2-ethoxybenzoic acid

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

AMES test;

Test chemical  failed to induce mutation in Salmonella typhimurium strains TA97, TA98, TA100, TA1535 both in the presence and absence of S9 metabolic activation system and hence does not classify for gene mutation in vitro.

In vitro Mammalian cell gene mutation assay

In vitro chromosome aberration test, Test material did not induce gene mutation in Chinese hamster lungs cells in the presence and absence of S9and hence 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 from handbook or collection of data

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:

Gene mutation toxicity study was performed to determine the mutagenic nature of Test chemical.

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine

Species / strain / cell type:

S. typhimurium, other: TA97, TA98, TA100, TA1535

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

not specified

Species / strain / cell type:

S. typhimurium TA 100

Additional strain / cell type characteristics:

not specified

Cytokinesis block (if used):

No data

Metabolic activation:

with and without

Metabolic activation system:

The S-9 (9000g supernatant) fractions was prepared from Aroclor 1254-induced, male Sprague- Dawley rat and male Syrian hamster livers

Test concentrations with justification for top dose:

1,0, 1.0, 3.0, 10.0, 33.0, 100.0 or 333.0 µg/plate
2,0.1, 1, 10, 100 or 1000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The 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: 4-nitro-o-phenylenediamine (TA98 + Without S9); 2-aminoanthracene (all strains + with S9)

Details on test system and experimental conditions:

METHOD OF APPLICATION: preincubation

DURATION
- Preincubation period: 20 min
- 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: At least five doses were tested in 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: No data
- Determination of endoreplication: No data
- Other: No data

OTHER: No data

Rationale for test conditions:

No data

Evaluation criteria:

Evaluations were made at both the individual trial and overall chemical levels. Individual trials were judged mutagenic (+), weakly mutagenic (+ W), questionable (?), or nonmutagenic (-), depending on the magnitude of the increase of his+ revertants, and the shape of the dose-response. A trial was considered questionable
(?) if the dose-response was judged insufficiently high to support a call of “ +W,” if only a single dose was elevated over the control, or if the increase seen was not dose related. The distinctions between a questionable mutagenic response and a non-mutagenic or weak mutagenic response, and between a weak mutagenic response and mutagenic response are highly subjective. It was not necessary for a response to reach two-fold over background for a chemical to be judged mutagenic.

A chemical was judged mutagenic (+) or weakly mutagenic (+ W) if it produced a reproducible dose-related response over the solvent control in replicate trials. A chemical was judged questionable (?) if the results of individual trials were not reproducible, if increases in his+ revertants did not meet the criteria for a “+W” response, or if only single doses produced increases in his+ revertants in repeat trials. Chemicals were judged nonmutagenic (-) if they did not meet the criteria for a mutagenic or questionable response.

Statistics:

Mean and SEM

Species / strain:

S. typhimurium, other: TA97, TA98, TA100, TA1535

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:

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 chemical was tested initially in a toxicity assay to determine the appropriate dose range for the mutagenicity assay. The toxicity assay was performed using TA100 or the sytem developed by Waleh et al. Toxic concentrations were those that produced a decrease in the number of his+ colonies, or a clearing in the density of the background lawn, or both.

COMPARISON WITH HISTORICAL CONTROL DATA: No data

ADDITIONAL INFORMATION ON CYTOTOXICITY: No data

Remarks on result:

other: No mutagenic potential

Conclusions:

Test chemical failed to induce mutation in Salmonella typhimurium strains TA97, TA98, TA100, TA1535 both in the presence and absence of S9 metabolic activation system and hence does not classify for gene mutation in vitro.

Executive summary:

AMES test;

Gene mutation toxicity study was performed to determine the mutagenic nature of test substance. Preincubation assay was performed as a dose level of 0, 1.0, 3.0, 10.0, 33.0, 100.0 or 333.0 µg/plate with and without S9 metabolic activation system. The chemical as dissolved in DMSO and concurrent solvent and positive controls were included in the study. Test chemical failed to induce mutation in Salmonella typhimurium strains TA97, TA98, TA100, TA1535 both in the presence and absence of S9 metabolic activation system and hence does not classify for gene mutation in vitro.

