Bis(4-(1,1,3,3-tetramethylbutyl)phenyl)amine

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames assay:

The test chemical did not induce gene mutation in Salmonella typhimurium strains TA98, TA1535, TA1537, TA100 and Escherichia Coli WP2uvrA in the presence and absence of S9 metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.

In vitro mammalian chromosome aberration study:

The test chemical did not induce chromosome aberration in mammalian cell line in the presence and absence of S9 metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.

In vitro mammalian cell gene mutation assay:

With S9 metabolic activation system:

In a gene toxicity test, Chinese Hamster Ovary (CHO) cells were exposed to the test chemical in the concentration of 0, 0.025, 0.05, 0.1 or 0.5 mM and S9-induced metabolic activation for 3 hours. The results showed that there was no evidence of cytotoxicity when CHO cells were treated with the test chemical. The results showed an indication of gene toxicity when treated with 0.5 mM when cells were exposed to the test chemical. Therefore, it is considered that the test chemical in the concentration of 0, 0.025, 0.05 or 0.1 mM does not cause genetic mutation(s) in the presence of metabolic activation, whereas treatment with 0.5 mM may, when CHO cells are exposed to the test chemical in the presence of metabolic activation.

Without S9 metabolic activation system:

In a gene toxicity test, Chinese Hamster Ovary (CHO) cells were exposed to the test chemical in the concentration of 0, 0.025, 0.05, 0.01 or 0.5 mM and without S9-induced metabolic activation. The results showed that there was no evidence of cytotoxicity when CHO cells were treated with the test chemical. Independently of treatment concentration, the results showed no evidence of gene toxicity when cells were exposed to the test chemical. Therefore, it is considered that the test chemical in the concentration of 0, 0.025, 0.05, 0.01 or 0.5 mM does not cause genetic mutation(s) in the absence of metabolic activation.

Overall conclusion

At time of exposure, the test chemical was added in the absence or presence of S9 liver microsomal fraction. The test chemical was added to each applicable well to give a final concentration of 0, 0.025, 0.05, 0.1 or 0.5 mM. Negative controls, solvent/vehicle controls and positive control substance(s) were also included in each experiment. pH and osmolality was not determined in the gene mutation test. The positive control ENU gave a clear indication of gene mutations occurring while no other treatment gave rise to gene toxicity except when treated with 0.5 mM in the presence of 4% S9 liver microsomal fraction. Thus, the results show evidence of genotoxicity when CHO cells are exposed to the test chemical in the highest tested concentrations, i.e. at 0.5 mM. It was concluded that the test chemical does not give rise to gene mutations when used at concentrations of ≤ 0.1 mM in organisms with a fully functioning metabolic activation, whereas concentrations > 0.1 mM may induce gene mutations. However, when treated with test chemical in the absence of S9 liver microsomal fraction, no evidence of occurring gene mutations were detected. Hence, test chemical does not give rise to gene mutations in organisms who has no or a non-functional metabolic activation at the above mentioned concentrations. When the mutation frequency was determined, a frequency of 3.67 x 10^-4was shown after a 3 hour exposure of ENU as the positive control and in the absence of S9 liver microsomal fraction, and a frequency of -1.92 x 10^-4was observed for 0.5 mM in the presence of S9 liver microsomal fraction. Since no other tested concentration of the test chemical and in the absence or presence of S9 liver microsomal fraction resulted in colonies, it was concluded that the test chemical does not give rise to gene mutations when CHO cells are exposedin vitroto the test chemical at ≤ 2.5 mM for 3 hrs.

