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Genetic toxicity: in vitro

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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 experimental phase of this study was performed between 17 April 2013 and 20 June 2013.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted to GLP and in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do no effect the quality of the relevant results.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report Date:
2013

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
Qualifier:
according to
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Version / remarks:
Meets the requirements of the Japanese Regulatory Authorities including METI, MHLW and MAFF, OECD Guidelines for Testing of Chemicals No. 471 "and the USA, EPA (TSCA) OPPTS harmonised guidelines.
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Date of Inspection: 10 July 2012. Date of Signature: 30 November 2012
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent

Method

Target gene:
Histidine for Salmonella.
Tryptophan for E.Coli
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell type (if applicable):
Not applicable.
Additional strain / cell type characteristics:
not applicable
Species / strain / cell type:
E. coli WP2 uvr A
Details on mammalian cell type (if applicable):
Not applicable.
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
phenobarbitone/beta­naphthoflavone induced rat liver, S9
Test concentrations with justification for top dose:
Experiment 1: 0, 1.5, 5, 15, 50, 150, 500, 1500, 5000 µg/plate.
Experiment 2: 0, 1.5, 5, 15, 50, 150, 500, 1500, 5000 µg/plate.
Vehicle / solvent:
Vehicle: dimethyl sulfoxide (DMSO)

Justification for choice of solvent/vehicle: The test item was immiscible in sterile distilled water but was fully miscible in DMSO at 50 mg/ml in solubility checks performed in-house. DMSO was therefore selected as the vehicle.

Controlsopen allclose all
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rates of TA100
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-Aminoanthracene: 1 µg/plate
Remarks:
With S9 mix
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rates of TA1535
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-Aminoanthracene: 2 µg/plate
Remarks:
With S9 mix
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rates of TA1537
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-Aminoanthracene: 2 µg/plate
Remarks:
With S9 mix
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rates of WP2uvrA
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-Aminoanthracene: 10 µg/plate
Remarks:
With S9 mix
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rates of TA98
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
With S9 mix Migrated to IUCLID6: Benzo(a)pyrene: 5 µg/plate
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rates of TA98
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
without S9 mix Migrated to IUCLID6: 4-Nitroquinoline-1-oxide: 0.2 µg/plate
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rates of TA1537
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
without S9 mix Migrated to IUCLID6: 9-Aminoacridine: 80 µg/plate
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rates of TA100
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
N-ethyl-N-nitro-N-nitrosoguanidine
Remarks:
without S9 mix Migrated to IUCLID6: N-ethyl-N'-nitro-N-nitrosoguanidine: 3 µg/plate
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rates of TA1535
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
N-ethyl-N-nitro-N-nitrosoguanidine
Remarks:
Without S9 mix Migrated to IUCLID6: N-ethyl-N'-nitro-N-nitrosoguanidine: 5 µg/plate
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rates of WP2uvrA
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
N-ethyl-N-nitro-N-nitrosoguanidine
Remarks:
Without S9 mix Migrated to IUCLID6: N-ethyl-N'-nitro-N-nitrosoguanidine: 2 µg/plate
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation) - Experiment 1

DURATION
- Preincubation period for bacterial strains: 10h
- Exposure duration: 48 - 72 hrs
- Expression time (cells in growth medium): Not applicable
- Selection time (if incubation with a selection agent): Not applicable

NUMBER OF REPLICATIONS: Triplicate plating.

DETERMINATION OF CYTOTOXICITY
- Method: plates were assessed for numbers of revertant colonies and examined for effects on the growth of the bacterial background lawn.

METHODS OF APPLICATION: in agar (pre-incubation) – Experiment 2

- Pre-incubation period for bacterial strains: 10hrs
- Exposure duration: 48-72hrs
- Expression time (cells in growth medium): Not applicable
- Selection time (in incubation with a selective agent): 20 minutes at 37 degrees C

NUMBER OF REPLICATIONS: Triplicate plating.

DETERMINATION OF CYTOTOXICITY
-Method: plates were assessed for numbers of revertant colonies and examined for effects on the growth of the bacterial background lawn.

Evaluation criteria:
Acceptance 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. Combined historical negative and solvent control ranges for 2009 and 2010 are presented in Appendix 3. All
tester strain cultures should be in the range of 0.9e9 to 9e9 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, both with or without metabolic activation. The historical ranges of the
positive controls for 2009 and 2010 are presented in Appendix 3. 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).
A test item will be considered non-mutagenic (negative) in the test system if the above criteria are not met.
Statistics:
Standard Deviation
Dunnetts Linear Regression Analysis

Results and discussion

Test resultsopen allclose all
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Remarks:
Tested up to maximum recommended dose of 5000 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
The test item caused a cytotoxic response to all of the Salmonella tester strains (except TA98 +S9-mix), initially from 50 µg/plate.
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECFIC CONFOUNDING FACTORS:
- Precipitation: No precipitation was seen up to 5000 µg/plate.
- Purity: No correction required.
- Cytotoxicity: The test item caused a visible reduction in the growth of the bacterial background lawns and/or a substantial reduction in the
frequency of revertant colonies of all of the Salmonella tester strains (except TA98 dosed in the presence of S9-mix), initially from 50 µg/plate. No toxicity was noted to Escherichia coli strain WP2uvrA at any test item dose level in either the absence or presence of S9-mix. 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. 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.

