Reaction mass of 3a,4,5,6,7,7a-hexahydro-4,7-methano-1H-inden-5-yl isobutyrate and 3a,4,5,6,7,7a-hexahydro-4,7-methano-1H-inden-6-yl isobutyrate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

- Gene mutation in bacteria (Bacterial Reverse Mutation Assay/Ames, OECD 471): not mutagenic.

- In vitro micronucleus test (OECD TG 487): negative 

- In vitro Mammalian Cell Gene Mutation Test (Read across from Cyclacet, OECD 476): not mutagenic.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

The executive summaries are presented of the Ames test with Cyclabute; MLA test with Cyclacet and Chromosomal aberrations with Cyclobutanate. Thereafter the read across justification is presented.

Cyclabute tested in the Ames test: A bacterial reverse mutation assay (Ames test) was performed with bacterial strains Salmonella typhimurium TA 1535, TA 1537, TA 98, TA 100 and Escherichia coli WP2uvrA‾. The test was performed under GLP conditions and according to OECD 471. A preliminary test was performed to determine the test range. The test item was dissolved in DMSO and applied once at test initiation with concentrations ranging from 1.5 -5000 µg/plate. The validity criteria of the test results were fulfilled. No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, at any dose level either with or without metabolic activation. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies thus confirming the activity of the S9 -mix and the sensitivity of the bacterial strains. The test material was considered to be non-mutagenic under the conditions of this test.

Cyclabute tested in the in vitro micronucleus test

Cyclabute is tested for cytogenicity in an in vitro micronucleus test according to OECD TG 487. Peripheral blood lymphocytes were obtained from a healthy non-smoking 31-year-old adult male and cultured. Test was performed with and without metabolic activation. Aroclor 1254-induced rat liver S9 was used as the metabolic activation system. The preliminary toxicity test showed cytoxicity >=220 ug/ml and >=66 ug/ml when incubated for 4 and 24 hours, respectively. With and without S9 and 4 h incubation the cytotoxicity was >=660 ug/ml. Dose levels without S9 and 4 hours incubation were: 25, 50, 100, 200, 220, 240, 260, 280 and 300 ug/ml. Dose levels without S9 and 24 hours incubation were 2.5, 5, 10, 25, 50, 60, 70, 80 and 100 ug/ml. Dose levels selected of the 4 hour without S9 were 100, 200 and 240 ug/l and the highest dose 57% cytotoxicity was seen. Without S9 and 24 hours incubation the dose levels were 10, 25 and 60 ug/ml. At the highest concentration cytotoxicity was 53% relative to control. With S9 and 4 hours incubation the dose levels selected for scoring were 100, 200 and 280 ug/ml, the cytotoxicity at the highest dose was 51%). The positive and vehicle controls fulfilled the requirements for a valid test. Under the conditions of this test the substance is negative for the induction of micronuclei with and without S9 in the in vitro mammalian micronucleus test. 

Cyclacet and its effects gene mutation in mammalian cells

The test article, Cyclacet, was tested for its potential to induce mutations at the thymidine kinase locus of L5178Y TK+mouse lymphoma cellsin 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 ug/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 ug/mL and above. Cultures treated with 50 ug/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 ug/mL and above. The culture treated with 100 ug/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 ug/mL without activation and from 109 to 300 ug/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 ug/rnL with 20 ug/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 of 1.0, 5.0, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, and 140 ug/mL. The cultures treated with 30 to 140 ug/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).

Cyclabute and its gene-mutation potential in mammalian cells using read across information from Cyclacet

Introduction and hypothesis for the analogue approach

Cyclabute is an isobutyl ester attached to a tricyclodecenyl fused ring backbone. For this substance an Ames test is available and in an in vitro micronucleus test but no test for gene mutations in 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 Cyclabute the analogue approach is selected because for one closely related analogue reliable gene-mutation study in human cells is available which can be used for read across.