Link to relevant study records

Reference

Reference 1

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

Remarks:

Experimental data from from various test chemicals

Justification for type of information:

Data for the target chemical is summarized based on the data from various test chemicals

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Principles of method if other than guideline:

WoE derived based on the experimental data from various test chemicals

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

not specified

Species / strain / cell type:

E. coli WP2 uvr A

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

not specified

Cytokinesis block (if used):

No data

Metabolic activation:

with and without

Metabolic activation system:

S9 prepared by enzyme induction of male Sprague-Dawley male rats of 7 weeks of age by combination administration of phenobarbital (PB) and 5, 6-benzoflavone (BF)

Test concentrations with justification for top dose:

1. Trial 1.1:
Without S9: 0, 1.56, 3.13, 6.25, 12.5, 25 or 50 µg/plate

With S9: 0, 6.25, 12.5, 25, 50, 100 or 200 µg/plate

Trial 1.2:
With and without S9: 0, 125, 250, 500, 1000 or 2000 µg/plate

Trial 2.1:
Without S9: 0, 1.56, 3.13, 6.25, 12.5, 25 or 50 µg/plate

With S9: 0, 6.25, 12.5, 25, 50, 100 or 200 µg/plate

Trial 2.2:
With and without S9: 0, 125, 250, 500, 1000 or 2000 µg/plate

2. 0, 15.6, 31.3, 62.5, 125, 250, 500 or 1000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test chemical was soluble in DMSO

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

9-aminoacridine

sodium azide

other: AF-2 (2- (2-furyl) -3- (5-nitro-2-furyl) acrylamide) and 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Preincubation period: No data
- Exposure duration: 48 hrs
- Expression time (cells in growth medium): 48 hrs
- Selection time (if incubation with a selection agent): No data
- Fixation time (start of exposure up to fixation or harvest of cells): No data

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

NUMBER OF REPLICATIONS: No data

NUMBER OF CELLS EVALUATED: No data

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

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

OTHER: No data

Rationale for test conditions:

No data

Evaluation criteria:

1. When the number of revertive mutant colonies on the flat plate containing the test substance increases more than twice as compared with that of the negative control and the reproducibility or dose dependency is recognized in the increase, the test substance It was decided to have mutagenicity (positive) in the system.

2. When the increase increased more than 2 times and the increase was found to be reproducible or dose dependent, it was decided that the test substance has mutagenicity (positive) in this test system.

Statistics:

No data

Species / strain:

S. typhimurium, other: TA98, TA1535, TA1537, TA100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Additional information on results:

1. TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No data
- Effects of osmolality: No data
- Evaporation from medium: No data
- Water solubility: No data
- Precipitation: No data
- Other confounding effects: No data

RANGE-FINDING/SCREENING STUDIES: When the test was carried out in the range of 50 to 5000 μg / plate, antibacterial activity was strongly observed in the direct test and metabolic activation test of all test bacteria. Antimicrobial activity was found more strongly than WP 2 in S. typhimurium test organisms, and was also remarkable in the direct test. For this reason,
additional tests were conducted on 4 tested bacteria of S. typhimurium at a dose of 20 to 200 μg / plate for TA 100 and TA 98, at a dose of 10 to 100 μg / plate for TA 1535 and TA 1537 only for direct test. Antimicrobial activity was observed in 2 to 3 dose groups of high dose in any of the test organisms.

From the above results, the maximum dose in this study was 50 μg / plate in the direct test of S. typhimurium 4 test bacteria, 200 μg / plate in the metabolic activation test, both direct test and metabolic activation test for WP 2 2000 μg / plate, 4 doses of S. typhimurium, 6 doses and 5 doses of WP 2 were established at a common ratio of 2.

COMPARISON WITH HISTORICAL CONTROL DATA: No data

ADDITIONAL INFORMATION ON CYTOTOXICITY: No data

2. TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No data
- Effects of osmolality: No data
- Evaporation from medium: No data
- Water solubility: No data
- Precipitation: No data
- Other confounding effects: No data

RANGE-FINDING/SCREENING STUDIES: When the test was carried out with the common ratio in the range of 50.0 to 5000 μg / plate being about 3, it was found that WP 2 uvrA was 1500 μg / plate or more in the S9 mix-free test and the antibacterial activity was observed in other cases at 500 μg /plate or more It was done. In the S9 mix addition test, antibacterial activity was confirmed at TA 98 and WP 2 uvr A above 1500 μg / plate, and others at doses above 500 μg / plate.

