3-phenylpropionic acid

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The gene mutation study was conducted according toL5178Y TK+/-Mouse Lymphoma Mutagenicity Assay to determine the mutagenic nature of the test compound 3-Phenylpropionic acid.

The Cells at a concentration of 1.2 X 10⁷cells/mL were exposed for 4 h to a range of concentrations from 500 -8091 µg/mL. The cells were then washed, resuspended in growth medium, and incubated at 37°C for 48h to allow recovery and mutant expression. Cells in the cultures were adjusted to 3 X 10⁵/mL at 24 h intervals. They were then cloned (1 X 10⁶cells/plate for mutant selection and 200 cells/plate for viable count determinations) in soft agar medium containing Fischer’s medium, 20% horse serum, 2 mM sodium pyruvate, 0.02% pluronic F-68, and 0.23% granulated agar. Resistance to trifluorothymidine (TFT) was determined by adding TFT (final concentration, 3µg/mL) to the cloning medium for mutant selection. The 100X stock solution of TFT in saline was stored at -70 °C and was thawed immediately before use. Plates were incubated at 37 ( 1 °C in 5% CO2 in air for 10-12 days and then counted with an Artek automated colony Counter. Only colonies larger than ~ 0.2 mm in diameter were counted. Mutant frequencies were expressed as mutants per 10⁶surviving cells.

3-Phenylpropionic acid failed to induce a doubling of the mutant frequency both in the presence and absence of S9 activation system and hence is not likely to be gene mutant in vitro.

Link to relevant study records

Reference

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

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 peer reviewed publication

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Principles of method if other than guideline:

The gene mutation study was conducted according to L5178Y TK+/- Mouse Lymphoma Mutagenicity Assay to determine the mutagenic nature of 3-Phenylpropionic acid

GLP compliance:

not specified

Type of assay:

other: Mouse lymphoma assay

Specific details on test material used for the study:

- Name of test material: 3-Phenylpropionic acid
- IUPAC name: 3-phenylpropanoic acid
- Molecular formula: C9H10O2
- Molecular weight: 150.176 g/mol
- Substance type: Organic
- Physical state: No data available.
- Purity: No data available
- Impurities (identity and concentrations): No data available

Target gene:

Thymidine kinase

Species / strain / cell type:

mouse lymphoma L5178Y cells

Remarks:

TK+/- 3.7.C

Details on mammalian cell type (if applicable):

- Type and identity of media:
The cells were grown in Fischer’s medium for leukemic cells of mice supplemented with 10% horse serum and 0.02% pluronic F-68.
- Properly maintained: No data available
- Periodically checked for Mycoplasma contamination: Yes
- Periodically checked for karyotype stability: No data available
- Periodically "cleansed" against high spontaneous background: No data available

Additional strain / cell type characteristics:

not specified

Cytokinesis block (if used):

No data

Metabolic activation:

with and without

Metabolic activation system:

liver S9 prepared from Aroclor 1254-induced male Sprague- Dawley rats.

Test concentrations with justification for top dose:

500 - 8091 µg/mL

Vehicle / solvent:

No data available

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

other: ethyl methylsulfonate, 3-methylcholanthrene and dimethylbenz[a]- anthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: No data available
- Exposure duration: 4 h
- Expression time (cells in growth medium):48 h
- 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): 1×106 cells/plate for mutant selection
SPINDLE INHIBITOR (cytogenetic assays): No data available
STAIN (for cytogenetic assays): No data available

NUMBER OF REPLICATIONS: Duplicate

NUMBER OF CELLS EVALUATED: 1X 106 cells/plate for mutant selection and 200
cells/plate for viable count determinations

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:

Results were interpreted using a doubling of the mutant frequency over the concurrent solvent-treated control value as an indication of a positive effect, together with evidence of a dose-related increase. Doubling of the mutant frequency was previously reported as representing a positive effect. Only doses yielding total growth values of 10% were used in the analysis of induced mutant frequency. Doses yielding less than 10% total growth were used in determining dose response.

Statistics:

No data

Species / strain:

mouse lymphoma L5178Y cells

Remarks:

TK+/- 3.7.C

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 available
- Effects of osmolality: No data available
- Evaporation from medium: No data available
- Water solubility: No data available
- Precipitation: No data available
- Other confounding effects: No data available

RANGE-FINDING/SCREENING STUDIES: No data available

COMPARISON WITH HISTORICAL CONTROL DATA: No data available

ADDITIONAL INFORMATION ON CYTOTOXICITY: The doses of chemical selected for testing were within the range yielding approximately 0-90% cytotoxicity.

Conclusions:

3-Phenylpropionic acid failed to induce a doubling of the mutant frequency both in the presence and absence of S9 activation system and hence is not likely to be gene mutant in vitro.

Executive summary:

The gene mutation study was conducted according toL5178Y TK+/-Mouse Lymphoma Mutagenicity Assay to determine the mutagenic nature of the test compound 3-Phenylpropionic acid.

The Cells at a concentration of 1.2 X 10⁷cells/mL were exposed for 4 h to a range of concentrations from 500 -8091 µg/mL. The cells were then washed, resuspended in growth medium, and incubated at 37°C for 48h to allow recovery and mutant expression. Cells in the cultures were adjusted to 3 X 10⁵/mL at 24 h intervals. They were then cloned (1 X 10⁶cells/plate for mutant selection and 200 cells/plate for viable count determinations) in soft agar medium containing Fischer’s medium, 20% horse serum, 2 mM sodium pyruvate, 0.02% pluronic F-68, and 0.23% granulated agar. Resistance to trifluorothymidine (TFT) was determined by adding TFT (final concentration, 3µg/mL) to the cloning medium for mutant selection. The 100X stock solution of TFT in saline was stored at -70 °C and was thawed immediately before use. Plates were incubated at 37 ( 1 °C in 5% CO2 in air for 10-12 days and then counted with an Artek automated colony Counter. Only colonies larger than ~ 0.2 mm in diameter were counted. Mutant frequencies were expressed as mutants per 10⁶surviving cells.

