Safety Assessment of Hydroxyethyl-3,4-Methylenedioxyaniline HCl as Used in Cosmetics

Transcription

1 Safety Assessment of Hydroxyethyl-3,4-Methylenedioxyaniline HCl as Used in Cosmetics Status: Draft Final Report for Panel Review Release Date: May 19, 2017 Panel Meeting Date: June 12-13, 2017 The 2017 Cosmetic Ingredient Review Expert Panel members are: Chair, Wilma F. Bergfeld, M.D., F.A.C.P.; Donald V. Belsito, M.D.; Ronald A. Hill, Ph.D.; Curtis D. Klaassen, Ph.D.; Daniel C. Liebler, 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 Lillian J. Gill, D.P.A. This safety assessment was prepared by Laura N. Scott, Scientific Writer/Analyst. Cosmetic Ingredient Review 1620 L Street, NW, Suite 1200 Washington, DC ph fax cirinfo@cir-safety.org

2 Commitment & Credibility since 1976 Memorandum To: CIR Expert Panel Members and Liaisons From: Laura N. Scott Senior Scientific Writer Date: May 19, 2017 Subject: Draft Final Report of the Safety Assessment of Hydroxyethyl-3,4-Methylenedioxyaniline HCl as Used in Cosmetics At the December 5 th -6 th, 2016 CIR Expert Panel meeting, the Panel issued a Tentative Report for public comment on the Safety Assessment of Hydroxyethyl-3,4-Methylenedioxyaniline HCl with the conclusion of safe for use as a hair dye ingredient in the present practices of use and concentrations described in this safety assessment; ingredients should not be used in cosmetic products in which N-nitroso compounds can be formed. The N-nitrosation caveat was added to the abstract and conclusion in this iteration of the report. Enclosed is the Draft Final Report of the Safety Assessment of Hydroxyethyl-3,4-Methylenedioxyaniline HCl as Used in Cosmetics (identified as HMA062017rep in the pdf document). Additionally, the CIR report history (HMA062017hist), literature search strategy (HMA062017strat), ingredient data profile (HMA062017prof), process flow chart (HMA062017flow), 2017 FDA VCRP data (HMA062017FDA), and minutes from the December 2016 Panel Meeting (HMA062017min) are included for the Panel s review. The main changes made to this Draft Final Report following the December 2016 Meeting were removing the acute oral exposure study containing an analog inappropriate for read-across, removing the unnecessary schematic and corresponding text in the Impurities and Nitrosation section, removing the information in the Non-Cosmetic Use section (applicable to hazard assessments used for the labeling and transport of raw materials, but not appropriate here), and adding the pka (Table 1) of the non-ionized form of the ingredient. Council comments on the Draft Report (HMA062017pcpc_1) and on the Tentative Report (HMA062017pcpc_2) have been addressed and are enclosed for the Panel s review. After reviewing the Draft Final Report, the Panel will need to verify the Discussion and Conclusion and be prepared to issue a Final Report L Street, NW Suite 1200, Washington, DC (Main) (Fax) ( ) cirinfo@cir-safety.org (Website)

3 SAFETY ASSESSMENT FLOW CHART INGREDIENT/FAMILY Hydroxyethyl-3,4-Methylenedioxyaniline HCl MEETING June 2017 Public Comment CIR Expert Panel Report Status Priority List INGREDIENT PRIORITY LIST 60 day public comment period SLR Sept 19, 2016 Draft Report DRAFT REPORT Dec 2016 Table Table IDA TR IDA Notice Draft TR IDA DRAFT TENTATIVE REPORT Table Table Tentative Report Dec 15, 2016 Issue TR 60 day Public comment period Draft FR DRAFT FINAL REPORT June 2017 Table Table Different Conclusion PUBLISH Final Report Issue FR

4 Report History-Hydroxyethyl-3,4-Methylenedioxyaniline HCl September 19 th, 2016-The Hydroxyethyl-3,4-Methylenedioxyaniline HCl Scientific Literature Review was posted at for public comment. December 5-6 th, 2016-This is the first time the Panel saw the Draft Report of the Safety Assessment of Hydroxyethyl-3,4-Methylenedioxyaniline HCl; The Panel issued a Tentative Report for public comment with the conclusion that Hydroxyethyl-3,4-Methylenedioxyaniline HCl is safe for use as a hair dye ingredient in the present practices of use and concentrations described in this assessment; this ingredient should not be used with N- nitrosating agents. December 15 th, 2016-The Tentative Report of the Safety Assessment of Hydroxyethyl-3,4-Methylenedioxyaniline HCl was posted online at for public comment. April 10 th -11 th, 2017-This report was not reviewed at the April Panel Meeting.

5 Dermal Penetration Hydroxyethyl-3,4-Methylenedioxyaniline HCl Data Profile for June 12-13, Writer Laura Scott Penetration Enhancement ADME Acute Toxicity Short-Term Toxicity Subchronic Tox DART Genotox Carcinogenicity Dermal Irr. Dermal Sen. Ocular Irr. Animal Human-QSAR Human-Market Analysis Animal Human Animal Animal In Vivo-IP In Vitro Animal-Oral Animal-Oral Animal-Inhalation Animal-Oral Animal-Dermal Animal-Inhalation Animal-Oral Animal-Dermal Animal-IV Animal-Oral Animal-Dermal In Vitro Human Animal In Vitro Animal In Vitro Safety Data Available? Used in Cosmetics? Hydroxyethyl-3,4- Y Y X X X X X X X X X X X X Methylenedioxyaniline HCl X X X X indicates available, relevant studies included in this safety assessment in each applicable category. Blank boxes indicate no available, relevant data were found in the literature or submitted.

6 Hydroxyethyl-3,4-Methylenedioxyaniline HCl-Search Strategy Info Ingredient Cas No. Prev Rev Hydroxyethyl-3,4- Methylenedioxyaniline HCl X indicates data were available; - indicates no relevant data were available; *Online International Cosmetic Ingredient Dictionary and Handbook; ** indicates ingredients are in the Australian Inventory of Chemical Substances (AICS) and secondary notification conditions do not apply PubMed: Searched: hydroxyethyl-3,4-methylenedioxyaniline HCl (no hits); (no hits) ; 2-(1,3-benzodioxol-5-ylamino)ethanol hydrochloride (no hits); (hydroxyethyl-3[all Fields] AND and 4-methylenedioxyaniline[All Fields] AND and HCl[All Fields]) OR 2-[All Fields] AND 1[UID] AND (("ethanol"[mesh Terms] OR "ethanol"[all Fields]) AND hydrochloride[all Fields]) AND [All Fields] (no hits) alert for potential future articles matching the search terms above was setup ( ). SciFinder: in Use Info base * NTIS FDA/ CFR NTP TOXNET WHO ECHA IUCLID EPA/ HPVIS Searched: (6 hits/ 5 potentially useful); Hydroxyethyl-3,4-methylenedioxyaniline HCl (no hits); 2-(1,3-benzodioxol-5-ylamino)ethanol hydrochloride (no hits); N1-Hydroxyethyl-3,4-methylenedioxy aniline hydrochloride (no hits) Keep Me Posted (started ) for updates when new articles (using search term CAS # ) become available Searched: (free base form, Hydroxyethyl-3,4-Methylenedioxyaniline; 8 hits/0 potentially useful) Keep Me Posted (started ) for updates when new articles (using search term CAS# ) become available. ECHA Citations Searched: and 1 hit appeared for 2-(1,3-benzodioxol-5-ylamino)ethanol hydrochloride (which is another name for Hydroxyethyl-3,4- methylenedioxyaniline HCl) This ingredient is pre-registered on the ECHA website, but no registration dossier exists. The information that was available indicates that the ingredient may be a skin and/or respiratory sensitizer and causes serious eye irritation and may cause an allergic skin reaction. OECD/ SIDS EU NICNAS Web No Yes X X X X** X

7 and Searched for the ingredient using , 2-(1,3-benzodioxol-5-ylamino)ethanol hydrochloride, and Hydroxyethyl-3,4- methylenedioxyaniline HCl in NTIS, NTP, FDA, TOXNET, NICNAS, HPVIS/EPA, OECD/SIDS, WHO, COSING, EU, FEMA***, FAO***, RIFM***, and IARC*** websites (available, relevant information is indicated in the table above). The ingredient was not found to be used as a food additive, flavor, or fragrance. ***No relevant information for the ingredient was located on this website Searched for ingredient by CAS# and names above at ; the ingredient above did not appear on prescription medication labels or as an inactive ingredient in FDA approved drugs at

8 MINUTES FROM DECEMBER 2016 PANEL MEETING-HYDROXYETHYL-3,4- METHYLENEDIOXYANILINE HCL-(Day 1) DR. MARKS TEAM DR. MARKS: Okay. So I'm still going to move final report 40 ingredients as safe but then I'm going to immediately say Ron comment on two ingredients which you had concerns about. So you can voice that and see what the Belsito team does with it, and we'll go from there. Any other comments? Move on to the next ingredients. Okay. Let's see. What's next? The hydroxyethyl-3, 4-methylenedioxyaniline hydrochloride is a hair colorant. This is the first time. So let me see here. So this is a -- as I said, it's a draft report, so it's the first time we've seen this specific ingredient. Does everybody like the ingredient? You don't want to eliminate it? I don't think we can do that. And Ron, Tom, do you have any needs? It's a known sensitizer, but with the other hair dye ingredients, it's exempted with the pretesting. DR. HILL: I'm going to make a motion that we table this ingredient unless we can refute

9 135 what I'm about to say. I don't see in the search strategies that we actually did the free hydroxyethyl lanolin [aniline]. Everything seems to pertain to hydrochloride. As soon as you dump that hydrochloride into a solution, it's no longer the hydrochloride. And in fact, given the PK of that aniline, 99 plus percent of it will be the free aniline in any physiological test solution. And so we need all the toxicology for the hydroxyethyl lanolin [aniline] which has a different CAS number. And it looks like the way the searches were done might not have picked that up. DR. MARKS: Tom Slag, Ron Shank, comments about that? DR. SHANK: I don't follow what you're saying. DR. HILL: Everything was searched on the hydrochlorides but if you were doing a toxicology study on that, you would not do a toxicology study on that aniline HCL. You would do a toxicology study on that hydroxy-n. Everything --

10 136 DR. SHANK: In the lab, what do you actually take off? DR. HILL: It doesn't matter. When you reported the data, when you published the data, you wouldn't publish it as the hydrochloride; you would publish it as the aniline because it is that aniline. It's just a salt form of it. That's all. So if all the searches were done strictly on that hydrochloride, we've missed probably 95 percent of whatever has been published on that, at least. And it not necessarily, depending on the nature of the publication, have even that CAS number in there which definitely wouldn't pick up in some of these kinds of sources. DR. SLAGA: It's the first time we've seen it so I want clarification. DR. HILL: Well, I'm just saying that we potentially are missing 95 percent of the information we need to get the tox evaluation done in a valid way. DR. SHANK: Laura, this is your -- MS. SCOTT: Yes.

