SAR Read -Across Justification_pFAoAc_Final Draft_15 - 04 -2013 TS#: TS01TX Clariant Produkte (DE) GmbH

April 2013 

### As **sessment of the validity of the read**  **- across approach for the human health**  **hazard assessment of C**  **16 -**  **18** 

### -**(ev en numbered)**  **- alky** **lamine acetates** 1. Introduction Based on Article 10 of the REACH Regulation (EC) 1907/2006, testing requirements for individual substances derive d from the application of Annexes VI to X have to b e fulfilled fo r registration purposes. However, article 13 of REACH also requires that “ information on intrinsic properties of substances may be generated by means other than tests, provided that the conditions set out in Annex XI are met. In particular for human toxicity, information shall be generated whenever possible by means other than vertebrate animal tests, through the use of alternative methods, for example, in vitro methods or qualitative or quantitative structure -activity relationship models or from information from structurally related substances (grouping or read-across”. In this respect, annex XI provides general rules for the adaptation of the standard testing regime and also promotes read - across and/or category approaches to fulfill a.m. REACH information requirements. Category and read - across approaches based on structure - activity- relationship (SAR) considerations, are regarded to be effective and valid tools in toxicology for comparing toxicological properties of structural closely related compounds. This is also recognized in the guidance documents of various HPV p rograms and category approaches have been successfully used at the OECD level, the US HPV Challenge Program as well as the EU Existing Substance Program . Exam ples of accepted categories comprise aliphatic aldehyds, alpha - olefins, long - chain alcohols and li near alkylbenzene sulfonates. According to Annex XI of REACH “ substances whose physic-chemical, toxicological and ecotoxicological properties are likely to be similar or follow a regular pattern as a result of structural similarity may be considered as a group, or `category` of substances” based on similarities of the following principles

- common functional group

- common precursor or breakdown products

- constant pattern in changing potency

- common constituents or chemical class According to ECHA guidance, sim ilar principles apply a lso to read - across approaches to fill data gaps. In this respect, endpoint information from appropriate source molecules is used to predict the same endpoint. Comparable approaches for the development of chemical categories and re ad - across strategies are used also in the EPA HPV Challen ge Program as described by Hefter et al, 1999 . In the following, a justification of the chosen read - across approach usin g analogues primary a lkyl am ines to address data gaps for the target mo lecule C 16- 18- (even numbered) -alkylamine acetates is given and reference is ma de to structural and functional similarities, common metabolic pathways, and a relation to an existing chemical class of compounds .SAR Read -Across Justification_pFAoAc_Final Draft_15 - 04 -2013 TS#: TS01TX

- 2 - 2. Read -Across Hypothesis The target mo lecule ` C 16- 18- (even numbered) -alkylamine acetates ` is the ` C 16 - 18- (even num bered) - alkyl ammonium salt` of acetic acid . P rimary a lkyl amines and derivatives (e.g . fatty am ine s and fatty am ine salts) have a close structural relationship and share common functio nalities . The mo lecular structure is characterized by t he hydrophobic aliphatic alkyl chain as well as a hydrophilic am ine group giving the whole mo lecule am phiphilic properties. The unprotonated and the protonated am ine are in equilibrium w hen dissolved i n water or physiological media, and their ratio in the me dium i s determined by the acid constant pKa of 10.6 and the pH. At pH 7 and lower the nitrogen of primary alkyl am ine s is almost com pletely (>> 99%) protonated, resulting in a positively charged catio nic amine structure. Beside close structural and functional similarities, primary a lkylam ines and primary a lkylam ine acetates are considered to be also biologically equivalent because they follow the same metabolic pathways of oxidation by monoamine oxidas es to generate the respective fatty acid and ammonia. The fatty acid would be metabolized by well - known pathways (ß - oxidation) to acetic acid, which may be used intermediary or is metabolized ultimately to carbon dioxide and water. Due to these comparabili ty’s , a category based approach for “N - alkyl - (C 8- C 18 ) - primary amines and Acetate Salts” was already used to establish an exemption from the requirement of a tolerance for residues of primary a mi nes and the respective acetate salts for materials where the a lkyl group is linear but may b e saturated and/or unsaturated (EPA, Final Rule, Federal Register 75, 2010 [EPA- HQ - OPP -2009 - 0046; FRL - 8836 -4]). Beside, a “ fatty nitrogen derived amines category ” was also established within EPA`s HPV Challenge Program (EPA, 2010) as well as by t he German GDCh -Advisory Committee on Existing Chemicals of Environmental Relevance (BUA- Report 177, 1994). Based on these similarities in structural, functional and metabolic behavior of primary a lkylam ines and alkylam ine salts , it is therefore reasonable and scientifically justified to u se analogue primary alkylam ines also as source molecules to inform endpoint related data gaps for the target molecule under discussion. It should be noted that the read - across hypothesis relies not only on structural considerations, i.e. incremental and constant change across an underlying category based on chain length, but also on common functional and metabolic princi ples across primary a lkyl am ines and primary a lkylam ine salts as well as on a close re lationship to an already established category of primary amines and acetate salts which in principle reflect the read - across approach as used herein . To ensure comparability with the target molecule `C 16- 18- (even numbered) -alkylamine acetates` and in line with the used approach by E PA, only

s ource mo lecules having a linear alkyl chain have been considered for the read - across. Generic structure of target molecule C 16 - 18- (even numbered) - alkylam ine acetates CAS No. 1273322 -45 -4SAR Read -Across Justification_pFAoAc_Final Draft_15 - 04 -2013 TS#: TS01TX

- 3 – Representative structure s of source molecule s from the alkyl am ines category used for read - across C 16 - 18- (even numbered) - alkylam ines CAS - No. 90640 - 32 - 7 C 12 - 18- (even numbered) - alkylam ines CAS - No. 68155 -27 - 1 C 16 - 18- (even numbered, C 18- unsaturated ) alkylam ines CAS - No. 68037 - 95 - 6 C 16 - 18- (even numbered, saturated and unsaturated) alkylam ines The applicability to read - across endpoint specific data from surrogate substances of the primary alkylam ine category is supported by t rend a nalysis performed by various groups which indicated consistency for various physical - chemical properties . In any case, from all appropriate source mo lecules those reflecting `worst- case`, e.g. lowest NOAEL, have been chosen for the read - across. 2. Source Chemicals used for Re ad -Across The following source molecules have been considered and endpoint specific data were used for informing data gaps of the target chemical ` C 16- 18- (even numbered) -alkylamine acetates` . The source chemicals were selected on the basis of structural com parability and data availability.

**Source Molecule CA** **S -**  **No. Synonym**  C16 - 18 - (even number) - alkylam ines 90640 - 32 - 7 Hydrogenated tallow alkylam ines C12 - 18 - (even numbered) - alkyl am ines 68155 -27 - 1 Coco alkyl am ines C16 - 18 - (even numbered, C18 - unsaturated) - alkylam ines 68037 - 95 - 6 Tallow alkylam ines C16 - 18 - (even numbered, saturated and unsaturated) - alkyl am ines 112 - 90 - 3 (Z) - Octadecy- 9 - enylamine, OleylamineSAR Read -Across Justification_pFAoAc_Final Draft_15 - 04 -2013 TS#: TS01TX

- 4 – The following endpoints are covered either by d ata from the target chemical itself or by read - across using data from a.m. source molecules: Endpoint considerations

**Endpoint Ap**  **proach used to inform endpoint**  Acute oral toxicity Data from target molecule Acute dermal toxicity Read - across from source molecules Skin irritation Data from target molecule Eye irritation Read - across from source mo lecule Skin sensitization Data from target molecule Repeated dose toxicity Read - across from source molecules. Screening data (14 - day

repeated dose) from target molecu le in support of used approach available and included. Mutagenicity / Genotoxicity Data from target molecule Reproductive toxicity Read - across from source molecules. Screening data (OECD 421 ) and dose - range -finder study for developmental toxicity from target molecule in support of used approach available and included.

Like the target mo lecule, all source mo lecules belong to the category of “N - alkyl (C 8- C 18 ) primary a mi nes and acetate salts” used by E PA for exem ption from the requirements of a tolerance. Prim ary a lkylam ines and primary

a lkyl am ine acetates exhibit a consistent toxicity profile as dem onstrated in the read - across justification below. Based hereupon not only comparability to the target mole cule ` C 16- 18- (even numbered) - alkylamine ace tates` is given , but the used data are also covering the required exposure durations and conditions with regard to the endpoints of interest. The underlying studies are of adequate reliability, well documented and thus allow proper classification and label ing of the target molecule.SAR Read -Across Justification_pFAoAc_Final Draft_15 - 04 -2013 TS#: TS01TX

- 5 – 3 . Read -Across Justification The basic chemical structure of primary alkylam ine acetates can be illustrated as follows:

with n as descriptor of the carbon chain length (for target molecule n = 15, 17). 3 .1 Ph ys ico - chemical properties Wh en considering target and source molecules, the available measured data and estimated values for the below listed physical - chemical parameters me lting and boiling points, vapor pressure, partition coefficient (m easured as hydrochlorid e ) and water solubility appear to be consistent with molecular ma ss and hence support the used read - across approach. All materials are in a comparable mo lecular weigh t range. Additionally, all compounds have comparable surfactant properties.

