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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:
11 August 2016 to ****
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018

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
Qualifier:
according to
Guideline:
other: Official notice of MHLW, METI and MOE (31 March 2011) YAKUSHOKUHATSU 0331 No 7 SEIKYOKU No 5 KANPOKIHATSU No 110331009
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Specific details on test material used for the study:
CAS RN: 15337-18-5
Purity: 96.6%
Description: Yellow viscous liquid

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 Rat Liver, induced with phenobarbitone/β-naphthoflavone
Test concentrations with justification for top dose:
Test 1 and Test 2: 5, 15, 50, 150, 500, 1500, 5000 µg.
5000 µg is the standard top dose recommended in the regulatory guidelines that this assay follows. Other concentrations used were a series of ca half-log10 dilutions of the highest concentration.
Vehicle / solvent:
dimethyl sulfoxide (DMSO)
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
sodium azide
benzo(a)pyrene
mitomycin C
other: 2-Nitrofluorene (in the absence of S9 Mix), 2-Aminoanthracene (in the presence of S9 Mix)
Details on test system and experimental conditions:
The strains of S. typhimurium and E. coli were obtained from established commercial sources and were stored at ca -80°C as aliquots of nutrient broth cultures. Each batch of frozen strain was tested for amino acid requirement and, where applicable, for cell membrane permeability (rfa mutation), sensitivity to UV light, and the pKM101 plasmid, which confers resistance to ampicillin. 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 ºC for 10 hours.

Preparation of S9 Fraction
S9 fraction 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
The 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 (Test 1)
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 ºC 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 (≤ 50% reduction), by a sparse or absent background bacterial lawn, or both.

Second Test (Test 2)
As a clear negative response was obtained in the first test, the pre-incubation assay was used for the second test, 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. No statistical analysis was performed.

Results and discussion

Test results
Key result
Species / strain:
other: TA1537, TA98, TA1535, TA100, and E. coli WP2 uvrA (pKM101)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
First Test (Test 1)
No evidence of toxicity was obtained following exposure to the test item at any concentration tested in all strains used. Precipitate was observed on all plates, in the presence and absence of S9 mix (metabolic activation), at 5000 µg/plate. A maximum exposure concentration of 5000 µg/plate was, therefore, selected for use in the second test.
No substantial increases in revertant colony numbers over control counts were obtained with any of the tester strains following exposure to the test item at any concentration up to and including 5000 µg/plate in either the presence or absence of S9 mix.

Second Test (Test 2)
Toxicity (observed as a reduction in revertant colony numbers) was seen following exposure to the test item in strain TA1537 at 50 µg/plate in the presence of S9 mix. This reduction in colony number was not dose dependent, appears to be anomalous when considered with the remaining cultures in each exposure condition and therefore, was considered an effect of experimental variation.
No evidence of toxicity was observed in any other strains at any concentration tested in either the absence or presence of S9 mix.
No substantial increases in revertant colony numbers over control counts were obtained with any of the tester strains following exposure to the test item at any concentration up to and including 5000 µg/plate in either the presence or absence of S9 mix.

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 was a standard plate incorporation assay; the second test included a pre-incubation stage.

 

Concentrations of the test item up to 5000 µg/plate were tested. Other concentrations used were a series of approximately half-log10 dilutions of the highest concentration.

 

In the first test no evidence of toxicity following exposure to the test item was observed at any concentration tested in any strains used. Precipitate was observed on all plates, in the presence and absence of S9 mix, at 5000 µg/plate. 

 

In the second test a pre-incubation method was used. Toxicity (observed as a reduction in revertant colony numbers) was seen following exposure to the test item in strain TA1537 at 50 µg/plate in the presence of S9 mix. This reduction in colony number was not dose dependent, appears to be anomalous when considered with the remaining cultures in each exposure condition, and therefore, was considered an effect of experimental variation.

 

No evidence of mutagenic activity was seen at any concentration of the test item 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 the test item showed no evidence of mutagenic activity in this bacterial system under the test conditions employed.