11 137 DR. SHANK: So do you remember how you searched? MS. SCOTT: Right. No, I searched with the CAS number, the and I searched for the salt because that's what the ingredient is. DR. SHANK: Salt? MS. SCOTT: The salt, the HCL. I didn't do any separate searches for any other versions or forms of it. So most of the data came from an SCCS report from So that's -- most of the data is from there. DR. SHANK: Okay. DR. HILL: And I'm sure they probably did capture on the free aniline, but I'm just asserting that that's a secondary source. MR. [DR.] BOYER: And you did a SciFinder search as well? MS. SCOTT: Yes. SciFinder and PubMed for the HCL, for the salt, with the CAS. MR.[DR.] BOYER: Okay. With SciFinder, I think SciFinder would have found it. DR. HILL: I think it won't if you only

12 138 search that CAS number and you only search it with the HCL. All these terms have hydrochloride or HCL in it. It won't find it. If you search by structure it will definitely find it and that's always the way I think it ought to be done with SciFinder in this kind of circumstance but I don't think you will have picked it up based on what I'm looking at. I mean, I use SciFinder every day all the time. I don't think if you search the way it's written in here you will have picked them up. MR.[DR.] BOYER: My understanding is with SciFinder, at least, you're searching for concepts, and there would be concepts within -- DR. HILL: No, not if you search it in quotes as you've got it. MS. SCOTT: I searched -- in SciFinder I searched the whole CAS number as it's written here. I don't recall if I actually put quotes on it or not. DR. HILL: No, with the CAS number it will just find that CAS number. MS. SCOTT: Right.

13 139 DR. HILL: It won't find alternative forms. It will just be that one. MR.[DR.] BOYER: Right. DR. HILL: It won't find the free aniline. MR.[DR.] BOYER: But the quotation, whether or not you search with the quotation marks -- DR. HILL: On the text-based search. On the text- based searches. MR.[DR.] BOYER: Right. DR. GILL: I was just going to say no mention of this in the SECS [SCCS] or the -- right, the SECS [SCCS] information at all? MS. SCOTT: No mention. DR. GILL: With the aniline being toxic? MS. SCOTT: The -- I'm sorry -- DR. GILL: I'm going back to Ron's question. MS. SCOTT: Okay. DR. GILL: You did not see anything related to that in the SECS [SCCS] discussion of this? MS. SCOTT: The only issue the SEC [SCC]

14 140 report -- SECS [SCCS] report mentioned was the nitrosation issue, and that's why they set a limit of less than 50 parts per billion. MS. FIUME: It does appear to be very well -- a lot of information in the report on the hydrochloride ingredient itself that was done in the SECS [SCCS] report, repro, genotox. DR. HILL: Just that we're only picking up things from that report, right? I mean, we wouldn't be picking up things that are coming from their cross-references. Are you looking directly at the cross-references in that report that pertain to this? Because I didn't get a clear sense of that. DR. EISENMANN: Most of it, I presume, is unpublished data that we've been trying to get, but this ingredient is tied up in a transfer from one company to another. DR. HILL: Yeah, but -- DR. EISENMANN: So we're trying to -- we're still trying to get it. Usually we can get these but because of the timing it's going -- it's

15 141 kind of in limbo, the original studies that are cited in the SECS [SCCS] opinion. DR. HILL: I was more disturbed by we don't have reaction schemes and kinetics, which you know how I feel about that but I feel like perhaps some of that might be available in the public domain literature if the search was done to expand that. And in fact, to the extent we do have anything absorbed, which if you use the hair dye according to strictly the instructions there won't be because it will never contact the skin, right? But if it's absorbed, it will definitely be converted to the hydroxyethyl group will come off of there as soon as it finds some P450 somewhere fairly quickly. And then you have a free aniline. DR. SHANK: When the developmental toxicity and reproductive toxicity studies were done, those were all oral studies. Wasn't the agent put into a solution in order to administer it so as you would say you'd lose the salt and -- DR. HILL: Yeah, sure. As soon as you

16 142 dissolve it it's not the hydrochloride anymore. DR. SHANK: Okay. And all of that data showed no problems, there were no observed adverse effect levels and that's an oral study which would be much more effective than skin application. DR. HILL: I dispute that claim because you've got a hydroxyethyl group and if it's in rodents you've got very aggressive first pass metabolizers and you wouldn't expect to get much of that substance into the skin. How high were the doses? DR. SHANK: DR. HILL: Right. Small. DR. SHANK: And that's an ingredient concentration. I don't know if that's applied -- if that's what is applied to the hair because this is mixed, is it not, as I recall? DR. HILL: Yeah, it's mixed and it should be consumed and it should be consumed fairly quickly. DR. SHANK: With a coupler and an oxidizer?

17 143 DR. MARKS: Right. Absolutely. Usually hydrogen peroxide is added. DR. SHANK: So the amount that actually gets to the scalp should be pretty small. DR. HILL: Yes, it should be zero if you used it according to strict guidelines, instructions on the package. DR. MARKS: Tom, are you worried about any tumor production by this in terms of skin contact? DR. SLAGA: No, it's going to be consumed timely. DR. MARKS: Yeah. DR. SLAGA: So I don't have any problem with it. DR. MARKS: We had the hair dye boilerplate we always put in. Ron, it sounds like you're satisfied with the oral, that you wouldn't be worried about systemic tox? DR. SLAGA: That's great. DR. MARKS: And we have taken care of the sensitizer so it sounds like we could issue a

18 144 tentative report with a safe conclusion. Ron Hill, do you want me to -- obviously, when we ask for a discussion you can go ahead and bring up your issues about the needing toxicity on the free aniline. DR. HILL: I was only questioning the thoroughness of the search strategy based on the way it was done. DR. MARKS: Well, why don't we -- we can certainly - - DR. HILL: And the lack of information. DR. MARKS: -- have that in -- we certain could get that. It will go out as a tentative report so we will see it again. DR. HILL: Okay. DR. MARKS: So I think we can move forward since it sounds like Ron Shank and Tom are okay with it. I'm okay with it with the exemption. I know it's a sensitizer but that's taken care of. Okay. And then -- DR. HILL: Wait.

19 145 DR. MARKS: Okay. DR. HILL: There's some information here on -- what is it? It's a compound that's not even an aniline. I'm trying to find the page number. Page 13. Yeah, that first deal under oral, oral toxicology animal, that's not the compound. It shouldn't be regarded functionally as an analog of that compound and the whole things needs to come out. DR. GILL: Are you talking the beta hydroxyl-3, 4- methylenedioxyaniline? DR. HILL: Yes, that needs to come out. That's not an analog. DR. SHANK: But the reference called it that. MS. SCOTT: Yes. The SECS [SCCS] report called it -- they used it for read-across but they didn't -- I don't believe they said why they used it. The rationale may not have been there. DR. HILL: That's okay because they couldn't come up with one if they tried. There is no valid rationale for using that as read-across

20 146 in this case. DR. SHANK: Okay. But it is in the literature. DR. HILL: Then put it in an appendix or a footnote or something but it's just -- that's goofy. DR. SHANK: Page 12. The bottom. DR. MARKS: I know what I -- are we settled with that? Where is the use? The number of uses and the concentration? Usually there's a table after the conclusion, and so I just want to be sure we're -- what the concentration of this being used is and how does that relate to the SEC [SCC] restriction? MS. SCOTT: So I didn't do a table because it was only one ingredient with when used. DR. MARKS: Okay. MS. SCOTT: But in the cosmetic use section, page 10. DR. MARKS: Page 10. MS. SCOTT: The first paragraph, the VCRP data from 2016 indicated 67 uses in hair dyes

21 147 and colors and the council industry survey lists deconcentrations [the concentrations] between 0.52 percent and 0.75 percent in hair dyes and colors. DR. MARKS: So it's higher than what the FCC [SCC] -- that's what I was getting at. I kind of had recalled that but I didn't know how to highlight it. Or no, it's not higher. DR. SHANK: That's before. MS. SCOTT: It's lower. Okay, I'm sorry. Yeah, so that's not an issue. DR. SHANK: And that's before a coupler and oxidizer -- DR. MARKS: Yes. Yes. I assume that's what they -- okay. Fine. Any other comments? If not, then I assume I'll be seconding a tentative report with safe conclusion tomorrow and we'll find out if there are any literature search on the toxicity of the free anilines. And Ron Hill, obviously, go ahead and comment tomorrow if you feel like it so that the other team is -- DR. HILL: As long we, yeah, I mean, as 148 long as we go -- I'd rather not.

22 DR. MARKS: Okay. Yeah. I'll just put it in my notes -- DR. HILL: I think Laura and Ivan can make sure everybody knows what the issue is but I will make a comment about we need to get this one compound out of there. DR. MARKS: Okay. DR. HILL: We'll see if others concur with me. Reading across from something that's just a benzene to an analyn [aniline] is craziness in this context. MS. SCOTT: For the discussion is there anything specific besides whatever is in the boilerplate that would normally go into a hair dye report? DR. MARKS: No, I just mentioned the FCC [SCC], the restriction that already exists that you have in your memo. MS. SCOTT: Okay. DR. MARKS: I think that's important to put in the discussion.

23 149 DR. HILL: I still would like to see, I mean, it looks like maybe this is used since it's in 67 different things, it probably doesn't always use the same coupler and always use the same oxidant, but we have information that it's sometimes used as coupler and oxidant. So I don't know why we can't get some slice of the chemistry that goes on here. I'm thinking somebody knows something but if it's tied up transferring between two companies and there's no public domain but I thought perhaps there might be public domain if we do the search more expansively. MS. SCOTT: I can check and see. I didn't see anything with the original searching. DR. HILL: So in those SCCS reports, they do have their cross references listed that they used to make the conclusion? MS. SCOTT: In the report themselves. DR. HILL: I seem to have trouble getting these reports sometimes. MS. SCOTT: Oh, I can you a copy --

24 150 DR. HILL: Okay. MS. SCOTT: -- if you like. DR. HILL: Okay, great. MS. SCOTT: Yes, all of the sources that are in there from the SCCS report are listed, but as I think Carol mentioned, most -- a lot of it is industry data so, I mean, we wouldn't have -- I don't think we would have access to that easily. DR. HILL: Okay. Yeah, if it's just coming from an industry -- DR. EISENMANN: We're trying but -- DR. HILL: Okay. Well, if we can get more -- MS. SCOTT: I can you a copy of the report that I used if that would be helpful to you. DR. HILL: That's -- and maybe your link will take me there and just for some reason where I was sitting at the time didn't work. MS. SCOTT: I'm not sure but -- DR. HILL: Okay. If you've got it in electrons.

25 151 MS. SCOTT: I can, yes. DR. HILL: As long as you're not you can do that, right, not getting in trouble with copyright? MS. SCOTT: I found it on the Internet. DR. HILL: Okay. You found it out there. Okay, great. MS. SCOTT: Yeah, this was something I found for the European Union. DR. HILL: I know you can't post it on the website. MS. SCOTT: Through the European Union. DR. HILL: Great. Okay, great. MS. SCOTT: Okay. DR. MARKS: Thank you, Laura. MS. SCOTT: You're welcome. DR. MARKS: Any other comments? If not we'll move on to the next ingredients. MINUTES FROM DECEMBER 2016 PANEL MEETING-HYDROXYETHYL-3,4- METHYLENEDIOXYANILINE HCL-(Day 1) DR. BELSITO S TEAM DR. BELSITO: Okay. Anything else? No? Very good Laura. You're doing a great job. Quick learner. Okay. Hydroxyethyl-3 4-methyledioxy aniline [Hydroxyethyl-3,4-Methylenedioxyaniline]. First time safety assessment of this hair dye and amine salt containing a free

26 secondary aromatic substituted amine or aniline derivative. According to the SCCS, the ingredient, it was referred to as both a precursor and a coupler. And they limit it to 1.5 percent on the head following mixing. Our maximum concentration is So, we're not in any conflict with them there. And I wondered whether nitrosation was a concern, just as a question out to Dan. The SCCS made mention of nitrosation because of the secondary amine and the primary amine in the aniline impurity and limited it to 50 ppb. No, that's fine. DR. LIEBLER: I'll deal with yours in a second. DR. BELSITO: Nitrosation, page 2 of the PDF. DR. LIEBLER: Yeah. So, page 9 of the PDF, Impurities and nitrosation? DR. BELSITO: Two. DR. LIEBLER: Two? Oh, the memo? DR. BELSITO: Yeah. The SCCS opinion. DR. LIEBLER: Right. Okay. DR. BELSITO: And page 9 has the impurities. DR. LIEBLER: So, it's a secondary amine. And so, there's a lengthy section -- relatively lengthy on impurities and nitrosation. And I commented on this, and I said that I think the point about nitrous amines [nitrosamines] is worth making,

27 but the scheme with the analog and the extended text is unnecessary. I think we have applicable boilerplate for possible nitrous amine formation. So, I think it's reasonable to acknowledge the possibility, but we don't need to devote as much as there is in this version. DR. BELSITO: Okay. DR. LIEBLER: So, you don't need that structure to illustrate nitrosation with another compound. DR. BELSITO: So, you would include all the verbiage, just get rid of the structure. Is that what I'm hearing? DR. LIEBLER: Actually, what I would do is -- DR. SNYDER: I mean we basically have our own boilerplate -- DR. LIEBLER: Yeah. DR. SNYDER: -- which states what the SCC report stated. DR. LIEBLER: Yeah, right. And you could substitute that for that second paragraph on nitrosation. DR. HELDRETH: Yeah, most of that second paragraph is our boilerplate -- DR. LIEBLER: Okay. DR. HELDRETH: -- until you get down to the sentence that starts with a recent study.

