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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:
From February 28 to March 13, 2020
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2020

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

1
Chemical structure
Reference substance name:
(S)-3-(benzoylthio)-2-methylpropionic acid
EC Number:
276-764-6
EC Name:
(S)-3-(benzoylthio)-2-methylpropionic acid
Cas Number:
72679-02-8
Molecular formula:
C11H12O3S
IUPAC Name:
(S)-3-(benzoylthio)-2-methylpropionic acid

Method

Target gene:
Histidine
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:
liver S9 fraction from rats pre-treated with phenobarbital and 5,6-benzoflavone
Test concentrations with justification for top dose:
On the basis of the results obtained in the preliminary toxicity test, inMain Assay I, using the plate incorporation method, the test item was assayed at 5000, 2500, 1250, 625 and 313 µg/plate with all tester strains. The additional dose level of 156 µg/plate was included for TA1537 in the presence of S9 metabolism..
As no relevant increase in revertant numbers was observed at any concentration tested, a
Main Assay II was performed.
Vehicle / solvent:
The test item was used as a solution in dimethylsulfoxide (DMSO).
Details on test system and experimental conditions:
A preliminary toxicity test was undertaken in order to select the concentrations of the test item to be used in theMain Assays. In this test a wide range of dose levels of the test item, set at half-log intervals, were used. Treatments were performed both in the absence and presence of S9 metabolism using the plate incorporation method; a single plate was used at each test point and positive controls were not included. Toxicity was assessed on the
basis of a decline in the number of spontaneous revertants, a thinning of the background lawn or a microcolony formation.

TwoMain Assays were performed including negative and positive controls in the absence and presence of an S9 metabolising system. Three replicate plates were used at each test point.
In addition, plates were prepared to check the sterility of the test item solutions and the S9 mix and dilutions of the bacterial cultures were plated on nutrient agar plates to establish the number of bacteria in the cultures.
The Main Assay I was performed using a plate-incorporation method. The components of the assay (the tester strain bacteria, the test item and S9 mix or phosphate buffer) were added to molten overlay agar and vortexed. The mixture was then poured onto the surface of a minimal medium agar plate and allowed to solidify prior to incubation.

The overlay mixture was composed as follows:
Overlay agar (held at 45°C) 2.0mL
Test or control item solution 0.1mL
S9 mix or phosphate buffer (pH 7.4, 0.1 M) 0.5mL
Bacterial suspension 0.1mL

The Main Assay II was performed using a pre-incubation method. The components were added in turn to an empty test-tube:
Bacterial suspension 0.1mL
Test item solution 0.05mL
or control item solution 0.05mL
S9 mix or phosphate buffer (pH 7.4, 0.1 M) 0.5mL
The incubate was vortexed and placed at 37°C for 30 minutes. Two mL of overlay agar was then added and the mixture vortexed again and poured onto the surface of a minimal medium agar plate and allowed to solidify.

Incubation and scoring
The prepared plates were inverted and incubated for approximately 72 hours at 37°C. After this period of incubation, plates were immediately scored by counting the number of revertant colonies on each plate of the Main Assays.
Evaluation criteria:
For the test item to be considered mutagenic, two-fold (or more) increases in mean revertant
numbers must be observed at two consecutive dose levels or at the highest practicable dose
level only. In addition, there must be evidence of a dose-response relationship showing
increasing numbers of mutant colonies with increasing dose levels.

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium, other: all strain tested
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Additional information on results:
No precipitation of the test item was observed at the end of the incubation period at any concentration tested, in the absence or presence of S9 metabolic activation.
In the absence of S9 metabolism a slight toxicity as indicated by thinning of background lawn and/or reduction of revertant numbers was seen at the highest dose level tested for all tester strains with the exception of WP2 uvrA. In the presence of S9 metabolic activation toxicity was seen at the highest dose level tested for TA1537 and TA98 tester strains, more remarkable for TA1537 where a slight thinning of background lawn was seen at the next
lower dose level of 1580 µg/plate.

In the absence of S9 metabolism toxicity as indicated by thinning of background lawn or reduction in the revertant numbers was seen for all tester strains at the highest dose level tested, more obviouos for TA1537 where thinning of background lawn and reduction of revertant numbers was also seen at the next lower dose level of 2500 µg/plate.
In the presence of S9, toxicity, as indicated by thinning of background lawn or reduction of revertant numbers, was seen for all tester strains at the highest dose level tested with the exception ofWP2uvrA. For TA1537 and TA100 tester strains thinning of the background lawn or reduction in revertant numbers was also seen at the next lower dose level of 2500 µg/plate.
As no relevant increase in revertant numbers was observed at any concentration tested, a Main Assay II was performed including a pre-incubation step for all treatments.
The following concentrations were employed:
TA1535 ± 5000, 2500, 1250, 625, 313
WP2 uvrA ± 5000, 2500, 1250, 625, 313
TA98 + 5000, 2500, 1250, 625, 313
TA98 − 5000, 2500, 1250, 625, 313, 156
TA100 − 5000, 2500, 1250, 625, 313, 156
TA1537 ± 2500, 1250, 625, 313, 15
TA100 + 2500, 1250, 625, 313, 15

Marked or severe toxicity was observed at the highest dose level tested where reduction of
revertant numbers and/or thinning of background lawn were noticed for all tester strains
in the absence and presence of S9. Toxicity was also observed at the next lower dose level
for all tester strains with the exception of TA1537 in the absence and presence of S9 and
TA100 in the presence of S9.
No precipitation of the test item was observed at the end of the incubation period at
any concentration tested in the absence or presence of S9 metabolic activation, in any
experiment.
The sterility of the S9 mix and of the test item solutions was confirmed by the absence of
colonies on additional agar plates spread separately with these solutions. Marked increases
in revertant numbers were obtained in these tests following treatment with the positive
control items, indicating that the assay system was functioning correctly.

Applicant's summary and conclusion

Conclusions:
It is concluded that the test item (S)-3-Benzoylthio-2-methylpropionic acid (ABP) does not induce reverse mutation in Salmonella typhimurium or Escherichia coli in the absence or presence of S9 metabolism, under the reported experimental conditions.