reactive and bifunctional, combining the properties of alcohols and amines. At temperatures of C, DEA will react with fatty acids to form ethanolamides. The reaction of ethanolamines and sulfuric acid produces sulfates and, under anhydrous conditions, DEA may react with carbon dioxide to form carbamates. DEA can act as an antioxidant in the autoxidation of fats of both animal and vegetable origin. From the Final Report on the Safety Assessment of Triethanolamine, Diethanolamine, and Monoethanolamine 1 2 Panel Book Page 35

39 f concern in cosmetics is the conversion (nitrosation) of secondary amines (R 1 -NH-R 2 ), such as DEA (wherein R 1 and R 2 are each ethanol), into N-nitrosamines that may be carcinogenic. f the approximately 209 nitrosamines tested, 85% have been shown to produce cancer in laboratory animals. 7 Nitrosation can occur under physiologic conditions. 8 Depending on the nitrosating agent and the substrate, nitrosation can occur under acidic, neutral, or alkaline conditions. Atmospheric N 2 may also participate in the nitrosation of amines in aqueous solution. 9 Accordingly, DEA and those ingredients in this report which readily dissociate to DEA should be formulated to avoid the formation of nitrosamines. Acid Salts The acid salts (inorganic acid salt, organic acid salts, and organo-substituted inorganic acid salts), as mentioned above, are ion pairs which freely dissociate in water (e.g., Figure 2). Therefore, these salts are closely related to the corresponding free acids and DEA. In other words, DEA Stearate is closely related to Stearic Acid and DEA. Accordingly, the potential formation of nitrosamines should be a consideration for the ingredients in this group Figure 2. DEA Stearate Alkyl Substituted Diethanolamines The alkyl substituted diethanolamines consist of covalent, tertiary amines, whereby two of the nitrogen substituents are ethanol and the third is an alkyl chain (i.e. a four carbon chain is the alkyl substituent on butyl diethanolamine; Figure 3). These ingredients are not salts, do not readily dissociate in water, and are not readily hydrolysed. Tertiary alkyl amines do not tend to react with nitrosating agents to form nitrosamines. Figure 3. Butyl Diethanolamine Diethanolamides The diethanolamides consist of covalent, tertiary amides, whereby two of the nitrogen substituents are ethanol (or at least an ethanol residue) and the third is a carbonyl attached substituent. For example, behenamide DEA is a tertiary amide wherein two of the nitrogen substituents are ethanol and the third is a twenty-two carbon, carbonyl attached chain (Figure 4). These ingredients are not salts and do not readily dissociate in water. However, amidases, such as fatty acid amide hydrolase (FAAH) which is known to be present in human skin, could potentially convert these amides to DEA and the corresponding fatty acids The potential for DEA generation is at least somewhat greater for the longer chain amides, as amide hydrolysis tends to occur more commonly with highly lipophilic amides. 13 Tertiary amides do not tend to react with nitrosating agents to form nitrosamides. CH 3 (CH 2 ) 20 N Figure 4. Behenamide DEA 3 Panel Book Page 36

40 The structures and definitions of DEA and all related ingredients are provided in Table 1, and available chemical and physical properties are provided in Table 3. Method of Manufacture Method of manufacture data of on most of the ingredients included in this assessment were not found. The information that was available is follows. Diethanolamine DEA is produced by reacting 2 moles of ethylene oxide with 1 mole of ammonia. 14 Typically, ethylene oxide is reacted with ammonia in a batch process to produce a crude mixture of approximately one-third each MEA, DEA, and TEA. The crude mixture is later separated by distillation. Diethanolamides Lauramide, oleamide, linoleamide, and cocamide DEA are produced by a condensation reaction at a 1:1 or 1:2 molar ratio of a mixture of lauric and myristic acid (for lauramide DEA), oleic acid (oleamide DEA), linoleic acid or its methyl ester (linoleamide DEA), or methyl cocoate, coconut oil, whole coconut acids, or stripped coconut fatty acids (cocamide DEA) to DEA. From the Final Report on the Safety Assessment of Cocamide, Lauramide, Linoleamide, and leamide DEA 2 Lauramide DEA Lauramide DEA is produced by the condensation of lauric acid methyl ester with DEA at elevated temperature and in the presence of a catalyst. 15 Cocamide DEA Cocamide DEA has been produced by the reaction of refined coconut oil with diethanolamide in the presence of sodium methoxide (catalyst), yielding cocamide DEA, 10% glycerine, and 5% coconut fatty acid amide. From the Amended Final Report on the Safety Assessment of Cocamide DEA. 3 Analytical Methods The amount of DEA in fatty acid diethanolamides was determined using a gas chromatographic method with flame ionization detection. 16 Diethanolamine Impurities Dow Chemical Company reports that DEA is commercially available with a minimum purity of 99.3%, containing 0.45% max. MEA and 0.25% max TEA. 17 Diethanolamides In the manufacture of the 1:2 mixture of fatty acid to DEA, ethylene glycol and free DEA residues are present. The 1:1 mixture contains much less free amine. Alkanolamides manufactured by base-catalyzed condensation of DEA, and the methyl ester of long chain fatty acids are susceptible to nitrosamine formation. From the Final Report on the Safety Assessment of Cocamide, Lauramide, Linoleamide, and leamide DEA 2 Lauramide DEA Various grades of Lauramide DEA are available for cosmetic use. The free amine value is (sic). From the Final Report on the Safety Assessment of Cocamide, Lauramide, Linoleamide, and leamide DEA 2 In National Toxicology Program (NTP) studies, the purity of lauramide DEA was approximately 90% for lauric acid DEA condensate, with approximately 5% amine (probably DEA) and approximately 5% other organic impurities. 15 NDELA was detected at a concentration of 3600 ppb. However, the report also stated that, based on data provided by the manufacturer, lauramide DEA contained 0.83% free DEA by weight, and approximately 9% other organic impurities. Commercial samples of lauramide DEA were analyzed for DEA. 16 from %. NDELA was not found in any of the samples. 4 The amount of DEA in the 9 samples ranged Panel Book Page 37

41 Stearamide DEA Stearamide DEA is characterized by 9-12% free fatty acids (as oleic acid) and 2-6% free amines (as DEA). From the Final Report on Isostearamide DEA & MEA, Myristamide DEA & MEA, and Stearamide DEA & MEA 4 leamide DEA leamide DEA contains % free fatty acids (as oleic acid). From the Final Report on the Safety Assessment of Cocamide, Lauramide, Linoleamide, and leamide DEA 2 In NTP studies of oleamide DEA, the oleic acid DEA condensate content was 47.5% Impurities were identified as other fatty acid alkanolamides (approximately 30%), other fatty acids, and unidentified impurities. Free DEA was estimated at 0.19%. NDELA was detected at a concentration of 68 ppb. Linoleamide DEA Commercial sample of linoleamide DEA were analyzed for DEA, and % was detected. 16 NDELA was not found in any of the samples. Cocamide DEA Various grades of cocamide DEA are available. Alkanolamines manufactured by base-catalyzed condensation of DEA and the methyl ester of long chain fatty acids are susceptible to nitrosamine formation. Cocamide DEA contains % free DEA. From the Amended Final Report on the Safety Assessment of Cocamide DEA. 3 In NTP studies, cocamide DEA contained approximately 18.2% free DEA by wt, alkanolamides of unsaturated acids, and amine salts of the acids. NDELA was detected at a concentration of 219 ppb. 19 Commercial samples of cocamide DEA were analyzed for DEA. 16 from %. NDELA was not found in any of the samples. DEA functions in cosmetics as a ph adjuster. 20 USE Cosmetic The amount of DEA in the 9 samples ranged While a few of the other ingredients might function as a ph adjuster, the majority have other functions, including surfactant, emulsifying agent, viscosity increasing agent, hair or skin conditioning agents, foam booster, or antistatic agent. In 1981, according to data provided through the Food and Drug Administration (FDA) Voluntary Cosmetic Registration Program (VCRP), DEA was used in 18 formulations, and all but one of those products were rinse-off formulations. 1 Twelve uses were in hair coloring products. Products containing DEA were used at concentrations of 5%. VCRP data obtained in 2010 indicate that DEA is used in 30 formulations; 15 are leave-on formulations and 15 are rinse-off, and 13 uses are in non-coloring hair formulations. 21 According to data submitted by Industry in response to a survey conducted by the Personal Care Products Council (Council), DEA is used at concentrations of %. 22 The highest concentration used in leave-on products is 0.06%, in moisturizing products. The complete current and historical use data for DEA and other previously reviewed ingredients are provided in Table 4a. The use data on ingredients being reviewed for the first time are provided in Table 4b. Ingredients not reported to be in use, according to VCRP data obtained in 2010, are listed in Table 4c. According to the Council, DEA per se is rarely if ever used in personal care products. 23 The potential for exposure to DEA exists from the use of alkanolamides of DEA (which are condensation products of DEA and fatty acids, i.e., diethanolamides). Some of the ingredients included in this assessment are present in aerosolized products, and potential effects on the lungs of aerosolized products containing this ingredient are of concern. The Expert Panel has previously determined that Panel Book Page 38

42 because the size of aerosol particles used in hair sprays are greater than 10µm in diameter, they are deposited in the nasopharyngeal region and are not inhalable. According to the opinion of the Scientific Committee on Consumer Safety of the European Commission (EC), dialkanolamines (e.g. DEA) and their salts (i.e., the acid salts listed previously) are on the list of substances which must not form part of the composition of cosmetic products. 24 According to the Commission s opinion paper, amines occur only in their salt form in all cosmetic products. This is because all amines are alkaline compounds which are always neutralized by an acid component to produce their salts. There is concern about the potential for nitrosamine formation; in principle, secondary amines are potential precursors of nitrosamines. The ingredients that are included in Annex II of the EC CosIng database (the list of substances prohibited in cosmetic products) based on this opinion are listed in Table 5 Fatty acid dialkanolamines (i.e., the alkyl substituted diethanolamines) are listed in Annex III of the EC CosIng database, which is a list of substances cosmetic products must not contain except subject to the restrictions laid down. The restrictions for these ingredients are: maximum secondary amine content of 0.5% in the finished product; do not use with nitrosating systems; maximum secondary amine content of 5% for raw materials; maximum nitrosamine content of 50 µg/kg; and keep in nitrite free containers. The ingredients listed in Annex III with these restrictions, as well as EC information for all other ingredients included in this report, are also provided in Table 5. In Canada, DEA is completely prohibited as per the Cosmetic Hotlist; Health Canada prohibits the use of dialkanolamines (e.g., DEA). 25 This prohibition is based on the EU prohibition in the Cosmetics Regulation, Annex II. The use of DEA in product formulations in Canada is being investigated due to reported use at concentrations of 3%. (Health Canada, personal communication). Non-Cosmetic Many of the ingredients included in this safety assessment have use as indirect food additives. 26 Diethanolamine DEA is used in the manufacture of emulsifiers and dispersing agents for textile specialties, agricultural chemicals, waxes, mineral and vegetable oils, paraffin, polishes, cutting oils, petroleum demulsifiers, and cement additives. It is an intermediate for resins, plasticizers, and rubber chemicals. DEA is used as a lubricant in the textile industry, a humectant and softening agent for hides, as an alkalizing agent and surfactant in pharmaceuticals, as an absorbent for acid gases, and in organic syntheses. From the Final Report on the Safety Assessment of Triethanolamine, Diethanolamine, and Monoethanolamine 1 Methyl Diethanolamine Methyl diethanolamine is used as a flocculent monomer in water treatment, as a sweetening agent in natural gas treatment, as a catalyst in urethane coating, and as a chemical intermediate in the manufacture of textile lubricants (fabric softener) and analgesic pharmaceuticals. 27 Cocamide DEA Cocamide DEA is used as a corrosion inhibitor in metalworking fluids and in polishing agents. From the Amended Final Report on the Safety Assessment of Cocamide DEA 3 TXICKINETICS Absorption, Distribution, Metabolism and Excretion In vitro absorption studies were performed using mouse, rat, and human skin. In in vitro studies using mouse and rat skin, 1.3 and 0.04%, respectively, of the applied dose of undiluted [14C]DEA was absorbed. In studies using human skin samples, the absorption of undiluted DEA, as well as concentrations of <1% DEA in combination with fatty acid dialkanolamides, was less than 1% of the applied dose. Penetration of DEA in aqueous solutions was greater than when DEA was undiluted. In studies using human liver slices, DEA was absorbed; the aqueous-extractable radioactivity was 6 Panel Book Page 39

43 primarily unchanged DEA, while analysis of the organic extracts suggested that DEA was incorporated into ceramides, and slowly methylated. Lauramide DEA was better absorbed in liver slices, and while the absorbed radioactivity was mostly unchanged lauramide DEA, 18-42% was present in the form of metabolites. In dermal studies with DEA, methyl DEA, and lauramide DEA, the applied doses were generally well absorbed through mouse and/or rat skin, and absorption increased with duration of exposure. In the tissues, the liver generally had the greatest disposition of radioactivity. Urine was the principal route of elimination. Upon dosing with methyl DEA, primarily metabolites, not unchanged methyl DEA, were found in the urine. Lauramide DEA absorption was not dose dependent and the parent compound and the half-acid amide metabolites were detected in the plasma, and disposition did not vary with time. In oral studies, DEA accumulated in the tissues, with the greatest disposition being in the liver; radioactivity was primarily as unchanged DEA. Urinary excretion was also primarily as unchanged DEA. In a repeated-dose study, steadstate for bioaccumulation occurred after 4 wks; however, DEA continued to bioaccumulate in blood throughout dosing. With lauramide DEA, 79% of the dose was excreted in the urine 72 h after dosing. Four percent of the dose was recovered in the tissue. After 6 hrs, only very polar metabolites, thought to be carboxylic acids, were found in the urine. In vitro percutaneous absorption studies of cosmetic preparations containing free DEA up to 0.6% showed some penetration occurred in human skin. Mice exposed orally to sodium nitrate were dosed orally and dermally with 4 mg/kg DEA. A small amount of NDELA was formed following a single oral dose of DEA. No NDELA was detected following dermal dosing with DEA. In-Vitro Diethanolamine The in vitro absorption of 2 mg/cm 2 [ 14 C]DEA was determined using fuzzy rat skin A total of 1.4% of the applied dose was absorbed over 24 h, with 1.9% of the dose remaining in both the stratum corneum and viable dermis/epidermis. Values were similar at 72 h. Three full thickness skin preparations from CD rats, CD-1 mice, New Zealand White (NZW) rabbits, and 6 from female mammoplasty patients, were used to compare the dermal penetration of DEA through the skin of different species. 29 [ 14 C]DEA (96.5% purity; sp. act mci/mmol) was applied to the skin sample undiluted, or as an aq. solution, at a dose of 20 mg/cm 2. Dose volumes of 35 µl undiluted [ 14 C]DEA or 95 µl of the aq. solution (37% w/w) were applied to the exposed surface of the skin (1.77 cm 2 ). These volumes maintained an infinite dose during the 6 h exposure period. Skin sample integrity was confirmed with the use of a reference chemical, [ 14 C]ethanol. With undiluted DEA, the cumulative dose absorbed was 0.04% in rat skin, 1.30% in mouse skin, 0.02% in rabbit skin, and 0.08% in human skin. With aq. DEA, an increase in the cumulative dose absorbed was seen in all species: 0.56% in rat skin, 6.68% in mouse skin, 2.81% in rabbit skin, and 0.23% in human skin. Penetration of undiluted and aq. DEA was greater through mouse skin than any other skin sample. With undiluted DEA, penetration was similar for rat, rabbit, and human skin samples. Penetration of DEA was greater for an aqueous solution than for undiluted DEA. The researchers hypothesized that this may be attributable to elevated skin hydration caused by the application of the aqueous solution to the skin. The percutaneous absorption of DEA in cosmetic formulations spiked with [ 14 C]DEA (95-99% purity) was examined using viable human skin. 30 Two shampoo formulations, both with a [ 14 C]DEA dose of 0.49 µci were applied as a 1:6 aq. dilution for 5 min; one shampoo contained cocamide DEA with a concentration of 0.092% free DEA, and the other contained lauramide DEA with a concentration of 0.28% free DEA. The amount applied of the shampoo was 1.2 mg (diluted 1:6), and the dose was 1.9 mg/cm 2. (The DEA dose was 4.2 µg/cm 2 for the first formulation, and 7.7 µg/cm 2 for the second shampoo formulation.) Two hair dye formulations, spiked with 0.49 or 0.43 µci DEA, were applied for 30 min. ne hair dye contained lauramide DEA and the other contained cocamide and oleamide DEA. The estimated concentration of free Panel Book Page 40

44 DEA in the hair dye products was 0.61%. An application of 11.2 mg was applied to the skin samples, with a dose of 17.5 mg/cm 2. (The DEA dose was µg/cm 2 for the first formulation, and µg/cm 2 for the second hair dye formulation.) Additionally, 3 mg/cm 2 of two body lotions (amount applied not stated) with 0.13 or 0.12 µci DEA was applied for 24 h. (The DEA dose was 1.0 µg/cm 2 for the first formulation, and 1.2 µg/cm 2 for the second lotion formulation.) These products contained % TEA, with 0.020% free DEA. Very little DEA was found in the receptor fluid, with only 0.1% of the applied dose recovered in the receptor fluid of the shampoo and hair dye formulations. While the amount absorbed was similar for these two product types, the distribution and localization were different, with most of the DEA (62-68%) penetrating from the shampoos being localized in the stratum corneum, and that from the hair dyes (52-64%) being found in deeper epidermal and dermal layers. With the lotion, % of the dose was recovered in the receptor fluid. Penetration from the lotions differed from each other. With the first lotion, 15.4% of the applied dose penetrated, with 0.6% found in the receptor fluid and 14.8% in the skin; approximately 65% of the DEA in the skin was in the epidermis and dermis. With the second lotion formulation, only 7.8% of the applied dose penetrated, with 1.2% found in the receptor fluid and 6.6% in the skin; approximately 56% of the DEA in the skin was in the epidermis and dermis. The researchers examined whether DEA was binding to skin. DEA did not appear to be covalently bound to skin proteins, and extending the times before analysis to 48 and 72 h did not result in any statistically significant difference when compared to the values obtained after 24 h. Repeat application studies with viable skin did not produce a significant change of dose absorbed into receptor fluid. However, with non-viable skin, absorption into the receptor fluid from the lotion increased each day, from 0.6% on the first day to 2.6% on the third day; testing showed that the skin barrier did not remain intact for the entire 72 h. Using a shampoo and a lotion formulation, non-viable skin gave penetration values similar to viable skin. The researchers concluded that most of the DEA that penetrated was not available for systemic absorption. Another study examining the percutaneous penetration of seven cosmetic formulations spiked with radiolabeled and unlabeled DEA was performed, mimicking simulated use conditions using fresh human skin samples. 31 Two shampoos both contained 0.98% DEA and 4.02% cocamide DEA, and two additional shampoo formulations both contained 0.25% DEA and 4.75% lauramide DEA; all were applied as a 1:10 aq dilution and a dose of 100 µl/cm 2 (equivalent to 100 mg/cm 2 ) for 10 min. A bubble bath containing 0.25% DEA and 4.75% lauramide DEA, applied as a 1:300 dilution and a dose of 100 µl/cm 2 (equivalent to 100 mg/cm 2 ) for 30 min, a moisturizer containing 0.008% DEA and 2% TEA, applied at a dose of 5 mg/cm 2 for 48 h, and a semi-permanent hair dye containing 0.075% DEA and 1.42% lauramide DEA and an oxidative hair dye containing 0.25% DEA and 4.74% lauramide DEA, both applied at a dose of 100 mg/cm 2 for 30 min, were also used. Very little DEA penetrated the skin; % of the applied dose of the shampoo formulations penetrated, % of the applied dose of the hair dye formulations, and 0.508% of the bubble bath formulation penetrated the skin. The moisturizer formulation was also applied to frozen skin samples. A total of 0.605% of the applied dose penetrated fresh skin, while 0.456% penetrated frozen skin. The researchers also examined the penetration of a simple aq. 1% solution of DEA through fresh and frozen human skin samples. The cumulative 24 h percutaneous absorption was approximately 5-fold greater in frozen skin compared to fresh skin, i.e., 8.87 µg/cm 2 compared to 1.73 µg/cm 2. The 24-h cumulative penetration values represented and 0.086% of the dose for frozen and fresh skin, respectively. The amounts of DEA remaining on and in the skin were 1.68 and 1.14% of the applied dose recovered from the frozen and fresh skin samples, respectively. The researchers further investigated the distribution of [ 14 C]DEA between aqueous and lipid fractions of viable skin strata. The radioactivity on the skin 8 Panel Book Page 41

45 samples from 2 donors (n=12) was determined after a 24 h exposure. At 24 h, the cumulative permeation value was 0.405% of the applied dose for one donor (abdominal skin) and 0.067% for the other (breast skin). (The researchers stated that it may be significant that one sample was abdominal skin and the other was breast skin.) Tape-strip profiles for both donors appeared to indicate that the DEA had become evenly distributed throughout the stratum corneum. The majority of DEA was recovered in aqueous extract, as opposed to organic extract, of the epidermal and dermal tissue, which suggested to the researchers that the material was in the free state and not associated with the lipid fraction. Human liver slices were incubated with 1 mm [ 14 C]DEA (>97% purity). 32 After 4 and 12 h, 11 and 29% of the DEA, respectively, absorbed into the liver slices. The radioactivity was comprised mostly of DEA (85-97%); four other metabolites were present at low concentrations. The liver slices were fractionated into aqueous, organic, and pellet fractions. The aqueous-extractable radioactivity was comprised primarily of DEA, with up to four other components. DEA-derived radioactivity in the organic extracts was predominately (>90%) comprised of phospholipids containing non-methylated headgroups. DEA was readily absorbed and incorporated into ceramides, forming mostly ceramide-phosphodiethanolamine, and it was slowly methylated therein. Lauramide DEA Human liver slices, and liver slices from diethylhexyl phthalate-(dehp) induced and untreated male F344 rats, were incubated with [ 14 C]lauramide DEA. 33 Lauramide DEA partitioned well into the liver slices, and approximately 70% of the radioactivity absorbed into the slices in 4 h. The absorbed radioactivity was present mostly as lauramide DEA. In the extracts, 18-42% of the radioactivity was present in the form of metabolites. The analytes present in the incubation media included half-acid amides, parent lauramide DEA, and three other metabolites that are products of ω- and ω-1 to 4 hydroxylation. The in vitro metabolism of [ 14 C]lauramide DEA, randomly labeled on the DEA moiety, was examined in liver and kidney microsomes from rats and humans to determine the extent of hydroxylation, and to determine the products formed. 34 Incubation of lauramide DEA with liver microsomes from control and DEHP-treated rats produced two major high performance liquid chromatography (HPLC) peaks that were identified as 11-hydroxy- and 12-hydroxy-lauramide DEA. Treatment with DEHP increased the 12-hydroxylation rate 5-fold, while the 11-hydroxylase activity was unchanged. Upon comparison of lauramide DEA hydroxylation rates from human liver microsomes with those from rat liver and kidney microsomes, the lauramide DEA 12-hydroxylase activity in human liver was similar to the rate found in liver microsomes of control rats. The rate was 3 times greater than that observed in rat kidney microsomes. Dermal Non-Human Diethanolamine [ 14 C]DEA was applied to a 2 cm 2 area of the intrascapular region of male F344/N rats at a dose of 2.1, 7.6, or 27.5 mg/kg bw. 35,36 After 48 h, 2.9, 10.5, or 16.2% of the dose, respectively, was absorbed. It was shown that a 10-fold increase in the concentration of DEA resulted in a 450-fold increase in the rate of absorption. A single occluded dose of [ 14 C]DEA was applied to a 19.5 cm 2 area on the back of rats for 6 h, and the treated site of 50% of the animals was rinsed. 37 In the animals that were not rinsed, 80% of the dose was found in the wrappings, and 3.6% found in the skin. In rinsed animals, rinsates from the wrappings contained 58% of the dose, and from the skin contained 26% of the dose. Unrinsed animals absorbed 1.4% of the dose, while those that were rinsed absorbed 0.64%. The majority of the radioactivity was found in the carcass, liver, and kidneys. 9 Panel Book Page 42

