Formaldehyde, oligomeric reaction products with phenol

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames assay:

The test chemical did not induce mutation in the Salmonella typhimurium strains both in the presence and absence of S9 metabolic activation system and hence is not likely to be mutagenic under the conditions of this study.

 

In vitro mammalian chromosome aberration study:

The test chemical did not induce chromosome aberrations in the mammalian cell line in the presence and absence of S9 metabolic activation system and hence it is not mutagenic in the chromosome aberration study performed.

In vitro gene mutation study in mammalian cells

Test chemical did not induce mutation in mammalian cell line in the presence and absence of metabolic activation and hence it is not likely to classify as a gene mutant in vitro.

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

Justification for type of information:

Data for the target chemical is summarized based on the structurally similar read across chemicals

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

other: As mention below

Principles of method if other than guideline:

WoE derived based on the experimental data from structurally and functionally similar read across chemicals for the target chemical.

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine

Species / strain / cell type:

S. typhimurium, other: TA 98,TA 100 and TA 102

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

not specified

Species / strain / cell type:

S. typhimurium, other: TA 7001, TA 7002, TA 7003, TA 7004, TA 7005, TA 7006,

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

not specified

Cytokinesis block (if used):

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9 prepared by using Aroclor 1254-induced rat liver.

Test concentrations with justification for top dose:

2,0.009-900mmol
3.50-5000 µg/ml

Vehicle / solvent:

2. Methanol
3. DMSO

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

Remarks:

Menthanol

True negative controls:

not specified

Positive controls:

yes

Remarks:

2-nitrofluorene, sodium azide, mitomycinC, 2-aminofluorene

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

not specified

Positive controls:

yes

Remarks:

Streptonigrin (TA 7003), methyl methanesulfonate (TA7002, TA7004, TA7005, TA7006), 4 amino cytidine (7001)

Details on test system and experimental conditions:

2.,Details on test system and conditions
METHOD OF APPLICATION: Preincubation Method
DURATION
- Preincubation period:
- Exposure duration: 3 days

3,Details on test system and conditions
METHOD OF APPLICATION: fluctuation (microtiter)
DURATION
- Preincubation period: 90min
- Exposure duration: 48hr
- Expression time (cells in growth medium):48hr

Rationale for test conditions:

Not specified

Evaluation criteria:

2.Mutation factors (MF) (induced/spontaneous revertants) were calculated at the dose levels that give the greatest effect.
3.Histidine revertent colonies were observed.

Statistics:

2.Yes ,Mean ±Standard deviation was observed.
3.Student ̔s t-test at p <0.05

Species / strain:

S. typhimurium, other: TA 98,TA 100 and TA 102

Remarks:

study 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

Species / strain:

S. typhimurium, other: TA 7001, TA 7002, TA 7003, TA 7004, TA 7005, TA 7006,

Remarks:

study 3

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

Remarks on result:

other: No mutagenic effct were observed

Conclusions:

Gene mutation toxicity study for test chemical as predicted using data from read across chemicals for Salmonella typhimurium bacterial strains in the presence and absence of S9 metabolic activation system is negative and hence the chemical is not likely to classify as a gene mutant in vitro.

Executive summary:

Genetic mutation in vitro;

Experimental data from read across chemical have been reviewed to determine the mutagenic nature of test chemical.The studies are as mentioned below

The read across substances share high similarity in structure and functional group .Therefore, it is acceptable to derive information on mutation from the analogue substance for test substance. Genetic toxicity in vitro study was assessed for test chemical. The test material was exposed to Salmonella typhimurium TA 98, TA 100 and TA 102 in the presence and absence of metabolic activation S9by Preincubation method. The test chemical was exposed at the concentration of0.009-900mmol. No mutagenic effects were observed in all strains, in the presence and absence of metabolic activation. Therefore test chemical was considered to be non mutagenic in Salmonella typhimurium TA 98, TA 100 and TA 102 by AMES test. Hence the substance cannot be classified as gene mutant in vitro.