28 DR. LIEBLER: Yeah. DR. HELDRETH: So, we could just delete the -- DR. LIEBLER: Yeah. DR. HELDRETH: -- a recent study -- DR. LIEBLER: Right. Okay. From there on, I'm deleting that here. Okay. And then the figure goes. And so, yes, it is a secondary aromatic substituted amine. DR. SNYDER: But then down below it says that that's the primary -- it says it's a primary. So, is that amine in parentheses below? DR. HELDRETH: Oh, the other one where it says a primary -- DR. SNYDER: Yeah. DR. HELDRETH: -- it's talking about an impurity - - DR. SNYDER: Oh, it's -- DR. HELDRETH: -- not the ingredient itself. DR. SNYDER: Not the ingredient. Okay. So, the impurity 3-methylendioxy aniline. Okay. Okay. DR. HELDRETH: Should we then keep the last sentence of that second paragraph of impurities where we say consequently, hereby formulations containing this, yada, yada, should be free of nitrous-aiding [nitrosating] agents. DR. LIEBLER: Yeah. DR. BELSITO: Okay.

29 DR. LIEBLER: And then under Use -- under Noncosmetic Uses, those really aren't noncosmetic uses. The Australian classification and labeling, it's not really about uses. So, if I move that. And I don't know if they're any noncosmetic uses. MS. SCOTT: That was all I could find. DR. LIEBLER: Yeah. Okay, so that's -- MS. SCOTT: And they're not really applicable. DR. LIEBLER: -- just might be that none were identified. MS. SCOTT: Okay. I can change that. DR. LIEBLER: And then one other point I just wanted to make, Laura, about PDF 5 where you list the data types that are available, and you actually have in the table, instead of just yes, no or check or not check or whatever, you actually have, the number of studies for that type. I haven't seen that before in our reports, I don't think. And I think it's a great idea. So, anyway, yeah, I would like to suggest that we do that for other representations of the data because it's a really good way to eyeball, not only what data we have but how deep it is. DR. BELSITO: Which table, Dan? DR. LIEBLER: It was on PDF page 5. DR. BELSITO: Oh. DR. LIEBLER: It's the -- DR. BELSITO: It's not a table.

30 DR. LIEBLER: No, it's a representation of the data, summary of the data that were found. DR. BELSITO: Yeah. Well, we usually get tick boxes that have that in them. DR. LIEBLER: This is better because it's got the numbers of the studies in it -- DR. BELSITO: Numbers of studies. I see, you're saying the depth -- DR. SNYDER: More information -- DR. BELSITO: Yeah. DR. SNYDER: -- rather than just a plus minus. DR. BELSITO: Okay. DR. LIEBLER: It tells you more -- DR. BELSITO: Okay. DR. LIEBLER: -- at a glance, which is nice. DR. BELSITO: Yeah. Glances are always nice. DR. SNYDER: I think Laura was (inaudible). DR. BELSITO: Yes. DR. LIEBLER: That's right. DR. BELSITO: Anything else on this? DR. LIEBLER: So, you're satisfied with the sensitization data? DR. BELSITO: It's a hair dye -- DR. LIEBLER: Oh --

31 DR. BELSITO: -- just needs labeler. DR. SNYDER: It's a sensitizer. DR. BELSITO: Yeah. DR. LIEBLER: Yeah, it is a sensitizer. DR. BELSITO: Yeah, but it's -- so is (11:13:33 NPPD?). So, I have the -- DR. LIEBLER: So, it's safe as used. DR. BELSITO: Yeah, with the usual hair dye labeling. DR. LIEBLER: Yeah, okay. Got it. DR. BERGFELD: Are you going to put that in your discussion? DR. BELSITO: Yeah, I mean it's a hair dye, it's going to have all the usual hair dye boilerplates with it. DR. BERGFELD: Is there anything else besides the usual boilerplate for the discussion? DR. BELSITO: No, I mean we've dealt with the nitrosation, right? So, that's not a part of the discussion at all. DR. BERGFELD. Okay. DR. BELSITO: I mean unless you felt that you wanted to point out that there was some concern about an impurity that doesn't, in fact, does not exist in this material, that's giving it too much emphasis. So, no, just the usual hair dye. DR. LIEBLER: That's fine.

32 DR. BELSITO: So, we're getting rid of nitrosation in the discussion and with just the usual hair dye. Okay. 249 MINUTES FROM DECEMBER 2016 PANEL MEETING-HYDROXYETHYL-3,4- METHYLENEDIOXYANILINE HCL-(Day 2) DR. BERGFELD: Okay. Thank you. That will be recorded in the minutes. Okay, so we're moving forward to reports advancing to the next level. We have a hair dye, Dr. Belsito, the Aniline dye. DR. BELSITO: So this is the first time we're looking at this, Hydroxyethyl-3,4-Methylenedioxyaniline Hydrochloride. Sixty-seven reported uses in hair dyes and colors, all of them requiring an cautionary statement regarding patch testing in uses at 0.52 to 0.75 percent. And my team felt that we could go ahead with a safe as used conclusion for this hair dye. DR. MARKS: Second DR. BERGFELD: Any further discussion regarding this hair dye? DR. BELSITO: The discussion needs to be developed, but it's the usual hair dye discussion and that brought up the discussion of updating the hair dye boilerplate. DR. BERGFELD: Ron Hill. DR. HILL: I raised the concern that I had great doubts that the search strategy as written picked up a good bit of data of the aniline itself because it looks like all of the

33 searching was done one the hydrochloride solvent. I don't know if staff made any further efforts to look and see how exhaustive our, I mean we were using a secondary source, I believe the SSCS review. So, we're getting everything arguably second hand. And so I'm going to abstain until I get the chance to find out if we can get additional information. Bart, would you like to comment? DR. HELDRETH: So, I believe that Laura's search strategy did actually cover the free-base, the aniline. But we did a quick check yesterday with SciFinder, compared the results, looking at the free-base and the HCL acid, and eliminating those that were in both camps. And a few patents came up, but no relevant safety data came up, or method of manufacture. There were some bench chemistry synthetic methods that you would possibly make this, but they don't relate to cosmetic method of manufacture. So although there were a few patents, there was nothing relevant to safety. DR. HLIL: And the other thing I raised is, I was completely disgruntled that we don't have information about the chemistry of what goes on in the hair dyeing process to get to the final product, in terms of kinetics and intermediates. And I feel like I'm not happy with that. DR. HELDRETH: We did search the literature for that and did not find it. DR. HILL: Did not find it. I realize that this is

34 in, what 67, is it 67 different reported formulations, and that those chemistries might vary, depending on what else is in there. And we were also informed yesterday that the ingredient is in the process of being transferred from one company to another, which is making it more difficult to get certain kinds of information. But, I feel like the kind of information we should get, I mean this under the Coal Tar exemption, and we know that going in. So from that point of view, and with that mindset, I don't think there are any new issues we can raise. But I believe that, in this day and age, the reporting the kinds of chemistry that happens, I don't know how much proprietary issue there might be, but don't they have to list the couplers and the oxidants on their label. So, I'm not sure what they would be giving away by providing that information. Maybe I'm missing something. My raised concerns, that we haven't fully addressed, but it's under the Coal Tar exemption, it's supposed to not be contacting the scalp at all really, or no more than transiently. But, I feel like we should be getting that kind of information. DR. BERGFELD: Bart, do you want to respond? DR. HELDRETH: Sure. In the process for using these hair dyes, you have to remember that this isn't necessarily a separate package that has labels on it. If it's used in the salon, it doesn't necessarily require the same labeling. And, like as you mentioned, you know, you're gonna have a coupler,

35 you're gonna have the developer, you're gonna have the different parts of the acidized or paradized solution. And those could be different. So, yes, we don't know which ones they're being mixed with, but that's certainly something that can be added here with this ingredient. DR. BELSITO: The salon won't have labeling, per se, but it will have material safety data sheets. DR. HELDRETH: Yes DR. BELSITO: All of the ingredients will not necessarily be captured, but any hazards will be identified. DR. HILL: So that would be salon use, but I'm assuming this is also probably available in home use. DR. BELSITO: Then it would have to be completely labeled. And you see it. It comes in parts, and there's one part that's labeled and then there's the developer part that's labeled. DR. HILL: It's been several years since I glanced at one of my wife's products, but. DR. BERGFELD: Does that satisfy you, Ron Hill, or not? DR. HILL: I'm just making a statement DR. BERGFELD: Okay DR. HILL: I'm not fully satisfied with the information here.

36 DR. BERGFELD: All right. Any other discussion? Yes, go ahead, Laura. MS. SCOTT: One question on read across. There's one study from the SSCS report, an oral tox study, which refers to one Beta Hydroxyethyl-3,4-Methylenedioxyaniline Benzene, and it's actually a read across ingredient. Is that something you would like to have stay in the report, or removed? DR. HILL: I wanted it removed. Just to let you know. I thought that was highly inappropriate read across. DR. LIEBLER: I think that the only time we would use, we should be really thinking about read across is if we have a clear data gap that's not satisfied by data from the ingredient we're reviewing. So, I don't know that it's necessary. DR. BERGFELD: So the consensus is to remove? DR. LIEBLER: Yeah DR. SHANK: Yes DR. BERGFELD: Any other questions, or comments? If not, I'm going to call the question. Dr. Belsito, do you want to re-state your conclusion? DR. BELSITO: Safe as used. DR. BERGFELD: Safe as used, has been second. All those in favor, please indicate by raising your hand. Opposed? One. Okay. Thank you. DR. LIEBLER: Wilma, I'd like to offer just one

37 comment. DR. BERGFELD: Sure DR. LIEBLER: In the preamble to the report on PDF page 5, there's a table that lists the data sources, kinda at a glance. And this is something that I think Laura did that I really like, which is adding the numbers of studies. So that you can really capture, you know, at a glance, what was done. And I just wanted to encourage the maybe broader adoption of that. It's a nice way to get a better depth of representation at a quick glance at what you've got coming in the report. DR. BERGFELD: Thank you. DR. HILL: I also really liked that. DR. BERGFELD: Yes. Okay. I think that that's going to happen. That's gonna happen. Good. Moving on to the next ingredient, Dr. Marks, the Acid Esters, Dialkyl Acid Esters.

