(2E)-2-(4-methylbenzylidene)heptanal

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames test (OECD TG 471): negative

Link to relevant study records

Reference

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The study was conducted between 13 July 2016 and 05 August 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

S. typhimurium: Histidine gene
E. coli: Tryptophan gene

Species / strain / cell type:

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

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

Rat liver S9-mix induced by a combination of Phenobarbital and β-Naphta flavone

Test concentrations with justification for top dose:

Experiment 1: Plate Incorporation Method:
The maximum concentration was 5000 μg/plate (the maximum recommended dose level). Eight concentrations of the test item (1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/plate) were assayed in triplicate against each tester strain, using the direct plate incorporation method.

Experiment 2: Pre-Incubation Method:
The initial dose range used for Experiment 2 was determined by the results of Experiment 1 and was as follows:
All bacterial strains (absence of S9-mix): 0.5, 1.5, 5, 15, 50, 150, 500, 1500 μg/plate.
All bacterial strains (presence of S9-mix): 1.5, 5, 15, 50, 150, 500, 1500, 5000 μg/plate.
Results from the second experiment showed that the toxicity of the test item yielded results that differed slightly from the Experiment 1 and consequently an insufficient number of non-toxic dose levels were attained for TA100 and TA1535 (absence of S9-mix only). Therefore, these strains were repeated employing an amended test item dose range of 0.015, 0.05, 0.15, 0.5, 1.5, 5, 15, 50 μg/plate.
Eight test item dose levels per bacterial strain were selected in the second mutation test in order to achieve both a minimum of four non-toxic dose levels and the toxic limit of the test item following the change in test methodology from plate incorporation to pre-incubation.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Dimethyl sulphoxide
- Justification for choice of solvent/vehicle: The test item was immiscible in sterile distilled water at 50 mg/mL but was fully miscible in dimethyl sulphoxide at the same concentration in solubility checks performed in-house. Dimethyl sulphoxide was therefore selected as the vehicle.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

N-ethyl-N-nitro-N-nitrosoguanidine

benzo(a)pyrene

other: 2-aminoanthracene

Details on test system and experimental conditions:

Study Controls:
The solvent (vehicle) control used was dimethyl sulphoxide. The negative (untreated) controls were performed to assess the spontaneous revertant colony rate. The solvent and negative controls were performed in triplicate.

The positive control items used demonstrated a direct and indirect acting mutagenic effect depending on the presence or absence of metabolic activation. The positive controls were performed in triplicate.

The sterility controls were performed in triplicate as follows:
Top agar and histidine/biotin or tryptophan in the absence of S9-mix;
Top agar and histidine/biotin or tryptophan in the presence of S9-mix; and
The maximum dosing solution of the test item in the absence of S9-mix only (test in singular only).

Microsomal Enzyme Fraction:
The S9 Microsomal fractions were pre-prepared using standardized in-house procedures (outside the confines of this study). Lot No. PB/βNF S9 10 April 2016 was used in this study.

S9-Mix and Agar:
The S9-mix was prepared before use using sterilized co-factors and maintained on ice for the duration of the test.
S9: 5.0 mL
1.65 M KCl / 0.4 M MgCl2: 1.0 mL
0.1 M Glucose-6-phosphate: 2.5 mL
0.1 M NADP: 2.0 mL
0.2 M Sodium phosphate buffer (pH 7.4): 25.0 mL
Sterile distilled water: 14.5 mL

A 0.5 mL aliquot of S9-mix and 2 mL of molten, trace histidine or tryptophan supplemented, top agar were overlaid onto a sterile Vogel-Bonner Minimal agar plate in order to assess the sterility of the S9-mix. This procedure was repeated, in triplicate, on the day of each experiment.

Media:
Top agar was prepared using 0.6% Bacto agar and 0.5% sodium chloride with 5 mL of 1.0 mM histidine and 1.0 mM biotin or 1.0 mM tryptophan solution added to each 100 mL of top agar. Vogel-Bonner Minimal agar plates were purchased from SGL Ltd.

Test System and Supporting Information:
Bacteria:
The five strains of bacteria used, and their mutations, are as follows:
Salmonella typhimurium
Strains/ Genotype/ Type of mutations indicated
TA1537/ his C 3076; rfa-; uvrB-: / frame shift
TA98/ his D 3052; rfa-; uvrB-;R-factor/ frame shift
TA1535/ his G 46; rfa-; uvrB-: / base-pair substitution
TA100/ his G 46; rfa-; uvrB-;R-factor/ base-pair substitution
Escherichia coli
Strain/ Genotype/ Type of mutations indicated
WP2uvrA/ trp-; uvrA-: / base-pair substitution

The bacteria used in the test were obtained from:
• University of California, Berkeley, on culture discs, on 04 August 1995.
• British Industrial Biological Research Association, on a nutrient agar plate, on 17 August 1987.