 

The read across substances share high similarity in structure and functional group .Therefore, it is acceptable to derive information on mutation from the analogue substance for target substance.SalmonellaMutagenicity Tests oftest chemicalwas performed inSalmonellaTyphimurium TA 7001, TA 7002, TA 7003, TA 7004, TA 7005, TA 7006,Both in the presence and absence of S9 metabolic activation system.Fluctuation (microtiter)methodwas followed. The test compound phenolwas dissolved in DMSOto made 50-5000µg/mlin triplicate for 48 hr.The S-9 fractions of Aroclor 1254-induced,rat liver were prepared.Streptonigrin (TA 7003), methyl methanesulfonate (TA7002, TA7004, TA7005, TA7006), 4 amino cytidine (7001) used as positive control.Mutagenicity evaluatedbyBromocresol purple turns yellow in revartant cell in absence of histidine. Astest chemicaldid not produce mutationinSalmonellaTyphimurium TA 7001, TA 7002, TA 7003, TA 7004, TA 7005, TA 7006,Therefore itis considered to be negative for gene mutation in vitro.

 

The data available for the target chemical based on its read across substance and applying weight of evidence the test chemical does not exhibit gene mutation in vitro. Hence the test chemical 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:

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:

Data for the target chemical is summarized based on the structurally similar read across chemical

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

other: As mention below

Principles of method if other than guideline:

WoE derived based on the experimental data from structurally similar read across chemcal

GLP compliance:

not specified

Type of assay:

other: In vitro mammalian chromosome aberration test

Target gene:

Not specified

Species / strain / cell type:

other: Chinese hamster CHL/IU cells

Details on mammalian cell type (if applicable):

CHL cells derived from Chinese hamster obtained from Research • Resource Bank (JCRB) (February 1988, obtained at passage 4) were used in the test within 10 years of thawing succession.

Additional strain / cell type characteristics:

not specified

Species / strain / cell type:

other: Chinese hamster ovary (CHO) cells

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

other: bromodeoxyuridine-substituted DNA

Cytokinesis block (if used):

not specified

Metabolic activation:

with and without

Metabolic activation system:

Rat liver, induced with phenobarbital and 5,6-benzoflavone

Test concentrations with justification for top dose:

5. -S9 (continuous treatment): 0, 0.09, 0.19, 0.37 mg/ml
-S9 (short-term treatment): 0, 1.3, 2.5, 5.0 mg/ml
+S9 (short-term treatment): 0, 1.3, 2.5, 5.0 mg/ml

6.,-S9; 0; 50; 100; 200 ug/ml
+S9;0,160,170,180 ug/ml

Vehicle / solvent:

Vehicle
5.- Vehicle(s)/solvent(s) used: 0.5% Carboxymethyl cellulose sodium
6. DMSO

Untreated negative controls:

yes

Remarks:

Not specified

Negative solvent / vehicle controls:

yes

Remarks:

0.5% Carboxymethyl cellulose sodium

True negative controls:

not specified

Positive controls:

yes

Remarks:

-S9, Mitomycin C +S9, Cyclophosph amide

Remarks:

WoE-5

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

not specified

Positive controls:

yes

Remarks:

-S9; Mitomycin-C +S9; Cyclophosphamide

Remarks:

WoE-6

Details on test system and experimental conditions:

1,Details on test system and conditions
Cells used
CHL cells derived from Chinese hamster obtained from Research • Resource Bank (JCRB) (February 1988, obtained at passage 4) were used in the test within 10 years of thawing succession.

2. Preparation of culture solution
Eagle MEM culture medium supplemented with 10% fetal bovine serum (FCS: JRH BIOSCIENCES, lot number: 1C2073) was used for the culture.

3. Culture conditions
2 × 10 4 CHL cells were seeded in a dish (6 cm in diameter, Corning) containing 5 ml of the culture solution and cultured in a 37 ° C. CO 2 incubator (5% CO 2).In the direct method, test substances were added on day 3 of cell seeding and treated for 24 hours and 48 hours. In the metabolic activation method, the cells were treated for 6 hours in the presence and absence of S9mix on the third day of cell seeding, and after completion of the treatment, the cells were cultured for 18 hours with fresh culture medium.

Method for preparing chromosome specimen
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 for about 10 minutes.

OTHER EXAMINATIONS:
- Determination of polyploidy: Yes
2,Details on test system and conditions
In the Abs test without S9, cells were incubated in McCoy’s 5A medium with test chemical for 10 hours; Colcemid was added, and incubation continued for 2 hours. The cells were then harvested by mitotic shake-off, fixed, and stained with Giemsa.

For the Abs test with S9, cells were treated with test chemical and S9 for 2 hours, after which the treatment medium was removed and the cells were incubated for 10 hours in fresh medium, with Colcemid present for the final 2 hours. Cells were harvested in the same manner as for the treatment without S9.