38 Safety Assessment of Hydroxyethyl-3,4-Methylenedioxyaniline HCl as Used in Cosmetics Status: Draft Final Report for Panel Review Release Date: May 19, 2017 Panel Meeting Date: June 12-13, 2017 The 2017 Cosmetic Ingredient Review Expert Panel members are: Chair, Wilma F. Bergfeld, M.D., F.A.C.P.; Donald V. Belsito, M.D.; Ronald A. Hill, Ph.D.; Curtis D. Klaassen, Ph.D.; Daniel C. Liebler, 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 Lillian J. Gill, D.P.A. This safety assessment was prepared by Laura N. Scott, Scientific Writer/Analyst. Cosmetic Ingredient Review 1620 L Street, NW, Suite 1200 Washington, DC ph fax cirinfo@cir-safety.org

39 ABSTRACT The Cosmetic Ingredient Review (CIR) Expert Panel (the Panel) reviewed the safety of Hydroxyethyl-3,4-Methylenedioxyaniline HCl, which functions as a hair dye ingredient. The Panel reviewed relevant data provided in this safety assessment, and concluded that Hydroxyethyl-3,4-Methylenedioxyaniline HCl is safe for use as a hair dye ingredient in the present practices of use and concentrations described in this report. The Expert Panel cautions that ingredients should not be used in cosmetic products in which N-nitroso compounds can be formed. INTRODUCTION This assessment reviews the safety of Hydroxyethyl-3,4-Methylenedioxyaniline HCl. According to the International Cosmetic Ingredient Dictionary and Handbook (Dictionary) this cosmetic ingredient functions as a hair colorant. 1 The majority of the data included in this safety assessment were gathered from a report titled European Commission Scientific Committee on Consumer Safety (SCCS) Opinion on Hydroxyethyl-3,4-Methylenedioxyaniline HCl, 2 the European Union s website (EUROPA), 2-4 and the Australian Government Department of Health National Industrial Chemicals Notification and Assessment Scheme (NICNAS) website. 5 The above references provide summaries of data generated by industry, and it is those summary data that are reported in this safety assessment when the references above are cited. CHEMISTRY Definition and Structure Hydroxyethyl-3,4-Methylenedioxyaniline HCl (CAS# ) is the bicyclic, ammonium salt that conforms to the structure in Figure 1. 1 Figure 1. Hydroxyethyl-3,4-Methylenedioxyaniline HCl Hydroxyethyl-3,4-Methylenedioxyaniline HCl is used as an oxidative hair coloring precursor ingredient. 2,6 In a 2009 SCCS report, an industry submission indicated that this oxidative coloring precursor and a developer (hydrogen peroxide) were mixed at ratios ranging from 1:1 to 1:3. 2 However, Hydroxyethyl-3,4-Methylenedioxyaniline HCl was also listed as a coupler in a different SCCS report from No reaction scheme was found in the literature. In a typical formulation, a precursor is activated via an oxidant, such as hydrogen peroxide. The resultant activated precursor proceeds to react with a coupler to form in-situ a product that is purported to be the actual dye that colors hair in this type of oxidative hair dye products. Physical and Chemical Properties Hydroxyethyl-3,4-Methylenedioxyaniline HCl is a beige crystal. 2 It is soluble in water, acetone/water, dimethyl sulfoxide (DMSO), and ethanol (Table 1). Method of Manufacture There were no method of manufacture data identified in the literature for Hydroxyethyl-3,4-Methylenedioxyaniline HCl. Impurities and Nitrosation Impurities that may be present in Hydroxyethyl-3,4-Methylenedioxyaniline HCl include 3,4-methylenedioxy-aniline (114 to 1097 ppm in 5 batches tested), 1,3-benzodioxol (below limit of detection [35 ppm] in 5 batches tested), and 1,2-methylenedioxy-4- nitrobenzene (below limit of detection [8 ppm] in 5 batches tested). 2 The following residual solvents were not identified at a 100 ppm detection limit: methanol, ethanol, isopropanol, acetone, ethyl acetate, cyclohexane, methyl ethyl ketone, and monochlorobenzene. Hydroxyethyl-3,4-Methylenedioxyaniline HCl contains a free, secondary aromatic substituted amine group (aniline derivative), thus raising a concern about potential N-nitrosation. A concern in cosmetics is the conversion (nitrosation) of nitrogen-containing ingredients, such as hair dyes, into N-nitroso chemicals that may be carcinogenic. In one study, 85% of the approximately 209

40 nitrosamines tested were 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 NO 2 may also participate in nitrosation in aqueous solution. 9 Consequently, hair dye formulations containing Hydroxyethyl-3,4- Methylenedioxyaniline HCl, and formulations intended for admixture with this ingredient (e.g., to generate an active hair dye), should be free of nitrosating agents. Furthermore, nitrosamines may be present in a cosmetic as an impurity of an ingredient. The Panel advised that manufacturers may avoid these issues by formulating ingredients in a way that reduces the formation of nitrosamines, and by eliminating the presence of impurities that are N-nitrosated or contain nitrosating agents. In an SCCS report, concern was raised about nitrosation of the impurity 3,4-methylenedioxy-aniline (a primary aromatic amine) that may occur in Hydroxyethyl-3,4-Methylenedioxyaniline HCl. 2 The total content of N-nitroso compounds for Hydroxyethyl-3,4- Methylenedioxyaniline HCl was determined to be < 10 µg/kg (ppb) in 3 batches tested; the SCCS concluded that nitrosamine content should be limited to < 50 ppb. The SCCS determined that Hydroxyethyl-3,4-Methylenedioxyaniline HCl should not be used in combination with nitrosating substances. 2 USE Cosmetic The CIR Panel evaluates the safety of the cosmetic ingredient included in this assessment based on the expected use of, and potential exposure to, the ingredient in cosmetics. The data received from the United States (US) Food and Drug Administration (FDA) are collected from manufacturers through the FDA Voluntary Cosmetic Registration Program (VCRP), and include the use of individual ingredients in cosmetics by cosmetic product category. The data received from the cosmetic industry are collected by the Personal Care Products Council (Council) in response to a survey of the maximum reported use concentrations by product category. VCRP data obtained from the FDA in 2017 indicate that Hydroxyethyl-3,4-Methylenedioxyaniline HCl has 67 reported uses in hair dyes and colors (all requiring caution statements and patch tests). 10 The 2016 Council industry survey indicated that Hydroxyethyl-3,4- Methylenedioxyaniline HCl was reported to be used at maximum concentrations of 0.52% to 0.75% (diluted or on head concentrations) in hair dyes and colors. 11 Hydroxyethyl-3,4-Methylenedioxyaniline HCl is considered to be a coal tar hair dye for which regulations require caution statements and instructions for patch testing to exempt the dye from adulteration and color additive provisions of the US Federal Food, Drug, and Cosmetics Act. To be exempt, the following caution statement must be displayed on all coal tar hair dye products: Caution- this product contains ingredients which may cause skin irritation on certain individuals and a preliminary test according to accompanying directions should be made. This product must not be used for dyeing the eyelashes or eyebrows; to do so may cause blindness. Product labels shall also bear patch test instructions for determining whether the product causes skin irritation. The CIR Expert Panel recommends, for products containing this type of ingredient, that an open patch test be applied and evaluated by the beautician and/or consumer for sensitization 48 hours after application of the test material and prior to the use of a hair dye formulation. In 2012, a report was published about such self-testing for contact sensitization to hair dyes. 12 The authors concluded that, in its present form, the hair dye self-test has severe limitations. The authors issued the warning that, if the use of a hair dye self-test to predict contact sensitization becomes widespread, there is severe risk that a tool has been marketed that may cause morbidity in European consumers. An accompanying editorial performed on behalf of the European Society of Contact Dermatitis (ESCD) asserted that industry is focusing on predicting the risks from exposure to hair dyes by having millions of European consumers perform a self-test prior to each hair dying and stated that it is the opinion of the ESCD that attention must be given to reducing the risks of serious allergic reactions by improving the safety of the products themselves. 13 The opinion issued by the European Commission SCCS on Hydroxyethyl-3,4-Methylenedioxyaniline HCl in 2009 expressed that Hydroxyethyl-3,4-Methylenedioxyaniline HCl is not considered a health risk to the consumer when the hair dye is used at a maximum concentration of 1.5% on the head after mixing under oxidative conditions. 2,6 The SCCS also concluded that Hydroxyethyl-3,4- Methylenedioxyaniline HCl has the potential to be a strong sensitizer. Additionally, the SCCS determined that genotoxicity/mutagenicity studies in finished hair dyes should be conducted in accordance with Scientific Committee on Consumer Products and Non-Food Products Intended for Consumers (SCCNFP/SCCP) opinions and Notes of Guidance. According to the European Commission, Hydroxyethyl-3,4-Methylenedioxyaniline HCl is allowed for restricted cosmetic use as regulated in the Annex III List of Substances which Cosmetic Products Must Not Contain Except Subject to the Restrictions Laid Down (European Union Reference #246), as stated above. 6 The European Commission stipulates that oxidative hair dye ingredients, including Hydroxyethyl-3,4-Methylenedioxyaniline HCl, must be labeled as such: The mixing ratio must be printed on the label. Hair colorants can cause severe allergic reactions. Read and follow instructions. This product is not intended for use on persons under the age of 16. Temporary black henna tattoos may increase your risk of allergy. Do not color your hair if you have a rash on your face or sensitive, irritated and