Supported by other AMES test . Gene mutation toxicity study was performed to determine the mutagenic nature of the test compound. The study was performed as part of mutagenicity produced by aqueous chlorination of organic compounds. Test compound was used at dose levels of 10-1, 100, 101, 102or 103µg/plate (0.1, 1, 10, 100 or 1000 µg/plate) without chlorination. Test compound did not induce reversion of mutant strains without chlorination and hence is not mutagenic in the bacterium Salmonella typhimurium TA 100 and hence does not classify as gene mutant in vitro.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

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 from handbook or collection of data

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:

Weight of evidence prepared from various studies mention below
To evaluate the mutagenic potential of test chemical in Chinese hamster lung(CHL)cells by in vitro mammalian chromosome aberration test.

GLP compliance:

not specified

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

Not specified

Species / strain / cell type:

other: Chinese hamster lung(CHL)cells

Details on mammalian cell type (if applicable):

CHL / IU cells derived from Chinese hamster lungs obtained from Dainippon Pharmaceutical Co., Ltd. (September 2003, passage: 14th generation,
frozen: 17th) were used for the test within 4 weeks after thawing succession

Additional strain / cell type characteristics:

not specified

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:

1,-S9 mix (6 hr short-term treatment); 100, 125, 150, 175, 200 μg/mL
+S9 mix (6 hr short-term treatment); 200, 225, 250, 275, 300 μg/mL
-S9 mix (24 hr continuous treatment; main test); 50, 75, 100, 125, 150 μg/mL
-S9 mix (24 hr continuous treatment; additional test); 100, 110, 120, 130, 140, 150 μg/mL
2,-S9 mix (6 hr short-term treatment); 100, 125, 150, 175, 200 μg/mL
+S9 mix (6 hr short-term treatment); 200, 225, 250, 275, 300 μg/mL
-S9 mix (24 hr continuous treatment; main test); 50, 75, 100, 125, 150 μg/mL
-S9 mix (24 hr continuous treatment; additional test); 100, 110, 120, 130, 140, 150 μg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Isotonic sodium chloride solution

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

Remarks:

Isotonic sodium chloride solution

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: -S9 mix, Mitomycin C +S9 mix, Benzo [a] pyrene

Details on test system and experimental conditions:

Details on test system and conditions
METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 3 days
- Exposure duration: short-time treatment method :6h
continuous treatment method:24h
- Expression time (cells in growth medium):
- Selection time (if incubation with a selection agent): No data
- Fixation time (start of exposure up to fixation or harvest of cells): No data

SPINDLE INHIBITOR (cytogenetic assays): No data

NUMBER OF CELLS EVALUATED: 200 metaphases were scored per experimental group.

OTHER EXAMINATIONS:
- Determination of polyploidy: yes

Evaluation criteria:

Structural abnormalities such as breakage and exchange of chromosome type or chromosome type, presence and absence of gap, and ploidy The presence or absence of cells (polyploid) was observed. The gap was not included in structural abnormality. Also analyzed 200 groups of metaphase cells per group for structural abnormalities and ploidy cells.

Species / strain:

other: Chinese hamster lung (CHL/IU) cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Species / strain:

other: Chinese hamster lung (CHL/IU) cells

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 effect were observed

Conclusions:

In vitro chromosome aberration test, Test material did not induce gene mutation in Chinese hamster lungs cells in the presence and absence of S9and hence is not likely to classify as a gene mutant in vitro.

