2H-Azepin-2-one, hexahydro-, polymer with 1,6-diisocyanatohexane

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Under the present test conditions the test item 2H-Azepin-2-one, hexahydro-, polymer with 1,6-diisocyanatohexane tested up to cytotoxic concentrations in the in vitro bacterial mutagenicity assay (Salmonella reverse mutation Assay, OECD 471), the in vitro mammalian mutagenicity assay (HPRT Assay, OECD 476) and the in vitro mammalian cytogenetic assay (Micronucleus Test,OECD 487) each assay carried out without and with metabolic activation, revealed no genotoxic activity.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2007-08-15 to 2007-08-28

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Principles of method if other than guideline:

Remark: Both TA 102 and E.coli WP2 were not tested (not required by applied version of guideline).

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

mutated gene loci responsible for histidine auxotrophy

Species / strain / cell type:

other: Salmonella typhimurium TA 98, TA100, TA 102, TA 1535, TA 1537

Additional strain / cell type characteristics:

other: histidine auxotroph

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced rat liver S9 fraction, prepared from  male Sprague Dawley rats

Test concentrations with justification for top dose:

20 to 5000 µg/plate

Vehicle / solvent:

dimethyl sulfoxide,  DMSO (CAS No. 67-68-5)

Untreated negative controls:

yes

Remarks:

untreated

Negative solvent / vehicle controls:

yes

Remarks:

Ethylen glycol dimethylether (EGDE)

True negative controls:

no

Positive controls:

yes

Remarks:

details see below

Positive control substance:

other: without metabolic activation: 4-Nitro-1,2-phenylene (TA 98; TA 1537), Nitrofurantoin (TA100), Sodium azide (TA 1535), Mitomycin C (TA 102); with metabolic activation: Aminoanthracene

Details on test system and experimental conditions:

Ames test
SYSTEM OF TESTING
- Metabolic activation system:  Aroclor 1254-induced rat liver S9 fraction, prepared from  male Sprague Dawley rats
ADMINISTRATION: 
- Dosing:    
Plate incorporation test: 50/158/500/1581/5000 µg/plate (+/- metabolic  activation)   
- Number of replicates: 2
- Repeat: preincubation
- Application: solvent ethylen glycol dimethylether (EGDE)
- Positive and negative control groups and treatment:    
positive without metabolic activation:   
TA 98: 4-Nitro-1,2-phenylene diamine (0.5 µg/plate in dimethyl sulfoxide)   
TA 100: Nitrofurantoin (0.2 µg/plate in dimethyl sulfoxide)   
TA 102: Mitomycin C (0.2 µg/platein deionized water)
TA 102: Cumene hydroperoxide (50 µg/plate in dimethyl sulfoxide) in plate preincubtion trials
TA 1535: Sodium azide (10 µg/plate in dimethyl sulfoxide)   
TA 1537: 4-Nitro-1,2-phenylene diamine (10 µg/plate in dimethyl sulfoxide)   
positive with metabolic activation and activity of metabolic system:   all strains: 2-aminoanthracene (2.5 µg/plate in dimethyl sulfoxide)   
negative: solvent control, 100 µl/plate (pre-incubation: 50 µl/plate)
- Pre-incubation: 20 minutes at 37 °C   incubation 48 hours at 37 °C

Evaluation criteria:

CRITERIA FOR EVALUATING RESULTS:    
- negative controls and to be within the expected range, as defined by published data
- positive controls had to show sufficient effects, as defined by the laboratories experience
- titer determinations had to demonstrate sufficient bacterial density in the suspension

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

doses up to and including 5000 µg per plate did not cause any bacteriotoxic effects

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

GENOTOXIC EFFECTS: 
- With metabolic activation: None
- Without metabolic activation: None   
The positive controls w ere functional.
PRECIPITATION CONCENTRATION: Precipitation occurred at the dose 1581 µ per plate and above
CYTOTOXIC CONCENTRATION (including effects on background lawn): 
- Doses up to and including 5000 µg per plate did not cause any bacteriotoxic effects

Remarks on result:

other: other:

Remarks:

Migrated from field 'Test system'.

no other results

Conclusions:

Interpretation of results (migrated information):
negative

The test item was considered to be non-mutagenic without and with S9 mix in the plate incorporation as well as in the preincubation modification of the Salmonella/microsome test.

Executive summary:

The test item was tested for its ability to induce reverse mutations in an in vitro bacterial system.