41 damaged scalp, you have ever experienced any reaction after coloring your hair, you have ever experienced any reaction to a temporary black henna tattoo in the past. 6 A Swedish market analysis was conducted on 122 oxidative hair dye products sold at typical retailers (i.e., grocery stores, beauty shops, hairdressing salons). 14 The hair dye products evaluated were marketed in Europe and internationally. Shades from light to dark were represented in this evaluation. Information about the ingredients comprising the hair dye formulations was gathered from the product label and the European Commission cosmetic ingredient database (COSING); no chemical testing was performed in this study. Of the 122 hair dye formulations examined, 120 contained ingredients known to be potent skin sensitizers. Notably, more than 80% of the hair dye products contained at least 4 potent skin sensitizers. The range in the numbers of potent skin sensitizers was 0 to 8 in light blonde shades, 2 to 12 in dark brown colors, and 3 to 7 in black dyes. Hydroxyethyl-3,4-Methylenedioxyaniline HCl was one of the 37 target hair dyes identified by the researchers conducting the analysis to be a potent skin sensitizer. It was reported to be used at the maximum authorized concentration of 3% or, when combined with hydrogen peroxide, at 1.5%, as determined by the Cosmetics Directive 76/768/EEC, consolidated version No additional information specific to Hydroxyethyl-3,4- Methylenedioxyaniline HCl was provided in this analysis. In Australia, there are no known restrictions for Hydroxyethyl-3,4-Methylenedioxyaniline HCl as reported by NICNAS, although it is recommended that consumers use products containing the ingredient as instructed on the label; the assessment suggested control measures for industry to minimize risk associated with dermal exposure to Hydroxyethyl-3,4-Methylenedioxyaniline HCl. 5 The NICNAS assessment mentions a recommendation, to be included in Schedule 6 of the Poisons Standard 2015-Standard for the Uniform Scheduling of Medicines and Poisons, that the concentration of use should be appropriately limited for use in hair dye products. Non-Cosmetic There was no relevant, non-cosmetic use information found in the literature. TOXICOKINETIC STUDIES Dermal Penetration In Vitro A percutaneous absorption study was conducted in accordance with Organization for Economic Co-operation and Development Test Guideline (OECD TG) 428 and Good Laboratory Practice (GLP). 2 Four-hundred mg of an oxidative hair dye formulation that consisted of 1.5% Hydroxyethyl-3,4-Methylenedioxyaniline HCl, 1.75 mg of an unspecified reaction partner, and 3% hydrogen peroxide were applied to a 4 cm 2 sample of Schweizer Edelschwein porcine skin in a diffusion Teflon-chamber. This resulted in a dermal exposure of 1.5 mg/cm 2 to 1.5% Hydroxyethyl-3,4-Methylenedioxyaniline HCl. A phosphate buffer containing sodium chloride and antibiotics was pumped through the receptor compartment at 5 ml/h. At 60 min post-application, the test site was washed twice with water (4 ml) followed by a shampoo formulation (4 ml) and then twice again with water. The portion of dye in the washing solutions was quantitatively determined by high-performance liquid chromatography (HPLC). At 16 and 24 hours post-application, fractions of receptor compartment fluid were collected, concentrated and analyzed. When the experiment was completed, the skin above the basal layer was mechanically separated from the basal layer skin down to the upper dermis by heat treatment. These skin compartments were analyzed and the amount of dye present was quantitated by HPLC. Test results concerning the integrity of the skin (using tritiated water) indicated that penetration of the applied amount ranged from 0.9% to 1.4%. 2 The total recovery of the applied amount of Hydroxyethyl-3,4-Methylenedioxyaniline HCl was 19.8%. The SCCS report states that the low recovery may have been caused in part by the reaction partner. The amount of Hydroxyethyl-3,4-Methylenedioxyaniline HCl that remained on the skin surface was 19.5% of the applied amount. By 24 hours the applied amount recovered was 0.047% in the upper skin, % in the lower skin, and 0.3% in the receptor compartment fluid. The skin penetration rate (i.e., the bioavailable amount, calculated by adding the portions in the skin compartments and the receptor fluid) was determined to be 5.8 µg/cm 2 during the 24-hour exposure duration. Animal A GLP study evaluating the percutaneous absorption of 14 C-Hydroxyethyl-3,4-Methylenedioxyaniline HCl (ring-labelled) was conducted in Sprague Dawley (Him:OFA, SPF) rats (n=3/sex/concentration). 2 The test substance ( 14 C-Hydroxyethyl-3,4- Methylenedioxyaniline HCl) was applied to a 9 to12 cm 2 area of skin clipped free of hair in anesthetized animals as follows: 3.33% test substance in a water solution; 1% test substance in a formulation with hydrogen peroxide (also containing p-toluenediamine, resorcinol, and m-aminophenol in this application); 1% test substance in a formulation without hydrogen peroxide. At 30 minutes post-application the test substance was removed and the skin washed with a shampoo formulation (100 ml) and warm water until no color was seen in the rinse solution. The skin was then covered with a gauze patch that was secured in place with adhesive tape (animals were fitted with an air-permeable plastic cone to prevent them from licking the treatment area), and the animals were placed in metabolism cages for 72 h (see Table 2 for metabolism results). Animals were killed 72 hours after application of the test substance. Results indicated that the washing solutions contained 95.9% to 97.1% of the applied radiolabeled concentration. The skin

42 contained 1.68% of the applied radioactivity from the formulation containing water, 0.56% from the formulation containing hydrogen peroxide, and 0.34% from the formulation without hydrogen peroxide. Absorption, Distribution, Metabolism, Excretion Absorption, distribution, metabolism, and excretion (ADME) studies are summarized below; details are presented in Table 2. Studies were conducted to evaluate the ADME of Hydroxyethyl-3,4-Methylenedioxyaniline HCl via dermal and oral exposure routes. Experiments were performed in which 1% to 3% 14 C-Hydroxyethyl-3,4-Methylenedioxyaniline HCl (ring-labeled) was applied to rat skin (clipped free of hair) for 30 minutes and then removed by washing. 2 The applied radioactivity was eliminated mainly in urine and to a lesser extent in feces; 0.05% to 0.345% of the applied radiolabel was recovered in urine and feces (combined) by 72 hours post-application. Dermal absorption ranged from 0.59 µg/cm 2 to 14.8 µg/cm 2 and no substantial bioaccumulation was observed. The biological half-life of 14 C-Hydroxyethyl-3,4-Methylenedioxyaniline HCl was determined from the blood analysis to be 1 hour. Experiments evaluating the effects of 14 C-Hydroxyethyl-3,4-Methylenedioxyaniline HCl (ring-labeled) in orally exposed rats (1 to 100 mg/kg single dosage) yielded results indicating that > 95% of the administered radioactivity was absorbed. 2,15 Blood, urine, and feces samples were collected up to 24 to 96 hours post-dosing. The radioactivity detected in urine and feces were 78% and 14%, respectively, of the administered radioactivity; 15 bioaccumulation was not observed. 2 Blood analysis showed the half-life of the test substance to be 1.5 hours. 2 The highest blood concentration detected following oral exposure was 70-fold greater than the highest blood concentration after dermal exposure. Animal Oral TOXICOLOGICAL STUDIES Subchronic Toxicity A 90-day study conducted in accordance with OECD TG 408 and GLP in Wistar HanBrl:WIST (SPF) rats (n=10/sex/dosage) was performed to evaluate the effects of Hydroxyethyl-3,4-Methylenedioxyaniline HCl (99.8% purity) following daily oral exposure, by gavage, at 0, 20, 100, and 350 mg/kg/day using a purified water vehicle. 2 One male died (350 mg/kg/day) on day 85; the SCCS report, in which the study was presented, indicated that the study researchers thought the death was unlikely to be treatment-related because no clinical symptoms were reported. In the 100 and 350 mg/kg/day groups, both sexes showed reduced locomotion. Treatment-related increases in mean absolute and relative reticulocyte counts were noted in males at 350 mg/kg/day after 13 weeks, and this effect was associated with splenic extramedullary hematopoiesis found during histopathology. Reduced red blood cell count and hematocrit and hemoglobin measurements, as well as elevated mean absolute and relative reticulocyte counts, were observed in females (350 mg/kg/day). There was a decrease in thymus weights, an increase in ovary and spleen weights, extramedullary hematopoiesis in the spleen, and corpus luteum hypertrophy in the ovaries of females (350 mg/kg/day). Elevated clinical biochemistry parameters, increased liver and kidney weights, increased urine volume, hepatocellular hypertrophy, and renal tubular damage were reported in both sexes (350 mg/kg/day). At the 100 mg/kg/day dosage rate, bilirubin and phospholipid levels (clinical biochemistry parameters) were elevated in females, increases in absolute and relative liver weights and hepatocellular hypertrophy were noted in males, and increases in blood cholesterol and urinary volume occurred in both sexes. A no-observed-adverse-effect level (NOAEL) of 20 mg/kg/day was reported. DEVELOPMENTAL AND REPRODUCTIVE TOXICITY (DART) STUDIES A teratogenicity study was conducted in accordance with OECD TG 414 and GLP to evaluate Hydroxyethyl-3,4-Methylenedioxyaniline HCl (purity 99.8%) in rats (HanBrl: WIST, SPF quality). 2 The test substance in bi-distilled water was administered by gavage to pregnant rats (n=22/ group) on days 6 to 20 of gestation at 0, 50, 250, and 1000 mg/kg/day. All animals were killed on day 21 of gestation and necropsies were performed. Instability of the test solution was reported in this study (assumed to have been caused by incorrect sample handling contributing to oxidation of the test substance), although previous experiments showed Hydroxyethyl-3,4-Methylenedioxyaniline HCl to be stable in water at room temperature for 7 days. 2 Therefore, dosage rates were re-calculated based on analytical results to be 26, 152, and 702 mg/kg/day. Following the first 3 days of dosing (day 9 to day 20 of gestation) the high dosage rate of 702 mg/kg/day was reduced to 688 mg/kg/day because of severe toxicity and mortality (5 animals died or were killed because of moribund conditions). 2 In the 688 mg/kg/day group, hypoactivity, hunched posture, lacrimation, and behavioral indications of discomfort were observed. Discomfort was also seen at 152 mg/kg/day. With 152 mg/kg/day and 688 mg/kg/day, reduced food consumption and body weight were noted. At the same dosage rates, severe stomach abnormalities and effects on adrenals, spleen, and kidneys were observed. An increase in fetal resorptions occurred with the 688 mg/kg/day group only, compared to controls. The 688 mg/kg/day treatment resulted in effects on litter size and fetal body weight (no further details provided), but not on the sex ratio of the fetuses. In the 152 and 688 mg/kg/day groups, skeletal and visceral abnormalities were seen (at 688 mg/kg/day: hemorrhages, dilated lateral brain ventricles, thymus, cranial

43 displacement and/or elongation; at 688 and 152 mg/kg/day: heart, aorta and pulmonary effects). At 152 mg/kg/day and 688 mg/kg/day, there was an increase in non-ossified and incompletely ossified bones of the cranium, vertebrae, ribs, sternebrae, and also of extremities and supernumerary ribs. Treatment-related effects were seen on cervical and thoracic vertebrae and on costal cartilage in the 152 mg/kg/day and 688 mg/kg/day groups. The SCCS report states that the severe reproductive and developmental effects at the 688 mg/kg/day and 152 mg/kg/day dosage rates were caused by maternal toxicity. 2 A reproductive and developmental NOAEL of 26 mg/kg/day was reported. GENOTOXICITY STUDIES Provided below is a summary of genotoxicity studies that are presented in detail in Table 3. Experiments examining Hydroxyethyl-3,4-Methylenedioxyaniline HCl were conducted in vitro. An Ames test using Salmonella typhimurium was negative for genotoxicity up to 5000 µg/plate. 2 A mammalian cell gene mutation assay using mouse lymphoma cells was non-mutagenic during a 4-hour incubation with concentrations up to 1100 µg/ml with metabolic activation and up to 1650 µg/ml without activation. In an in vitro micronucleus test performed in human peripheral blood lymphocytes, there was a statistically significant, concentration-dependent increase in relevant micronucleated binucleate cells in the presence of metabolic activation. This indication of chromosomal damage resulted from a 3-hour treatment (at concentrations up to 2177 µg/ml) performed 48 hours after mitogen stimulation (cells were harvested 96 hours after mitogen stimulation); cytotoxicity (13% to 65%) was noted at concentrations ranging from 700 to 2177 µg/ml in several of the micronucleus experiments. Palpebral closure and lethargy were observed 1 hour post-administration in an in vivo bone marrow micronucleus test conducted in mice that were intraperitoneally exposed to Hydroxyethyl-3,4-Methylenedioxyaniline HCl (up to 250 mg/kg). 2 No chromosomal aberrations or damage to the mitotic apparatus in bone marrow cells were observed from treatment with the test substance. CARCINOGENICITY STUDIES There were no carcinogenicity studies for Hydroxyethyl-3,4-Methylenedioxyaniline HCl found in the literature, and unpublished data were not provided. Animal DERMAL IRRITATION AND SENSITIZATION STUDIES Irritation A non-glp study was conducted in female, Pirbright White (SPF) guinea pigs (n=15) to evaluate the effect of Hydroxyethyl-3,4- Methylenedioxyaniline HCl from dermal exposure. 2 A 5% aqueous solution of Hydroxyethyl-3,4-Methylenedioxyaniline HCl (purity not specified) was applied (non-occlusive) to a 12 cm 2 area of clipped flank skin one time per day for 5 consecutive days. The skin was examined for signs of irritation 5 hours following each application and on the third day following the final application. The results indicated that the test substance was non-irritating. Animal Sensitization A local lymph node assay (LLNA) was performed in accordance with OECD TG 429 and GLP. 2 To examine skin sensitization potential, the cell proliferation in the draining lymph nodes was measured in CBA/J mice (n=5 females/ concentration) following dermal exposure to Hydroxyethyl-3,4-Methylenedioxyaniline HCl (99.7% purity). Twenty-five microliters of 0 (vehicle only), 0.5, 1.5, 5, and 10% Hydroxyethyl-3,4-Methylenedioxyaniline HCl (in DMSO, or in a 3:1 mixture of water/acetone (1:1) and olive oil) were applied to the ear skin of mice for 3 consecutive days. A hair dryer was used to dry the ears after application. At the 1.5% test concentration (DMSO vehicle), slight-to-severe skin desquamation on the ears was observed in all animals by day 5, and erythema was noted in one animal. The SCCS report indicated that these effects were attributed to the low ph of the test solution. 2 The stimulation indices for all the test concentrations in either vehicle were greater than 3 (dose-dependent with DMSO vehicle), and an EC3 ( equal to the concentration inducing a stimulation index of 3 ) was not estimated. Positive controls performed as expected. The SCCS report, in which this study was presented, indicated that the results using either vehicle indicated that dermally applied Hydroxyethyl-3,4-Methylenedioxyaniline HCl has strong sensitization potential in mice. Quantitative Structure-Activity Relationship (QSAR) Analysis A study conducted in 2004 applied a previously published, quantitative structure-activity relationship (QSAR) model to estimate the skin sensitization potential of all European registered hair dye substances, including Hydroxyethyl-3,4-Methylenedioxyaniline HCl. 16 Topological sub-structural molecular descriptors (TOPS-MODE) were correlated with LLNA experimental data (based on LLNAs of 93 chemicals) to construct a general QSAR model, unrestrictive to chemical class. This QSAR model was not specifically designed for hair dye substances, but deemed by the researchers conducting the study to be suitable for this application. The allergic contact

