Butanoic acid, 3a,4,5,6,7,7a-hexahydro-4,7-methano-1H-indenyl ester

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The substance is negative in the Ames test (OECD TG 471)

The substance is negative in the chromosome aberration test (OECD TG 473)

The substance is negative in the in vitro mammalian cell gene mutation test (OECD TG 476) based on read across from Cyclacet

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

For Cyclobutanate two out of three experimental tests required for Annex VIII and higher marketed tonnages are available: Ames and chromosomal aberrations (CAB). To assess the gene-mutations in mammalian cells the experimental data of Cyclacet set is used for read across. A full read across document is presented at the end of the section and is also added as an attached document.

AMES test with Cyclobutanate

A bacterial reverse mutation assays (Ames test) was performed according to OECD 471 using Salmonella thyphimurium strains TA1535, TA1537, TA98 and TA100, and Escherichia coli strain WP2uvrA‾. The bacterial cultures were treated according to the Ames plate incorporation method at up to six dose level, in triplicate, both with and without the addition of a rat liver homogenate metabolising system (10% liver S9). The dose range was determined in a preliminary toxicity assay and ranges between 1.5 and 5000 µg/plate, depending on bacterial tester strain type and presence or absence of S9 -mix.

The mean number of revertants observed in the negative controls (DMSO) for each of the test strains was within acceptable historical negative control ranges. Furthermore, all of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with and without metabolic activation. All test strains demonstrated appropriate phenotypic characteristics.

No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation. Therefore, the testing material was considered to be non-mutagenic under the conditions of this test.

Chromosomal aberration test (cytogenicity) with Cyclobutanate

This study was conducted according to a method which was designed to assess the potential chromosomal mutagenicity of Cyclobutanate on the metaphase chromosomes of the Chinese Hamster Lung (CHL) cell line according to the requirements of OECD Guideline 473. Duplicate cultures of CHL cells were treated with Cyclobutanate at several dose levels, together with vehicle (DMSO) and positive controls. Five exposure groups were used: Experiment 1 included a 6(18)-hour exposure, both with and without the addition of an induced rat liver homogenate metabolising system; Experiment 2 included a 24-hour continuous exposure without metabolic activation, a 48-hour continuous exposure without metabolic activation and a repeat of the 6(18)-hours exposure with metabolic activation.The dose levels evaluated in the main experiments were selected from a range of dose levels based on the results of a preliminary toxicity test and were in the range of 8.70 to 34.5 µg/ml for the 6(18)-hour exposure, without S9, 137.7 to 550.8 µg/ml for the with-S9 exposure, in both Experiment 1 and 2, and 4.35 to 34.5 µg/ml for the 24 and 48-hour treatments. The vehicle (solvent) controls gave frequencies of cells with aberrations within the range expected for the CHL cell line. All the positive control chemicals induced highly significant increases in the frequency of cells with aberrations indicating the satisfactory performance of the test and of the activity of the metabolising system. Cyclobutanate did not induce any toxicologically significant increases in the frequency of cells with aberrations in any of the exposure groups. Cyclobutanate was shown to be toxic to CHL cells in vitro and optimal levels of toxicity were achieved in all exposure groups. Cyclobutanate was shown to be non-clastogenic to CHL cells in vitro.

For the key study with regards to gene mutation in mammalian cells, read-across was done to the structurally related Cyclacet. Cyclobutanate has the same tricyclodecenyl fused ring backbone as Cyclacet. Cyclobutanate, however, has a butyl ester, while Cyclacet has an acetic ester attached to this backbone. This longer alkyl chain of Cyclobutanate is considered minimally relevant for the genotoxicity profile in the in vitro Mammalian Cell Gene Mutation Test, which is performed according to OECD 476, because it does not present additional reactivity, which is a key parameter for genotoxicity.

MLA-information from the analogue Cyclacet

The test article, Cyclacet, was tested for its potential to induce mutations at the thymidine kinase locus of L5178Y TK+mouse lymphoma cells in vitro. The concentrations of test article tested with and without S-9 activation in the Range Finding Test were 0.1, 0.5, 1.0, 5.0, 10, 50, 100, 500, 1000, and 5000 µg/mL. Relative Suspension Growth (RSG) was used to measure toxicity. The RSG for cultures treated with Cyclacet without activation indicated that Cyclacet was toxic at 50 µg/mL and above. Cultures treated with 50 µg/mL had 16% RSG. The cultures treated with higher concentrations had 0% RSG. The RSG for cultures treated with Cyclacet with S-9 activation indicated that Cyclacet was completely toxic, i.e., 0% RSG, at 500 µg/mL and above. The culture treated with 100 µg/mL had 74% RSG.