46 The researchers also conducted a repeated-dose percutaneous-absorption study in which as a pre-exposure, 1500 mg/kg/day non-radiolabeled DEA was applied occlusively under a 2 in x 2 in gauze square to the backs of rats 6 h/day for 3 or 6 days. [ 14 C]DEA, 1500 mg/kg/day, was then applied for 3-6 consecutive days under occlusion; each dose was left in contact with the skin for 48 h. Totals of 21 and 41% of the dose were absorbed by the animals dosed for 3 and 6 days, respectively. The majority of the recovered radioactivity was in the wrapping. The carcass, liver, and kidneys contained most of the radioactivity in the animals. Totals of 4.3 and 13% of the radioactivity were recovered in the urine of animals dosed with non-radiolabeled DEA for 3 and 6 days, respectively. Pre-exposure to DEA resulted in a 1.5- to 3.0-fold increase in the absorption rate. Groups of 4-5 male B6C3F 1 mice were used in dermal studies of the absorption, distribution, metabolism, and excretion (ADME) of DEA. 38 The total volume for mice was 15 µl/dose, and the dose was applied to a 1 cm 2 area of skin using a non-occlusive covering. Absorption through mouse skin was greater than through rat skin. At 48 h after dermal application of 8 and 23 mg/kg [ 14 C]DEA, 26.8 and 33.8% of the dose was absorbed in the mice. A statistically significant increase in the amount absorbed after dosing with 81 mg/kg, 58.1%, was observed. The amount of radioactivity found at the site of application was only 4.0, 3.1,and 2.2% of the dose following application of 8, 23, and 81 mg/kg [ 14 C]DEA, respectively, and the amount excreted in the urine 48 h after application was 7.5%, 10.4, and 16.4%, respectively. The tissue/blood ratio was greatest in the liver and kidneys. Groups of 4-5 male Fischer 344 rats were used to evaluate the ADME of DEA (99% purity) following dermal administration. 38 The dose, which contained 6-20 µci radiolabel, unlabeled DEA, and ethanol, for a total volume of 25 µl per dose, was applied to a 2 cm 2 area of skin under a non-occlusive covering. Absorption increased significantly with increasing dose. After 48 h, only 2.9% of the radioactivity was absorbed following dermal application of 2.1 mg/kg, while 10.5 and 16.2% of the radioactivity was absorbed with doses of 7.6 and 27.5 mg/kg, respectively. f the amount absorbed, 1.2, 4.3, and 4.5% of the radioactivity was recovered in the skin at the dose site following application of 2.1, 7.6, and 27.5 mg/kg [ 14 C]DEA, respectively. The greatest tissue/blood ratios were found in the liver and lung. DEA, 80 mg/kg bw in acetone, was applied to a 2 cm 2 area on the backs 7 female C57BL/6 mice for 11 days. 39 DEA and its methylated metabolites accumulated in the liver and plasma of mice. Also, a statistically significantly decrease in hepatic concentrations of choline and its metabolites were reported. Methyl Diethanolamine A dose of 500 mg/kg, uniformly labeled, [ 14 C]methyl DEA, 25 µci, was applied to a 2 cm x 4 cm area of 4 male, and a 2 cm x 3 cm area of 4 female, Fischer 344 rats. 40 The occlusive patches were applied for 6 or 72 h; the number of animals used per duration was not specified. With the 6-h exposure, 17-21% of the applied dose was absorbed. With the 72 h exposure, 41-50% of the applied dose was absorbed. The greatest amounts of radioactivity were found in the livers and kidneys of animals dosed for 6 and 72 h. The principal route of elimination was the urine, but the rates of urinary excretion were slow. With the 6 h exposure, approximately % of the total dose was excreted in the urine in 72 h, and with the 72 h exposure, approximately % of the total dose was excreted. The major component in the urine was metabolites, rather than unchanged methyl DEA. In the animals dosed for 6 h, radioactivity was systemically available after the dose was washed. The concentrations of radioactivity in the plasma in the plasma of animals of this group were greatest at 60 h, with values of µg/g. In the animals dosed for 72 h, the amounts of radioactivity in the plasma were also greatest at 60 h, with values of µg/g. Unchanged methyl DEA was available at 72 h for both exposure groups. 10 Panel Book Page 43

47 Lauramide DEA Groups of four male B6C3F 1 mice and four F344 rats were dosed dermally with randomly labeled [ 14 C]lauramide DEA, 2-17 µci/dose for mice and µci/dose for rats. 33 The vehicle was ethanol. A non-occlusive application was made to a 0.5 in 2 or 1 in 2 area of skin of mice and rats, respectively. At the end of the study, the excised skin was rinsed with ethanol. Mice were dosed with mg/kg [ 14 C]lauramide DEA. At 72 h, 50-70% of the applied radioactivity was absorbed, and absorption was similar for all the doses. Approximately 32-55% of the radioactivity was excreted in the urine. In rats dosed with 25 or 400 mg/kg lauramide DEA, 21-26% of the radioactivity penetrated the skin in 72 h, and 3-5% was recovered at the site of application. Approximately 20-24% of the radioactivity was recovered in the urine. The tissue/blood ratio was greatest in the liver and kidney. Lauramide DEA and the half-acid amide metabolites were detected in the plasma, with maximum levels found 24 h after dosing. The researchers also applied 25 mg/kg/day lauramide DEA, 5 days/wk for 3 wks, to a group of 5 rats. Disposition did not vary much at the different collection time points. Human Three female subjects applied a lotion containing 1.8 mg DEA/g lotion to their entire body 2x/day for 1 mo. 39 Blood samples were collected 1 day prior to the start of dosing, at 1 wk, and at 1 mo. Two of the subjects completed 1 mo of application, while the third completed 3 wks. Application of the DEA-containing lotion for 1 mo resulted in increased concentrations of DEA and dimethyl DEA in plasma, when compared to blank samples. (As a reference point, however, it was calculated that the concentration of DEA and metabolites in the plasma of humans after a few weeks of application of the lotion were only 0.5-1% of the concentrations achieved in mice, where 80 mg/kg/day was applied for 11 days.) ral Non-Human Diethanolamine Four male Fischer 344 rats were dosed orally by gavage with 7 mg/kg aq. [ 14 C]DEA to examine the ADME of DEA (>97% purity). 32 The amount of radioactivity excreted in the urine at 24 and 48 h was 9 and 22%, respectively, and the amount in the feces was 1.6 and 2.4%, respectively. At 48 h after dosing, 27% of the radioactivity accumulated in the liver, 5% in the kidneys, and 0.32, 0.27, 0.19, and 0.18% in the spleen, brain, heart, and blood, respectively. As measured in the liver and the brain, 87-89% of the radioactivity distributed into the aq. phase, and 70-80% of that total radioactivity was as unchanged DEA. In the liver, the remaining radioactivity in the aqueous phase was distributed between three methylated metabolite fractions, while in the brain, only one minor, non-methylated metabolite was present in the aqueous extract. With repeat oral dosing with [ 14 C]DEA, radioactivity continued to accumulate in tissues, reaching steady states at 4-8 wks. The levels of DEA equivalents in the blood, brain, and liver were much higher following 8 weeks of repeated oral doses of 7 mg/kg/day, when compared to the single dose post-48-h values. Again, DEA was the major radioactive component. Using HPLC, almost all of the organic-extractable hepatic radioactivity eluted with the phosphatidylcholine fraction, with greater than 95% of the material in the form of phospholipids containing an N,N-dimethyl-DEA headgroup. In the brain, the entire organic-extractable radioactivity eluted in the phosphatidylethanolamine fraction, and it was almost entirely comprised of DEA-containing headgroups. According to the researchers, the results of this study demonstrated that DEA is -phosphorylated and N-methylated, and that these metabolites are incorporated as the polar headgroups in aberrant phospholipids. The researchers felt this was evidence that DEA and MEA, a naturally-occurring alkanolamine, share common biochemical pathways of transforma- 11 Panel Book Page 44

48 tion. Retention and bioaccumulation of DEA-derived radioactivity was attributed partly to aberrant phospholipids being incorporated into tissues, most probably in cell membranes. Groups of 3-5 male Fischer 344 rats were used to evaluate the ADME of a single oral administration of [ 14 C]DEA (99% purity). 38 The dose administered contained µci radiolabeled, unlabeled DEA, and the amount of water needed to achieve a target dose of 5 ml/kg bw. Gastrointestinal absorption was nearly complete after doses up to 200 mg/kg DEA. At 48 h after dosing with 7 mg/kg, 22% of the radioactivity was excreted in the urine and 2.4% in the feces, primarily as unchanged DEA. Radioactivity was not detected in carbon dioxide. Excretion was mostly unchanged DEA. A total of 57% of the radioactivity was found in the body 48 h after dosing with 7 mg/kg [ 14 C]DEA. Disposition of radioactivity was greatest in the liver (27.3%), muscle (16.3%), skin (5.1%), and kidneys (5%); only 0.2% of the radioactivity was found in the blood after 48 h. Dose did not affect distribution in the tissues. The researchers then evaluated the ADME of DEA upon repeat oral exposure. Four rats were dosed orally with 7 mg/kg/day [ 14 C]DEA for 5 days. Approximately 40% of the total radioactivity was excreted during dosing. At 48 h after the last dose, a total of 42% of the radioactivity was found in the tissues, with the greatest distribution in the liver (18.2%), muscle (12.4%), kidneys (5.56%), and skin (4.18%). To further investigate DEA bioaccumulation, rats were dosed with 7 mg/kg/day DEA, 5 days/wk, for 2, 4, or 8 wks. During the last week of each dosing period, 69, 79, and 92% of the dose was excreted, respectively. After 8 wks of dosing, most of the radioactivity was recovered as unchanged DEA; however there were also significant amounts of poorly retained metabolites, N-methylDEA, and another metabolite that was tentatively identified as a quaternized lactone. The % radioactivity recovered in the liver after 2, 4, and 8 wks of dosing was 12.3, 7.9, and 4.12%, respectively. It was estimated that steady-state for bioaccumulation occurred after 4 wks of repeat dosing, except for the blood, which continued to bioaccumulate DEA throughout dosing. Clearance of bioaccumulated DEA appears to be a first-order process, with a whole body elimination half-life of ~6 days. Lauramide DEA Three male F344 rats were dosed orally with randomly labeled [ 14 C]lauramide DEA, µci/dose, formulated to give a target dose of 5 ml/kg bw. 33 After oral dosing with 1000 mg/kg [14C]lauramide DEA, approximately 10, 60,and 79% of the dose was recovered in the urine after 6, 24 h, and 72 h, respectively. Approximately 4% of the dose was recovered in the tissues after 72 h, with almost 3% found in adipose tissue and 1.3% in the liver. After 6 h, no DEA, DEA metabolites, or unchanged lauramide DEA were present in the urine; only very polar metabolites were found. The researchers postulated that the metabolites were carboxylic acids, and that the acid function was formed from the lauryl chain. Intravenous Non-Human Diethanolamine Groups of Sprague-Dawley rats (number per group not specified) were given an intravenous (i.v.) dose of 10 or 100 mg/kg DEA in physiological saline. 37 Animals were killed 96 h after dosing. Peak blood concentrations appeared 5 min after dosing. Elimination from the blood was biphasic. Totals of 25 and 36% of the dose were excreted in the urine with 10 and 100 mg/kg DEA, respectively. Groups of 3-5 male Fischer 344 rats were used to evaluate the ADME of [ 14 C]DEA (99% purity) in phosphatebuffered saline after i.v. administration. 38 After 48 h, 28% of the dose was excreted in the urine, 0.6% in the feces, and only 0.2% in carbon dioxide. Using HPLC, it was determined that most of the radioactivity in the urine was present as unchanged DEA. A total of 54% of the dose was found in the body 48 h after dosing with 7 mg/kg [ 14 C]DEA, and as with oral dosing, 12 Panel Book Page 45

49 the greatest disposition was found in the liver (27%), muscle (15%), skin (4.5%), and kidneys (4%). nly 0.2% of the radioactivity was found in the blood after 48 h. Groups of 5 female Sprague-Dawley rats were dosed i.v., via the cannulated jugular vein, with 10 or 100 mg/kg [ 14 C]DEA (97.4% purity). 41 The dose volume was 2 ml/kg, and each rat received ~4.2 µci 14 C. Blood samples were taken at various intervals up to 84 h after dosing. The peak concentrations of radioactivity in both the plasma and the red blood cells were observed 5 min after dosing. Clearance of radioactivity from the plasma was calculated to be approximately 50 and 93 ml/h/kg for the low and high dose, respectively. In blood, these values were 84 and 242 ml/h/kg, respectively Urine and feces were collected for 96 h after dosing. During this time, the major route of excretion was urinary; 25 and 36% of the dose was recovered in the urine. Urinary excretion was rapid at the high dose level, with 23% of the dose recovered in the first 12 h. nly 8.5% of the dose was recovered with the low dose during this time frame. The majority of the radioactivity was recovered in the carcass; 35 and 28% for the low and high dose, respectively. At 96 h after dosing with 10 and 100 mg/kg DEA, approximately 21 and 17% of the dose was recovered in the liver, 7 and 5% in the kidneys, and 5 and 5% in the skin, respectively. The researchers stated that because the majority of the applied dose (administered radioactivity) was recovered in the tissues, particularly in the liver and kidneys, this indicated a propensity for bioaccumulation. There was some evidence that the bioaccumulation was dose-dependent. Methyl Diethanolamine Groups of 4 male Fischer 344 rats were given a single i.v. dose of 50 or 500 mg/kg, uniformly labeled, [ 14 C]methyl DEA, 10 µci, at a dose volume of 2 ml/kg. The principal route of elimination was the urine, but the rates of urinary excretion were slow. In the 50 mg/kg group, approximately 60% of the total dose was excreted in the urine in 72 h; with the 500 mg/kg dose, approximately 67.5% of the total dose was excreted. The major components in the urine of animals dosed with 50 mg/kg were metabolites. Conversely, in urine of animals dosed with 500 mg/kg, the major component was unchanged methyl DEA. In the plasma, a rapid distribution phase was followed by a slower elimination phase; 9 and 44% of the total radioactivity in the plasma was unchanged methyl DEA at 1 h after dosing with 50 and 500 mg/kg, respectively. Lauramide DEA Three male B6C3F 1 mice and four F344 rats were dosed intravenously with randomly labeled [ 14 C]lauramide DEA, 3-5 µci and µci, respectively, formulated to deliver a target dose of 4 ml/kg in mice and 1 ml/kg in rats. 33 The dose for mice was 50 mg/kg and for rats was 25 mg/kg. In mice, lauramide DEA was quickly metabolized and eliminated. At 24 h after dosing, approximately 95% of the dose was excreted, with 90% found in the urine. The highest concentrations and total amounts of the lauramide DEA were in adipose tissue. In rats, 50% of the dose was excreted in the urine within the first 6 h, and more than 80% was excreted in the urine by 24 h. The rats were killed at 72 h after dosing, and only 3% of the dose was recovered in the tissues; 1% of the dose was in the adipose tissue and 0.67% was found in the liver. N-Nitrosodiethanolamine (NDELA) Formation The formation of NDELA upon dermal dosing with DEA (99.7% purity), with and without supplemental oral sodium nitrite, was determined in male B6C3F 1 mice. 42 Groups of 5-6 mice were dosed orally with aq. DEA or dermally with DEA in acetone. DEA, 160 mg/kg/day, was applied for 7 days/wk for 2 wks. ne group of mice dosed dermally was allowed access to the application site, while the other was not. Studies were performed both with and without ~40 mg/kg/day supplemental sodium nitrite in drinking water. NDELA was not found in the urine or blood of mice dosed with DEA without nitrite or in the blood or gastric contents of those given supplemental nitrite with DEA. 13 Panel Book Page 46

50 NDELA formation from DEA (>99% purity) and nitrite was also examined in another study. 43 Female B6C3F 1 mice were dosed dermally or orally with 4 mg/kg DEA, in conjunction with oral exposure to sodium nitrite. Following 7 days of dermal dosing, no NDELA was detected in the blood, ingesta, or urine of test, vehicle control, or sodium nitrite control mice. (The limits of detection for the blood, ingesta, and urine were 0.001, 0.006, and 0.47 µg/ml, respectively.) With a single oral dose, NDELA was formed in all of the animals; the amounts of NDELA detected in the blood and ingesta of mice 2 h post-dosing were very small; ± µg/g and ± µg/g, respectively. TXICLGICAL STUDIES Acute dermal testing with methyl diethanolamine,50% lauramide DEA, and undiluted and 10% aq linoleamide DEA, acute oral testing with DEA, methyl DEA, butyl DEA, and several fatty acid diethanolamides, and acute inhalation testing with methyl DEA did not result in significant toxicity. In repeat dermal testing with DEA, lauramide DEA, and cocamide DEA in mice and/or rats, irritation was observed at the site of application. Increases in liver and kidney weights were observed in most studies, while decreases in body weight were observed sporadically. The LAEL for DEA in a 2-wk study in mice was 160 mg/kg bw. Repeat dermal dosing with methyl DEA in rats also caused skin lesions, but it did not seem to affect liver weights or body weights, and an increase in kidney weights was observed in 1 of 3 studies. The NEL for methyl DEA in a 13-wk study in rats was 100 mg/kg day. A formulation containing 3% linoleamide DEA was not a cumulative systemic toxicant in a 13-wk dermal study. In repeat oral testing with DEA, increases in liver and kidney weights and decreases in body weights were seen in mice and rats. Deaths, believed to be test-article related, occurred in most of the studies, and included a mouse given 100 mg/kg DEA by gavage. With repeat oral dosing of lauramide DEA, the NEL was 250 mg/kg/day in one study using rats. The NEL for Beagle dogs fed lauramide DEA for 12 wks was 5000 ppm. In inhalation studies with DEA in rats, liver and kidney weights were again increased. In 13-wk studies with 400 mg/m 3 DEA, microscopic effects were observed in the larynx. The 90-day NAEC was 1.5 mg/m 3 DEA. Acute (Single Dose) Toxicity Dermal Methyl Diethanolamine The percutaneous LD 50 of undiluted methyl DEA was determined in NZW rabbits, using occlusive patches. 27 The LD 50 values were 9.85 ml/kg (10.2 g/kg) for males and 10.9 ml/kg (11.3 g/kg) for females. In other studies, the dermal LD 50 in rabbits ranged from g/kg bw. 44 Lauramide DEA In an acute dermal toxicity study using guinea pigs, 50% lauramide DEA in corn oil was non-toxic. From the Final Report on the Safety Assessment of Cocamide, Lauramide, Linoleamide, and leamide DEA 2 Linoleamide DEA Linoleamide DEA, tested as 10% aq. and undiluted, was nontoxic in acute studies with guinea pigs. From the Final Report on the Safety Assessment of Cocamide, Lauramide, Linoleamide, and leamide DEA 2 ral Diethanolamine The acute oral toxicity of DEA was determined using guinea pigs and rats. Using groups of 2-3 guinea pigs, all guinea pigs survived dosing with 1 g/kg, but none survived dosing with 3 g/kg DEA gum arabic solution. Using groups of 5 rats, the oral LD 50 of undiluted DEA was ml/kg, and for a group of 6 rats, the LD 50 of DEA in water was 1.82 g/kg. Using rats, 20% aq. DEA had an acute LD 50 of g/kg, based on results of testing performed over a 10 yr time period. With groups of 10 rats, a hair preparation containing 1.6% DEA had an LD 50 of 14.1 g/kg when diluted and 12.9 ml/kg when undiluted. From the Final Report on the Safety Assessment of Triethanolamine, Diethanolamine, and Monoethanolamine 1 14 Panel Book Page 47

51 Methyl Diethanolamine Groups of 5 male and 5 female Sprague-Dawley rats were used to determine the LD 50 value for undiluted methyl DEA. The oral LD 50 for male and female rats was 1.87 ml/kg (1.95 g/kg). 27 from g/kg bw. 44 Butyl Diethanolamine Lauramide DEA The oral LD 50 of butyl DEA in rats was 4.25 g/kg In other oral studies in rats, the LD 50 ranged In rats, the oral LD 50 of 25% lauramide DEA in corn oil was >5 g/kg, of 10% aq. was 2.7 g/kg, of a shampoo formulation containing 8% lauramide DEA was 9.63 g/kg, and of a bubble bath containing 6% lauramide DEA was >15 g/kg. From the Final Report on the Safety Assessment of Cocamide, Lauramide, Linoleamide, and leamide DEA 2 Stearamide DEA The oral LD 50 of a mixture containing 35-40% stearamide DEA was >20 g/kg in CFW mice. From the Final Report on Isostearamide DEA & MEA, Myristamide DEA & MEA, and Stearamide DEA & MEA 4 leamide DEA In rats, the oral LD 50 of undiluted oleamide DEA was 12.4 ml/kg. From the Final Report on the Safety Assessment of Cocamide, Lauramide, Linoleamide, and leamide DEA 2 Linoleamide DEA In rats, the oral LD 50 of undiluted and 10% aq. linoleamide DEA was >5 g/kg, and the LD 50 of a product containing 1.5% linoleamide in formulation was 3.16 g/kg. From the Final Report on the Safety Assessment of Cocamide, Lauramide, Linoleamide, and leamide DEA 2 Cocamide DEA The acute oral toxicity of cocamide DEA was determined using groups of 3 male and 3 female Wistar rats. Three or more animals per group died with doses of 6.3 g/kg. 46 Inhalation Methyl Diethanolamine Five male and 5 female Sprague-Dawley albino rats were exposed for 6 h to a saturated methyl DEA vapor. 27 of the rats died. ther Diethanolamine The intraperitoneal (i.p.) LD 50 of DEA was 2.3 g/kg for mice. From the Final Report on the Safety Assessment of Triethanolamine, Diethanolamine, and Monoethanolamine 1 Methyl Diethanolamine Reported i.p. LD 50 values for methyl DEA in mice ranged from g/kg bw. 44 Repeated Dose Toxicity Dermal Diethanolamine In a 13-wk study, 1 mg/kg of a hair dye formulation containing 2.0% DEA was applied to the backs of 12 rabbits for 1 h, twice weekly. The test site skin was abraded for half of the animals. No systemic toxicity was observed, and there was no histomorphologic evidence of toxicity From the Final Report on the Safety Assessment of Triethanolamine, Diethanolamine, and Monoethanolamine 1 DEA was applied dermally to groups of 5 male and 5 female B6C3F1 mice, 5x/wk for 2 wks, at doses of mg/kg bw in 95% ethanol. 47 All of the male and 3 of the female high-dose test animals died during the study. Ulceration, None Panel Book Page 48