Salmonella typhimurium strains TA 98, TA 100, TA 102, TA 1535 and TA 1537 were treated with 2H-Azepin-2-one, hexahydro-, polymer with 1,6-diisocyanatohexane by the Ames test plate incorporation as well as the preincubation method. Five dose levels covering the range between 50 and 5000 µg/plate, in triplicate both with and without the addition of a metabolizing system were employed.

All five bacterial strains exhibited mutagenic response to the appropriate positive control substances. Solvent controls were also tested with each strain and the mean numbers of spontaneous revertants were in an acceptable range.

Mutagenic activity of the test compound to one or more of the tester strains was not observed in either experiment with and without metabolic activation. Therefore, the test item was considered to be non-mutagenic without and with S9 mix in the plate incorporation as well as in the preincubation modification of the Salmonella/microsome test.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2011-08-17 to 2011-11-25

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study; GLP study without deviations

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 chromosome aberration test

Target gene:

mammalian cell system( Chinese hamster Ovary cells)

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:

The test item was completely dissolved in acetone as the test item was not soluble in other solvents recommended. The test item was heated to 50°C shortly before use. Preparations of the test item made on the day of use were employed.

Untreated negative controls:

yes

Remarks:

solvent control

Negative solvent / vehicle controls:

yes

Remarks:

acetone

True negative controls:

no

Positive controls:

yes

Remarks:

clastogen

Positive control substance:

cyclophosphamide

Remarks:

+S9-mix

Untreated negative controls:

yes

Remarks:

solvent control

Negative solvent / vehicle controls:

yes

Remarks:

acetone

True negative controls:

no

Positive controls:

yes

Remarks:

clastogen

Positive control substance:

mitomycin C

Remarks:

-S9-mix

Untreated negative controls:

yes

Remarks:

solvent control

Negative solvent / vehicle controls:

yes

Remarks:

acetone

True negative controls:

no

Positive controls:

yes

Remarks:

aneugen

Positive control substance:

other: colchicine

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
- No. of metaphases analyzed: first 100 consecutive from each culture if  possible; mitotic index based on 1000 cell nuclei counted per count
ADMINISTRATION: 
- Solubility: test item was completely dissolved in acetone as the test item was not soluble in other solvents recommended. The test item was heated
to 50°C shortly before use. Preparations of the test item made on the day of use were employed.
- Preliminary experiment: precipitation was noted starting at a concentration of 100 µg test item/mL. Hence, 100 µg /mL were employed as the top
concentration for the mutagenicity tests without and with metabolic activation.
- Dosing:  12.5, 25, 50 and 100 µg/mL
Both for presence and absence of S9-mix, 3 relevant concentration levels per test were analyzed for chromosomal aberrations. The highest  level 
selected should have reduced the mitotic index by 50-70 %.
- Number of replicates: 2
- Application:    
- Test 1: after 4 hours exposure visual inspection; replace medium with  test substance by medium without test substance;  
12 hours later (i.e. at 16 hours) visual inspection, addition of  colcemid (resulting concentration 0.1 mg/l);  harvesting time was 20 hours after end of exposure  
- Test 2: similar to Test 1, except continuos exposure for 20 hours (i.e.  no replacement of medium) of samples without S9-mix 
- Positive and negative control groups and treatment:    
negative: acetone
positive (+S9): cyclophosphamide 
positive (-S9): mitomycin C 
positive (-S9): colchicine

Evaluation criteria:

The study is considered valid if:
(1) the positive controls show a significant increase in the number of aberrant cells and   
(2) the negative controls are within historical ranges.

A response is considered to be positive if:
a statistically significant increase in the number of aberrant cells is observed which is   
(1) either concentration-related   
(2) or reproduced in the second experiment at a similar dose level.

A response is considered to be equivocal if:
0.05

Statistics:

Fisher's exact probability test (two-sided) for significant differences between treated and control cultures

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

precipitation was noted starting at a concentration of 100 µg test item/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 2H-Azepin-2-one, hexahydro-, polymer with 1,6-diisocyanatohexane at concentrations from 12.5 to 100 µg/mL medium (4 h and 20-h exposure) in the absence of metabolic activation ranged from 2.0 to 13.0 micronuclei per 1000 binucleated cells. The results obtained are considered to be within the normal range of the negative control acetone where a mean incidence of micronucleus frequencies of 8.5 or 3.0 micronuclei per 1000 binucleated cells were observed after a 4-hour and 20-hour exposure, respectively. The micronucleus frequency of the untreated controls was 7.0 or 4.0 micronuclei per 1000 binucleated cells after a 4-hour and 20-hour exposure, respectively.
Test with metabolic activation (4-hour exposure)
The micronucleus frequencies of cultures treated with 2H-Azepin-2-one, hexahydro-, polymer with 1,6-diisocyanatohexane at concentrations from 12.5 to 100 µg/mL medium in the presence of metabolic activation ranged from 3.0 to 9.0 micronuclei per 1000 binucleated cells. The results obtained are considered to be within the normal range of the negative control acetone where a mean incidence of micronucleus frequencies of 5.5 or 5.0 micronuclei per 1000 binucleated cells were observed in the first and second experiment, respectively. The micronucleus frequencies of the untreated controls were 7.0 or 3.5 micronuclei per 1000 binucleated cells in the first and second experiment, respectively.