COMPARISON WITH HISTORICAL CONTROL DATA: No data

ADDITIONAL INFORMATION ON CYTOTOXICITY: No data

Remarks on result:

other: No muatgenic potential

Conclusions:

The test chemical did not induce gene mutation in Salmonella typhimurium strains TA98, TA1535, TA1537, TA100 and Escherichia Coli WP2uvrA in the presence and absence of S9 metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.

Executive summary:

Data available for the various test chemicals was reviewed to determine the mutagenic nature of the test chemical. The studies are as mentioned below:

Bacterial reverse mutation assay was performed to determine the mutagenic nature of the test chemical. The study was performed using Salmonella typhimurium strains TA98, TA1535, TA1537, TA100 and Escherichia Coli WP2uvrA in the presence and absence of S9 metabolic cativation system. The test chemical was dissolved in DMSO and used at dose level of 0, 1.56, 3.13, 6.25, 12.5, 25 or 50µg/plate without S9 and 0, 6.25, 12.5, 25, 50, 100 or 200µg/plate with S9 in trial 1.1 And 2.1 and at dose level of 0, 125, 250, 500, 1000 or 2000µg/plate in trial 1.2 and 2.2 with and without S9. Concurrent solvent and positive controls were also included in the study.The test chemical did not induce gene mutation inSalmonella typhimurium strains TA98, TA1535, TA1537, TA100 and Escherichia Coli WP2uvrA in the presence and absence of S9 metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.

In another study, bacterial reverse mutation assay was performed to determine the mutagenic nature of the test chemical. The study was performed using Salmonella typhimurium strains TA98, TA1535, TA1537, TA100 and Escherichia Coli WP2uvrA in the presence and absence of S9 metabolic cativation system. The test chemical was dissolved in DMSO and used at dose level of 0, 15.6, 31.3, 62.5, 125, 250, 500 or 1000µg/plates with and without S9. Concurrent solvent and positive controls were also included in the study.The test chemical did not induce gene mutation inSalmonella typhimurium strains TA98, TA1535, TA1537, TA100 and Escherichia Coli WP2uvrA in the presence and absence of S9 metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.

Based on the data available, the test chemical did not induce gene mutation in Salmonella typhimurium strains TA98, TA1535, TA1537, TA100 and Escherichia Coli WP2uvrA in the presence and absence of S9 metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

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

Remarks:

Experimenal data from various test chemicals

Justification for type of information:

Data for the target chemical is summarized based on the data from various test chemicals

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Principles of method if other than guideline:

WoE derived based on the experimental data from various test chemicals

GLP compliance:

not specified

Type of assay:

other: In vitro mammalian chromosome aberration study

Target gene:

No data

Species / strain / cell type:

mammalian cell line, other: Chinese hamster cultured cells (CHL)

Remarks:

1

Details on mammalian cell type (if applicable):

- Type and identity of media: Eagle MEM culture medium supplemented with 10% fetal bovine serum
- Properly maintained: No data
- Periodically checked for Mycoplasma contamination: No data
- Periodically checked for karyotype stability: No data
- Periodically "cleansed" against high spontaneous background: No data

Additional strain / cell type characteristics:

not specified

Species / strain / cell type:

lymphocytes: Rat

Remarks:

2

Details on mammalian cell type (if applicable):

No data

Additional strain / cell type characteristics:

not specified

Cytokinesis block (if used):

No data

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

1. 0, 4, 8 or 16 µg/mL
2. -S9 (20-hour cultures): 1.0 – 125 µg/mL
+S9 (20-hour cultures): 1.88 –120 µg/mL
+S9 (30-hour cultures): 1.88 – 60 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test chemical was soluble in DMSO

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Remarks:

1

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

cyclophosphamide

ethylmethanesulphonate

Remarks:

2

Details on test system and experimental conditions:

1. METHOD OF APPLICATION: in medium
Cells at the start of the experiment: 20000 cells

DURATION
- Preincubation period: No data
- Exposure duration: Direct method: 24 and 48 hrs
Short term treatment method with S9: 6 hrs
- Expression time (cells in growth medium):
Direct method: 24 and 48 hrs
Short term treatment method with S9: 18 hrs
- Selection time (if incubation with a selection agent): No data
- Fixation time (start of exposure up to fixation or harvest of cells): No data

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

NUMBER OF REPLICATIONS: No data

NUMBER OF CELLS EVALUATED: For structural abnormalities, 200 metaphase cells per group and 800 division metastatic cells for multiplicative cells were analyzed.