44 dermatitis that may develop from the use of skin-sensitizing compounds involves the formation of an antigenic complex, consisting of a protein covalently bonded to a hapten (low-molecular-weight molecule). This type of biochemical reaction is amenable to developing predictive tools, such as QSAR models. A literature review was also conducted as part of this study to retrieve relevant LLNA data (up to July 2003) and human evidence (up to August 2003) to help validate the QSAR predictions or elucidate areas in need of improvement. Hair dyes that were studied in this analysis were classified as strong/moderate, weak, or extremely weak sensitizers or non-sensitizing. Values indicating rank were assigned to each chemical studied, reflecting the predicted relative sensitization potential within each classification group. Approximately 75% of the 229 hair dye substances assessed were classified as strong/moderate sensitizers based on the QSAR model prediction. Hydroxyethyl-3,4-Methylenedioxyaniline HCl was predicted to be a moderate or strong sensitizer with an assigned numerical value of 0.2. For comparison, the highest value assigned to the hair dye substances categorized as strong/moderate sensitizers was 16.3 for Direct Red 80. OCULAR IRRITATION STUDIES A non-glp study was conducted in Pirbright White (SPF) guinea pigs (n=10 females) evaluating 2% (water vehicle) Hydroxyethyl- 3,4-Methylenedioxyaniline HCl. 2 A single, 0.1 ml application of the test substance was instilled into the conjunctival sac of the right eye, and the left eye served as the control; the eyes were not rinsed following application. Eyes were examined 0.5, 1, 2, 3, 4, 6, and 7 hours after the instillation of the test substance. At 24 hours following treatment, a fluorescein-instillation reading was performed. Edema, slight conjunctival redness, and corneal opacity were observed in 2 animals 3 hours post-instillation. The SCCS report indicated that 2% Hydroxyethyl-3,4-Methylenedioxyaniline HCl caused transient irritation to guinea pig eyes. EPIDEMIOLOGY STUDIES Hydryoxyethyl-3,4-Methylenedioxyaniline HCl is used as an oxidative (permanent) hair dye ingredient. While the safety of individual hair dye ingredients is not addressed in epidemiology studies that seek to determine links, if any, between hair-dye use and disease, such studies do provide broad information. Currently available epidemiology studies provided insufficient evidence to support a causal association between personal hair dye use and a variety of tumors and cancers. A detailed summary of the available hair dye epidemiology data is available at SUMMARY Hydroxyethyl-3,4-Methylenedioxyaniline HCl functions in cosmetics as a hair colorant. VCRP data received in 2017 indicate that there are 67 reported uses of Hydroxyethyl-3,4-Methylenedioxyaniline HCl in hair dyes and colors; a Council industry survey received in 2016 reported maximum use concentrations to be 0.52% to 0.75% (diluted or on head concentrations) in hair dyes and colors. Hydroxyethyl-3,4-Methylenedioxyaniline HCl contains a free, secondary aromatic substituted amine (aniline derivative), which raises a concern about possible N-nitrosation. The SCCS report specified a limit of < 50 ppb for nitrosating content in hair dye formulations containing Hydroxyethyl-3,4-Methylenedioxyaniline HCl. An in vitro percutaneous absorption study evaluated 1.5 mg/cm 2 (1.5%) Hydroxyethyl-3,4-Methylenedioxyaniline HCl as part of an oxidative hair dye formulation that was applied to 4 cm 2 porcine skin mounted on a diffusion Teflon-chamber. The test substance was washed off the skin 60 min post-application. At 16 and 24 hours post-application, fractions of receptor compartment fluid were collected, concentrated and analyzed. By 24 hours the applied amount recovered was 0.047% in the upper skin (above the basal layer), % in the lower skin (basal layer skin down to upper dermis), and 0.3% in the receptor compartment fluid. The skin penetration rate was determined to be 5.8 µg/cm 2. An in vivo percutaneous absorption experiment in rat skin was conducted to evaluate 14 C-Hydroxyethyl-3,4-Methylenedioxyaniline HCl (ring-labeled) in the following formulations: 3.33% pure dye in water; 1% formulation with hydrogen peroxide (also containing p-toluenediamine, resorcinol, and m-aminophenol in this application); 1% formulation without hydrogen peroxide. At 30 minutes post-application the test substance was removed and the skin washed. Results indicated that the washing solutions contained 95.9% to 97.1% of the applied radiolabeled concentration. The skin contained 1.68% of the applied radioactivity for the formulation containing water, 0.56% for the formulation containing hydrogen peroxide, and 0.34% for the formulation without hydrogen peroxide. In dermal ADME studies (in vivo), 1% to 3% 14 C-Hydroxyethyl-3,4-Methylenedioxyaniline HCl (ring-labeled) was applied to rat skin for 30 minutes and then removed by washing. The applied radioactive concentration detected in urine and feces (combined percentages) by 72 hours post-application were 0.05% to 0.345%. Dermal absorption rates ranged from 0.59 µg/cm 2 to 14.8 µg/cm 2 and no substantial bioaccumulation was observed. Oral ADME studies were performed in rats administered single dosages by gavage of 1 to 100 mg/kg 14 C-Hydroxyethyl-3,4- Methylenedioxyaniline HCl (ring-labeled). Results indicated that >95% of the radioactivity was absorbed. Blood, urine, and feces samples were collected up to 24 to 96 hours post-dosing. The radioactivity detected in urine and feces were 78% and 14%,

45 respectively; bioaccumulation was not observed. The highest blood concentration detected following oral exposure was 70-fold greater than the highest blood concentration after dermal exposure. A subchronic (90-day) oral exposure study was conducted in rats to evaluate the effects of Hydroxyethyl-3,4-Methylenedioxyaniline HCl following administration by gavage of 0, 20, 100, and 350 mg/kg/day in purified water. In the 100 and 350 mg/kg/day groups, both sexes showed reduced locomotion. Treatment-related abnormalities in red blood cells were observed in males at 350 mg/kg/day after 13 weeks, and were associated with splenic extramedullary hematopoiesis found during histopathology. Abnormalities in red blood cells and organs (thymus, ovary, spleen) were observed in females (350 mg/kg/day). In both sexes elevated clinical biochemistry parameters and liver and kidney abnormalities were reported (350 mg/kg/day). At 100 mg/kg/day elevated bilirubin levels and phospholipids were seen in females, liver abnormalities were noted in males, and increased cholesterol and urinary volume occurred in both sexes. A NOAEL of 20 mg/kg/day was reported. A teratogenicity study was conducted to evaluate the effects of Hydroxyethyl-3,4-Methylenedioxyaniline HCl in pregnant rats that were exposed by gavage to the following dosage rates (on days 6 to day 20 of gestation): 0, 26, 152, and 702 mg/kg/day. Following the first 3 days of dosing (days 9 to day 20 of gestation), 702 mg/kg/day was reduced to 688 mg/kg/day because of severe toxicity and mortality. In the 688 mg/kg/day group, hypoactivity, hunched posture, lacrimation, and behavioral indications of discomfort (discomfort also seen with 152 mg/kg/day) were observed. At 152 and 688 mg/kg/day reduced food consumption and body weight were noted. At the same dosage rates, stomach abnormalities and effects on adrenals, spleen, and kidneys were observed. An increase in fetal resorptions occurred in the 688 mg/kg/day group only. The 688 mg/kg/day treatment resulted in effects on litter size and fetal body weight, but not on sex ratio of fetuses. For the 152 and 688 mg/kg/day groups, skeletal and visceral abnormalities were seen. The SCCS report states the severe reproductive and developmental effects observed with the 152 and 688 mg/kg/day dosage rates were caused by maternal toxicity. A reproductive and developmental NOAEL of 26 mg/kg/day was reported. Hydroxyethyl-3,4-Methylenedioxyaniline HCl was negative in an Ames test (using Salmonella typhimurium) up to 5000 µg/plate. Results from a mammalian cell gene mutation assay using mouse lymphoma cells indicated that the ingredient was non-mutagenic during a 4-hour incubation at concentrations up to 1100 µg/ml with metabolic activation and up to 1650 µg/ml without activation. An in vitro micronucleus test performed in human peripheral blood lymphocytes showed a statistically significant, concentrationdependent increase in relevant micronucleated binucleate cells in the presence of metabolic activation. This indication of chromosomal damage resulted from a 3-hour treatment (at concentrations up to 2177 µg/ml) performed 48 hours after stimulation with a mitogen. No chromosomal aberrations or damage to the mitotic apparatus in bone marrow cells were observed in an in vivo bone marrow micronucleus test conducted in mice intraperitoneally exposed to Hydroxyethyl-3,4-Methylenedioxyaniline HCl (up to 250 mg/kg). There were no carcinogenicity studies for Hydroxyethyl-3,4-Methylenedioxyaniline HCl found in the literature. Hydroxyethyl-3,4-Methylenedioxyaniline HCl (5% aqueous solution) was not irritating when applied (non-occlusive) one time per day for 5 consecutive days to guinea pig skin clipped free of hair. Hydroxyethyl-3,4-Methylenedioxyaniline HCl (up to 10% in DMSO or in a 3:1 mixture of aqua/acetone (1:1) and olive oil) was shown to have strong sensitization potential in mouse skin in an LLNA test. In a Swedish market analysis (no chemical testing was conducted in this study) Hydroxyethyl-3,4-Methylenedioxyaniline HCl was one of 37 target hair dyes in 122 products examined. Hydroxyethyl-3,4-Methylenedioxyaniline HCl was identified by the researchers conducting the analysis to be a potent skin sensitizer. It was reported to be used at the maximum authorized concentration of 3% or when combined with hydrogen peroxide at 1.5%. A study was conducted applying a previously published QSAR model (based on TOPS-MODE and LLNA experimental data) to estimate the skin sensitization potential of all European registered hair dye substances in 2004, including Hydroxyethyl-3,4- Methylenedioxyaniline HCl. Hydroxyethyl-3,4-Methylenedioxyaniline HCl was predicted to be a moderate or strong sensitizer. An study was conducted in guinea pigs to evaluate the ocular irritation potential of 2% (water vehicle) Hydroxyethyl-3,4- Methylenedioxyaniline HCl. A single, 0.1 ml application of the test substance was instilled into the conjunctival sac of the right eye while the left eye served as the control; the eyes were not rinsed following application. Edema, slight conjunctival redness, and corneal opacity were observed in 2 animals, 3 hours after treatment. The SCCS report indicated that 2% Hydroxyethyl-3,4- Methylenedioxyaniline HCl caused transient irritation to guinea pig eyes. The most recent, comprehensive review of available epidemiology studies concluded that there is insufficient evidence to support a causal association between personal hair dye use and a variety of tumors and cancers. A summary of the available hair dye epidemiology data is available at DISCUSSION The Panel reviewed the data presented in this assessment to determine the safety of Hydroxyethyl-3,4-Methylenedioxyaniline HCl as used in hair dyes. Although no carcinogenicity data were found in the literature, the Panel concluded that the animal studies evaluating percutaneous dermal absorption, toxicokinetics (dermal and oral exposure), subchronic oral exposure, teratogenicity, and genotoxicity adequately support a low toxicity profile for this hair dye ingredient when used according to the present practices of use

