Cyclohexane, 5-isocyanato-1-(isocyanatomethyl)-1,3,3-trimethyl-, homopolymer, acetone oxime-blocked

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / micronucleus study

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2011-09-26 to 2011-12-23

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: OECD Guidelines for Testing of Chemicals: In Vitro Mammalian Cell Micronucleus Test (MNvit), No. 487, Guideline July 22, 2010

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian cell micronucleus test

Test material

Test material information

Constituent 1

Reference substance name:

(propan-2-ylidene)amino N-(3,3,5-trimethyl-5-{[2,4,6-trioxo-3,5-bis({1,3,3-trimethyl-5-[({[(propan-2-ylidene)amino]oxy}carbonyl)amino]cyclohexyl}methyl)-1,3,5-triazinan-1-yl]methyl}cyclohexyl)carbamate; (propan-2-ylidene)amino N-(3,3,5-trimethyl-5-{[2,4,6-trioxo-3-({1,3,3-trimethyl-5-[({[(propan-2-ylidene)amino]oxy}carbonyl)amino]cyclohexyl}methyl)-5-({1,3,3-trimethyl-5-[2,4,6-trioxo-3,5-bis(3,3,5-trimethyl-5-{[({[(propan-2-ylidene)amino]oxy}carbonyl)amino]methyl}cyclohexyl)-1,3,5-triazinan-1-yl]cyclohexyl}methyl)-1,3,5-triazinan-1-yl]methyl}cyclohexyl)carbamate

EC Number:

600-028-9

Cas Number:

1001254-87-0

Molecular formula:

Exact identification is not feasible

IUPAC Name:

(propan-2-ylidene)amino N-(3,3,5-trimethyl-5-{[2,4,6-trioxo-3,5-bis({1,3,3-trimethyl-5-[({[(propan-2-ylidene)amino]oxy}carbonyl)amino]cyclohexyl}methyl)-1,3,5-triazinan-1-yl]methyl}cyclohexyl)carbamate; (propan-2-ylidene)amino N-(3,3,5-trimethyl-5-{[2,4,6-trioxo-3-({1,3,3-trimethyl-5-[({[(propan-2-ylidene)amino]oxy}carbonyl)amino]cyclohexyl}methyl)-5-({1,3,3-trimethyl-5-[2,4,6-trioxo-3,5-bis(3,3,5-trimethyl-5-{[({[(propan-2-ylidene)amino]oxy}carbonyl)amino]methyl}cyclohexyl)-1,3,5-triazinan-1-yl]cyclohexyl}methyl)-1,3,5-triazinan-1-yl]methyl}cyclohexyl)carbamate

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

White solid powder
Approximately 99.5% cyclohexane, 5-isocyanato-1-(isocyanatomethyl)-1,3,3-trimethyl-, homopolymer, acetone oxime-blocked
0.1% cetone
< 0.5% blocked isophorone diisocyanate

Method

Target gene:

mammalian cell system( Chinese hamster Ovary cells)

Species / strain

Species / strain / cell type:

other: Chinese hamster ovary (CHO-K1) cells

Details on mammalian cell type (if applicable):

Species/cell type: CHO cells as originally derived from the ovary of Chinese hamster, obtained from ATCC
CHO-K1, modal chromosome number of 20

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9-mix based on liver homogenate fraction  from male rats, induced with Aroclor 1254 (i.p.)

Test concentrations with justification for top dose:

12.5, 25, 50 and 100 µg/mL

Vehicle / solvent:

Acetone, Fluka Chemie AG, Fresh preparations of the test item were used.

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

Acetone

True negative controls:

no

Positive controls:

yes

Remarks:

clastogen

Positive control substance:

cyclophosphamide

Remarks:

+S9-mix

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

Acetone

True negative controls:

no

Positive controls:

yes

Remarks:

clastogen

Positive control substance:

mitomycin C

Remarks:

-S9-mix

Details on test system and experimental conditions:

SYSTEM OF TESTING
- Species/cell type: CHO-K1 BH4 cell line, cell cycle length 12 hours
- Metabolic activation system: male rat liver S9 from  Aroclor 1254 induced animals
ADMINISTRATION: 
- Solubility: completely dissolved in acetone. Fresh preparations were used.
- Preliminary experiment: without and with metabolic activation test item precipitation was noted starting at a concentration of 100 µg
test item/mL. Hence, 100 µg test item/mL were employed as the top concentration for the genotoxicity tests
without and with metabolic activation.
- Dosing:  12.5, 25, 50 and 100 µg/mL
- Positive and negative control groups and treatment:    
negative: the vehicle acetone served as the negative reference item
positive (+S9): 20 µg/mL cyclophosphamide 
positive (-S9): 0.8 µg/mL mitomycin C 

DURATION
- most aneugens and clastogens are detected by a short term treatment period of 4 hours in the presence and absence of S9, followed by removal of
the test item and a growth period of 1.5 – 2.0 cell cycles. Cells were sampled at a time equivalent to about 1.5 – 2.0 times the normal (i.e. untreated)
cell cycle length either after the beginning or at the end of treatment. Because of the potential cytotoxicity of S9 preparations for cultured
mammalian
cells, an extended exposure treatment of 1.5 – 2.0 normal cell cycles was used only in the absence of S9.
Cell treatment and harvest times for the used CHO cell line see table below.
As both initial tests of the short 4-h treatment are negative or equivocal, a subsequent, extended exposure treatment without S9 was used.
- Harvesting time: harvesting time was 20 hours after the end of exposure

STAIN (for cytogenetic assays): Each culture was harvested and processed separately. High-quality cell preparations for scoring were obtained. Cell
cytoplasm were retained to allow the detection of micronuclei and (in the cytokinesis-block method) reliable identification of binucleate cells. The
slides were stained using Giemsa.