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

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

OTHER: No data

2. METHOD OF APPLICATION: in medium

Rationale for test conditions:

No data

Evaluation criteria:

1. The presence or absence of structural abnormality such as chromosome type or chromosome type gap, The presence or absence of cells (polyploid) was also observed.

2. The cell line was observed for chromosome aberration

Statistics:

1. With respect to the frequency of occurrence of chromosomal abnormal cells, a significant difference test between the solvent control group and the test substance treated group and between the solvent control group and the positive control group was carried out by Fisher's "Exact probability test" method. According to the criteria of Ishikan et al, the frequency of chromosomal abnormalities is negative, less than 5% negative, less than 10% false positive, less than 10% Was regarded as positive.

2. No data

Species / strain:

mammalian cell line, other: Chinese hamster cultured cells (CHL)

Remarks:

1

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Species / strain:

lymphocytes: Rat

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

With metabolic activation: 60.0 µg/ml and more dose (20- and 30-hour cultures) Without metabolic activation: 30.0 µg/ml and more dose

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Additional information on results:

1. TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No data
- Effects of osmolality: No data
- Evaporation from medium: No data
- Water solubility: No data
- Precipitation: No data
- Other confounding effects: No data

RANGE-FINDING/SCREENING STUDIES: To determine the treatment concentration of the test substance used for the chromosomal aberration test, the influence of the test substance on cell proliferation was investigated.
The proliferation inhibitory effect of the test substance on CHL cells was evaluated by measuring the proliferation level of each group using a monolayer cultured cell densitometer and using the ratio of cell proliferation to the solvent control group of the test substance treated group as an indicator. As a result, the concentration showing growth inhibition of about 50% in the direct method calculated from the regression line equation was 16 μg / ml, and in the metabolic activation method it was 40 μg / ml

COMPARISON WITH HISTORICAL CONTROL DATA: No data

ADDITIONAL INFORMATION ON CYTOTOXICITY: No data

2. No data

Remarks on result:

other: No mutagenic potential

Conclusions:

The test chemical did not induce chromosome aberration in mammalian cell line in the presence and absence of S9 metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.

Executive summary:

Data available for the test chemicals was reviewed to determine the mutagenic nature of the test chemical. The studies are as mentioned below:

In vitro mammalian chromosome aberration study was performed to determine the mutagenic nature of the test chemical. The study was performed using Chinese hamster cultured cells (CHL) in the presence and absence of S9 metabolic activation system. The test chemical was dissolved in DMSO and used at dose level of 0, 4, 8 or 16µg/mL. The doses for the main study were based on data from preliminary dose range finding study. Concurrent solvent and positive control plates were also included in the study. The cells were exposed to the test chemical for 24 or 48 hrs in the direct method and for 6 hrs in the short term treatment method. 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 a conventional method. Giemsa stained six slide specimens were prepared for each petri dish. The presence or absence of structural abnormality such as chromosome type or chromosome type gap, the presence or absence of cells (polyploid) was also observed.The test chemical did not induce chromosome aberration inChinese hamster cultured cells (CHL) in the presence and absence of S9 metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.

In another vitro mammalian chromosome aberration study was performed to determine the mutagenic nature of the test chemical. The study was performed using rat lymphocytes (CHL) in the presence and absence of S9 metabolic activation system. The test chemical was dissolved in DMSO and used at dose level of 1.0 – 125 µg/mL -S9 (20-hour cultures), 1.88 –120 µg/mL +S9 (20-hour cultures) and 1.88 – 60 µg/mL +S9 (30-hour cultures). Concurrent solvent and positive control plates were also included in the study. Based on the observations made,the test chemical did not induce chromosome aberration inrat lymphocytes in the presence and absence of S9 metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.