STERILITY, VEHICLE AND POSITIVE CONTROL DATA:
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 all experiments was shown to be sterile. The culture density for each bacterial strain was also checked and considered acceptable. These data are not given in the report.
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 individual plate counts, the mean number of revertant colonies and the standard deviations, for the test item, positive 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.
A history profile of vehicle/untreated and positive control values (reference items) for 2011 and 2012 are presented in Appendix 2.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

RESULTS

Whilst the test item did not induce a reduction in the bacterial background lawns, substantial decreases in TA100 revertant colony frequency were noted from 500 µg/plate (absence of S9-mix) and at 5000 µg/plate (presence of S9-mix). No toxicity was noted to WP2uvrA. The test item formulation and S9‑mix used in this experiment were both shown to be sterile.

 

Mutation Test

Table 1: Spontaneous Mutation Rates (Concurrent Negative Control)

Experiment 1

 

Number of revertants (mean number of colonies per plate)

Base-pair substitution type

Frameshift type

TA100

TA1535

WP2uvrA

TA98

TA1537

92

 

15

 

27

 

41

 

13

 

104

(98)

20

(15)

23

(23)

23

(30)

12

(13)

98

 

11

 

20

 

27

 

15

 

 

 

Experiment 2

Number of revertants (mean number of colonies per plate)

Base-pair substitution type

Frameshift type

TA100

TA1535

WP2uvrA

TA98

TA1537

69

 

19

 

35

 

24

 

8

 

112

(96)

24

(21)

23

(27)

25

(23)

7

(7)

106

 

19

 

23

 

21

 

7

 

 

FOR TABLES OF RESULTS FOR MUTATION TEST: Please see attached in overall remarks, attachments

 

References:

Ames B N, McCann J and Yamasaki E (1975), Methods for detecting carcinogens and mutagens with the Salmonella/mammalian microsome mutagenicity test, Mutation Research, 31, 347-364.

Maron D M and Ames B N (1983), Revised Methods for the Salmonella mutagenicity test, Mutation Research, 113, 173 - 215.

Mortelmans K and Zeiger E (2000), The Ames Salmonella/microsome mutagenicity assay, Mutation Research, 455, 29-60.

Green M H L and Muriel W J (1976), Mutagen Testing Using TRP+ Reversion in Escherichia coli, Mutation Research, 38, 3- 32.

De Serres F J and Shelby M D (1979), Recommendations on data production and analysis using theSalmonella/microsome mutagenicity assay,Environmental Mutagenesis, 1, 87-92.

Mahon G A T et al (1989) Analysis of data from microbial colony assays. In: Statistical Evaluation of Mutagenicity Test Data, UKEMS sub-committee on guidelines for mutagenicity testing (Kirkland D J Ed.), Cambridge University Press Report, 26-65.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

The test item, 2-ethyl-hexyl nitrate, was considered to be non-mutagenic under the conditions of this test.
Executive summary:

Introduction. The test method was designed to be compatible with the guidelines for bacterial mutagenicity testing published by the major Japanese Regulatory Authorities including METI, MHLW and MAFF, the OECD Guidelines for Testing of Chemicals No. 471 "Bacterial Reverse Mutation Test", Method B13/14 of Commission Regulation (EC) number 440/2008 of 30 May 2008 and the USA, EPA (TSCA) OPPTS harmonised guidelines.

Methods. Salmonella typhimurium strains TA1535, TA1537, TA98, TA100 and Escherichia coli strain WP2uvrA were treated with the test item, 2-ethyl-hexyl nitrate, using both the Ames plate incorporation and pre-incubation methods at eight dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolising system (10% liver S9 in standard co-factors). The dose range for the first experiment was 1.5 to 5000 µg/plate. The experiment was repeated on a separate day (pre-incubation method) using the same dose range, fresh cultures of the bacterial strains and fresh test item formulations.

Additional dose levels and an expanded dose range were selected in both experiments in order to achieve four non-toxic dose levels and the toxic limit of the test item. 

Results.The vehicle (DMSO) control plates gave counts of revertant colonies within the normal range. All of the positive controls used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

The test item caused a visible reduction in the growth of the bacterial background lawns and/or a substantial reduction in the frequency of revertant colonies of all of the Salmonella tester strains (except TA98 dosed in the presence of S9-mix), initially from 50 µg/plate. No toxicity was noted to Escherichia coli strain WP2uvrAat any test item dose level in either the absence or presence of S9-mix. 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. 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 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. 

Conclusion.The test item,2 -ethyl-hexyl nitrate, was considered to be non-mutagenic under the conditions of this test.