Benzoic acid, 4-hydroxy-, C18-22-alkyl esters

Endpoint summary

Additional information

Justification for grouping of substances and read-across

No data are available on the acute toxicity of Benzoic acid, 4-hydroxy-, C18-22-alkyl esters (CAS 201305-16-0). In order to fulfil the standard information requirements set out in Annex VIII in accordance with Annex XI, 1.5, of Regulation (EC) No 1907/2006, read-across from structurally related substances is conducted.

In accordance with Article 13 (1) of Regulation (EC) No 1907/2006, "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, which includes the use of information from structurally related substances (grouping or read-across).

Having regard to the general rules for grouping of substances and read-across approach laid down in Annex XI, Item 1.5, of Regulation (EC) No 1907/2006, whereby physicochemical, toxicological and ecotoxicological properties may be predicted from data for reference substance(s) by interpolation to other substances on the basis of structural similarity, the substances depicted in the table below are selected as source substances for assessment.

The read-across is based on the identified structural similarities and the likelihood of common breakdown products by biological processes (metabolism). A detailed analogue approach justification is provided in the technical dossier (see IUCLID Section 13).

CAS	201305-16-0 TARGET SUBSTANCE	68411-27-8	99-76-3
Chemical Name	Benzoic acid, 4-hydroxy, C18-22-alkyl esters	Benzoic acid, C12-15-alkyl esters	Methyl 4-hydroxybenzoate
MW	390.60-446.71 g/mol	311 g/mol	152.15 g/mol
Genetic Toxicity in vitro: gene mutation in bacteria	RA: CAS 68411-27-8	Experimental result: Negative	--
Genetic Toxicity in vitro: cytogenicity in mammalian cells	RA: CAS 99-76-3	--	Experimental result: Ambiguous
Genetic Toxicity in vitro: gene mutation in mammalian cells	--	--	--
Genetic Toxicity in vivo	RA: CAS 99-76-3	--	Experimental result: Negative

Lack of data is indicated by --

Discussion

In vitro

- Gene mutation in bacteria

No studies on the induction of gene mutations in bacteria are available for Benzoic acid, 4-hydroxy, C18-22-alkyl esters (CAS 201305-16-0). However, there are two reliable studies for the structurally related substance Benzoic acid, C12-15-alkyl esters (CAS 68411-27-8) available, which are used for read-across based on the analogue approach.

A reverse mutation assay (Ames test) was conducted with Benzoic acid, C12-15-alkyl esters (CAS 68411-27-8) according to OECD guideline 471 and in compliance with GLP (Bowles, 2005). The tester strains Salmonella typhimurium TA 1535, TA 1537, TA 98 and TA 100 and Escherichia coli WP2 uvrA- were treated with the test material using the plate incorporation method at five concentrations, in triplicate, both with and without metabolic activation (rat liver S9-mix).Based on the results of a preliminary toxicity test, the two independent main experiments were performed with test concentrations from 50 to 5000 µg/plate. Concurrent negative, vehicle (acetone) and positive controls were included in the test, which all yielded the expected results, thus validating the sensitivity of the assay and the efficacy of the S9-mix. The test material caused no visible reduction in the growth of the bacterial background lawn at any concentration. The test material was, therefore, tested up to the maximum recommended concentration of 5000 µg/plate. A precipitate (oily in appearance) was observed at and above 1500 µg/plate (with and without S9-mix), without preventing the scoring of revertant colonies. No biologically significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any concentration, either with or without metabolic activation. A small increase in revertant colony frequency was observed in tester strain TA 100, with S9-mix only, at 50 µg/plate in Experiment 1. However, this increase was not reproduced in Experiment 2 and was considered to be of no biological relevance because there was no evidence of a dose-response relationship or reproducibility. Furthermore, the revertant counts at 50 µg/plate were well within the in-house range of the testing facility for the tester strain, and the fold increase was only 1.32 times the concurrent vehicle control. In conclusion, the test material was considered to be not mutagenic under the conditions of this test.

A second Ames test is available for Benzoic acid, C12-15-alkyl esters (CAS 68411-27-8), conducted similarly to OECD guideline 471, but not in compliance with GLP (Phelps, 1994). The test item was found to be not mutagenic in Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and TA 1538 in the plate incorporation assay both with and without metabolic activation (rat liver S9-mix).

- Gene mutation in mammalian cells

No data are available on gene mutation in mammalian cells in vitro.

 

- Cytogenicity in mammalian cells

No studies on the induction of cytogenicity in mammalian cells in vitro are available for Benzoic acid, 4-hydroxy, C18-22-alkyl esters (CAS 201305-16-0). However, there are two studies for the structurally related Methyl 4-hydroxybenzoate (CAS 99-76-3) available, which are used for read-across based on the analogue approach.

