p-cymene

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2020-06-22 to 2020-08-27

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Version / remarks:

2008

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

2020

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

p-cymene

EC Number:

202-796-7

EC Name:

p-cymene

Cas Number:

99-87-6

Molecular formula:

C10H14

IUPAC Name:

1-isopropyl-4-methylbenzene

Method

Target gene:

The Salmonella typhimurium histidine (his) and the Escherichia coli tryptophan (trp) reversion system measures his- → his+ and trp- → trp+ reversions, respectively. The strains are constructed to differentiate between base pair (E.coli WP2, TA 1535, TA 100) and frameshift (TA 1537, TA 98) mutations.

Species / strain

Species / strain 1

Species / strain / cell type:

E. coli WP2 uvr A

Additional strain / cell type characteristics:

not applicable

Species / strain 2

Species / strain / cell type:

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

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- Source of S9: Phenobarbital/β-naphthoflavone induced rat liver
- Method of preparation of S9 mix: The S9 was prepared and stored according to the currently valid version of the SOP for rat liver S9 preparation
- Concentration or volume of S9 mix and S9 in the final culture medium: 50 µl S9 and 500 µL S9 mix in the final culture medium (2700 µl total)
- Quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): Each batch of S9 was routinely tested for its capability to activate the known mutagens benzo[a]pyrene and 2-aminoanthracene in the Ames test

Test concentrations with justification for top dose:

Experiment I (with and without S9 mix): 3, 10; 33; 100; 333; 1000; 2500 and 5000 μg/plate;

Experiment II (with and without S9 mix):
- Strain TA98: 3, 10; 33; 100; 333; 1000; 2500 and 5000 μg/plate
- All remaining strains: 10; 33; 100; 333; 1000; 2500; and 5000 μg/plate

Justification for top dose: tested up to recommended maximum test concentration according to OECD 471

Vehicle / solvent:

- Vehicle/solvent used: DMSO (test item, positive controls),deionised water (positive controls)

- Justification for choice of solvent/vehicle: The solvent was chosen because of its solubility properties and its relative nontoxicity to the bacteria (Maron et al.; 1981).

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

- with S9
- 2.5 µg/plate (TA 1535, TA 1537, TA 98, TA 100) and 10 µg/plate (WP2 uvrA)

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

deionised water

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

- without S9
- 2 µg/plate (WP2 uvrA)

Controls 3

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 4-nitro-o-phenylene-diamine, 4-NOPD

Remarks:

- without S9
- 10 µg/plate (TA98), 50 µg/plate (TA1537)

Controls 4

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

deionised water

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

- without S9
- 10 µg/plate (TA1535 and TA100)

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: Triplicate
- Number of independent experiments: 2

METHOD OF TREATMENT/ EXPOSURE:
- Experiment I: Plate incoporation method
- Experiment II: Pre-incubation method

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period: 60 minutes
- Duration of treatment: For at least 48 hours

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: Reduction in the number of spontaneous revertants (below a factor of 0.5) or a clearing of the bacterial background lawn

METHODS FOR MEASUREMENTS OF GENOTOXICIY
- Counting of revertant colonies using a Petri Viewer Sorcerer Colony Counter 3.0 (Instem, Suffolk IP33 3TA, UK) with the software program Ames Study Manager (v1.24) and Ames Archive Manager (v1.01).

Rationale for test conditions:

In experiment I, the concentration range of the test item was 3 – 5000 μg/plate. Since toxic effects were observed in experiment 1, seven concentrations were tested in experiment II, except of strain TA 98, in which eight concentrations were tested. 5000 μg/plate were chosen as maximal concentration (recommended maximum test concentration according to OECD 471). The concentration range included two logarithmic decades.

Precipitation:
- from 1000 to 5000 μg/plate in the experiment I (in the overlay agar)
- from 2500 to 5000 μg/plate in experiment II
- at 5000 μg/plate in the overlay agar in both experiments

Evaluation criteria:

1) A test item is considered as a mutagen if a biologically relevant increase in the number of revertants of twofold or above (strains TA 98, TA 100, and WP2 uvrA) or threefold or above (strains TA 1535 and TA 1537) the spontaneous mutation rate of the corresponding solvent control is observed.

2) A dose dependent increase is considered biologically relevant if the threshold is reached or exceeded at more than one concentration.

An increase of revertant colonies equal or above the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.

3) A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.

Statistics:

According to the OECD guideline 471, a statistical analysis of the data is not mandatory.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 100

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 examined

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 98

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 examined

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 1537

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 examined

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

S. typhimurium TA 1535

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 examined

Positive controls validity:

valid

Applicant's summary and conclusion

Conclusions:

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

Executive summary:

This study was performed to investigate the potential of the test item to induce gene mutations according to the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and the Escherichia coli strain WP2 uvrA. The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate. The test item was tested at the following concentrations:

 

Pre-Experiment/Experiment I: 3; 10; 33; 100; 333; 1000; 2500; and 5000 μg/plate

Experiment II:

Strain TA 98: 3, 10; 33; 100; 333; 1000; 2500 and 5000 μg/plate

The remaining strains: 10; 33; 100; 333; 1000; 2500; and 5000 μg/plate

 

The test item precipitated in the overlay agar in the test tubes from 1000 to 5000 μg/plate in experiment I and from 2500 to 5000 μg/plate in experiment II. Precipitation of the test item in the overlay agar on the incubated agar plates was observed at 5000 μg/plate in both experiments. The undissolved particles had no influence on the data recording. The plates incubated with the test item showed reduced background growth up to 5000 μg/plate in strains TA 1537, TA 98 and TA 100 in experiment I and in strains TA 1535, TA 1537 and TA 98 in experiment II. Toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), were observed in all strains, except of strains TA 1535 and WP2 uvrA in both experiments. No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with the test item at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance. Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies.