Diphenylacetonitrile

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames Assay:

Based on data of the target chemical and of strucutral analogues, the target chemical is not considered to be mutagenic in bacteria.

In vitro chromosomal aberration study:

Based on read-across data, the target chemical is not considered to be clastogenic in mammalian cell lines.

In vitro mammalian cell gene mutation assay:

Based on the read-across data, the target chemical is not expected to be mutagenic at the TK locus in L5178Y mouse lymphoma cells.

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:

weight of evidence

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

significant methodological deficiencies

Justification for type of information:

Data is from peer reviewed publication

Principles of method if other than guideline:

Bacterial reverse mutation assay using several strains of Salmonella typhimurium.

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine gene

Species / strain / cell type:

S. typhimurium, other:

Details on mammalian cell type (if applicable):

Not Specified

Additional strain / cell type characteristics:

other: Histidine-requiring mutants

Metabolic activation:

without

Test concentrations with justification for top dose:

No details provided.

Vehicle / solvent:

Water or first dissolved in a small amount of aceton and then brought up to volume with distilled water.

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

not specified

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: N-methyl-N’-nitro-N-nitrosoguanidine, ICR-191 and diethyl sulfate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)
DURATION
- Preincubation period: No data available
- Exposure duration: No data available
- Expression time (cells in growth medium): No data available
- Selection time (if incubation with a selection agent): No data available
- Fixation time (start of exposure up to fixation or harvest of cells): No data available

SELECTION AGENT (mutation assays): No data available
SPINDLE INHIBITOR (cytogenetic assays): No data available
STAIN (for cytogenetic assays): No data available

NUMBER OF REPLICATIONS: No data available

NUMBER OF CELLS EVALUATED: No data available

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: No data available

OTHER EXAMINATIONS:
- Determination of polyploidy: No data available
- Determination of endoreplication: No data available
- Other: No data available

OTHER: No data available

Evaluation criteria:

Not specified

Statistics:

Not specified

Key result

Species / strain:

other: Salmonella typhimurium

Metabolic activation:

not specified

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

True negative controls validity:

not specified

Positive controls validity:

valid

Additional information on results:

No data available

Remarks on result:

other: The test chemical showed no evidence of mutagenicity in the assay

Conclusions:

The test chemical showed no evidence of mutagenic potential in Salmonella typhimurium in the absence of metabolic activation. The study lacked important methodological details e.g. the identity of strains, the dose levels at which the chemical was tested for mutagenicity, and the justification for top dose level. Therefore, the study can only be used as weight of evidence that the test chemical is non-mutagenic in Salmonella typhimurium in the absence of a metabolic activation system.

Executive summary:

The chemical was tested for mutagenicity in multiple strains of Salmonella typhiumurium in the absence of a metabolic activation system. The bacteria were exposed to the test compound on petri plates which were prepared by mixing 0.2 ml of freshly grown cultures (2 x 10 bacteria per milliliter) of the mutants with 2 ml of 0.6% agar at 45°C. The soft agar, which contained a trace (0.20 µmol) of histidine as well as the bacterial inoculum, was then poured onto plates of histidine free minimal agar medium. Approximately 1 to 5 µL of liquid test chemicals or small crystals of solid test chemicals were applied directly to the surface of each plate after the top layer of agar, containing the mutant bacteria, had solidified. The rates of spontaneous reversion of the eight mutants to form no longer requiring L-histidine for growth were also determined and was found to be low, ranging from 0 to 20 colonies per plate. N-methyl-N’-nitro-N-nitrosoguanidine, ICR-191, and diethyl sulfate served as positive controls. The test chemical showed no evidence of mutagenic potential in Salmonella typhimurium in the absence of metabolic activation. The study lacked important methodological details e.g. the identity of strains, the dose levels at which the chemical was tested for mutagenicity, and the justification for top dose level. Therefore, the study can only be used as weight of evidence that the test chemical is non-mutagenic in Salmonella typhimurium in the absence of a metabolic activation system.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Justification for type of information:

Data is from a handbook and collection of data.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Target gene:

His: S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Trp: E. coli WP2 uvr A

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells: Salmonella typhimurium strains were used
- Suitability of cells: No Data Available
- Normal cell cycle time (negative control): No Data Available

MEDIA USED
- 1) Top agar
The following aqueous solutions (A) and (B) were mixed at a volume ratio of 10: 1.
(A) Bacto Agar (Difco): 0.6%
Sodium chloride: 0.5%
(B) L-histidine: 0.5 mM
Biotin: 0.5 mM

2) Synthetic medium
The minimal agar medium was used as the medium. The composition per liter of medium is as follows.
Magnesium sulfate heptahydrate: 0.2g
Citric acid monohydrate: 2g
Dipotassium hydrogen phosphate: 10g
Monoammonium phosphate: 1.92g
Sodium hydroxide: 0.66g
glucose: 20g
Bacto Agar (Difco): 15g
30 ml of 90 mm diameter petri dish is poured and hardened.

Additional strain / cell type characteristics:

not specified

Species / strain / cell type:

E. coli WP2 uvr A

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells: E.Coli WP2 uvr A
- Suitability of cells: No Data Available
- Normal cell cycle time (negative control): No Data Available

MEDIA USED
- 1) Top agar
The following aqueous solutions (A) and (B) were mixed at a volume ratio of 10: 1.
(A) Bacto Agar (Difco): 0.6%
Sodium chloride: 0.5%
(B) 0.5 mM L-tryptophan aqueous solution was used for WP2 uvrA.
Biotin: 0.5 mM

2) Synthetic medium
The minimal agar medium was used as the medium. The composition per liter of medium is as follows.
Magnesium sulfate heptahydrate: 0.2g
Citric acid monohydrate: 2g
Dipotassium hydrogen phosphate: 10g
Monoammonium phosphate: 1.92g
Sodium hydroxide: 0.66g
glucose: 20g
Bacto Agar (Difco): 15g
30 ml of 90 mm diameter petri dish is poured and hardened.