OTHER: Cells were selected for scoring on the basis of good morphology and completeness of karyotype
(21 ± 2 chromosomes). All slides were scored blind, and those from a single test were read by the same person.

Rationale for test conditions:

Not specified

Evaluation criteria:

1,Among the five test bacteria used, in the direct method or metabolic activation method of one or more test bacteria, the number of revertive mutant colonies on the flat plate containing the test substance is more than twice that of the negative control, And when the increase was found to be reproducible or dose-dependent, it was decided that the test substance had mutagenicity (positive) in this test system.
2,Two hundred first-division metaphase cells were scored at each dose level. Classes of aberrations included simple (breaks and terminal deletions), complex (rearrangements and translocations), and other (pulverized cells, despiralized chromosomes, and cells containing 10 or more aberrations).

Statistics:

1,Analysis results for untreated control, solvent, positive control group and test substance treated group are tabulated for the observed cell number, type and number of structural abnormality, and number of ploidy cells, and the values of each group are entered on the recording paper did. With regard 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 judgment criteria of Ishikan et al.2), the frequency of cells with chromosomal abnormality is negative, less than 5% negative, less than 10% false positive, and more than 10% Positive.

2,To arrive at a statistical call for a trial, analyses were conducted on both the dose response curve and individual dose points. For a single trial, a statistically significant (P≤0.05) difference for one dose point and a significant trend (P≤ 0.015) were considered
weak evidence for a positive response; significant differences for two or more doses indicated the trial was positive. A positive trend test in the absence of a statistically significant increase at any one dose resulted in an equivocal call (Galloway et al., 1987). Ultimately, the trial calls were based on a consideration of the statistical analyses as well as the biological information available to the reviewers.

Species / strain:

other: Chinese hamster CHL/IU cells

Remarks:

WoE-5

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

other: Chinese hamster ovary (CHO) cells

Remarks:

WoE-6

Metabolic activation:

with and without

Genotoxicity:

not specified

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:

5.,Additional information on results
Lowest concentration producing cytogenetic effects in vitro:
without metabolic activation (continuous treatment ): > 0.37 mg/ml
without metabolic activation (short-term treatment): > 5.0 mg/ml
with metabolic activation (short-term treatment): > 5.0 mg/ml
other;
No polyploidy was also observed.

6. Cytotoxicity was observed at 200 ug/ml

Remarks on result:

other: No mutagenic effect were observed.

Conclusions:

Gene mutation toxicity study for Phenol-formaldehyde resin (9003-35-4)as predicted using data from read across chemicals for mammmalian cell gene mutation assay.Test chemical did not induced genetoxic effect with and Without S9 activation in Cytogenetic assay.

Executive summary:

Genetic toxicity in vitro study was assessed for Calcium salt of 3-hydroxy-4 - [(4-methyl-2-sulfophenyl) azo] -2-naphthalenecarboxylic acid (5281-04-9). For this purpose chromosomal aberration test was performed according to Guidelines for Screening Mutagenicity Testing of Chemicals (Japan).The test material was exposed to Chinese hamster CHL/IU cells in the presence and absence of metabolic activation S9. The concentration of test material used in the presence and absence of metabolic activation weremention as fallow

-S9 (continuous treatment): 0, 0.09, 0.19, 0.37 mg/ml

-S9 (short-term treatment): 0, 1.3, 2.5, 5.0 mg/ml

+S9 (short-term treatment): 0, 1.3, 2.5, 5.0 mg/ml

 

 No significant statistical mutagenic effects were observed in chromosome, in the presence and absence of metabolic activation. Therefore test chemical was considered to be non mutagenic in Chinese hamster CHL/IU cells by chromosomal aberration test. Hence the substance cannot be classified as gene mutant in vitro.

Reference 3

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

Remarks:

Experimental data of read across substances

Justification for type of information:

Data for the target chemical is summarized based on the structurally similar read across chemicals.

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

other: as mentioned below

Principles of method if other than guideline:

WoE derived based on the experimental data from structurally and functionally similar read across chemicals i.e. WoE-8 and WoE-9.