46 and concentration described in this safety assessment. Hydroxyethyl-3,4-Methylenedioxyaniline HCl was non-irritating to guinea pig skin and transiently irritating to guinea pig eyes at concentrations greater than those expected in human use. Animal tests and QSAR analysis, predictive of potential human response, indicated that Hydroxyethyl-3,4-Methylenedioxyaniline HCl may be a skin sensitizer. Hair dye ingredients are known to be potential skin irritants/sensitizers. The Panel emphasized that labeling instructions included for the use of Hydroxyethyl-3,4-Methylenedioxyanline HCl in hair dye formulations should be carefully followed. Hair dyes containing Hydroxyethyl-3,4-Methylenedioxyaniline HCl, as a coal tar hair dye ingredient, are exempt from certain adulteration and color additive provisions of the Federal Food, Drug, and Cosmetic Act, when the label includes a caution statement and consumer patch test instructions for determining whether the product causes skin irritation. The Expert Panel expects that following this procedure will identify prospective individuals who would have an irritation/sensitization reaction and allow them to avoid significant exposures. The Panel considered concerns that such self-testing might induce sensitization, but agreed that there was not a sufficient basis for changing this advice to consumers at this time. The Panel concluded that hair dye formulations containing Hydroxyethyl-3,4-Methylenedioxyaniline HCl should not be used with nitrosating agents because of the potential for N-nitrosation of the free, secondary aromatic substituted amine in the ingredient and the possible presence of nitrosating impurities (i.e., 3,4-methylenedioxy-aniline). The Panel supported the limit established in the SCCS report, stating that nitrosating content for hair dye formulations containing Hydroxyethyl-3,4-Methylenedioxyaniline HCl should be < 50 ppb. In considering hair dye epidemiology data, the Panel concluded that the available epidemiology studies are insufficient to scientifically support a causal relationship between hair dye use and cancer or other toxicologic endpoints, based on lack of strength of the associations and inconsistency of findings. The Panel noted that the use of oxidative hair dye formulations involves exposure to precursors and coupling agents, as well as to their reaction products. While reaction intermediates are formed, human exposure is primarily to the precursors, coupling agents, and reaction products, not the reaction intermediates. Exposures to the precursors and couplers are low, because they are consumed in the color forming reaction, and exposures to reaction products are even lower, because they are adsorbed onto and physically retained on the hair shaft. Therefore, it was the consensus of the Panel that safety assessments of oxidative hair dyes are primarily determined by the toxicological evaluation of the ingredients (i.e., precursors and coupling agents), and not of the reaction intermediates or products formed during use. CONCLUSION The CIR Expert Panel concluded that Hydroxyethyl-3,4-Methylenedioxyaniline HCl is safe for use as a hair dye ingredient in the present practices of use and concentrations. The Expert Panel cautions that ingredients should not be used in cosmetic products in which N-nitroso compounds can be formed.

47 TABLES Table 1. Physical and Chemical Properties of Hydroxyethyl-3,4-Methylenedioxyaniline HCl Property Value Reference Physical Form Crystals 2 Color Beige 2 Formula Weight (g/mol) Density 20 C Vapor Pressure 20 C 5.5 to Melting Point ( C) (decomposition) 2 Water Solubility (ph 1.5) 20 C 408 (saturated solution) 2 Water Solubility (ph 6.0) 20 C < 20 2 Other Solubility (ph 7.0) (g/l) Acetone/water (1:1) >100 2 Other Solubility (g/l) DMSO > Other Solubility (g/l) Ethanol Log P ow (ph 36 C (this value was determined using the free-base form of 2,17-19 the ingredient in accordance with EC Method A.8. Partition Coefficient and OECD TG 107) 25 C 5.11±0.20 (calculated for proton on nitrogen atom, free-base form) 20 EC=European Commission; OECD=Organization for Economic Cooperation and Development

48 Table 2.Toxicokinetics Studies (ADME) for Hydroxyethyl-3,4-Methylenedioxyaniline HCl Test Substance(s) Species/ Strain Sample Type/Test Population-Sex Concentration or Dosage (Vehicle) 14 C-Hydroxyethyl-3,4- Methylenedioxyaniline HCl (ring-labelled, 100% purity) 14 C-Hydroxyethyl-3,4- Methylenedioxyaniline HCl (ring-labelled) Rat/ Wistar Kyoto WKY/ NR Crl BR (inbred) Rat/ Sprague Dawley Him:OFA, SPF n=4 females in Exp 1 (urine, feces, metabolite analysis performed in this mass balance experiment) n=5 females in Exp 2 (urine, feces, blood, tissues, carcass examined in toxicokinetics analysis) n=3/sex/ concentration group Exp 1 & Exp 2: 1.5% 14 C- labeled (10 mg/kg, 0.15 mg/cm 2 ); vehicle=acetone/water, 1:1; ph adjusted to Exp 1: 1% (1.1 mg/cm 2 ) formulation without hydrogen peroxide (urine, feces, and carcass analysis) Exp 2: 1% (1.1 mg/cm 2 ) formulation with hydrogen peroxide, p-toluenediamine, resorcinol, m-aminophenol (urine, feces, and carcass analysis) Exp 3: 3.33% (1.1 mg/cm 2 ) pure dye in water (urine, feces, and carcass analysis) Exp 4: 3.33% (1.1 mg/cm 2 ) pure dye in water (blood analysis) Procedure Results Reference ANIMAL Dermal A GLP study was conducted in accordance with OECD TG 417 and 427; immediately before treating the skin it was cleaned with 10% shampoo and water solution and dried; test substance (in Exp 1 & 2) was applied 1x to skin shaved free of hair on the back (2.5 x 4 cm 2 ); a 4 x 4 cm 2 area of shaved skin on the abdomen was used as a negative control; animals were anesthetized during application, exposure, and removal of test substance; 30 min post-application, skin was washed with an aqueous shampoo solution; in Exp 1, urine and feces were collected 5x for the first 96 h post-administration; metabolite analysis was conducted via HPLC; in Exp 2 blood was collected at 10, 20, 40 min and at 1, 2, 4, 8, 24, 48, and 72 h post-administration (urine, feces, blood, tissues, and carcass were analyzed for radioactivityno further details provided as to when the sampling occurred); animals in Exp 1 & 2 were killed 96 h post-administration In a GLP study the test substance was applied (animals under anesthesia) 1 x to skin clipped free from hair as indicated in Experiments 1-4; skin surface area to which test substance was applied in Exp 1 was cm 2 and for all other experiments was 9 cm 2 ; at 30 min post-application the test substance was removed and skin washed with shampoo formulation (100 ml) and then by warm water until no color was seen; skin was then covered with gauze patch and secured with adhesive and an air permeable plastic cone to prevent animals from licking treated area during the 72-hour study duration (carried out in metabolism cages); Exp 1, 2, & 3 were used to evaluate radioactivity eliminated in urine and feces and Exp 4 was used to evaluate administered radioactivity in blood; blood was sampled several times within 24 h post-dosing (animals were under light anesthesia during blood collection from the peri-orbital plexus); in all experiments animals were killed 72 h postapplication Exp 1: Recovery of applied radioactivity was 97% to 99%; in urine and feces only the metabolized test substance was detected and no conjugates were found; SCCS report indicated that metabolites may have been excreted in bile and any that were glucuronide conjugates may have been de-conjugated in intestines Exp 2: Absorption rate based on radioactive content in carcass, urine, and feces was 5% (8 µg/cm 2 ); if the radioactive content of the application site is added to the total of what was potentially absorbed the result is 8% (14.8 µg/cm 2 ) of applied radioactivity; maximum concentration of radioactivity in blood was observed 1 h post-application (no further details provided); SCCS report noted that elimination of the radioactive concentration was excreted in the greatest amounts in urine and much less in feces and that bioaccumulation was not observed Dermal penetration results are described in the text of this assessment; applied radioactivity was eliminated within 72 h, mainly in urine and to a lesser extent in feces, in the following combined (urine + feces) amounts: 0.314% (Exp 1), 0.05% (Exp 2), and 0.345% (Exp 3); amount of applied radioactivity remaining in the carcass 72 h post-application was 0.005% for Exp 1 & 3 and 0.002% for Exp 2; cutaneous absorption rates of 3.5 µg/cm 2 (Exp 1), 0.59 µg/cm 2 (Exp 2), and 3.95 µg/cm 2 (Exp 3) were reported for bioavailable amounts in carcass, urine, and feces combined; in Exp 4, highest blood concentration (no further details provided) of test substance was observed at the first sampling (35 min post-application) then declined, half-life was determined to be 1 h 2 2

49 Table 2.Toxicokinetics Studies (ADME) for Hydroxyethyl-3,4-Methylenedioxyaniline HCl Test Substance(s) Species/ Strain Sample Type/Test Population-Sex Concentration or Dosage (Vehicle) 14 C-Hydroxyethyl-3,4- Methylenedioxyaniline HCl (ring-labeled) 14 C-Hydroxyethyl-3,4- Methylenedioxyaniline HCl (ring-labeled, purity 100%) Rat/ Sprague Dawley Him:OFA, SPF Rat/ Wistar Kyoto WKY/ NR Crl BR (inbred) n=3/sex n=4 females/experiment in Exp 1 & 2 (urine, feces, metabolite analysis performed in this mass balance experiment) n=6 females/ experiment in Exp 3 & 4 (urine, feces, blood, tissues, carcass examined in toxicokinetics analysis) mg/kg (3.3%) pure dye in water (blood analysis) Exp 1 & 3: 14 C-labeled 1 mg/kg in water Exp 2 & 4: 14 C-labeled 100 mg/kg in water Procedure Results Reference Oral In a GLP test performed in conjunction with the 4 dermal experiments summarized in the previous row of this table, the test substance was administered 1 x by gavage; blood was sampled several times within 24 h post-dosing (animals were under light anesthesia during blood collection from the periorbital plexus); animals were killed 72 h postadministration A GLP study was conducted in accordance with OECD TG 417 and 427; single dosages were administered by gavage to fasted animals (18 h prior to and 4 h after dosing); in Exp 1 & 2 urine and feces were collected 5 x for the first 96 h postadministration; metabolite analysis was conducted via HPLC; in Exp 3 & 4 blood was collected at 10, 20, 40 min and at 1, 2, 4, 8, 24, 48, and 72 h postadministration (urine, feces, blood, tissues, and carcass were analyzed for radioactivity-no further details provided as to when the sampling occurred); all animals were killed 96 h post-administration Highest blood concentration (no further details specified) of test substance was observed at the first sampling (35 min post-application) then declined, half-life was determined to be 1.5 h; the highest blood concentration following oral exposure was approximately 70-fold greater than highest observed blood concentration following dermal application in the study summarized above (in the preceding row) Exp 1 & 2: Recovery of administered radioactivity was 97% to 99%; in urine and feces, only the metabolized test substance was detected and no conjugates were found; SCCS report indicated that metabolites may have been excreted in bile and any that were glucuronide conjugates may have been de-conjugated in intestines Exp 3 & 4: > 95% of dosed radioactivity was absorbed (no further details provided); blood kinetics showed fast absorption for low and high dosages; highest concentration of radioactivity in blood was observed 10 min post-dosing; highest residual amount of radioactivity was located in carcass, liver, kidney and thyroid but was only 1% of administered radioactive dosage; SCCS report noted that elimination of the radioactive dosage was excreted in the greatest amounts in urine and much less in feces, and the radioactivity in feces represented metabolites of test substance that were excreted through bile; bioaccumulation was not observed 2 2