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9 : Seven-week-old Sprague-Dawley male rats were used.
- method of preparation of S9 mix: In a small test tube, mix 0.1 ml of the test substance preparation solution, 0.5 ml of phosphate buffer (S9 mix 0.5 ml in the S9 mix addition test), 0.1 ml of the test bacterial solution and 2 ml of top agar and then on the synthetic medium plate.
- concentration or volume of S9 mix and S9 in the final culture medium:
1 ml contains the following ingredients
S9: 0.1ml
Magnesium chloride: 8 μmol
Potassium chloride: 33 μmol
Glucose-6-phosphate: 5 μmol
NADH: 4 μmol
NADPH: 4 μmol
Sodium-phosphate buffer (pH 7.4): 100μmol

- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): No Data Available

Test concentrations with justification for top dose:

Doses chosen for Salmonella typhimurium and E.coli WP2 uvr A strains with and without metabolic activation system:
0, 313, 625, 1250, 2500, 5000 ug/plate.

Vehicle / solvent:

Acetone

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

Acetone

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

9-aminoacridine

sodium azide

furylfuramide

other: 2-aminoanthracene with metabolic activation (all bacteria strains)

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: Duplicate
- Number of independent experiments : Two

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): 0.1 ml of the test bacterial solution was added to the top agar medium and then to the synthetic medium.
- Test substance added in agar (plate incorporation)

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: No Data Available
- Exposure duration/duration of treatment: 48 hours
- Harvest time after the end of treatment (sampling/recovery times): No Data Available

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method, e.g.: background growth inhibition

METHODS FOR MEASUREMENTS OF GENOTOXICITY

- OTHER: No Data Available

Rationale for test conditions:

No Data Available

Evaluation criteria:

The doubling of the number of revertant colonies over the solvent control data was taken as an indication of a positive effect if the effect was dose-dependent or reproducible.

Statistics:

No Data Available

Species / strain:

other: Salmonella typhimurium TA100, TA1535, TA98 and TA1537 and Escherichia coli WP2 uvrA

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not specified

Positive controls validity:

valid

Remarks on result:

other: No evidence of mutagenic potential was observed under the experimental conditions.

Conclusions:

The test chemical tested negative for mutagenicity in S. typhimurium TA 1535, TA 1537, TA 98 and TA 100 and E. coli WP2 uvr A strains in presence and absence of S9 metabolic activation system.

Executive summary:

An in vitro study similar or equivalent to OECD guideline 471 was performed to study the mutagenic potential of the test chemical in Salmonella and E.coli strains. The chemical was tested at 0 (solvent control) and up to 5 mg/plate in Salmonella typhimurium strains TA98, TA100, TA1535 and TA1537 and Escherichia coli WP2 uvrA with and without of metabolic activation (rat-liver S9). 2-aminoanthracene (all strains) with metabolic activation and furylfuramide (TA98, TA100 and WP2 uvr A), sodium azide (TA1535) and 9-aminoacridine (TA1537) without metabolic activation served as positive controls. The doubling of the number of revertant colonies over the solvent control data was taken as an indication of a positive effect if the effect was dose-dependent or reproducible. The test chemical failed to produce any significant increase in the number of revertant colonies compared to the solvent control data. There was no trend of increased number of revertant colonies with increased dosing of the test chemical. The positive control produced marked increases in the number of revertant colonies, thus confirming the validity of the assay.

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

Not tested for mutagenicity in TA102 or E coli WP2 uvr A

GLP compliance:

not specified

Type of assay:

bacterial forward mutation assay

Target gene:

Genes in the histidine operon

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

Liver S-9 was prepared from male Sprague-Dawley rats (RLI) and Syrian hamsters (HLI) that were induced with Aroclor 1254

Test concentrations with justification for top dose:

The chemical was tested at 0 (solvent control), 3.0, 10.0, 33.3, 100.0 and 333.3 µg/plate in Salmonella typhimurium strains TA98, TA100, TA1535 and TA1537 with and without metabolic activation (Aroclor-1254-induced rat- and hamster-liver S9). Dose levels were selected based on the results from a pre-experiment in which toxicity of the test chemical was evaluated in TA100 with and without metabolic activation. Based on the results from the pre-experiment 333.3 µg/plate was selected as top dose in the main study in order to evaluate the chemical for mutagenic effects up to a dose that exhibited some degree of toxicity.

Vehicle / solvent:

DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

sodium azide

other: 2-aminoanthracene with metabolic activation (all bacteria strains), 4-nitro-o-phenylenediamine without metabolic activation (TA98).

Details on test system and experimental conditions:

The test chemical was tested according to the pre-incubation method.

Rationale for test conditions:

In brief, 0.5 ml of S9 mix or 0.1 M of PO4 buffer was dispensed into an appropriate number of 13 X 100 mm culture tubes maintained at 37°C in a dry-bath. After this, 0.05 ml of cells and 0.05 ml of solvent or test chemical dilution were added to each tube. The mixture was mixed and allowed to incubate with shaking in the early tests or standing for 20 min at 37°C. Following preincubation, 2.5 ml (EGG) or 2.0 ml (CWR, SRI) of molten top agar (45°C) supplemented with 0.5 mM L-histidine and 0.5 mM d-biotin was added into the tubes, which were immediately mixed, and their contents poured onto 25 ml of minimal glucose
bottom agar. After the overlay solidified, the plates were inverted and incubated at 37°C for 48 h. At least five doses of test chemical, in addition to the concurrent solvent and positive controls, were tested on each strain in the presence of S-9 mix or buffer. Three plates were used, and the experiment was repeated no less than 1 week after completion of the initial test.