52 irritation, and crusting were observed at the application site of male mice of the 1250 and 2500 mg/kg groups and females of the 2500 mg/kg group. Microscopically, moderate to marked epidermal ulceration and inflammation were observed in these animals. Ulcerative necrosis extended into the underlying dermis. Minimally severe acanthosis, without inflammation, was seen in the 160, 320, and 630 mg/kg dose groups. Absolute and relative liver weights increased in a dose-dependent manner in males and females. The lowest observable adverse effect level (LAEL) was 160 mg/kg bw. Repeated dermal exposure of DEA (99.6% purity) in 96% ethanol was applied to the shaved backs of groups of B6C3F 1 mice for the following time periods: 8 males and 8 females were dosed daily with 0 or 160 mg/kg bw (dose volume 2.13 ml/kg bw) for 1 wk, followed by a 3-wk recovery period; groups of 10 males were dosed 5 days/wk with 0 or 160 mg/kg bw for 1, 4, or 13 wks; and groups of 8 males were dosed 5 days/wk with mg/kg for 1 or 13 wks. 48 With the last dosing scheme, application of 630 and 1250 mg/kg DEA was discontinued, and the animals killed, after 1 wk due to severe skin lesions; the high dose was then 160 mg/kg. In the other animals, including controls, some erythema and/or focal crust formation was observed and attributed to the procedure and/or the vehicle, but not to DEA. No DEA-related deaths were observed. Body weights were not affected by dosing. Statistically significant increases in liver weights were seen in mice dosed with 10 mg/kg bw/day DEA for 1 or more weeks. Microscopically in the liver, eosinophilia was found in animals dosed with 40 mg/kg DEA for 1 or more weeks, and hepatocellular giant cells were seen in animals dosed with 160 mg/kg for 13 wks. Unoccluded dermal applications of mg/ml DEA (purity >99%) in acetone were applied to the backs of 10 male and 10 female B6C3F 1 mice, 5 days/wk for 13 wks, at doses of mg/kg bw. 49 (The area of the dose site was not provided.) Two male mice and 4 female mice dosed with 1250 mg/kg DEA were killed in moribund condition. Final mean body weights of male mice dosed with 1250 mg/kg were statistically significantly decreased compared to controls. Clinical signs of toxicity, observed in males and females dosed with 630 and 1250 mg/kg DEA, were irritation, crust formation, and thickening at the application site. Ulceration and inflammation were observed in the 630 and 1250 mg/kg dose groups. Dose-dependent increases in absolute and relative liver weights, associated with hepatocellular cytological changes, were observed, and hepatocellular necrosis was seen in males dosed with mg/kg DEA. Absolute kidney weights were statistically significantly increased in males and females of all test groups and relative kidney weights were increased in males of all test groups and females dosed with 630 or 1250 mg/kg DEA; nephropathy was not found. Heart weights were increased in high dose males and females, and degeneration was reported. Unoccluded dermal applications of mg/ml DEA (purity >99%) in 95% ethanol were applied to the backs of 10 male and 10 female F344/N rats, 5 days/wk for 13 wks, at doses of mg/kg bw. 50 (The area of the dose site was not provided.) ne male and 2 females given 500 mg/kg died or was killed in moribund condition during the study. Clinical signs of toxicity, observed in males and females given mg/kg DEA, were irritation and crusting at the application site. Final mean body weights were statistically significantly decreased in males dosed with 250 and 500 mg/kg and females dosed with mg/kg DEA. Increases in absolute and relative kidney weights were observed with increased incidence of renal lesions. Increases in absolute and relative liver weights were not accompanied by an increase in hepatic lesions. Demyelination in the medulla oblongata was seen in all high dose animals and 7 females given 250 mg/kg DEA; this lesion was minimal in severity. Methyl Diethanolamine The dermal toxicity of methyl DEA was evaluated in a two 9-day studies using groups of 20 male and 20 female Fischer 344 rats. 51 In both studies, nine 6-hr occlusive applications using a 2 x 2 gauze pad were made to a shaved area of 16 Panel Book Page 49

53 the trunk over an 11-day period. The animals were killed the day after dosing termination. In the first study, the rats were dosed with 0, 260, 1040, or 2080 mg/kg/day in deionized water. The dose volume was 2.0, 0.25, 1.0, and 2.0 ml/kg/day, respectively; methyl DEA was undiluted. None of the animals died during the study, and no clinical signs of toxicity were observed. Transient, barely perceptible erythema was observed for one male and one female of the mid and high dose groups, and barely perceptible edema was observed for on female of the low dose group on day 5. Dose-related exfoliation, excoriation, and necrosis were observed, and fissuring were observed in female rats. Body weight gains were reduced in a dose-related fashion, being statistically significant for males. Feed consumption was also reduced dose-dependently in males, being statistically significant in the mid and high dose groups. Females of the high dose group had statistically significant decreases in hemoglobin concentration, hematocrit, and mean corpuscular hemoglobin, with increased segmented neutrophils. Absolute kidney and adrenal gland weights were statistically significantly increased in females of the 2080 mg/kg group, and relative kidneys to body weights were statistically significantly increased in the 1040 and 2080 mg/kg groups In the second study, the rats were dosed with 0, 100, 500, or 750 mg/kg/day methyl DEA. The dose volume was 1.0 ml/kg/day for all doses.. None of the animals died during the study, and no clinical signs of toxicity were observed. Similar dermal results were reported. In this study, there were no statistically significant differences in body weights or feed consumption between treated and control animals, and there were no differences in hematology. No treatment-related differences in organ weights were found for any dose. At the application site, dose-related increases in the incidence and severity of acanthosis and hyperkeratosis were observed, and multifocal dermatitis and exocytosis of polymorphonuclear leukocytes into the stratum corneum were observed. The researchers also performed a 13 wk study in which Fischer 344 rats received 65 occlusive applications of methyl DEA, 6 h/day, 5 days/wk. Twenty male and 20 female rats were dosed with 0 or 750 mg/kg/day, and groups of 10 male and 10 female rats were dosed with 100 or 250 mg/kg/day. Patches were applied as described previously. All rats of the low and mid dose groups, and 10 rats/gender of the control and high dose groups, were killed the day after dosing termination. The remaining control and high dose animals were killed after a 4-wk recovery period. As in the 9-day studies, no animals died and no clinical signs of toxicity were observed. Slight, transient, erythema was only seen in the high dose group. Dose-related incidences of desquamation, excoriation, ulceration, necrosis, and eschar were observed. Microscopically, acanthosis, hyperkeratosis, and parakeratosis were observed at the site of application. There were no significant differences in body weights, and no effects on organ weights. The no-observed effect level (NEL) was 100 mg/kg/day. Lauramide DEA The dermal toxicity of lauramide DEA was evaluated in two 13-wk studies using Sprague-Dawley rats. A 0.45% aq. solution of a cream cleanser containing 4.0% lauramide DEA, tested in 15 females, and a medicated liquid cleanser containing 5.0% lauramide DEA, tested in 10 males and 10 females, did not have any systemic toxic effects. From the Final Report on the Safety Assessment of Cocamide, Lauramide, Linoleamide, and leamide DEA 2 The dermal toxicity of lauramide DEA (90% purity; 0.83% free DEA by wt) was evaluated in mice and rats. Groups of 10 male and 10 female B6C3F 1 mice were dosed with mg/kg bw lauramide DEA in ethanol, 5 days/wk, for 14 wks. 15 All animals survived until study termination. Dermal irritation was observed at the application site in males and females dosed with 400 or 800 mg/kg lauramide DEA. Final mean body weights and mean body weight gains were similar for test and control animals. The absolute kidney weights of males of the 10, 400 and 800 mg/kg bw groups, the relative kidney to body weights of all dosed males, and the liver weights of females of the 200, 400, and 800 mg/kg bw groups, were 17 Panel Book Page 50

54 significantly greater than those of the control mice. The absolute thymus weights of males of the 400 and 800 mg/kg groups were significantly less than those of the controls. There were no significant differences in reproductive tissue evaluation or estrous cycle between the treated and control groups. At the application site, incidences of non-neoplastic lesions of the skin, including hyperplasia of the epidermis and sebaceous gland, chronic inflammation, parakeratosis, and ulceration, were increased in males and females dosed with 200 mg/kg lauramide DEA.. Groups of 20 male and 20 female F344/N rats were administered mg/kg bw lauramide DEA in ethanol, 5 days/wk for 14 wks; 10 rats per group were used for clinical pathology. 15 All animals survived until study termination. Dermal irritation was observed at the application site of males dosed with 100 mg/kg and in females dosed with 200 or 400 mg/kg lauramide DEA. Final mean body weights and mean body weight gains of males of the 200 and 400 mg/kg bw group were significantly less than those of the control group. Kidney weights of females dosed with 200 or 400 mg/kg bw were significantly greater, and absolute liver weights of males dosed 400 mg/kg lauramide DEA were significantly less, than those of the control groups. There were no significant differences in reproductive tissue evaluation or estrous cycle between the treated and control groups. At the application site, incidences of non-neoplastic lesions of the skin, including hyperplasia of the epidermis and sebaceous gland, chronic inflammation, parakeratosis, and ulceration, were significantly increased with increasing dose. leamide DEA The dermal toxicity of oleamide DEA (47.5% oleic acid DEA condensate content; 0.19% free DEA) was evaluated using mice and rats. Groups of 10 male and 10 female B6C3F 1 mice were dosed with mg/kg bw oleamide DEA in ethanol (0-320 mg/ml), 5 days/wk, for 13 wks. 18 All animals, except one high dose male, survived until study termination. Final mean body weighs and body weight gains of males of the 800 mg/kg group and females of the 400 mg/kg group were significantly less than those of controls. Dermal irritation was observed at the application site of all treated males and most females dosed with 100 mg/kg oleamide DEA. Lesions included epidermal hyperplasia, parakeratosis, suppurative epidermal and chronic active dermal inflammation, sebaceous gland hypertrophy, and ulcer; severity generally increased with increased dose. Heart weights of females of the 200 mg/kg and males and females of the 400 and 800 mg/kg groups, kidney weights of males of the 50, 100, and 400 mg/kg groups, and liver weights of all dose groups were significantly greater than those of controls. The incidences of hematopoietic cell proliferation of the spleen of males of the 800 mg/kg group and females of the 400 and 800 mg/kg groups were significantly greater than the controls. Sperm motility and vaginal cytology parameters of dosed mice were similar to those of the controls. Groups of 20 male and 20 female F344/N rats were administered mg/kg bw oleamide DEA in ethanol (0-485 mg/ml) for 5 days/wk for 13 wks; 10 rats per group were used for clinical chemistry and hematology evaluation. 18 All animals survived until study termination. Dermal irritation was observed at the application site of most males dosed with 100 mg/kg and all females dosed with 50 mg/kg oleamide DEA. Lesions included epidermal hyperplasia, parakeratosis, suppurative epidermal and chronic active dermal inflammation, and sebaceous gland hypertrophy; severity general increased with increased dose. The final mean body weights and mean body weight gains of males of the 200 and 400 mg/kg groups and mean body weight gains of females of the 400 mg/kg group were significantly less than controls; some associated lower organ weights were observed. Kidney weights were significantly greater for females of the 200 and 400 mg/kg groups as compared to controls. Some increases in segmented neutrophil counts and alkaline phosphatase concentrations were reported. There were no biologically significant differences in sperm motility or vaginal cytology parameters between treated and control rats. 18 Panel Book Page 51

55 Linoleamide DEA The dermal toxicity of a shampoo formulation containing 3.0% linoleamide DEA was evaluated in a 13-wk study. The test article was applied as a 2.5% solution, a 25% solution, or a 25% solution that was rinsed after 15 min, to groups of 10 male and 10 female Sprague-Dawley rats. Dermal irritation was observed, but the formulation containing 3% linoleamide DEA was not a cumulative systemic toxicant. From the Final Report on the Safety Assessment of Cocamide, Lauramide, Linoleamide, and leamide DEA 2 Cocamide DEA The dermal toxicity of cocamide DEA (containing 18.2% free DEA by wt) was evaluated using mice and rats. Groups of 10 male and 10 female B6C3F 1 mice were dosed with mg/kg bw cocamide DEA in ethanol (0-320 mg/ml), 5 days/wk, for 14 wks. 19 All animals survived until study termination. Dermal irritation was observed at the application site of males and females of the 800 mg/kg dose group. Epidermal and sebaceous gland hyperplasia, parakeratosis, chronic active inflammation, and ulcer were observed; severity general increased with increased dose. Final mean body weights and mean body weight gains were similar for test and control animals. The absolute and relative liver and kidney weights to body weights of males and females of the 800 mg/kg group, relative liver weights to body weights of females of the 400 mg/kg group, and absolute and relative lung weights to body weights of females of the 800 mg/kg group were significantly greater than for those of the controls. The epididymal spermatozoal concentration was significantly greater in males of the 800 mg/kg dose group. Groups of 20 male and 20 female F344/N rats were dosed dermally with mg/kg/bw cocamide DEA in ethanol (0-485 mg/ml), 5 days/wk, for 14 wks; 10 rats per group were used for clinical chemistry and hematology evaluation. 19 All animals survived until study termination. Dermal irritation was observed at the application site of 2 males and one female of the 100 mg/kg group and nearly all males and females of the 200 and 400 mg/kg dose groups. Lesions included epidermal and sebaceous gland hyperplasia, parakeratosis, chronic active inflammation, and ulcer; incidence and severity general increased with increasing dose. Final mean body weights and mean body weight gains of males and females of the 200 and 400 mg groups were significantly less than those of the controls. Kidney weights of females of the 50 mg/kg group were significantly greater than those of the controls. Decreases in epididymal weights in 200 and 400 mg/kg males were attributed to decreased body weights. Changes in some hematology and clinical chemistry parameters were noted, and the researchers stated there was an indication of altered lipid metabolism, as evidenced by decreased cholesterol and triglyceride concentrations. The incidences of renal tubule regeneration were greater in females of the 100 dose group, and the incidences and severities were greater in females of the 200 and 400 mg/kg dose groups, as compared to controls. ral Diethanolamine ral studies were conducted in which neonatal rats were dosed with 1-3 mm/kg/day DEA, as a neutralized salt, on days 5-15 after birth, male rats were dosed with 4 mg/ml neutralized DEA in drinking water for 7 wks, and rats were fed g/kg/day DEA in feed for 90 days. Repeated oral ingestion of DEA produced evidence of hepatic and renal damage. Deaths occurred in the 7 wk and 90 day studies. Administration of neutralized DEA in the drinking water at doses of 490 mg/kg/day for 3 days or of 160 mg/kg/day for 1 wk produced alterations of hepatic mitochondrial function. ral and administration of DEA my affect, directly or indirectly, the serum enzyme levels, isozyme patterns, and concentrations of some amino acids and urea in the male rat liver and kidney. Repeated DEA administration in the drinking water increased male rat hepatic mitochondrial ATPase and altered mitochondrial structure and function. From the Final Report on the Safety Assessment of Triethanolamine, Diethanolamine, Monoethanolamine 1 Female CD-1 mice, 3 per group, were dosed orally, by gavage, with mg/kg bw DEA in distilled water for 7 days. 52 ne animal of the 100 mg/kg group died, and the death was considered test article-related. (Two deaths in the 1000 mg/kg group were attributed to gavage error.) 19 Panel Book Page 52

56 Groups of 48 female B6C3F 1 mice were dosed orally, by gavage, with mg/kg bw DEA in distilled water for 14 days. 53 No effect on body weight gain was observed for any group. Liver weights were increased in a dose-dependent manner; no effects were seen in thymus, spleen, or kidney weights. DEA exposure resulted in an increased in the number of B-cells and a decreased in the number of CD4+CD8- (18%) T-cell subset; total T-cells and other T-cell subsets were not affected. A dose-dependent decreased in the antibody-forming response to sheep erythrocytes was observed with 600 mg/kg DEA, and a dose-dependent decrease in the cytotoxic T-lymphocyte response was observed, which was statistically significant at the lowest dose. The natural killer cell response was not affected. In a 13-wk study, 10 male and 10 female B6C3F 1 mice were dosed orally, by gavage, 5 times/wk, with mg/kg DEA in deionized water. 54 Two males of the high dose group died during the study. Body weights and body weight gains of high dose males were reduced. No clinical signs of toxicity were noted. Treatment-related renal lesions were found, but details were not provided. Groups of 10 male and 10 female B6C3F 1 mice were given 0-10,000 ppm DEA (>99% purity) in the drinking water for 13 wks. 49 The ph of the solution was adjusted to 7.4. All males and females of the 5000 and 10,000 ppm groups and 3 females of the 2500 ppm group died during the study. Body weight gains were decreased in males of the 2500 ppm group and females of the 1250 and 2500 ppm groups. No significant gross effects were noted at necropsy. Statistically significant, dose-dependent, increases in absolute and relative liver weights were observed, with hepatocellular cytological alterations and necrosis in animals given 2500 pm DEA. Absolute and relative kidney weights also increased dose-dependently, with statistically significant increases in mice given 1250 or 2500 ppm DEA, and neuropathy was observed in all male test groups and female test groups given 2500 or 5000 ppm DEA. Heart weights were increased in female mice given 2500 ppm DEA, and relative heart weights were increased in males of the 2500 ppm group and females of the 1250 and 2500 ppm groups. Groups of 48 F344 female rats were dosed orally, by gavage, with mg/kg bw DEA in distilled water for 14 days. 55 Rats exposed to 10 and 200 mg/kg DEA had significant decreases in body weight and/or body weight gains. Liver and kidney weights were increased in a dose-dependent manner. Exposure to DEA did not alter the number of B-cells, T- cells, or T-cell subsets. The proliferative response to allogenic cells, as measured by the mixed leukocyte response, was increased in a dose-dependent manner; the increase in the high dose was statistically significant. Natural killer cell response and cytotoxicity of resident macrophages were decreased in DEA-treated animals. Groups of 10 male albino rats were given feed containing mg/kg bw/day DEA (99% purity) for 32 days. 56 Nine of the 10 high dose animals died during the study. All test animals had decreased hemoglobin and hematocrits, with an increased white blood cell count. The relative liver weights of animals fed 0.01 and 0.1% DEA, and the absolute liver weights of animals of the 0.1% group, were increased. In a repeat 30-day study using the same procedure and dose levels, 7 of the 10 high dose animals died, and the remaining 3 high-dose animals killed, prior to study termination; body weights and feed consumption were significantly decreased in this group. Again, hemoglobin and hematocrit were decreased in the 0.1% group, and the hemoglobin value was reduced in the 0.1% group. Groups of 10 male F344/N rats were given ppm and 10 female F344/N rats were given ppm DEA in the drinking water for 13 wks. 50 Two males of the high dose group died during the study. The following dose-related effects were observed: decreases in body weight gains in males and females; hematological effects; increases in kidney weights accompanied by renal lesions; and increases in relative liver weights in males and females. Demyelination of the medulla of the brain and of the spinal cord was observed in all males of the 2500 and 5000 ppm groups and all females of the 1250 and 2500 ppm groups. 20 Panel Book Page 53

57 To determine whether repeated dermal administration of DEA induced cell proliferation and/or apoptosis in the livers of mice, male and female B6C3F1/Crl mice were dosed dermally with 0 and 160 mg/kg bw DEA (99.6% purity) in 96% ethanol for 1 wk followed by a 3-wk recovery period, and male mice were exposed dermally to 0 or 160 mg/kg for 1, 4, or 13 wks. 48 A dose response relationship was examined with application of mg/kg DEA to male mice for 1 or 13 wks. After 1 wk of dosing, increased cell proliferation in the liver was observed in males and females; this effect was reversible. Repeated application of 10 mg/kg DEA to male B6C3F 1 mice caused increased liver cell proliferation. DEA had no effect on the number of apoptotic cells in the liver. Lauramide DEA The oral toxicity of lauramide DEA was evaluated in two 13-wk dietary studies. In the first study, 0-2% lauramide DEA was evaluated using groups of 15 male and 15 female SPF rats. A reduction in growth was associated with reduced feed intake at dose of 0.5% lauramide DEA. There were no treatment-related gross or microscopic lesions. The no-effect dose was 0.1% lauramide DEA. In the second study, groups of 20 male and 20 female Wistar rats were fed mg/kg/day. No adverse effects were reported, and the no-effect dose for rats was 250 mg/kg/day. Groups of 4 male and 5 female Beagle dogs were fed ppm lauramide DEA for 12 wks. No adverse effects were reported, and the no-effect dose for dogs was 5000 ppm lauramide DEA. From the Final Report on the Safety Assessment of Cocamide, Lauramide, Linoleamide, and leamide DEA 2 Inhalation Diethanolamine Short-term inhalation of 200 ppm DEA vapor or 1400 ppm DEA aerosols produced respiratory difficulties and some deaths in male rats. Inhalation of 25 ppm for 216 continuous hours resulted in increased liver and kidney weights, while exposure of male rats to 6 ppm DEA following a workday schedule for 13 wks caused decreased growth rate, increased lung and kidney weights, and some deaths. From the Final Report on the Safety Assessment of Triethanolamine, Diethanolamine, and Monoethanolamine 1 In a 2-wk dose-range finding inhalation study, 10 male and 10 female Wistar rats were exposed, nose only, to target concentrations of mg/m 3 DEA (>99% pure) for 6 h/day, 5 days/wk Mass median aerodynamic diameter (MMAD) was µm. Test article-related effects were not seen with 100 or 200 mg/m 3 DEA. With 400 mg/m 3 DEA, decreased body weights and body weight gains were seen in males and relative and absolute liver weights were seen in females. Microscopically, no effects were seen in the respiratory tract; the larynx was not examined. Based on the results of the dose-range finding study, target concentrations of 0, 15, 150, and 400 mg/m 3 DEA were used in a 90-day study, in which groups of 13 male and 13 female rats were exposed via inhalation to 65 exposures, 6-h/day 5 days/wk. The MMAD was µm. A functional observational battery was conducted using 10 rats/gender/group. Body weight gains were reduced in males of the high dose group. Some statistically significant effects on clinical chemistry values were seen in the mid and high dose group. Males and females of the high dose group had statistically significant increases in blood content in the urine, and males of the mid and high dose groups excreted significantly elevated amounts of renal tubular epithelial cells. Relative liver weights were statistically significantly increased in males and females of the high dose group and females of the mid-dose group, and relative kidney weights were statistically significantly increased in males and females of the mid and high dose groups. Focal squamous metaplasia of the ventral laryngeal epithelium was observed in test animals of all groups, and a concentration-dependent increase in laryngeal squamous hyperplasia, and in the incidence and severity of local inflammation of the larynx and trachea, were observed. There were no indications of neurotoxicological effects. In a third study, test groups of 10 male and 10 female Wistar rats were exposed to target concentrations of 0, 1.5, 3, and 8 mg/m 3 DEA using the same dosing schedule as above, and recovery groups of 10 females were exposed to 3 or 8 mg/m 3, with a post-exposure period of 3 mos. No dose-related clinical signs were observed. Liver weights of test, but not Panel Book Page 54

58 recovery, females dosed with 8 mg/m 3 were statistically significantly increased. Laryngeal effects were similar to those described above. No microscopic changes were observed in the upper respiratory tract of recovery animals. The 90-day no observed effect concentration (NAEC) was determined to be 1.5 mg/m 3 DEA. In inhalation studies, Sprague-Dawley rats, Hartley guinea pigs and Beagle dogs (number per species and sex not specified) were each exposed to 0.5 ppm DEA for 6 h/day, 5days/wk, for a total of 45 exposures. 56 All animals survived until study termination. There were no clinical signs of toxicity, and no evidence of irritation. No gross or microscopic lesions were observed at necropsy. REPRDUCTIVE AND DEVELPMENTAL TXICITY In a study in which gravid mice were dosed with mg/kg DEA from day 6 of gestation through PND 21, no effects on skeletal formation were observed, but dose-dependent effects on some growth and developmental parameters were observed. In a study in which parental mice were treated with DEA for 4 wks prior to dosing, sperm motility was decreased in a dose-dependent manner. In rats and rabbits, dermal dosing with up to 1500 mg/kg, and 350 mg/kg DEA, respectively, during gestation, did not have any fetotoxic or teratogenic effects. The NEL for embryonal/fetal toxicity was 380 mg/kg/day for rats and 350 mg/kg/day for rabbits. In dermal reproductive studies with up to 1000 mg/kg/day methyl DEA in rats, the NEL for developmental toxicity was 1000 mg/kg/day. In an oral reproductive study in which rats were dosed with up to 1200 mg/kg/day DEA on days 6-15 of gestation, maternal mortality was observed at doses of 50 mg/kg; the NEL for embryonal/fetal toxicity was 200 mg/kg/day. In a study in which gravid rats were dosed orally with up to 300 mg/kg/day DEA, the dams of the 300 mg/kg group were killed due to excessive toxicity; the LD 50 was calculated to be 218 mg/kg. The LAEL for both maternal toxicity and teratogenicity was 125 mg/kg/day. In a reproductive study in which rats were exposed by inhalation to DEA on days 6-15 of gestation, the NAEC for both maternal and developmental toxicity was 0.05 mg/l, and the NAEC for teratogenicity was.0.2 g/ml Dermal Diethanolamine Hair dyes containing up to 2% DEA were applied topically to the shaved skin of groups of 20 gravid rats on days, 1, 4, 7, 10, 13, 16, and 19 of gestation, and the rats were killed on day 20 of gestation. No developmental or reproductive effects were observed. From the Final Report on the Safety Assessment of Triethanolamine, Diethanolamine, and Monoethanolamine 1 A reproductive and developmental toxicity study was performed in which mg/kg DEA (98.5% purity) in ethanol was applied to a 2 cm 2 area on the back of 15 male C57BL/6 mice, 15 per group, for 4 wks. 58 (It was not stated whether the test area was covered.) These males were then mated with untreated females. In the parental male mice, sperm motility was significantly decreased in a dose-dependent manner. In male pups, a significant decrease in epididymis weight was seen in the 80 mg/kg group at postnatal day (PND) 21, and reductions in male reproductive weights of high-dose pups was seen at PND 70. There were no significant differences in skeletal formation, and differences in growth and development parameters were not significant. Doses of mg/kg DEA (98.5% purity) in ethanol were applied to a 2 cm 2 area on the backs of groups of 10 gravid female C57BL/6 mice on day 6 of gestation through PND 21. The body weights of male and female pups of the high dose group were statistically significantly decreased compared to controls. No specific differences in organ weights were observed in pups of the test groups as compared to controls. There were no significant differences in skeletal formation. Some dose-dependent effects on growth and developmental parameters were noted. Sperm motility was decreased in male pups, but this result was not statistically significant. CD rats and NZW rabbits were used to evaluate the potential of DEA ( 99.4% purity) to produce developmental toxicity with dermal exposure. 59 Groups of 25 gravid CD rats were dosed dermally with mg/kg/day DEA in deion- 22 Panel Book Page 55