GLP compliance:

not specified

Type of assay:

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

Target gene:

8. thymidine kinase (TK)
9. TK +/-

Species / strain / cell type:

mouse lymphoma L5178Y cells

Remarks:

WoE-8

Details on mammalian cell type (if applicable):

- Type and source of cells: The mouse lymphoma cell line, L5178Y TK+/- - 3.7.2C, used in this assay was derived from the Fischer L5178Y line of Dr. Donald Clive.
- Suitability of cells: Stocks were maintained in liquid nitrogen

MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature, if applicable: The cells were maintained in RPMI 1640 medium supplemented with pluronic solution, L-glutamine, sodium pyruvate, antibiotics, and horse serum (10% by volume). Cloning medium consisted of the pre-ceding growth medium (minus pluronic) with the addition of agar to a final concentration of 0.35% to achieve a semisolid state. Selection medium was cloning medium containing 3 μg/ml of TFT.

Species / strain / cell type:

mouse lymphoma L5178Y cells

Remarks:

WoE-9

Cytokinesis block (if used):

Not Specified

Metabolic activation:

with and without

Metabolic activation system:

8. The S9 activation mix prepared from Aroclor 1254-induced adult male rat livers
9.S9-MIX of rat liver was used as metabolic activation system

Test concentrations with justification for top dose:

8. with activation: 0.256 ug/ml, 0.511 ug/ml, 0.767 ug/ml, 1.02 ug/ml, 1.53ug/ml, and 3.07 ug/ml.
without activation: 51.1 ug/ml, 102 ug/ml, 153 ug/ml, 204 ug/ml, 307 ug/l, and 409 ug/ml.

9.0, 52.0, 78.0, 104, 156, 260, 312, 416, 520 ug/ml

Vehicle / solvent:

8. The doses were selected for each test condition of the mutation experiments in order to cover a toxicity range from little or no survival to eighty to one hundred percent growth compared to the solvent control.
9.- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle:
Test chemical was solube in DMSO

Untreated negative controls:

yes

Remarks:

WoE-8

Negative solvent / vehicle controls:

yes

Remarks:

Dimethylsulfoxide (DMSO)

True negative controls:

not specified

Positive controls:

yes

Remarks:

other: ethylmethane sulfonate (without activation), 3-methylcholantren (with activation)

Untreated negative controls:

not specified

Remarks:

WoE-9

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Positive controls:

yes

Positive control substance:

3-methylcholanthrene

other: ethylmethanesulphonate

Details on test system and experimental conditions:

WoE-8:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration (single, duplicate, triplicate) : single


METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): approximately 6x10E6 cells
- Test substance added in medium.

8. TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: Not specified
- Exposure duration/duration of treatment: four hours at approximately 37°C
- Harvest time after the end of treatment (sampling/recovery times): 2 days



FOR GENE MUTATION:
- Expression time (cells in growth medium between treatment and selection): 4 hours
- Selection time (if incubation with a selective agent): 12 days
- Fixation time (start of exposure up to fixation or harvest of cells): not specified
- Method used: microwell plates for the mouse lymphoma assay.
- If a selective agent is used (e.g., 6-thioguanine or trifluorothymidine), indicate its identity, its concentration and, duration and period of cell exposure.
selective agent: 5-trifluorothymidine (TFT).
concentration: 3 μg/ml
- Number of cells seeded and method to enumerate numbers of viable and mutants cells: 3x106 cells; The mutant frequency was calculated by dividing the total number of colonies in each set of three mutant selection dishes by the total count in the set of three viable count dishes and multiplying by 2x10-4 .
- Criteria for small (slow growing) and large (fast growing) colonies: The smallest detectable colony is between 0.2 and 0.3mm in diameter, depending on its position in the agar matrix.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method, e.g.: cloning efficiency
- Any supplementary information relevant to cytotoxicity: The measurement of the toxicity of each treatment is the relative suspension growth of the cells over the two-day expression period multiplied by the cloning efficiency, relative to the average solvent control. Although not strictly a measure of cell survival, this parameter (called percent relative growth) provides a measure of the effect1veness of treatment and is used as the basis for selecting doses for any necessary repeat trials.

METHODS FOR MEASUREMENTS OF GENOTOXICIY

- OTHER: The two assays (with and without activation system) are identical except for the addition of the S9 fraction of rat liver homogenate and necessary cofactors {CORE) during the four-hour treatment period in activation assay. Each batch of S9 was checked for sterility and assayed for AHH activity and protein content. Normal activation of the MCA positive control treatment must be achieved in the mouse lymphoma assay before accepting a particular batch for test article screening.