The Definitive Mutation Assay was performed using a 4-hour treatment period at test article concentrations ranging from 19 to 170 µg/mL without activation and from 109 to 300 µg/mL with S-9 activation. Cultures were selected for cloning for mutant selection based on their RSG. All of the cloned cultures, both with and without activation, had Mutant Frequencies (MF) that were similar to the average MF of their concurrent solvent control cultures. The Relative Total Growth (RTG) for the cloned cultures ranged from 17% to 99% for cultures treated without activation and from 42% to 90% for cultures treated in conjunction with exogenous activation.

Since it is ideal to have some cloned cultures that have between 10% and 30% RTG for evaluating a test articles mutagenic potential, a repeat assay with a 4-hour exposure period with activation was conducted. Cultures were treated with concentrations ranging from 200 to 500 µg/mL with 20 µg/mL increments between doses. The results for the repeat of the with S-9 activation portion of the Definitive Mutation Assay also showed that the treated cultures all had MFs that were similar to the average MF of the solvent control cultures. The RTG for these cultures ranged from 0% to 104%. Therefore the validity criteria for cytotoxicity were met. Under the test conditions, the results of the Definitive Mutation Assay are considered negative.

The Confirmatory Mutation Assay was conducted without activation with a 24-hour exposure period. Cultures were treated with concentrations of 1.0, 5.0, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, and 140 µg/mL. The cultures treated with 30 to 140 µg/mL were cloned for mutant selection. All of the cultures had MFs that were similar to the average MF of the solvent controls. The RTG for the cloned cultures ranged from 3% to 59%.

Under the test conditions, the results of the Definitive and Confirmatory Mutation Assays are considered negative (i.e. the test substance is not mutagenic).

Cyclobutanate (Cas no 113889-23-9; 5-yl) and its genemutations in mammalian cells using read across information from Cyclacet (2500-83-6)

Introduction and hypothesis for the analogue approach

Cyclobutanate is a butyl ester attached to a tricyclodecenyl fused ring backbone. For this substance an Ames test is available and a chromosomal aberration test but not a gene mutation assay with mammalian cells. In accordance with Article 13 of REACH, lacking information can be generated by means of applying alternative methods such as in vitro tests, QSARs, grouping and read-across. For assessing the genotoxicity of Cyclobutanate the analogue approach is selected because for one closely related analogue reliable gene-mutation study in human cells is available.

Hypothesis: Cyclobutanate has similar genotoxicity compared to Cyclacet. This source chemical is very similar in structure, having two methyl groups less than Cyclobutanate in the alkyl chain. In addition, the similarity in genetic profile is supported with non-genotoxicity in the Ames test performed in both substances. Therefore the experimental in vitro genemutation information from Cyclacet can be used for Cyclobutanate.

Available experimental information: The target and the source chemicals are both negative in well conducted Ames tests including Salmonella typhimurium strain TA102, receiving Klimisch codes 1. Cyclobutanate is negative in a chromosomal aberration assay (OECD TG 473).

The source substance Cyclacet is negative in the mouse lymphoma assay including scoring small colonies, being indicative for the absence of chromosomal aberrations (OECD TG 476). The information from the source substance Cyclacet has a sufficient reliability and receives a Klimisch code of 1.

Target chemical and source chemical(s)

Chemical structures of the target chemical and the source chemical are shown in the attachment. Physico-chemical properties thought relevant for genotoxicity are listed in the data matrix (see Data matrix).

Both esters have a tricyclodecenyl fused ring structure with an unsaturated bond in the right ring. On the left ring an ester bond is attached with an alkyl chain (see data matrix).

Purity / Impurities

The constituents and impurities of the source and the target substance are all similar and do not indicate a genotoxic potential. The impurities are all below < 10%. The unsaturated bond in the right ring of both substances can be at the 5-yl or the 6-yl position, which is not thought to make a difference for the genotoxic potential because such a double bond at 5-yl or at 6- yl is not expected to be reactive as such.

