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Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames test: not mutagenic;

HPRT test performed with CHO cells: not mutagenic;

Chromosome aberration test performed with V79 cells: not clastogenic

Link to relevant study records

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Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2012-10-09 to 2012-11-06
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Qualifier:
according to
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
GLP compliance:
yes (incl. certificate)
Type of assay:
other: mammalian gene mutation assay in vitro
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: DEBR 012553
Target gene:
hypoxanthine-guanine phosphoribosyl transferase enzyme locus (hprt) in cultured Chinese hamster ovary cells
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
- Type and identity of media: Ham´s F12 medium
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9-mix prepared from incuded rat liver
Test concentrations with justification for top dose:
Experiment I (without S9-mix, 5 hours treatment):
53.8, 107.5, 215, 430 and 860 µg/mL
Experiment I (with S9-mix, 5-hours treatment):
53.8, 107.5, 215, 430 and 860 µg/mL
Experiment II (without S9-mix, 20 hour treatment):
53.8, 107.5, 215, 430, 860 µg/mL
Experiment II (with S9-mix, 5-hour treatment):
53.8, 107.5, 215, 430, 860 µg/mL

The highest test concentration of 860 µg/mL corresponds to 10 mM.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Ham´s F12 medium
- Justification for choice of solvent/vehicle: suitable solvent to dissolve test item
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9,10-dimethylbenzanthracene
ethylmethanesulphonate
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DOSE SELECTION EXPERIMENT
In a pre-test on cytotoxicty no cytotoxicity was observed up to concentrations of 5000 µg/mL in the presence or absence of S9. As the highest test concentration according to guideline is 10 mM or 5000 µg/mL, whichever is obtained first, 10 mM (corresponding to 860 µg/mL) was selected as the highest test concentration for the main experiment.

MAIN EXPERIMENT

DURATION
- Exposure duration: 5 hours (with and without S9) and 20 hours (without S9)
- Expression time (cells in growth medium): 8 days

SELECTION and FIXATION
At the end of the expression period the cultures from each of the dose levels were resided at 2x105 cells per 100-mm dish (five dishes) in selection medium. After the selection period, the colonies were fixed, stained with Giemsa and counted for mutant selection and cloning efficiency determination.

SELECTION AGENT (mutation assays): 6-thioguanine

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: 500000

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency

Evaluation criteria:
The test item would have been considered to be mutagenic in this assay if all the following criteria were met:
• The assay is valid.
• The mutant frequency at one or more doses is significantly greater than that of the relevant control.
• Increase of the mutant frequency is reproducible.
• There is a clear dose-response relationship.
The test item would have been considered to have shown no mutagenic activity if no increases were observed which met the criteria listed above.

ASSAY ACCEPTANCE CRITERIA
• The mutant frequency in the negative (solvent) control cultures is within the range (min-max) of historical laboratory control data.
• The positive control chemicals induce a statistically significant and biologically relevant increase in mutant frequency.
• The cloning efficiency of the negative controls is between the range of 60% to 140% on Day 1 and 70% to 130% on Day 8.

Statistics:
Statistical analysis was done with SPSS PC+ software for the following data:
• Mutant frequency between the negative (solvent) and the test item or positive control item treated groups.

The heterogeneity of variance between groups was checked by Bartlett's homogeneity of variance test. Where no significant heterogeneity was detected, a one-way analysis of variance was carried out. If the obtained result was positive, Duncan's Multiple Range test was used to assess the significance of inter-group differences.
Where significant heterogeneity was found, the normal distribution of data was examined by Kolmogorov-Smirnov test. In case of a none-normal distribution, the non-parametric method of Kruskal-Wallis One-Way analysis of variance was used. If there was a positive result, the inter-group comparisons were performed using the Mann-Whitney U-test.
Key result
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
CYTOTOXICITY
Comparable toxicity was observed in treatment groups when compared to the negative (solvent) controls, both in the absence and in the presence of the metabolic activation, confirming the response seen in the dose selection cytotoxicity assays, that the Crotonic acid is a non-cytotoxic item.

MUTATION DATA
In Experiment 1, there were no biologically or statistically significant increases in mutation frequency at any concentration tested, either in the absence or in the presence of metabolic activation. There were no statistical differences between treatment and control groups and no dose-response relationships noted. In Experiment 2, the mutant frequencies of the cells did not show biologically or statistically significant alterations compared to the concurrent control, when the test item was tested without S9 mix over a prolonged treatment period (20 hours). Furthermore, a five-hour treatment in the presence of S9 mix did not cause significant increases in mutant frequency.