Based on the data available, the test chemical did not induce chromosome aberration in mammalian cell line in the presence and absence of S9 metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

The study was conducted in accordance with the Good Laboratory Practice Principles as Published by OECD in 1998, No 1 ENV/MC/CHEM(98)17

Qualifier:

according to guideline

Guideline:

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

Principles of method if other than guideline:

In vitro mammalian cell gene mutation assa ywas performed to determine the mutagenic nature of the test chemical

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Target gene:

Cells deficient in hypoxanthine-guanine phosphoribosyl transferase (HPRT) due to the mutation HPRT+/- to HPRT-/- are resistant to cytotoxic effects of 6-thioguanine (TG). HPRT proficient cells are sensitive to TG (which causes inhibition of cellular metabolism and halts further cell division since HPRT enzyme activity is important for DNA synthesis), so mutant cells can proliferate in the presence of TG, while normal cells, containing hypoxanthine-guanine phosphoribosyl transferase cannot.

This in vitro test is an assay for the detection of forward gene mutations at the in hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus on the X chromosomes of hypodiploid, modal No. 20, CHO cells. Gene and chromosome mutations are considered as an initial step in the carcinogenic process.
The hypodiploid CHO cells are exposed to the test item with and without exogenous metabolic activation. Following an expression time the descendants of the treated cell population are monitored for the loss of functional HPRT enzyme.
HPRT catalyses the transformation of the purine analogues 6-thioguanine (TG) rendering them cytotoxic to normal cells. Hence, cells with mutations in the HPRT gene cannot phosphoribosylate the analogue and survive treatment with TG.

Therefore, mutated cells are able to proliferate in the presence of TG whereas the non-mutated cells die. However, the mutant phenotype requires a certain period of time before it is completely expressed. The phenotypic expression is achieved by allowing exponential growth of the cells for 7 days.

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

- Cell line used: Chinese Hamster Ovary (CHO) cells
- Type and identity of media: Ham's F-12K (Kaighn's) Medium containing 2 mM L-Glutamine supplemented with 10% Fetal Bovine Serum and 1% Penicillin-Streptomycin (10,000 U/mL).
- Properly maintained: Yes
- Periodically checked for Mycoplasma contamination: Not applicable
- Periodically checked for karyotype stability: Not applicable

Additional strain / cell type characteristics:

other: Hypodiploid, modal No. 20

Cytokinesis block (if used):

No data

Metabolic activation:

with and without

Metabolic activation system:

S9 liver microsomal fraction obtained from Arcolor 1254-induced male Sprague-Dawley rats

Test concentrations with justification for top dose:

0, 0.025, 0.05, 0.1 or 0.5 mM

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Dimethyl sulfoxide (DMSO)
- Justification for choice of solvent/vehicle: The test chemical was dissolved in DMSO

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

Remarks:

N-ethyl-N-nitrosourea (ENU) was the positive control substance in the tests done without S9

Details on test system and experimental conditions:

METHOD OF APPLICATION: In medium with pre-incubation

DURATION
-Pre-incubation: One week involving 3 days of incubation with Hypoxanthine-aminopterin-thymidine (HAT) in medium as a mutant cleansing stage, followed by overnight incubation with hypoxanthine-thymidine (HT) in medium prior to a 3-4 days incubation in regular cell medium. After seeding and prior to treatment, the mutant-free cells were incubated for an additional of 24 hours.
- Exposure duration: 3 hours
-Expression time: 7 days
- Selection time: 14 days
- Fixation time: 7 days (harvest of cells)

SELECTION AGENT (mutation assays): 6-thioguanine (TG)
SPINDLE INHIBITOR (cytogenetic assays): Not applicable
STAIN (for cytogenetic assays): Crystal violet

NUMBER OF REPLICATIONS: A minimum of 2 replicates per dose concentration including negative and positive control.

NUMBER OF CELLS EVALUATED: 5 x 10 E5 cells were plated 7 days after treatment and whatever cells left, after 14 days of incubation with the selection medium, were evaluated

DETERMINATION OF CYTOTOXICITY
- Cytotoxicity test: After being exposed to the test chemical for 3 hours, in the absence or presence of S9, cells were trypsinized and 0.5 x 10 E5 cells per well was seeded in duplicates from two parallel duplicate cultures into 6-well plates in fresh medium. The relative total growth and cytotoxicity was evaluated 24 and 48 hours after seeding.

