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Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
26 February 2009 - 12 March 2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009
Report Date:
2009

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
other: Standards Established by the Minister of Labour in Accordance with the Industrial Safety and Health Law, Article 57-2-1
Version / remarks:
Notification No. 77, September 1, 1988, and Notification No. 67, June 2 1997, Ministry of Labour, Japan
Deviations:
no
Qualifier:
according to
Guideline:
other: Study Methods on New Chemical Substances, etc.
Version / remarks:
November 21, 2003; YakuShokuHatsu No. 1121002, Heisei 15-11-13 SeiKyoku No. 2, and KanHoKiHatsu No. 031121002, Revised finally on November 20, 2006
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Details on test material:
- Physical appearance:
- Storage conditions: at room temperature in an airtight container under nitrogen gas
Specific details on test material used for the study:
Solubility in water: insoluble
Solubility in DMSO: 50 mg/mL

Method

Target gene:
- S. typhimurium: Histidine gene
- E. coli: Tryptophan gene
Species / strainopen allclose all
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 from male rats induced by phenobarbital and 5,6-benzoflavone
Test concentrations with justification for top dose:
Dose-range finding study (all tester strains, with and without S9): 1.22, 4.88, 19.5, 78.1, 313, 1250 and 5000 µg/plate

Concentrations used in the main studies were selected based on the results of the dose-range finding study. As the maximum dose, the minimum dose at which growth inhibition was observed was selected.

First main study (without S9)
TA100: 0.61, 1.22, 2.44, 4.88, 9.77 and 19.5 µg/plate
TA1535: 0.15, 0.31, 0.61, 1.22, 2.44 and 4.88 µg/plate
WP2uvrA: 2.44, 4.88, 9.77, 19.5, 39.1 and 78.1 µg/plate
TA98: 0.15, 0.31, 0.61, 1.22, 2.44 and 4.88 µg/plate
TA1537: 0.04, 0.08, 0.15, 0.31, 0.61 and 1.22 µg/plate

First main study (with S9)
TA100: 0.61, 1.22, 2.44, 4.88, 9.77 and 19.5 µg/plate
TA1535: 0.61, 1.22, 2.44, 4.88, 9.77 and 19.5 µg/plate
WP2uvrA: 2.44, 4.88, 9.77, 19.5, 39.1 and 78.1 µg/plate
TA98: 2.44, 4.88, 9.77, 19.5, 39.1 and 78.1 µg/plate
TA1537: 0.15, 0.31, 0.61, 1.22, 2.44 and 4.88 µg/plate

Second main study (without S9)
TA100: 0.61, 1.22, 2.44, 4.88, 9.77 and 19.5 µg/plate
TA1535: 0.15, 0.31, 0.61, 1.22, 2.44 and 4.88 µg/plate
WP2uvrA: 2.44, 4.88, 9.77, 19.5, 39.1 and 78.1 µg/plate
TA98: 0.15, 0.31, 0.61, 1.22, 2.44 and 4.88 µg/plate
TA1537: 0.04, 0.08, 0.15, 0.31, 0.61 and 1.22 µg/plate

Second main study (with S9)
TA100: 0.61, 1.22, 2.44, 4.88, 9.77 and 19.5 µg/plate
TA1535: 0.61, 1.22, 2.44, 4.88, 9.77 and 19.5 µg/plate
WP2uvrA: 2.44, 4.88, 9.77, 19.5, 39.1 and 78.1 µg/plate
TA98: 2.44, 4.88, 9.77, 19.5, 39.1 and 78.1 µg/plate
TA1537: 0.15, 0.31, 0.61, 1.22, 2.44 and 4.88 µg/plate
Vehicle / solvent:
- Solvent: DMSO
- Justification for choice of solvent: the test item was soluble in DMSO at and above 50mg/mL (according to solubility test at Bozo Research Center Inc.)
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
benzo(a)pyrene
other: ICR-191; 2-aminoanthracene; 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide (AF-2)
Remarks:
For more details on positive controls see 'any other information on materials and methods'
Details on test system and experimental conditions:
A dose-range finding study and two independent main studies were performed.