POSITIVE CONTROL/NEGATIVE CONTROL
The sensitivity of the tests and the efficacy of the S9 mix were demonstrated by large and statistically significant (p < 0.01) increases in mutation frequency in the positive control cultures. The mutation frequencies of the positive and negative control cultures were consistent with the historical control data from the previous studies performed at this laboratory. In the Experiment 1 the mutation frequencies of EMS were higher (1374.67 and 1421.62 ) than the historical control (1204.29) value, but this deviation did not influence the quality or integrity of the study.

PH AND OSMOLALITY
The pH and osmolality of control and treatment solutions did not show any alterations compared to the concurrent control groups in Experiments 1 and Experiment 2
Conclusions:
It is concluded that the test item, Crotonic acid, was not mutagenic in this in vitro mammalian cell gene mutation test performed with in Chinese hamster ovary cells under the conditions of the present study.
Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2012-11-27 to 2012-12-10
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
GLP compliance:
yes (incl. certificate)
Type of assay:
other: in vitro chromosome aberration test
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: DEBR 012553
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Metabolic activation system:
rat liver microsome preparations (S9 mix)
Test concentrations with justification for top dose:
215, 430, 860 µg/mL (860 µg/mL corresponds to 10 mM, which represents the maximum test concentration acc. to guideline)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DME (Dulbecco’s Modified Eagle’s) medium
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
ethylmethanesulphonate
Remarks:
ethylmethanesulphonate (without metabolic activation); cyclophosphamide (with metabolic activation)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DOSE SELECTION EXPERIMENT
In a pre-test on cytotoxicty no cytotoxicity was observed up to concentrations of 5000 µg/mL in the presence or absence of S9. As the highest test concentration according to guideline is 10 mM or 5000 µg/mL, whichever is obtained first, 10 mM (corresponding to 860 µg/mL) was selected as the highest test concentration for the main experiment.

MAIN EXPERIMENT

DURATION
- Preincubation time: 24 hours (10 mL of DME medium containing 10 % foetal bovine serum)
- Exposure duration and Expression time (cells in growth medium):
Experiment A
3 hours treatment (with and without S9-mix), harvest and cell counting 20 hours from the beginnung of treatment
Experiment B
20 hours treatment (without S9-mix), harvest and cell counting 20 hours and 28 hours from the beginnung of treatment
3 hours treatment (with S9-mix), harvest and cell counting 28 hours from the beginnung of treatment

SPINDLE INHIBITOR (cytogenetic assays): colchicine (0.2 µg/mL, added 2 hours prior to harvest)
STAIN (for cytogenetic assays): 5 % Giemsa

NUMBER OF REPLICATIONS:
Two independent experiments, each in the presence and in the absence of S9 mix, duplicate cultures per concentration or control.

NUMBER OF CELLS EVALUATED:
Cytotoxicity
For concurrent measures of cytotoxicity for all treated and negative control cultures, 5 x 10*5 cells were set up
Analysis of metaphase cells
At least 200 metaphase cells containing 2 N ± 2 centromeres were evaluated for structural aberrations from each experimental group

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: yes
Evaluation criteria:
Interpretation of Results

The criteria for determining a positive result are:
–a concentration-related increase or a reproducible increase in the number of cells with aberrations.
–biological relevance of the results should be considered first, however, for the interpretation of the data both biological and statistical significance should be considered together.
–an increase in the number of polyploid cells may indicate that the test item has the potential to inhibit mitotic processes and to induce numerical chromosome aberrations.
–an increase in the number of cells with endoreduplicated chromosomes may indicate that the test item has the potential to inhibit cell cycle progression.

A test item for which the results do not meet the above criteria is considered as non mutagenic in this system.
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: There were no significant differences between treatment and negative control groups
- Effects of osmolality: There were no significant differences between treatment and negative control groups

RANGE-FINDING/SCREENING STUDIES: The test item is considered as relatively non-cytotoxic up to concentrations of 5000 µg/mL

HISTRORICAL CONTROL DATA: The observed chromosome aberration rates of the controls were within the ranges of historical control data

- Experiment A:

Crotonic acid did not induce an increase in the number of cells with aberrations at any examined concentration, either in the absence or in the presence of metabolic activation. There were no statistically significant differences between treatment and control groups and no dose‑response relationship was noted.

