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

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Under the present test conditions the test item 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 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

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

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:

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

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

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.

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.

see attached document

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.

Reference 2

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:

2011-09-26 to 2012-01-26

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

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: 15.63; 31.3; 62.5; 125; 250 µg/mL

Vehicle / solvent:

Test item 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 DMSO (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 pronounced to complete cytotoxicity and test item precipitation were noted at
concentrations of 1000 µg test item/mL and higher. Hence, 625 or 1250 µg test item per mL were employed as the top concentrations for the
mutagenicity tests without and with metabolic activation, respectively.

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.

Key result

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 250 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 pronounced to complete cytotoxicity and test item precipitation were noted at concentrations of 250 µg test item/mL and higher.
Hence, 250 µg test item/mL were employed as the top concentrations for the mutagenicity tests without and with metabolic activation, respectively .


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.

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 [1]. 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:

Under the present test conditions, the test item tested up to a concentration of 250 µg/mL, that led to test item precipitation 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:

The test item 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 250 µg test item/100/mL and higher. Hence, 250 µg test item/100/mL were employed as the top concentration for the mutagenicity tests without and with metabolic activation.

 

Main study

Five concentrations ranging from 15.63 to 250 µg test item/100/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 250 µg/mL in the absence and presence of metabolic activation. No signs of cytotoxicityin form of decreased plating efficiency (PE1) and (PE2) were noted in the first andsecond experiments up to the top concentration 250 µg/mL in the absence and presence of metabolic activation.

 

Experiments without metabolic activation

The mutation frequency of the negative control acetone was 10.80 and 10.03 x 10-6clonable cells. Hence, the negative controls were well within the expected range (see below).

The mutation frequency of the cultures treated with concentrations of 15.63, 31.3, 62.5, 125 or 250 µg test item/100/mL culture medium ranged from 8.02 to 15.88 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 10.44 and 15.24 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 15.63, 31.3, 62.5, 125 or 250 µg test item/100/mL culture medium ranged from 8.72 to 17.29 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 171.61 to 1262.73 x 10-6 clonable cells in the case of EMS and ranging from 304.58 to 798.83 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 10E6 clonable cells for DMBA.

 

Under the present test conditions, the test item tested up to a concentration of 250 µg/mL, that led to test item precipitation 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.

 

 

Reference 3

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:

2011-09-26 to 2011-10-31

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Version / remarks:

2005

Qualifier:

according to guideline

Guideline:

other: ICH Guideline S2A:'Genotoxicity: Specific Aspects of Regulatory gentoxocoty tests for Pharmaceuticals (CPMP/ICH/141/95)'

Qualifier:

according to guideline

Guideline:

other: ICH Giudeline S2B: 'A Standard Battery for Genotoxicity Testing of Pharmaceuticals (CPMP/ICH/174/95)'

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

mutated gene loci resposible for histidine auxotropy

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Additional strain / cell type characteristics:

other: histidine auxotroph

Metabolic activation:

with and without

Metabolic activation system:

Arochlor 1254 induced rat liver S9; male rats

Test concentrations with justification for top dose:

Experiment I and II:
10.0, 31.6, 100, 316, 1000 and 3160 µg per plate.

Vehicle / solvent:

Test item was completely dissolved in dimethyl sulfoxide (DMSO).

Untreated negative controls:

no

Remarks:

solvent test will be used as negative reference item

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Remarks:

for details see below

Positive control substance:

other: without metabolic activation: sodium azide in aqua ad iniectabilia for TA 1535 and TA 100, 2-nitroflurene in DMSO for TA 98, 9-amino-acridine in ethanol abs. for TA 1537, Cumene hydroperoxide in DMSO for TA 102

Remarks:

with metabolic activation: 2-aminoanthracene for TA 98, TA 102, TA 1537, Cyclophosphamide for TA 100, TA 1535

Details on test system and experimental conditions:

Bacterial Reverse Mutation Test
SYSTEM OF TESTING
- Pre-Experiment: plate incorporation cytotoxicity test (+/- metabolic activation) with strain TA 100,
10 concentrations ranging from 0.316 to 5000 µg/plate were tested. Cytotoxicity was noted in the plate incorporation test without and with
metabolic activation at concentrations of 3160 and 5000 µg/plate. In addition, test item precipitation was noted at the top concentration of
5000 µg/plate.
- Main test: 1st - Standard plate incorporation method, 2nd - Preincubation method
- Metabolic activation assay: Arochlor 1254 induced rat liver S9 fraction, the protein content of the S9 fraction was 34.2 mg/mL S9, cytochrome
P-450: 0.36 nmol/mg protein
ADMINISTRATION
- Dosing: 10.0, 31.6, 100, 316, 1000 and 3160 µg per plate.
- Data : 2 independent experiments with and without metabolic activation
- Number of replicates: 3 per concentration and experiment
- Positive and negative control groups and treatment:
- without metabolic activation:
sodium azide in aqua ad iniectabilia for TA 1535 and TA 100, 10 µg/plate
2-nitroflurene in DMSO for TA 98, 10 µg/plate
9-amino-acridine in ethanol abs. for TA 1537, 100 µg/plate
Cumene hydroperoxide in DMSO for TA 102, 10 µg/plate
- with metabolic acivation
2-aminoanthracene in DMSO for TA 98, TA 102, TA 1537, 2 µg/plate
Cyclophosphamide in aqua ad iniectabilia for TA 100, TA 1535, 1500 µg/plate
- negative control: solvent control: DMSO for all strains
- Incubation time: 48 h to 72 h at 37 °C in the dark
- Pre-incubation time: 20 min at 37 °C;

Evaluation criteria:

The test item is considered to show a positive response if:
- the number of revertants is significantly increased (p ≤ 0 .05, U-test according to MANN and WHITNEY) compared to the solvent control to at least
2-fold of the solvent control for TA 98, TA 100 and TA 102 and 3-fold of the solvent control for TA 1535 and TA 1537 in both independent
experiments.
- additionally, a significant (p ≤ 0.05) concentration (log value)-related effect (Spearman's rank correlation coefficient) is observed.
- positive results have to be reproducible and the histidine independence of the revertants has to be confirmed by streaking random samples on
histidine-free agar plates.

Statistics:

According to the OECD Guideline 471, a statistical analysis of the data is not mandatory

Key result

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

without and with metabolic activation at concentrations of 3160 and 5000 µg/plate, precipitation was noted at 5000 µg/plate.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

GENTOXIC EFFECTS:
- With metabolic activation: negativ
- Without metabolic activation: negativ

see attchached document

Conclusions:

Under the present test conditions test item tested up to a cytotoxic concentration of 3160 µg/plate, caused no mutagenic effect in the Salmonella typhimurium strains TA 98, TA 100, TA 102, TA 1535 and TA 1537 neither in the plate incorporation test nor in the preincubation test each carried out without and with metabolic activation.
 

Executive summary:

The purpose of this study was to evaluate the test item for mutagenic activity (gene mutation) in bacteria without and with the addition of a mammalian metabolic activation system as originally described by AMES et al. (1973, 1975) and revised by MARON and (1983).

The test item was examined in the five Salmonella typhimurium strains TA 98, TA 100, TA 102, TA 1535 and TA 1537 in two independent experiments, each carried out without and with metabolic activation (a microsomal preparation derived from Aroclor 1254-induced rat liver). The first experiment was carried out as a plate incorporation test and the second as a preincubation test.

Six concentrations ranging from 10.0 to 3160 µg test item per plate were employed in two independent experiments each carried out without and with metabolic activation.

In the plate incorporation test and in the preincubation test, each carried out without and with metabolic activation, cytotoxicity (scarce background lawn and/or reduction of the number of revertants)were noted at the top concentration of 3160 µg/plate in all test strains.

No mutagenic effect (no increase in revertant colony numbers as compared with control counts) was observed for the test item, tested up to a cytotoxic concentration of 3160 µg/plate, in any of the 5 test strains in two independent experiments without and with metabolic activation, respectively (plate incorporation and preincubation test).

 

In conclusion, under the present test conditions test item tested up to a cytotoxic concentration of 3160 µg/plate, caused no mutagenic effect in the Salmonella typhimurium strains TA 98, TA 100, TA 102, TA 1535 and TA 1537 neither in the plate incorporation test nor in the preincubation test each carried out without and with metabolic activation.

 

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

Since all three in vitro genotoxicity studies showed no genotoxic effects, no in vivo genetic toxicity study was performed.

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Cyclohexane, 5-isocyanato-1-(isocyanatomethyl)-1,3,3-trimethyl-, homopolymer, acetone oxime-blocked did not induce gene mutations in bacteria (OECD TG 471; LPT, 2012) 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) either with or 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 three 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 cyclohexane, 5-isocyanato-1-(isocyanatomethyl)-1,3,3-trimethyl-, homopolymer, acetone oxime-blocked is not genotoxic in vitro and therefore must not be classified according to the criteria of EC Regulation 1272/2008.