3-Phenylpropionic acid failed to induce a doubling of the mutant frequency both in the presence and absence of S9 activation system and hence is not likely to be 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

Gene mutatioin in vitro:

Variuos peer reviewed publications for the target chemical and its read across and prediction based data for the target chemical were reviewed to determine the mutagenic nature of 3-Phenylpropionic acid . The studies are as mentioned below:

The gene mutation study was conducted by Seifried et al ( Chem. Res. Toxicol., 2006) according toL5178Y TK+/-Mouse Lymphoma Mutagenicity Assay to determine the mutagenic nature of the test compound 3-Phenylpropionic acid (CAS no 501 -52 -0). The Cells at a concentration of 1.2 X 10⁷cells/mL were exposed for 4 h to a range of concentrations from 500 -8091 µg/mL. The cells were then washed, resuspended in growth medium, and incubated at 37°C for 48h to allow recovery and mutant expression. Cells in the cultures were adjusted to 3 X 10⁵/mL at 24 h intervals. They were then cloned (1 X 10⁶cells/plate for mutant selection and 200 cells/plate for viable count determinations) in soft agar medium containing Fischer’s medium, 20% horse serum, 2 mM sodium pyruvate, 0.02% pluronic F-68, and 0.23% granulated agar. Resistance to trifluorothymidine (TFT) was determined by adding TFT (final concentration, 3µg/mL) to the cloning medium for mutant selection. The 100X stock solution of TFT in saline was stored at -70 °C and was thawed immediately before use. Plates were incubated at 37 ( 1 °C in 5% CO2 in air for 10-12 days and then counted with an Artek automated colony Counter. Only colonies larger than ~ 0.2 mm in diameter were counted. Mutant frequencies were expressed as mutants per 10⁶surviving cells. 3-Phenylpropionic acid failed to induce a doubling of the mutant frequency both in the presence and absence of S9 activation system and hence is not likely to be gene mutant in vitro.

Gene mutation toxicity was predicted for 3-Phenylpropionic acid (CAS no 501 -52 -0) using the battery approach from Danish QSAR database (2017). The study assumed the use of Salmonella typhimurium bacteria in the Ames test. The end point for gene mutation has been modeled in the Danish QSAR using the three software systems Leadscope, CASE Ultra and SciQSAR. Based on predictions from these three systems, a fourth and overall battery prediction is made. The battery prediction is made using the so called Battery algorithm. With the battery approach it is in many cases possible to reduce “noise” from the individual model estimates and thereby improve accuracy and/or broaden the applicability domain. 3-Phenylpropionic acid was assumed to not induce mutation in Salmonella typhimurium by the Ames assay performed and hence the chemical is predicted to not classify as a gene mutant in vitro.

In another study by Zeiger et al (1988) for 50 -60% structurally similar read across chemical, Acetylsalicylic acid (RA CAS no 50 -78 -2; IUPAC name: 2-acetoxybenzoic acid) was studied for its ability to induce gene mutations in strains of Salmonella typhimurium. The test compound was tested at concentration of 0, 33, 100, 333, 1000 or 2000 µg/plate using Salmonella typhimurium TA100, TA1535, TA97 and TA98 in the presence and absence of 10 % and 30 % rat and hamster liver S9 metabolic activation system. Preincubation assay was performed. Concurrent solvent and vehicle control were incorporated in the study. The plates were observed an increase in the number of revertants. Acetylsalicylic acid did not induce an increase in the number of revertants and hence is not mutagenic to the Salmonella typhimurium TA100, TA1535, TA97 and TA98 in the presence and absence of rat and hamster liver S9 metabolic activation system.

Gene mutation toxicity study was performed by Zeiger et al (1992) to determine the mutagenic nature of 50 -60% structurally and functionally similar read across chemical Phthalic acid (RA CAS no 88 -99 -3; IUPAC name: Phthalic acid) . The study was performed using Salmonella typhimurium strains TA97, TA98, TA100, TA1535 in the presence and absence of S9 metabolic activation system. The chemical was dissolved in DMSO and used at dose levels of 0, 33, 100, 333, 1000, 3333 or 10000 µg/plate by the preincubation method. Concurrent solvent and postive control chemicals were used in the study. The plates were observed for a dose dependent increase in the number of Histidine- independent (his+) colonies. Phthalic acid failed to induce mutation a dose dependent increase in the number of revertants in Salmonella typhimurium strains TA97, TA98, TA100, TA1535 in the presence and absence of S9 metabolic activation system and hence is not likely to classify as a gene mutant in vitro.)

Based on the available data for the target chemical and its read across, 3-Phenylpropionic acid does not exhibit gene mutation is vitro. Thus, the chemical is not likely to classify as a gene mutant as per the criteria mentioned in CLP regulation.

Justification for classification or non-classification

Based on the available data for the target chemical and its read across, 3-Phenylpropionic acid does not exhibit gene mutation is vitro. Thus, the chemical is not likely to classify as a gene mutant as per the criteria mentioned in CLP regulation.