4,4'-methylenedi-2,6-xylenol

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 mutations in Salmonella typhimurium and Escheria coli WP2 uvrA strains both in the presence and the absence of metabolic activation system, therefore the test chemical not likley classify as mutagenic substance in vitro.

In vitro mammalian chromosome aberration study:

The test chemical did not induce the increase in the number of cells with chromosome aberattions or polyploid cells in CHL / IU cells in the presence and absence of metabolic activation system, therefore it is not likely to classify as mutagenic substance in vitro.

In vitro mammalian cell gene mutation assay:

In an in vitro Mammalian Cell Gene Mutation test, Chinese Hamster Ovary (CHO) cells were exposed to the test chemical in the concentration of 0, 12.5, 25, 50 or 100 µM for 3hour with or without metabolic activation system. As seen by the results no tested concentration of the test chemical resulted in colonies thus the test chemical did not give a rise to gene mutation when CHO cells were exposed to at ≤ 100µM for 3 hours.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

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

Remarks:

Experimental 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:

other: Refer below principle

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 gene mutation assay

Target gene:

Histidine

Species / strain / cell type:

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

Remarks:

1

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

not applicable

Species / strain / cell type:

E. coli WP2 uvr A

Remarks:

1

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

not specified

Species / strain / cell type:

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

Remarks:

LT-2 strains / 2

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 metabolic activation system isolated from 7 week old male Sprague-Dawley male rats administered concomitant administration of phenobarbital (PB) and 5,6-benzoflavone (BF).

Test concentrations with justification for top dose:

1. 0, 313, 625, 1250, 2500 or 5000 µg/plate
2. 0 or 3 µmole/plate

Vehicle / solvent:

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


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

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

Remarks:

Acetone

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

9-aminoacridine

sodium azide

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

Remarks:

1

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

Remarks:

Ethanol

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: N-methyl-N'-nitro-N-nitrosoguanidin (without metabolic activation) and 2-aminoanthracene (with activation)

Remarks:

2

Details on test system and experimental conditions:

1. 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: Triplicate

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:Yes
- Determination of endoreplication: No data
- Other: No data

OTHER: No data

2. METHOD OF APPLICATION: Spot test (in agar)

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

Rationale for test conditions:

No data

Evaluation criteria:

1. When the increase in the number of revertants/plate 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.
2. 1. Increase in the number of spontaneous revertants
2. The presence of the rfa-mutation was checked by crystal violet inhibition;
3. The presence of the plasmid pKM 101 in strains TA 98 and TA 100 was checked by resistance to ampicillin

Statistics:

No data

Species / strain:

S. typhimurium, other: TA100 and TA98

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:

not specified

Species / strain:

S. typhimurium, other: LT-2 strains TA 98, TA 100, TA 1535, and TA 1537

Remarks:

2

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 with a common ratio of about 3 in the range of 50 to 5000 μg / plate for the test chemical, it was confirmed that in all test bacteria, no antibacterial activity was observed in any of the addition tests.

COMPARISON WITH HISTORICAL CONTROL DATA: No data

ADDITIONAL INFORMATION ON CYTOTOXICITY: No data

Remarks on result:

other: No mutagenic potential

Conclusions:

The test chemical did not induce reversion of mutations in the Salmonella typhimurium strains both in the presence and absence of S9 metabolic activation system and hence is not likley 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 mentioned below:

Bacterial gene mutation assay was performed to determine the mutagenic nature of the test chemical. The study was performed using Salmonella typhimurium TA 100, TA 1535, TA 98, TA 1537, and Escherichia coli WP 2 uvrA in the presence and absence of S9 metabolic activation system. The test chemical was dissolved in acetone and used at dose level of 0, 313, 625, 1250, 2500 or 5000µg/plate. No increase in the number of mutant colonies that were more than twice the solvent control value in the S9 mix non-addition test and addition test of the five test bacteria used was observed. Based on the observations made, thetest chemical did not induce gene mutation inSalmonella typhimurium TA 100, TA 1535, TA 98, TA 1537, and Escherichia coli WP 2 uvrA in the presence and absence of S9 metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.

Gene mutation toxicity study was also performed to determine the mutagenic nature of the test chemical. The material was dissolved in ethanol and applied at a concentration of 0 or 3 µmole/plate in the spot test performed. The plates were observed for an increase in the number of spontaneous revertants. The test chemical did not induce an increase in the number of spontaneous revertants and hence is not mutagenic in the bacteriumSalmonella typhimuriumLT-2 strains TA98, TA100, TA1537 and TA1535 with and without S9 metabolic activation system and hence is not likely to classify as gene mutant in vitro.