Remarks on result:

other: other: Chinese hamster Ovary (CHO)

Remarks:

Migrated from field 'Test system'.

see attached documents

Conclusions:

Interpretation of results (migrated information):
negative

Under the present test conditions, 2H-Azepin-2-one, hexahydro-, polymer with 1,6-diisocyanatohexane 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 mutagenic 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 acentric 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 samples of 2H-Azepin-2-one, hexahydro-, polymer with 1,6-diisocyanatohexane were assayed in an in vitro micronucleus test using CHO cell cultures both in the presence and absence of metabolic activation by a ratliver post-mitochondrial fraction (S9 mix) from Aroclor 1254 induced animals.

The test was carried out employing 2 exposure times without S9 mix: 4and 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 starting 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 /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 /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.

Under the present test conditions, 2H-Azepin-2-one, hexahydro-, polymer with 1,6-diisocyanatohexane 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.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2011-08-17 to 2011-12-16

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP Guideline study.

Qualifier:

according to guideline

Guideline:

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

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Target gene:

Hypoxanthine-guanine phosphoribosyl transferase (HPRT)

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

Cells were maintained in Dulbecco's modified Eagle-Mediumsupplemented with 10% fetal calf serum, penicillin (100 U/mL) and streptomycin
(100 µg/mL) called DMEM-FCS; Cells were periodically checked for the absence of mycoplasma contamination by using the HOECHST stain 33258;
Spontaneous mutation rate was continuously monitored

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

post-mitochondrial supernatant fraction derived from livers of Aroclor 1254-treated rats (S9 mix)

Test concentrations with justification for top dose:

Five concentrations: 6.25; 12.5; 25; 50; 100µg/mL

Vehicle / solvent:

The test item was completely dissolved in acetone . Preparations of the test item made on the day of use were employed.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Acetone

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: ethyl methanesulphonate (EMS) in direct mutagenicity experiment; 9,10-dimethyl-1,2-benzanthracene (DMBA) in S9 mix mediated assay; both EMS and DMBA were dissolved in DMSO. The applied concentrations were 600 or 700 µg EMS/mL medium or 20 or 30 µg DMBA/mL

Details on test system and experimental conditions:

CELLS AND TISSUE CULTURE MEDIA
- V79 cells were maintained in Dulbecco's modified Eagle-Mediumsupplemented with 10% fetal calf serum, penicillin 3 (100 U/mL) and streptomycin (100 µg/mL) called DMEM-FCS
- Incubation of cultures: at 37°C in a humidified atmosphere (90%) containing 10% CO2
- For subculturing, a trypsin (0.05%)-EDTA (ethylenediaminetetraacetic acid, 0.02%) solution in modified Puck's salt solution A was used.

METHOD OF APPLICATION:
- Exposure to the test item in the presence of S9 mix was performed in Dulbecco's phosphate buffered saline (PBS) which additionally contained 20 mM HEPES (N'-2-hydroxyethylpiperazine-N'-2-ethane-sulfonic acid) pH 7.4 (PBS-HEPES).


DURATION
- Preincubation period: 1 day (in 30 mL DMEM-FCS)
- Exposure duration:
* 4 hours (1st experiment) and 24 hours (2nd experiment) without S9 mix, respectively;
* in the experiments with S9 mix, the medium was replaced by 18 mL S9 mix and the exposure limited to 4 hours.
* the negative control was treated with acetone (the vehicle) in the same way
* After removal of the test item and washing of the plates with PBS cells were trypsinised and a relative plating efficiency was determined for each
dose to obtain an accurate measure of the toxic effect of the chemical
- Expression time (cells in growth medium):
* Three replicate plates (60 mm diameter) were used with a known number of cells.
* Remaining cells were replated and the culture incubation continued until day 8 with 30 mL normal DMEM-FCS with one subcultivation on day 5.
* Afterwards cells were harvested by trypsinisation and replated at a density of 1 000 000 per 150mm diameter dish in DMEM-FCS containing
6-thioguanine (10 µg/mL) for selection of mutants (5 replicate plates), or at approx. 100 to 150 cells (exact number known) per 60 mm diameter
dish in medium without 6-thioguanine for the estimation of plating efficiencies (PE 2), (3 replicate plates).
* Plates were fixed and stained after about 8 days (plating efficiency plates) or 12 days (6-thioguanine plates).
- Positive control:
* ethyl methanesulphonate (EMS) in direct mutagenicity experiment;
* 9,10-dimethyl-1,2-benzanthracene (DMBA) in S9 mix mediated assay
both EMS and DMBA were dissolved in DMSO. The applied concentrations were 600 or 700 µg EMS/mL medium or 20 or 30 µg DMBA/mL


