Phenylacetic acid

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames assay:

The given test chemical shows negative result in in-vitro bacterial reverse mutation assay test on Salmonella typhimurium strains TA 1538; TA 1535; TA 1537; TA 100; TA 98 with and without S9 metabolic activation. This indicates that the substance is not mutagenic.

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 mammalian cell gene mutation assay:

The given test chemical shows negative result in Mouse Lymphoma Forward Mutation Assay on Mouse lymphoma L5178Y tk ± cells with and without S9 metabolic activation. This indicates that the substance is not mutagenic

In vitro DNA damage and/or repair study

The given test chemical shows negative result in unscheduled DNA synthesis on Rat hepatocytes. This indicates that the substance is not mutagenic

 

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Justification for type of information:

Data is from publication.

Qualifier:

according to guideline

Guideline:

other: As mentioned below

Principles of method if other than guideline:

To evaluate mutagenic effects of the given test chemical by the method of Ames reverse mutation assay.

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine

Species / strain / cell type:

other: Salmonella strains TA1535, TA1537, TA1538, TA98, and TA100.

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:

Aroclor 1254-induced rat liver S9 activation system

Test concentrations with justification for top dose:

1000 μg/plate

Vehicle / solvent:

No data

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

not specified

True negative controls:

not specified

Positive controls:

not specified

Details on test system and experimental conditions:

No data

Rationale for test conditions:

No data

Evaluation criteria:

No data

Statistics:

No data

Species / strain:

other: Salmonella strains TA1535, TA1537, TA1538, TA98, and TA100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

True negative controls validity:

not specified

Positive controls validity:

not specified

Additional information on results:

No data

Remarks on result:

other: No mutagenic potential

Conclusions:

The given test chemical shows negative result in in-vitro bacterial reverse mutation assay test on Salmonella typhimurium strains TA 1538; TA 1535; TA 1537; TA 100; TA 98 with and without S9 metabolic activation. This indicates that the substance is not mutagenic.

Executive summary:

In-vitro bacterial reverse mutation test was conducted by using the given test chemical to evaluate mutagenic effects on Salmonella typhimurium strains TA 1538; TA 1535; TA 1537; TA 100; TA 98 with and without Aroclor 1254-induced rat liver S9 activation system at the concentration of 1000 μg/plate.

The given test chemical shows negative result in Salmonella typhimurium strains TA 1538; TA 1535; TA 1537; TA 100; TA 98 with and without S9 metabolic activation. This indicates that the substance is not mutagenic.

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

Remarks:

Experimental data from various test chemicals

Justification for type of information:

Data for the target chemical is summarized based on the 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:

other: As mentioned below

Principles of method if other than guideline:

WoE for the target CAS is summarized based on data from various test chemicals.

GLP compliance:

not specified

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

5. No data
6. Not specified

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Remarks:

5

Details on mammalian cell type (if applicable):

- Type and identity of media: Mc- Coy’s 5a medium with 10% fetal calf serum, L-glutamine, and antibiotics
- Properly maintained: No data available
- Periodically checked for Mycoplasma contamination: No data available
- Periodically checked for karyotype stability: No data available
- Periodically "cleansed" against high spontaneous background: No data available

Additional strain / cell type characteristics:

not applicable

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Remarks:

6

Details on mammalian cell type (if applicable):

not specified

Additional strain / cell type characteristics:

not specified

Cytokinesis block (if used):

5. No data
6. not specified

Metabolic activation:

with and without

Metabolic activation system:

5. The S9 mix consisted of 15 pl/ml liver homogenate (from male Sprague-Dawley rats, induced with Aroclor 1254), 2.4 mg/ ml NADP, and 4.5 mg/ml isocitric acid in serum-free medium.
6. induced male Sprague Dawley rat liver S9

Test concentrations with justification for top dose:

5. Without S9: 160-1600 µg/ ml
With S9: 500-5000 µg/ ml
6. -S9;0,6.4,12.8 and 18.3 µg/mL (Long duration)
0,6.02,7.96 and 10.21 µg/mL (short duration)
+S9; 0,50.2,174.8and 100.3 µg/mL

Vehicle / solvent:

5. Water, dimethyl sulfoxide (DMSO), ethanol, or Acetone (in the order of preference)
6. Vehicle
- Vehicle(s)/solvent(s) used: Dimethyl Sulfoxide
- Justification for choice of solvent/vehicle: The test substance is soluble in DMSO.

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

other: Triethylenemelamine

Remarks:

5

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: -S9 mix; Triethylenemelamine +S9 mix; Cyclophosphamide

Remarks:

6

Details on test system and experimental conditions:

5. METHOD OF APPLICATION: in medium

DURATION
- Preincubation period:
- Exposure duration: With S9: 2 hrs
Without S9: Apprx. 8.5-9 hrs
- Expression time (cells in growth medium): 8.5-9 hrs
- Selection time (if incubation with a selection agent): after 18-26 hrs
- Fixation time (start of exposure up to fixation or harvest of cells): 8-12 hr after the beginning of treatment.

