Cyclohexane, 1-methyl-4-(1-methylethyl)-, tetradehydro deriv.

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Depanol I was tested and found negative for the various endpoints in a genotoxicity battery, including a key study for bacterial gene reverse mutation (Ames test), a key study for mammalian gene mutation (HPRT study) and a key study for chromosomal aberration (in vitro Micronucleus study). Further, there was a supporting Ames study with limited number of strains and cytotoxicity at lower concentrations.

Link to relevant study records

Reference

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study was conducted according to GLP and valid methods, therefore it is considered relevant, reliable and adequate for classification.

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

Salmonella typhimurium strains TA98, TA100, TA1535 and TA1537 demonstrated the requirement of histidine amino acid for their growth. Escherichia coli strain WP2uvrA (pKM101) demonstrated the requirement of tryptophan amino acid for its growth. Ampicillin resistance was demonstrated by the strains TA98, TA100 and WP2uvrA (pKM101) which carry R-factor plasmids. The presence of characteristic mutations like the rfa mutation was demonstrated by all the Salmonella typhimurium strains by their sensitivity to crystal violet. The uvrA mutation in the Escherichia coli strain and the uvrB mutation in the Salmonella typhimurium strains was demonstrated through their sensitivity to ultraviolet light. Finally, all these tester strains produced spontaneous revertant colonies which were within the frequency ranges of the test facility’s historical control data.

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

S9-mix

Test concentrations with justification for top dose:

Depanol I was tested for its toxicity to the Salmonella typhimurium tester strain TA100 at 50, 100, 200, 400, 800, 1600, 3200 and 5000 μg/plate test concentrations along with a DMSO control.
Based on the observations of the preliminary toxicity test, the following test doses were selected (with approximately half-log dose interval) for testing in the initial mutation assay: (a) 50, (b) 158, (c) 500, (d) 1581 and (e) 5000 μg/plate.
In the confirmatory assay, the test item was exposed in triplicate to concentrations of 100, 266, 707, 1880 and 5000 µg/plate in the presence and absence of metabolic activation using pre-incubation procedure.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Depanol I formed a solution in DMSO at 500 mg/mL.
DMSO is one of the organic vehicles compatible with this test system. Hence, DMSO was selected as the vehicle of choice to prepare the stock and dilutions of the test item as well as the positive controls.

Negative solvent / vehicle controls:

yes

Remarks:

100 µL of DMSO was used as the vehicle control

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

TA98 (-S9, 2 µg/plate)

Negative solvent / vehicle controls:

yes

Remarks:

100 µL of DMSO was used as the vehicle control

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

TA98, TA100, TA1535,TA1537 and WP2uvrA (+S9, 4 µg/plate)

Negative solvent / vehicle controls:

yes

Remarks:

100 µL of DMSO was used as the vehicle control

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

TA1537 (-S9, 50 µg/plate)

Negative solvent / vehicle controls:

yes

Remarks:

100 µL of DMSO was used as the vehicle control

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

TA100 and TA1535 (-S9, 1 µg/plate)

Details on test system and experimental conditions:

METHOD OF APPLICATION:
- Initial mutation assay: in agar (plate incorporation)
- Confirmatory mutation assay: preincubation.

DURATION
- Preincubation period: 30 min (confirmatory mutation assay)
- Exposure duration: 67 hours (initial and confirmatory mutation assay)
- Selection time (if incubation with a selection agent): 67 hours (initial and confirmatory mutation assay)
- Fixation time (start of exposure up to fixation or harvest of cells):

NUMBER OF REPLICATIONS:
- triplicate
- two independent experiments

DETERMINATION OF CYTOTOXICITY
- Method: other: number of revertant colonies; evaluation of the bacterial background lawn

Evaluation criteria:

To determine a positive result, there should be a dose related increase in the mean revertants per plate of at least one tester strain over a minimum of two increasing concentrations of the test item either in the presence or absence of the metabolic activation system.
The test will be judged positive, if the increase in mean revertants at the peak of the dose response is equal to or greater than 2 times the mean solvent control value for strains TA98, TA100 and WP2uvrA (pKM101) or equal to or greater than 3 times the mean solvent control value for strains TA1535 and TA1537.
An equivocal response is a biologically relevant increase in a revertant count that partially meets the criteria for evaluation as positive. This could be a dose responsive increase that does not achieve the respective threshold cited above or a non dose responsive increase that is equal to or greater than the respective threshold cited. A response will be evaluated as negative, if it is neither positive nor equivocal.