NUMBER OF REPLICATIONS: three
NUMBER OF CELLS EVALUATED: 1 500 000

DETERMINATION OF CYTOTOXICITY (same procedure was used as employed for the mutagenicity experiments, except that no mutant selection was carried out)
- Method: survival
- A concentration of the test item which produces a low level of survival (10 to 20%) would be used as highest concentration and the survival in the
lowest concentration being approximately the same as that in the negative control.
- Five adequately spaced concentrations are employed
- In this preliminary experiment without and with metabolic activation test item precipitation was noted at concentrations of 100 µg
test item/mL and higher. Hence, 100 µg test item per mL were employed as the top concentrations for the mutagenicity tests without and with
metabolic activation.

Evaluation criteria:

The following pre-determined descriptive criteria are used for interpretation of the results:
- If in both independent experiments solvent and positive controls show results within the norm and if the test item does not increase the mutation
frequency 2-fold above the mean of the solvent controls under any condition, or if the mutation frequency is always lower than 40 x 10^-6 and if at least 1 000 000 cells per condition have been evaluated, the item is considered as negative in the test.
- In case of a dose-dependent increase of the mutation frequency in both independent experiments (at similar concentrations) to at least 2-fold
solvent control and at least 40 x 10^-6 both in the presence and/or absence of S9 mix, the item is considered as positive in the test.

Statistics:

No satisfactory mathematical methods are available for the statistical analysis of mammalian cell mutagenicity experiments.

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

precipitation were noted at concentrations of 100 µg test item/mL

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES (Preliminary cytotoxicity test):
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 at concentrations of 100 µg copolymer of test item/mL and higher. Hence, 100 µg test item per mL were employed as the top concentrations for the mutagenicity tests without and with metabolic activation.


COMPARISON WITH HISTORICAL CONTROL DATA:
All the mutation frequencies obtained for the test item are within the negative control ranges. The mean mutation frequency of the control
background data is 14.11 ± 7.42 x 10^-6 clonable cells with a range of 1.30 - 34.80 x 10^-6 clonable cells for the experiments without metabolic
activation and 14.88 ± 8.20 x 10^-6 clonable cells with a range of 2.18 - 38.36 x 10^-6 clonable cells for the experiments with metabolic activation.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Criteria for assay acceptance

Solvent control: As the total number of colonies is normally low and as a single mutation may cause several colonies due to cell division during the expression period, a relatively large variation of the mutation frequency may result. This is especially true, if a low spontaneous mutation frequency is forced by cloning (in order to achieve a high sensitivity of the test).

The historical background mutation frequency in this system has been reported to be 1 to 44 mutants per 10⁶survivors in non-activation solvent controls and 6 to 46 per 10⁶survivors in S9 activation solvent controls. The background data obtained at LPT are given at the end of this chapter. The spontaneous mutation frequency may be variable from experiment to experiment, but should normally lie within the above-mentioned range. The positive controls(600 and 700 µg/mL) and DMBA (20 and 30 µg/mL) should cause a 10-fold or greater increase in mutation frequency.

The background mutation frequency at LPT ranges from 1.30 to 38.36 x 10^-6clonable cells for the negative controls. The mutation frequency of the positive controls at LPT ranges from 112.1 to 1708.4 x 10^-6clonable cells forand 130.0 to 2693.3 x 10^-6clonable cells for DMBA (see table below).