SELECTION AGENT (mutation assays): Geimsa stain

NUMBER OF CELLS EVALUATED: 100 cells

OTHER EXAMINATIONS:
- Determination of polyploidy: Yes
6. Details on test system and conditions
METHOD OF APPLICATION: In medium
DURATION
- Fixation time (start of exposure up to fixation or harvest of cells):
-S9; 11 hours (Long duration)
10.5 hours(short duration)
+S9; 10.5 hours

Rationale for test conditions:

5. No data
6. No data

Evaluation criteria:

5. Chromosomal aberrations were noted; Cells were selected for scoring on the basis of good morphology and completeness of karyotype (21 ± 2 chromosomes).

Classes of aberrations included simple (breaks and terminal deletions), complex (rearrangements and translocations), and other (pulverized chromosomes). Gaps and endo-reduplications were recorded but were not included in the totals. Aberrations were not scored in polyploidy cells but metaphases with 19-23 chromosomes were used (the modal number being 21).
6. The mammalian cells were observed for chromosome aberration, Chromosome gaps and breaks.

Statistics:

5. For chromosome aberrations, linear regression analysis of the percentage of cells with aberrations vs the log-dose was used as the test for trend. To examine absolute increases over control levels at each dose, a binomial sampling assumption was used. The P values were adjusted to take into account the multiple dose comparisons. For data analysis, we used the “total” aberration category, and the criterion for a positive response was that the adjusted P value be < 0.05.
6. Yes, SD ± Mean was observed.

Species / strain:

Chinese hamster Ovary (CHO)

Remarks:

5

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

True negative controls validity:

not specified

Positive controls validity:

valid

Species / strain:

Chinese hamster Ovary (CHO)

Remarks:

6

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

True negative controls validity:

not specified

Positive controls validity:

valid

Additional information on results:

5. RANGE-FINDING/SCREENING STUDIES: doses were chosen for the aberration test based on a preliminary test of cell survival 24 hr after treatment. Doses were based on observations of cell confluence and mitotic cell availability in the SCE test.
6. No data

Remarks on result:

other: No mutagenic effects were observed

Conclusions:

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.

Executive summary:

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

 

In vitro mammalian chromosome aberration test was performed to evaluate the mutagenic nature of the test chemical. Cloned Chinese hamster ovary cells (CHO-W-B1) were cultured in Mc-Coy’s 5a medium with 10% fetal calf serum, L-glutamine, and antibiotics. Tests were carried out with and without an in vitro metabolic activation system (S9 mix). In tests without metabolic activation, the test chemical was left in culture until colcemid addition, whereas with activation the test chemical was added along with S9 mix for only 2 hr at the beginning of the test period. The doses used for the study were 160-1600µg/mL without S9 and 500-5000µg/mL with S9. The test chemical did not induce chromosome aberrations in the Chinese hamster ovary 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 another study, the chromosomal aberration induction potential was determined by performing the gene mutation study of the test chemical. For this purpose in vitro mammalian chromosome aberration test was performed .The test material was exposed toChinese hamster ovary cells inthe presence and absence of metabolic activation S9. The concentration of test material used in the presence and absence of metabolic activation were mention below – S9; 0, 6.4, 12.8 and 18.3 µg/mL (Long duration); 0, 6.02, 7.96 and 10.21 µg/mL (short duration) and +S9; 0, 50.2, 174.8 and 100.3 µg/mL. No chromosome aberration, Chromosome gaps and breaks were observed in the presence or absence of metabolic activation. Therefore test chemical was considered to be non-mutagenic inChinese hamster ovary cells by in vitro mammalian chromosome aberration test. Hence the substance cannot be classified as non -mutagenic in vitro.

 

Thus, based on the above summarized studies on test chemical, it can be concluded that the given 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.

Reference 3

Endpoint:

in vitro gene mutation 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

Justification for type of information:

Data is from publication.

Qualifier:

according to guideline

Guideline:

other: As mentioned below

Principles of method if other than guideline:

To evaluate mutagenic effects by using the given test chemical in mammalian cell line.