METHOD OF APPLICATION: in agar

DURATION
- Preincubation period: 20 minutes (while shaking)
- Exposure duration: 48 hours (dose-range finding), 50 hours (first main study) and 48.5 hours (second main study)

NUMBER OF REPLICATIONS: 2

METHOD OF SLIDE PREPARATION: D-biotin, L-histidine or L-tryptophan solutions were added to a soft agar solution to prepare top agar. Top agar were kept in a therostatic chamber set at 45°C to prevent fixation.

DETERMINATION OF CYTOTOXICITY
- Method: number of revertant colonies, counted with an automatic colony counter

- OTHER: the presence or absence of growth inhibition was observed using a stereoscopic microscope.
Evaluation criteria:
If a two-fold or more increase in the number of revertant colonies compared to that of spontaneous revertant colonies (solvent control) and dose-response and reproducibility were noted, or even if no clear dose-response was observed but there was at least a two-fold increase in comparison with the number of spontaneous revertant colonies and reproducibility was observed, the test item was considered to be positive.
Statistics:
No statistical method applied.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 4.88 µg/plate without S9-mix and at 19.5 µg/plate with S9-mix
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
not valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at and above 1.22 µg/plate without S9-mix and at 4.88 µg/plate with S9-mix
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
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
Remarks:
4.88 µg/plate without S9-mix and at 78.1 µg/plate with S9-mix
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
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
Remarks:
at and above 9.77 µg/plate with and without S9-mix
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
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:
cytotoxicity
Remarks:
at and above 39.1 µg/plate without S9-mix and at 78.1 µg/plate with S9-mix
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
Precipitation: in all tester strains, at 5000 µg/plate without metabolic activation and at and above 1250 µg/plate with metabolic activation.
Cytotoxicity: in tester strain TA1537 at and above 1.22 µg/plate without S9-mix and at 4.88 µg/plate with S9-mix; in tester strain TA98 at 4.88 µg/plate without S9-mix and at 78.1 µg/plate with S9-mix; in tester strain TA100 at and above 9.77 µg/plate with and without S9-mix; in tester strain TA1535 at 4.88 µg/plate without S9-mix and at 19.5 µg/plate with S9-mix; in tester strain WP2uvrA at and above 39.1 µg/plate without S9-mix and at 78.1 µg/plate with S9-mix.
Mutagenicity: in all strains, in the absence and in the presence of S9-mix, no increase in the number of revertants was observed.

ACCEPTABILITY
- in the positive control, the increase in the number of revertant colonies was more than two-fold, compared to the solvent control.
- the number of revertant colonies in the plates in the solvent control group and in the positive control group were within the historical data range.
- no contaminants such as other bacteria were seen in this test system.

Any other information on results incl. tables

See attached illustration for more details.

Applicant's summary and conclusion

Conclusions:
In an AMES test, 1,4-bis(isocyanatomethyl)cyclohexane was found to be not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay with and without metabolic activation.
Executive summary:

The mutagenic potential of 1,4-bis(isocyanatomethyl)cyclohexane was assessed in an Ames test, performed under GLP principles. The test item was tested in several strains of Salmonella typhimurium (TA98, TA100, TA1535 and TA1537) and in one strain of Escherichia coli (WP2uvrA) in the absence and in the presence of a metabolic activation system (S9 -mix). A dose-range finding study and two independent main studies were conducted. Concentrations up to and including 5000 µg/plate were used in the dose-range finding study. Based on the results of the dose-range finding study, the concentrations were determined for the main studies: the top dose was the minimum dose at which growth inhibition was observed in the dose-range finding study.

The test item precipitated on the plates at the top dose of 5000 μg/plate in the absence of S9 -mix and at a dose of 1250 µg/plate in the presence of S9 -mix. Cytotoxicity, as evidenced by a decrease in the number of revertants, was observed in all tester strains at different dose levels, with and without S9 -mix. In all strains, in the absence and in the presence of S9-mix, no increase in the number of revertants was observed. The results of the solvent control and the positive controls were within the historical range of the test facility.

In conclusion, 1,4 -bis(isocyanamethyl)cyclohexane is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay, with and without metabolic activation.