Evaluation criteria:

A positive response was indicated by a reproducible, dose-related increase, whether it be twofold over background or not. Based on all findings, the test chemical could be concluded to be "positive", "negative" or "inconclusive".

Statistics:

Not specified but according to previous work.

Species / strain:

other: TA98, TA100, TA1535 and TA1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: The test chemical showed no evidence of mutagenic potential under the experimental conditions.

Conclusions:

The test chemical tested negative for mutagenicity in Salmonella typhimurium strains TA98, TA100, TA1535 and TA1537 with and without metabolic activation.

Executive summary:

The chemical was tested at 0 (solvent control), 3.0, 10.0, 33.3, 100.0 and 333.3 µg/plate in Salmonella typhimurium strains TA98, TA100, TA1535 and TA1537 with and without metabolic activation (Aroclor-1254-induced rat- and hamster-liver S9). Dose levels were selected based on the results from a pre-experiment in which toxicity of the test chemical was evaluated in TA100 with and without metabolic activation. Based on the results from the pre-experiment 333.3 µg/plate was selected as top dose in the main study in order to evaluate the chemical for mutagenic effects up to a dose that exhibited some degree of toxicity. 2-aminoanthracene, 4-nitro-o-phenylenediamine, sodium azide and 9-aminoacridine severed as positive control chemicals. The test chemical failed to produce any significant increase in the number of revertant colonies compared to the solvent control data. There was no trend of increased number of revertant colonies with increased dosing of the test chemical. The positive control produced marked increases in the number of revertant colonies, thus confirming the validity of the assay.

Reference 4

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Justification for type of information:

Data is from a handbook and collection of data.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Target gene:

His: S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Trp: E. coli WP2 uvr A

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells: Salmonella typhimurium strains were used
- Suitability of cells: No Data Available
- Normal cell cycle time (negative control): No Data Available

MEDIA USED
- 1) Top agar
The following aqueous solutions (A) and (B) were mixed at a volume ratio of 10: 1.
(A) Bacto Agar (Difco): 0.6%
Sodium chloride: 0.5%
(B) L-histidine: 0.5 mM
Biotin: 0.5 mM

2) Synthetic medium
The minimal agar medium was used as the medium. The composition per liter of medium is as follows.
Magnesium sulfate heptahydrate: 0.2g
Citric acid monohydrate: 2g
Dipotassium hydrogen phosphate: 10g
Monoammonium phosphate: 1.92g
Sodium hydroxide: 0.66g
glucose: 20g
Bacto Agar (Difco): 15g
30 ml of 90 mm diameter petri dish is poured and hardened.

Additional strain / cell type characteristics:

not specified

Species / strain / cell type:

E. coli WP2 uvr A

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells: E.Coli WP2 uvr A
- Suitability of cells: No Data Available
- Normal cell cycle time (negative control): No Data Available

MEDIA USED
- 1) Top agar
The following aqueous solutions (A) and (B) were mixed at a volume ratio of 10: 1.
(A) Bacto Agar (Difco): 0.6%
Sodium chloride: 0.5%
(B) 0.5 mM L-tryptophan aqueous solution was used for WP2 uvrA.
Biotin: 0.5 mM

2) Synthetic medium
The minimal agar medium was used as the medium. The composition per liter of medium is as follows.
Magnesium sulfate heptahydrate: 0.2g
Citric acid monohydrate: 2g
Dipotassium hydrogen phosphate: 10g
Monoammonium phosphate: 1.92g
Sodium hydroxide: 0.66g
glucose: 20g
Bacto Agar (Difco): 15g
30 ml of 90 mm diameter petri dish is poured and hardened.

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9 : Seven-week-old Sprague-Dawley male rats were used.
- method of preparation of S9 mix: In a small test tube, mix 0.1 ml of the test substance preparation solution, 0.5 ml of phosphate buffer (S9 mix 0.5 ml in the S9 mix addition test), 0.1 ml of the test bacterial solution and 2 ml of top agar and then on the synthetic medium plate.
- concentration or volume of S9 mix and S9 in the final culture medium:
1 ml contains the following ingredients
S9: 0.1ml
Magnesium chloride: 8 μmol
Potassium chloride: 33 μmol
Glucose-6-phosphate: 5 μmol
NADH: 4 μmol
NADPH: 4 μmol
Sodium-phosphate buffer (pH 7.4): 100μmol

- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): No Data Available

Test concentrations with justification for top dose:

Doses chosen for Salmonella typhimurium and E.coli WP2 uvr A strains with and without metabolic activation system:
0, 312.5, 625, 1250, 2500, 5000 ug/plate.

Vehicle / solvent:

DMSO

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

9-aminoacridine

sodium azide

furylfuramide

other: 2-aminoanthracene with metabolic S9 activation (all strains).

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: Duplicate
- Number of independent experiments : Two

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): 0.1 ml of the test bacterial solution was added to the top agar medium and then to the synthetic medium.
- Test substance added in agar (plate incorporation)

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: No Data Available
- Exposure duration/duration of treatment: 48 hours
- Harvest time after the end of treatment (sampling/recovery times): No Data Available

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method, e.g.: background growth inhibition

METHODS FOR MEASUREMENTS OF GENOTOXICITY

- OTHER: No Data Available

Rationale for test conditions:

No Data Available

Evaluation criteria:

The doubling of the number of revertant colonies over the solvent control data was taken as an indication of a positive effect if the effect was dose-dependent or reproducible.

Statistics:

No Data Available

Species / strain:

other: Salmonella typhimurium TA100, TA1535, TA98 and TA1537 and Escherichia coli WP2 uvrA

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not specified

Positive controls validity:

valid

Remarks on result:

other: The test chemical showed no evidence of mutagenic potential under the experimental conditions.

