Reaction mass of bis(2-methylpropyl) (1R,2R,3R,6S)-3,6-dimethylcyclohexane-1,2-dicarboxylate and bis(2-methylpropyl) (1S,2S,3R,6S)-3,6-dimethylcyclohexane-1,2-dicarboxylate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Gene mutation in bacteria: Negative

Chromosome aberration in Chinese Hamster Lung cells: Negative

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:

31 July 2007 - 03 September 2007

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: Japanese Ministry of Labor, "Standards for testing of mutagenicity study in bacteria" Notification No.77, 1988

Version / remarks:

Partially revised, Notification No. 67, 1997

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: KU0704

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Stability: stable at room temperature
- Stability in solvent: insoluble in water; soluble (stable) in DMSO and acetone

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: Japan bioasay research centre

Reason for selection of bacterial strains:
The selected bacterial strains are hypersensitive to mutagenic chemicals and generally used in the bacterial mutation test.

Species / strain / cell type:

E. coli WP2 uvr A

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: Japan bioasay research centre

Reason for selection of bacterial strains:
The selected bacterial strains are hypersensitive to mutagenic chemicals and generally used in the bacterial mutation test.

Metabolic activation:

with and without

Metabolic activation system:

S9-mix

Test concentrations with justification for top dose:

Dose range-finding assay: 1.2, 4.9, 20, 78, 313, 1250, 5000 µg/plate
Main assay: 313, 625, 1250, 2500 and 5000 µg/plate

Precipitation of test material was observed at 5000 µg/plate and at 2500 µg/plate in the absence of S9 mix

Vehicle / solvent:

- Vehicle used: DMSO;
- Justification for choice of vehicle: substance was soluble and stable, the preliminary test on solvent selection in the testing facility revealed that the test substance was uniformly dispersed and stable but was undissolved at 5% in DMSO.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

sodium azide

other: 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide, 6-Chloro-9-[3-(2-chloroethylamino)-propylamino]-2-methoxyarcidine dihydrochloride, A-Aminoanthracene

Details on test system and experimental conditions:

S9-mix preparation:
Prepared on: 20 April 2007
Lot No.: 07042001

A pre-incubation step was employed which involved a 20-minute incubation (37°C) with shaking of the test solution with the bacterial culture (plus S9 mix in the case of metabolic activation assays) prior to the addition of agar. Cultures were incubated for 48 hours at 37°C. Dose levels were analysed in duplicate and solvent controls were analysed in triplicate.

An automatic colony counter was used to count the number of revertant colonies.

Evaluation criteria:

When the number of relevant colonies on test substance treatment plates is statistically significant increased compared to the spontaneous rate of mutation and the increase is a dose-dependent and reproducible between the dose-finding assay and the main study, the test substance is judged to be positive for mutagenicity. Otherwise, the test substance is judged as negative.

Statistics:

No statistical analysis is performed for data analysis.

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

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:

no cytotoxicity, but tested up to precipitating concentrations

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:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Conclusions:

Based on the results, it was concluded that DMCHIBU was negative for mutagenicity under the test conditions of this study.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

31 May 2010 - 01 September 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

Adopted 21 July 1997

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: KM0901D

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature (18.2 - 22.9 °C)

- Purity: 99.7%

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

Modal chromosome number: 25
Stored in liquid nitrogen after subculture
Supplemented with 10 vol% calf serum, 10% CS/MEM, in a humidified CO2 incubator (5 % CO2; 37 °C)

Metabolic activation:

with and without

Metabolic activation system:

S9

Test concentrations with justification for top dose:

Growth inhibation test:
0.0015, 0.0030, 0.0061, 0.012, 0.024, 0.049, 0.098, 0.20, 0.39, 0.78, 1.6 and 3.1 mg/mL
The highest dose was set at 3.1 mg/ml (10 mmol/L) according to the guideline.

Cytogenetic test:
6 hour treatment without S9 mix: 0.0088, 0.013, 0.020, 0.030, 0.044, 0.067, 0.10 mg/mL (Severe cytotoxicity seen at 0.067 and 0.10 mg/mL)
6 hour treamtent with S9 mix: 0.20, 0.39, 0.78, 1.6, 3.1 mg/mL
24 hour continuous treatment without S9 mix: 0.0088, 0.013, 0.020, 0.030, 0.044, 0.067, 0.10 mg/mL (Severe cytotoxicity seen at 0.044, 0.067, and 0.10 mg/mL)
24 hour continuous treatment without S9 mix (additional test): 0.028, 0.032, 0.034, 0.036, 0.038, 0.040, 0.042, 0.044, 0.048 mg/mL (No observation at 0.042 or 0.044 mg/mL due to small metaphase cells; severe cytotoxicity seen at 0.048 mg/mL).

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

For short-term treatment without S9 mix

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

For short-time treatment with S9-mix

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium
- Cell density at seeding: 4 x 10^3 cells/mL

DURATION
- Exposure duration: 6 hours or 24 hours
- Expression time: 18 hours (6-hour exposure samples only)

SPINDLE INHIBITOR: Colcemid

STAIN: 3% (v/v) Giemsa

NUMBER OF REPLICATIONS: Two slides per sample

NUMBER OF CELLS EVALUATED: 200 per sample (100 cells per slide)

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells):

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index;

OTHER EXAMINATIONS:
- Determination of polyploidy: Yes

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Positive controls validity:

valid

During the test period, there were no unexpected situations that might have affected the quality of the study, nor were there any deviations from the protocol.

Conclusions:

From the results, it was concluded that SIBE138 did not induce chromosomal aberrations in CHL/IU cells under the present test conditions.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

An in-vitro gene mutation test in bacteria was conducted to assess the mutagenic potential of SIBE138 (Koei Tachno, 2007). No indication of mutagenic potential was observed in the four strains of S. Typhimurium or one strain of E. Coli tested, either in the presence or absence of metabolic activation.

An in-vitro chromosome aberration test was conducted to determine the cytogenetic potential of SIBE138 (Hatano Research, 2010). The test material was found not to have induced chromosomal aberrations under the test conditions used.

On the basis of the above results it is concluded that there is no evidence which supports the classification of SIBE138 for gene mutation.