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

At the top dose of 5000 µg/plate,there was marginal decrease in the mean revertant colonies compared to the DMSO control, followed by a slight decrease in the intensity of bacterial background lawn, both in the presence and absence of metabolic activation

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

At the top dose of 5000 µg/plate,there was marginal decrease in the mean revertant colonies compared to the DMSO control, followed by a slight decrease in the intensity of bacterial background lawn, both in the presence and absence of metabolic activation

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: The test item did not cause precipitation on the basal agar plates at any of the tested doses either in the presence or in the absence of metabolic activation.

RANGE-FINDING/SCREENING STUDIES:
- Depanol I was tested for its toxicity to the Salmonella typhimurium tester strain TA100 at 50, 100, 200, 400, 800, 1600, 3200 and 5000 µg/plate test concentrations along with a DMSO control.
- The results indicated that, the mean number of revertant colonies was more or less comparable to the DMSO control plates up to 3200 μg/plate with comparable bacterial background lawn intensity, both in the presence and absence of metabolic activation. However, at the top dose of 5000 μg/plate, there was a marginal decrease in the mean revertant colonies compared to the DMSO control, followed by a slight decrease in the intensity of bacterial background lawn, both in the presence and absence of metabolic activation. The test item did not cause precipitation on the basal agar plates up to 5000 μg/plate either in the presence or absence of metabolic activation.

COMPARISON WITH HISTORICAL CONTROL DATA:
- Initial mutation Assay: the mean numbers of revertant colonies/plate in the DMSO control was within the range of the in-house spontaneous revertant counts for all the tester strains;
- Confirmatory Assay: the mean numbers of revertant colonies/plate in the DMSO control was within the range of in-house spontaneous revertant counts for all the tester strains .

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Initial mutation Assay: No toxicity of the test item was seen up to 1581 µg/plate as the intensity of the bacterial background lawn was comparable to that of the DMSO control. However, at the top dose of 5000 µg/plate, there was a marginal decrease in the mean revertant colonies compared to the DMSO control, followed by a slight decrease in the intensity of bacterial background lawn, both in the presence and absence of metabolic activation.
- Confirmatory mutation Assay: No toxicity of the test item was seen up to 1880 µg/plate as the intensity of the bacterial background lawn was comparable to that of the DMSO control. However, at the top dose of 5000 µg/plate, there was a marginal decrease in the mean revertant colonies compared to the DMSO control, followed by a slight decrease in the intensity of bacterial background lawn, both in the presence and absence of metabolic activation.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

- Initial mutation Assay: The tested doses showed no positive mutagenic increase in the mean number of revertant colonies for any of the tester strains when compared to the respective vehicle control plates, either in the presence or absence of metabolic activation.   Positive control chemicals tested simultaneously produced more than a 3-fold increase in the mean numbers of revertant colonies for all the strains when compared to the respective vehicle control plates. No toxicity was observed in the positive controls as the intensity of the bacterial background lawn of all the tester strains was comparable to that of the respective vehicle control plates.

- Confirmatory mutation Assay: The tested doses showed no positive mutagenic increase in the mean number of revertant colonies for any of the tester strains when compared to the respective vehicle control plates, either in the presence or absence of metabolic activation.  

Positive control chemicals tested simultaneously produced more than a 3-fold increase in the mean numbers of revertant colonies for all the strains when compared to the respective vehicle control plates. No toxicity was observed in the positive controls as the intensity of the bacterial background lawn of all the tester strains was comparable to that of the respective vehicle control plates.

Conclusions:

Interpretation of results (migrated information):
negative with metabolic activation
negative without metabolic activation

It is concluded that the test item, Depanol I, was not mutagenic in this bacterial reverse mutation test at the tested concentrations and under the conditions of testing employed.