GLP compliance:

not specified

Type of assay:

other: Mouse Lymphoma cell mutagenesis (MLY) Assay

Target gene:

Thymidine kinase (Tk)

Species / strain / cell type:

mouse lymphoma L5178Y cells

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:

Aroclor 1254-induced rat liver S9 activation system

Test concentrations with justification for top dose:

1000, 1500 μg/ml

Vehicle / solvent:

No data

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

not specified

True negative controls:

not specified

Positive controls:

not specified

Details on test system and experimental conditions:

No data

Rationale for test conditions:

No data

Evaluation criteria:

No data

Statistics:

No data

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

True negative controls validity:

not specified

Positive controls validity:

not specified

Additional information on results:

No data

Remarks on result:

other: No mutagenic potential

Conclusions:

The given test chemical shows negative result in Mouse Lymphoma Forward Mutation Assay on Mouse lymphoma L5178Y tk ± cells with and without S9 metabolic activation. This indicates that the substance is not mutagenic

Executive summary:

Mouse Lymphoma cell mutagenesis (MLY) Assay was carried out to evaluate the mutagenic effects of the given test chemical in Mouse lymphoma L5178Y tk ± cells with and without Aroclor 1254-induced rat liver S9 activation system at 1000, 1500 μg/ml.

The given test chemical shows negative result in Mouse Lymphoma Forward Mutation Assay on Mouse lymphoma L5178Y tk ± cells with and without S9 metabolic activation. This indicates that the substance is not mutagenic

Reference 4

Endpoint:

in vitro DNA damage and/or repair study

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Justification for type of information:

Data is from publication.

Qualifier:

according to guideline

Guideline:

other: As mentioned below

Principles of method if other than guideline:

To evaluate mutagenic effects by using the given test chemical performing Unscheduled DNA synthesis

GLP compliance:

not specified

Type of assay:

other: Unscheduled DNA synthesis (UDS)

Target gene:

No data

Species / strain / cell type:

hepatocytes:

Details on mammalian cell type (if applicable):

No data

Additional strain / cell type characteristics:

not specified

Cytokinesis block (if used):

No data

Metabolic activation:

not specified

Metabolic activation system:

No data

Test concentrations with justification for top dose:

500 μg/ml

Vehicle / solvent:

No data

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

not specified

True negative controls:

not specified

Positive controls:

not specified

Details on test system and experimental conditions:

No data

Rationale for test conditions:

No data

Evaluation criteria:

No data

Statistics:

No data

Species / strain:

hepatocytes:

Metabolic activation:

not specified

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

True negative controls validity:

not specified

Positive controls validity:

not specified

Additional information on results:

No data

Remarks on result:

other: No mutagenic potential

Conclusions:

The given test chemical shows negative result in unscheduled DNA synthesis on Rat hepatocytes. This indicates that the substance is not mutagenic

Executive summary:

The mutagenic effect was determine by using the given test chemical by performing Unscheduled DNA synthesis (UDS) on Rat hepatocytes at 500 μg/ml.

The given test chemical shows negative result in unscheduled DNA synthesis on Rat hepatocytes. This indicates that the substance is not mutagenic

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Gene mutation in vitro:

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

Ames assay:

In-vitro bacterial reverse mutation test was conducted by using the given test chemical to evaluate mutagenic effects on Salmonella typhimurium strains TA 1538; TA 1535; TA 1537; TA 100; TA 98 with and without Aroclor 1254-induced rat liver S9 activation system at the concentration of 1000 μg/plate. The given test chemical shows negative result in Salmonella typhimurium strains TA 1538; TA 1535; TA 1537; TA 100; TA 98 with and without S9 metabolic activation. This indicates that the substance is not mutagenic.

 

The above study is supported with the gene mutation toxicity study performed to determine the mutagenic nature of the given test chemical. The study was performed as per the preincubation modification of the Salmonella/mammalian microsome mutagenicity (Ames) test. The chemicals were tested in a preincubation procedure in strains TA98 and TA100 without metabolic activation and with activation provided by Aroclor induced rat and hamster liver homogenates (S9). If a positive response was seen in one of these two strains, the strain/metabolic activation combination producing that response was repeated, and no further testing was performed. If no positive responses were seen, the chemical was tested in strains TA97 and TA1535. The plates were observed for a dose dependent increase in the number of revertants/plate. The combination of a questionable (?) and negative (-) response was considered negative (-); the combination of a weakly positive (+w) and negative response was considered questionable (?). The given test chemical did not induce a dose dependent increase in the number of revertants in Salmonella typhimurium TA98, TA100, TA97 and TA1535 in the presence and absence of S9 metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.

 

In another study, mutagenic nature of the test chemical was determined by performing gene mutation toxicity study. The material was dissolved in ethanol and applied at a concentration of 3 µmole/plate in the spot test performed to Salmonella typhimurium LT-2 strains TA 98, TA 100, TA 1535, and TA 1537 with and without S9 metabolic activation system. The test chemical did not induce reversion of mutant strains and hence is not mutagenic in the bacterium Salmonella typhimurium LT-2 strains TA 98, TA 100, TA 1535, and TA 1537 with and without S9 metabolic activation system and hence the chemical is not likely to classify as gene mutant in vitro.