Conclusions:

The test chemical tested negative for mutagenicity in S. typhimurium TA 1535, TA 1537, TA 98 and TA 100 and E. coli WP2 uvr A, in presence and absence of S9 metabolic activation system.

Executive summary:

An in vitro study similar or equivalent to OECD guideline 471 was performed to study the mutagenic potential of the test chemical in Salmonella and E.coli strains. The chemical was tested at 0 (solvent control) and up to 5 mg/plate in Salmonella typhimurium strains TA98, TA100, TA1535 and TA1537 and Escherichia coli WP2 uvrA with and without of metabolic activation (rat-liver S9). 2-aminoanthracene (all strains) with metabolic activation and furylfuramide (TA98, TA100 and WP2 uvr A), sodium azide (TA1535) and 9-aminoacridine (TA1537) without metabolic activation served as positive controls. The doubling of the number of revertant colonies over the solvent control data was taken as an indication of a positive effect if the effect was dose-dependent or reproducible. The test chemical failed to produce any significant increase in the number of revertant colonies compared to the solvent control data. There was no trend of increased number of revertant colonies with increased dosing of the test chemical. The positive control produced marked increases in the number of revertant colonies, thus confirming the validity of the assay.

Reference 5

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Justification for type of information:

Weight of evidence approach based on the available data of the read-across chemicals.

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

GLP compliance:

not specified

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Remarks:

Study 6

Details on mammalian cell type (if applicable):

No Data Available

Additional strain / cell type characteristics:

not specified

Species / strain / cell type:

Chinese hamster lung (CHL/IU)

Remarks:

Study 7

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells:
Chinese hamster-derived CHL / IU cells obtained from Research Resource Bank (JCRB)
- Suitability of cells:
Cells were suitable for the test.
- Normal cell cycle time (negative control):
No Data Available

For cell lines:
- Absence of Mycoplasma contamination:
No Data Available
- Number of passages if applicable:
The cells were used after 10th passage.
- Methods for maintenance in cell culture:
No Data Available
- Cell cycle length, doubling time or proliferation index :
No Data Available
- Modal number of chromosomes:
No Data Available
- Periodically checked for karyotype stability: No Data Available
- Periodically ‘cleansed’ of spontaneous mutants: No Data Available

MEDIA USED
- 2 × 10^4 CHL / IU cells were seeded in a dish (diameter 6 cm) containing 5 ml of the culture solution and cultured in a 37 ° C CO2 incubator (5% CO2). In the continuous treatment, the test substance was added on the 3rd day of cell seeding and treated for 24 and 48 hours. For short-term treatment, the cells were treated for 6 hours in the presence and absence of S9 mix on the 3rd day of cell seeding, and then cultured for 18 hours in fresh medium after the treatment.

Additional strain / cell type characteristics:

not specified

Cytokinesis block (if used):

Study 6 and 7: Colcemid

Metabolic activation:

with and without

Metabolic activation system:

Study 6: Rat liver S9 was used in the study.
Study 7: Type and composition of metabolic activation system:
- source of S9
: No Data Available
- method of preparation of S9 mix: No Data Available
- concentration or volume of S9 mix and S9 in the final culture medium: No Data Available
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability)

Test concentrations with justification for top dose:

Study 6:
Test Concentrations used:
Without S9: 10, 50, 100 & 150 ug/ml.
WIth S9: 5, 30, 50 ug/ml.
Justification: Dose levels in the study were based on the results from a pre-experiment in which the test chemical was tested at doses of 5, 50, 100, 250, 500, 750, 1000, 2500 and 5000 µg/ml with and without metabolic activation. Without metabolic activation, 150 µg/ml was selected as top dose as no cells survived treatment at 250 µg/ml in the pre-experiment. With metabolic activation, 50 µg/ml was selected as the top dose because 50 µg/ml in the pre-experiment produced a 80.85% increase in the average proliferation time as compared to the solvent control data.

Study 7:
Continuous experiment (Without metabolic activation system): 0, 0.33, 0.65 and 1.3 mg/ml.
Short Term experiment (With and without metabolic activation system): 0, 0.33, 0.65 and 1.3 mg/ml.
Justification: The top dose of 1.3 mg/ml (10 mM) was selected as top dose as there were no signs of cell growth inhibition in the pre-experiment.

Vehicle / solvent:

Study 6 and 7: Aceton

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

Acetone

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: N-nitrosodimethylamine and MNNG were used as positive controls.

Remarks:

Study 6

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

Acetone

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Remarks:

Study 7

Details on test system and experimental conditions:

Study 6: In the definitive study, the test chemical was incubated in CHO cell cultures, both with and without metabolic activation. Each evaluation was performed with cells from duplicate flasks. Based on the preliminary study results of proliferation kinetics and cytotoxic effects, the test chemical was evaluated with and without metabolic activation at optimized concentrations with 5 hour exposure followed by washing and then 18 hrs of additional incubation; After cell harvest, Giesma-stained chromosomal preparations were prepared on slides and at least 50 cells/flask (100 cells/dosage) were evaluated. Number of Replicates Used: 2

Study 7:
NUMBER OF REPLICATIONS:
- Number of cultures per concentration: duplicates
- Number of independent experiments
: Two

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable):
2×10^4 CHL / IU cells were seeded
- Test substance added in medium

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable:
No Data Available
- Exposure duration/duration of treatment:
24 hours
- Harvest time after the end of treatment (sampling/recovery times):
No Data Available