59 ized water, 6 h/day, on days 6-15 of gestation, under an occlusive covering. Dosing volume was 4 ml/kg/day. Control animals were dosed with vehicle only. Teratogenic effects were not seen at any dose. Dermal application of 500 mg/kg DEA resulted in alterations of maternal hematological parameters, but did not affect embryonal/fetal development. ther signs of maternal toxicity were seen at this dose and with 1500 mg/kg/day. The NEL for embryonal/fetal toxicity was estimated to be 380 mg/kg/day, which incorporates an adjustment for the 10-24% deficit in expected dose that occurred on days of gestation. Groups of 15 mated rabbits were dosed dermally with mg/kg/day DEA in deionized water, 6 h/day, on days 6-18 of gestation, under an occlusive covering. Dosing volume was 2 ml/kg/day, and controls were dosed with vehicle only. Dermal administration of 350 mg/kg/day DEA produced severe skin irritation in rabbits, and signs of maternal toxicity were observed at this dose. No developmental toxicity was observed, and there was no evidence of teratogenicity at any dose. The NEL for maternal toxicity of DEA in rabbits was 35 mg/kg/day, and the embryonal/fetal NEL was 350 mg/kg/day DEA. Methyl Diethanolamine Five groups of 8 gravid female CD rats were used in a dermal dose-range finding study with methyl DEA in water (99.5% purity). 60 Doses of mg/kg/day, at a volume of 4 ml/kg, were applied to a 20 cm 2 shaved area of the back with an occlusive patch on days 6-15 of gestation, and the dams were killed on day 21 of gestation. Significant dermal irritation, including exfoliation, excoriation, crusting, and ecchymoses, was observed at the dosing site of dams of the 750 and 1000 mg/kg groups. Erythema increased in severity during dosing; no erythema was observed at 4 days after termination of dosing. Barely perceptible to moderate edema was also observed in several dams of these groups. There was no effect on maternal body weights, body weight gains, and liver or kidney weights. There were no signs of reproductive or fetal toxicity, and there was no increased incidence of malformations. Groups of 25 gravid female CD rats were used in the definitive study, and doses of 0, 250, 500, or 1000 mg/kg/day were applied using the same procedure described previously. Dermal reactions, including erythema, exfoliation, and crusting in the 500 mg/kg group and erythema, exfoliation, excoriation, crusting, ecchymoses, necrosis, and edema in the 1000 mg/kg group, were observed at the site of application. There was no effect on maternal body weights, body weight gains, or liver or kidney weights; erythrocyte and hematocrit count were decreased in the 1000 mg/kg group. There were no signs of reproductive or fetal toxicity, and there was no increased incidence of malformations. The NELs for maternal toxicity was 250 mg/kg/day and for developmental toxicity was 1000 mg/kg/day. ral Diethanolamine In a Chernoff-Kavlock screening test, groups of 4 gravid CD-1 mice were dosed orally, by gavage, with mg/kg bw DEA in distilled water on days 6-15 of gestation. 52 Two, 3, and 4/4 animals of the 720, 1370, and 2605 mg/kg dose groups died during the study. Rough hair coats were observed at all dose levels. Group of 50 female gravid CD-1 female mice were dosed orally, by gavage, with 0 or 450 mg/kg bw DEA in distilled water on days 6-15 of gestation. No animals died during the study. The reproductive index and average number of live litters on day 0 were not affected by dosing, but the average number of live litters on day 3 was decreased. Mean body weights and body weight gains of pups were also decreased on PND 3. Gravid Sprague-Dawley rats were dosed orally, by gavage, with mg/kg/day DEA on days 6-15 of gestation. 61 Maternal mortality was observed at doses of mg/kg/day. At doses of 50 and 200 mg/kg/day, no differences in gross developmental endpoints were found between test and control animals. The NEL for embryonal/fetal toxicity was 23 Panel Book Page 56

60 200 mg/kg/day DEA. However, maternal weight gains were significantly decreased at that dose. (Additional details were not provided.) Groups of 12 gravid female Sprague-Dawley rats were dosed orally, by gavage, with mg/kg bw/day DEA (>98% purity) distilled water on days 6-19 of gestation, while controls were dosed with vehicle only. 62 Dosing volume was 5 ml/kg. Surviving dams and pups were killed on PND 21. All females dosed with 300 mg/kg DEA were killed prior to study termination due to excessive toxicity. Toxicity was also observed for one dam dosed with 200 mg/kg, and only 5 dams of the 250 mg/kg group delivered live litters and survived until study termination. The calculated LD 50 was 218 mg/kg bw/day. No significant maternal or developmental toxicity was seen with 50 mg/kg bw/day DEA. Signs of maternal and developmental toxicity were seen at doses of 125 mg/kg bw/day and included decreased maternal weight gains, increased kidney weights in dams, increased post-implantation and postnatal mortality, and reduced live pup weights. The no observable adverse effect level for maternal toxicity and teratogenicity was 0.05 mg/l, and the LAEL for these parameters were 125 mg/kg/day. Inhalation Diethanolamine In a range-finding study, groups of 10 gravid Wistar rats were exposed, nose-only, to target concentrations of mg /l DEA, 6 h/day, on days 6-15 of gestation. 63 (DEA purity was >98.7%.) All animals survived until study termination. Relative liver weights were increased in animals of the 0.2 mg/l group, and absolute and relative liver weights were increased in animals of the 0.4 mg/l group. No treatment-related effects were observed with 0.1 mg/l DEA. Groups of 25 gravid Wistar rats were exposed, nose-only, to target concentrations of mg DEA aerosol/l air, 6 h/day, on days 6-15 of gestation. 64,65 (DEA purity >98.7%.) Maternal toxicity, as indicated by vaginal hemorrhage, was seen at the highest dose level. No treatment-related malformations were observed at 0.2 mg/m 3. The NAEC for both maternal and developmental toxicity was 0.05 mg/l, and the NAEC for teratogenicity was >0.2 mg/l, which was the highest dose tested in this study. Effect on Hippocampal Neurogenesis and Apoptosis Diethanolamine The effect of DEA on neurogenesis was investigated using C57BL/6 mice. 66 DEA, mg/kg bw in ethanol, was applied to a 2 cm 2 area on the backs of gravid female mice, 6 per group, on days 7-17 of gestation. A dose-related decrease in litter size was observed at doses >80 mg/kg, and the decrease was statistically significant at doses of mg/kg bw/day. The livers of the maternal mice were analyzed on day 17 of gestation, and hepatic concentrations of choline and its metabolites were statistically significantly decreased. In the fetal brain, treatment with 80 mg/kg bw/day DEA diminished the proportion of cells that were in the mitotic phase to 50% of controls. The number of apoptotic cells of the hippocampal area was >70% higher in fetuses of DEA-treated mice compared to controls. The researchers stated that the effect observed in the mouse fetal brain after administration of DEA was likely to be secondary to diminished choline levels. The researchers also hypothesized that a potential mechanism for the effect of choline deficiency, and maybe DEA, on progenitor cell proliferation and apoptosis involves abnormal methylation of promoter regions of genes. The doses used in the above study were based on expected concentrations of 1-25% DEA in cosmetic formulations. However, based on comments from the Cosmetic, Toiletry, and Fragrance Association (now known as the Personal Care Products Council) indicating the over-estimation of the amount of DEA contained in consumer products, 23 the researchers tested lower doses to establish a dose-response relationship. Groups of 7 mice were dosed dermally with 0-80 mg/kg bw DEA (purity >99.5%) as described previously, with the exception that acetone was used as the vehicle. While the results 24 Panel Book Page 57

61 reported in the earlier study with 80 mg/kg bw DEA were confirmed, no differences were seen between treated and control groups with <80 mg/kg bw. In a study to identify the potential mechanism for the alterations described above, mouse neural precursor cells were treated in vitro with DEA. 67 Cells exposed to 3 mm DEA had less cell proliferation at 48 h and had increased apoptosis at 72 h. DEA treatment decreased choline uptake into the cells, resulting in diminished choline and phosphocholine. A three-fold increase in choline concentration prevented the effects of DEA exposure on cell proliferation and apoptosis; intracellular phosphocholine levels remained low. The researcher hypothesized that DEA interferes with choline transport and choline phosphorylation in neural precursor cells. Additionally, it was suggested that DEA acts by altering intracellular choline availability. GENTXICITY DEA, methyl DEA, oleamide DEA, and cocamide DEA were, generally, non-genotoxic in a number of assays. Exceptions were positive results for DEA in an in vitro assay of DNA strand break in isolated rat, hamster, and pig hepatocytes, the induction of SCEs in CH cells by lauramide DEA, and an increase in the frequency of micronucleated erythrocytes in mice by cocamide DEA. Diethanolamine In Vitro DEA, with and without metabolic activation using liver preparations from rats induced with a polychlorinated biphenyl mixture, was not mutagenic to Salmonella typhimurium TA100 or TA1535. From the Final Report on the Safety Assessment of Triethanolamine, Diethanolamine, and Monoethanolamine 1 With or without metabolic activation, DEA was not mutagenic in Ames test using S. typhimurium TA98, TA100, TA1535, or TA1537, was negative in a mouse lymphoma assay, and did not induce sister chromatid exchanges (SCEs) or chromosomal aberrations in a Chinese hamster ovary (CH) cell cytogenetic assay. 35 DEA was not clastogenic in a mouse micronucleus test. Positive results were reported in an in vitro assay for induction of DNA single-strand breaks in isolated hepatocytes for rats, hamsters (both at 25 µmol/tube), and pigs ( 12.5 µmol/tube). In studies examining species selectivity of effects caused by DEA, increases in DNA synthesis were observed in mouse and rat, but not human, hepatocytes following treatment with DEA. 68 Additionally, when the hepatocytes were incubated in medium containing reduced choline, DNA synthesis was increased in mouse and rat hepatocytes, but not human hepatocytes. Conversely, choline supplementation reduced DEA-induced DNA synthesis in mouse and rat hepatocytes. Methyl Diethanolamine The genotoxic potential of methyl DEA was evaluated in an Ames test, the CH/HGPRT forward mutation test, an SCE in CH cells, with and without metabolic activation, and in an in vivo micronucleus test in Swiss-Webster mice. 69 Methyl DEA did not produce any significant response in any of these assays. Lauramide DEA Lauramide DEA was not mutagenic or genotoxic in multiple Ames assays, a DNA damage assay using Bacillus subtilis, an in vitro transformation assay using Syrian golden hamster embryo cells, or an in vivo transformation assay using hamster embryo cells. Lauramide DEA was mutagenic in the spot test with two strains of S. typhimurium, but quantitative results were not provided. From the Final Report on the Safety Assessment of Cocamide, Lauramide, Linoleamide, and leamide DEA 2 Lauramide DEA ( µg/plate) was not mutagenic in the Ames test with or without metabolic activation, and it was negative in a L5178Y mouse lymphoma assay, did not increase the number of chromosomal aberrations in CH cells, 25 Panel Book Page 58

62 with or without metabolic activation, and was not clastogenic in a mouse micronucleus test Lauramide DEA induced SCEs in CH cells, in the presence and absence of metabolic activation. leamide DEA leamide DEA was not mutagenic in an Ames test ( µg/plate) with or without metabolic activation, and it did not induce mutations in L5178Y mouse lymphoma cells, with or without metabolic activation. 18 Cocamide DEA Cocamide DEA was not mutagenic in an Ames assay, did not induce mutations in L5178Y mouse lymphoma cells, SCEs or chromosomal aberrations in CH cells; all tests were performed with and without metabolic activation. 19 However, at the end of a 14-wk repeated dose study (described earlier), significant increases in the frequencies of micronucleated normochromatic erythrocytes were found in peripheral blood of male and female mice. CARCINGENICITY The carcinogenic potential of dermally applied DEA and lauramide, oleamide, and cocamide DEA was evaluated by the NTP in B6C3F 1 mice and F344/N rats. The doses tested are included in parentheses. DEA produced clear evidence of carcinogenic activity in male and female mice (0-160 mg/kg) and no evidence in male and female rats (0-64 mg/kg); lauramide DEA produced some evidence of carcinogenic activity in female mice (0-200 mg/kg) and no evidence in male mice or male and female rats (0-100 mg/kg); oleamide DEA produced no evidence of carcinogenic activity in male or female mice (0-30 mg/kg) or male or female rats (0-100 mg/kg); and cocamide DEA produced clear evidence of carcinogenic activity in male and female mice (0-200 mg/kg), equivocal evidence in female rats (0-100 mg/kg),and no evidence in male rats (0-100 mg/kg). According to the IARC Working Group, their overall evaluation is that there is inadequate evidence in humans for the carcinogenicity of DEA and that DEA is not classifiable as to its carcinogenicity in humans. Because DEA is not mutagenic or clastogenic, a non-genotoxic mode of tumorigenic action is indicated. A plausible mode of action for DEA carcinogenicity in rodents involves cellular choline deficiency. Dermal Table 6 summarizes the conclusions of the NTP dermal studies on DEA, lauramide DEA, oleamide DEA, and cocamide DEA. Diethanolamine The NTP evaluated the carcinogenic potential of DEA (>99% purity) in ethanol using B6C3F 1 mice and F344/N rats. 35 Groups of 50 male and 50 female mice were dosed dermally with mg/kg/day, 5 days/wk, for 103 wks. Survival of dosed females, but not males, was significantly decreased in a dose-dependent manner. Mean body weights of test animals were decreased at various intervals throughout the study. In male mice, the incidences of hepatocellular adenoma and of hepatocellular adenoma and carcinoma (combined) in all dose groups, and the incidence of hepatocellular carcinoma and hepatoblastoma in the 80 and 160 mg/kg group, were statistically significantly increased compared to controls. In female mice, the incidence of hepatocellular neoplasms was significantly increased. Male mice also had a dose-related increase in the incidences of renal tubule hyperplasia and renal tubule adenoma or carcinoma (combined), and an increase in the incidence of renal tubule adenoma. In male and female mice, incidences of thyroid gland follicular cell hyperplasia were increased. Hyperkeratosis, acanthosis, and exudate were treatment-related changes observed at the application site. It was concluded that there was clear evidence of carcinogenic activity of DEA in male and female B6C3F 1 mice based on increased incidences of liver neoplasms in males and females and increased incidences of renal tubule neoplasms in males. In the rats, groups of 50 males were dosed dermally with 0-64 mg/kg bw DEA, and groups of 50 females with 0-32 mg/kg bw, 5 days/wk, for 103 wks. The only treatment-related clinical finding was irritation at the application site. Minimal to mild non-neoplastic lesions were found at the site of application of dosed males and females. The incidence and severity Panel Book Page 59

63 of nephropathy in dosed females, but not males, was significantly greater in the treated groups compared to controls. There was no evidence of carcinogenic activity of DEA in male or female F344/rats. The carcinogenic potential of DEA was evaluated using a Tg AC (zetaglobin v-ha-ras) transgenic mouse model. 70,71 Groups of female homozygous mice were dosed dermally with 0-20 mg DEA/mouse in 95% ethanol, 5x/wk, for 20 wks. DEA was inactive in Tg AC mice. According to a review of DEA by the International Agency for Research on Cancer (IARC) Working Group, there is inadequate evidence in humans, for the carcinogenicity of DEA. 72 There is limited evidence in experimental animals for the carcinogenicity of DEA. The overall evaluation of the IARC is that DEA is not classifiable as to its carcinogenicity to humans (Group 3). Lauramide DEA The NTP evaluated the carcinogenic potential of lauramide DEA (90% purity; 0.83% free DEA by wt) using B6C3F 1 mice and F344/N rats. 15 Groups of 50 male and 50 female mice were dosed dermally with 0, 100, or 200 mg/kg/day DEA in ethanol (0, 50, or 100 mg/ml, respectively), 5 days/wk, for wks. There were no clinical findings attributable to lauramide DEA. In female mice, the incidences of hepatocellular adenoma and carcinoma (combined) were significantly increased in all dose groups, of hepatocellular adenoma was significantly increased in females of the 100 mg/kg group, and of eosinophilic foci was significantly increased in the 200 mg/kg group. The incidences of these lesions in male mice were not significantly different from controls. Incidences of non-neoplastic lesions of the skin at the site of application were significantly increased in treated males and females; the lesions were mostly epidermal and sebaceous gland hyperplasia. The incidence of focal hyperplasia of thyroid gland follicular cells was significantly greater in males of the 200 mg/kg group compared to controls; there were not corresponding increases in the incidences of follicular cell neoplasms. There was no evidence of carcinogenic activity in male mice, and there was some evidence of carcinogenic activity in female B6C3F 1 mice. This conclusion for female mice was based on increased incidences of hepatocellular neoplasms; these researchers stated these increases were associated with free DEA, which was present as a contaminant. Groups of 50 male and 50 female rats were dosed dermally with 0, 50, or 100 mg/kg bw lauramide DEA in ethanol (0, 85, or 170 mg/ml, respectively), 5 days/wk, for wks. Survival and mean body weights of test animals were similar to controls. The only treatment-related clinical finding was minimal to moderate irritation at the application site; epidermal and sebaceous gland hyperplasia, hyperkeratosis, and chronic inflammation were significantly increased compared to controls. The incidence of neoplasms was similar for treated and control rats. The incidence of forestomach ulcer in the 100 mg/kg group males, of inflammation of the nasal mucosa in all test males, and of chronic inflammation of the liver in 100 mg/kg females was significantly lower than in the controls. There was no evidence of carcinogenic activity of lauramide DEA in male or female F344/rats. leamide DEA The NTP also examined the carcinogenic potential of dermally applied oleamide DEA (47.5% oleic acid DEA condensate content; 0.19% free DEA) using B6C3F 1 mice and F344/N rats. 18 Groups of 55 male and 55 female mice were dosed dermally with 0, 15, or 30 mg/kg oleamide DEA in ethanol (0, 7.5, or 15 mg/ml, respectively), 5 days/wk, for 105 wks; 5 males and 5 females per group were used for a 3-mos interim evaluation. Survival was similar for treated and control mice. Mean body weights of females of the 30 mg/kg group were less than controls as of wk 76 of the study. Increased incidence of dermal irritation was observed at the application site of males of the 30 mg/kg dose group. The incidences of epidermal and sebaceous gland hyperplasia were significantly increased in all males and female dose groups, as compared to controls, at 27 Panel Book Page 60

64 both the 3-mos and 2-yr evaluation. Additional dermal lesions were observed, but a dose-related increase in neoplasms was not observed. The incidence of malignant lymphoma in female mice increased with increasing dose, and the increase was significant in the high dose group. However, the researchers noted that the incidence in the high-dose group was similar to the incidences observed in other studies that used ethanol as the vehicle. There was no evidence of carcinogenic activity in male or female mice dosed dermally with 30 mg/kg oleamide DEA. The researchers also dosed dermally groups of 50 male and 50 female rats with 0, 50, or 100 mg/kg oleamide DEA in ethanol (0, 85, or 170 mg/ml, respectively), 5 days/wk, for 104 wks. Survival was similar for treated and control rats. Mean body weights of males of the 100 mg/kg group were slightly less than controls throughout the study, while in the females of this dose group, a decrease in body weights was observed from wk 24 on. Mild to moderate irrigation was observed at the application site of doses rats. Skin lesions observed at the application site, including, significant increases in epidermal and sebaceous hyperplasia, were considered indicative of local irritation, with no neoplastic or preneoplastic changes. The researchers did not consider increased incidences of lesions in the forestomach, testis, and thyroid gland test article-related. There was no evidence of carcinogenic activity in male or female rats dosed dermally with 100 mg/kg oleamide DEA. Cocamide DEA The carcinogenic potential of dermally applied cocamide DEA (containing 18.2% free DEA by wt) was also assayed by the NTP, using B6C3F 1 mice and F344/N rats. 19 Groups of 50 male and 50 female mice were dosed dermally with 0, 100, or 200 mg/kg cocamide DEA in ethanol, 5 days/wk, for wks. There were no significant differences in survival between the test animals and the controls. Mean body weights of 100 and 200 mg/kg females were less than controls from wks 93 and 77, respectively. Dermal irritation was observed at the application site of 200 mg/kg males. The incidences of epidermal and sebaceous gland hyperplasia and hyperkeratosis were significantly greater in all dose groups compared to the controls, and the incidences of ulceration, in 200 mg/kg males and inflammation and parakeratosis in 200 mg/kg females were increased.. The incidences of hepatic neoplasms were significantly greater in dosed male and female mice compared to controls. The incidences of eosinophilic foci in dosed groups of males were increased compared to controls. The incidence of nephropathy was significantly less than that of the controls. The incidences of renal tubule adenoma and of renal tubule adenoma or carcinoma (combined) in 200 mg/kg males were significantly greater than controls and exceeded the historical control ranges for these neoplasms. In the thyroid gland, the incidences of follicular cell hyperplasia in all dosed groups of males and females were significantly greater than the controls. The researchers concluded there was clear evidence of carcinogenic activity in male B6C3F 1 mice, based on increased incidences of hepatic and renal tubule neoplasms, and in female B6C3F 1 mice, based on increased incidences of hepatic neoplasms. The researchers stated these increases were associated with the concentration of free DEA present as a contaminant in the DEA condensate. In the rats, groups of 50 males and 50 females were dosed dermally with 0, 50, or 100 mg/kg bw cocamide DEA in ethanol (0, 85, or 170 mg/ml, respectively), 5 days/wk for 104 wks. Survival and mean body weights were similar in test and control animals. Dermal irritation was observed at the application site of 100 mg/kg females. The incidences of epidermal and sebaceous gland hyperplasia, parakeratosis, and hyperkeratosis were significantly greater in all dose groups compared to the controls; the severity of the lesions generally increased with increasing dose and ranged from minimal to mild. Incidences of renal tubule hyperplasia in dosed females and of renal tubule adenoma or carcinoma (combined) in females of the 50 mg/kg group were significantly greater than in the controls Incidences of nephropathy were similar between test and control rats; severity in females increased with increasing dose. In the forestomach, the incidences of chronic, active inflam- 28 Panel Book Page 61