An in vitro mammalian chromosome aberration test similar to OECD guideline 473 was performed with Methyl 4-hydroxybenzoate (CAS 99-76-3) in cells of a clonal sub-line derived from the lung of a male Chinese hamster (CHL) (Matsuoka et al., 1979). Triplicate cell cultures were treated with the test substance at 3 concentrations including 125 µg/mL for a period of 3 h both with and without metabolic activation. In the absence of metabolic activation, no significant increase in the induction of chromosomal aberrations (1%) was observed after treatment with 125µg/mL test item. In the presence of metabolic activation, the induction of chromosomal aberrations was significantly increased (13%) as compared to solvent controls. Chromosomal aberrations observed consisted of gaps, breaks, exchanges and ring formation. The authors concluded methyl p-hydroxybenzoate to be positive for clastogenicity with metabolic activation under the conditions of this test. However, a sample was only concluded to be negative, if less than 4.9% of the aberration was detected even when doses elevated to sub-lethal amounts and where almost no mitosis was observed. No raw data are given for the test materials, and no information is given on the results of the solvent and the negative controls. Furthermore, no cytotoxicity data are given. It is therefore unclear, whether the induction of chromosome aberrations is the consequence of cytotoxicity rather than a real genotoxic effect. Thus, the result with metabolic activation is ambiguous and the data are not sufficient for current regulatory purposes. The negative result obtained in the absence of metabolic activation is reliable, as a sufficient number of positive control substances proved the sensitivity of this test.

In a further in vitro mammalian chromosome aberration test, Methyl 4-hydroxybenzoate (CAS 99-76-3) induced chromosomal aberrations in CHL cells in the absence of a metabolic activation system (Ishidate et al., 1978).

 

In vivo

No studies on the induction of gene mutations in mammals are available for Benzoic acid, 4-hydroxy, C18-22-alkyl esters (CAS 201305-16-0). However, there are two studies for the structurally related Methyl 4-hydroxybenzoate (CAS 99-76-3) available, which are used for read-across based on the analogue approach.

A dominant lethal test, indicative of chromosomal aberrations in germ cells, was performed with Benzoic acid, 4-hydroxy, C18-22-alkyl esters (CAS 201305-16-0) (Litton Bionetics Inc., 1974). In the experiment conducted similarly to OECD guideline 478, 10 male Sprague-Dawley rats were orally administered the test substance via gavage at doses of 5, 50, 500 and 5000 mg/kg bw. The animals were treated either in a single exposure or in a repeated exposure on 5 consecutive days, 24 h apart. Over a period of 8 weeks post dosing, the males were sequentially mated to 2 females per week for 8 weeks (7 weeks in the subacute study). Females were killed 14 days after separating from the males, and at necropsy the uterus was examined for deciduodimata, late foetal deaths and total implantations. Corpora lutea, early foetal deaths, late foetal deaths and total implantations per uterine horn were recorded. Significant decreases in average Corpora lutea and preimplantation losses were seen in the experimental groups of Weeks 1, 4 and 5 after single exposure to 5, 50, and 500 mg/kg bw. Significant increases in average implantations and Corpora lutea were seen in the experimental groups at week 4. In contrast, significant, dose-related increases were seen in average resorptions at 500 mg/kg bw/day at Weeks 1 and 6 after repeated exposure to 5, 50, and 500 mg/kg bw. Animals treated with 5000 mg/kg bw (both single and repeated exposure) did not significantly vary from those of the vehicle control group, except for a significant increase in the number of dead implants per total of weeks 2 and 6. In these respective examples, 1 and 2 were found with 6 or more dead embryos. Omission of these animals from the sample would bring the calculated values into line with those determined for the vehicle controls. Whether these are sporadic phenomena or due to compound effects could not be determined from the data, although the former is suggested by the lack of effect on the other parameters. The positive control substance Triethylene Melamine caused significant preimplatation loss and embryo resorptions during the first 5 weeks. Comparing the values obtained by application with 5000 mg/kg bw/day with those of the animals treated with 5, 50, and 500 mg/kg bw/day revealed no dose response or time trend patterns. Based on the results of this study, the test item is considered to be not genotoxic to mammals under the conditions of this test.

 

A chromosome aberration assay similar to OECD guideline 475 was conducted with Benzoic acid, 4-hydroxy, C18-22-alkyl esters (CAS 201305-16-0) (Litton Bionetics Inc., 1974), in which the test substance was applied by oral gavage to 5 male Sprague-Dawley rats at doses of 5, 50 and 500 mg/kg bw. The animals were treated either in a single exposure or in a repeated exposure on 5 consecutive days, 24 h apart. Concurrent control animals were treated either with the vehicle (physiological saline, vehicle control) or with Triethylene Melamine (0.3 mg/kg bw via intraperitoneal injection, positive control). 6, 24 and 48 hours after treatment, the animals were sacrificed and bone marrow from both femurs was prepared for chromosome analysis. No increase in chromosome aberrations was observed with the test substance. The concurrent positive and vehicle controls gave the expected results. Based on the results of this study, the test item is considered to be not genotoxic to mammals under the conditions of this test.

 

Conclusion for genetic toxicity

In conclusion, assessment of the available data together with the lack of functional groups associated with genetic toxicity indicates that Benzoic acid, 4-hydroxy, C18-22-alkyl esters (CAS 201305-16-0) is not genotoxic to mammals.

Justification for selection of genetic toxicity endpoint
Hazard assessment is conducted by means of read-across from structural analogues/surrogates. No study was selected, since none of the available in vitro genetic toxicity studies was positive. All available studies are adequate and reliable based on the identified similarities in structure and intrinsic properties between source and target substance and overall quality assessment (refer to the endpoint discussion for further details).

Short description of key information:
OECD Guideline 471 (Ames test): negative
similar to OECD Guideline 473: ambiguous
similar to OECD Guideline 478: negative

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Based on read-across from structurally similar substances, the available data on genetic toxicity of Benzoic acid, 4-hydroxy, C18-22-alkyl esters (CAS 201305-16-0) do not meet the criteria for classification according to Regulation (EC) 1272/2008 or Directive 67/548/EEC, and are therefore conclusive but not sufficient for classification.