3-Isocyanatomethyl-3,5,5-trimethylcyclohexyl isocyanate, oligomers, 2-Hydroxy-1(2)- methylethyl carbamate blocked

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell micronucleus test

Test material

Test material information

Constituent 1

Reference substance name:

2-(carbamoyloxy)propyl N-[(3-{[3,5-bis({5-[({[2-(carbamoyloxy)propoxy]carbonyl}amino)methyl]-1,3,3-trimethylcyclohexyl}methyl)-2,4,6-trioxo-1,3,5-triazinan-1-yl]methyl}-3,5,5-trimethylcyclohexyl)methyl]carbamate

EC Number:

921-910-2

IUPAC Name:

2-(carbamoyloxy)propyl N-[(3-{[3,5-bis({5-[({[2-(carbamoyloxy)propoxy]carbonyl}amino)methyl]-1,3,3-trimethylcyclohexyl}methyl)-2,4,6-trioxo-1,3,5-triazinan-1-yl]methyl}-3,5,5-trimethylcyclohexyl)methyl]carbamate

Test material form:

solid

Details on test material:

- Analytical purity: 83.9 g/100g
- Composition of test material, percentage of components: 0.2g/100g water, 13.4g/100g n-butyl acetate, 2.5g/100g 1,2-propanediol, monocarbamate
- Lot/batch No.: 389-48
- Expiration date of the lot/batch: 2019-05-23

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 16/0437-1, 389-48
- Expiration date of the lot/batch: 2019-05-23

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature

Method

Species / strain

Species / strain / cell type:

lymphocytes: human lymphocytes

Details on mammalian cell type (if applicable):

blood samples were drawn from healthy non-smoking donors not receiving medication.

Metabolic activation:

with and without

Metabolic activation system:

Mammalian Microsomal Fraction S9 mix

Test concentrations with justification for top dose:

1st Run: 3.0, 5.3, 9.2, 16.1, 28.2, 49.4, 86.5, 151, 265, 795, 2384µg/plate
2nd Run: 4.2, 7.3, 12.8, 22.4, 39.2, 68.6, 120, 300µg/plate
With regard to the purity (83.9%) and the solubility properties of the test item, 2384 μg/mL were applied as top concentration for treatment of the cultures in the pre-test. Test item concentrations ranging from 3.0 to 2384 μg/mL (with and without S9 mix) were chosen for the evaluation of cytotoxicity. Clear toxic effects were observed after 4 hours treatment only with 795 μg/mL in the presence of S9 mix. Considering the precipitation data of Experiment I, 300 μg/mL (without S9 mix) were chosen as top concentration in Experiment II.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

Controls

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

other: Demecolcin

Details on test system and experimental conditions:

NUMBER OF CELLS EVALUATED: 1000 binucleate cells

Evaluation criteria:

The micronucleus assay will be considered acceptable if it meets the following criteria:
− The concurrent solvent control will normally be within the laboratory historical solvent control data range (95% control limit realized as 95% confidence interval).
− The concurrent positive controls should produce a statistically significant increase in the micronucleus frequency and should be within the laboratory historical positive control data range.
− Cell proliferation criteria in the solvent control are considered to be acceptable.
− All experimental conditions described earlier were tested unless one exposure condition resulted in a clearly positive result.
− The quality of the slides must allow the evaluation of an adequate number of cells and concentrations.

Statistics:

Statistical significance was confirmed by the Chi square test (α < 0.05), using a validated test script of “R”, a language and environment for statistical computing and graphics. Within this test script a statistical analysis was conducted for those values that indicated an increase in the number of cells with micronuclei compared to the concurrent solvent control.

Results and discussion

Test results

Species / strain:

lymphocytes: Human lymphocytes

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no relevant effect observed
- Effects of osmolality: no relevant effect observed
- Precipitation: observed at 49.4 µg/ml and above in the absence of S9 mix and at 86.5µg/ml and above in the presence of S9 mix at the end of treatment.

Applicant's summary and conclusion

Conclusions:

In conclusion, it can be stated that under the experimental conditions reported, the test item did not induce micronuclei as determined by the in vitro micronucleus test in human lymphocytes.
Therefore, the test item is considered to be non-mutagenic in this in vitro micronucleus test, when tested up to precipitating concentrations.

Executive summary:

The test item, dissolved in DMSO, was assessed for its potential to induce micronuclei in human lymphocytes in vitro in two independent experiments. In each experimental group two parallel cultures were analyzed. Per culture 1000 binucleated cells were evaluated for cytogenetic damage. The highest applied concentration in this study (2384 μg/mL of the test item) was chosen with regard to the purity (83.9%) and the solubility properties of the test item and with respect to the current OECD Guideline 487. Dose selection of the cytogenetic experiment was performed considering the toxicity data and the occurrence of test item precipitation in accordance with OECD Guideline 487. In the absence and presence of S9 mix, no cytotoxicity was observed up to the highest evaluated concentrations, which showed precipitation. In the absence and presence of S9 mix, no relevant increases in the numbers of micronucleated cells were observed after treatment with the test item. Appropriate mutagens were used as positive controls. They induced statistically significant increases in cells with micronuclei.

In conclusion, it can be stated that under the experimental conditions reported, the test item did not induce micronuclei as determined by the in vitro micronucleus test in human lymphocytes.

Therefore, the test item is considered to be non-mutagenic in this in vitro micronucleus test, when tested up to precipitating concentrations.