65 mation, epithelial hyperplasia, and epithelial ulcer were significantly increased in 100 mg/kg females. The incidence of pancreatic acinar atrophy was significantly greater in the 100 mg/kg males than in the controls. The researchers concluded there was no evidence of carcinogenic activity in male F344/N rats dosed dermally with 50 or 100 mg/kg cocamide DEA. There was equivocal evidence of carcinogenic activity in female F344/N rats, based on a marginal increase in the incidences of renal tubule neoplasms. Possible Mode of Action for Carcinogenic Effects A non-genotoxic mode of tumorigenic action is indicated, because DEA is not mutagenic or clastogenic. Choline deficiency has been shown to increase spontaneous carcinogenesis in rodents, and choline deficiency may promote liver tumor formation. 6 Since disposition data indicate that DEA is less readily absorbed across rat skin than mouse skin, resulting in lower blood and tissue levels of DEA in rats than in mice, it is suggested that, in rats, the levels of DEA that occur are not high enough to markedly alter choline homeostasis. If true, species differences observed in tumor susceptibility could be a function of the internal dose of DEA. Alternatively, species differences in tumor susceptibility may explain the increased incidence of hepatocarcinogenesis in B6C3F 1 mice compared to rats exposed to DEA.. Additionally, rats and mice are reported to be much more susceptible to choline deficiency than humans. 73 A species-selective inhibition of gap junctional intercellular communication by DEA in mouse and rat, but not human, hepatocytes with medium containing reduced choline concentrations provided additional support that the mechanism for DEA-induced carcinogenicity in rodents involves cellular choline deficiency. 74 Also, Bachman et al. have hypothesized the DEA-induced choline deficiency leads to altered DNA methylation patterns, which facilitates tumorigenesis. 75 Two other hypotheses as to the mode of action of DEA carcinogenicity as possible alternatives to the intracellular choline deficiency hypothesis have been proposed. 76 ne involves the nitrosation of DEA to NDELA and the other the formation of DEA-containing phospholipids. The researcher did not find these likely for the following reasons. Regarding the first alternate hypothesis, nitrosation to NDELA; NDELA was not detected in mouse plasma or urine after cotreatment of mice with DEA and nitrite. In regards to the second, altered phospholipids; while DEA has been shown to be incorporated into phospholipids, without qualitative or quantitative differences between rats and mice, carcinogenic effects are seen only in mice, making it unlikely that incorporation of DEA into the phospholipids is a major determinant of carcinogenic response. Leung et al reviewed the information available and also felt that choline deficiency is the mechanism responsible for liver tumor promotion in mice. 77 The localization of β-catenin protein in hepatocellular neoplasms and hepatoblastomas in B6C3F1 mice exposed dermally to mg/kg bw DEA for 2 yrs were characterized, and genetic alterations in the Catnb and H-ras genes were evaluated. 78 A lack of H-ras mutations in hepatocellular neoplasms and hepatoblastomas led the researchers to suggest that the signal transduction pathway is not involved in the development of liver tumors following DEA administration. IRRITATIN AND SENSITIZATIN Undiluted DEA was moderately irritating to rabbit skin, and methyl DEA was non to slightly irritating to rabbit skin. The dermal irritation of fatty acid diethanolamides, in non-human and human testing, varied greatly with formulation and test conditions. Similar observations were made with ocular irritation testing of DEA and fatty acid ethanolamides. Undiluted methyl DEA was moderately irritating to rabbit eyes. DEA and methyl DEA were not sensitizers in guinea pig maximization studies. DEA and lauramide DEA, and linoleamide DEA were not sensitizers in humans. Cocamide DEA, %, produced positive results in provocative sensitization studies. Lauramide DEA was not phototoxic in humans. 29 Panel Book Page 62

66 Skin Irritation Non-Human Diethanolamine The primary skin irritation potential of DEA was determined using rabbits. Undiluted DEA, applied to an unspecified number of rabbits using 10 open applications of 0.1 ml to the ears and 10 semi-occluded applications to the abdomen, was moderately irritating. No irritation was observed with 10% aq. DEA following the same protocol. Using groups of 6 rabbits, application of 30 and 50% DEA using semi-occlusive patches to intact and abraded skin produced essentially no irritation. From the Final Report on the Safety Assessment of Triethanolamine, Diethanolamine, and Monoethanolamine 1 Methyl Diethanolamine cclusive patches with 0.5 ml undiluted methyl DEA were applied to a shaved area of the trunk of NZW rabbits (number not specified) for 4 h. 27 scattered ecchymoses were also observed, without necrosis. albino rabbits. 44 Methyl DEA produced mild erythema and edema, which subsided after 2 days; a few The primary skin irritation of methyl DEA was evaluated by applying 0.01 ml of the test substance to the skin of 5 Methyl DEA was slightly irritating to rabbit skin, with a score of 2/10. ther sources reported that methyl DEA was not irritating to rabbit skin. 44 Lauramide DEA The dermal irritation potential of lauramide DEA was evaluated in numerous tests using rabbits and guinea pigs. In immersion tests using guinea pigs, lauramide DEA, applied as % aq solutions, was minimally to mildly irritating in, a shampoo formulation containing 8% lauramide DEA, tested as a 0.5% solution, was a slight irritant, and a bubble bath containing 6% lauramide DEA, tested as a 0.5% aq. solution, was practically non-irritating.. In rabbits, lauramide DEA, tested as a % aq solution, was practically non- to slightly irritating, while a 20% aq. solution was a severe irritant. In a cumulative irritation test using rabbits, a 1% aq. solution of lauramide DEA was not an irritant, a 5% solution was a moderate irritant, and a 25% solution was a severe irritant. Liquid soap formulations containing 10% lauramide DEA ranged from mildly to severely irritating in rabbit skin. From the Final Report on the Safety Assessment of Cocamide, Lauramide, Linoleamide, and leamide DEA 2 Stearamide DEA A mixture containing 35-40% stearamide DEA had a primary irritation score of 0 in a dermal study using rabbits. From the Final Report on Isostearamide DEA & MEA, Myristamide DEA & MEA, and Stearamide DEA & MEA 4 leamide DEA leamide DEA, tested at 5 and 70% in propylene glycol, was mildly and moderately irritating, respectively, to rabbit skin. From the Final Report on the Safety Assessment of Cocamide, Lauramide, Linoleamide, and leamide DEA 2 Linoleamide DEA Linoleamide DEA, tested as a % aq., was non- to slightly irritating in immersion tests with guinea pigs, and a formulation containing 1.5% linoleamide DEA, tested as a 0.5% aq. solution, was a slight irritant in an immersion test. In primary irritation tests using rabbits, 5-10% aq. linoleamide DEA was non to mildly irritating, while an aq. solution of 20% linoleamide DEA was a severe dermal irritant in rabbits.. A formulations containing 1.5% linoleamide DEA, tested as a 2.5% aq. solution, was a minimal dermal irritant in rabbits. From the Final Report on the Safety Assessment of Cocamide, Lauramide, Linoleamide, and leamide DEA 2 Cocamide DEA The irritation potential of a solution containing 10% cocamide DEA and 20% sodium lauryl sulfate was evaluated in 15 subjects in conjunction with 5 other cosmetic-grade surfactant solutions. Adverse reactions were not observed. The researchers concluded that skin irritation was not simply related to the total concentration of the surfactants in contact with the skin, but rather the combination of surfactants present. From the Amended Final Report on the Safety Assessment of Cocamide DEA 3 30 Panel Book Page 63

67 Ricinoleamide DEA NZW rabbits. 79 The surfactant glyceryl ricinoleate + ricinoleamide DEA was evaluated for dermal irritation in a Draize test using A semi-occlusive patch with 0.5 g of the test material was applied to a 6 cm 2 shaved site on the dorsal area of the trunk for 4 h. No signs of irritation were observed, and the surfactant was non-irritating to rabbit skin. Human Diethanolamine In a study in which an undiluted formulation containing 1.6% DEA was applied to the back of 12 female subjects for 23 h/day for 21 days, the formulation was considered an experimental cumulative irritant. No irritation was reported during the induction phase of sensitization studies for a formulation containing 2% DEA, tested as a 10% solution in distilled water (165 subjects), for a formulation containing 2.7% DEA, tested undiluted (100 subjects), or for a formulation containing 1.6% DEA, tested undiluted (25 subjects). From the Final Report on the Safety Assessment of Triethanolamine, Diethanolamine, and Monoethanolamine. 1 Lauramide DEA Numerous studies were conducted in humans to evaluate the dermal irritation potential of lauramide DEA. In primary irritation tests (single patch) using subjects of a shampoo containing 8% and a bubble bath containing 6% lauramide DEA, both tested as a 1.25% aq solution, and an unspecified product containing 5% tested as a 1% aq. solution, minimal to mild irritation was observed. In three cumulative irritation, soap chamber, tests using subjects, liquid soap formulations containing 10% lauramide DEA and tested as 8% aq solutions were essentially non- to mildly irritating. In a 21-day cumulative irritation study, a medicated liquid soap containing 5% lauramide DEA, tested as a 25% solution, was a moderate skin irritant. In use studies, a liquid soap containing 10% lauramide DEA, evaluated in 114 subjects for 4 wks, was minimally irritating under normal use and an acne liquid cleanser containing 5% lauramide DEA, evaluated in 50 subjects with twice daily use for 6 wks, was a mild irritant. From the Final Report on the Safety Assessment of Cocamide, Lauramide, Linoleamide, and leamide DEA 2 Linoleamide DEA In a primary irritation (single patch) study, a product containing 1.5% linoleamide DEA, tested as a 1.25% aq. solution in 20 subjects, was a mild skin irritant. From the Final Report on the Safety Assessment of Cocamide, Lauramide, Linoleamide, and leamide DEA 2 Cocamide DEA An aq. solution of 12.5 mmol/l cocamide DEA was applied to the forearm of 15 volunteers. 80 Twice a day, 5 days/wk, 0.3 ml of the test material was applied for 45 min/exposure, using a plastic chamber, for a total of 28 applications. The mean transepidermal water loss (TEWL) with cocamide DEA was 7.0 g/m 2 l; as a point of comparison, the TEWL with sodium lauryl sulfate was 15.2 g/m 2 l. Mucosal In Vitro Myristamide DEA The irritation potential of various concentrations of myristamide DEA was evaluated in a neutral red assay. The IC 50 values in Chinese hamster fibroblast V79 cells, rabbit corneal cells, and human epidermal keratinocytes were 15.2, 23.9, and 6.2 µg/ml, respectively. The DS 20 (concentration predicted to produce a Draize score of 20/110) was 14.4% w/w myristamide DEA. From the Final Report on Isostearamide DEA & MEA, Myristamide DEA & MEA, and Stearamide DEA & MEA 4 Cocamide DEA The ocular irritation potential of cocamide DEA was evaluated in the MTT (not defined) cytotoxicity assay, and the irritation classification was compared to the results of a Draize test. 81 In the MTT assay, a 10% solution was classified as a non- to minimal ocular irritant. This result was similar to a non-irritant score obtained in the Draize test. Non-Human Diethanolamine The ocular irritation potential of % DEA was evaluated using rabbits. As a 30% aq. solution, DEA was 31 Panel Book Page 64

68 essentially non-irritating, while a 50% aq. solution was a severe irritant. Instillation of 0.02 ml undiluted DEA produced severe injury to rabbit eyes. A hair preparation containing 1.6% DEA had a maximum avg. irritation score of 0.7/110 for rinsed and unrinsed eyes. From the Final Report on the Safety Assessment of Triethanolamine, Diethanolamine, and Monoethanolamine 1 Methyl Diethanolamine The ocular irritation potential of methyl DEA was evaluated by instilling undiluted test material into the conjunctival sac of rabbits. 44 Methyl DEA was moderately irritating to rabbit eyes, with a score of 4/10. In a study in which ml was instilled into the conjunctival sac of 6 rabbits, slight to moderate conjunctival irritation was observed in all 6 rabbits. 27 Iritis was seen in all rabbits, and corneal opacity was observed in the eye of one rabbit. All effects resolved by day 3. Lauramide DEA Five ocular irritation studies were performed in rabbits with lauramide DEA at concentrations of 1-25% ne percent aq. lauramide DEA was mildly irritating, 5% was slightly to moderately irritating, 10-20% was moderately irritating, and 25% was moderately to severely irritating. ne bubble bath formulations containing 6% lauramide DEA was practically nonirritating, while another was moderately irritating, and three shampoo formulations containing 8% lauramide DEA were non- to moderately irritating. In a mucous membrane irritation test, a soap containing 10% lauramide DEA was significantly more irritating than water to vaginal mucosa of rabbits. From the Final Report on the Safety Assessment of Cocamide, Lauramide, Linoleamide, and leamide DEA 2 Stearamide DEA A mixture containing 35-40% stearamide DEA was not-irritating to rabbit eyes. From the Final Report on Isostearamide DEA & MEA, Myristamide DEA & MEA, and Stearamide DEA & MEA 4 Isostearamide DEA A formulation containing 8.0% isostearamide DEA was a moderate irritant in rabbit eyes. From the Final Report on Isostearamide DEA & MEA, Myristamide DEA & MEA, and Stearamide DEA & MEA 4 leamide DEA Undiluted oleamide DEA was practically non-irritating to rabbit eyes. From the Final Report on the Safety Assessment of Cocamide, Lauramide, Linoleamide, and leamide DEA 2 Linoleamide DEA Linoleamide DEA, 10% aq, was practically non-irritating to rabbit eyes, while the undiluted test article was minimally to moderately irritating. A product containing 1.5% linoleamide DEA, applied as a 25% aq solution, and a formulation containing 15%, were moderate eye irritants in rabbits, while a formulation containing 15%, applied as a 25% aq. solution, was mildly irritating. From the Final Report on the Safety Assessment of Cocamide, Lauramide, Linoleamide, and leamide DEA 2 Cocamide DEA A substance composed of >64% cocamide DEA and <29% DEA was a severe irritant in rabbit eyes. From the Amended Final Report on the Safety Assessment of Cocamide DEA 3 Ricinoleamide DEA The surfactant glyceryl ricinoleate + ricinoleamide DEA was evaluated for ocular irritation using NZW rabbits. 79 No signs of irritation were observed, and the surfactant was a non-irritant. Non-Human Diethanolamine Sensitization DEA had an EC3 value of 40% in a mouse local lymph node assay (ECD guideline 429), resulting in a categorization of weak potency of for skin sensitization Panel Book Page 65

69 In a maximization study using 15 Dunkin-Hartley guinea pigs, intradermal and epicutaneous induction used 1% and 17.6% aq. DEA, respectively, after 10% sodium lauryl sulfate pre-treatment. 83 At challenge with 0.7, 3.5, or 7% DEA, 1/15 animals reacted to the lowest and highest challenge concentrations after 2, but not 3 days. In a second maximization test using 20 Himalayan spotted guinea pigs, the intradermal induction, epicutaneous induction, and epicutaneous challenge concentrations were 5%, 75%, and 25% DEA in physiological saline, respectively. 83,84 Freund s complete adjuvant (FCA) was used at intradermal induction. Two animals had mild erythema at day 1, and one animals had mild erythema at day 2. DEA was not a sensitizer. Methyl Diethanolamine The sensitization potential of methyl DEA was evaluated in a guinea pig maximization study using 10 male and 10 female Dunkin Hartley guinea pigs. 85 FCA and sodium lauryl sulfate were used in the study. A concentration of 5% in propylene glycol was used for the intradermal induction, and undiluted methyl DEA was used for the topical induction and at challenge. Dermal responses were seen at challenge in both test and control animals; therefore, a re-challenge was performed using 10 and 50% methyl DEA. No dermal responses were observed at re-challenge, and methyl DEA was not considered a sensitizer in guinea pigs. Human Diethanolamine Formulations containing 1.6 and 2.7% DEA, tested undiluted, and a formulations containing 2% DEA, tested at a 10% solution in distilled water, were not sensitizing in clinical studies. From the Final Report on the Safety Assessment of Triethanolamine, Diethanolamine, and Monoethanolamine 1 Lauramide DEA Six repeat insult patch tests (RIPTs) using subjects were performed on formulations containing 4-10% lauramide DEA, as % solutions. Lauramide DEA was not a sensitizer in any of the studies. However, a liquid soap containing 10% lauramide DEA, tested as a 1% aq. solution on 159 subjects, a shampoo containing 8%, tested as a 0.5% aq. solution in 99 subjects, and a skin cleanser containing 5%, tested as a 0.25% aq. solution in 86 subjects, all had reactions that were considered irritating. From the Final Report on the Safety Assessment of Cocamide, Lauramide, Linoleamide, and leamide DEA 2 Linoleamide DEA In an RIPT conducted with 100% linoleamide DEA on 100 subjects, no irritation or sensitization reactions were observed. A dandruff shampoo containing 1.5% linoleamide DEA, tested as a 1% aq. solution in a RIPT using 101 subjects, was an irritant, but not a sensitizer. From the Final Report on the Safety Assessment of Cocamide, Lauramide, Linoleamide, and leamide DEA 2 Cocamide DEA Numerous studies were performed in provocative studies, mostly using patients with occupational exposure to cocamide DEA, to evaluate the sensitization potential of cocamide DEA. Concentrations of % were tested. Positive results were seen in all eight studies. However, during their Discussion, the Panel noted that there is a need to recognize that while occupational exposure to cocamide DEA can result in sensitization, cosmetic use does not present the same concerns. From the Amended Final Report on the Safety Assessment of Cocamide DEA 3 Co-Reactivity Cocamide DEA Thirty-five patients that had positive patch tests to cocamidopropyl betaine, amidoamine, or both, were tested for coreactivity with cocamide DEA. 86 Two of the patients (5.7%) had positive reactions to cocamide DEA. 33 Panel Book Page 66

70 Provocative Testing Diethanolamine ver a 15-yr period, provocative patch testing using DEA was performed on 8791 patients. 83 There were 157 (1.8%) positive reactions to DEA, and most of the reactions (129; 1.4%) were weak positives. There were 17 (0.2%) irritant reactions reported. Cosensitization was reported; 77% of the patients that reacted to DEA also tested positive to MEA. ccupational sensitization was reported; of 7112 male patients, 1.0% that did not work in the metal industry had positive reactions to DEA, as opposed to 3.1 and 7.5% of those working in the metal industry and those exposed to water-based metalworking fluids, respectively. Phototoxicity/Photosensitivity Human Lauramide DEA A liquid soap containing 10% lauramide DEA, tested as a 10% aq. solution in 25 subjects, was not phototoxic. In a photosensitivity study of a liquid soap containing 10% lauramide DEA, tested as a 1% aq. solution in 25 subjects, slight irritation was seen in 9 subjects at induction and 4 at challenge, but the test substance was not a photosensitizer. From the Final Report on the Safety Assessment of Cocamide, Lauramide, Linoleamide, and leamide DEA 2 Case Studies Undecylenamide DEA ne patient with dermatitis of the hands and axillae had positive test reaction to a liquid soap. 87 Testing with undecylenamide DEA, an ingredient in the soap, at 0.1 and 1% aq., gave positive reactions. In 10 control subjects, testing with 0.1% undecylenamide DEA was negative. Cocamide DEA ne patient with dermatitis on the hands and face, and two with dermatitis on the hands and forearms, were patch tested using the North American Contact Dermatitis Group standard tray and some additional chemicals. 88 The three patients had either personal or industrial exposure to cocamide DEA-containing products. All three had positive patch test results (2+) to cocamide DEA, and two had reactions to several other chemicals. In all patients, the dermatitis cleared with avoidance of DEA-containing products. MISCELLANEUS STUDIES Diethanolamine In male albino rats, following repeated oral administration of 320 mg/kg/day in drinking water of radiolabeled DEA, a decrease was seen in the amount of choline incorporated in the liver and the kidneys after 1, 2, and 3 wks as compared to 0 wks. MEA and choline phospholipid derivatives were synthesized faster and in greater amounts, and were catabolized faster than DEA phospholipid derivatives. This was not seen with a single 250 mg/kg injection of DEA. From the Final Report on the Safety Assessment of Triethanolamine, Diethanolamine, and Monoethanolamine 1 Diethanolamine Inhibition of Choline Uptake The ability of DEA to alter cellular choline levels was examined in a number of studies. In the study described previously in which B6C3F 1 mice were dosed orally and dermally with 160 mg/kg/day DEA in conjunction with oral sodium nitrite, a pronounced decrease in choline and its metabolites was observed in the livers. 42 The smallest decreases were seen in the mice that were dosed dermally and not allowed access to the test site, and the greatest increase was seen in the mice dosed orally. 34 Panel Book Page 67

71 In Syrian hamster embryo (SHE) cells, DEA inhibited choline uptake at concentrations 50 µg/ml, reaching a maximum 80% inhibition at µg/ml. 6 DEA also reduced phosphatidylcholine in the phospholipds, was incorporated into SHE lipids, and transformed SHE cells in a concentration-dependent manner. Excess choline blocked these biochemical effects and inhibited cell transformation, and the researchers hypothesized there is a relationship between the effect of DEA on intracellular choline availability and utilization and its ability to transform cells. In a study performed to test the hypothesis that DEA treatment could produce biochemical changes consistent with choline deficiency in mice, it was found that DEA treatment caused a number of biochemical changes consistent with choline deficiency in mice. 89 Hepatic concentrations of S- adenosymethionine (SAM) decreased. Biochemical changes were seen without fatty livers, an observation often associated with choline deficiency. To study the dose-response, reversibility and strain-dependence of DEA effects, B6C3F 1 mice were dosed dermally with DEA in ethanol for 4 wks. 89 Control animals were either not dosed or dosed with ethanol only. The pattern of changes observed in choline metabolites after DEA treatment was very similar to that observed in choline-deficient mice, and the NEL for DEA-induced changes in choline homeostasis was 10 mg/kg/day. Fatty livers were not observed. (Lehman- McKeeman hypothesized that the lack of fatty livers is the result of an age-dependence mechanism. 76 ) The reactions were dose-dependent, strain-dependent, and reversible. Dermal application of 95% ethanol decreased hepatic betaine levels, suggesting that used of ethanol as a vehicle for dermal application of DEA could exacerbate the biochemical effects of DEA. CCUPATINAL EXPSURE Diethanolamine The National Institute for ccupational Safety and Health recommended exposure limits time-weight average for DEA is 3 ppm (15 mg/m 3 ). 90 The ccupational Safety and Health Administration does not have a permissible exposure limit for DEA. SUMMARY This report assesses the safety of DEA and 68 additional DEA-containing ingredients as used in cosmetics. Some of these ingredients have been previously reviewed by the CIR, and are included here to create a report on the complete family of ingredients. The acid salt ingredients would be expected to dissociate into DEA and the corresponding acid. The covalent DEA ingredients, however, are not salts and do not readily dissociate in water. However, amidases, such as fatty acid amide hydrolase which is known to be present in human skin, could potentially convert the diethanolamides to DEA and the corresponding fatty acids. In the case of these covalent ingredients, DEA may be of concern as an impurity, but not as a major component. Because the acid salts can dissociate, there is a potential of the formation of nitrosamines. However, tertiary alkyl amines do not tend to react with nitrosating agents to form nitrosamines, and tertiary amides do not tend to react with nitrosating agents to form nitrosamides. DEA functions in cosmetic formulations as a ph adjuster. While a few of the other ingredients function as a ph adjuster, the majority have other functions, including surfactant, emulsifying agent, viscosity increasing agent, hair or skin conditioning agents, foam booster, or antistatic agent. DEA typically contains some amount of MEA or TEA; according to one supplier, DEA has a minimum purity of 99.3%, with 0.045% max MEA and 0.25% max TEA. The diethanolamides generally have some amount of free DEA, and 35 Panel Book Page 68