9.FOR GENE MUTATION:
- Criteria for small (slow growing) and large (fast growing) colonies:
no differentiation between small and large colonies

Rationale for test conditions:

8. Thymidine kinase (TK) is a cellular enzyme that allows cells to salvage thymidine from the surrounding medium for use in DNA synthesis. If the thymidine analog TFT is included in the growth medium, the analog will be phosphorylated via the TK pathway and cause cell death by inhibiting DNA synthesis. Cells which are heterozygous at the TK locus (TK+/-) may undergo a single-step forward mutation to the TK-/- genotype in which little or no TK activity remains. Such mutants are as viable as the heterozygotes in normal medium because DNA synthesis proceeds by de novo synthetic pathways that do not involve thymidine as an intermediate. The basis for selection of the TK-/- mutants is the lack of any ability to utilize toxic analogs of thymidine, which enables only the TK-/- mutants to grow in the presence of TFT. Cells which grow to form colonies in the presence of TFT are therefore assumed to have mutated, either spontaneously or by the action of a test substance, to the TK-/- genotype.

Evaluation criteria:

WoE-8. 1.Mutation assays are initiated by exposing cell cultures to a range of concentrations of test material that is expected, on the basis of preliminary toxicity studies, to span the cellular responses from no observed toxicity to growth to complete lethality within twenty-four hours of treatment. Then five or six dose levels are selected for completion of the mutation assay. The doses are selected to cover a range of toxicities to growth with emphasis on the most toxic doses. An assay may need to be repeated with different concentrations in order to properly evaluate a test material.

2.The minimum criterion considered necessary to demonstrate mutagenesis for any given treatment is a mutant frequency that is ≥2 times the concurrent background frequency. The background frequency is defined as the average mutant frequency of the solvent controls. The minimum increase is based on extensive experience which indicates that assay variability increases with higher backgrounds and the calculated minimum increase as defined above is often a repeatable result; statistical analysis for the confidence limits is not yet available.

WoE-9.a solitive response was indicated by a > two-fold increase of mutant frequency over the concurrent background frequencies

Statistics:

WoE.8. The mutant frequency was calculated by dividing the total number of colonies in each set of three mutont selection dishes by the total count in the set of three viable count dishes and
multiplying by 2x10-4.

WoE.9.statistical evaluation not mentioned

Species / strain:

mouse lymphoma L5178Y cells

Remarks:

WoE 8

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 specified

Positive controls validity:

valid

Species / strain:

mouse lymphoma L5178Y cells

Remarks:

WoE 9

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

with and without S9-mix: 520 ug/ml

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

True negative controls validity:

not specified

Positive controls validity:

valid

Additional information on results:

WoE-8. In the preliminary cytotoxicity assay, the test material was lethal at approximately 1020 μg/m1 and highly toxic at 511 μg/ml without activation. In the presence of metabolic activation, the test material was converted to a more toxic form or forms. Treatments at and above 128 μg/ml were lethal and the test material was highly toxic as low as 7.98 μg/ml. The nonactivation portion of the mutation assay was initiated with treatments from 6.39 μg/ml to 818 μg/ml and the activation assay with treatments from 0.128 μg/ml to 40.9 μg/ml in an attempt to obtain a good range of toxicities for analysis. One trial of the mutation assay was performed.

Under nonactivation conditions , the test material was lethal at and above 511 μg/ml. Six treatments from 51.1 μg/ml to 409 μg/ml were therefore chosen for the analysis of mutant induction and a good range of toxicities was induced (percent relative growths, 72.6% to 3.6%). The minimum criterion for mutagenesis in this assay was a mutant frequency exceeding 43.3 x 10E-6 and none of the assayed treatments induced this level of mutant action. The test material was therefore considered non-mutagenic without activation in this assay.

In the presence of metabolic activation, the test material was excessively toxic above 3.07 μg/ml. Six treatments from 0.256 μg/ml to 3.07 μg/ml were analyzed for mutant induction and nondetectable to very high toxicities were induced (percent relative growths, 106.7% to 4.8%). No evidence for mutagenicity by the test material was observed. The minimum criterion for mutagenicity by the test material was 49.4 x 10E-6. and the assayed treatments induced mutant frequencies ranging from 22.1 x 10E-6 to 29.5 x 10E-6. The test material was therefore considered non-mutagenic with activation in this assay.