OTHER EXAMINATIONS: Not applicable
- Determination of polyploidy:
- Determination of endoreplication:
- Other:

Rationale for test conditions:

No data

Evaluation criteria:

The cell line was observed for gene mutation at the HGPRT locus

Statistics:

No data

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No data
- Effects of osmolality: No data
- Evaporation from medium: No data
- Water solubility: No data
- Precipitation: No data
- Definition of acceptable cells for analysis: No data
- Other confounding effects: No data

RANGE-FINDING/SCREENING STUDIES: Completed without S9 metabolic activation. A range of test concentrations (0, 0.01, 0,05, 0.1, 0.5, 1, 5, 10 or 50mM) was applied 24 hours after seeding to single cultures in fresh medium in 96-well plates. The cell population (control and treated cells) were assessed 24 and 48 hours after treatment using the colorimetric assayMTTand the BCA assay to assess cell viability and total protein concentration, respectively. From the basis of these results, the test concentrations of the chemical was chosen to be included in the gene toxicity test. Since no cytotoxicity was evident at the tested concentrations in this preliminary dose-finding test further testing concentrations were adapted to have a maximum test concentration of 0.5 mM. Since the test chemical was dissolved in DMSO, higher concentrations of the test chemical than the concentration mentioned above would result in a toxic effect of DMSO. The test chemical could only be dissolved in 100% DMSO.

CYTOKINESIS BLOCK (if used)
- Distribution of mono-, bi- and multi-nucleated cells: No data

NUMBER OF CELLS WITH MICRONUCLEI
- Number of cells for each treated and control culture:
- Indication whether binucleate or mononucleate where appropriate: No data

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: No data
- Negative (solvent/vehicle) historical control data: No data

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: No data
- Other observations when applicable: No data

Remarks on result:

other: No mutagenic potential

Conclusions:

When CHO cells were exposed to the test chemical in the concentration of 0, 0.025, 0.05, 0.01 or 0.5 mM the results did not show any evidence of gene toxicity without metabolic activation. CHO cells when exposed to the test chemical in the presence of metabolic activation at the concentration of 0, 0.025, 0.05 or 0.1 mM did not show any evidence of gene toxicity. However, treatment with 0.5 mM showed an indication of genetic toxicity.

Executive summary:

With S9 metabolic activation system:

In a gene toxicity test, Chinese Hamster Ovary (CHO) cells were exposed to the test chemical in the concentration of 0, 0.025, 0.05, 0.1 or 0.5 mM and S9-induced metabolic activation for 3 hours. The results showed that there was no evidence of cytotoxicity when CHO cells were treated with the test chemical. The results showed an indication of gene toxicity when treated with 0.5 mM when cells were exposed to the test chemical. Therefore, it is considered that the test chemical in the concentration of 0, 0.025, 0.05 or 0.1 mM does not cause genetic mutation(s) in the presence of metabolic activation, whereas treatment with 0.5 mM may, when CHO cells are exposed to the test chemical in the presence of metabolic activation.

Without S9 metabolic activation system:

In a gene toxicity test, Chinese Hamster Ovary (CHO) cells were exposed to the test chemical in the concentration of 0, 0.025, 0.05, 0.01 or 0.5 mM and without S9-induced metabolic activation. The results showed that there was no evidence of cytotoxicity when CHO cells were treated with the test chemical. Independently of treatment concentration, the results showed no evidence of gene toxicity when cells were exposed to the test chemical. Therefore, it is considered that the test chemical in the concentration of 0, 0.025, 0.05, 0.01 or 0.5 mM does not cause genetic mutation(s) in the absence of metabolic activation.