Hypothesis: Cyclabute has similar gene mutations in mammalian cells compared to Cyclacet based on similarity in chemical structure, bioavailability and Mode of Action. The isobutyl chain of Cyclabute has the same reactivity compared to the straight butyl chain of Cyclacet. Therefore the experimental in vitro gene-mutation information from Cyclacet can be used for Cyclabute.

Available experimental information: The target and source chemicals are both negative in well conducted Ames tests including Salmonella typhimurium strain TA102, receiving Klimisch codes 1 (OECD TG 471. Cyclabute is negative in the in vitro micronucleus test (OECD TG 487). The source substance Cyclacet is negative in the gene-mutations in mammalian cells (OECD TG 476).

Target chemical and source chemical(s)

Chemical structures of the target chemical and the source chemicals are shown in the data matrix below. 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 in both substances.

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.

Structural similarities and differences: The target and the source chemical 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 Cyclabute (C4) is two methyl groups shorter than Cyclacet (C2) and contains an isobutanoic instead of an acetic chain. These chain differences are not expected to behave differently in relation to genotoxicity.

Toxico-kinetic: Cyclabute (target) and Cyclacet (source) 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. These are both liquids. Their vapour pressures are similar around 1 Pa. It can be seen that the water solubility of the Cyclabute 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 an isobutyl one. The target and the source chemicals will both be metabolised into the same alcohol (CAS no: 3385-61-3) and the respective acid, isobutanoic-acid for the target, acetic acid for Cyclacet. This metabolisation is illustrated in the toxico-kinetic section.

Uncertainty of the prediction: The absence of gene mutations in mammalian cells predicted for Cyclabute has a high certainty because of the already negative result of the Ames test and the in vitro micronucleus close. The similarity between Cyclabute (target) and Cyclacet (source) is only the small differences in the alkyl chains, which have no activating fragments. Both substances, target and source will metabolise into the respective Cycla-alcohol and their respective acids. Therefore the reliability of this read across is high 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 per endpoint for hazard identification

For Cyclabute no gene-mutation information in mammalian cells is available. These data are available for the analogue Cyclacet for which a negative OECD TG 487 is available and read across can be applied because the data are applicable for C&L and/or risk assessment and adequate and reliable documentation is provided. Therefore also Cyclabute is considered to be negative for in vitro gene-mutation in mammalian cells because the substance has only two additional methyl groups, which do not influence the genotoxic potential.

Final conclusion: Cyclabute is negative for gene-mutations in mammalian cells.

Data matrix of Cyclabute for gene-mutations in mammalian cells using read across from Cyclacet

Common names	Cyclabute (Target)	Cyclacet (Source)
Chemical structures
Cas no of the main isomer Cas no of the generic	67634-20-2 (5-yl) 93941-73-2 C14H20O2	2500-83-6 (5-yl) 54830-99-8 C12H16O2
EC number	916-331-7	441-420-8
Molecular weight	220	192
Physico-chemical data
Physical state	liquid	liquid
Melting point (°C)	< -20	< -20
Boiling point (°C)	275	247
Vapour pressure (Pa) (measured)	0.61	2.1
Water solubility (mg/l) (measured)	16	186
Log Kow (measured)	5.1	3.9
Human health endpoints
Genotoxicity -Ames test	Negative (Read across from Cyclobutanate)	Negative (OECD TG 471)
Genotoxicity in vitro-Chromosomal aberrations	Negative (Read across from Cyclobutanate)	Read across from Cyclobutanate
Genotoxicity in vitro MLA	Negative (Read across Cyclacet)	Negative (OECD TG 476)

*Vapour pressure value is expected to be too high due to experimental error in view of the value of the other Cycla-esters and the EpiSUITE predicted value of 0.6.

Justification for classification or non-classification

Cyclabute did not show any genotoxic potential in the Ames test and the in vitro micronucleus test. No mutagenicity in mammalian cells is expected based on read across to structural similar Cyclacet. Therefore, it can be concluded that Cyclabute is not mutagenic and therefore does not need to be classified for mutagenicity according to the criteria outlined in EU CLP (EC No. 1272/2008/EC and its updates).