FOR CHROMOSOME ABERRATION AND MICRONUCLEUS:
- Spindle inhibitor (cytogenetic assays): indicate the identity of mitotic spindle inhibitor used : Colchicine, (0.1 μg / ml)
- If cytokinesis blocked method was used for micronucleus assay: indicate the identity of cytokinesis blocking substance (e.g. cytoB), its concentration, and duration and period of cell exposure: No data Available
- Methods of slide preparation and staining technique used including the stain used (for cytogenetic assays):
Two hours before the end of the culture, colcemid was added to the culture solution to a final concentration of about 0.1 μg / ml. Chromosome specimens were prepared according to conventional methods. Six slide specimens were prepared for each dish. The prepared specimen was stained with 3% Giemsa solution.
- Number of cells spread and analysed per concentration (number of replicate cultures and total number of cells scored):
200 metaphases were scored.
- Criteria for scoring micronucleated cells (selection of analysable cells and micronucleus identification):
No Data Available
- Methods, such as kinetochore antibody binding, to characterize whether micronuclei contain whole or fragmented chromosomes (if applicable):
No Data Available
- Criteria for scoring chromosome aberrations (selection of analysable cells and aberration identification):
The cells were scored on the basis of structural and numerical chromosomal aberrations.
- Determination of polyploidy:
No Data Available
- Determination of endoreplication:
No Data Available

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method, e.g. other:
To determine the treatment concentration of the test substance used in the chromosomal aberration test, the effect of the test substance on cell proliferation was investigated. The growth inhibitory effect of the test substance on CHL / IU cells was measured using a monolayer culture cell densitometer. to measure the degree of proliferation of each group, and the solvent control group of the test substance treatment group. The ratio of the cell growth to that was used as an index.
- Any supplementary information relevant to cytotoxicity:

METHODS FOR MEASUREMENTS OF GENOTOXICITY: No Data Available

- OTHER: No Data Available

Evaluation criteria:

Study 6: The chemical was evaluated for its ability to statistically increase the number of aberrations over the solvent control data and for its ability to produce a dose-response.
Study 7 :The results were evaluated statistically for trend and peak responses. Both responses had to be statistically secured in order to conclude a mutagenic effect.

Statistics:

Study 6: All slides were scored blind and statistically analyzed using a t-test to compare pairwise each treatment group with the control group using aberrants per cell. The proportion of aberrant metaphases were analyzed using Chi-square analysis. Significance was generally determined at the p<0.05 probability level.

Study 7: Regarding the frequency of cells with chromosomal abnormalities, referring to the method of Hayashi 2), Fisher's exact method 3) between the solvent background data and the test substance treatment group 3) Significant difference test was carried out. If there was a significant difference by Fisher's exact method, Cochrane Armitage tendency test4) (p <0.05) was performed for dose dependence.

Species / strain:

Chinese hamster Ovary (CHO)

Remarks:

Study 6

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

Chinese hamster lung (CHL/IU)

Remarks:

Study 7

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Conclusions:

Based on read-across data, the target chemical is considered to be non-clastogenic in mammalian cell lines.

Executive summary:

Data for read-across chemicals were evaluated to determine if the target chemical should be considered to be clastogenic or non-clastogenic in mammalian cells.

Study 6:

The read-across chemical was tested in Chinese hamster ovary cells at 10, 50, 100 and 150 µg/ml without metabolic activation and at 5, 30 and 50 µg/ml with metabolic activation. Treatment with the solvent alone served as negative control data. Dose levels in the study were based on the results from a pre-experiment in which the test chemical was tested at doses of 5, 50, 100, 250, 500, 750, 1000, 2500 and 5000 µg/ml with and without metabolic activation. Without metabolic activation, 150 µg/ml was selected as top dose as no cells survived treatment at 250 µg/ml in the pre-experiment. With metabolic activation, 50 µg/ml was selected as the top dose because 50 µg/ml in the pre-experiment produced a 80.85% increase in the average proliferation time as compared to the solvent control data. N-methyl-N-nitro-N-nitroguanidine without metabolic activation and N-nitrosodimethylamine with metabolic activation served as positive control chemicals. After treatment with colcemid and staining in Giemsa, 100 cells per slide were analysed microscopically for structural aberrations (chromatid and chromosome breaks and exchanges) and the total number of aberrations. Gaps were recorded but not included in the total number of aberrations. The chemical was evaluated for its ability to statistically increase the number of aberrations over the solvent control data and for its ability to produce a dose-response. Without metabolic activation, there were no surviving cells following treatment with the test chemical at 150 µg/ml. At no other dose level, with or without metabolic activation, did the test chemical produced a significant increase in the number of aberrations per cell. The positive controls produced marked increases in the number of aberrations per cells, thus confirming the validity of the assay.

Study 7:

The read-across chemical was tested in Chinese hamster lung (CHL) cells at 0, 0.33, 0.65 and 1.3 mg/ml in two series of experiments. The first series of experiments were performed by short-term treatment (6 hrs) in the presence and absence of a metabolic activation system (rat-liver S9). The second series of experiments were performed by long-term treatment (24 and 48 hours respectively) in the absence of metabolic activation. The top dose of 1.3 mg/ml (10 mM) was selected as top dose as there were no signs of cell growth inhibition in the pre-experiment. Mitomycin C and cyclophosphamide served as positive control chemicals. After treatment with colcemid and staining in Giemsa, 200 cells per slide were analysed microscopically for structural aberrations (chromatid and chromosome gaps, breaks and exchanges), total number of cells with aberrations, total number of cells with aberrations except gaps, and polyploidy. The results were evaluated statistically for trend and peak responses. Both responses had to be statistically secured in order to conclude a mutagenic effect. The test chemical failed to produce any significant increase in the number of chromosomal aberrations or in the number of polyploid cells. No trend of increased number of aberrations with increased dosing of the test chemical was observed. The positive controls produced marked increased in the number of aberrations, thus confirming the validity of the assay.

Based on the above data, the target chemical is not considered to be clastogenic in mammalian cells.

Reference 6

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Justification for type of information:

Weight of evidence approach based on the data of the read-across chemicals.