Executive summary:

Depanol I was tested for its mutagenic potential in the bacterial reverse mutation assay using TA98, TA100, TA1535 and TA1537 strains of Salmonella typhimurium and WP2uvrA (pKM101) strain of Escherichia coli in te presence and absence of metabolic activation. The study consisted of a preliminary toxicity test and two mutation assays comprising four independent experiments. Depanol I formed a solution in dimethyl sulfoxide (DMSO) at 500 mg/mL and was found to be stable in DMSO for 24 hours at room temperature.

In a preliminary toxicity test, the mean number of revertant colonies was more or less comparable to the DMSO control plates up to 3200 µg/plate, with a comparable bacterial background lawn intensity, both in the presence and absence of metabolic activation. However, at the top dose of 5000 µg/plate, there was a marginal decrease in the mean revertant colonies compared to the DMSO control, followed by a slight decrease in the intensity of bacterial background lawn, both in the presence and absence of metabolic activation. The test item did not cause precipitation on the basal agar plates up to 5000 µg/plate either in the presence or absence of metabolic activation.

In the initial mutation assay, the test item was exposed in triplicate to 50, 158, 500, 1581 and 5000 µg/plate with and without metabolic activation by direct plate incorporation. In the confirmatory assay, the test item was exposed in triplicate to concentrations of 100, 266, 707, 1880 and 5000 µg/plate with and without metabolic activation by pre-incubation. The DMSO control and the appropriate positive controls were tested simultaneously. The results from the initial as well as from the confirmatory assays, indicate the tested doses showed no positive mutagenic increase in the mean numbers of revertant colonies for all tester strains when compared to the respective vehicle control plates, either in the presence or absence of metabolic activation. The mean numbers of revertant colonies neither doubled for strains TA98, TA100 and WP2uvrA (pKM101) nor tripled for strains TA1535 and TA1537 when compared to the respective vehicle control plates, either in the presence or in the absence of the metabolic activation at any of the tested doses. Under identical test conditions, there was a more than 3-fold increase in the mean numbers of revertant colonies in the positive controls, demonstrating the sensitivity of the assay procedure used.

The study indicated that Depanol I was not mutagenic in this Bacterial Reverse Mutation Assay up to the highest tested concentration of 5000 µg/plate.

 

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vitro:

Depanol I was tested for its mutagenic potential in a key bacterial reverse mutation assay using TA98, TA100, TA1535 and TA1537 strains of Salmonella typhimurium and WP2uvrA (pKM101) strain of Escherichia coli in the presence and absence of metabolic activation (Advinus, 2012d). In a preliminary toxicity test, the mean number of revertant colonies was more or less comparable to the DMSO control plates up to 3200 µg/plate, with a comparable bacterial background lawn intensity, both in the presence and absence of metabolic activation. However, at the top dose of 5000 µg/plate, there was a marginal decrease in the mean revertant colonies compared to the DMSO control, followed by a slight decrease in the intensity of bacterial background lawn, both in the presence and absence of metabolic activation. The test item did not cause precipitation on the basal agar plates up to 5000 µg/plate either in the presence or absence of metabolic activation.

In the initial mutation assay, the test item was exposed in triplicate to 50, 158, 500, 1581 and 5000 µg/plate with and without metabolic activation by direct plate incorporation. In the confirmatory assay, the test item was exposed in triplicate to concentrations of 100, 266, 707, 1880 and 5000 µg/plate with and without metabolic activation by pre-incubation.The results from the initial as well as from the confirmatory assays, indicate the tested doses showed no positive mutagenic increase in the mean numbers of revertant colonies for all tester strains when compared to the respective vehicle control plates, either in the presence or absence of metabolic activation.The study indicated that Depanol I was not mutagenic in this Bacterial Reverse Mutation Assay up to the highest tested concentration of 5000 µg/plate.

In a supporting bacterial mutagenicity, Depanol I was tested in the strains TA 100, TA 1535, TA 1537 and TA 98 of Salmonella typhimurium in the presence and absence of metabolic activation (Hoechst Marion Roussel Deutschland GmbH, 1997). Concentrations were variable across bacterial strains to account for varying susceptibilities to toxic effects: low doses ranged from 0.032 to 100 µg/plate (TA 98) and high doses ranged from 0.16 to 500 µg/plate (TA 100, TA 1535 and TA 1537). Toxicity was observed with and without metabolic activation at concentrations of 100 µg/plate and above. A toxicity test using histidine-enriched agar plates and a dilution of the tester strain TA 100 (designated TA 100 D) was performed in parallel with the second experiment. Toxicity was found at concentrations of 100 µg/plate and above in the presence of metabolic activation and at 20 µg/plate and above in the absence of metabolic activation. In the absence and in the presence of the metabolic activation system Depanol I did not result in relevant increases in the number of revertants in any of the bacterial strains.