 

In vitro mammalian chromosome aberration study:

 

In vitro mammalian chromosome aberration test was performed to evaluate the mutagenic nature of the test chemical. Cloned Chinese hamster ovary cells (CHO-W-B1) were cultured in Mc-Coy’s 5a medium with 10% fetal calf serum, L-glutamine, and antibiotics. Tests were carried out with and without an in vitro metabolic activation system (S9 mix). In tests without metabolic activation, the test chemical was left in culture until colcemid addition, whereas with activation the test chemical was added along with S9 mix for only 2 hr at the beginning of the test period. The doses used for the study were 160-1600µg/mL without S9 and 500-5000µg/mL with S9. The test chemical did not induce chromosome aberrations in the Chinese hamster ovary 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 another study, the chromosomal aberration induction potential was determined by performing the gene mutation study of the test chemical. For this purpose in vitro mammalian chromosome aberration test was performed .The test material was exposed toChinese hamster ovary cells inthe presence and absence of metabolic activation S9. The concentration of test material used in the presence and absence of metabolic activation were mention below – S9; 0, 6.4, 12.8 and 18.3 µg/mL (Long duration); 0, 6.02, 7.96 and 10.21 µg/mL (short duration) and +S9; 0, 50.2, 174.8 and 100.3 µg/mL. No chromosome aberration, Chromosome gaps and breaks were observed in the presence or absence of metabolic activation. Therefore test chemical was considered to be non-mutagenic inChinese hamster ovary cells by in vitro mammalian chromosome aberration test. Hence the substance cannot be classified as non -mutagenic in vitro.

 

 

In vitro mammalian cell gene mutation assay:

An in vitro mammalian cell gene mutation study was designed and conducted to determine the genotoxicity profile of the given test chemical by performing Mouse Lymphoma cell mutagenesis (MLY) Assay in Mouse lymphoma L5178Y tk ± cells with and without Aroclor 1254-induced rat liver S9 activation system at 1000, 1500 μg/ml. The given test chemical shows negative result in Mouse Lymphoma Forward Mutation Assay on Mouse lymphoma L5178Y tk ± cells with and without S9 metabolic activation. This indicates that the substance is not mutagenic

 

In another study, Chinese Hamster Ovary (CHO) cells were exposed to the test chemical in the concentration of 0, 0.5, 1.0, 2.5 or 5.0 mM and without metabolic activation for 3 hours. The results showed that there was no evidence of cytotoxicity after treatment. Independently of tested concentration, the results showed no evidence of gene toxicity. Therefore, it is considered that the test chemical in the concentration of 0, 0.5, 1.0, 2.5 or 5.0 mM does not cause genetic mutation(s) when CHO cells are exposed to the test chemical in the absence of metabolic activation and hence does not classify for gene mutation in vitro.

 

These test results were supported with an in vitro mammalian cell gene mutation study was designed and conducted to determine the genotoxicity profile of the given test chemical when administered to Chinese Hamster Ovary (CHO) cells. A preliminary dose-finding study was conducted prior to the main study. A range of different concentrations were tested in 96-well plates and analyzed by two commonly used assays, i.e. the colorimetric assay of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and the bicinchoninic acid (BCA) assay to assess cell viability and protein concentration, respectively. From the basis of the results from the MTT and BCA assays, test concentrations of the test chemical was chosen to be included in the gene toxicity test. In the genotoxicity test, test chemical was administered to CHO cells for 3 hrs at the dose levels of 0, 0.5, 1.0, 2.5 or 5.0 mM and in the absence or presence of exogenous metabolic activation. CHO cells representing the negative controls were exposed to the vehicle. Positive controls, such as N-ethyl-N-nitrosourea (ENU) experiments without metabolic activation and 7,12-dimethylbenz(a) anthracene in experiments with metabolic activation, were also included in each test. The results showed indication of gene mutations occurring in the positive controls ENU and 7,12-dimethylbenz(a) anthracene while no other treatment gave rise to gene toxicity. No cytotoxic effects were observed when CHO cells were exposed to Phenethylphenylacetate for 3 hrs. Based on the results of the current study, it can be concluded that the given test chemical does not give rise to gene mutations when exposed at ≤ 5.0 mM for 3 hrs or more, and hence it is not induce cytotoxic effects at concentrations of ≤ 5.0mM.

 

In vitro DNA damage and/or repair study

The mutagenic effect was determined by using the given test chemical by performing Unscheduled DNA synthesis (UDS) on Rat hepatocytes at 500 μg/ml. The given test chemical shows negative result in unscheduled DNA synthesis on Rat hepatocytes. This indicates that the substance is not mutagenic

 

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, In vitro mammalian cell gene mutation assay and In vitro DNA damage and/or repair study. Hence the test chemical is not likely to classify as a gene mutant as per the criteria mentioned in CLP regulation.