All of the strains were stored at approximately -196 °C in a Statebourne liquid nitrogen freezer, model SXR 34.

In this assay, overnight sub-cultures of the appropriate coded stock cultures were prepared in nutrient broth (Oxoid Limited; lot number 1758279 10/20)) and incubated at 37 °C for approximately 10 hours. Each culture was monitored spectrophotometrically for turbidity with titres determined by viable count analysis on nutrient agar plates.

Experimental Design and Study Conduct:
Test Item Preparation and Analysis:
The test item was accurately weighed and approximate half-log dilutions prepared in dimethyl sulphoxide by mixing on a vortex mixer on the day of each experiment. No correction was made for test item purity. Prior to use, the solvent was dried to remove water using molecular sieves i.e. 2 mm sodium alumino-silicate pellets with a nominal pore diameter of 4 x 10^-4 microns.

All formulations were used within four hours of preparation and were assumed to be stable for this period. Analysis for concentration, homogeneity and stability of the test item formulations is not a requirement of the test guidelines and was, therefore, not determined. This is an exception with regard to GLP and has been reflected in the GLP compliance statement.

Test for Mutagenicity: Experiment 1 - Plate Incorporation Method:
Without Metabolic Activation:
0.1 mL of the appropriate concentration of test item, solvent vehicle or appropriate positive control was added to 2 mL of molten, trace amino-acid supplemented media containing 0.1 mL of one of the bacterial strain cultures and 0.5 mL of phosphate buffer. These were then mixed and overlayed onto a Vogel-Bonner agar plate. Negative (untreated) controls were also performed on the same day as the mutation test. Each concentration of the test item, appropriate positive, vehicle and negative controls, and each bacterial strain, was assayed using triplicate plates.

With Metabolic Activation:
The procedure was the same as described previously except that following the addition of the test item formulation and bacterial culture, 0.5 mL of S9-mix was added to the molten, trace amino-acid supplemented media instead of phosphate buffer.

Incubation and Scoring:
All of the plates were incubated at 37 ± 3 °C for approximately 48 hours and scored for the presence of revertant colonies using an automated colony counting system. The plates were viewed microscopically for evidence of thinning (toxicity). Several manual counts were required, predominantly due to interference in the base agar and colonies on the edge of plates slightly distorting the counts.

Test for Mutagenicity: Experiment 2 – Pre-Incubation Method:
As Experiment 1 was deemed negative, Experiment 2 was performed using the pre-incubation method in the presence and absence of metabolic activation.

Without Metabolic Activation:
0.1 mL of the appropriate bacterial strain culture, 0.5 mL of phosphate buffer and 0.1 mL of the test item formulation, solvent vehicle or 0.1 mL of appropriate positive control were incubated at 37 ± 3 °C for 20 minutes (with shaking) prior to addition of 2 mL of molten, trace amino-acid supplemented media and subsequent plating onto Vogel-Bonner plates. Negative (untreated) controls were also performed on the same day as the mutation test employing the plate incorporation method. All testing for this experiment was performed in triplicate.

With Metabolic Activation:
The procedure was the same as described previously except that following the addition of the test item formulation and bacterial strain culture, 0.5 mL of S9-mix was added to the tube instead of phosphate buffer, prior to incubation at 37 ± 3 °C for 20 minutes (with shaking) and addition of molten, trace amino-acid supplemented media. All testing for this experiment was performed in triplicate.

Incubation and Scoring:
All of the plates were incubated at 37 ± 3 °C for approximately 48 hours and scored for the presence of revertant colonies using an automated colony counting system. The plates were viewed microscopically for evidence of thinning (toxicity).

Acceptability Criteria:
The reverse mutation assay may be considered valid if the following criteria are met:
- All bacterial strains must have demonstrated the required characteristics as determined by their respective strain checks according to Ames et al., (1975), Maron and Ames (1983) and Mortelmans and Zeiger (2000).

