Registration Dossier

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
16 August 2016 - 12 September 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report Date:
2016

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1997
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
2008
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
no
Qualifier:
according to
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Test material form:
other: liquid

Method

Target gene:
- S. typhimurium: Histidine gene
- Escherichia coli: Tryptophan gene
Species / strain
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Metabolic activation system:
Rat liver S9-mix
Test concentrations with justification for top dose:
1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/plate for both experiments (pre-determined dose range)
Vehicle:
- Vehicle(s)/solvent(s) used: Dimethyl sulfoxide (DMSO )
- 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.
Controls
Negative controls:
no
Solvent controls:
yes
Remarks:
DMSO
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 (TA100, TA1535, WP2uvrA, TA1537; with metabolic activation)
Details on test system and conditions:
METHOD OF APPLICATION
- Experiment 1: in agar (plate incorporation)
- Experiment 2: (independent repeat) preincubation

DURATION
- Preincubation period: 20 minutes
- Exposure duration: 48 hours

NUMBER OF REPLICATIONS
- Doses of the test substance were tested in triplicate in each strain (in all experiments)

DETERMINATION OF CYTOTOXICITY
- Method: on the basis of a decline in the number of spontaneous revertants, a thinning of the background lawn or a microcolony formation

ACCEPTABILITY OF THE ASSAY
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. The historical ranges of the positive control reference items for 2014 and 2015 are presented in Appendix 1.
- There should be a minimum of four non-toxic test item dose levels.
- There should be no evidence of excessive contamination.
Evaluation criteria:
A test item will be considered non-mutagenic (negative) in the test system if the following criteria are not met. 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)).
Statistics:
Dunnetts Regression Analysis (p < 0.05) was performed.

Results and discussion

Test results
Key result
Species / strain:
other: S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
yes
Vehicle controls valid:
yes
Negative controls valid:
not applicable
Positive controls valid:
yes
Remarks on result:
other: No significant increases in the frequency of revertant colonies for any of the bacterial strains, with any dose of the test item, with or without metabolic activation (S9-mix) in Experiment 1 & 2.
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No precipitation was observed at the start or at the end of the incubation period.

RANGE-FINDING/SCREENING STUDIES: No results reported.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- 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 at 5000 μg/plate in both the absence and presence of S9-mix.
- In the second mutation test (pre-incubation method), the test item induced a stronger toxic response, with weakened bacterial background lawns noted in the absence of S9-mix from 500 μg/plate (TA100, TA1537 and TA1535) and 1500 μg/plate (TA98 and WP2uvrA). In the presence S9-mix weakened bacterial background lawns were noted from 1500 μg/plate (TA100, TA1535, TA98 and TA1537) and 5000 μg/plate (WP2uvrA).

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%):
-Positive historical control data (values 2015):
Strain TA100 TA1535 TA102 WP2uvrA TA98 TA1537 WP2uvrA pKM101 WP2pKM101
S9-Mix -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9
Values 276 280 252 264 13 13 231 227 262 276 253 261 20 35 20 35
Min 222 250 79 118 953 673 116 103 100 78 164 97 430 494 745 325
Max 2266 2402 2779 457 3140 1655 2769 550 502 705 2318 823 1696 2264 3662 1174
Mean 614 927 472 246 2303 1093 792 266 222 218 911 336 761 1461 2257 569
SD 260.6 452.5 434.8 55.7 815.2 376.5 342.1 97.7 70.2 107.6 412.4 135.7 350.0 382.0 790.7 220.3

-Negative (solvent/vehicle) historical control data (values 2015):
Strain TA100 TA1535 TA102 WP2uvrA TA98 TA1537 WP2uvrA pKM101 WP2pKM101
S9-Mix -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9
Values 274 278 504 285 26 13 461 229 526 299 506 282 42 51 39 49
Min 60 61 7 7 222 278 10 12 11 10 4 6 87 98 89 93
Max 166 175 31 29 376 388 58 43 45 46 27 27 237 254 174 177
Mean 91 95 16 14 286 333 24 27 21 24 12 13 156 164 123 137
SD 19.3 19.1 4.5 4.0 48.7 37.6 5.6 5.9 6.2 6.1 3.8 3.4 42.2 35.6 23.1 21.2

SD = Standard deviation
n = Number of observations

Applicant's summary and conclusion

Conclusions:
Under the conditions of this study, the test substance was determined to be not mutagenic.
Executive summary:

The mutagenic activity of Muguesia was evaluated in accordance with OECD TG 471 and according to GLP principles. The test was performed in two independent experiments: at first a direct plate assay was performed and secondly a pre-incubation assay, both in the absence and presence of S9-mix up to and including 5000 μg/plate. The dose levels were selected based on a dose range finding test with all tester strains. Cytotoxicity, as evidenced by a decrease in the number of revertants, or reduced background lawn was observed in the second mutation test (pre-incubation method), with weakened bacterial background lawns noted in the absence of S9-mix from 500 μg/plate (TA100, TA1537 and TA1535) and 1500 μg/plate (TA98 and WP2uvrA). In the presence of S9-mix, weakened bacterial background lawns were noted from 1500 μg/plate (TA100, TA1535, TA98 and TA1537) and 5000 μg/plate (WP2uvrA). The negative and positive controls were found to be adequate. There were no significant (dose-related) 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) or Experiment 2 (pre-incubation method). Based on these results, it is concluded that Muguesia is not mutagenic under the conditions of this test.