- Experiment B:

Crotonic acid was examined without S9 mix, over a long treatment period and the sampling was made at approximately 1.5 cell cycles (20 hours after treatment start). The cells with structural chromosome aberrations did not show significant alterations compared to the concurrent solvent controls. There was no increase in the number of cells with aberrations without S9 mix following exposure over a period of 20 hours and sampling at approximately 2 cell cycles (28 hours after treatment start). A 3-hour treatment in the presence of S9 mix with 28-hour harvest from the beginning of treatment did not cause an increase in the number of cells with structural chromosome aberrations over concurrent solvent control. In conclusion, as in Experiment A, in Experiment B no statistically significant differences between treatment and control groups and no dose-response relationships were noted.

- No increase in the rate of polyploid and endoreduplicated metaphases was found after treatment with the different concentrations of Crotonic acid.

- In the concurrent solvent control group the percentage of cells with structural aberration(s) without gap was equal to or less than 5 %, proving the suitability of the cell line used.

- The concurrent positive controls Ethyl methanesulfonate (0.4 and 1.0 µL/mL) and Cyclophosphamide(5 µg/mL) caused the expected biologically relevant increases of cells with structural chromosome aberrations as compared to solvent controls. Thus, the study is considered as valid.

Conclusions:
Crotonic acid tested up to the maximum recommended concentration of 860 µg/mL, both with and without mammalian metabolic activation system, did not induce structural chromosome aberrations in Chinese hamster lung cells. Therefore, Crotonic acid is considered as not clastogenic in this system.
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
Please refer to read-across justification document attached in IUCLID Section 13.
Reason / purpose:
read-across source
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
Positive controls validity:
valid
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
Positive controls validity:
valid
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
Positive controls validity:
valid
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
Positive controls validity:
valid
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

In vitro bacterial reverse mutation assay (Ames test)

The Scientific Committee on Food reported in an opinion (SCF, 2002) that Crotonic acid was clearly found to be not mutagenic in the gene mutation assay in Salmonella typhimurium strains TA 1535, TA 1537, TA 98 and TA 100 and in Escherichia coli strain WP2uvrA. As no further information is available on this assay, the bacterial reverse mutation assay performed with Crotonic acid anhydride (Hoechst AG, 1996) was chosen as key study. The read-across approach is justified because Crotonic acid anhydride hydrolyzes to Crotonic acid in aqueous media. Its t1/2 in water is 63 hours at 20°C and 10.2 hours at 25°C (Hoechst AG, 1995 and 1996), indicating that Crotonic acid is rapidly formed and represents the actual test substance under the test conditions employed in the Ames test (incubation at 37°C for 48 hours).

Crotonic acid anhydride was tested in the Salmonella typhimurium reverse mutation assay (Hoechst AG, 1996) with four histidine-requiring strains of Salmonella typhimurium (TA1535, TA1537, TA100 and TA98) and in Escherichia coli reverse mutation assay with a tryptophan-requiring strain of Escherichia coli WP2uvrA according to OECD guideline 471 and GLP. Two independent mutagenicity studies were conducted (plate incorporation method), each in the absence and in the presence of a metabolising system derived from a rat liver homogenate. For both studies, the compound was dissolved in DMSO, and each bacterial strain was exposed to 6 dose levels. Doses for the first study ranged from 4 to 5000 µg/plate. Because of cytotoxicity in the first experiment doses from 0.8 to 2500 µg/plate in the present of metabolic activation and doses from 0.16 to 500 µg/plate in the absence of metabolic activation were chosen for the second experiment. Control plates without mutagen showed that the number of spontaneous revertant colonies was within the historical control range and similar to that described in the literature. All the positive control compounds gave the expected increase in the number of revertant colonies. In the mutagenicity experiments cytotoxicity was observed without metabolic activation at concentrations of 100 µg/plate and above. In the present of metabolic activation the test compound proved to be toxic at all bacterial strains at concentrations of 500 µg/plate and above. In the toxicity test using histidine enriched agar plates and a dilution of the strain TA100 (designated TA 100 D), which was performed in parallel with the second experiment, toxicity was found at concentrations of 2500 µg/plate and above in the presence of metabolic activation and 100 µg/plate and above in the absence of metabolic activation. In the absence and in the presence of the metabolic activation system Crotonic acid anhydride did not result in relevant increases in the number of revertants and any of the bacterial strains. Summarising, it can be stated that Crotonic acid anhydride and thus, Crotonic acid is not mutagenic in this bacterial test system either with or without exogenous metabolic activation.

