Cyclohexane, 5-isocyanato-1-(isocyanatomethyl)-1,3,3-trimethyl-, homopolymer, 3,5-dimethyl-1H-pyrazole-blocked

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

March 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

(1997)

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

3-Isocyanatomethyl-3,5,5-trimethylcyclohexyl isocyanate, oligomers, reaction products with 3,5-dimethyl-1H-pyrazole

EC Number:

606-423-2

Cas Number:

200295-52-9

Molecular formula:

>= C51H78N12O6 (IPDI-Isocyanurate n=3, blocked with 3,5-dimethylpyrazole)

IUPAC Name:

3-Isocyanatomethyl-3,5,5-trimethylcyclohexyl isocyanate, oligomers, reaction products with 3,5-dimethyl-1H-pyrazole

Test material form:

solid

Specific details on test material used for the study:

- Stability under test conditions: The stability of the substance in the vehicle was analytically verified for at least 4 hours (see Ames-Test AT03677).

Method

Target gene:

HPRT locus

Species / strain

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

- Type and identity of media: MEM containing Hank's salts supplemented with 10 % FBS, neomycin (5 µg/mL) and amphotericin B (1%); for the selection of mutant cells the complete medium was supplemented with 11 µg/mL 6-thioguanine.
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes

Metabolic activation:

with and without

Metabolic activation system:

S9-Mix from the liver of phenobarbital/beta-naphthoflavone induced rats.

Test concentrations with justification for top dose:

Exp. I: 4 hours exposure with and without S9-mix 0.91, 2.7, 8.2, 24.7, 74.0, and 222 µg/mL
Exp. II: 24 hours exposure without S9-mix 0.91, 2.7, 8.2, 24.7, 74.0, and 222 µg/mL; 4 hours exposure with S9-mix 0.91, 2.7, 8.2, 24.7, 74.0, and 222 µg/mL µg/mL

The following concentrations were selected for reading: Exp. I without S9-mix 2.7, 8.2, 24.7, 74.0, and 222 µg/mL and with S9-mix 0.91, 2.7, 8.2, 24.7, and 74.0 µg/mL; Exp. II without S9-mix (24 hours exposure) 2.7, 8.2, 24.7, 74.0, and 222 µg/mL, and with S9-mix (4 hours exposure) 0.91, 2.7, 8.2, 24.7, and 74.0 µg/mL

Vehicle / solvent:

DMSO

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

ethylmethanesulphonate

Remarks:

Ethylmethane sulphonate (1.2 mM) used without metabolic activation; 7,12-dimethylbenz(a)anthracene (Exp. I: 4.3, Exp. II: 8.6 µM) used with metabolic activation

Details on test system and experimental conditions:

A pre-test was performed in order to determine the concentration range for the mutagenicity experiments. The general culture conditions and experimental conditions in this pre-test were the same as for the mutagenicity experiment. In this pre-test the colony forming ability of approximately 500 single cells (duplicate cultures per concentration level) after treatment with the test item was observed and compared to the controls. Toxicity of the test item is indicated by a reduction of the cloning efficiency (CE).

The study was performed in two independent experiments, using identical experimental procedures. In the first experiment the treatment period was 4 hours with and without metabolic activation. The second experiment was performed with a treatment time of 4 hours with and 24 hours without metabolic activation.

TREATMENT PROTOCOL
Approximately 1.5 x 10exp6 (single culture) and 5 x 10exp2 cells (in duplicate) were seeded in plastic culture flasks. The cells were grown for 24 hours prior to treatment. After 24 hours the medium was replaced with serum-free medium containing the test item, either without S9 mix or with 50 µl/mL S9 mix. Concurrent solvent and positive controls were treated in parallel. After 4 hours this medium was replaced with complete medium following two washing steps. In the second experiment the cells were exposed to the test item for 24 hours in complete medium, supplemented with 10 % FBS, in the absence of metabolic activation. The colonies used to determine the cloning efficiency (survival) were fixed and stained approx. 7 days after treatment. Three or four days after treatment 1.5 x 10exp6 cells per experimental point were sub-cultivated in 175 cm² flasks containing 30 mL medium. Following the expression time of 7 days five 80 cm² cell culture flasks were seeded with about 3 - 5 x 10exp5 cells each in medium containing 6-TG. Two additional 25 cm² flasks were seeded with approx. 500 cells each in non-selective medium to determine the viability.
The cultures were incubated at 37 °C in a humidified atmosphere with 1.5 % CO2 for about 8 days. The colonies were stained with 10 % methylene blue in 0.01 % KOH solution.
The stained colonies with more than 50 cells were counted. In doubt the colony size was checked with a preparation microscope.