- All tester strain cultures should exhibit a characteristic number of spontaneous revertants per plate in the vehicle and untreated controls (negative controls). Acceptable ranges are presented as follows:
TA1535: 7 to 40
TA100: 60 to 200
TA1537: 2 to 30
TA98: 8 to 60
WP2uvrA: 10 to 60

- All tester strain cultures should be in the range of 0.9 to 9 x 10^9 bacteria per mL.
- Diagnostic mutagens (positive control chemicals) must be included to demonstrate both the intrinsic sensitivity of the tester strains to mutagen exposure and the integrity of the S9-mix. All of the positive control chemicals used in the study should induce marked increases in the frequency of revertant colonies, which are at least the minimum positive control value over the previous two years, both with or without metabolic activation.

- There should be a minimum of four non-toxic test item dose levels.

- There should be no evidence of excessive contamination.

Evaluation criteria:

There are several criteria for determining a positive result. Any, one, or all of the following can be used to determine the overall result of the study:
1. A dose-related increase in mutant frequency over the dose range tested (De Serres and Shelby, 1979).
2. A reproducible increase at one or more concentrations.
3. Biological relevance against in-house historical control ranges.
4. Statistical analysis of data as determined by UKEMS (Mahon et al., 1989).
5. Fold increase greater than two times the concurrent solvent control for any tester strain (especially if accompanied by an out-of-historical range response (Cariello and Piegorsch, 1996)).

A test item will be considered non-mutagenic (negative) in the test system if the above criteria are not met.

Although most experiments will give clear positive or negative results, in some instances the data generated will prohibit making a definite judgment about test item activity. Results of this type will be reported as equivocal.

Statistics:

Statistical significance was confirmed by using Dunnetts Regression Analysis (* = p < 0.05) for those values that indicate statistically significant increases in the frequency of revertant colonies compared to the concurrent solvent control.

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 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

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:

E. coli WP2 uvr A

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

Additional information on results:

Mutation test:
Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory). The amino acid supplemented top agar and the S9-mix used in both experiments was shown to be sterile. The test item formulation was also shown to be sterile.

Results for the negative controls (spontaneous mutation rates) were considered to be acceptable. These data are for concurrent untreated control plates performed on the same day as the Mutation Test.

The maximum dose level of the test item in the first experiment was selected as the maximum recommended dose level of 5000 μg/plate. In the first mutation test (plate incorporation method), the test item caused a visible reduction in the growth of the bacterial background lawns of all of the tester strains from 500 μg/plate in the absence of S9-mix and 1500 μg/plate in the presence S9-mix. Consequently the toxic limit or the maximum recommended dose level of the test item was employed in the second mutation test, depending on presence or absence of S9-mix. In the second mutation test (pre-incubation method), the test item induced a much stronger toxic response with weakened bacterial background lawns noted in the absence of S9-mix from 1.5 μg/plate (TA100 and TA1535), 150 μg/plate (TA98 and TA1537) and 500 μg/plate (WP2uvrA). In the presence S9-mix weakened bacterial background lawns were noted from 150 μg/plate (TA100), 1500 μg/plate (TA1535, TA98 and TA1537) and 5000 μg/plate (WP2uvrA). The sensitivity of the bacterial tester strains to the toxicity of the test item varied slightly between strain type, exposures with or without S9-mix and experimental methodology. A test item precipitate (greasy in appearance) was noted at and above 1500 μg/plate, this observation did not prevent the scoring of revertant colonies.

There were no toxicologically significant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in Experiment 1 (plate incorporation method). Similarly, no toxicologically significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in Experiment 2 (pre-incubation method). Small, statistically significant increases in WP2uvrA revertant colony frequency were observed in the first mutation test at 500 μg/plate (absence of S9-mix) and at 5000 μg/plate (presence of S9-mix) in the second mutation test. These increases were considered to have no biological relevance because weakened bacterial background lawns were also noted. Therefore the response would be due to additional tryptophan being available to Trp- bacteria allowing these cells to undergo several additional cell divisions and presenting as non-revertant colonies.

The vehicle (dimethyl sulphoxide) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies in excess of the minimum positive control values over the previous two years, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

Remarks on result:

other: No mutagenic potential

Conclusions:

The test substance was considered to be non-mutagenic under the conditions of this test.