72 that amount can vary greatly be ingredient. For example, it was estimated that oleamide DEA contained 0.19% free DEA, while cocamide DEA contained 18.2% free DEA by weight. NDELA may also be present in DEA or DEA-containing ingredients. In 2010, DEA was reported to be used in 30 formulations at concentrations of %. The highest leave-on concentration reported was 0.06%. The ingredients with the greatest reported frequency of use were cocamide DEA and lauramide DEA, with 850 and 545 reported used in 2010; the majority of the uses were in rinse-off products. In Europe, dialkanolamines and their salts (i.e., DEA and the acid salts) are on the list of substances which must not form part of the composition of cosmetic products, and in Canada, the use of dialkanolamines is prohibited, based on the European ruling. Fatty acid dialkanolamines (i.e., the alkyl substituted diethanolamines) are allowed in use for use in products in Europe, with restrictions. In vitro absorption studies were performed using mouse, rat, and human skin. In in vitro studies using mouse and rat skin, 1.3 and 0.04%, respectively, of the applied dose of undiluted [14C]DEA was absorbed. In studies using human skin samples, the absorption of undiluted DEA, as well as concentrations of <1% DEA in combination with fatty acid dialkanolamides, was less than 1% of the applied dose. Penetration of DEA in aqueous solutions was greater than when DEA was undiluted. In studies using human liver slices, DEA was absorbed; the aqueous-extractable radioactivity was primarily unchanged DEA, while analysis of the organic extracts suggested that DEA was incorporated into ceramides, and slowly methylated. Lauramide DEA was better absorbed in liver slices, and while the absorbed radioactivity was mostly unchanged lauramide DEA, 18-42% was present in the form of metabolites. In dermal studies with DEA, methyl DEA, and lauramide DEA, the applied doses were generally well absorbed through mouse and/or rat skin, and absorption increased with duration of exposure. In the tissues, the liver generally had the greatest disposition of radioactivity. Urine was the principal route of elimination. Upon dosing with methyl DEA, primarily metabolites, not unchanged methyl DEA, were found in the urine. Lauramide DEA absorption was not dose dependent and the parent compound and the half-acid amide metabolites were detected in the plasma, and disposition did not vary with time. In oral studies, DEA accumulated in the tissues, with the greatest disposition being in the liver; radioactivity was primarily as unchanged DEA. Urinary excretion was also primarily as unchanged DEA. In a repeated-dose study, stead-state for bioaccumulation occurred after 4 wks; however, DEA continued to bioaccumulate in blood throughout dosing. With lauramide DEA, 79% of the dose was excreted in the urine 72 h after dosing. Four percent of the dose was recovered in the tissue. After 6 hrs, only very polar metabolites, thought to be carboxylic acids, were found in the urine. In vitro percutaneous absorption studies of cosmetic preparations containing free DEA up to 0.6% showed some penetration occurred in human skin. Mice exposed orally to sodium nitrate were dosed orally and dermally with 4 mg/kg DEA. A small amount of NDELA was formed following a single oral dose of DEA. No NDELA was detected following dermal dosing with DEA. Acute dermal testing with methyl diethanolamine,50% lauramide DEA, and undiluted and 10% aq linoleamide DEA, acute oral testing with DEA, methyl DEA, butyl DEA, and several fatty acid diethanolamides, and acute inhalation testing with methyl DEA did not result in significant toxicity. In repeat dermal testing with DEA, lauramide DEA, and cocamide DEA in mice and/or rats, irritation was observed at the site of application. Increases in liver and kidney weights were observed in most studies, while decreases in body weight were observed sporadically. The LAEL for DEA in a 2-wk study in mice was 160 mg/kg bw. Repeat dermal dosing with methyl DEA in rats also caused skin lesions, but it did not seem to affect liver weights or body weights, and an 36 Panel Book Page 69

73 increase in kidney weights was observed in 1 of 3 studies. The NEL for methyl DEA in a 13-wk study in rats was 100 mg/kg day. A formulation containing 3% linoleamide DEA was not a cumulative systemic toxicant in a 13-wk dermal study. In repeat oral testing with DEA, increases in liver and kidney weights and decreases in body weights were seen in mice and rats. Deaths, believed to be test-article related, occurred in most of the studies, and included a mouse given 100 mg/kg DEA by gavage. With repeat oral dosing of lauramide DEA, the NEL was 250 mg/kg/day in one study using rats. The NEL for Beagle dogs fed lauramide DEA for 12 wks was 5000 ppm. In inhalation studies with DEA in rats, liver and kidney weights were again increased. In 13-wk studies with 400 mg/m 3 DEA, microscopic effects were observed in the larynx. The 90-day NAEC was 1.5 mg/m 3 DEA. In a study in which gravid mice were dosed with mg/kg DEA from day 6 of gestation through PND 21, no effects on skeletal formation were observed, but dose-dependent effects on some growth and developmental parameters were observed. In a study in which parental mice were treated with DEA for 4 wks prior to dosing, sperm motility was decreased in a dose-dependent manner. In rats and rabbits, dermal dosing with up to 1500 mg/kg, and 350 mg/kg DEA, respectively, during gestation, did not have any fetotoxic or teratogenic effects. The NEL for embryonal/fetal toxicity was 380 mg/kg/day for rats and 350 mg/kg/day for rabbits. In dermal reproductive studies with up to 1000 mg/kg/day methyl DEA in rats, the NEL for developmental toxicity was 1000 mg/kg/day. In an oral reproductive study in which rats were dosed with up to 1200 mg/kg/day DEA on days 6-15 of gestation, maternal mortality was observed at doses of 50 mg/kg; the NEL for embryonal/fetal toxicity was 200 mg/kg/day. In a study in which gravid rats were dosed orally with up to 300 mg/kg/day DEA, the dams of the 300 mg/kg group were killed due to excessive toxicity; the LD 50 was calculated to be 218 mg/kg. The LAEL for both maternal toxicity and teratogenicity was 125 mg/kg/day. In a reproductive study in which rats were exposed by inhalation to DEA on days 6-15 of gestation, the NAEC for both maternal and developmental toxicity was 0.05 mg/l, and the NAEC for teratogenicity was.0.2 g/ml. DEA, methyl DEA, oleamide DEA, and cocamide DEA were, generally, non-genotoxic in a number of assays. Exceptions were positive results for DEA in an in vitro assay of DNA strand break in isolated rat, hamster, and pig hepatocytes, the induction of SCEs in CH cells by lauramide DEA, and an increase in the frequency of micronucleated erythrocytes in mice by cocamide DEA The carcinogenic potential of dermally applied DEA and lauramide, oleamide, and cocamide DEA was evaluated by the NTP in B6C3F 1 mice and F344/N rats. The doses tested are included in parentheses. DEA produced clear evidence of carcinogenic activity in male and female mice (0-160 mg/kg) and no evidence in male and female rats (0-64 mg/kg); lauramide DEA produced some evidence of carcinogenic activity in female mice (0-200 mg/kg) and no evidence in male mice or male and female rats (0-100 mg/kg); oleamide DEA produced no evidence of carcinogenic activity in male or female mice (0-30 mg/kg) or male or female rats (0-100 mg/kg;, and cocamide DEA produced clear evidence of carcinogenic activity in male and female mice (0-200 mg/kg), equivocal evidence in female rats (0-100 mg/kg),and no evidence in male rats (0-100 mg/kg). According to the IARC Working Group, their overall evaluation is that there is inadequate evidence in humans for the carcinogenicity of DEA and that DEA is not classifiable as to its carcinogenicity in humans. Because DEA is not mutagenic or clastogenic, a non-genotoxic mode of tumorigenic action is indicated. A plausible mode of action for DEA carcinogenicity in rodents involves cellular choline deficiency. Undiluted DEA was moderately irritating to rabbit skin, and methyl DEA was non- to slightly irritating to rabbit skin The dermal irritation fatty acid diethanolamides, in non-human and human testing, varied greatly with formulation and 37 Panel Book Page 70

74 test conditions. Similar observations were made with ocular irritation testing of DEA and fatty acid ethanolamides. Undiluted methyl DEA was moderately irritating to rabbit eyes. DEA and methyl DEA were not sensitizers in guinea pig maximization studies. DEA and lauramide DEA, and linoleamide DEA were not sensitizers in humans. Cocamide DEA, %, produced positive results in provocative sensitization studies. Lauramide DEA was not phototoxic in humans. DISCUSSIN primarily to be developed at the Panel meeting, but to include In 1983, the Expert Panel reviewed the safety of DEA in an assessment that also included TEA and MEA. The CIR Expert Panel has determined that DEA, TEA, and MEA should be updated separately to allow incorporation of new data, and to add related ingredients. Accordingly, this report assesses the safety of DEA and 68 DEA-containing ingredients. The potential adverse effects of inhaled aerosols depend on the specific chemical species, the concentration and the duration of the exposure and their site of deposition within the respiratory system. In practice, aerosols should have at least 99% of their particle diameters in the µm range and the mean particle diameter in a typical aerosol spray has been reported as ~38 µm. Particles with an aerodynamic diameter of 10 µm are respirable. Inhalation data are available for DEA, but even in the absence of inhalation toxicity data, the Panel determined that DEA and related DEA-containing ingredients can be used safely in aerosol products, because the product size is not respirable. 38 Panel Book Page 71

75 Table 1. Definitions and Structures TABLES Ingredient CAS No. Definition Function(s) Formula/structure Diethanolamine and inorganic salt Diethanolamine Diethanolamine Bisulfate a secondary amine with two ethanol functional groups the diethanolamine salt of sulfuric acid Diethanolamine organic acid salts DEA-Myristate the diethanolamine salt of myristic acid ph adjuster Buffering agent; ph adjuster Surf. - Cleansing Ag. H HS 4 H NH NH 2 DEA Stearate the diethanolamine salt of stearic acid In VCRP/not in Council Database DEA-Isostearate the diethanolamine salt of isostearic acid Surf. - Cleansing Ag. ne example of an iso DEA-Linoleate diethanolamine salt of linoleic acid Surf. - Cleansing Ag. DEA- Lauraminopropionate the diethanolamine salt of lauraminopropionic acid Hair Cond. Ag. Surf. - Foam Boosters Diethanolamine organo-substituted inorganic acid salts -Alkyl sulfate esters DEA-Lauryl Sulfate DEA-C12-13 Alkyl Sulfate DEA-Myristyl Sulfate DEA-C12-15 Alkyl Sulfate the diethanolamine salt of lauryl sulfate the diethanolamine salt of the sulfate of C12-13 alcohols the diethanolamine salt of myristyl sulfate the diethanolamine salt of the sulfate of C12-15 alcohols Surf. - Cleansing Ag. Surf. - Cleansing Ag. Surf. - Cleansing Ag. Surf. - Cleansing Ag. CH 3 (CH 2 ) 11 S NH 2 H R S NH 2 H wherein R is a 12 to 13 carbon alkyl chain CH 3 (CH 2 ) 13 S NH 2 H wherein R is a 12 to 15 carbon alkyl chain 39 Panel Book Page 72

76 Table 1. Definitions and Structures (continued) Ingredient CAS No. Definition Function(s) Formula/structure DEA-Cetyl Sulfate the diethanolamine Surf salt of cetyl sulfate Cleansing Ag. CH 3 (CH 2 ) 15 S H DEA-Laureth Sulfate DEA-C12-13 Pareth-3 Sulfate DEA-Myreth Sulfate DEA- Dodecylbenzenesulfonate DEA-Methyl Myristate Sulfonate PEG sulfate esters the diethanolamine salt of an ethoxylated lauryl sulfate the diethanolamine salt of the sulfate ester of C12-13 Pareth-3 the diethanolamine salt of ethoxylated myristyl sulfate -Sulfonate esters the diethanolamine salt of dodecylbenzene sulfonic acid the diethanolamine salt of an α-sulfonated fatty acid ester Surf. - Cleansing Ag. Surf. - Cleansing Ag. Surf. - Cleansing Ag. Surf. - Cleansing Ag. Surf. - Cleansing Ag. CH 3 (CH 2 ) 11 CH 3 (CH 2 ) 13 S n H wherein n 1-4 NH 2 NH 2 wherein R is a 12 to 13 carbon alkyl chain S n H wherein n 1-4 NH 2 DEA-Cetyl Phosphate DEA-Ceteareth-2 Phosphate -Alkyl and PEG phosphate esters the diethanolamine salt of cetyl phosphate the diethanolamine salt of ceteareth-2 phosphate Surf. - Emuls. Ag. Surf. - Cleansing Ag.; Surf. - Emuls. Ag. CH 3 (CH 2 ) 15 P H NH 2 DEA-leth-3 Phosphate [generic CAS No. for all DEA-leth-n Phosphates] the diethanolamine salt of oleth-3 phosphate Surf. - Emuls. Ag. wherein R is a 16 or 18 carbon alkyl chain CH 3 (CH 2 ) 7 CH CH(CH 2 ) 8 P 3 DEA-leth-5 Phosphate [generic CAS No. for all DEA-leth-n Phosphates] the diethanolamine salt of a complex mixture of esters of oleth-5 phosphate Surf. - Cleansing Ag.; Surf. - Emuls. Ag. CH 3 (CH 2 ) 7 CH CH(CH 2 ) 8 H 5 NH 2 P DEA-leth-10 Phosphate [generic CAS No. for all DEA-leth-n Phosphates] the diethanolamine salt of a complex mixture of oleth-10 phosphate Surf. - Emuls. Ag. CH 3 (CH 2 ) 7 CH CH(CH 2 ) 8 H 10 NH 2 P H NH 2 40 Panel Book Page 73

77 Table 1. Definitions and Structures (continued) Ingredient CAS No. Definition Function(s) Formula/structure DEA-leth-20 Phosphate the diethanolamine Surf [generic CAS salt of a complex Cleansing No. for all DEA-leth-n mixture of oleth-20 Ag.; Surf. - CH 3 (CH 2 ) 7 CH CH(CH 2 ) 8 Phosphates] phosphate Emuls. Ag. 20 P DEA-Hydrolyzed Lecithin -Disubstituted phosphate esters the diethanolamine salt of partially hydrolyzed lecithin Hair Cond. Ag.; Skin- Cond. Ag. - Misc. H NH 2 DEA-Di(2- Hydroxypalmityl) - Phosphate [More likely to be INCI named: DEA-Di(2- Hydroxycetyl) Phosphate] the diethanolamine salt of di(2-hydroxycetyl)phosphate Alkyl substituted diethanolamines Methyl Diethanolamine [ ] Butyl Diethanolamine a tertiary amine with one methyl group and two ethanol groups. a tertiary amine with one butyl group and two ethanol groups. In VCRP/ not in Council Database ph Adj. wherein R is an 8-18 carbon alkyl chain, which may be partially unsaturated CH 3 (CH 2 ) 13 P NH 2 H R N-Lauryl Diethanolamine [ ] a tertiary amine with one lauryl group and two ethanol groups. Not Reported Diethanolamides -Alkyl amides Capramide DEA a mixture of ethanolamides of capric acid Surf. - Foam Boosters; Visc. Incr. Ag. - Aq. CH 3 (CH 2 ) 8 N Undecylenamide DEA [Structure in this CAS file is saturated] Lauramide DEA a mixture of ethanolamides of undecylenic acid a mixture of ethanolamides of lauric acid Hair Cond. Ag.; Surf. - Foam Boosters; Visc. Incr. Ag. - Aq. Surf. - Foam Boosters CH 2 CH(CH 2 ) 8 CH 3 (CH 2 ) 10 N N Myristamide DEA a mixture of ethanolamides of myristic acid Surf. - Foam Boosters; Visc. Incr. Ag. - Aq. CH 3 (CH 2 ) 12 N Lauramide/ Myristamide DEA a mixture of ethanolamides of a blend of lauric and myristic acids Surf. - Foam Boosters; Visc. Incr. Ag. - Aq. R N wherein RC() represents a 12 or 14 carbon fatty acid residue 41 Panel Book Page 74

78 Table 1. Definitions and Structures (continued) Ingredient CAS No. Definition Function(s) Formula/structure Palmitamide DEA Stearamide DEA Behenamide DEA Lactamide DEA Isostearamide DEA leamide DEA [CAS file is specific to Z isomer] -α-hydroxy -Branched a mixture of ethanolamides of palmitic acid. a mixture of ethanolamides of stearic acid. a mixture of ethanolamides of behenic acid the diethanolamide of lactic acid a mixture of ethanolamides of isostearic acid -Partially unsaturated a mixture of ethanolamides of oleic acid Surf. - Foam Boosters; Visc. Incr. Ag. - Aq. Surf. - Foam Boosters; Visc. Incr. Ag. - Aq. Hair Cond. Ag.; Surf. - Foam Boosters; Visc. Incr. Ag. - Aq. Skin-Cond. Ag. - Humectant Surf. - Foam Boosters; Visc. Incr. Ag. - Aq. Surf. - Foam Boosters; Visc. Incr. Ag. - Aq. H 3 C H 3 C CH(CH 2 ) 14 CH 3 (CH 2 ) 14 CH 3 (CH 2 ) 16 CH 3 (CH 2 ) 20 H 3 C CH 3 (CH 2 ) 7 CH N N N N N CH(CH 2 ) 7 one example of an iso N Linoleamide DEA Almondamide DEA Apricotamide DEA Avocadamide DEA a mixture of ethanolamides of linoleic acid -Natural source mixtures a mixture of ethanolamides of the fatty acids derived from almond oil a mixture of ethanolamides of the fatty acids derived from Prunus Armeniaca (Apricot) Kernel il a mixture of ethanolamides of the fatty acids derived from Persea Gratissima (Avocado) il Hair Cond. Ag.; Surf. - Foam Boosters; Visc. Incr. Ag. - Aq.; Hair Cond. Ag.; Surf. - Foam Boosters; Visc. Incr. Ag. - Aq. Surf. - Foam Boosters; Visc. Incr. Ag. - Aq. Surf. - Foam Boosters; Visc. Incr. Ag. - Aq. Surf. - Foam Boosters; Visc. Incr. Ag. - Aq. R R R CH 3 (CH 2 ) 4 CH N N CHCH 2 CH CH(CH 2 ) 7 N wherein RC() represents the fatty acid residues derived from almond oil wherein RC() represents the fatty acid residues derived from Prunus Armeniaca (Apricot) Kernel il N wherein RC() represents the fatty acid residues derived from Persea Gratissima (Avocado) il 42 Panel Book Page 75

79 Table 1. Definitions and Structures (continued) Ingredient CAS No. Definition Function(s) Formula/structure Babassuamide DEA Cocamide DEA Cornamide DEA Cornamide/ Cocamide DEA Hydrogenated Tallowamide DEA Lanolinamide DEA [ ] Lecithinamide DEA Minkamide DEA livamide DEA Palm Kernelamide DEA Palmamide DEA a mixture of ethanolamides of the fatty acids derived from rbignya leifera (Babassu) il a mixture of ethanolamides of Coconut Acid a mixture of ethanolamides of Corn Acid the diethanolamide of a mixture of coconut acid and the fatty acids obtained from Zea Mays (Corn) il a mixture of ethanolamides of the fatty acids derived from hydrogenated tallow a mixture of ethanolamides of Lanolin Acid the mixture of reaction products of diethanolamine and the fatty acids of lecithin. a mixture of ethanolamides of the fatty acids derived from mink oil. a mixture of ethanolamides of the fatty acids derived from olive oil a mixture of ethanolamides of the fatty acids derived from Elaeis Guineensis (Palm) Kernel il a mixture of ethanolamides of the fatty acids derived from Elaeis Guineensis (Palm) il Hair Cond. Ag.; Surf. - Foam Boosters; Visc. Incr. Ag. - Aq. Surf. - Foam Boosters; Visc. Incr. Ag. - Aq. Surf. - Foam Boosters; Visc. Incr. Ag. - Aq. Surf. - Foam Boosters; Visc. Incr. Ag. - Aq. Surf. - Foam Boosters; Visc. Incr. Ag. - Aq. Surf. - Foam Boosters; Visc. Incr. Ag. - Aq. Hair Cond. Ag.; Surf. - Foam Boosters; Visc. Incr. Ag. - Aq. Surf. - Foam Boosters; Visc. Incr. Ag. - Aq. Surf. - Foam Boosters; Visc. Incr. Ag. - Aq. Surf. - Foam Boosters; Visc. Incr. Ag. - Aq. Surf. - Foam Boosters; Visc. Incr. Ag. - Aq. 43 R R R R R R R R R R R N N N N wherein RC() represents the fatty acid residues derived from rbignya leifera (Babassu) il wherein RC() represents the fatty acid residues derived from Coconut Acid wherein RC() represents the fatty acid residues derived from Corn Acid wherein RC() represents the fatty acid residues derived from Coconut Acid and Zea Mays (Corn) il N N N N N N N wherein RC() represents the fatty acid residues derived from Hydrogenated Tallow wherein RC() represents the fatty acid residues derived from Lanolin Acid wherein RC() represents the fatty acid residues derived from Lecithin wherein RC() represents the fatty acid residues derived from mink oil wherein RC() represents the fatty acid residues derived from olive oil wherein RC() represents the fatty acid residues derived from Elaeis Guineensis (Palm) Kernel il wherein RC() represents the fatty acid residues derived from Elaeis Guineensis (Palm) il Panel Book Page 76

80 Table 1. Definitions and Structures (continued) Ingredient CAS No. Definition Function(s) Formula/structure Ricebranamide DEA a mixture of ethanolamides of Rice Bran Boosters; Surf. - Foam Acid Visc. Incr. R N Ag. - Aq. Ricinoleamide DEA Sesamide DEA Shea Butteramide/Castoramide DEA Soyamide DEA Tallamide DEA Tallowamide DEA Wheat Germamide DEA PEG-2 Tallowamide DEA PEG-3 Cocamide DEA a mixture of ethanolamides of ricinoleic acid a mixture of diethanolamides of the fatty acids derived from Sesamum Indicum (Sesame) il a mixture of diethanolamides of the fatty acids derived from Butyrospermum Parkii (Shea Butter) and Ricinus Communis (Castor) Seed il a mixture of ethanolamides of soy acid a mixture of ethanolamides of the fatty acids derived from tall oil acid a mixture of ethanolamides of tallow acid a mixture of diethanolamides of wheat germ acid -Glycol ethers the polyethylene glycol amine derived from tallow acid the polyethylene glycol derivative of cocamide DEA with an average of 3 moles of ethylene oxide Surf. - Foam Boosters; Visc. Incr. Ag. - Aq. Surf. - Foam Boosters; Visc. Incr. Ag. - Aq. Visc. Incr. Ag. - Aq. Surf. - Foam Boosters; Visc. Incr. Ag. - Aq. Surf. - Foam Boosters; Visc. Incr. Ag. - Aq. Surf. - Foam Boosters; Visc. Incr. Ag. - Aq. Surf. - Foam Boosters; Visc. Incr. Ag. - Aq. Surf. - Cleansing Ag. Surf. - Emuls. Ag. R R N N wherein RC() represents the fatty acid residues derived from Rice Bran Acid wherein RC() represents the ricinoleic acid residue wherein RC() represents the fatty acid residues derived from Sesamum Indicum (Sesame) il R N wherein RC() represents the fatty acid residues derived from Butyrospermum Parkii (Shea Butter) and Ricinus Communis (Castor) Seed il R R R R R R N N N N N N wherein RC() represents the fatty acid residues derived from Soy Acid wherein RC() represents the fatty acid residues derived from Tall il Acid wherein RC() represents the fatty acid residues derived from Tallow Acid wherein RC() represents the fatty acid residues derived from Wheat Germ Acid wherein RC() represents the fatty acid residues derived from Tallow Acid wherein RC() represents the fatty acid residues derived from Coconut Acid 44 Panel Book Page 77

81 Table 1. Definitions and Structures (continued) Ingredient CAS No. Definition Function(s) Formula/structure -Amidoethyl Stearamidoethyl an amidoamine Antistatic Diethanolamine Ag. N CH 3 (CH 2 ) 16 NH Stearamidoethyl Diethanolamine HCl DEA- Cocoamphodipropionate -thers Diethanolaminooleamide DEA a substituted amine salt an amphoteric organic compound a substituted oleoyl diethanolamide containing a tertiary alkanolamine on the carbon chain Antistatic Ag.; Hair Cond. Ag. Hair Cond. Ag.; Surf. - Cleansing Ag.; Surf. - Foam Boosters; Surf. Hydrotropes Surf. - Foam Boosters; Visc. Incr. Ag. - Aq. R NH CH 3 (CH 2 ) 16 N NH wherein RC() represents the fatty acid residues derived from coconut oil H H N NH Cl H ne example a Diethanolaminooleamide DEA H NH N Stearamide DEA- Distearate a substituted ethanolamide pacifying Ag.; Surf. - Foam Boosters; Visc. Incr. Ag. - Aq.; Visc. Incr. Ag. - NonAq. wherein the DEA substitution could occur anywhere along the carbon chain (CH 2 ) 16 CH 3 CH 3 (CH 2 ) 16 N (CH 2 ) 16 CH 3 Cocoyl Sarcosinamide DEA a mixture of diethanolamides of N-cocoyl sarcosine Hair Cond. Ag.; Surf. - Foam Boosters; Visc. Incr. Ag. - Aq. R N CH 3 N wherein RC() represents the fatty acid residues derived from coconut oil 45 Panel Book Page 78