In vitro mammalian cell gene mutation test

The test item, Crotonic acid was tested in a mammalian gene mutation test in CHO-K1 cells (HPRT assay) according to OECD guideline 476 and GLP (Toxi-Coop Zrt. (d), 2012). The test item was dissolved in medium and the following concentrations were selected on the basis of cytotoxicity investigations made in a preliminary study (without and with metabolic activation using S9 mix).

Two independent main experiments (both run in duplicate) were performed at the concentrations and treatment intervals given below:

Experiment 1, 5-hour treatment period without S9 mix:

53.8, 107.5, 215, 430 and 860 µg/mL

Experiment 1, 5-hour treatment period with S9 mix:

53.8, 107.5, 215, 430 and 860 µg/mL

Experiment 2, 20-hour treatment period without S9 mix:

53.8, 107.5, 215, 430 and 860 µg/mL

Experiment 2, 5-hour treatment period with S9 mix:

53.8, 107.5, 215, 430 and 860 µg/mL

The highest test concentration of 860 µg/mL corresponds to 10 mM. In Experiment 1, there were no biologically or statistically significant increases in mutation frequency at any concentration tested, either in the absence or in the presence of metabolic activation. There were no statistical differences between treatment and control groups and no dose-response relationships were noted. In Experiment 2, the mutant frequencies of the cells did not show biologically or statistically significant alterations compared to the concurrent control, when the test item was tested without S9 mix over a prolonged treatment period (20 hours). Furthermore, a five-hour treatment in the presence of S9 mix did not cause significant increases in mutant frequency.

As in Experiment 1, in Experiment 2 no statistical differences between treatment and solvent control groups and no dose response relationships were noted. The sensitivity of the tests and the efficacy of the S9 mix were demonstrated by large increases in mutation frequency in the positive control cultures.

Crotonic acid tested both without and with metabolic activation (S9 mix), did not induce increases in mutant frequency in this test in Chinese hamster ovary cells. Crotonic acid was not mutagenic in this in vitro mammalian cell gene mutation test performed with CHO-K1 cells.

In vitro mammalian chromosome aberration test

Crotonic acid was assessed to its ability to induce chromosomal aberrations in Chinese Hamster lung fibroblasts (V79) according to OECD guideline 473 and GLP (Toxi-Coop Zrt. (e), 2012). The test item was dissolved in DME (Dulbecco´s Modified Eagle´s) medium and the concentrations were selected on the basis of cytotoxicity investigations made in a preliminary study (without and with metabolic activation using S9 mix). In the two independent experiments of the Chromosome Aberration Assay (Experiments A and B, both run in duplicate) at least 200 well-spread metaphase cells were analysed at concentrations and incubation/expression intervals given below:

Experiment A with 3/20 h treatment/sampling time without and with S9 mix: 215, 430 and 860 µg/mL

Experiment B with 20/20 h treatment/sampling time without S9 mix: 215, 430 and 860 µg/mL

Experiment B with 20/28 h treatment/sampling time without S9 mix: 215, 430 and 860 µg/mL

Experiment B with 3/28 h treatment/sampling time with S9 mix: 215, 430 and 860 µg/mL

The highest concentration of 860 µg/mL corresponds to 10 mM. The positive control substances were Ethylmethanesulphonate ( without metabolic activation) and Cyclophoshamide (with metabolic activation). In Experiments A and B, there were no biologically and statistically significant increases in the number of cells showing structural chromosome aberrations, either in the absence or in the presence of metabolic activation, up to the maximum recommended concentration as compared with concurrent solvent controls. No dose-response relationships were noted.There were no biologically relevant increases in the rate of polyploid or endoreduplicated metaphases in either experiment in the presence or absence of metabolic activation. There was no precipitation of the test item at any dose level tested. No biologically relevant changes in pH or osmolality of the test system were noted at the dose levels tested. The observed chromosome aberration rates of the controls were within the ranges of the historical control data. The validity of the test was demonstrated as the concurrent positive controls induced biologically and statistically significant increases in the number of cells with chromosome aberration(s) over background. Crotonic acid tested up to the maximum recommended concentration of 860 µg/mL, both with and without mammalian metabolic activation system, did not induce structural chromosome aberrations in Chinese Hamster lung cells, and is thus considered as not clastogenic in this system.

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008
The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Based on available data on genotoxicity in vitro, the test item is does not need to be classified as genotoxic according to Regulation (EC) No 1272/2008 (CLP), as amended for the tenth time in Regulation (EU) No 2017/776.