SAR Read-Across Justification_pFAoAc_Final Draft_15-04-2013 TS#: TS01TX

Clariant Produkte (DE) GmbH

April 2013

Assessment of the validity of the read-across approach for the human health

hazard assessment of

C16-18-(even numbered)-alkylamine acetates

1. Introduction

Based on Article 10 of the REACH Regulation (EC) 1907/2006, testing requirements for individual 

substances derived from the application of Annexes VI to X have to be fulfilled for 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 programs

and category approaches have been successfully used at the OECD level, the US HPV Challenge 

Program as well as the EU Existing Substance Program. Examples of accepted categories comprise

aliphatic aldehyds, alpha-olefins, long-chain alcohols and linear 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, similar principles apply also 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 read-across strategies are used 

also in the EPA HPV Challenge Program as described by Hefter et al, 1999.

In the following, a justification of the chosen read-across approach using analogues primary alkylamines

to address data gaps for the target molecule C16-18-(even numbered)-alkylamine acetates is given and 

reference is made to structural and functional similarities, common metabolic pathways, and a relation to

an existing chemical class of compounds.
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2. Read-Across Hypothesis

The target molecule `C16-18-(even numbered)-alkylamine acetates` is the `C16-18-(even numbered)-

alkylammonium salt` of acetic acid. Primary alkyl amines and derivatives (e.g. fatty amines and fatty 

amine salts) have a close structural relationship and share common functionalities. The molecular 

structure is characterized by the hydrophobic aliphatic alkyl chain as well as a hydrophilic amine group 

giving the whole molecule amphiphilic properties. The unprotonated and the protonated amine are in 

equilibrium when dissolved in water or physiological media, and their ratio in the medium is determined by 

the acid constant pKa of 10.6 and the pH. At pH 7 and lower the nitrogen of primary alkylamines is almost 

completely (>> 99%) protonated, resulting in a positively charged cationic amine structure.

Beside close structural and functional similarities, primary alkylamines and primary alkylamine acetates 

are considered to be also biologically equivalent because they follow the same metabolic pathways of 

oxidation by monoamine oxidases 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 comparability’s, a category based approach for “N-alkyl-(C8-C18)-primary amines and Acetate 

Salts” was already used to establish an exemption from the requirement of a tolerance for residues of 

primary amines and the respective acetate salts for materials where the alkyl group is linear but may be 

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 the 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 alkylamines and 

alkylamine salts, it is therefore reasonable and scientifically justified to use analogue primary alkylamines

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 principles across primary alkylamines and primary alkylamine salts as 

well as on a close relationship 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 `C16-18-(even numbered)-alkylamine acetates` and in line 

with the used approach by EPA, only source molecules having a linear alkyl chain have been considered 

for the read-across.

Generic structure of target molecule

C16-18-(even numbered)-alkylamine 

acetates

CAS No. 1273322-45-4
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Representative structures of source molecules from the alkylamines category used for read-across

C16-18-(even numbered)-alkylamines

CAS-No. 90640-32-7

C12-18-(even numbered)-alkylamines

CAS-No. 68155-27-1

C16-18-(even numbered,C18-unsaturated) 

alkylamines

CAS-No. 68037-95-6

C16-18-(even numbered, saturated and 

unsaturated) alkylamines

The applicability to read-across endpoint specific data from surrogate substances of the primary 

alkylamine category is supported by trend analysis performed by various groups which indicated 

consistency for various physical-chemical properties. In any case, from all appropriate source molecules 

those reflecting `worst-case`, e.g. lowest NOAEL, have been chosen for the read-across. 

2. Source Chemicals used for Read-Across

The following source molecules have been considered and endpoint specific data were used for informing 

data gaps of the target chemical `C16-18-(even numbered)-alkylamine acetates`. The source chemicals 

were selected on the basis of structural comparability and data availability.

Source Molecule CAS-No. Synonym

C16-18-(even number)-alkylamines 90640-32-7 Hydrogenated tallow alkylamines

C12-18-(even numbered)-alkylamines 68155-27-1 Coco alkylamines

C16-18-(even numbered, C18-unsaturated)-

alkylamines 68037-95-6 Tallow alkylamines

C16-18-(even numbered, saturated and 

unsaturated)-alkylamines 112-90-3

(Z)-Octadecy-9-enylamine, 

Oleylamine
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The following endpoints are covered either by data from the target chemical itself or by read-across using 

data from a.m. source molecules:

Endpoint considerations

Endpoint Approach 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 molecule

Skin sensitization Data from target molecule

Repeated dose toxicity Read-across from source molecules. Screening 

data (14-day repeated dose) from target molecule 

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 molecule, all source molecules belong to the category of “N-alkyl (C8-C18) primary amines 

and acetate salts” used by EPA for exemption from the requirements of a tolerance. Primary alkylamines 

and primary alkylamine acetates exhibit a consistent toxicity profile as demonstrated in the read-across 

justification below. Based hereupon not only comparability to the target molecule `C16-18-(even numbered)-

alkylamine acetates` 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 labeling of the target molecule. 
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3. Read-Across Justification

The basic chemical structure of primary alkylamine acetates can be illustrated as follows:

with n as descriptor of the carbon chain length (for target molecule n = 15, 17).

3.1 Physico-chemical properties

When considering target and source molecules, the available measured data and estimated values for the

below listed physical-chemical parameters melting and boiling points, vapor pressure, partition coefficient 

(measured as hydrochloride) and water solubility appear to be consistent with molecular mass and hence 

support the used read-across approach. All materials are in a comparable molecular weight range. 

Additionally, all compounds have comparable surfactant properties. 

- Molecular weight: 185 - 330

- Melting point: increases with carbon chain length

- Boiling point: increases with carbon chain length

- Vapour pressure: decreases with carbon chain length

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

- Log POW: comparable (approximately 2 – 3; measured as hydrochloride))

An inherent common property of all source and target molecules is that they accumulate at the interface 

between hydrophobic and hydrophilic phases rather than equilibrating between the two phases which 

complicates measurement and predictions of their physico-chemical properties. However, consistent with 

the size and nature of these molecules, measured and modeled vapor pressures are very low, so that all 

molecules can be considered to be essentially nonvolatile. Overall it is noted that the data and knowledge 

of primary alkylamines and respective salts support the conclusion that the source and target molecules

behave similarly or as a function of carbon chain length from the perspective of physico-chemical 

properties.

3.2 Human health aspect

From the structural and functional characteristics, the target molecule `C16-18-(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 molecule itself but also supported by missing of 

structural alerts for protein and/or DNA binding which is valid also for the category of primary alkylamines. 
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Additionally, acute 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 `C16-18-(even 

numbered)-alkylamine acetates` based on experimental data on the target molecule itself. Likewise, no 

indications of significant acute systemic dermal and acute systemic inhalation toxicity were derived, 

independent of carbon chain length and saturation degree.

For skin irritation, results from experimental data using reconstituted three-dimensional human epidermis 

models with the target molecule `C16-18-(even numbered)-alkylamine acetates` have been interpreted as 

dermal irritative properties which is in good agreement with comparable results from skin 

irritation/corrosion testing in vivo with the corresponding primary `C16-18-(even numbered)-alkylamine`.

With 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, readacross from the corresponding primary alkylamines is justified.

Repeated dose toxicity studies with the target molecule 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 alkylamines. In this respect, the comparison of the observed toxicity 

profiles (i.e. effects, affected organs/tissues, dose-response) from analogous repeated dose studies 

between the target molecule `C16-18-(even numbered)-alkylamine acetates` and the source molecule `C16-

18-(even numbered)-alkylamines` on one hand, and between saturated and unsaturated source molecules 

on the other hand 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 justified because all available data on this endpoint with primary alkylamines and salts

have shown a consistent toxicity profile independent on their 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 common metabolic principles valid for the entire category of N-alkyl (C8-C18) primary amines 

and acetate salts. Primary amines and primary amine acetates are biologically equivalent and follow the 

same metabolic pathways of oxidation by monoamine oxidases to generate the C8–C10 fatty acid and 

ammonia. The fatty acid would be degraded 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 may also be conjugated, whether by glucuronidation or 

sulfonation, and excreted directly.

4. Overall Assessment and Conclusions

According to REACH Annex XI, read-across for informing data gaps are appropriate if the result is 

adequate for the purpose of classification and labeling and/or risk assessment, if there is adequate and 

reliable coverage of the key parameters 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 data base on the target and source molecules in general and for the endpoints 

relevant for read-across in particular, a.m. conditions are met. The provided rational on structural, physicochemical, toxicological and toxicokinetic data, support the appropriateness of endpoint specific readacross to inform data gaps of the target molecule `C16-18-(even numbered)-alkylamine acetates` by using 

available data from source molecules from the primary alkylamines category. The provided read-across 

thus is robust and scientifically justified and additional safety factors in the risk characterization are 

considered not necessary.
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5. References

BUA (1994), Report 177, German GDCh-Advisory Committee on Existing Chemicals of Environmental 

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)