50 Table 2.Toxicokinetics Studies (ADME) for Hydroxyethyl-3,4-Methylenedioxyaniline HCl Test Substance(s) Species/ Strain Sample Type/Test Population-Sex Concentration or Dosage (Vehicle) 14 C-Hydroxyethyl-3,4- Methylenedioxyaniline HCl (position of label not specified) Rat/ Wistar CRL:WI BR n=? 1 mg/kg (Mass balance group) 1 mg/kg (Toxicokinetics group) No further details on groups provided Procedure Results Reference Animals were fasted for 18 h before and 4 h after dosing, then food and water were freely available; a single dosage was administered; for mass balance group urine and feces were collected prior to treatment and 6 x up to 96 h post-dosing; for toxicokinetics, grouped blood samples were collected at 0.25, 0.5, 1, 4, 8, and 24 h post-dosing; animals were killed 72 or 96 h post-dosing; urine, feces, and blood samples were extracted and analyzed by LC-MS/MS; oral absorption of total radiolabeled parent compound and metabolites was calculated based on % recovered in urine following oral exposure divided by the sum of % recovered in urine and % recovered in feces following oral exposure, yielding the normalized amount excreted in urine; the fraction absorbed following oral exposure was calculated as the normalized amount in urine (after oral exposure) divided by normalized amount in urine following iv exposure (calculated similarly to calculation using the oral exposure data); calculations were performed using plasma data to estimate oral absorption (by dividing the dose normalized 14 C area under the curve following oral exposure by dose normalized 14 C area under the curve following iv exposure) Fraction of administered radioactivity detected in urine was 77.8% ± 8.2% and in feces was 14.3% ± 4.4% of the administered radioactivity; Fraction of radioactivity absorbed following oral exposure was calculated to be 99% from total radioactivity in the urine (normalized to the amount recovered), and 91% from the normalized plasma AUCs after oral and i.v. exposures. DMSO=Dimethyl Sulfoxide; GLP=Good Laboratory Practice; HPLC=High Performance Liquid Chromatography; LC-MS/MS=Liquid Chromatography-Mass Spectrometry/Mass Spectrometry; OECD TG=Organization for Economic Co-operation and Development Test Guideline; P app=apparent permeability values: TEER=transepithelial electrical resistance 15

51 Table 3. Genotoxicity Studies for Hydroxyethyl-3,4-Methylenedioxyaniline HCl Species/ Strain Sample Type/ Test Population Concentration (Vehicle) Procedure Results Reference Salmonella typhimurium Mouse Human TA98, TA100, TA102, TA1535, TA1537 Thymidine kinase locus in mouse lymphoma L5178Y cells Peripheral blood lymphocytes 33, 100, 333, 1000, 2500, 5000 µg/plate (vehicle=de-ionized water); purity of test substance was 99.8% Exp. 1: 34.4, 68.8, 137.5, 206.3, 275, 412.5, 550, 825, 1100 µg/ml (4 h incubation, with activation); Exp. 2: 68.8, 137.5, 206.3, 275, 412.5, 550, 825, 1100, and 1650 µg/ml (4 h incubation, without activation); Exp. 3: 12.5, 25, 50, 100, 150, 200, 250, and 300 µg/ml (24 h incubation without activation) Purity of test substance was 99.8% (vehicle=de-ionized water) Exp. 1: 500, 1100, 1800 µg/ml (3 h treatment 24 h after mitogen stimulation, with activation); 200, 400, 700 µg/ml (20 h treatment 24 h after mitogen stimulation) Exp. 2: 1200, 1500, 1800, 2177 µg/ml (3 h treatment 48 h after mitogen stimulation, with activation); 650, 750, 950 µg/ml (20 h treatment 48 h after mitogen stimulation) Purity of test substance was 99.8% (vehicle=sterile water) IN VITRO Bacterial reverse mutation assay (Ames Test) was performed, with and without metabolic activation, in accordance with GLP and OECD TG 471 (Bacterial Reverse Mutation Assay); both the plate incorporation method and pre-incubation methods were used in two separate experiments using the same concentrations listed; negative, vehicle, and positive controls were used Range-finding test was performed using a concentration range of 17.2 to 2200 µg/ml; Concentration ranges in Exp. 1 & 2 were limited by the toxicity of test substance; Mammalian cell gene mutation assay was performed, with and without metabolic activation, in accordance with GLP and OECD TG 476 (In vitro Mammalian Cell Gene Mutation Test); solvent and positive controls were used Micronucleus test was performed in accordance with OECD TG 487 and GLP; blood was pooled from 2 male donors in each test; mitogen stimulation was performed with phytohemagglutinin; 72 h and 96 h after mitogen stimulation, cells from Exp 1 and 2, respectively, were harvested; 500 cells per replicate (1000/concentration) were examined for mono-, bi-, and multinucleated cells used to calculate the replication index; 1000 binucleate cells from each culture were evaluated for number of micronuclei; solvent and positive controls were used Negative; controls performed as expected Non-mutagenic; no biologically relevant increases in mutations were observed Cytotoxicity reported in Exp. 1 was 65% (700 µg/ml) and 53% (1800 µg/ml) and in Exp. 2 was 62% (950 µg/ml) and 13% (2177 µg/ml); in Exp. 2 a clear concentration-dependent, statistically significant increase in relevant micronucleated binucleate cells in the presence of metabolic activation was, indicating chromosomal damage as noted in the SCCS report; the same effects were not seen in the absence of metabolic activation; positive controls performed as expected 2 2 2

52 Table 3. Genotoxicity Studies for Hydroxyethyl-3,4-Methylenedioxyaniline HCl Species/ Strain Sample Type/ Test Population Concentration (Vehicle) Procedure Results Reference Mouse/ NMRI Pre-Experiment: n=3/sex/dosage Main Experiment: n=5/sex/dosage/time Pre-Experiment: 250 to 1000 mg/kg Main Experiment: 25, 125, 250 mg/kg Purity of test substance was 99.7% (vehicle=distilled water) IN VIVO A bone marrow micronucleus test was performed in accordance with OECD TG 474 and GLP; a pre-experiment was performed for dosage selection; in the main experiment, single dosage was administered intraperitoneally 24 h (all dosage levels) or 48 h (250 mg/kg) before animals were killed; at least 2000 polychromatic erythrocytes with micronuclei from each animal were analyzed; the ratio between polychromatic and total erythrocytes was determined for each animal; solvent and positive controls were used GLP=good laboratory practice; OECD TG=Organization for Economic Co-operation and Development Test Guideline Pre-Experiment: 3 male and 3 female mice died in 24 h (1000 mg/kg); 1 st h post-administration palpebral closure and lethargy (for up to 6 h) were observed, but there was no mortality (250 mg/kg) Main Experiment: Similar toxic effects with 250 mg/kg as noted in pre-experiment at 250 mg/kg; ratio between polychromatic erythrocytes and total erythrocytes unaffected by test substance; SCCS report noted the observed systemic toxicity indicated systemic distribution and bioavailability of test substance; there was no statistically significant increase in micronuclei/2000 polychromatic erythrocytes in treated vs. control animals; positive controls performed as expected; test substance did not induce chromosome aberrations or damage to mitotic apparatus in bone marrow cells 2

53 REFERENCES 1. Nikitakis J (ed) and Lange B (ed). International Cosmetic Ingredient Dictionary and Handbook. 16 ed. Washington, D.C.: Personal Care Products Council, European Commission SCCS (Scientific Committee on Consumer Safety). Scientific Committee on Consumer Safety Opinion on Hydroxyethyl-3,4-methylenedioxyaniline HCl (SCCS/1269/09) Date Accessed European Commission SCCS (Scientific Committee on Consumer Safety). Opinion On Reaction Products of Oxidative Hair Dye Ingredients Formed During Hair Dyeing Processes Date Accessed European Chemicals Agency (ECHA). Substance Information for 2-(1,3-benzodioxol-5-ylamino)ethanol hydrochloride; CAS# Last Updated Date Accessed Australian Government Department of Health National Industrial Chemicals Notification and Assessment Scheme (NICNAS). Human Health Teir II Assessment for Ethanol,2-(1,3-benzodioxol-5-ylamino)-,hydrochloride; CAS# Last Updated Date Accessed European Commission. COSING Database Search Results for Hydroxyethyl-3,4-Methylenedioxyaniline HCl, Following Cosmetic Regulation 1223/ Last Updated Date Accessed Shank RC and Magee PN. Mycotoxins and N-Nitroso Compounds: Environmental Risks. Chapter: 1. Boca Raton, FL: CRC Press, Inc.; 1981: Rostkowska K, Zwierz K, Rozanski A, 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: Food and Drug Administration (FDA). Frequency of use of cosmetic ingredients. FDA Database Personal Care Products Council Concentration of Use by FDA Product Category: Hydroxyethyl-3,4- Methylenedioxyaniline HCl (Dated September 20th). Unpublished data submitted by Personal Care Products Council. 12. Thyssen JP, Sosted H, Uter W, Schnuch A, Gimenez-Arnau AM, Vigan M, Rustemeyer T, Granum B, McFadden J, White JM, White IR, Goossens A, Menne T, Liden C, and Johansen JD. Self-testing for contact sensitization to hair dyes-scientific considerations and clinical concerns of an industry-led screening programme. Contact Dermatitis. 2012;66: Goossens A. Self-testing for contact sensitization to hair dyes. Contact Dermatitis. 2012;66: Yazar K, Boman A, and Liden C. Potent skin sensitizers in oxidative hair dye products on the Swedish market. Contact Dermatitis. 2009;61: Obringer C, Manwaring J, Goebel C, Hewitt NJ, and Rothe H. Suitability of the in vitro Caco-2 assay to predict the oral absorption of aromatic amine hair dyes. Toxicology in Vitro. 2016;32:1-7.

54 16. Sosted H, Basketter DA, Estrada E, Johansen JD, and Patlewicz GY. Ranking of hair dye substances according to predicted sensitization potency: quantitative structure-activity relationships. Contact Dermatitis. 2004;51: European Commission. IV Notices From European Union Institutions, Bodies, Offices and Agencies (Partition Coefficient n-octanol/water method). Official Journal of the European Union. 2013;C 95: Organization for Economic Cooperation and Development (OECD). OECD Guideline for the Testing of Chemicals 107 Partition Coefficient (n-octanol/water): Shake Flask Method D33CA A04051C8E48. Date Accessed pp European Commission. Testing Methods and Directives (lists EC Method A.8 in a Summary table) Date Accessed pp Advanced Chemistry Development (ACD/Labs) Software V11.02 ( ACD/Labs)

55 2017 VCRP Data For Hydroxyethyl-3,4-Methylenedioxyaniline HCl Hydroxyethyl-3,4- Methylenedioxyaniline HCl 06A - Hair Dyes and Colors (all types requiring caution statements and patch tests) 67

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