Executive summary:

The mutagenic activity of the substance was evaluated in accordance with OECD TG 471 (1997) and according to GLP principles. The test was performed in two experiments. Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA were treated with the test item using both the Ames plate incorporation and pre-incubation methods at up to eight dose levels, in triplicate, both with and without the addition of metabolizing system (S9-mix). The dose range for Experiment 1 (plate incorporation method) was predetermined and was 1.5 to 5000 μg/plate. The dose range was amended for Experiment 2 (pre-incubation method) and ranged between 0.5 and 5000 μg/plate depending on presence or absence of S9-mix. Results from the second experiment showed that the toxicity of the test item yielded results that differed slightly from Experiment 1 and consequently an insufficient number of non-toxic dose levels were attained for TA100 and TA1535 (absence of S9-mix only). Therefore, these strains were repeated employing an amended test item dose range of 0.015 to 50 μg/plate. In the first mutation test, the test item caused a visible reduction in the growth of the bacterial background lawns of all of the tester strains from 500 μg/plate in the absence of S9-mix and 1500 μg/plate in the presence of S9-mix. In the second mutation test, the test item induced a much stronger toxic response with weakened bacterial background lawns noted in the absence of S9-mix from 1.5 μg/plate (TA100 and TA1535), 150 μg/plate (TA98 and TA1537) and 500 μg/plate (WP2uvrA). In the presence S9-mix weakened bacterial background lawns were noted from 150 μg/plate (TA100), 1500 μg/plate (TA1535, TA98 and TA1537) and 5000 μg/plate (WP2uvrA). The sensitivity of the bacterial tester strains to the toxicity of the test item varied slightly between strain type, exposures with or without S9-mix and experimental methodology. A test item precipitate (greasy in appearance) was noted at and above 1500 μg/plate, this observation did not prevent the scoring of revertant colonies. There were no toxicologically significant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation in Experiment 1 and 2. Considered not biologically relevant, small statistically significant increases in WP2uvrA revertant colony frequency were observed in the first mutation test at 500 μg/plate (absence of S9-mix) and at 5000 μg/plate (presence of S9-mix) in the second mutation test. These increases were considered to have no biological relevance because weakened bacterial background lawns were also noted. Therefore the response would be due to additional tryptophan being available to Trp- bacteria allowing these cells to undergo several additional cell divisions and presenting as non-revertant colonies. Adequate negative and positive controls were included. Under the conditions of this test the test substance was considered to be non-mutagenic.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Ames test:

The mutagenic activity of the substance was evaluated in accordance with OECD TG 471 (1997) and according to GLP principles. The test was performed in two experiments. Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA were treated with the test item using both the Ames plate incorporation and pre-incubation methods at up to eight dose levels, in triplicate, both with and without the addition of metabolizing system (S9-mix). The dose range for Experiment 1 (plate incorporation method) was predetermined and was 1.5 to 5000 μg/plate. The dose range was amended for Experiment 2 (pre-incubation method) and ranged between 0.5 and 5000 μg/plate depending on presence or absence of S9-mix. Results from the second experiment showed that the toxicity of the test item yielded results that differed slightly from Experiment 1 and consequently an insufficient number of non-toxic dose levels were attained for TA100 and TA1535 (absence of S9-mix only). Therefore, these strains were repeated employing an amended test item dose range of 0.015 to 50 μg/plate. In the first mutation test, the test item caused a visible reduction in the growth of the bacterial background lawns of all of the tester strains from 500 μg/plate in the absence of S9-mix and 1500 μg/plate in the presence of S9-mix. In the second mutation test, the test item induced a much stronger toxic response with weakened bacterial background lawns noted in the absence of S9-mix from 1.5 μg/plate (TA100 and TA1535), 150 μg/plate (TA98 and TA1537) and 500 μg/plate (WP2uvrA). In the presence S9-mix weakened bacterial background lawns were noted from 150 μg/plate (TA100), 1500 μg/plate (TA1535, TA98 and TA1537) and 5000 μg/plate (WP2uvrA). The sensitivity of the bacterial tester strains to the toxicity of the test item varied slightly between strain type, exposures with or without S9-mix and experimental methodology. A test item precipitate (greasy in appearance) was noted at and above 1500 μg/plate, this observation did not prevent the scoring of revertant colonies. There were no toxicologically significant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation in Experiment 1 and 2. Considered not biologically relevant, small statistically significant increases in WP2uvrA revertant colony frequency were observed in the first mutation test at 500 μg/plate (absence of S9-mix) and at 5000 μg/plate (presence of S9-mix) in the second mutation test. These increases were considered to have no biological relevance because weakened bacterial background lawns were also noted. Therefore the response would be due to additional tryptophan being available to Trp- bacteria allowing these cells to undergo several additional cell divisions and presenting as non-revertant colonies. Adequate negative and positive controls were included. Under the conditions of this test the test substance was considered to be non-mutagenic.

Justification for classification or non-classification

Based on the results of the Ames test, the substance does not have to be classified for mutagenicity in accordance with EU CLP and its amendments (1272/2008).