cis-2-tert-butylcyclohexyl acetate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Verdox gave negative results in the Ames test with Salmonella typhimurium strains TA97a, TA98, TA100, TA102 and TA1535, both with and without metabolic activation. Negative results were observed for its read-across candidate Coniferan in the in vitro mouse lymphoma assay and chromosome aberration assay in Chinese hamster ovary (CHO) cells, both with and without metabolic activation. Based on these results, Verdox is not genotoxic.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

15 Aug 2000 to 5 Oct 2000

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Qualifier:

according to guideline

Guideline:

other: EEC-Guideline 92/69 L 383 A, B.14

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

his

Species / strain / cell type:

S. typhimurium, other: TA97a, Ta98, TA100, TA102 and TA1535

Metabolic activation:

with and without

Metabolic activation system:

rat S9

Test concentrations with justification for top dose:

Study 1:
Strains TA971, TA100, TA1535, -S9: 0.016, 0.05, 0.16, 0.5 and 1.6 mg/plate
Strains TA97a (+S9), TA98 (+/-S9), TA100 (+S9), TA1535 (+S9), TA102 (+/-S9): 0.05, 0.16, 0.5, 1.6 and 5 mg/plate

Study 2:
Strains TA97 and TA1535 (-S9): 0.016, 0.05, 0.16, 0.5 and 2.6 mg/plate
Strains TA97 (+ S9), TA98 (+/-S9), TA100 (+/-S9), TA1535 (+S9) and TA102 (+/-S9): 0.05, 0.16, 0.5, 1.6 and 5 mg/plate.

Vehicle / solvent:

- Vehicle used: DMSO

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: ICR 191, 4-nitro-1,2-phenylenediamine, nitrofurantoine, natrium azide, 2-aminoanthracene, cumene hydroperoxide, danthron.

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Incubation period: 18 hours
- Exposure duration: 48 hours

NUMBER OF REPLICATIONS: 3 replicates per concentration level and control

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth

Evaluation criteria:

For evaluation of the results the induction rate of the mean values was calculated: induction rate = (revertant colonies of the test item)/(revertant colonies of the corresponding control). The test item is to be interpretated mutagenic if there is a concentration effect relationship and the induction rate is ≥ 2.

Key result

Species / strain:

S. typhimurium TA 97a

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

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:

valid

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:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

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:

valid

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES: The range of test concentrations was determined in a preliminary (non-GLP) test with the concentration rage 0.05-5 mg/plate (factor 10).

ADDITIONAL INFORMATION ON CYTOTOXICITY: the lowest cytotoxic concentrations were 1.6 mg/plate for TA97a (-S9), 5 mg/plate for TA100 (+/- S9), 1.6 mg/plate for TA1535 (-S9) and 5 mg/plate for TA1535 (+ S9). No cytotoxicity was observed in strains TA97a (+ S9), TA98 (+/-S9) and TA102 (+/-S9).

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Conclusions:

The substance gave negative results in the Ames test with Salmonella typhimurium strains TA97a, TA98, TA100, TA102 and TA1535, with and without metabolic activation, in a concentration range of 0.016-5 mg/plate.

Executive summary:

The ability of the test substance to induce gene mutations in Ames test was tested in the GLP-compliant OECD Guideline 471 study in Salmonella typhimurium strains TA97a, TA98, TA100, TA102 and TA1535, with and without metabolic activation. The tested concentration ranges were 0.05 -5 mg/plate for strains TA97a (+S9), TA98 (+/-S9), TA100 (+S9), TA102 (+/-S9) and TA1535 (+S9), and 0.016 -1.6 mg/plate for TA97a (-S9), TA100 (-S9) and TA1535 (-S9). The lowest cytotoxic concentrations were 1.6 mg/plate for TA97a (-S9) and TA1535 (-S9), and 5 mg/plate for TA 100 (+/-S9) and TA1535 (+ S9). No cytotoxicity was observed for strains TA97a (+S9), TA98 (+/-S9) and TA102 (+/-S9). The tests were performed in triplicate. No increased number of revertants was observed in any case. Based on the results of the study, the substance is concluded to be negative in Amest test.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

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

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: read-across from a study which is equivalent to a guideline study

Justification for type of information:

The genetic toxicity in vitro CA is based on read-across from Coniferan. The read across rationale is presented in the Genetic toxicity Endpoint summary. The accompanying file is also attached there.

Reason / purpose for cross-reference:

read-across source

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

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

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: read-across information from an analogue is used

Justification for type of information:

The genetic toxicity in vitro MLA is based on read-across from Coniferan, which is documented in the overall Genetic toxicity endpoint. The accompanying files are also attached there.