82 Table 2. Conclusions of previously reviewed ingredients and components Ingredient Conclusion Reference PREVIUSLY REVIEWED INGREDIENTS DEA safe for use in cosmetic formulations designed for discontinuous, brief use followed by thorough rinsing from the surface of the skin; in products intended for prolonged contact with the skin, the concentration of DEA should not exceed 5%; should not be used with products containing N-nitrosating agents. Cocamide DEA safe as used in rinse-off products; safe at concentrations 10% in leave-on products; should not be used as an ingredient in cosmetic products in which N-nitroso compounds are formed DEA Dodecylbenzenesulfonate safe as used when formulated to be non-irritating Isostearamide DEA safe for use in rinse-off products; in leave-on products, safe for use at a concentration that will limit the release of free ethanolamines to 5%, with a maximum use concentration of 40% Lauramide DEA safe as used; should not be used in products containing nitrosating agents 2 4 Linoleamide DEA safe as used; should not be used in products containing nitrosating agents 2 Myristamide DEA safe for use in rinse-off products; in leave-on products, safe for use at a concentration that will limit the release of free ethanolamines to 5%, with a maximum use concentration of 40% leamide DEA safe as used; should not be used in products containing nitrosating agents 2 4 Stearamide DEA safe for use in rinse-off products; in leave-on products, safe for use at a concentration that will limit the release of free ethanolamines to 5%, with a maximum use concentration of 40% 4 CMPNENTS Ammonium Laureth Sulfate safe as used when formulated to be non-irritating 91 Ammonium Lauryl Sulfate safe in formulations designed for discontinuous, brief use followed by thorough rinsing from the surface of the skin; in products intended for prolonged contact with skin, concentrations should not exceed 1% Ammonium Myreth Sulfate safe as used when formulated to be non-irritating Ammonium Myristyl Sulfate safe as used 93 Butyrospermum Parkii (Shea) Butter safe as used 94 C12-13 Pareth-3 safe as used when formulated to be non-irritating 95 Cocoamphodipropionate safe as used 96 Coconut Acid safe as used 94 Cocoyl Sarcosine safe as used in rinse-off products, safe for use in leave-on products at concentrations of 5%, and the data were insufficient to determine the safety for use in products where cocoyl sarcosine is likely to be inhaled; should not be used in cosmetic products 97 in which N-nitroso compounds may be formed Corn Acid safe as used 94 Elaeis Guineensis (Palm) Kernel il safe as used 94 Elaeis Guineensis (Palm) il safe as used 94 Isostearic Acid safe as used Panel Book Page 79

83 Table 2. Conclusions of previously reviewed ingredients and components (continued) Ingredient Conclusion Reference Lactic Acid safe for use in cosmetic products at concentrations 5 lo%, at final formulation ph > 3.5, when formulated to avoid increasing sun sensitivity or when directions for use include the daily use of sun protection. These ingredients are safe for use in salon products at concentrations 5 30%, at final formulation ph 2 3.0, in products designed for brief, discontinuous use followed by thorough rinsing from the skin, when applied by trained professionals, and when application is accompanied by directions for the daily use of sun protection. Lanolin Acid safe as used in topical applications Lauric Acid safe as used Lecithin safe as used in rinse-off products; safe for use in leave-on products at concentrations of 15%; and the data were insufficient to determine the safety for use in products where lecithin is likely to be inhaled; should not be used in cosmetic products in which N-nitroso compounds may be formed Mink il safe as used Myristic Acid safe as used 104 lea Europaea (live) Fruit il safe as used 94 leic Acid safe as used 101 rbignya leifera (Babassu) il safe as used 94 Palmitic Acid safe as used 101 PEGs safe as used 105 Persea Gratissima (Avocado) il safe as used 94 Prunus Amygdalus Dulcis (Sweet Almond) il safe as used 94 Prunus Armeniaca (Apricot) Kernel il safe as used 94 Rice Bran Acid safe as used 94 Ricinoleic Acid safe as used 106 Ricinus Communis (Castor) Seed il safe as used 106 Sesamum Indicum (Sesame) il safe as used 94 Sodium Cetyl Sulfate safe as used 93 Sodium Laureth Sulfate safe as used when formulated to be non-irritating 91 Sodium Lauryl Sulfate safe in formulations designed for discontinuous, brief use followed by thorough rinsing from the surface of the skin; in products intended for prolonged contact with skin, concentrations should not exceed 1% Sodium Myreth Sulfate safe as used when formulated to be non-irritating Sodium Myristyl Sulfate safe as used Soy Acid safe as used 94 Stearic Acid safe as used 101 Tall il Acid safe as used 107 Tallow safe as used 47 Panel Book Page 80

84 Table 2. Conclusions of previously reviewed ingredients and components (continued) Ingredient Conclusion Reference Wheat Germ Acid safe as used 94 Zea Mays (Corn) il safe as used Panel Book Page 81

85 Table 3. Physical and chemical properties Property Value Reference DEA Physical Form clear viscous liquid 1 white crystalline solid at room temperature 57 viscous liquid above 28 C Color colorless 1 dor ammoniacal 1 Molecular Weight Specific Gravity 20 C 108 Melting Point 28.0 C 108 Boiling Point C 108 Water Solubility soluble 108 ther Solubility soluble in alcohol, ethanol, and benzene 108 log K ow 25 C 108 Disassociation Constant ( pka) 25 C 108 Diethanolamine Bisulfate Molecular Weight Density 1.21 g/cm Methyl Diethanolamine Physical Form liquid 51 Molecular Weight Density (predicted) ±0.06 g/cm 3 (20 C) 111 Water Solubility completely soluble 51 Boiling Point C 112 log P Butyl Diethanolamine Molecular Weight Density (predicted) 0.990±0.06 g/cm Boiling Point 275 C 113 Vapor Pressure (predicted) x Lauryl Diethanolamine Molecular Weight Density g/cm 3 (25 C) g/cm 3 (25 C) log P (predicted) ± (25 C) 111 Capramide DEA Molecular Weight Density (predicted) ± 0.06 g/cm Boiling Point (predicted) ±30.0 C 111 log P (predicted) ± Undecylenamide DEA Molecular Weight Density (predicted) ± 0.06 g/cm Boiling Point (predicted) ±40.0 C 111 Lauramide DEA Physical Form viscous liquid or waxy solid 15 Color light yellow (liquid) or white to light yellow (solid) 2 dor faint, characteristic 2 Molecular Weight Density ± 0.06 g/cm 3 (at 20 C) 111 Refractive Index (n30/l) 2 Melting Point C 2 Boiling Point ± C 111 Water Solubility dispersible 2 ph (10% aq. dispersion) Acid Value Alkaline Value log P (predicted) ± (at 25 C) 111 pk a (at 25 C) 111 pk b (at 25 C) Myristamide DEA Physical Form waxy solid 4 Color white to off-white 4 Melting Point C 4 Water Solubility dispersible 4 49 Panel Book Page 82

86 Table 3. Physical and chemical properties (continued) Property Value Reference ther Solubility soluble in alcohol, chlorinated hydrocarbons, and aromatic hydrocarbons; dispersible in mineral spirits, kerosene, white mineral oils, and natural fats and oils ph (10% aq. dispersion) log P (predicted) 5.025±0.270 Acid Value 1 (max) Alkaline Value Palmitamide DEA Molecular Weight Density (predicted) ± 0.06 g/cm 3 (20 C) Boiling Point (predicted) ±30.0 C log P (predicted) ±0.270 Stearamide DEA Physical Form wax-like solid Color white to pale yellow Molecular Weight Density (predicted) ± 0.06 g/cm 3 (20 C) ph (1% aq. dispersion) 9-10 log P (predicted) ±0.270 Behenamide DEA Molecular Weight Density (predicted) ± 0.06 g/cm 3 (20 C) Boiling Point (predicted) ±30.0 C log P (predicted) ±0.270 leamide DEA Physical Form liquid Color amber Molecular Weight Specific Gravity 0.99 (25/25 C) Phase Transition congeals at -8 C Boiling Point (predicted) ±45.0 C Water Solubility dispersible ther Solubility soluble in alcohols, glycols, ketones, esters, benzenes, chlorinated solvents, and aliphatic hydrocarbons ph 9-10 log P (predicted) ±0.275 Linoleamide DEA Physical Form syrup-like liquid or waxlike mass Color light yellow (liquid) or white to yellow (mass) dor characteristic Specific Gravity (25 /25 C) Water Solubility slightly soluble Boiling Point (predicted) ±50.0 C ther Solubility soluble in ethanol, propylene glycol, and glycerin; insoluble in mineral oil Acid Value 2.0 (max) Alkaline Value (calculated as DEA) log P (predicted) ±0.275 Cocamide DEA Physical Form clear viscous liquid Color amber or yellow dor faint coconut Molecular Weight Melting Point C Water Solubility soluble in water ph (10% aq. solution) Acid Value 3.0 max Ricinoleamide DEA Molecular Weight Density (predicted) 1.007± 0.06 g/cm 3 (20 C) Boiling Point (predicted) ±50.0 C log P (predicted) ± ,19 3,19, Panel Book Page 83

87 Table 4a. Historical and current frequency and concentration of use according to duration and type of exposure # of Uses Conc. of Use (%) # of Uses Conc. of Use (%) # of Uses Conc. of Use (%) # of Uses Conc. of Use (%) # DEA Lauramide DEA Myristamide DEA Stearamide DEA Totals* NR Duration of Use Leave-n NR NR Rinse ff NR Diluted for Use 4 NR NR NR NR NR NR NR NR Exposure Type Eye Area NR NR NR NR 2 NR NR NR NR NR NR NR Possible Ingestion NR NR NR NR NR NR NR NR NR NR NR NR NR Inhalation NR 3 NR NR NR NR NR NR NR NR Dermal Contact NR Deodorant (underarm) NR NR NR NR NR 1 NR NR NR 15 NR 1 15 Hair - Non-Coloring NR NR 2-6 Hair-Coloring NR NR NR NR NR NR NR Nail 1 NR NR NR 4 NR 1-5 NR NR NR NR NR NR Mucous Membrane NR NR NR 1 NR NR Bath Products 4 NR NR NR NR NR NR NR NR Baby Products NR NR NR NR 13 NR NR NR NR NR NR NR # of Uses Conc. of Use (%) # of Uses Conc. of Use (%) # of Uses Conc. of Use (%) # of Uses Conc. of Use (%) Isostearamide DEA leamide DEA Linoleamide DEA Cocamide DEA Totals ** 850 NA Duration of Use Leave-n NA Rinse-ff 3 NR NA Diluted for Use 3 NR NR NA Exposure Type Eye Area 1 NR NR NR NR NR NR NR NR NR 1 NA Possible Ingestion NR NR NR NR NR NR NR NR NR NR NR NA Inhalation NR NR NR NR NR NR NR 1 NR 1 NR NA Dermal Contact NA Deodorant (underarm) NR NR 15 NR NR NR NR NR NR 4 NR NA Hair - Non-Coloring 3 NR NA Hair-Coloring NR NR NR 99 NR NA Nail NR NR NR NR NR NR NR NR NR 2 NR NA Mucous Membrane NR NR NR 5 NR NA Bath Products 3 NR NR NR 8 NR NA Baby Products NR NR NR NR NR NR NR NR NR 1 13 NA * Because each ingredient may be used in cosmetics with multiple exposure types, the sum of all exposure types my not equal the sum of total uses. # Concentration of use survey in process. NR no reported uses 51 Panel Book Page 84

88 Table 4b. Frequency (2010) and concentration of use according to duration and type of exposure Diethanolamine Bisulfate DEA Stearate DEA Linoleate # of Uses 21 Conc of Use (%)# # of Uses 21 Conc of Use (%) # of Uses 21 Conc of Use (%) Totals* Duration of Use Leave-n NR 1 NR Rinse-ff 6 NR 1 Diluted for Use NR NR NR Exposure Type Eye Area NR NR NR Possible Ingestion NR NR NR Inhalation NR NR NR Dermal Contact 6 1 NR Deodorant (underarm) NR NR NR Hair - Non-Coloring NR NR 1 Hair-Coloring NR NR NR Nail NR NR NR Mucous Membrane 6 NR NR Bath Products NR NR NR Baby Products NR NR NR DEA Lauryl Sulfate DEA Laureth Sulfate DEA-Cetyl Phosphate # of Uses 21 Conc of Use (%)# # of Uses 21 Conc of Use (%) # of Uses 21 Conc of Use (%) Totals* Duration of Use Leave-n NR NR 46 Rinse ff 3 NR 4 Diluted for Use NR 1 NR Exposure Type Eye Area NR NR 5 Possible Ingestion NR NR NR Inhalation NR NR NR Dermal Contact Deodorant (underarm) NR NR NR Hair - Non-Coloring NR NR NR Hair-Coloring NR NR NR Nail NR NR NR Mucous Membrane 1 NR NR Bath Products NR 1 NR Baby Products NR NR NR DEA-leth-3 Phosphate DEA-leth-10 Phosphate DEA-(Di(2-Hydroxypalmityl) Phosphate # of Uses 21 Conc of Use (%)# # of Uses 21 Conc of Use (%) # of Uses 21 Conc of Use (%) Totals* Duration of Use Leave-n Rinse-ff Diluted for Use NR NR NR Exposure Type Eye Area NR NR NR Possible Ingestion NR NR NR Inhalation NR NR NR Dermal Contact 2 NR 3 Deodorant (underarm) NR NR NR Hair - Non-Coloring 5 8 NR Hair-Coloring NR NR NR Nail NR NR NR Mucous Membrane NR NR NR Bath Products NR NR NR Baby Products NR NR NR 52 Panel Book Page 85

89 Table 4b. Frequency (2010) and concentration of use according to duration and type of exposure (continued) Capramide DEA Lauramide/Myristamide DEA Palm Kernelamide DEA # of Uses 21 Conc of Use (%)# # of Uses 21 Conc of Use (%) # of Uses 21 Conc of Use (%) Totals* Duration of Use Leave-n NR NR NR Rinse ff Diluted for Use NR NR NR Exposure Type Eye Area NR NR NR Possible Ingestion NR NR NR Inhalation NR NR NR Dermal Contact NR 1 NR Deodorant (underarm) NR NR NR Hair - Non-Coloring 1 NR 4 Hair-Coloring NR NR NR Nail NR NR NR Mucous Membrane NR NR NR Bath Products NR NR NR Baby Products NR NR NR Totals* 19 Duration of Use Leave-n 2 Rinse-ff 17 Diluted for Use NR Exposure Type Eye Area NR Possible Ingestion NR Inhalation NR Dermal Contact NR Deodorant (underarm) NR Hair - Non-Coloring 19 Hair-Coloring NR Nail NR Mucous Membrane NR Bath Products NR Baby Products NR Soyamide DEA # of Uses 21 Conc of Use (%)# * Because each ingredient may be used in cosmetics with multiple exposure types, the sum of all exposure types my not equal the sum of total uses. # Concentration of use survey in process. NR no reported uses 53 Panel Book Page 86

90 Table 4c. Ingredients not reported to be in use DEA Myristate DEA-Isostearate DEA Lauraminopropionate DEA-C12-13 Alkyl Sulfate DEA-Myristyl Sulfate DEA-C12-15 Alkyl Sulfate DEA-Cetyl Sulfate DEA C12-13 Pareth-3 Sulfate DEA-Myreth Sulfate DEA-Dodecylbenzenesulfonate DEA Methyl Myristate Sulfonate DEA-Ceteareth-2 Phosphate DEA-leth-5 Phosphate DEA-leth-20 Phosphate DEA-Hydrolyzed Lecithin Methyl Diethanolamine Butyl Diethanolamine N-Lauryl; Diethanolamine Undecylenamide DEA Myristamide DEA Palmitamide DEA Behenamide DEA Lactamide DEA Almondamide DEA Apricotamide DEA Avocadamide DEA Babassuamide DEA Cornamide DEA Cornamide/Cocamide DEA Hydrogenated Tallowamide DEA Lanolinamide DEA Lecithinamide DEA Minkamide DEA livamide DEA Palmamide DEA Ricebranamide DEA Ricinoleamide DEA Sesamide DEA Shea Butteramide/Castoramide DEA Tallamide DEA Tallowamide DEA Wheat Germamide DEA PEG-2 Tallowamide DEA PEG-3 Cocamide DEA Stearamidoethyl Diethanolamine Stearamidoethyl Diethanolamine HCl DEA Cocoamphodipropionate Diethanolaminooleamide DEA Stearamide DEA-Distearate Cocoyl Sarcosinamide DEA 54 Panel Book Page 87

91 Table 5. Status for use in Europe according to the EC CosIng Database Dialkanolamines and Their Salts (i.e., DEA and its acid salts) listed in Annex II - prohibited DEA DEA-C12-13 Pareth-3 Sulfate Diethanolamine Bisulfate DEA-Myreth Sulfate DEA-Myristate DEA-Dodecylbenzenesulfonate DEA-Isostearate DEA-Methyl Myristate DE-Linoleate DEA-Cetyl Phosphate DEA-Lauraminopropionate DEA-Ceteareth-2 Phosphate DEA-Lauryl Sulfate DEA-leth-3 Phosphate DEA-C12-13 Alkyl Sulfate DEA-leth-5 Phosphate DEA-Myristyl Sulfate DEA-leth-10 Phosphate DEA-C12-15 Alkyl Sulfate DEA-leth-20 Phosphate DEA-Cetyl Sulfate DEA Hydrolyzed Lecithin DEA-Laureth Sulfate Fatty Acid Dialkanolamides (i.e., the alkyl substituted diethanolamines) listed in Annex III - restrictions N-Lauryl Diethanolamine Cornamide DEA Capramide DEA Cornamide/Cocamide DEA Undecylenamide DEA Hydrogenated Tallowamide DEA Lauramide DEA Lanolinamide DEA Myristamide DEA Lecithinamide DEA Lauramide/Myristamide DEA Minkamide DEA Palmitamide DEA livamide DEA Stearamide DEA Palm Kernelamide DEA Behenamide DEA Palmamide DEA Isostearamide DEA Ricebranamide DEA leamide DEA Ricinoleamide DEA Linoleamide DEA Sesamide DEA Almondamide DEA Soyamide DEA Apricotamide DEA Tallamide DEA Avocadamide DEA Tallowamide DEA Babassuamide DEA Wheat Germamide DEA Cocamide DEA Listed in EC Inventory Annex III (restrictions) Butyl Diethanolamine (maximum of 2.5% in ready for use preparations; do not use with nitrosating systems; minimum purity 99%; maximum secondary amine content in raw material, 0.5%; maximum nitrosamine content, 50 µg/kg; keep in nitrite-free containers) In EC Inventory no annex specified Lactamide DEA Shea Butteramide/Castoramide DEA PEG-2 Tallowamide DEA PEG-3 Cocamide DEA Stearamidoethyl Diethanolamine Stearamidoethyl Diethanolamine HCl Diethanolaminooleamide DEA Stearamide DEA-Distearate Cocoyl Sarcosinamide DEA Not Listed in EC Inventory DEA Stearate (also not in INCI Dictionary) DEA-Di(2-Hydroxypalmityl)Phosphate (also not in INCI Dictionary) Methyl Diethanolamine DEA Cocoamphodipropionate 55 Panel Book Page 88

92 Table 6. Conclusions of NTP dermal carcinogenicity studies DEA Lauramide DEA leamide DEA Cocamide DEA amount of free DEA >99% pure 0.83% 0.19% 18.2% B6C3F 1 mice 0, 40, 80, and 160 mg/kg 0, 100, or 200 mg/kg 0, 15, or 30 mg/kg 0, 100, or 200 mg/kg Males clear evidence of carcinogenic activity no evidence of carcinogenic activity no evidence of carcinogenic activity clear evidence of carcinogenic activity Basis increased incidences of liver neoplasms and renal tubule neoplasms increased incidences of hepatic and renal tubule neoplasms Females clear evidence of carcinogenic activity some evidence of carcinogenic activity no evidence of carcinogenic activity clear evidence of carcinogenic activity Basis increased incidence of liver neoplasms increased incidences of hepatocellular neoplasms F344/N rats 0, 16, 32, and 64 mg./kg 0, 50, or 100 mg/kg 0, 50, or 100 mg/kg 0, 50, or 100 mg/kg increased incidences of hepatic neoplasms Males no evidence of carcinogenic activity no evidence of carcinogenic activity no evidence of carcinogenic activity no evidence of carcinogenic activity Basis Females no evidence of carcinogenic activity no evidence of carcinogenic activity no evidence of carcinogenic activity equivocal evidence of carcinogenic activity Basis marginal increase in the incidences of renal tubule neoplasms 56 Panel Book Page 89

93 REFERENCES 1. Elder RL (ed). Final Report on the Safety Assessment of Triethanolamine, Diethanolamine, and Monoethanolamine. J Am Coll Toxicol. 1983;2:(7). 2. Elder RL (ed). Final report on the safety assessment of cocamide DEA, lauramide DEA, linoleamide DEA, and oleamide DEA. J Am Coll Toxicol. 1986;5:(5): Andersen FA (ed). Amended final report on the safety assessment of cocamide DEA. J Am Coll Toxicol. 1996;15:(6): Pang S. Isostearamide DEA & MEA, Myristamide DEA & MEA, Stearmide DEA & MEA Available from the CIR, th Street, NW, Ste 412, Washington DC Becker LC. Amended safety assessment of dodecylbenzenesulfonate, decylbenzenesulfonate, and tridecylbenezenesulfonate salts as used in cosmetics Available from the CIR, th Street, NW, Ste 412, Washington DC Lehman-McKeeman LD and Gamsky EA. Choline supplementation inhibits diethanolamine-induced morphological transfomation in Syrian hamster embryo cells: Evidence for a carcinogenic mechanism. Toxicol Sci. 2000;55: Toxicity and carcinogenicity of N-nitroso compounds Chapter: 1. Mycotoxins and N-Nitroso Compounds:Environmental Risks. Shank RC. Boca Raton, FL: CRC Press, Inc. 8. Rostkowski K, Zwierzl K, Ró añski J, Moniuszko-Jakoniuk J, and Roszczenko A. Formation and Metabolism of N- Nitrosamines. Polish Journal of Environmental Studies. 1998;7:(6): Challis BC, Shuker DE, Fine DH, Goff EU, and Hoffman GA. Amine nitration and nitrosation by gaseous nitrogen dioxide. IARC Sci Publ. 1982;41: Bíró T, Tóth BI, Haskó G, Paus R, and Pacher P. The endocannabinoid system of the skin in health and disease: novel perspectives and therapeutic opportunities. Trends Pharmacol Sci. 2009;30:(8): Bisogno T, De Petrocellis L, and Di Marzo V. Fatty acid hydrolase, an enzyme with many bioactive substrates. Possible therapeutic implications. Current Pharmaceutical Design. 2002;8: Gray GM, Tabiowo A, and Trotter MD. Studies on the soluble membrane-bound amino acid 2-naphthylamidases in pig and human epidermis. Biochem J. 1977;161: Lemke TL. Review of organic functional groups: introduction to medicinal organic chemistry. 4th ed Dow Chemical Company. The alkanolamines handbook Midland, MI: The Dow Chemical Company.Secondary reference in Knaak et al National Toxicology Program. NTP Technical report on the toxicology and carcinogenesis studies of lauric acid diethanolamine condensate (CAS No ) in F344/N rats and B6C3F 1 mice. (Dermal studies.) NTP TR Chou, H. J. Determination of diethanolamine and N-nitrosodiethanolamine in fatty acid diethanolamides. J Assoc ff Anal Chem Intl. 1998;81:(5): Dow Chemical Company. Sales Specification: Diethanolamine Secondary reference in IARC National Toxicology Program. NTP Technical report on the toxicology and carcinogenesis studies of oleic acid diethanolamine condensate (CAS No ) in F344/N rats and B6C3F 1 mice. (Dermal studies.) NTP TR National Toxicology Program. NTP Technical report on the toxoicology and carcinogenesis studies of coconut oil acid diethanolamine condensate (CAS No ) in F344/N rats and B6C3F 1 mice. (Dermal studies.) NTP TR Gottschalck T.E. and Bailey, J. E. eds. International Cosmetic Ingredient Dictionary and Handbook. Washington, DC: Personal Care Products Council, Panel Book Page 90

