Registration Dossier

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
20 July 2016 to 24 October 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report Date:
2016

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 Agriculture, Forestry and Fisheries. Test Data for Registration of Agricultural Chemicals, 12 Nohsan No. 8147, Guideline 2-1-19-1, Agricultural Production Bureau, November 24, 2000.
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Test material form:
liquid: viscous
Specific details on test material used for the study:
- Purity: >99%
- Description: Brown paste

Method

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 pKM 101
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
Test 1 (in the absence and presence of metabolic activation): 5, 15, 50, 150, 500, 1500, 5000 µg/plate

Additional Test 1 (TA98; in the absence of metabolic activation): 0.5, 1.5, 5, 15, 50, 150, 500, 1500, 5000 µg/plate

Test 2; TA98 (in the absence and presence of metabolic activation): 0.5, 1.5, 5, 15, 50, 150, 500, 1500, 5000 µg/plate

Test 2; TA100, TA1535, TA1537 and WP2 uvrA (pKM101) (in the absence and presence of metabolic activation): 5, 15, 50, 150, 500, 1500, 5000 µg/plate

The highest concentration tested in this study was 50 mg/mL in the chosen vehicle, which provided a final concentration of 5000 μg/plate.

This is the standard limit concentration recommended in the regulatory guidelines that this assay follows. The highest concentration in each test was diluted with DMSO to produce a series of lower concentrations, separated by approximately half-log10 intervals.
Vehicle / solvent:
Dimethyl sulfoxide (DMSO)
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
2-acetylaminofluorene
4-nitroquinoline-N-oxide
9-aminoacridine
2-nitrofluorene
sodium azide
benzo(a)pyrene
Details on test system and experimental conditions:
Strains used to detect base changes and frameshift mutations:
Base change mutagens: S. typhimurium TA1535 and TA100, and E. coli WP2 uvrA (pKM101).
Frameshift mutagens: S. typhimurium TA1537 and TA98.

The strains of S. typhimurium and E. coli are obtained from an established commercial source and were stored at ca -80°C as aliquots of nutrient broth cultures. Dimethyl sulfoxide (DMSO) was added to the cultures at 8% v/v as a cryopreservative. Each batch of frozen strain was tested for amino acid requirement and, where applicable, for cell membrane permeability (rfa mutation), deficiency in DNA excision repair system (uvrA/uvrB mutation), and the pKM101 plasmid that confers resistance to antibiotics. The responses of the strains to a series of reference mutagens were also assessed.

For use in tests, an aliquot of frozen culture was added to 25 mL of nutrient broth and incubated, with shaking, at 37°C for 10 hours.

Preparation of S9 Fraction
S9 fraction was purchased from a commercial source and was prepared from male Sprague-Dawley derived rats, dosed with phenobarbital/5,6-benzoflavone to stimulate mixed-function oxidases in the liver and stored at approximately -80°C.

Preparation of S9 Mix
S9 mix contained: S9 fraction (10% v/v), MgCl2 (8 mM), KCl (33 mM), sodium phosphate buffer pH 7.4 (100 mM), glucose-6-phosphate (5 mM) and NADP (4 mM) in water.

First Test
Aliquots of the test item solutions, positive control or vehicle control were placed in glass tubes. S9 mix (0.5 mL) or 0.1 M pH 7.4 sodium phosphate buffer (0.5 mL) was added, followed by 0.1 mL of a 10-hour bacterial culture and 2 mL of agar containing histidine (0.05 mM), biotin (0.05 mM) and tryptophan (0.05 mM). The mixture was thoroughly shaken and overlaid onto previously prepared Petri dishes containing 25 mL minimal agar. Each Petri dish was individually labelled with a unique code, identifying the contents of the dish. Three Petri dishes were used for each treatment. Plates were also prepared without the addition of bacteria in order to assess the sterility of the test item, S9 mix and sodium phosphate buffer. All plates were incubated at approximately 37°C for between 48 and 72 hours. After this period, the appearance of the background bacterial lawn was examined and revertant colonies counted using an automated colony counter (Perceptive Instruments Sorcerer). Some plates were scored manually because of the presence of precipitate. Any toxic effects of the test item may be detected by a substantial reduction in mean revertant colony counts, by a sparse or absent background bacterial lawn, or both.