Based on the observations made, the test chemical did not induce reversion of mutations in the Salmonella typhimurium strains both in the presence and absence of S9 metabolic activation system and hence is not likley 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:

Experimental 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:

equivalent or similar 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:

in vitro mammalian chromosome aberration test

Target gene:

No data

Species / strain / cell type:

mammalian cell line, other: CHL / IU cells

Remarks:

1

Details on mammalian cell type (if applicable):

- Type and identity of media: Eagle MEM culture broth supplemented with 10% fetal bovine serum was used for the culture.
- Properly maintained: Yes
- 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:

mammalian cell line, other: CHL/IU

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 metabolic activation system

Test concentrations with justification for top dose:

1. Continuous treatment:
0, 0.0020, 0.0040, 0.0080 mg/mL

Short term treatment:
With S9: 0, 0.0075, 0.015 or 0.030 mg/mL
Without S9: 0, 0.00050, 0.0010 or 0.0020 mg/mL

2. 0, 250, 500 or 1000 µg/mL

Vehicle / solvent:

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

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

Remarks:

Acetone

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:

Acetone

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: 1-methyl-3-nitro-1-nitrosoguanidine, 3,4-benzo[a]pyrene

Details on test system and experimental conditions:

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

DURATION
- Preincubation period: No data
- Exposure duration:
Continuous treatment: 24 or 48 hrs
Short term treatment: 6 hrs
- Expression time (cells in growth medium): 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): 3% Giemsa solution

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:Yes
- Determination of endoreplication: No data
- Other: No data

OTHER: No data

2. METHOD OF APPLICATION: in medium
Cells at the start of experiment: 4000 cells

DURATION
- Preincubation period: No data
- Exposure duration:
Continuous treatment: 24 hrs
Short term treatment: 6 hrs
- Expression time (cells in growth medium): 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): No data
STAIN (for cytogenetic assays): No data

NUMBER OF REPLICATIONS: No data

NUMBER OF CELLS EVALUATED: 200 metaphase cells were evaluated

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

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

OTHER: No data

Rationale for test conditions:

No data

Evaluation criteria:

1. The cell line was observed for the presence or absence of structural abnormality such as chromosome type or chromosome type gap, The presence or absence of cells (polyploid) was observed.
2. The clastogenicity was judged to be positive when the frequency of the structural chromosomal aberration detected was 10% or more.

Statistics:

1. Yes, With reference to the method of Hayashi, with reference to the method of the forest, the Fisher's direct probability method between the solvent background data and the test substance treated group (refer to the groupware significance level in consideration of multiplicity 5%), a significant difference test was carried out. In addition, when significant differences were found by Fisher's exact stochastic method, Cochran-Armitage's trend test (p <0.05) was carried out for dose dependency. In principle, a case where a significant difference was observed in both of the above two tests was regarded as positive. When there was no significant difference in the trend test, it was judged as false positives. The case where the number of observed cells was less than 100 for structural abnormality and less than 400 for polyploid cells was determined as undetectable due to cytotoxicity.

2. No data

Species / strain:

mammalian cell line, other: CHL / IU cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

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: The doses for the main study were based on cell proliferation inhibition test

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: A preliminary cell proliferation inhibition test was conducted using doses of 15.6-1000 ug/mL and two dishes/dose. Based on the results of the cell proliferation inhibition test, the highest dose level of the test substance was established at 1000 µg/mL (upper limit of the preparable dose of the test solution) with a total of three doses including 500 and 250 µg/mL at a common ratio of two both in the short-term and continuous treatment methods.

COMPARISON WITH HISTORICAL CONTROL DATA: No data

ADDITIONAL INFORMATION ON CYTOTOXICITY: No data

Remarks on result:

other: No mutagenic potential

Conclusions:

The test chemical did not induce chromosome aberration in CHL / IU cells 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 test was performed to determine the mutagenic nature of the test chemical. The test was performed using CHL / IU cells in the presence and absence of S9 metabolic activation system. The test chemical was dissolved in acetone as solvent and used at dose level of 0, 0.0020, 0.0040, 0.0080 mg/mL in continuous treatment and 0, 0.0075, 0.015 or 0.030 mg/mL with S9 and 0, 0.00050, 0.0010 or 0.0020 mg/mL without S9 in short term treatment method. The study was performed by short term treatment method for 6 hrs and continuous treatment method for 24 and 48 hrs. The cell line was allowed to express further for 18 hrs in shirt 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. Six slide specimens were prepared for each dish. The prepared specimens were stained with 3% Giemsa solution. The cell line was observed for the presence or absence of structural abnormality such as chromosome type or chromosome type gap, The presence or absence of cells (polyploid) was observed. Based on the observations made, the test chemicaldid not induce chromosome aberration inCHL / IU cells 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 test was performed to determine the mutagenic nature of the test chemical. The test was performed using CHL / IU cells in the presence and absence of S9 metabolic activation system. The test chemical was dissolved in acetone as solvent and used at dose level of 0, 250, 500 or 1000µg/mL.The study was performed by short term treatment method for 6 hrs and continuous treatment method for 24 hrs. The cell line was allowed to express further for 18 hrs in short term treatment method. According to the results of the study, no increase in the number of chromosomally aberrant cells was observed in the short-term and continuous treatment method with and without S9 mix. Hence thetest chemical did not induce chromosome aberration inCHL / IU cells 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 CHL / IU cells 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

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Data is from study report

Qualifier:

according to guideline

Guideline:

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

Principles of method if other than guideline:

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.

GLP compliance:

no

Type of assay:

in vitro mammalian cell gene mutation test using the Hprt and xprt genes

Target gene:

Hypoxanthine-guanine phosphoribosyl transferase (HGPRT)

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

Metabolic activation system:

S9 liver microsomal fraction obtained from Arcolor 1254-induced male Sprague-Dawley rats (Supplier: Molecular Toxicology Inc. via Trinova Biochem GmbH, Giessen, Germany)

Test concentrations with justification for top dose:

0, 12.5, 25, 50 or 100 µM

Vehicle / solvent:

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

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

Ethanol

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: N-ethyl-N-nitrosourea (ENU)

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)

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 CYTOTOXICITYAfter 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: No data available

Rationale for test conditions:

No data available

Evaluation criteria:

The number of mutant colonies were counted and compared to the negative and positive controls. The mutation frequency was calculated.

Statistics:

The Standard deviation (± SD) was calculated.

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with

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

Additional information on results:

No data available

Remarks on result:

other: No mutagenic potential

Conclusions:

When CHO cells were exposed to the test chemical in the concentration of 0, 12.5, 25, 50 or 100 µM the results did not show any evidence of gene toxicity.

Executive summary:

An in vitro Mammalian Cell Gene Mutation study was conducted to determine the potential of the given test chemical inducing gene mutations using Chinese Hamster Ovary (CHO) cell line. The cells were exposed to the test chemical at the doses of 0, 12.5, 25.0, 50.0 or 100.0 µM for 3 hours in the presence of S9 metabolic activation.The results showed no evidence of cytotoxicity when CHO cells were exposed to the test chemical. In addition, no tested concentration of test chemical, in the presence of S9 metabolic activation resulted in colonies. Thus, it was concluded that the test chemical in the concentration of ≤100 µM does not cause genetic mutation(s) in the presence of metabolic activation.

Reference 4

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Data is from study report

Qualifier:

according to guideline

Guideline:

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

Principles of method if other than guideline:

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.

GLP compliance:

no

Type of assay:

in vitro mammalian cell gene mutation test using the Hprt and xprt genes

Target gene:

Hypoxanthine-guanine phosphoribosyl transferase (HGPRT)

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:

without

Metabolic activation system:

S9 liver microsomal fraction obtained from Arcolor 1254-induced male Sprague-Dawley rats (Supplier: Molecular Toxicology Inc. via Trinova Biochem GmbH, Giessen, Germany)

Test concentrations with justification for top dose:

0, 12.5, 25, 50 or 100 µM

Vehicle / solvent:

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

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

Ethanol

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylnitrosurea

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)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 CYTOTOXICITYAfter 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: No data available

Rationale for test conditions:

No data available

Evaluation criteria:

The number of mutant colonies were counted and compared to the negative and positive controls. The mutation frequency was calculated.

Statistics:

The Standard deviation (± SD) was calculated.

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:

No data available

Remarks on result:

other: No mutagenic potential

Conclusions:

When CHO cells were exposed to the test chemical in the concentration of 0, 12.5, 25, 50 or 100 µM the results did not show any evidence of gene toxicity.