WoE-9. not specified

Remarks on result:

other: No mutagenic Potential

Conclusions:

The test material was not mutagenic in the mouse lymphoma forward mutation assay with and without metabolic activation.

Executive summary:

The test chemical was assayed for mutagenic activity at the thymidine kinase (TK) locus in cultured mouse lymphoma cells (L5178Y TK +/-) by looking for an increase in the frequency of colonies growing in the presence of 5-trifluorothymidine. The test chemical was dissolved in dimethylsulfoxide and diluted into culture medium to obtain an applied concentration range of 6.39 μg/ml to 818 μg/ml without activation and 0.128 μg/ml to 40.9 μg/ml with S9 activation. The test material appeared to remain soluble for all applied concentrations in culture medium.

Under nonactivation test conditions, test chemical was excessively toxic at 611 μg/ml, but six concentrations in the dose range selected for mutant analysis (51.1 μg/ml to 409 μg/ml} caused a wide range of relative growth values (72.6% to 3.6%). In the presence of the S9 metabolic activation system, the test chemical was converted to a more toxic form(s) and treatment with 5.11 μg/ml was excessively toxic. six doses from 0.256 μg/ml to 3.07 μg/ml were chosen for mutant analysis. These treatments resulted in a relative growth range of 106.7% to 4.8%.

In the assays used in this evaluation, the average cloning efficiencies for the solvent controls varied from 92.6% without activation to 86.5% with activation which demonstrated very good cloning conditions for the assays. The solvent control mutant frequencies were all in the expected range and the positive control compounds yielded mutant frequencies that were greatly in excess of the background.

None of the treatments under either test condition caused increases in mutant frequency greater than 2-fold over the average solvent control mutant frequency, and no dose related trend in mutant frequency was evident. Thus, the test chemical was considered to have no significant effect on the mutant frequency and was evaluated as non-mutagenic in the mouse lymphoma cell system.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Genetic mutation in vitro;

AMES assay

Experimental data from read across chemical have been reviewed to determine the mutagenic nature of test chemical.The studies are as mentioned below

The read across substances share high similarity in structure and functional group .Therefore, it is acceptable to derive information on mutation from the analogue substance for test substance. Genetic toxicity in vitro study was assessed for test chemical. The test material was exposed to Salmonella typhimurium TA 98, TA 100 and TA 102 in the presence and absence of metabolic activation S9by Preincubation method. The test chemical was exposed at the concentration of0.009-900mmol. No mutagenic effects were observed in all strains, in the presence and absence of metabolic activation. Therefore test chemical was considered to be non mutagenic in Salmonella typhimurium TA 98, TA 100 and TA 102 by AMES test. Hence the substance cannot be classified as gene mutant in vitro.

 

The read across substances share high similarity in structure and functional group .Therefore, it is acceptable to derive information on mutation from the analogue substance for target substance.SalmonellaMutagenicity Tests oftest chemicalwas performed inSalmonellaTyphimurium TA 7001, TA 7002, TA 7003, TA 7004, TA 7005, TA 7006,Both in the presence and absence of S9 metabolic activation system.Fluctuation (microtiter)methodwas followed. The test compound phenolwas dissolved in DMSOto made 50-5000µg/mlin triplicate for 48 hr.The S-9 fractions of Aroclor 1254-induced,rat liver were prepared.Streptonigrin (TA 7003), methyl methanesulfonate (TA7002, TA7004, TA7005, TA7006), 4 amino cytidine (7001) used as positive control.Mutagenicity evaluatedbyBromocresol purple turns yellow in revartant cell in absence of histidine. Astest chemicaldid not produce mutationinSalmonellaTyphimurium TA 7001, TA 7002, TA 7003, TA 7004, TA 7005, TA 7006,Therefore itis considered to be negative for gene mutation in vitro.

 

Chromosome abbreviation;

The read across substances share high similarity in structure and functional group .Therefore, it is acceptable to derive information on mutation from the analogue substance for target substance. Genetic toxicity in vitro study was assessed for test chemical. For this purpose chromosomal aberration test was performed according to Guidelines for Screening Mutagenicity Testing of Chemicals (Japan).The test material was exposed to Chinese hamster CHL/IU cells in the presence and absence of metabolic activation S9. The concentration of test material used in the presence and absence of metabolic activation weremention as fallow

-S9 (continuous treatment): 0, 0.09, 0.19, 0.37 mg/ml

-S9 (short-term treatment): 0, 1.3, 2.5, 5.0 mg/ml

+S9 (short-term treatment): 0, 1.3, 2.5, 5.0 mg/ml

 

 No significant statistical mutagenic effects were observed in chromosome, in the presence and absence of metabolic activation. Therefore test chemical was considered to be non mutagenic in Chinese hamster CHL/IU cells by chromosomal aberration test. Hence the substance cannot be classified as gene mutant in vitro.

