3a,4,5,6,7,7a-hexahydro-4,7-methano-1H-indenyl propionate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Cyclaprop tested for gene mutation in bacteria (OECD 471, GLP): Not mutagenic

Cyclaprop for gene mutations in mammalian cells is based on read across from Cyclacet, which was tested in OECD 476: Not mutagenic

Cyclaprop for cytogenicity is based on read across from Cyclobutanate, which was tested in OECD 473: Non-clastogenic.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

In this section the experimental information on genotoxicity of Cyclaprop, Cyclacet and Cyclobutanate is presented first. Thereafter the read across justification is presented.

Cyclaprop in Ames
The test substance was evaluated in a bacterial reverse mutation assay performed according to OECD 471 and following GLPin two independent studies employing Salmonella typhimurium strains TA97a, TA98, TA100, TA102 and TA1535 both in the presence and absence of an exogenous metabolic activation system. Positive and negative controls were included in each study. The duration of each study was 48 hours. The test item was dissolved in DMSO and applied once at test initiation with concentrations ranging from 0.0016 mg/plate - 1.6 mg/plate. The validity criteria of the test were fulfilled. Cyclaprop did not induce any mutagenicity. Cytotoxicity occurred at the highest concentrations tested, therefore these concentrations were not included in the evaluation. The findings of this study show the test substance to be negative for the induction of mutagenicity in the bacterial reverse mutation test.

Cyclacet in vitro gene mutation assay with mammalian cells

No gene mutation study was available for Cyclaprop. However, an available study on the structural similar substance Cyclacet could be used for read across.

The test article was tested for its potential to induce mutations at the thymidine kinase locus of L5178Y TK+ mouse lymphoma cells in an OECD 476 study performed according to GLP. 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 without activation indicated that the test substance 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 S-9 activation indicated that the test substance 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%. 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 between 1.0 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 In vitro cytogenicity assay with mammalian cells

No cytogenicity study was available for Cyclaprop. However, an available study on the structural similar substance Cyclobutanate could be used for read across.

In this in vitro chromosome aberration test performed according to OECD 473 and following GLP, the potential chromosomal mutagenicity of the test substance on the metaphase chromosomes of the Chinese Hamster Lung (CHL) cell line was determined. Duplicate cultures of CHL cells were treated with the test substance 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. The test substance did not induce any toxicologically significant increases in the frequency of cells with aberrations in any of the exposure groups. The test substance was shown to be toxic to CHL cells in vitro and optimal levels of toxicity were achieved in all exposure groups. The test substance was shown to be non-clastogenic to CHL cells in vitro.

Cyclaprop and its genotoxic potential using read across information from Cyclacet and Cyclobutanate:

Introduction and hypothesis for the analogue approach

Cyclaprop is a propylester attached to a tricyclodecenyl fused ring structure. For this substance an Ames test is available but not a chromosomal aberration test or 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 Cyclaprop the analogue approach is selected because for one closely related analogue reliable chromosomal aberration data are available and for another analogue a reliable gene-mutation study in human cells is available.

Hypothesis: Cyclaprop has similar genotoxicity compared to Cyclacet and Cyclobutanate, because these are the same substances only differing a methyl group in the alkyl chain: Cyclacet containing one methyl less and Cyclobutanate one methyl group extra in the alkyl chain compared to Cyclaprop.

Available experimental information: The target and the source chemicals are all negative in well conducted Ames tests including E.coli strains or Salmonella typhimurium strain TA102 receiving Klimisch codes of 1. In addition, 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. The source substance Cyclobutanate is negative in the chromosomal aberration test (OECD TG 473). The information of Cyclobutanate is sufficiently reliable and receives Klimisch code 1.

Target chemical and source chemical(s)

Chemical structures of the target chemical and the source chemicals are shown in the data matrix, including physico-chemical properties and toxicological information, thought relevant for genotoxicity.

Purity / Impurities

Cyclaprop is a reaction mass containing a mixture of two very similar isomers (5yl and 6-yl). The impurities are all below 1%.

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, which is presented below.

Analogue justification:For Cyclaprop the two nearest neighbours are used. Cyclacet with one methyl group shorter in the alkyl chain and Cyclobutanate with one methyl group longer in this alkyl chain and for these sources necessary genotoxicity information was available.

Structural similarities and differences: The target and the source chemicals, the Cycla-esters, 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 Cyclaprop (C3) is one methyl group longer than Cyclacet (C2) and Cyclobutanate (C4) is one methyl group longer than of Cyclaprop (C4). These differences between the target and source chemical are not expected to behave differently in relation to genotoxicity because the alkyl side chains (propyl versus ethyl and butyl) are not expected to influence genotoxicity of these chemicals.

Toxico-kinetic: The source chemical and the target chemical have similar toxico-kinetic characteristics based on the same backbone and the ester as a functional group. The one methyl group difference between Cyclaprop and both source chemical result in sufficient similar physico-chemical properties as presented in the data matrix below relevant for genotoxicity.

Genotoxic reactivity: Cyclaprop, Cyclacet and Cyclobutanate have the same genotoxic reactivity. The experimental Ames test is negative for all three substances. The OECD Toolbox show the same genotoxicity profile (data not shown).

Remaining uncertainties: There are no remaining uncertainties other than those addressed above.

Data matrix

The relevant information on physico-chemical properties and toxicological characteristics are presented in the Data Matrix.

Conclusions for genotoxicity

For Cyclaprop an experimental Ames test is available (OECD TG 471, Rel. 1) but no experimental information is available on cytogenicity and genemutations in mammalian cells. For two nearest neighbours such information is available and these can be used to read across to Cyclaprop. 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, which is presented here. For Cyclacet (ethyl side chain) 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. For the source chemical Cyclobutanate (butyl side chain) well conducted OECD TG 473 tests is available, which is negative. Based on these data Cyclaprop (propyl-side chain) is negative for genotoxicity.

Final conclusion: Cyclaprop is negative for cytogenicity and for genemutation in mammalian cells.

Data matrix for Cycla-esters

Common names	Cyclaprop	Cyclacet	Cyclobutanate
	Target	Source	Source
Chemical structures
Cas no 5-yl Cas no of the generic	- 68912-13-0	2500-83-6 54830-99-8	1361017-07-3 113889-23-9
EC number	272-805-7	911-369-0	441-420-8
Empirical formula	C13H18O2	C12H16O2	C14H20O2
REACH registration	Registered	Registered	Registered
Molecular weight	206	192	220
Physico-chemical data
Physical state	liquid	liquid	liquid
Melting point (°C)	< -20	< -20	< -20
Boiling point (°C)	263	247	275
Vapour pressure (Pa)	0.67	2.1	11.2
Water solubility (mg/l)	57	186	11.5
Log Kow	4.4	3.9	4.48
Human health endpoints
Genemutations in bacterial cells (Ames)	Negative (+ T102) (OECD TG 471	Negative (+ E. coli) (OECD TG 471)	Negative (+ E.coli) (OECD TG 471)
Genemutations in mammalian cells	RA from Cyclacet	Negative (OECD TG 476)	RA from Cyclacet
In vitro cytogenicity (chromosomal aberration)	RA from Cyclobutanate	No small colonies found in the MLA test being an indicator for the absence of chromosomal aberrations.	Negative (OECD TG 473)

 

Justification for classification or non-classification

Based on the information presented above the substance is not genotoxic and therefore does not need to be classified according to EU CLP (EC 1272/2008 and its amendments).