Reaction mass of ethyl dodecanoate and ethyl hexadecanoate and ethyl tetradecanoate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Oenanthic ether is negative for genemutations in bacteria based on read across from Zenoldie, which was tested in an Ames test (OECD TG 471)

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

2018

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Read-across information.

Justification for type of information:

See endpoint summary

Reason / purpose for cross-reference:

read-across source

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Conclusions:

The substance is not mutagenic in the Salmonella typhimurium and Escherichia coli reverse mutation assay, based on the results of the source substance.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1997

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

The information is reliable and adequate for covering the endpoint.

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Qualifier:

according to guideline

Guideline:

OECD Guideline 472 (Genetic Toxicology: Escherichia coli, Reverse Mutation Assay)

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

S. typhimurium TA1535 hisG46 rfa uvrB; S. typhimurium TA1537 hisC3076 rfa uvrB; S. typhimurium TA 1538 hisD3052 rfa uvrB; S. typhimurium TA98 hisD3052 rfa uvrB pKMl01; S. typhimurium TA100 hisG46 rfa uvrB pKM101; E. coli WP2 uvrA trp

Species / strain / cell type:

S. typhimurium, other: TA 1535, TA 1537, TA 1538, TA 98 and TA 100

Additional strain / cell type characteristics:

not applicable

Species / strain / cell type:

E. coli WP2 uvr A

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced rats (S9 mix)

Test concentrations with justification for top dose:

Exp 1: with and without S9-mix, 10 dose levels between 9.8 and 5000 µg/plate
Exp 2 with and without S9-mix
for TA1535, TA1537 and WP2 uvrA: 6 dose levels between 39.1 and 12500 µg/plate
for TA1538, TA98 and TA100: 8 dose levels between 39.1 and 12500 µg/plate

Vehicle / solvent:

dimethyl sulphoxide (DMSO)

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

and 9-Aminoacridine, 2-Nitrofluorene and 2-Aminoanthracene

Details on test system and experimental conditions:

An aliquot of 0.1 ml of a 10 hour bacterial culture and 0.5 ml S9 mix or 0.5 ml 0.1 M phosphate buffer (pH 7.4) were placed in glass bottles. An aliquot of 100 µl of the test solution was added, followed immediately by 2 ml of histidine/tryptophan deficient agar. The mixture was thoroughly shaken and overlaid onto previously prepared petri dishes containing 25 ml minimal agar. Each petri dish was individually labelled with a unique code corresponding to a sheet, identifying the dish's contents. Plates were also prepared without the addition of bacteria in order to assess the sterility of the test substance, 59 mix and phosphate buffer. All plates were incubated at 37°C for 3 days. After this period the appearance of the background bacterial lawn was examined and revertant colonies counted using a Seescan Automatic Colony Counter.

Evaluation criteria:

Please see below.

Statistics:

no

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

Toxicity was observed towards all the strains at the top three dose levels, down to 625 µg/plate for TA1538 and down to 312.5 µg/plate for TA98 and TA100. In the second mutation test toxicity was observed towards all the strains at the top dose level and down to 312.5 µg/plate for TA1538, TA98 and TA100.
No substantial increases in revertant colony numbers of any of the tester strains were observed following treatment at any dose level, in the presence or absence of S9 mix, in either mutation test.

Conclusions:

It is concluded that the results obtained with the test substance in Salmonella typhimurium strains TA 1535, TA 1537, TA 1538, TA 98 and TA 100, and in the Escherichia coli strain WP2 uvrA, in the absence and presence of the S9-mix, indicate that the test substance is not mutagenic under the conditions of this test.

Executive summary:

In a study, performed in accordance with GLP and OECD guideline 471 and 472, the test substance was examined for its possible mutagenic activity in the bacterial reverse mutation test using the Salmonella typhimurium strains TA 1535, TA 1537, TA 1538, TA 98, TA 100 and the Escherichia coli strain WP2uvrA, in the absence and presence of a liver fraction of Aroclor 1254-induced rats for metabolic activation (S9-mix).The test substance was diluted in dimethylsulphoxide (DMSO). Two independent mutation tests were performed. A top dose level of 5000 µg/plate, based on a preliminary test, was chosen for the first mutation study. Other dose levels used were a series of 2-fold dilutions of the highest concentration. A total of ten dose levels were used in the first test to ensure that sufficient non-toxic concentrations were assessed. Toxicity was observed towards all the strains at the top three dose levels, down to 625 µg/plate for TA1538 and down to 312.5 µg/plate for TA98 and TA100. For the second mutation test a top dose level of 12500 µg/plate was chosen and the bottom two dose levels not included for TA1535, TA1537 and WP2 uvrA. Toxicity was observed towards all the strains at the top dose level and down to 312.5 µg/plate for TA1538, TA98 and TA100. No evidence of mutagenic activity was seen at any dose level in either mutation test. The concurrent positive control compounds demonstrated the sensitivity of the assay and the metabolising activity of the liver preparations. It is concluded that the test substance shows no evidence of mutagenic activity in this bacterial system.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Genetic toxicity is assessed based on read-across from Zenolide to Oenanthic ether. The executive summary of the source information on the substance is presented below, followed by the read-across rationale.