The mutation frequencies of the solvent controls andthe positive controlswithout and with metabolic activation for the last 58 experiments (most recent background data, not audited by the QAU-department) are given as follows:

Mutation frequency per 10^6 clonable cells
	Without metabolic activation (24-h exposure)		With metabolic activation (4-h exposure)
Solvent control (n = 58)
mean	14.11		14.88
SD	7.42		8.20
range	1.30 - 34.80		2.18 - 38.36
Positive control (µg/mL) (n = 58)
	EMS (600)	EMS (700)	DMBA (20)	DMBA (30)
mean	449.1	468.4	347.1	563.8444.2
SD	444.2	268.6	241.8	700.1
range	112.1 – 1708.4	152.0 – 976.9	130.0 – 844.8	151.3 – 2693.3

SD     = Standard deviation

EMS   = ethyl methanesulfonate

DMBA = 9,10-dimethyl-1,2-benzanthracene

Conclusions:

Interpretation of results (migrated information):
negative

Under the present test conditions, 2H-Azepin-2-one, hexahydro-, polymer with 1,6-diisocyanatohexane tested up to a concentration of 100 µg/mL, in the experiments without and with metabolic activation, respectively, was negative in the HPRT-V79 mammalian cell mutagenicity test under
conditions where positive controls exerted potent mutagenic effects.
 

Executive summary:

2H-Azepin-2-one, hexahydro-, polymer with 1,6-diisocyanatohexane was tested for mutagenic potential in a gene mutation assay in cultured mammalian cells (V79, genetic marker HPRT) both in the presence and absence of metabolic activation by a rat liver post-mitochondrial fraction (S9 mix) from Aroclor 1254-induced animals. The duration of the exposure with the test item was 4 hours or 24 hours in the experiments without S9 mix and 4 hours in the experiments with S9 mix.

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 at concentrations of 100 µg /mL and higher. Hence, 100 µg test item/mL were employed as the top concentration for the mutagenicity tests without and with metabolic activation.

Main study

Five concentrations ranging from 6.25 to 100 µg /mL were selected for the experiments without and with metabolic activation.

Cytotoxicity

In the main study, test item precipitation was noted in the first and second experiments at the top concentration of 100 µg/mL in the absence and presence of metabolic activation. No signs of cytotoxicity in form of decreased plating efficiency (PE₁) and (PE₂)were noted in the first and second experiments up to the top concentration 100 µg/mL in the absence and presence of metabolic activation.

Experiments without metabolic activation

The mutation frequency of the negative control acetone was 9.58 and 8.28 x 10-6 clonable cells. Hence, the negative controls were well within the expected range (see below).

The mutation frequency of the cultures treated with concentrations of 6.25, 12.5, 25, 50 or 100 µg test item/mL culture medium ranged from 7.25 to 13.10 x 10‑6 clonable cells. These results are within the normal range of the negative controls.

Experiments with metabolic activation

The mutation frequency of the negative control acetone was 15.43 and 9.62 x 10-6 clonable cells. Hence, the negative controls were well within the expected range (see below).

The mutation frequency of the cultures treated with concentrations of 6.25, 12.5, 25, 50 or 100 µg test item/mL culture medium ranged from 7.54 to 13.25 x 10‑6 clonable cells. These results are within the normal range of the negative controls.

The positive controls EMS (ethyl methanesulfonate) in the direct test and DMBA (9,10-dimethyl-1,2-benzanthracene), a compound which requires metabolic activation, caused a pronounced increase in the mutation frequencies ranging from

235.79 to 913.57 x 10-6 clonable cells in the case of EMS and ranging from 365.45 to 1102.99 x 10-6 clonable cells in the case of DMBA, indicating the validity of this test system.

The background mutation frequency at LPT ranges from 1.30 to 38.36 x 10-6 clonable cells for the negative controls. The mutation frequency of the positive controls at LPT ranges from 112.1 to 1708.4 x 10-6 clonable cells forand 130.0 to 2693.3 x 106 clonable cells for DMBA.

Under the present test conditions, 2H-Azepin-2-one, hexahydro-, polymer with 1,6-diisocyanatohexane tested up to a concentration of 100 µg/mL, in the experiments without and with metabolic activation, respectively, was negative in the HPRT-V79 mammalian cell mutagenicity test under conditions where positive controls exerted potent mutagenic effects.  

 

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

2H-Azepin-2-one, hexahydro-, polymer with 1,6-diisocyanatohexane did not induce gene mutations in bacteria (OECD TG 471; Bayer AG, 2007) or in mammalian cells (OECD TG 476; LPT, 2012) and demonstrate no potential to induce micronuclei in Chinese Hamster Ovary cells in vitro (OECD TG 487; LPT, 2012) neither with nor without metabolic activation.

Results from genetic toxicity tests in vivo are not available.

Justification for selection of genetic toxicity endpoint
No study was selected, since all in vitro genotoxicity studies showed no genotoxic effects.

Justification for classification or non-classification

Because all in vitro genotoxicity studies revealed clearly negative results, it can be concluded that 2H-Azepin-2-one, hexahydro-, polymer with 1,6-diisocyanatohexane is not genotoxic in vitro and therefore must not be classified according to the criteria of EC Regulation 1272/2008.