Reason / purpose for cross-reference:

read-across source

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

In the section below the executive summaries of the experimental tests are presented followed by the read across rationale

 

Verdox - Ames study

The ability of the test substance to induce gene mutations in Ames test was tested in the GLP-compliant OECD Guideline 471 study in Salmonella typhimurium strains TA97a, TA98, TA100, TA102 and TA1535, with and without metabolic activation (Dr. U. Noack-Laboratorium, 2000). The tested concentration ranges were 0.05 -5 mg/plate for strains TA97a (+S9), TA98 (+/-S9), TA100 (+S9), TA102 (+/-S9) and TA1535 (+S9), and 0.016 -1.6 mg/plate for TA97a (-S9), TA100 (-S9) and TA1535 (-S9). The lowest cytotoxic concentrations were 1.6 mg/plate for TA97a (-S9) and TA1535 (-S9), and 5 mg/plate for TA 100 (+/-S9) and TA1535 (+ S9). No cytotoxicity was observed for strains TA97a (+S9), TA98 (+/-S9) and TA102 (+/-S9). The tests were performed in triplicate. No increased number of revertants was observed in any case. Based on the results of the study, the substance is concluded to be negative in Ames test.

 

Coniferan genemutations in mammalian cells

The ability of Coniferan to induce gene mutations in mammalian cells was studied in the GLP-compliant OECD Guideline 476 study using L5178Y TK^+/-mouse lymphoma assay. The study was performed using a 4 -hour treatment period at test concentrations 2.0 -47 μg/mL without metabolic activation and 25 -100 μg/mL with metabolic activation. Cultures were selected for cloning for mutant selection based on their relative suspension growth. Only cultures treated without activation were cloned. The cultures treated in the presence of metabolic activation were not cloned because all concentrations were relatively nontoxic. All of the cloned cultures without activation had mutation frequencies similar to the average mutation frequencies of their concurrent controls. The relative total growth for the cloned cultures ranged from 47 to 63%. The assay with metabolic activation was performed at concentrations 25 -250 μg/mL. All of the cloned cultures had mutation frequencies similar to the average mutation frequencies of the solvent controls. The relative total growth for the cloned cultures ranged from 14% to 88%.

The confirmatory assay was conducted without activation with a 24 -hour exposure. Cultures were treated with concentrations ranging from 5.0 to 50 μg/mL. The cultures treated with 5.0 to 26 μg/mL were cloned for mutant selection. All of the cultures had mutation frequencies similar to the average mutation frequencies of the solvent controls. The relative total growth for the cloned cultures ranged from 14% to 73%.

Based on the results of the assay, the substance was considered to be negative in the mouse lymphoma assay.

 

Coniferan and its cytogenicity

The ability of Coniferan to induce chromosome aberrations in mammalian cells was tested in the GLP-compliant guideline study with the Chinese hamster ovary (CHO) cells, with and without metabolic activation. Significant cytotoxicity was observed at 5 0 μg/mL and above without metabolic activation and at 100 μg/mL and above with metabolic activation. The study was performed at concentration levels 10, 20, 30, 35, 40 and 45 μL without metabolic activation and 25, 50, 60, 70, 80 and 90 μg/mL with metabolic activation, using 3 hours exposure. Chromosome aberrations were scored from the cells treated with the concentrations of 10, 20 and 30 μg/mL without activation and 50, 60 and 70 μg/mL with activation, based on cytotoxicity data. The confirmatory repeat was performed without metabolic activation at concentrations 10, 20, 25, 30 and 35 μg/mL without metabolic activation, using 18 hours exposure. Chromosome aberrations were scored for cells treated at concentrations of 25, 30 and 35 μg/mL, based on cytotoxicity data. The results from both the main study and the confirmatory assay indicated that the test article did not induce a statistically significant increase in the percentage of cells with aberrations both with and without metabolic activation compared to the solvent controls, at all concentrations tested. Based on the results, the substance is considered to be negative in chromosome aberrations CHO assay.

 

 

Verdox and its genotoxicity in mammalian cells and cytogenicity based on read-across from Coniferan

Hypothesis for the analogue approach

For assessing the genotoxic profile of Verdox, data of Coniferan can be used because the two substances are close structural analogues; they both have a cyclohexyl ring, the backbone, with an acetate ester and a tert butyl group in ortho position. Coniferan has one additional methyl group attached to the tert-butyl group, which is not thought to influence the reactivity and thus the genotoxic potential.

For Verdox only an Ames test is available (OECD TG 471) whereas for Coniferan an Ames test (OECD TG 471), a chromosomal aberration test (CAB)(OECD TG 473) and an MLA test (OECD TG 476) is available. The results from the CAB and MLA test will be read across to Verdox.

The availablein vitrogenetic toxicity data in mammalian cells of Coniferan used for read across to Verdox are presented first.