Depanol I was tested for its genotoxic potential in a key mammalian gene mutation assay solved in DMSO and using Chinese Hamster ovary (CHO) cells (Advinus, 2012e). The study consisted of a preliminary toxicity test, an initial gene mutation assay, and a confirmatory gene mutation assay, each comprising two independent experiments with and without metabolic activation system. In a preliminary cytotoxicity test Depanol I did not show evidence of significant cell growth inhibition up to 44 µg/mL. However, at and higher than 88 µg/mL, there was no cell survival in the presence and absence of metabolic activation. The test item did not cause precipitation of the test solution at any of the tested concentrations and did not cause any appreciable change in the pH and osmolality of the test solutions at the end of the 3-hour exposure to treatment either in the presence or in the absence of metabolic activation. Dose levels to which CHO cells were exposed with and without metabolic activation were 9.5, 19, 38, 55, 65 and 76 µg/mL in the initial test and 7.5, 15, 30 and 60 µg/mL in the confirmatory test. There was no evidence of induction of gene mutations in any of the test material treated cultures either in the presence or absence of metabolic activation. Stability and concentration were also tested and found to be compliant. In each of these experiments, the respective positive controls produced a statistically significant increase in the frequencies of mutants, under identical conditions and concurrent vehicle control cultures values were within laboratory historical controls. The results of the forward gene mutation assay at the hprt locus with Depanol I indicate that under the conditions of this study, the test item was non-mutagenic when evaluated in the presence or absence of an externally supplied metabolic activation (S9) system.

Depanol I was tested for chromosome aberration potential in a key in vitro micronucleus test using human peripheral lymphocytes both in the presence and absence of metabolic activation (LPT, 2012). The test was carried out using acetone as vehicle and employing 2 exposure times without S9 mix: 4 and 20 hours, and 1 exposure time with S9 mix: 4 hours. In a preliminary experiment, test item concentrations of 10, 25, 100, 250, 1000, 2500 and 5000 µg/mL medium were employed. Cytotoxicity and haemolysis were noted starting at a concentration of 250 µg Depanol I/mL. Hence, 250 µg/mL was employed as the top concentration for the mutagenicity tests without and with metabolic activation. In the main study cytotoxicity and haemolysis were also noted at the top concentration of 250 µg of Depanol I/mL in the experiments without and with metabolic activation. The micronucleus frequencies of cultures treated with Depanol I at concentrations of 15.63, 31.3, 62.5, 125 or 250 µg/mL medium (4 h and 20 h exposure) in the absence of metabolic activation ranged from 5.5 to 14.0 micronuclei per 1000 binucleated cells without any dose related increase in micronuclei up to the cytotoxic concentration. The micronucleus frequencies of cultures treated with Depanol I at concentrations of 15.63, 31.3, 62.5, 125 and 250 µg/mL medium (4 h exposure) in the presence of metabolic activation ranged from 3.0 to 11.5 micronuclei per 1000 binucleated cells without any dose related increase in micronuclei up to the cytotoxic concentration. Therefore Depanol I tested up to a cytotoxic concentration of 250 µg/mL medium, in the absence and in the presence of metabolic activation employing two exposure times (without S9) and one exposure time (with S9) revealed no indications of genotoxic properties in the in vitro micronucleus test. In the same test, Mitomycin C, Colchicine and cyclophosphamide induced significant damage.

Justification for selection of genetic toxicity endpoint
The Ames test was selected as main key study with Depanol I, however other study such as the mammalian gene mutation test and in vitro Micronucleus test with Depanol I were also key studies for these endpoints.

Justification for classification or non-classification

Based on the available data, Depanol I does not have to be classified for genotoxicity according the EU labelling regulations Commission Directive 93/21/EEC or CLP regulation No. 1272/2008 of 16 December 2008.