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Qualifier:

equivalent or similar to guideline

Guideline:

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

GLP compliance:

not specified

Type of assay:

in vitro mammalian cell gene mutation tests using the thymidine kinase gene

Target gene:

Study 9 and 10: TK locus

Species / strain / cell type:

mouse lymphoma L5178Y cells

Remarks:

Study 9 and 10

Details on mammalian cell type (if applicable):

3.7.C mouse lymphoma cells were grown in Fischer’s medium for leukemic cells of mice supplemented with 10% horse serum and 0.02% pluronic F-68. Cells were screened
for the presence of mycoplasma after cryopreservation. New cultures were initiated at approximately 3 month intervals from cells stored in liquid N2.

Study 7: Heterozygous L5178Y TK+ /- 3.7.2.C cells were obtained. Stock cultures were grown in Fischer's medium containing 10% horse serum with additions. The pH of the culture medium was adjusted to 7.2 which was found to improve the growth rate of the cells compared with pH 6.8. Cells to be used for mutagenicity assays were cleansed of TK+/- mutants by treatment with thymidine, hypoxanthine, methotrexate and glycine.

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

Study 9 and 10: The toxicity of test chemical was determined both with and without liver S9 prepared from Aroclor 1254-induced male Sprague-Dawley rats.

Test concentrations with justification for top dose:

Study 9: 0.39-1.21 ug/ml was the tesed concentrations. The dose range was expected to yield a toxicity range of 0 to 90% based on the results from a pre-experiment.
Study 10: 600-1000 ug/ml was the tested concentrations. The dose range was expected to yield a toxicity range of 0 to 90% based on the results from a pre-experiment.

Vehicle / solvent:

Study 9 and 10: DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: Study 9 and 10: 3-methylcholanthrene at 1.86 × 10-6 M (or dimethylbenz[a]anthracene at 0.5-4 µg/ml) with S9 and ethyl methylsulfonate at 4.7 × 10-6 M (or methyl methanesulfonate at 10-20 µg/ml) without S9 served as pos controls

Remarks:

Study 9 and 10

Details on test system and experimental conditions:

Study 9 and 10:
NUMBER OF REPLICATIONS:
- Number of cultures per concentration: Duplicate
- Number of independent experiments: One

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): Cells at a concentration of 1.2 × 107 were seeded.
- Test substance added in medium

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: No Data Available
- Exposure duration/duration of treatment: 4 hours
- Harvest time after the end of treatment (sampling/recovery times): No Data Available

FOR GENE MUTATION:
- Expression time (cells in growth medium between treatment and selection): A total of 1.2 × 107 cells in duplicate cultures were exposed to the test chemical. Cells in the cultures were adjusted to 3 × 105/mL at 24 h intervals. They were then cloned (1 × 106 cells/plate for mutant selection and 200
cells/plate for viable count determinations) in soft agar medium containing Fischer’s medium.
- Selection time (if incubation with a selective agent): 24 hours interval.
- Fixation time (start of exposure up to fixation or harvest of cells): 52 hours
- Method used: agar or microwell plates for the mouse lymphoma assay: Soft Agar medium was used.
- If a selective agent is used (e.g., 6-thioguanine or trifluorothymidine), indicate its identity, its concentration and, duration and period of cell exposure: TFT
(trifluorothymidine) was used a selective agent at the concentration of 3 µg/mL. The 100× stock solution of TFT in saline was stored at -70 °C and was thawed immediately before use. Plates were incubated at 37 +/-1 °C in 5% CO2 in air for 10-12 days.
- Number of cells seeded and method to enumerate numbers of viable and mutants cells: 1 × 106 cells/plate were seeded and evaluation of viable and mutant cells was done by a cell counter.
- Criteria for small (slow growing) and large (fast growing) colonies: No Data Available

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: No Data Available
- Any supplementary information relevant to cytotoxicity: No Data Available

METHODS FOR MEASUREMENTS OF GENOTOXICITY
: No Data Available

- OTHER: No Data Available

Evaluation criteria:

Study 9 and 10: The doubling of the mutant frequency over the solvent control data together with evidence of dose-dependency was taken as indication of a positive effect. The size of each mutant colony was also determined in the assay. Taken all data into consideration, a five-graded scale was used to conclude the outcome of the assay (P, positive effect; N, negative effect; WP, weak positive effect; E, equivocal and I, inconclusive).

Statistics:

Study 9 and 10: Not specified.

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

The test chemical tested negative for mutagenicity at the TK locuse in L5178Y mouse lymphoma cells in the presence and absence of metabolic activation in both studies

Conclusions:

Based on the read-across data, the target chemical is not expected to be mutagenic at the TK locus in L5178Y mouse lymphoma cells in the presence and absence of metabolic activation.

Executive summary:

Data for read-across chemicals were evaluated to determine if the target chemical should be considered to be mutagenic or non-mutagenic in mammalian cells.

Study 9:

The test chemical was tested for mutagenicity at the TK locus in L5178Y mouse lymphoma cellsat a dose range of 0.39 to 1.21 µg/ml with and without metabolic activation (Aroclor-1254-induced rat-liver S9). The dose range of 0.39 to 1.21 µg/ml was expected to yield a toxicity range of 0 to 90% based on the results from a pre-experiment. 3-methylcholanthrene at 1.86 × 10^-6M (or dimethylbenz[a]anthracene at 0.5-4 µg/ml) in the presence of metabolic activation and ethyl methylsulfonate at 4.7 × 10^-6M (or methyl methanesulfonate at 10-20 µg/ml) in the absence of metabolic activation served as positive control chemicals. The doubling of the mutant frequency over the solvent control data together with evidence of dose-dependency was taken as indication of a positive effect. The size of each mutant colony was also determined in the assay. Taken all data into consideration, a five-graded scale was used to conclude the outcome of the assay (P, positive effect; N, negative effect; WP, weak positive effect; E, equivocal and I, inconclusive). The test chemical showed no evidence of mutagenic potential and was therefore concluded to be non-mutagenic at the TK locus in mouse lymphoma cells. The positive controls produced marked increases in the number of mutations per 10⁶surviving cells, thus confirming the validity of the assay.