Executive summary:

In vitro Mammalian cell gene mutation assay

In vitro chromosome aberration study was performed to determine the mutagenic nature of test material.SHE cells (5x105) in tertiary culture were plated into 75-cm2flasks, incubated overnight, and treatedat dose levels from 0, 0.8,3,8,30µMfor 24 h.The test chemical was dissolved in distilled, deionized water .SHE cells have the doubling time of 16 h. The cells were harvested with 0.1% trypsin for chromosome preparation. Three hours before harvest, Colcemid was administered at 0.2µg/ml and metaphase chromosomes were prepared. For determination of chromosome aberrations, 100 metaphases were scored per experimental group. Achromatic lesions greater than the width of the chromatid were scored as gaps unless there was displacement of the broken piece of chromatid. If there was displacement, they were scored as breaks. Also, the chromosome aberration assay was carried out in the presence of exogenous metabolic activation with rat liver post-mitochondrial supernatant (PMS), Cells (5x105) were plated on 100-mm dishes and after overnight incubation, they were treated with chemical agents for 3 h in a 5% PMS mixture.). Cells were washed twice with 5 ml PBS (-) and incubated with fresh medium for 18 h followed by chromosome preparations., Test material did not induce gene mutation inSyrian hamster embryo (SHE) cells inthe presence and absence ofmetabolic activation with rat liver post-mitochondrial supernatant (PMS)and hence is not likely to classify as a gene mutant in vitro.

In vitro chromosome aberration study was performed to determine the mutagenic nature of test material. CHL / IU cells derived from Chinese hamster lungs. 2 × 10 4 CHL / IU cells were seeded in a dish (6 cm in diameter) containing 5 mL of the culture solution and cultured in a 37 ° C. CO 2 incubator (5% CO 2) for 3 days. After the preculture, in the short-time treatment method, the test substance was treated for 6 hours in the presence and absence of S9 mix and then cultured for 18 hours in fresh culture medium. In the continuous treatment method, the test substance was continuously treated for 24 hours. The test substance was dissolved in a Physiological saline to prepare a stock solution, and then the stock solution was serially diluted with a solvent to prepare a test substance preparation solution of a 0,100,125,150,200 µg/mL concentration without S9 and 2,200,225,250,275,300 µg/mL with S9 in the short-time treatment method while 0,50,75,100,125,150 µg/mL concentration in In the continuous treatment method were used . The test substance preparation solution was added so as to be 10 % Volume of the culture solution in all tests. As a positive control group, benzo [a] pyrene was added at a concentration of 20 μg / mL in the presence of S 9 mix in a short time treatment method, and mitomycin C at a concentration of 0.1 μg / mL, and in continuous treatment, mitomycin C was set at a concentration of 0.05 μg / mL. Chromosome analysis based on structural abnormalities such as breakage and exchange of chromosome type or chromosome type, presence and absence of gap, and ploidy. The presence or absence of cells (polyploid) was observed. The gap was not included in structural abnormality. Also analyzed 200 groups of metaphase cells per group for structural abnormalities and ploidy cells. The appearance frequency of chromosomal structural abnormalities and polyploid cells was less than 5% in both the short treatment method S9 mix presence and in the absence of S9 mix and in the treatment group treated with continuous treatment method 24 hours. Hence test material did not induce gene mutation inChinese hamster lungs cells inthe presence and absence of S9and hence is not likely to classify as a gene mutant in vitro

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Data for the various test chemicals was reviewed to determine the mutagenic nature of 2-Ethoxybenzoic acid (134-11-2). The studies are as mentioned below:

AMES test;

Gene mutation toxicity study was performed to determine the mutagenic nature of test substance. Preincubation assay was performed as a dose level of 0, 1.0, 3.0, 10.0, 33.0, 100.0 or 333.0 µg/plate with and without S9 metabolic activation system. The chemical as dissolved in DMSO and concurrent solvent and positive controls were included in the study. Test chemical failed to induce mutation in Salmonella typhimurium strains TA97, TA98, TA100, TA1535 both in the presence and absence of S9 metabolic activation system and hence does not classify for gene mutation in vitro.

Supported by other AMES test. Gene mutation toxicity study was performed to determine the mutagenic nature of the test compound. The study was performed as part of mutagenicity produced by aqueous chlorination of organic compounds. Test compound was used at dose levels of 10-1, 100, 101, 102or 103µg/plate (0.1, 1, 10, 100 or 1000 µg/plate) without chlorination. Test compound did not induce reversion of mutant strains without chlorination and hence is not mutagenic in the bacterium Salmonella typhimurium TA 100 and hence does not classify as gene mutant in vitro.