Zenolide Ames test information

In a study, performed in accordance with GLP and OECD guideline 471 and 472, the test substance was examined for its possible mutagenic activity in the bacterial reverse mutation test using the Salmonella typhimurium strains TA 1535, TA 1537, TA 1538, TA 98, TA 100 and the Escherichia coli strain WP2uvrA, in the absence and presence of a liver fraction of Aroclor 1254-induced rats for metabolic activation (S9-mix).The test substance was diluted in dimethylsulphoxide (DMSO). Two independent mutation tests were performed. A top dose level of 5000 µg/plate, based on a preliminary test, was chosen for the first mutation study. Other dose levels used were a series of 2-fold dilutions of the highest concentration. A total of ten dose levels were used in the first test to ensure that sufficient non-toxic concentrations were assessed. Toxicity was observed towards all the strains at the top three dose levels, down to 625 µg/plate for TA1538 and down to 312.5 µg/plate for TA98 and TA100. For the second mutation test a top dose level of 12500 µg/plate was chosen and the bottom two dose levels not included for TA1535, TA1537 and WP2 uvrA. Toxicity was observed towards all the strains at the top dose level and down to 312.5 µg/plate for TA1538, TA98 and TA100. No evidence of mutagenic activity was seen at any dose level in either mutation test. The concurrent positive control compounds demonstrated the sensitivity of the assay and the metabolising activity of the liver preparations. It is concluded that the test substance shows no evidence of mutagenic activity in this bacterial system.

The endpoint mutagenic properties in bacteria of Oenanthic ether using read across from Zenolide (CAS 54982-83-1)

 

Introduction and hypothesis for the read across

Oenanthic ether consists of 3 main constituents and a number of impurities. All are ethyl esters, despite the name ether, of long chain carboxylic acids. The major constituent has a C12 chain, the two minor ones have C14 and C16 saturated carbon chain. For the target substanceOenanthic ethergene mutation data in bacteria (Ames) are not available.In accordance with Article 13 of REACH, lacking information can be generated by other means, i.e. applying alternative methods such as QSARs, grouping and read-across. For assessing the mutagenicity properties of Oenanthic ether, the analogue approach is selected because for the closely related analogue Zenolide Ames information is available which can be used to read across.

Hypothesis: Oenanthic etheris negative in the Ames test based on the negative results of the analogue Zenolide in the Ames test.

Available information:In an Ames study (OECD guideline 471, Rel. 1), Zenolide was examined for its possible mutagenic activity in the bacterial reverse mutation test using the Salmonella typhimurium strains TA 1535, TA 1537, TA 1538, TA 98, TA 100 and the Escherichia coli strain WP2uvrA, in the absence and presence of metabolic activation (S9-mix). Two independent mutation tests were performed, Zenolide was tested up to and including cytotoxic levels. No evidence of mutagenic activity was seen at any dose level in either mutation test and Zenolide was considered negative for mutagenicity.

Target and Source chemical:

Chemical structures of the target chemical and the source chemical(s) are shown in the data matrix, including physico-chemical properties and available toxicologicalinformation. Furthermore, a full list of constituents of Oenanthic ether, including information relevant for read-across, is given in the data matrix.

Purity / Impurities:

The purity and unidentified impurities of the target chemical and source chemical are not expected to influence the potential for bacterial mutagenicity.

Analogue justification

According to Annex XI 1.5 read across can be used to replace testing when the similarity can be based on a common backbone and a common functional group. When using read across the result derived should be applicable for C&L and/or risk assessment and it should be presented with adequate and reliable documentation. The reasoning below fulfils this documentation.

Analogue selection: For Oenanthic etherZenolide was considered an appropriate analogue because Zenolide has a similar fatty acid type of acetic ester as Oenanthic ether and for Zenolide skin Ames test information is available. 