Additional First Test
The results of the first test in strain TA98 in the absence of S9 mix did not fulfill the test validity requirements due to the toxicity seen. An additional test was performed that repeated the exposure conditions of the first test for TA98 in the absence of S9 mix, but included two additional low doses (0.5 and 1.5 μg/plate).

Second Test
As a clear negative response was obtained in the first test(s), a variation to the test procedure was used for the second test. The variation used was the pre-incubation assay in which the tubes containing mixtures of bacteria, buffer or S9 mix and test dilution, were incubated at 37°C for 30 minutes with shaking before the addition of the agar overlay (2 mL). The maximum concentration chosen was again 5000 μg/plate.
Evaluation criteria:
If exposure to a test item produces a reproducible increase in mean revertant colony numbers of at least twice (three times in the case of strains TA1535 and TA1537) that of the concurrent vehicle controls, with some evidence of a positive concentration-response relationship, it is considered to exhibit mutagenic activity in this test system.

If exposure to a test item does not produce a reproducible increase in mean revertant colony numbers, it is considered to show no evidence of mutagenic activity in this test system.
Statistics:
Not performed.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no 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:
no 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:
no 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:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A pKM 101
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid

Applicant's summary and conclusion

Conclusions:
It was concluded that the test item showed no evidence of mutagenic activity in this bacterial system under the test conditions employed.
Executive summary:

Histidine-dependent auxotrophic mutants of Salmonella typhimurium, strains TA1535, TA1537, TA98 and TA100, and a tryptophan-dependent mutant of Escherichia coli, strain WP2 uvrA (pKM101), were exposed to the test item diluted in dimethyl sulfoxide (DMSO). DMSO was also used as a vehicle control.

 

Two independent mutation tests were performed in the presence and absence of liver preparations (S9 mix) from rats treated with phenobarbital and 5,6 -benzoflavone. The first test and an additional first test (in strain TA98 without S9 mix) were standard plate incorporation assays; the second included a pre-incubation stage.

 

Concentrations of test material up to 5000 μg/plate were tested. This is the standard limit concentration recommended in the regulatory guidelines that this assay follows. Other concentrations used were a series of ca half-log10 dilutions of the highest concentration. In the first test, toxicity (observed as a reduction in revertant colony numbers) was seen in strains TA98 at 150 μg/plate and above, and TA1535 at 500 μg/plate in the absence of S9 mix, and in strain TA100 and WP2 uvrA (pKM101) at 5000 μg/plate in the presence of S9 mix.

 

The first test in strain TA98 in the absence of S9 mix did not provide a minimum of four nontoxic concentrations. Therefore an additional test was performed in TA98 in the absence of S9 mix that included two additional low doses (0.5 and 1.5 μg/plate). No signs of toxicity towards the tester strain were observed in the additional mutation test following exposure.

 

In the second test, toxicity (observed as a reduction in revertant colony numbers) was seen in strains TA100 at 500 and 1500 μg/plate and TA1537 at 150 and 1500 μg/plate and above in the absence of S9 mix, and in strain TA100 at 5000 μg/plate in the presence of S9 mix.

 

Precipitate was observed on all plates containing test material at 1500 μg/plate and above in both tests.

 

No evidence of mutagenic activity was seen at any concentration of test material in either mutation test.

 

The concurrent positive controls verified the sensitivity of the assay and the metabolizing activity of the liver preparations. The mean revertant colony counts for the vehicle controls were within or close to the current historical control range for the laboratory.

 

It was concluded that test material showed no evidence of mutagenic activity in this bacterial system under the test conditions employed.