Study 10:

The test chemical was tested for mutagenicity at the TK locus in L5178Y mouse lymphoma cells at a dose range of 600 to 1000 µg/ml with and without metabolic activation (Aroclor-1254-induced rat-liver S9). The dose range of 600 to 1000 µg/ml was expected to yield a toxicity range of 0 to 90% based on the results from a pre-experiment. 3-methylcholanthrene at 1.86 × 10^-6M (or dimethylbenz[a]anthracene at 0.5-4 µg/ml) in the presence of metabolic activation and ethyl methylsulfonate at 4.7 × 10^-6M (or methyl methanesulfonate at 10-20 µg/ml) in the absence of metabolic activation served as positive control chemicals. The doubling of the mutant frequency over the solvent control data together with evidence of dose-dependency was taken as indication of a positive effect. The size of each mutant colony was also determined in the assay. Taken all data into consideration, a five-graded scale was used to conclude the outcome of the assay (P, positive effect; N, negative effect; WP, weak positive effect; E, equivocal and I, inconclusive). The test chemical showed no evidence of mutagenic potential and was therefore concluded to be non-mutagenic at the TK locus in mouse lymphoma cells. The positive controls produced marked increases in the number of mutations per 10⁶surviving cells, thus confirming the validity of the assay.

Based on the above data, the target chemical is not expected to be mutagenic at the TK locus in L5178Y mouse lymphoma cells in the presence and absence of metabolic activation.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Ames studies

Study 1:

The chemical was tested for mutagenicity in multiple strains of Salmonella typhiumurium in the absence of a metabolic activation system. The bacteria were exposed to the test compound on petri plates which were prepared by mixing 0.2 ml of freshly grown cultures (2 x 10 bacteria per milliliter) of the mutants with 2 ml of 0.6% agar at 45°C. The soft agar, which contained a trace (0.20 µmol) of histidine as well as the bacterial inoculum, was then poured onto plates of histidine free minimal agar medium. Approximately 1 to 5 µL of liquid test chemicals or small crystals of solid test chemicals were applied directly to the surface of each plate after the top layer of agar, containing the mutant bacteria, had solidified. The rates of spontaneous reversion of the eight mutants to form no longer requiring L-histidine for growth were also determined and was found to be low, ranging from 0 to 20 colonies per plate.N-methyl-N’-nitro-N-nitrosoguanidine, ICR-191, and diethyl sulfate served as positive controls. The test chemical showed no evidence of mutagenic potential inSalmonella typhimurium in the absence of metabolic activation. The study lacked important methodological details e.g. the identity of strains, the dose levels at which the chemical was tested for mutagenicity, and the justification for top dose level. Therefore, the study can only be used as weight of evidence that the test chemical is non-mutagenic in Salmonella typhimurium in the absence of a metabolic activation system.

Study 2:

An in vitro study similar or equivalent to OECD guideline 471 was performed to study the mutagenic potential of the test chemical in Salmonella and E.coli strains.The chemical was tested at 0 (solvent control) and up to 5 mg/plate in Salmonella typhimurium strains TA98, TA100, TA1535 and TA1537 and Escherichia coli WP2 uvrA with and without of metabolic activation (rat-liver S9). 2-aminoanthracene (all strains) with metabolic activation and furylfuramide (TA98, TA100 and WP2 uvr A), sodium azide (TA1535) and 9-aminoacridine (TA1537) without metabolic activation served as positive controls. The doubling of the number of revertant colonies over the solvent control data was taken as an indication of a positive effect if the effect was dose-dependent or reproducible. The test chemical failed to produce any significant increase in the number of revertant colonies compared to the solvent control data. There was no trend of increased number of revertant colonies with increased dosing of the test chemical. The positive control produced marked increases in the number of revertant colonies, thus confirming the validity of the assay.

Study 3:

The chemical was tested at 0 (solvent control), 3.0, 10.0, 33.3, 100.0 and 333.3 µg/plate in Salmonella typhimurium strains TA98, TA100, TA1535 and TA1537 with and without metabolic activation (Aroclor-1254-induced rat- and hamster-liver S9). Dose levels were selected based on the results from a pre-experiment in which toxicity of the test chemical was evaluated in TA100 with and without metabolic activation. Based on the results from the pre-experiment 333.3 µg/plate was selected as top dose in the main study in order to evaluate the chemical for mutagenic effects up to a dose that exhibited some degree of toxicity. 2-aminoanthracene, 4-nitro-o-phenylenediamine, sodium azide and 9-aminoacridine severed as positive control chemicals. The test chemical failed to produce any significant increase in the number of revertant colonies compared to the solvent control data. There was no trend of increased number of revertant colonies with increased dosing of the test chemical. The positive control produced marked increases in the number of revertant colonies, thus confirming the validity of the assay.

Study 4:

An in vitro study similar or equivalent to OECD guideline 471 was performed to study the mutagenic potential of the test chemical in Salmonella and E.coli strains.The chemical was tested at 0 (solvent control) and up to 5 mg/plate in Salmonella typhimurium strains TA98, TA100, TA1535 and TA1537 and Escherichia coli WP2 uvrA with and without of metabolic activation (rat-liver S9). 2-aminoanthracene (all strains) with metabolic activation and furylfuramide (TA98, TA100 and WP2 uvr A), sodium azide (TA1535) and 9-aminoacridine (TA1537) without metabolic activation served as positive controls. The doubling of the number of revertant colonies over the solvent control data was taken as an indication of a positive effect if the effect was dose-dependent or reproducible. The test chemical failed to produce any significant increase in the number of revertant colonies compared to the solvent control data. There was no trend of increased number of revertant colonies with increased dosing of the test chemical. The positive control produced marked increases in the number of revertant colonies, thus confirming the validity of the assay.