Overall conclusion

At time of exposure, the test chemical was added in the absence or presence of S9 liver microsomal fraction. The test chemical was added to each applicable well to give a final concentration of 0, 0.025, 0.05, 0.1 or 0.5 mM. Negative controls, solvent/vehicle controls and positive control substance(s) were also included in each experiment. pH and osmolality was not determined in the gene mutation test. The positive control ENU gave a clear indication of gene mutations occurring while no other treatment gave rise to gene toxicity except when treated with 0.5 mM in the presence of 4% S9 liver microsomal fraction. Thus, the results show evidence of genotoxicity when CHO cells are exposed to the test chemical in the highest tested concentrations, i.e. at 0.5 mM. It was concluded that the test chemical does not give rise to gene mutations when used at concentrations of ≤ 0.1 mM in organisms with a fully functioning metabolic activation, whereas concentrations > 0.1 mM may induce gene mutations. However, when treated with test chemical in the absence of S9 liver microsomal fraction, no evidence of occurring gene mutations were detected. Hence, test chemical does not give rise to gene mutations in organisms who has no or a non-functional metabolic activation at the above mentioned concentrations. When the mutation frequency was determined, a frequency of 3.67 x 10^-4 was shown after a 3 hour exposure of ENU as the positive control and in the absence of S9 liver microsomal fraction, and a frequency of -1.92 x 10^-4 was observed for 0.5 mM in the presence of S9 liver microsomal fraction. Since no other tested concentration of the test chemical and in the absence or presence of S9 liver microsomal fraction resulted in colonies, it was concluded that the test chemical does not give rise to gene mutations when CHO cells are exposed in vitro to the test chemical at ≤ 2.5 mM for 3 hrs.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Gene mutation in vitro:

Data available for the test chemical was reviewed to determine the mutagenic nature of the test chemical. The studies are mentioned below:

Ames test:

Bacterial reverse mutation assay was performed to determine the mutagenic nature of the test chemical. The study was performed using Salmonella typhimurium strains TA98, TA1535, TA1537, TA100 and Escherichia Coli WP2uvrA in the presence and absence of S9 metabolic cativation system. The test chemical was dissolved in DMSO and used at dose level of 0, 1.56, 3.13, 6.25, 12.5, 25 or 50µg/plate without S9 and 0, 6.25, 12.5, 25, 50, 100 or 200µg/plate with S9 in trial 1.1 And 2.1 and at dose level of 0, 125, 250, 500, 1000 or 2000µg/plate in trial 1.2 and 2.2 with and without S9. Concurrent solvent and positive controls were also included in the study.The test chemical did not induce gene mutation inSalmonella typhimurium strains TA98, TA1535, TA1537, TA100 and Escherichia Coli WP2uvrA in the presence and absence of S9 metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.

In another study, bacterial reverse mutation assay was performed to determine the mutagenic nature of the test chemical. The study was performed using Salmonella typhimurium strains TA98, TA1535, TA1537, TA100 and Escherichia Coli WP2uvrA in the presence and absence of S9 metabolic cativation system. The test chemical was dissolved in DMSO and used at dose level of 0, 15.6, 31.3, 62.5, 125, 250, 500 or 1000µg/plates with and without S9. Concurrent solvent and positive controls were also included in the study.The test chemical did not induce gene mutation inSalmonella typhimurium strains TA98, TA1535, TA1537, TA100 and Escherichia Coli WP2uvrA in the presence and absence of S9 metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.

In vitro mammalian chromosome aberration study:

In vitro mammalian chromosome aberration study was performed to determine the mutagenic nature of the test chemical. The study was performed using Chinese hamster cultured cells (CHL) in the presence and absence of S9 metabolic activation system. The test chemical was dissolved in DMSO and used at dose level of 0, 4, 8 or 16µg/mL. The doses for the main study were based on data from preliminary dose range finding study. Concurrent solvent and positive control plates were also included in the study. The cells were exposed to the test chemical for 24 or 48 hrs in the direct method and for 6 hrs in the short term treatment method. 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 a conventional method. Giemsa stained six slide specimens were prepared for each petri dish. The presence or absence of structural abnormality such as chromosome type or chromosome type gap, the presence or absence of cells (polyploid) was also observed.The test chemical did not induce chromosome aberration inChinese hamster cultured cells (CHL) in the presence and absence of S9 metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.