NUMBER OF REPLICATIONS: 2, duplicate cultures were used for each test item concentration and for the solvent control cultures.

NUMBER OF CELLS EVALUATED: The micronucleus frequencies were analysed in at least 2000 binucleated cells per concentration (at least 1000
binucleated cells per culture; two cultures per concentration).

DETERMINATION OF CYTOTOXICITY
- Method: evaluation of cytotoxicity was based on the Cytokinesis-Block Proliferation Index (CBPI) or the Replicative Index (RI).
The CBPI indicates the average number of cell cycles per cell during the period of exposure to cytoB, and is used to calculate cell proliferation.
The RI indicates the relative number of nuclei in treated cultures compared to control cultures and can be used to calculate the % cytostasis:

OTHER EXAMINATIONS: 1000 binucleated cells per duplicate cell culture were scored to assess the frequency of cells with one, two, or more than
two micronuclei. Additionally, the cells were classified as mononucleates, binucleates or multinucleates to estimate the proliferation index as a
measure of toxicity.

Evaluation criteria:

The assay demonstrates its ability to reliably and accurately detect substances of known aneugenic and clastogenic activity, with and without
metabolic activation.
Solvent/vehicle control and untreated cultures give reproducibly low and consistent micronuclei frequencies, typically 5 – 25 micronuclei per 1000
cells according to OECD 487. Data from negative and positive controls are used to establish historical control ranges. These values are used in
deciding the adequacy of the concurrent negative/positive controls for an experiment .

Statistics:

The assessment was carried out by a comparison of the samples with the positive and the vehicle control, using a chi-square test corrected for
continuity according to YATES.
If a test item induces a concentration-related increase or a statistical significant and reproducible increase in the number of cells containing
micronuclei, it is classified as a positive result.

Results and discussion

Test results

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

precipitation at 100 µg/mL

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Tests without metabolic activation (4- and 20-hour exposure)
The micronucleus frequencies of cultures treated with test item at concentrations from 12.5 to 100 µg/mL medium (4 h and 20-h
exposure) in the absence of metabolic activation ranged from 7.0 to 25.0 micronuclei per 1000 binucleated cells. The results obtained are
considered to be within the normal range of the vehicle acetone where a mean incidence of micronucleus frequencies of 12.0 or 18.0 micronuclei per 1000 binucleated cells was observed after a 4-hour and 20-hour exposure, respectively. The micronucleus frequency of the untreated controls was 2.0 micronuclei per 1000 binucleated cells.
Test with metabolic activation (4-hour exposure)
The micronucleus frequencies of cultures treated with test item at concentrations from 12.5 to 100 µg/mL medium in the presence
of metabolic activation ranged from 10.5 to 20.5 micronucleus per 1000 binucleated cells. The results obtained are considered to be within the
normal range of the vehicle acetone where a mean incidence of micronucleus frequencies of 10.0 or 22.0 micronucleus per 1000 binucleated cells
was observed in the first and second experiment, respectively. The micronucleus frequencies of the untreated controls were 0.5 or 2.0 micronuclei
per 1000 binucleated cells.

Any other information on results incl. tables

see attached document

Applicant's summary and conclusion

Conclusions:

Under the present test conditions, the test item tested up to a concentration of 100 µg/mL, that led to test item precipitation 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 mutagenic properties in the in vitro micronucleus test.

Executive summary:

The in vitro micronucleus assay is a genotoxicity test system for the detection of chemicals which induce the formation of small membrane bound DNA fragments i.e. micronuclei in the cytoplasm of interphase cells. These micronuclei may originate from a centric fragments (chromosome fragments lacking a centromere) or whole chromosomes which are unable to migrate with the rest of the chromosomes during the anaphase of cell division. The purpose of the micronucleus assay is to detect those agents which modify chromosome structure and segregation in such a way as to lead to induction of micronuclei in interphase cells.

Test item were assayed in an in vitro micronucleus test using CHO cell cultures both in the presence and absence of metabolic activation by a rat liver post-mitochondrial fraction (S9 mix) from Aroclor 1254 induced animals.

The test was carried out employing two exposure times without S9 mix: 4 and 20 hours, and 1 exposure time with S9 mix: 4 hours. The experiment with S9 mix was carried out twice. The harvesting time was 20 hours after end of exposure. The study was conducted in duplicate.

The test item was completely dissolved in acetone. The concentrations employed were chosen based on the results of a cytotoxicity study. In this preliminary experiment without and with metabolic activation test item precipitation was noted starting at a concentration of 100 µg test item/mL. Hence, 100 µg test item/mL were employed as the top concentration for the mutagenicity tests without and with metabolic activation. In the main study test item precipitation was noted at the top concentration of 100 µg test item/mL in the experiments without and with metabolic activation.

Mitomycin C and cyclophosphamide were employed as positive controls in the absence and presence of metabolic activation, respectively, both induced significant damage.

Under the present test conditions, the test item tested up to a concentration of 100 µg/mL, that led to test item precipitation 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 mutagenic properties in the in vitro micronucleus test.