DETERMINATION OF CYTOTOXICITY: Toxicity of the test item is indicated by a reduction of the cloning efficiency.

Evaluation criteria:

A test item is classified as positive if it induces either a concentration-related increase of the mutant frequency or a reproducible and positive response at one of the test points.
A test item producing neither a concentration-related increase of the mutant frequency nor a reproducible positive response at any of the test points is considered non-mutagenic in this system.
A positive response is described as follows:
A test item is classified as mutagenic if it reproducibly induces a mutation frequency that is three times above the spontaneous mutation frequency at least at one of the concentrations in the experiment.
The test item is classified as mutagenic if there is a reproducible concentration-related increase of the mutation frequency. Such evaluation may be considered also in the case that a threefold increase of the mutant frequency is not observed.
However, in a case by case evaluation this decision depends on the level of the corresponding solvent control data. If there is by chance a low spontaneous mutation rate within the laboratory´s historical control data range, a concentration-related increase of the mutations within this range has to be discussed. The variability of the mutation rates of solvent controls within all experiments of this study was also taken into consideration.

Statistics:

A linear regression (least squares, calculated using Sum_neu_v2.xltm, version 2.0) was performed to assess a possible dose dependent increase of mutant frequencies. The number of mutant colonies obtained for the groups treated with the test item were compared to the solvent control groups. A trend is judged as significant whenever the p-value (probability value) is below 0.05. However, both, biological and statistical significance were considered together.

Results and discussion

Test results

Species / strain:

Chinese hamster lung fibroblasts (V79)

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:

not applicable

Positive controls validity:

valid

Additional information on results:

No relevant and reproducible increase in mutant colony numbers/106 cells was observed in the main experiments up to the maximum concentration. The mutant frequency did not exceed the historical range of solvent controls. The threshold of three times the mutation frequency of the corresponding solvent control was not reached or exceeded.
EMS (150 μg/mL) and DMBA (1.1 μg/mL in the first experiment, 2.2 μg/mL in the second) were used as positive controls and showed a distinct increase in induced mutant colonies.

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH and osmolality: There was no relevant shift of pH and osmolarity of the medium even at the maximum concentration of the test item.
- Precipitation: Precipitation occurred at 74.0 μg/mL and above in experiment I and II without metabolic activation and at 24.7 μg/mL and above with metabolic activation.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
No relevant cytotoxic effects indicated by a relative cloning efficiency I or cell density below 50 % was observed in experiment I and II after 4 and 24 hours treatment with and without metabolic activation.

Applicant's summary and conclusion

Executive summary:

A study according to OECD TG 476 was conducted in order to investigate the potential of the substance to induce gene mutations at the HPRT locus in mammalian cells. For that purpose, V79 cells were exposed to the test item for 4 hours with and without metabolic activation in the first experiment and for 4 hours with and for 24 hours without metabolic activation in the second experiment. The maximum concentration of the pre-experiment (2666.7 μg/mL) was based on the solubility properties of the test item in the vehicle (DMSO). The concentration range of the main experiments was limited by precipitation of the test item in aqueous medium.

No substantial and reproducible dose dependent increase of the mutation frequency was observed in both main experiments. Appropriate reference mutagens, used as positive controls, induced a distinct increase in mutant colonies and thus, showed the sensitivity of the test system and the activity of the metabolic activation system.

In conclusion it can be stated that under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in mammalian cells.