In vitro Mammalian cell gene mutation assay

In vitro chromosome aberration study was performed to determine the mutagenic nature of test material.SHE cells (5x105) in tertiary culture were plated into 75-cm2flasks, incubated overnight, and treatedat dose levels from 0, 0.8,3,8,30µMfor 24 h.The test chemical was dissolved in distilled, deionized water .SHE cells have the doubling time of 16 h. The cells were harvested with 0.1% trypsin for chromosome preparation. Three hours before harvest, Colcemid was administered at 0.2µg/ml and metaphase chromosomes were prepared. For determination of chromosome aberrations, 100 metaphases were scored per experimental group. Achromatic lesions greater than the width of the chromatid were scored as gaps unless there was displacement of the broken piece of chromatid. If there was displacement, they were scored as breaks. Also, the chromosome aberration assay was carried out in the presence of exogenous metabolic activation with rat liver post-mitochondrial supernatant (PMS), Cells (5x105) were plated on 100-mm dishes and after overnight incubation, they were treated with chemical agents for 3 h in a 5% PMS mixture.). Cells were washed twice with 5 ml PBS (-) and incubated with fresh medium for 18 h followed by chromosome preparations., Test material did not induce gene mutation inSyrian hamster embryo (SHE) cells inthe presence and absence ofmetabolic activation with rat liver post-mitochondrial supernatant (PMS)and hence is not likely to classify as a gene mutant in vitro.

In vitro chromosome aberration study was performed to determine the mutagenic nature of test material. CHL / IU cells derived from Chinese hamster lungs. 2 × 10 4 CHL / IU cells were seeded in a dish (6 cm in diameter) containing 5 mL of the culture solution and cultured in a 37 ° C. CO 2 incubator (5% CO 2) for 3 days. After the preculture, in the short-time treatment method, the test substance was treated for 6 hours in the presence and absence of S9 mix and then cultured for 18 hours in fresh culture medium. In the continuous treatment method, the test substance was continuously treated for 24 hours. The test substance was dissolved in a Physiological saline to prepare a stock solution, and then the stock solution was serially diluted with a solvent to prepare a test substance preparation solution of a 0,100,125,150,200 µg/mL concentration without S9 and 2,200,225,250,275,300 µg/mL with S9 in the short-time treatment method while 0,50,75,100,125,150 µg/mL concentration in In the continuous treatment method were used . The test substance preparation solution was added so as to be 10 % Volume of the culture solution in all tests. As a positive control group, benzo [a] pyrene was added at a concentration of 20 μg / mL in the presence of S 9 mix in a short time treatment method, and mitomycin C at a concentration of 0.1 μg / mL, and in continuous treatment, mitomycin C was set at a concentration of 0.05 μg / mL. Chromosome analysis based on structural abnormalities such as breakage and exchange of chromosome type or chromosome type, presence and absence of gap, and ploidy. The presence or absence of cells (polyploid) was observed. The gap was not included in structural abnormality. Also analyzed 200 groups of metaphase cells per group for structural abnormalities and ploidy cells. The appearance frequency of chromosomal structural abnormalities and polyploid cells was less than 5% in both the short treatment method S9 mix presence and in the absence of S9 mix and in the treatment group treated with continuous treatment method 24 hours. Hence test material did not induce gene mutation inChinese hamster lungs cells inthe presence and absence of S9and hence is not likely to classify as a gene mutant in vitro.

 

Based on the data summarized, 2-Ethoxybenzoic acid (134-11-2)did not induce gene mutation .Hence it is not likely to be mutagenic in vitro.

Justification for classification or non-classification

Thus based on the above annotation and CLP criteria for target substance 2-Ethoxybenzoic acid (134-11-2) did not exhibit gene mutation in vitro. Hence the test chemical is not likely to classify as a gene mutant in vitro.