- Molec ular weight: 185 - 330

- Melting point: inc reases with carbon chain length

- Boiling point: increases with carbon chain length

- Vapour pressure: decreases with carbon chain length

- Water solubility: com parable for considered molecules (based on hydrochloride)

- Log P OW : com parable ( approximately 2 – 3; measured as hydrochloride)) An inherent common property of all source and target mo lecules is that they a ccu mu late at the interface between hydrophobic and hydrophilic phases rather than equilibrating betwee n the two phases which com plicates me asurement and predictions of thei r physico - chemical properties . However, consistent with the size and nature of these mo lecules, me asured and mo deled vapor pressures are very l ow, so that all mo lecules can be considered to be essentially nonvolatile. Overall it is noted that the data and knowledge of primary

a lkylam ines and respective salts support the conclusion that the source and ta rget mo lecules behave similarly or as a function of carbon chain length from t he perspective of physico - chemical properties. 3 .2 Human health aspect From t he structural and functional characteristics, the target mo lecule ` C 16- 18- (even numbered) -alkylamine acetates` is not considered to exhibit skin sensitizing, genotoxic/mutagenic or carcinogenic properties. This is based on experimental data for the target mo lecule itself but also supported by m issing of structural alerts for protein and/or DNA binding which is valid also for the category of primary alkylam ines .SAR Read -Across Justification_pFAoAc_Final Draft_15 - 04 -2013 TS#: TS01TX

- 6 – Additionally , a cute systemic toxicity following oral, dermal or inhalation uptake is of low concern. Especially for the oral route no indications of a respective toxicity potential exist for ` C 16- 18- (even numbered) -alkylamine acetates` based on experimental data on the target mo lecule itself . Likewise, no indications of significant acute systemic derm al and ac ute systemic inhalation toxicity were derived , independent of carb on chain length and saturation degree . For skin irritation, results from experim ental data using reconstituted three - dim ensional hum an epidermis mo dels with the target mo lecule ` C 16- 18- (even numbered) -alkylamine acetates` have been interpreted as dermal irritative properties which is in good agreement with comparable results from skin irritation/corro sion testing in vivo with the corresponding primary `C16 - 18 - (even numbered) - alkyl am ine`. Wi t h regard to eye irritation, structure - activity relationship considerations are pointing to severe irritative properties , which can als be assumed based on general experience with surfactants . From this , read across from the corresponding primary alkylam ines is justified. Repeated dose toxicity studies with the target mo lecule have been conducted only in form of a 14 - day screening study , which is used for comparison reasons to available data from similar screening studies of structural closely related primary a lkylam ines . In this respect, the comparison of the observed toxicity profiles (i.e. effects, affected organs/tissues, dose - respons e) from analogous repeated dose studies between the target mo lecule ` C 16- 18- (even numbered) -alkylamine acetates` and the source mo lecule `C 16- 18- (even numbered) -alkylamines` on one hand, and between saturated and unsaturated source molecules on the other h and revealed no distinct differences independent of the respective saturation degree which supports the use of a read - across approach for bridging endpoint data for repeated dose toxicity . This approach is further justifi ed because all available data on th is endpoint with primary a lkylam ines and salts have shown a consistent toxicity profile independent on th eir carbon chain length and/or degree of saturation / unsaturation . As indicated, the performed read - across is not only based on common structural and functional similarities but also on co mmon metabolic principles valid for the entire categ ory of N-alkyl (C8- C 18 ) primary amines and acetate salts . Prim ary a mi nes and primary

a mi ne acetates are biologically equivalent and follow the sam e metabolic path wa ysof oxidation by m onoam ine oxidases to generate the C8–C10 fatty acid and ammonia. The fatty

a cid would be de graded by well - known pathways (ß - oxidation) to successive releases of acetic acid, which enters into intermediary metabolism or is metabolized ultimately to carbon dioxide and water (EPA 2010). Alternatively, they m ay a lso be conjugated, whether by g lucuronidation or sulfonation, and excreted directly. 4. Overall Assessment and Conclusions According to REACH Annex XI , read - across for informing d ata gaps are appropriate if the result is adequate for the purpose of classification and label ing and/or risk assessment, if there is adequate and reliable coverage of the key p arameters addressed in the corresponding test methods and if exposure durations comparable to or longer than the corresponding test method for endpoint testing are considered . Based on the available d ata base on the target and source mo lecules in general and for the endpoints relevant for read - across in particular , a.m. conditions are met. The provided rational on structural, physico chemical, toxicological and toxicokinetic data, support the appropriateness of endpoint specific read across to inform data gaps of the target mo lecule ` C 16- 18- (even numbered) -alkylamine acetates` by u si ng available data from source mo lec ules from the primary a lkylam ines category. The provided read - across thus is robust and scientifically justified and additional safety factors in the risk characterization are considered not necessary.SAR Read -Across Justification_pFAoAc_Final Draft_15 - 04 -2013 TS#: TS01TX

- 7 – 5. References BUA (1994), Report 177, German GDCh -Advisory Committee on Existing Chemicals of E nvironm ental Relevance EPA (2010), Final Rule, Federal Register 75, 2010 [EPA- HQ - OPP -2009 - 0046; FRL - 8836 -4] Hefter, R., Hernandez, O. and Sayre, P.: Development of chemical categories in the HPV challenge program; EPA, Office of Pollution Prevention Toxics (1999)