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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
14 Jun - 14 Jul 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2017

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:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
no
Qualifier:
according to
Guideline:
other: Japanese Ministry of Economy, Trade and Industry, Japanese Ministry of Health, Labour and Welfare and Japanese Ministry of Agriculture, Forestry and Fisheries
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Specific details on test material used for the study:
Identification: Lumière Pink S.M. 8135N
Physical state/Appearance: Red powder
Batch: 021340
Purity: Preparation containing ≥90% UVCB (treated as 100%)
Expiry Date: 28 April 2022
Storage Conditions: Room temperature in the dark

The vehicle control used was as follows:
Identity: Dimethyl sulphoxide
Batch number (purity): 1690734 (>99%), Expiry: 03/2022

Direct acting compounds in the absence of S9-mix:
Identity: N-ethyl-N'-nitro-N-nitrosoguanidine (ENNG)
Batch number: 67F-3700
Expiry date: 18 September 2017
Solvent: DMSO
Concentration: 2 μg/plate for WP2uvrA
3 μg/plate for TA100
5 μg/plate for TA1535

Identity: 9-Aminoacridine (9AA)
Batch number: S32398-438
Purity: 99.9%
Expiry date: 01 October 2017
Solvent: DMSO
Concentration: 80 μg/plate for TA1537

Identity: 4-Nitroquinoline-1-oxide (4NQO)
Batch number: 030M1206
Purity: 100%
Expiry date: 08 October 2017
Solvent: DMSO
Concentration: 0.2 μg/plate for TA98

Direct acting compounds in the presence of S9-mix:
Identity: 2-Aminoanthracene (2AA)
Batch number: STBB1901M9
Purity: 97.5%
Expiry date: 08 October 2017
Solvent: DMSO
Concentration: 1 μg/plate for TA100
2 μg/plate for TA1535 and TA1537
10 μg/plate for WP2uvrA

Identity: Benzo(a)pyrene (BP)
Batch number: 090M1400V
Purity: 96%
Expiry date: 12 October 2017
Solvent: DMSO
Concentration: 5 μg/plate for TA98

Method

Target gene:
histidine or tryptophan synthesis
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 fraction
Test concentrations with justification for top dose:
0, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/plate

The test item was insoluble in sterile distilled water at 50 mg/mL but was fully soluble in dimethyl sulphoxide at the same concentration in solubility checks performed in-house. Dimethyl sulphoxide was therefore selected as the vehicle.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO;
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
no
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
N-ethyl-N-nitro-N-nitrosoguanidine
other: 2-Aminoanthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium; in agar (plate incorporation)

DURATION
- Preincubation period: 20 minutes (Experiment 2)
- Exposure duration: 48 h (Experiment 1 & 2)

NUMBER OF REPLICATIONS: 3
Evaluation criteria:
The test material may be considered positive in this test system if it induces a reproducible, dose-related
and statistically significant increase in the revertant count in at least one strain of bacteria.
Statistics:
Dunnetts Regression Analysis (* = p < 0.05)

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not determined
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:
not determined
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not determined
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:
not determined
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:
not determined
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
A pink test item induced colouration was noted from 50 μg/plate becoming red at and above 1500 μg/plate. A test item precipitate (powdery in appearance) was also observed from 1500 μg/plate. These observations did not prevent the scoring of revertant colonies.

Any other information on results incl. tables

Spontaneous Mutation Rates (Concurrent Negative Controls)

Experiment 1 (Plate Incorporation)

Number of revertants (mean number of colonies per plate)

Base-pair substitution type

Frameshift type

TA100

TA1535

WP2uvrA

TA98

TA1537

130

27

8

31

13

116 (127)

9 (18)

10 (9)

20 (26)

13 (14)

134

18

8

28

15

 

Experiment 2 (Pre-Incubation)

Number of revertants (mean number of colonies per plate)

Base-pair substitution type

Frameshift type

TA100

TA1535

WP2uvrA

TA98

TA1537

76

18

35

23

14

96 (85)

19 (15)

41 (31)

13 (21)

8 (10)

82

9

31

26

9

 

Applicant's summary and conclusion

Conclusions:
The test item was considered to be non-mutagenic under the conditions of this test.