In a mouse lymphoma assay, L5178Y cells were treated for 4 hours at concentration levels of 2 - 47 μg/mL without metabolic activation and 25 -100 μg/mL with metabolic activation. The assay with metabolic activation was repeated at concentration levels of 25 - 250μg/mL. A confirmatory repeat was performed without metabolic activation at concentration levels of 5 - 50μg/mL, using 24 hours exposure.

All cultures treated with the substance had mutant frequencies that were similar to that of their corresponding solvent control cultures. Therefore, the substance is considered to be negative in the mouse lymphoma assay (Sitek Research Laboratories, 2009). The study is performed under GLP and according to OECD Guideline 476, and therefore has a reliability of 1.

In a chromosome aberration test, Chinese hamster ovary cells were treated for 3 hours at concentration levels of 10, 20, 30, 35, 40 and 45μg/mL without metabolic activation and 25, 50, 60, 70, 80 and 90μg/mL with metabolic activation. A confirmatory repeat was performed without metabolic activation at concentration levels of 10, 20, 25, 30 and 35μg/mL, using 18 hours exposure.

Significant cytotoxicity was observed at 50μg/mL and above without metabolic activation and at 100μg/mL and above with metabolic activation. A statistically significant increase in the percentage of cells with aberrations both with and without metabolic activation compared to the solvent controls was not observed, at all concentrations tested. Based on the results, the substance is considered to be negative in the chromosome aberration test (Sitek Research Laboratories, 2009). The study is performed under GLP and according to OECD Guideline 473, and therefore has a reliability of 1.

Target chemical and source chemical(s)

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

Purity / Impurities

The configuration and the impurities (< 0.1%) of Verdox are not expected to affect the genetic toxicity and thus the read-across (see table 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 functional group. When using the read across the result derived should be applicable for C&L and/or risk assessment, cover an exposure period duration comparable or longer than the corresponding method and be presented with adequate and reliable documentation. The read across will be based on similarities in backbone and functional groups, physico-chemical properties and , acute toxicity andin vitrogenetic toxicity.

Analogue justification: For Verdox Coniferan, with only methyl group extra in its backbone was considered the most close analogue for which genotoxicity information was available.

Structural similarities and differences: Verdox and Coniferan have a similar backbone. Both have a cyclohexyl group with at the 2-position a tert-butyl group. Both also have an acetate ester group on the 1^stposition. The difference between Coniferan (the source chemical) is the additional methyl group on the tert-butyl group. This additional methyl group is not expected to influence the electrophilicity / reactivity of the substance compared to Verdox.

Bioavailability:The bioavailability of the substance is considered very similar based on the similarities in chemical structure. It can be seen that Verdox with a methyl group less has slightly lower log Kow and slightly higher water solubility.

Genotoxic reactivity: Verdox and Coniferan have a similar absence of genotoxic reactivity: there are no structural indications for genotoxicity.

Other similarities: Verdox and Coniferan are both negative in the Ames test.

filled by using the data of Coniferan.

Data matrix

The relevant information on physico-chemical properties and toxicological characteristics are presented in the Data Matrix in Table 1.

Conclusions on genotoxicity

For Verdox information is present on genotoxicity in bacterial cells but not on mammalian cells or cytogenicity. Therefore information from Coniferan is used. 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 in the current document. From the analogue justification section it is shown that Verdox is sufficiently similar to Coniferan. Coniferan is negative the genemutations in mammalian cells and for cytogenicity. Therefore also Verdox is negative for these endpoints.

Conclusion: Verdox is negative for genotoxicity in mammalian cells and for cytogenicity.

 

Data Matrix supporting the Genotoxicity profile of Verdox using read across from Coniferan.

Name	Verdox	Coniferan
	Target	Source
Chemical structures
Cas no	20298-69-5	67874-72-0
EC no.	243-718-1	267-500-0
REACH registration	Registered	Registered
Molecular weight	198	212
Physico-chemical properties
Physical state	Handled as liquid	Liquid
Melting point in oC	29.8	-20
Boiling point in oC	232	252
Vapour pressure at 20oC in Pa	9.7	4.2
Partition coefficient: log Kow	4.75	5.4
Water solubility at 20˚C in mg/L	10	7.6
Human health endpoints
In vitro gene mutation study in bacteria - Ames test	Negative (OECD TG 471)	Negative (OECD TG 471
In vitro gene mutation study in mammalian cells	Read across	Negative (OECD TG 476)
In vitro cytogenicity study in mammalian cells (chromosome aberrations)	Read across	Negative (OECD TG 473)

 

Justification for classification or non-classification

Verdox is negative for genemutations in bacterial and mammalian cells and for cytogenicity, therefore it does not need to be classified and labelled for genotoxicity according to EU CLP (EC No. 1272/2008 and its updates).