In another vitro mammalian chromosome aberration study was performed to determine the mutagenic nature of the test chemical. The study was performed using rat lymphocytes (CHL) in the presence and absence of S9 metabolic activation system. The test chemical was dissolved in DMSO and used at dose level of 1.0 – 125 µg/mL -S9 (20-hour cultures), 1.88 –120 µg/mL +S9 (20-hour cultures) and 1.88 – 60 µg/mL +S9 (30-hour cultures). Concurrent solvent and positive control plates were also included in the study. Based on the observations made,the test chemical did not induce chromosome aberration inrat lymphocytes in the presence and absence of S9 metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.

In vitro mammalian cell gene mutation assay:

With S9 metabolic activation system:

In a gene toxicity test, Chinese Hamster Ovary (CHO) cells were exposed to the test chemical in the concentration of 0, 0.025, 0.05, 0.1 or 0.5 mM and S9-induced metabolic activation for 3 hours. The results showed that there was no evidence of cytotoxicity when CHO cells were treated with the test chemical. The results showed an indication of gene toxicity when treated with 0.5 mM when cells were exposed to the test chemical. Therefore, it is considered that the test chemical in the concentration of 0, 0.025, 0.05 or 0.1 mM does not cause genetic mutation(s) in the presence of metabolic activation, whereas treatment with 0.5 mM may, when CHO cells are exposed to the test chemical in the presence of metabolic activation.

Without S9 metabolic activation system:

In a gene toxicity test, Chinese Hamster Ovary (CHO) cells were exposed to the test chemical in the concentration of 0, 0.025, 0.05, 0.01 or 0.5 mM and without S9-induced metabolic activation. The results showed that there was no evidence of cytotoxicity when CHO cells were treated with the test chemical. Independently of treatment concentration, the results showed no evidence of gene toxicity when cells were exposed to the test chemical. Therefore, it is considered that the test chemical in the concentration of 0, 0.025, 0.05, 0.01 or 0.5 mM does not cause genetic mutation(s) in the absence of metabolic activation.

Overall conclusion

At time of exposure, the test chemical was added in the absence or presence of S9 liver microsomal fraction. The test chemical was added to each applicable well to give a final concentration of 0, 0.025, 0.05, 0.1 or 0.5 mM. Negative controls, solvent/vehicle controls and positive control substance(s) were also included in each experiment. pH and osmolality was not determined in the gene mutation test. The positive control ENU gave a clear indication of gene mutations occurring while no other treatment gave rise to gene toxicity except when treated with 0.5 mM in the presence of 4% S9 liver microsomal fraction. Thus, the results show evidence of genotoxicity when CHO cells are exposed to the test chemical in the highest tested concentrations, i.e. at 0.5 mM. It was concluded that the test chemical does not give rise to gene mutations when used at concentrations of ≤ 0.1 mM in organisms with a fully functioning metabolic activation, whereas concentrations > 0.1 mM may induce gene mutations. However, when treated with test chemical in the absence of S9 liver microsomal fraction, no evidence of occurring gene mutations were detected. Hence, test chemical does not give rise to gene mutations in organisms who has no or a non-functional metabolic activation at the above mentioned concentrations. When the mutation frequency was determined, a frequency of 3.67 x 10^-4was shown after a 3 hour exposure of ENU as the positive control and in the absence of S9 liver microsomal fraction, and a frequency of -1.92 x 10^-4was observed for 0.5 mM in the presence of S9 liver microsomal fraction. Since no other tested concentration of the test chemical and in the absence or presence of S9 liver microsomal fraction resulted in colonies, it was concluded that the test chemical does not give rise to gene mutations when CHO cells are exposedin vitroto the test chemical at ≤ 2.5 mM for 3 hrs.

Based on the data available and applying the weight of evidence approach, the test chemical does not exhibit gene mutation in vitro. Hence the test chemical is not likely to classify as a gene mutant as per the criteria mentioned in CLP regulation.

Justification for classification or non-classification

Based on the data available and applying the weight of evidence approach, the test chemical does not exhibit gene mutation in vitro. Hence the test chemical is not likely to classify as a gene mutant as per the criteria mentioned in CLP regulation.