Analogue approach 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 as is presented below.

Structural similarities and differences: The target and the source chemicals have a tricyclodecenyl fused ring structure with an unsaturated bond in the right ring. On the left side of the ring an ester bond is attached with a short alkyl chain (C4). The alkyl chain of Cyclobutanate (C4) is two methyl groups longer than Cyclacet (C2). This difference between the target and source chemical are not expected to behave differently in relation to genotoxicity because the alkyl side chains (butyl versus ethyl) are not expected to influence significantly genotoxicity of these chemicals.

Toxico-kinetic: The source chemical and the target chemical have similar toxico-kinetic characteristics based on the similarity in chemical structure and physico-chemical properties. The molecular weight of the target chemical is 220, whereas for Cyclacet it is 192. They are both liquids. Their vapour pressures 11.2 and 0.67, respectively, differ somewhat but is not crucial for their genotoxic potential. It can be seen that the water solubility of the Cyclobutanate is somewhat lower and the log Kow somewhat higher compared to Cyclacet which is expected because the latter being an acetic (ethyl) ester instead of a butyl one. For the toxico-kinetic behaviour of both chemicals these physico-chemical differences are expected to be minimal. The target and the source chemical will both be metabolised into the same alcohol (3385-61-3) and the respective acid, butanoic-acid for the target and acetic acid for the source chemical. This metabolisation is illustrated in the toxico-kinetic section.

Uncertainty of the prediction: The genotoxicity profile has a high certainty because of close similarity between Cyclobutanate and Cyclacet, the target chemical and the source chemical. The differences in the alkyl chains are not expected to have any impact on the genotoxic potential because of absence of activating fragments. The available genotoxicity data e.g. Ames show similar results. Both these chemicals are expected to metabolise into the respective Cycla-alcohol and their acids. Therefore this read across is highly certain and adequate for this purpose.

Data matrix

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

Conclusions

For Cyclobutanate no information is available for mutations in mammalian cells, while for a close analogue such information is available, which 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. For the target substance, Cyclobutanate, a well conducted Ames test and a chromosomal aberration test is available which are both clearly negative (OECD TG 471). For the source substance Cyclacet (ethyl side chain) a negative Ames test and a well conducted mammalian in vitro genotoxicity assay is available (OECD TG 476) (Reliability 1) showing absence of gene-mutations in mammalian cells. Furthermore, no small colonies were found in this MLA test indicating that Cyclacet does not induce chromosomal aberrations. Therefore read across to Cyclobutanate from Cyclacet is adequate and Cyclobutanate is considered to be negative for in vitro gene mutation in mammalian cells based on similarity in structure and similarity in results for Ames and cytogenicity.

Final conclusion on hazard: Cyclobutanate is negative for genotoxicity for mutagenicity in bacterial and mammalian cells and cytogenicity.

Data matrix of Cyclobutanate for genotoxicity using read across from Cyclacet

Common names	Cyclobutanate (Target)	Cyclacet (Source)
Chemical structures
Cas no of the main isomer Cas no of the generic Reach registration no Empirical formula	113889-23-9 (5-yl) 113889-23-9 441-420-8 C14H20O2	2500-83-6 (5-yl) 54830-99-8 911-369-0 C12H16O2
Molecular weight Physico-chemical data	220	192
Physical state	liquid	liquid
Boiling point (°C)	275	247
Vapour pressure (Pa) (measured)	11.2	2.1
Water solubility (mg/l) (measured)	11.5	186
Log Kow (measured)	4.48	3.9
Human health endpoints
Genotoxicity -Ames test	Negative (OECD TG 471)	Negative (OECD TG 471)
Genotoxicity in vitro-Chromosomal aberrations	Negative (OECD TG 473)	Negative, no small colonies found in the MLA test being an indicator for the absence of chromosomal aberrations.
Genotoxicity in vitro MLA	Read across	Negative (OECD TG 476)

 

 

 

Justification for classification or non-classification

Based on the results of the genemutations in bacterial cells (Ames test), cytogenicity information (chromosome aberration test) and the genemutations in mammalian cells as read across from the structurally similar source substance Cyclacet, the substance is not genotoxic and therefore does not have to be classified for genotoxicity in accordance with EU CLP (EC no. 1272/2008 and its amendments).