Structural similarities and differences:All constituents of Oenanthic ether and Zenolide contain ethyl esters and a long alkane chain (C8-C1 and C12, respectively) and therefore have a similar backbone and functional group: i.e. esters of long chain carboxylic acids. The difference is that all constituents of Oenanthic ether have linear alkyl chains, whereas Zenolide has a cyclic aliphatic alkyl chain, connected by Ethylene glycol. Zenolide has therefore a double ester which is expected to be more electrophilic.

Toxico-kinetic, absorption: Oenanthic ether and Zenolide have chemical indicate similar absorption because of similar molecular weights and log KowsMetabolism: Fatty acid ethyl esters will be hydrolysed/metabolised by carboxyl esterases abundantly available throughout the body (Toxicological handbooks).

Toxico-dynamics: The reactive site of Oenanthic Ether and Zenolide is the ester bond which has only a non-branched alkyl group in its vicinity and therefore is expected to react very similar. Zenolide has a double ester in each other’s vicinity and may therefore be slightly more reactive and more ‘conservative’ as is also indicated in the OECD Toolbox (4.2) where there is a highlight for Hacceptor-path3-Hacceptor. This is not applicable for Zenolide, because also Ethylene glycol is active for this alert in the Toolbox and the latter is negative for genotoxicity.

Uncertainty of the prediction:There are no remaining uncertainties other than already discussed above. 

Data matrix

The relevant information on physico-chemical properties and toxicological characteristics are presented in the data matrix below.

Conclusions per endpoint for hazard in the risk assessment

For Oenanthic ether no Ames information is available. From the analogue Zenolide such information is available and this can be used for read across. When using read across, the result derived should be applicable for C&L and/or risk assessment and be presented with adequate and reliable documentation. This latter documentation is presented here in the current document. For Zenolide a well conducted negative Ames test according to OECDTG 471, Rel. 1, is available. Therefore also Oenanthic ether will be negative in the Ames test.

Final conclusion for hazard and risk assessment: Oenanthic ether is negative in the Ames test.

 

Data matrix to support the read across to Oenanthic ether from Zenolide for mutagenicity (Ames)

Chemical names for   Oenanthic ether#	ethyl octanoate (C8*)	ethyl decanoate (C10*)	ethyl dodecanoate (C12*)	ethyl tetra-decanoate (C14*)	ethyl hexa-decanoate (C16*)	ethyl octa-decanoate (C18*)	ethyl (9Z)-octadec-9-enoate (C18*)	ethyl (9Z,12Z)‐ octa-deca‐,12‐di enoate (C18*)	Zenolide (‘C12’)
Target									Source
CAS#	106-32-1	110-38-3	106-33-2	124-06-1	628-97-7	111-61-5	544-35-4	111-62-6	5982-83-1
Structure
% in product	<10	<10	35-55	15-30	5-15	<10	<10	<10
EC No. 945-734-0#									259-423-6
Registration 2018	EpiSuite	EpiSuite	EpiSuite	EpiSuite	EpiSuite	EpiSuite	EpiSuite	EpiSuite	Registered
Vp (Pa) 0.12 meas)#	31.4 (est.)	5.70 (est.)	1.17 (est.)	0.34 (est.)	0.036 (est.)	0.0084 (est.)	0.0081 (est.)	0.0067 (est.)	0.028  (exp.)
WS (mg/L) 1.6 meas)#	45.6 (est.)	4.8 (est.)	0.41 (est.)	0.037 (est.)	0.0037 (est.)	0.0004 (est.)	0.0006 (est.)	0.0009 (est.)	75  (exp.)
Log Kow 4.6 meas)#	3.8 (est.)	4.8 (est.)	5.8 (est.)	6.8 (est.)	7.7 (est.)	8.4 (est.)	8.5 (est.)	8.3 (est.)	3.65 (exp.)
Human health
Mutagenicity (Ames)	Negative (Read across)	Negative (Read across)	Negative (Read across)	Negative (Read across)	Negative (Read across)	Negative (Read across)	Negative (Read across)	Negative (Read across)	Negative (OECD TG 471)

*The C’s are related to the chain length not the overall number of Cs (as would be presented in the Empirical formula); (est.) = estimated using EpiSuite; (exp.) = experimental;#In this column the values are for Oenanthic ether as such.

 

Justification for classification or non-classification

Based on the results, the substance does not need to be classified for genetic toxicity according to EU CLP (EC No. 1272/2008 and its amendments).