The read across substances share high similarity in structure and functional group .Therefore, it is acceptable to derive information on mutation from the analogue substance for target substance. Genetic toxicity in vitro study was assessed for test chemical. The test material was exposed to Chinese hamster ovary (CHO) cells in the presence and absence of metabolic activation S9 by Cytogenetic assay. The test chemical was exposed at the given concentration

-S9; 0; 50; 100; 200 ug/ml

+S9;0,160,170,180 ug/ml

 No chromosomes Aberration were observed in the presence and absence of metabolic activation. Therefore test chemical was considered to be non mutagenic in Chinese hamster ovary (CHO) cells. Hence the substance cannot be classified as gene mutant in vitro.

 

The data available for the target chemical based on its read across substance and applying weight of evidence the test chemical does not exhibit gene mutation in vitro. Hence the test chemical is not likely to classify as a gene mutant in vitro.

In vitro gene mutation study in mammalian cells

In different studies, the given test chemical has been investigated for the mutagenic nature. The studies are as mentioned below:

 

The test chemical was assayed for mutagenic activity at the thymidine kinase (TK) locus in cultured mouse lymphoma cells (L5178Y TK +/-) by looking for an increase in the frequency of colonies growing in the presence of 5-trifluorothymidine. The test chemical was dissolved in dimethylsulfoxide and diluted into culture medium to obtain an applied concentration range of 6.39 μg/ml to 818 μg/ml without activation and 0.128 μg/ml to 40.9 μg/ml with S9 activation. The test material appeared to remain soluble for all applied concentrations in culture medium. Under nonactivation test conditions, test chemical was excessively toxic at 611 μg/ml, but six concentrations in the dose range selected for mutant analysis (51.1 μg/ml to 409 μg/ml} caused a wide range of relative growth values (72.6% to 3.6%). In the presence of the S9 metabolic activation system, the test chemical was converted to a more toxic form(s) and treatment with 5.11 μg/ml was excessively toxic. six doses from 0.256 μg/ml to 3.07 μg/ml were chosen for mutant analysis. These treatments resulted in a relative growth range of 106.7% to 4.8%. In the assays used in this evaluation, the average cloning efficiencies for the solvent controls varied from 92.6% without activation to 86.5% with activation which demonstrated very good cloning conditions for the assays. The solvent control mutant frequencies were all in the expected range and the positive control compounds yielded mutant frequencies that were greatly in excess of the background. None of the treatments under either test condition caused increases in mutant frequency greater than 2-fold over the average solvent control mutant frequency, and no dose related trend in mutant frequency was evident. Thus, the test chemical was considered to have no significant effect on the mutant frequency and was evaluated as non-mutagenic in the mouse lymphoma cell system.

 

The another Mouse lymphoma assay was performed to evaluate the mutagenic potential of the given test chemical on L 5178 Y (TK +/-) cells in the presence and absence of S9-MIX of rat liver at the dose concentrations of 0, 52.0, 78.0, 104, 156, 260, 312, 416, 520 ug/ml in DMSO. Ethylmethane sulfonate and 3-methylcholantrene served as positive cotrols. A solitive response was indicated by a > two-fold increase of mutant frequency over the concurrent background frequencies. No differentiation between small and large colonies was given. Statistical evaluation not mentioned. The given test chemical failed to induce mutation in L 5178 Y (TK +/-) cells with and without S9-mix and hence was evaluated as non-mutagenic in nature.

 

Thus, based on the above summarized studies on test chemical, it can be concluded that the given test chemical did not induce mutation in mammalian cell line in the presence and absence of metabolic activation and hence it is not likely to classify as a gene mutant in vitro.

Justification for classification or non-classification

Based on the data available and applying weight of evidence approach, the given test chemical does not exhibit gene mutation in vitro by Ames assay, In vitro mammalian chromosome aberration study and In vitro gene mutation study in mammalian cells. Hence, the test chemical is not likely to classify as a gene mutant as per the criteria mentioned in CLP regulation.