In vitro chromosomal aberration studies

Study 1:

The chemical was tested in Chinese hamster ovary cells at 10, 50, 100 and 150 µg/ml without metabolic activation and at 5, 30 and 50 µg/ml with metabolic activation. Treatment with the solvent alone served as negative control data. Dose levels in the study were based on the results from a pre-experiment in which the test chemical was tested at doses of 5, 50, 100, 250, 500, 750, 1000, 2500 and 5000 µg/ml with and without metabolic activation. Without metabolic activation, 150 µg/ml was selected as top dose as no cells survived treatment at 250 µg/ml in the pre-experiment. With metabolic activation, 50 µg/ml was selected as the top dose because 50 µg/ml in the pre-experiment produced a 80.85% increase in the average proliferation time as compared to the solvent control data. N-methyl-N-nitro-N-nitroguanidine without metabolic activation and N-nitrosodimethylamine with metabolic activation served as positive control chemicals. After treatment with colcemid and staining in Giemsa, 100 cells per slide were analysed microscopically for structural aberrations (chromatid and chromosome breaks and exchanges) and the total number of aberrations. Gaps were recorded but not included in the total number of aberrations. The chemical was evaluated for its ability to statistically increase the number of aberrations over the solvent control data and for its ability to produce a dose-response. Without metabolic activation, there were no surviving cells following treatment with the test chemical at 150 µg/ml. At no other dose level, with or without metabolic activation, did the test chemical produced a significant increase in the number of aberrations per cell. The positive controls produced marked increases in the number of aberrations per cells, thus confirming the validity of the assay.

Study 2:

The chemical was tested in Chinese hamster lung (CHL) cells at 0, 0.33, 0.65 and 1.3 mg/ml in two series of experiments. The first series of experiments were performed by short-term treatment (6 hrs) in the presence and absence of a metabolic activation system (rat-liver S9). The second series of experiments were performed by long-term treatment (24 and 48 hours respectively) in the absence of metabolic activation. The top dose of 1.3 mg/ml (10 mM) was selected as top dose as there were no signs of cell growth inhibition in the pre-experiment. Mitomycin C and cyclophosphamide served as positive control chemicals. After treatment with colcemid and staining in Giemsa, 200 cells per slide were analysed microscopically for structural aberrations (chromatid and chromosome gaps, breaks and exchanges), total number of cells with aberrations, total number of cells with aberrations except gaps, and polyploidy. The results were evaluated statistically for trend and peak responses. Both responses had to be statistically secured in order to conclude a mutagenic effect. The test chemical failed to produce any significant increase in the number of chromosomal aberrations or in the number of polyploid cells. No trend of increased number of aberrations with increased dosing of the test chemical was observed. The positive controls produced marked increased in the number of aberrations, thus confirming the validity of the assay.

In vitro mammalian cell gene mutation assays:

Study 1:

The test chemical was tested for mutagenicity at the TK locus in L5178Y mouse lymphoma cellsat a dose range of 0.39 to 1.21 µg/ml with and without metabolic activation (Aroclor-1254-induced rat-liver S9). The dose range of 0.39 to 1.21 µg/ml was expected to yield a toxicity range of 0 to 90% based on the results from a pre-experiment. 3-methylcholanthrene at 1.86 × 10^-6M (or dimethylbenz[a]anthracene at 0.5-4 µg/ml) in the presence of metabolic activation and ethyl methylsulfonate at 4.7 × 10^-6M (or methyl methanesulfonate at 10-20 µg/ml) in the absence of metabolic activation served as positive control chemicals. The doubling of the mutant frequency over the solvent control data together with evidence of dose-dependency was taken as indication of a positive effect. The size of each mutant colony was also determined in the assay. Taken all data into consideration, a five-graded scale was used to conclude the outcome of the assay (P, positive effect; N, negative effect; WP, weak positive effect; E, equivocal and I, inconclusive). The test chemical showed no evidence of mutagenic potential and was therefore concluded to be non-mutagenic at the TK locus in mouse lymphoma cells. The positive controls produced marked increases in the number of mutations per 10⁶surviving cells, thus confirming the validity of the assay.

Study 2:

The test chemical was tested for mutagenicity at the TK locus in L5178Y mouse lymphoma cells at a dose range of 600 to 1000 µg/ml with and without metabolic activation (Aroclor-1254-induced rat-liver S9). The dose range of 600 to 1000 µg/ml was expected to yield a toxicity range of 0 to 90% based on the results from a pre-experiment. 3-methylcholanthrene at 1.86 × 10^-6M (or dimethylbenz[a]anthracene at 0.5-4 µg/ml) in the presence of metabolic activation and ethyl methylsulfonate at 4.7 × 10^-6M (or methyl methanesulfonate at 10-20 µg/ml) in the absence of metabolic activation served as positive control chemicals. The doubling of the mutant frequency over the solvent control data together with evidence of dose-dependency was taken as indication of a positive effect. The size of each mutant colony was also determined in the assay. Taken all data into consideration, a five-graded scale was used to conclude the outcome of the assay (P, positive effect; N, negative effect; WP, weak positive effect; E, equivocal and I, inconclusive). The test chemical showed no evidence of mutagenic potential and was therefore concluded to be non-mutagenic at the TK locus in mouse lymphoma cells. The positive controls produced marked increases in the number of mutations per 10⁶surviving cells, thus confirming the validity of the assay.

Justification for classification or non-classification

The chemical is regarded to be classified as Not Classified for Germ cell mutagenicity.