94 21. Food and Drug Administration (FDA). Frequency of use of cosmetic ingredients. FDA Database Washington, DC: FDA.Updated May Personal Care Products Council. Concentration of use data - submitted by industry in response to a Council survey Unpublished data submitted by the Council. 23. Bailey J. DEA in consumer products is safe. FASEB J. 2007;21:(1): European Commission. Cosing Database. Annex II. List of substances which must not form part of the composition of cosmetic products. Secondary alkyl- and alkanolamines and their salts, including diethanolamine Health Canada.Cosmetic Ingredient Hotlist Accessed Food and Drug Administration.Everything Added to Food in the United States (EAFUS) Accessed Ballantyne B and Leung H-W. Acute toxicity and primary irritancy of alkylalkanolamines. Vet Human Toxicol. 1992;38:(6): Yourick JJ, Marks A, Lockhead RY, and Bronaugh RL. Diethanolamine (DEA) in vitro dermal absorption in Fuzzy rat skin. Unpublished manuscript. Secndary reference in ECD Sun JD, Beskitt JL, Tallant MJ, and Frantz SW. In vitro skin penetration of monoethanolamine and diethanolamine using excised skin from rats, mice, rabbits, and humans. J Toxicol -- Cut & cular Toxicol. 1996;15:(2): Kraeling MEK, Yourick JJ, and Bronaugh RL. In vitro human skin penetration of diethanolamine. Food Chem Toxicol. 2004;42: Brain KR, Walter KA, Green DM, Brain S, Loretz LJ, Sharma RK, and Dressler WE. Percutaneous penetration of diethanolamine through human skin in vitro: Application from cosmetic vehicles. Food Chem Toxicol. 2005;43: Mathews JM, Garner CE, and Matthews HB. Metabolism, bioaccumulation, and incorporation of diethanolamine into phospholipids. Chem Res Toxicol. 1995;8:(5): Mathews JM, DeCosta K, and Thomas BF. Lauramide diethanolamine absorption, metabolism, and disposition in rats and mice after oral, intravenous, and dermal administration. Drug Metabolism and Disposition. 1996;24:(7): Merdink, J., Decosta, K., Mathews, J. M., Jones, C. B., kita, J. R., and kita, R. T. Hydroxylation of lauramide diethanolamine by liver microsomes. Drug Metabolism and Disposition. 1996;24:(2): National Toxicology Program. Toxicology and carcinogenesis studies of diethanolamine (CAS No ) in F344/N rats and B6C3F 1 mice. (Dermal studies.) NTP TR Mathews JM and Jeffcoat AR. Absorption and disposition of diethanolamine (DEA) in rats and mice after oral, dermal, and intravenous administration. RTI/3662/00-12P Secondary reference in Knaak et al Waechter JM, Bormett GA, and Stewart HS. Diethanolamine: pharmacokinetics in Sprague-Dawley rats following dermal or intravenous administration Secondary reference in Knaak et al Mathews JM, Garner CE, Black SL, and Matthews HB. Diethanolamine absorption, metabolism and disposition in rat and mouse following oral, intravenous and dermal administration. Xenobiotica. 1997;27:(7): Craciunescu CN, Niculescu MD, Guo Z, Johnson AR, Fischer L, and Zeisel SH. Dose response effects of dermally applied diethanolamine on neurogenesis in fetal mouse hippocampus and potential exposure of humans. Toxicol Sci. 2009;107:(1): Leung H-W, Ballantyne B, and Frantz SW. Pharmacokinetics of N-methyldiethanolamine following acute cutaneous and intravenous dosing in the rat. J Toxicol -- Cut & cular Toxicol. 1996;15:(4): Panel Book Page 91

95 41. Mendrala AL, Waechter JM, Bormett GA, Bartels MJ, and Stott WT. The pharmacokinetics of diethanolamine in Sprague- Dawley rats following intravenous administration. Food Chem Toxicol. 2001;39: Stott WT, Bartels MJ, Brzak KA, Mar M-H, Markham DA, Thorton CM, and Zeisel SH. Potential mechanisms of tumorigenic action of diethanolamine in mice. Toxicol Lett. 2000;114: Saghir SA, Brzak A, Markham DA, Bartels MJ, and Stott WT. Investigation of the formation of N-nitrosodiethanolamine in B6C3F1 mice following topical administration of triethanolamine. REg Toxicol Pharmacol. 2005;43: European Commission.IUCLID Data set for methyl diethanolamine Accessed Smyth HF. Arch Indus Hyg ccupmed. 1954;10: Consumer Product Testing.Acute oral toxicity study on cocamide DEA in rats Summary 1.pdf. Accessed National Toxicology Program. Toxicity studies of diethanolamine (CAS No ) administered topically and in drinking water to F344/N rats and B6C3F1 mice. Tech. Rep. Series No Seconary reference in ECD Mellert W, Kaufmann W, Rossbacher R, and van Ravenzwaay B. Investigations on cell proliferation in B6C3F 1 mouse liver by diethanolamine. Food Chem Toxicol. 2004;42: Melnick RL, Mahler J, Bucher JR, Hejtmancik M, Singer A, and Persing RL. Toxicity of diethanolamine. 2. Drinking water and topical application exposures in B6C3F 1 mice. J Appl Toxicol. 1994;14:(1): Melnick RL, Mahler J, Bucher JR, Thompson M, Hejtmancik M, Ryan MJ, and Mezza LE. Toxicity of diethanolamine. 1. Drinking waer and topical application exposures in F344 rats. J Appl Toxicol. 1994;14:(1): Werley MS, Ballantyne B, Leung H-W, Hermansky SJ, and Fowler EH. Acute and subchronic repeated cutaneous application of N-methyldiethanolamine in the Fischer 344 rat. J Toxicol -- Cut & cular Toxicol. 1997;16: Environmental Health Research and Testing. Screening of priority chemicals for reproductive hazards. Monoethanolamin, diethanolamine, and triethanolamine, ETX PB Secondary reference in ECD Munson AE, White KL, and McCay JA. Immunotoxicity of diethanolamine in female B6C3F1 mice. Report to the NTP Secondary reference in ECD Gulf South Research Institiute. Subchronic test of diethanolamine (C55174) in B6C3F1 mice and Fischer 344 rats Secondary reference in ECD Munson AE, White KL, and McCay JA. Immunotoxicity of diethanolamine in female Fischer 344 rats. Report to the NTP Secondary reference in ECD Eastman Kodak Company. Health and safety studies for diethanolamine, with cover letter. TSCATS, TC , Doc ID Secondary reference in ECD Gamer A, Rossbacher R, Kaufmann W, and van Ravenzwaay B. The inhalation toxicity of di- and triethanolamine upon repeated exposure. Food Chem Toxicol. 2008;46: Lee ID, Lee GS, Moon HJ, Seok JH, Yang JY, Chae SY, Lee YW, Kim SM, Kim SH, and Jeong SY. A study of the reproductive and developmental toxicity of diethanolamine. (Unofficial translation.). The Annual Report of KFDA : EHQ A; Submission by Dow Chemical Co. 59. Marty MS, Neeper-Bradely TL, Neptun DA, and Carney EW. Developmental toxicity of diethanolamine applied cutaneously to CD rats and New Zealand White rabbits. REg Toxicol Pharmacol. 1999;30: Leung W-W and Ballantyne B. Developmental toxicity study with N-methyldiethanolamine by repeated cutaneous application to CD rats. J Toxicol -- Cut & cular Toxicol. 1998;17:(4): Panel Book Page 92

96 61. Gulati DK, Grimes LK, Heindel J, and Schwetz BA. Range finding studies: Developmental toxicity of diethanolamine when administered via gavage in CD Sprague-Dawley rats, NTP 89-RF/DT Secondary reference in Marty et al Center for Life Science and Technology. Final study report. Developmental toxicity screen for diethanolamine (CAS No ) administered by gavage to Sprague-Dawley (CD) rats on gestational days 6 through 19: Evaluation of dams and pups through postnatal day Prepared for the National Toxicology Program; NTP Study No. TER BASF AG. Range-finding study on the prenatal inhalation toxicity of diethanolamin in pregnant rats. BASF Project No 11R0233/ Secondary reference in ECD Gamer A, Hellwig J, and Hildebrand B. Study of the prenatal toxicity of diethanolamin n rats after inhalation, Project No. 31R0233/90010; BASF BASF AG. Study of the prenatal inhalation toxicity of diethanolamine in rats after inhalation. BASF Project No. 31R0233/ Secondary reference in ECD Craciunescu CN, Wu R, and Zeisel SH. Diethanolamine alters neurogenesis and induces apoptosis in fetal mouse hippocampus. FASEB J. 2006;20: Niculescu MD, Wu R, Guo Z, da Costa KA, and Zeisel SH. Diethanolamine alters proliferation and choline metabolism in mouse neural precursor cells. Toxicol Sci. 2007;96:(2): Kamendulis LM and Klaunig JE. Species differences in the induction of hepatocellular DNA synthesis by diethanolamine. Toxicol Sci. 2005;87:(2): Leung H-W and Ballantyne B. Evaluation of the genotoxic potential of alkylalkanolamines. Mutat Res. 1997;393: Tennant RW, French JE, and Spalding JW. Identifying chemical carcinogens and assessing potential risk in short-term bioassays using transgenic mouse models. Environ Health Perspect. 1995;103: Spalding JW, French J, Stasiewicz S, Furedi-Machacek M, Conner F, Tice RR, and Tennant RW. Responses of transgenic mouse lines p53 +/- and Tg AC to agents tested in conventional carcinogenicity bioassays. Toxicol Sci. 2000;53: International Agency for Research on Cancer (IARC). Diethanolamine. IARC Monographs on the evaluation of the carcinogenic risk of chemicals to humans. 2000;77: Zeisel SH and Blusztajn JK. Choline and human nutrition. Annu Rev Nutr. 1994;14: Kamendulis LM, Smith DJ, and Klaunig JE. Species differences in the inhibition of gap junctional intercellular communication (GJIC) by diethanolamine. Toxicologist :(S-1): 75. Bachman AN, Kamednulis LM, and Goodman JI. Diethanolamine and phenobarbital produce an altered pattern of methylation in GC-rich regions of DNA in B6C3F1 mouse hepatocytes similar to that resulting from choline deficiency. Toxicol Sci. 2006;90:(2): Lehman-McKeeman LD. Incorporating mechanistic data into risk assessment. Toxicology. 2002; : Leung H-W, Kamendulis LM, and Stott WT. Review of carcinogenic activity of diethanolamine and evidence of choline deficiency as a plausible mode of action. REg Toxicol Pharmacol. 2005;43: Hayashi S-M, Ton VT, Hong H-HL, Irwin RD, Haseman JK, Devereux TR, and Sills RC. Genetic alterations in the Catnb gene but not the H-ras gene in hepatocellular neoplasms and hepatoblastomas of B6C3Fa mice following exposure to diethanolamine for 2 years. Chemico-Biological Interactions : Secondary reference in ECD Corsini, E., Marinovich, M., Marabini, L., Chiesara, E., and Galli, C. L. Interleukin-1 production after treatment with non-ionic surfactants in a murine keratinocytes cell line. Toxicology in Vitro. 1994;8:(3): Tupker, R. A., Pinnagoda, J., Coenraads, P.-J., and Nater, J. P. The influence of repeated exposure to surfactants on the human skin as determined by transepidermal water loss and visual scoring. Contact Dermatitis. 1989;20:(2): Panel Book Page 93

97 81. Stern, M., Klausner, M., Alvarado, R., Renskers, K., and Dickens, M. Evaluation of the Epicular tissue model as an alternative to the Draize eye irritation test. Toxicology in Vitro. 1998;12:(4): Kimber I, Basketter DA, Butler M, Gamer A, Garrigue JL, Gerberick GR, Newsome C, Steiling W, and Vohr HW. Classification of contact allergens to potency. Proposals, Food Chemical Toxciol : Lessmann H, Uter W, Schnuch A, and Geier J. Skin sensitizing properties of ethanolamines mono-, di-, and triethanolamine. Data analysis of a multicentre surveillance network (IVDK) and review of literature. Contact Derm. 2009;60: RCC. Contact hypersensitivity to Diethanolamin, rein in albino Guinea pigs, Maximization test, RCC Project Unpublished report Secondary reference in ECD Leung H-W and Blaszcak DL. The skin sensitization potential of four alkylalkanolamines. Vet Human Toxicol. 1998;40: Brey NL and Fowler JF. Relevance of positive patch-test reactions tp cocamidopropyl betaine and amidoamine. Dermatitis. 2004;15:(1): Christersson, S. and Wrangsjo, K. Contact allergy to undecylenamide diethanolamide in a liquid soap. Contact Dermatitis. 1992;27:(3): Fowler JF. Allergy to cocamide DEA. Am J of Contact Derm. 1998;9:(1): Lehman-McKeeman LD, Gamsky EA, Hicks SM, Vassalo JD, Mar M-H, and Zeisel SH. Diethanolamine induces hepatic choline deficiency in mice. Toxicol Sci. 2002;67: NISH Pocket Guide to Chemical Hazards Accessed Robinson VC, Bergfeld WF, Belsito DV, Hill RA, Klaassen CD, Marks JG, Shank RC, Slaga TJ, Snyder PW, and Andersen FA. Final report of the amended safety assessment of sodium laureth sulfate and related salts of sulfated ethoxylated alcohols. Int J Toxicol. 2010;29:(Supple 3):151S-161S Elder RL (ed). Final report on the safety assessment of sodium lauryl sulfate and ammonium lauryl sulfate. J Am Coll Toxicol. 1983;2:(7): Fiume M, Bergfeld WF, Belsito DV, Klaassen CD, Marks JG, Shank RC, Slaga TJ, Snyder PW, and Andersen FA. Final report on the safety assessment of sodium cetearyl sulfate and related alkyl sulfates as used in cosmetics. Int J Toxicol. 2010;29:(Suppl 2):115S-132S. 94. Burnett CL and Fiume MM. Tentative report of the CIR Expert Panel on the safety of plant-derived fatty acid oils and used in cosmetics Available from the CIR, th Street, NW, Ste 412, Washington DC Fiume MM and Heldreth BA. CIR Expert Panel final amended report on alkyl PEG ethers as used in cosmetics Available from the CIR, th Street, NW, Ste 412, Washington DC Elder RL (ed). Final report on the safety assessment of cocamphoacetate, cocoamphopropionate, cocoamphodiacetate, and cocoamphodipropionate. J Am Coll Toxicol. 1990;9:(2): Andersen FA (ed). Final report on the safety assessment of cocoyl sarcosine, lauroyl sarcosine, myristoyl sarcosine, oleoyl sarcosin, stearoyl sarcosine, sodium cocoyl sarcosinate, sodium lauroyl sarcosinate, sodium myristoyl sarcosinate, ammonium cocoyl sarcosinate, and ammonium lauroyl sarcosinate. Int J Toxicol. 2001;20:(Suppl 1): Elder RL (ed). Final report on the safety assessment of isostearic acid. J Am Coll Toxicol. 1986;2:(7): Andersen FA (ed). Final report on the safety assessment of glycolic acid, ammonium, calcium, potassium, and sodium glycolates, methyl, ethyl, propyl, and butyl glycolates, and lactic acid, ammonium, calcium, potassium, sodium, and TEA-lactates, methyl, ethyl, isoprpyl, and butyl lactates, and lauryl, myristyl, and cetyl lactates. Int J Toxicol. 1998;17:(Suppl 1): Elder RL (ed). Final report on the safety assessment for acetylated lanolin alcohol and related compounds. JEPT. 1980;4:(4): Panel Book Page 94

98 101. Elder RL (ed). Final report on the safety assessment of oleic acid, lauric acid, palmitic acid, myristic acid, and stearic acid. J Am Coll Toxicol. 1987;6:(3): Andersen FA (ed). Final report on the safety assessment of lecithin and hydrogenated lecithin. Int J Toxicol. 2001;20:(Suppl 1): Andersen FA (ed). Final amended report on the safety of mink oil. Int J Toxicol. 2005;24:(Suppl 3): Becker LC, Bergfeld WF, Belsito DV, Hill RA, Klaassen CD, Marks JG, Shank RC, Slaga TJ, Snyder PW, and Andersen FA. Final report on the amended safety assessment of myristic acid and its salts and esters as used in cosmetics. Int J Toxicol. 2010;29:(Suppl 3):162S-186S Andersen FA. Final Report of the CIR Expert Panel - Amended Safety Assessment of Triethylene Glycol and Polyethylene Glycols (PEGs)-4, -6, -7, -8, -9, -10, -12, -14, -16, -18, -20, -32, -33, -40, -45, -55, -60, -75, -80, -90, -100, -135, -150, - 180, -200, -220, -240, -350, -400, -450, -500, -800, -2M, -5M, -7M, -9M, -14M, -20M, -23M, -25M, -45M, -65M, - 90M, -115M, -160M and -180M and any PEGs = 4 as used in Cosmetics Available from the CIR, th Street, NW, Ste 412, Washington DC Andersen FA (ed). Final Report on the Safety Assessment of Ricinus Communis (Castor) Seed il, Hydrogenated Castor il, Glyceryl Ricinoleate, Glyceryl Ricinoleate SE, Ricinoleic Acid, Potassium Ricinoleate, Sodium Ricinoleate, Zinc Ricinoleate, Cetyl Ricinoleate, Ethyl Ricinoleate, Glycol Ricinoleate, Isopropyl Ricinoleate, Methyl Ricinoleate, and ctyldodecyl Ricinoleate. Int J Toxicol. 2011;26:(Suppl 3): Robinson V, Bergfeld WF, Belsito DV, Klaassen CD, Marks JG, Shank RC, Slaga TJ, and Andersen FA. Amended safety assessment of tall oil acid, sodium tallate, potasstium tallate, and ammonium tallate. Int J Toxicol. 2009;28:(Suppl 3):252S-258S Toxicology of mono-, di-, and triethanolamine Ware GW. New York: Springer-Verlag Environmental Protection Agency.Substance Registry Services - Ethanol, 2,2'-iminobis-, sulfate (1:1) (salt) TN&p_value= Accessed Zhao C. Journal of Physical Chemistry B :(23): btained from CAPLUS Advanced Chemistry Development (ACD/Labs). Advanced Chemistry Development software v ((C) ACD/Labs) Nikolaev AF. Zhurnal bshchei Khimii. 1963;33: Syracuse Research Corporation. PhysProp data Syracuse, NY: 114. Nikawitz EJ. US btained from CAPLUS Komori S. Technology Reports of the saka University. 1956;6: Panel Book Page 95

99 What The Where CSMETIC Personal Care Products Council Committed to Safety, Qualify & Innovation Memorandum T: FRM: F. Alan Andersen, Ph.D. Director - INGREDIENT REVIEW (CIR) John Bailey, Ph.D. Industry Liaison to the CIR Expert Panel DATE: December 10, 2010 SUBJECT: Comments on the Draft Reports on Triethanolamine, Diethanolamine and Ethanolamine Prepared for the December 13-14, 2010 CIR Expert Panel Meeting Memo - Rather than Acute (Single Dose) Toxicity and Repeated Dose Toxicity, the sections should be titled Acute (Single) Dose Exposure and Repeated Dose Exposure. Acute and Repeated Dose describe the exposure rather than toxicity. Triethanolamine p.1 - is missing from the following sentence? The crude is later separated by distillation. p.1 - It would be helpful to indicate where in the report the in vivo studies of NDELA formation are presented. p.2 - The meaning of the following sentence is not clear, Accordingly, depending on storage and application conditions, aerosolized TEA may be a liquidlvapor instead of a particle. Aerosol products will produce aerosols. For compounds that are part of the formulation that have relatively high vapor pressures, the more important exposure will likely be inhalation of a vapor rather than inhalation of the aerosol. p.2 - Where did the information on use from Health Canada come from? The website listed in reference 13 (the Canadian Hotlist) was checked and use information for individual ingredients is not included on this website. p.3 - first two paragraphs of the Absorption, Distribution, Metabolism and Excretion study are describing the same study. Reference 16 is the unpublished version of the dermal study described in reference 17. p.4- - was the TEA-glucuronide found (reference 20)? p.6 - How was the 90-day NAEC for local irritation calculated, e.g., using safety factors or modeling? p.7 - Searching the internet indicates that syntanol DC-b is CAS alcohols Cb0-18 ethoxylated. p.9 - LLNA s are not in vitro studies. They are considered alternatives because they reduce distress. As LLNAs are useful for quantitative risk assessment., please include the doses used in this study th Street, N.W., Suite 3 Washington, D.C (fax) Panel Book Page 96

100 As The In Were It The Vehicle It These p.10 - the human studies described in the summary of the original report single patch tests or repeated patch tests? Were the subjects patients or volunteers with no dermal conditions? p.10 - Were the subjects tested in reference 15 patients with dermatological conditions? p.11 - would be helpful if the information on in vivo N-nitrosodiethanolamine formation were presented as a subsection under Absorption, Distribution, Metabolism and Excretion. p.11 - Please present the carcinogenicity mechanism information as a subsection under Carcinogenicity. p.12 - exposure information should be presented in the Cosmetic Use section and the cancer evaluation should be the last item presented in the Carcinogenicity section. p.13, Table 1 - Please provide the references for this Table. p.14, 15 reference 19 and reference 38 - two references are the same. Diethanolamine p.2 - there are inhalation data on Diethanolamine, is the aerosol boilerplate information necessary? p.2-3, It is not clear why the in vitro dermal penetration data is presented in two different subsections. p.4 - Please defined PC and PE the first time they appear. p.s - In the description of reference 20 should 5 mi/kg bw be 5 mg/kg bw as the units for the rest of the doses are mg/kg? p.s - i.v. study described in reference 22 appears to be the same study as that described in reference 20. Please provide the dose used in reference 20. = p.6 - Please change The percutaneous absorption of cosmetic formulations... to The percutaneous absorption of DEA in cosmetic formulations... p.8 - In the description of reference 30, should ppm be mg/kg? The units in the rest of the paragraph are mg/kg. p.9 - the summary of the inhalation data from the original report, please give the duration of the short-term inhalation exposure. p.10 - It would be helpful if the 45-day inhalation study were presented before the 90-day inhalation studies. p.10 - In reference 35, were any dermal effects observed in the male mice treated with Diethanolamine? Were there any effects on the number of offspring? p.11 - not specified is not necessary for reference 40, 41, an inhalation study. p.13 - LLNA s are not in vitro studies. They are considered alternatives because the reduce distress. As LLNAs are useful for quantitative risk assessment, please include the doses used in this study. p.13 - The internet indicates that FRAFAC 1203 is an additive used in portable fire extinguishers. As it is not possible to tell which component resulted in the sensitization, this study is not very helpful and can be deleted. p.14 - would be helpful if the information on in vivo N-nitrosodiethanolamine formation were presented as a subsection under Absorption, Distribution, Metabolism and Excretion. p.16 - Please present the carcinogenicity mechanism information as a subsection under Carcinogenicity. 2 Panel Book Page 97

101 Where LLNA s If The - Please p.17 - the SHA and ACGIH values are presented, they should be cited to SHA and ACGIH references, respectively. The NISH Safety Card (link from the n-line) indicates that SHA does not have a Permissible Exposure limit for Diethanolamine. NISH has a recommendation of 3 ppm. p.17 - IARC cancer review should be moved to the end of the Carcinogenicity section. p.18, Table 1 provide references for this table. Ethanolamine p.1 - did the information on use from Health Canada come from? The website listed in reference 5 (the Canadian Hotlist) was checked and use information for individual ingredients is not included on this website. p.6 - are not in vitro studies. They are considered alternatives because the reduce distress. p.6 - Please provide SHA and ACGIII references for the exposure limits. p.7, Table 1 - Please provide references for this table. 3 Panel Book Page 98