Executive summary:

In a Mammalian Cell Gene Mutation test, Chinese Hamster Ovary (CHO) cells were exposed to the test chemical in the concentration of 0, 12.5, 25, 50 or 100 µM in the absence S9-induced metabolic activation. The results showed no evidence of cytotoxicity when CHO cells were exposed to the test chemical. In addition, no tested concentration of test chemical, in the absence of S9 metabolic activation resulted in colonies. Thus, it was concluded that the test chemical in the concentration of ≤ 100 µM does not cause genetic mutation(s) in the 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 assay:

 

Bacterial gene mutation assay was performed to determine the mutagenic nature of the test chemical. The study was performed using Salmonella typhimurium TA 100, TA 1535, TA 98, TA 1537, and Escherichia coli WP 2 uvrA in the presence and absence of S9 metabolic activation system. The test chemical was dissolved in acetone and used at dose levels of 0, 313, 625, 1250, 2500 or 5000 µg/plate. No increase in the number of mutant colonies that were more than double of the control value was observed nor in the present neither in the absence of metabolic activation system. Based on the observations made, the test chemical did not induce gene mutation in the tested S. typhimurium and Escherichia coli WP 2 uvrA strains at the dose of ≤ 5000 µg/plate therefore it is not likely to classify as a mutagenic substrate in vitro.

Bacterial reverse mutation test was performed to determine the mutagenic nature of the test chemical. The test substance was dissolved in ethanol and applied at the concentrations of 0 or 3 µmol/plate in the spot test performed. The plates were observed for an increase in the number of His+ mutant colonies. As seen by the results, the test chemical did not induce an increase in the number of spontaneous revertant colonies thus it is not mutagenic in the S. typhimurium strains employed. Hence, test substance not likely classified as mutagenic substance in vitro.

In vitro mammalian chromosome aberration study:

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 test was performed to determine the mutagenic nature of the test chemical. The test was performed using CHL / IU cells in the presence and in the absence of S9 metabolic activation system. The cells were exposed to 2.0, 4.0 and 8.0 µg/ml of test chemical for 24 or 48 hrs in the absence of metabolic activation (in the continuous treatment). In the short time treatment CHL /IU cells were exposed at the dose of 7.5, 15.0 and 30.0 µg/ml in the presence of S9 mix or 0.5, 1.0 and 2.0 µg/ml in the absence of S9 for 6 hours followed by 18 hours recovery time. The test chemical was dissolved in acetone. Two hours before the end of the cell treatment, 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. Six slide specimens were prepared for each dish. The specimens were stained with 3% Giemsa solution and observed for the presence or absence of structural chromosome abnormality such as chromatid gap, chromosome gap, chromatid break and exchange, polyploid cells. As seen by the results, no chromosomal structural aberration and polyploid cell inducing effect were observed in any treatment group when cells were exposed to the test chemical for 24 hrs in the absence of S9 mix. Similarly, the results of analysis by short-time treatment has revealed no structural abnormality of chromosome and/or induction of polyploid cells nor in the presence neither in the absence of metabolic activation. Thus, that the test chemical did not induce chromosomal abnormalities in vitro in the CHL/ IU cells under the certain test conditions, hence it is not likely to classify as a mutagen in vitro.

In vitro mammalian chromosome aberration test was performed to determine the mutagenic nature of the test chemical. The test was performed using CHL / IU cells in the presence and absence of S9 metabolic activation system. The test chemical was dissolved in acetone and applied at dose level of 0, 250, 500 or 1000 µg/ml. The experiment was performed either using a short-term treatment method with 6 hours of exposure time followed by 18 hours recovery time or a continuous treatment of 24 hours exposure duration. Based on the observations made, the given test chemical did not induce an increase in the number of cells with chromosome aberrations using any of the treatment methods in CHL/IU cell line. Hence, test chemical did not induce chromosome aberration in CHL / IU cells in the presence and absence of S9 metabolic activation system and thus it is not likely to classify as a mutagen in vitro.

In vitro mammalian cell gene mutation assay:

In a mammalian cell gene mutation test, Chinese Hamster Ovary (CHO) cells were exposed to the test chemical in the concentration of 0, 12.5, 25, 50 or 100 µM for 3 hours with and without S9-induced metabolic activation Results showed no evidence of cytotoxicity when CHO cells were treated with the test chemical. Similarly, no evidence of gene toxicity was observed independently of the treatment concentrations. Therefore, it is considered that the test chemical in the concentration of ≤ 100 µM does not cause genetic mutation(s) in the presence and absence of metabolic activation in vitro. Hence the test chemical is not likely to classify as a mutagenic substance as per the criteria mentioned in CLP regulation.

Justification for classification or non-classification

The test chemical didnot exhibit mutagenic activityinbacterial reverse mutation testand mammalian chromosome aberration test in vitro. Results of mammalian cell gene mutation test are lacking and hence the test chemical can not be classified for mutagenicity endpoint, currently.