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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
The experiment phase of this study was performed between 14 March 2012 and 27 April 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2012

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
GLP compliance:
yes (incl. certificate)
Remarks:
No analysis was carried out to determine homogeneity, concentration or stability of the test item formulation. This exception is considered not to affect the purpose or integrity of the study.
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): (z)-hex-3-enyl acetate (LEAC)
- Physical state: clear colourless liquid
- Analytical purity: 98.45 %
- Lot/batch No.: 1Z00151
- Expiration date of the lot/batch: 13 June 2015
- Storage condition of test material: room temperature in the dark under nitrogen

Method

Target gene:
Salmonella typhimurium: histidine
Escherichia coli: tryptophan operon
Species / strainopen allclose all
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:
S9-mix
Test concentrations with justification for top dose:
Preliminary toxicity test: 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate
Experiment 1: 50, 150, 500, 1500 and 5000 µg/plate
Experiment 2, Salmonella strains (without S9): 5, 15, 50, 500, 1500 and 5000 µg/plate
Experiment 2, Salmonella strains (with S9) and E.coli strain WP2uvrA (with and without S9): 50, 150, 500, 1500, 5000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: the test item was fully miscible in dimethyl sulphoxide at 50 mg/mL in solubility checks
Controlsopen allclose all
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rates
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
N-ethyl-N-nitro-N-nitrosoguanidine
Remarks:
2 µg/plate for WPuvrA, 3µg/plate for TA 100, 5 µg/plate for TA1535
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rates
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
80 µg/plate for TA1537
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rates
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
0.2 µg/plate for TA98
Details on test system and experimental conditions:
METHOD OF APPLICATION
Experiment 1: in agar (plate incorporation)
Experiment 2: preincubation

NUMBER OF REPLICATIONS: 3

PRELIMINARY TOXICITY TEST
In order to select appropriate dose levels for use in the main test, a preliminary test was carried out to determine the toxicity of the test item. the test was performed by mixing 0.1 mL of bacterial culture (TA100 or WP2uvrA), 2 mL of molten, trace histidine or tryptophan supplemented, top agar, 0.1 mL of test item formulation and 0.5 mL of S9-mix or phosphate buffer and overlaying onto sterile plates of Vogel-Bonner Minimal agar (30 mL/plate). Ten concentrations of the test item formulation and a vehicle control (DMSO) were tested. In addition, 0.1 mL of the maximum concentration of the test item and 2 mL of molten, trace histidine or tryptophan supplemented, top agar were overlaid onto a sterile nutrient agar plate in order to assess the sterility of the test item. After approximately 48 h incubation at 37 °C the plates were assessed for numbers of revertant colonies using a Domino colony counter and examined for effects on the growth of the bacterial background lawn.

MUTATION TEST-EXPERIMENT 1
Measured aliquots (0.1 mL) of 1 of the bacterial cultures were dispensed into sets of test tubes followed by 2 mL of molten, trace histidine or trypophan supplemented, top agar, 0.1 mL of the test item formulation, vehicle or positive control and either 0.5 mL of S9-mix or phosphate buffer. The contents of each test tube were mixed and equally distributed onto the surface of Vogel-Bonner Minimal agar plates (1 tube per plate). This procedure was repeated for each bacterial strain and for each concentration of the test item both with and without S9-mix.

All plates were incubated at 37 °C for approximately 48 h and the frequency of revertant colonies assessed using a Domino colony counter.

MUTATION TEST- EXPERIMENT 2
The second experiment was performed using fresh bacterial cultures, test item and control solutions. In the initial second experiment, the toxicity of the test item yielded results that differed from Experiment 1 (due to the change in test methodology) and consequently, there were an insufficient number of non-toxic dose levels for the Salmonella strains dosed in the absence of S9-mix. Therefore, a repeat experiment was performed employing additional dose levels and an expanded dose range for each strain in order to achieve 4 non-toxic doses and the toxic limit.

As it is good scientific practice to alter 1 condition in the replicate assay, the exposure condition was changed from plate incorporation to pre-incubation. The test formulations and vehicle control were therefore dosed as follows:

Measured aliquots (0.1 mL) of 1 of the bacterial cultures were dispensed into sets of test tubes followed by 03.5 mL of S9-mix or phosphate buffer and 0.1 mL of the vehicle or test item formulation and incubated for 20 min at 37 °C with shaking at approximately 130 rpm prior to the addition of 2 mL of molten, trace histidine or tryptophan supplemented, top agar. The contents of the tube were then mixed and equally distributed on the surface of Vogel-Bonner Minimal agar plates (1 tube per plate). This procedure was repeated for each bacterial strain and for each concentration of test item both with and without S9-mix. The positive and untreated controls were dosed using the standard plate incorporation method described in Experiment 1.
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:
- A dose-related increase in mutant frequency over the dose range tested
- A reproducible increase against in-house historical control ranges
- Statistical analysis of data as determined by UKEMS
- Fold increase greater than 2 times the concurrent solvent control for any tester strain (especially if accompanied by an out-of-historical range response)

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

Results and discussion

Additional information on results:
Preliminary toxicity test
The test item was non-toxic to the strains of bacteria used (TA100 and WP2uvrA). The test item formulation and S9-mix used in this experiment were both shown to be sterile.

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 S9-mix used in both experiments was shown to be sterile. The culture density for each bacterial strain was also checked and considered acceptable.

Results for the negative controls were considered to be acceptable.
The individual plate counts, the mean number of revertant colonies and the standard deviations, for the test item, reference item and vehicle controls, both with and without metabolic activation are presented in Table 2 and Table 3 for Experiment 1 and Table 4 and Table 5 for Experiment 2, as attached.

In the first experiment (plate incorporation methodology), the test item caused no visible reduction in the growth of the bacterial background lawns at any dose level either in the absence or presence of S9-mix. In the second experiment (pre-incubation methodolody), the test item induced toxicity as weakened bacterial background lawns to all the Salmonella strains dosed in the absence of S9-mix from 1500 µg/plate and at 5000 µg/plate to the Salmonella strains dosed in the presence of S9-mix and Escherichia coli strain WP2uvrA dosed in both the absence and presence of S9-mix. These results were not indicative of toxicity sufficiently severe enough to prevent the test item being tested up to the maximum recommended dose level of 5000 µg/plate. No test item precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix.

No 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 or exposure method.

All the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies thus confirming the activity of the S9-mix and the sensitivity of the bacterial strains.

Any other information on results incl. tables

Preliminary toxicity test, the numbers of revertant colonies for the toxicity assay were:

With (+) or without (-) S9-mix

Strain

Dose (µg/plate)

0

0.15

0.5

1.5

5

15

50

150

500

1500

5000

-

TA100

136

112

120

111

119

129

124

134

124

113

101

+

TA100

110

119

110

106

110

89

116

115

115

105

119

-

WP2uvrA

24

27

18

31

15

22

20

17

22

22

17

+

WP2uvrA

17

19

31

15

22

28

16

25

26

24

26

 

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

The mutagenic potential of the test item was assessed according to the OECD guideline 471. The test item was determined to be non-mutagenic under the conditions of this test.