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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:
7 February 2017 - 10 April 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)
Version / remarks:
21 July 1997
Deviations:
no
Qualifier:
according to
Guideline:
other: ''Regulation on Test Methods for Chemical Substances”, Notification No. 2016-27, National Institute of Environmental Research, Republic of Korea
Version / remarks:
19 December 2016
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
Details on test material:
Appearance: colourless or light yellow viscous liquid
Storage conditions: at room temperature, container tightly closed, flushed with nitrogen.
Specific details on test material used for the study:
- No correction for purity was made

Method

Target gene:
Salmonella typhimurium: histidine gene
Escherichia coli: tryptophan gene
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Metabolic activation system:
S9-mix derived from male rats induced with phenobarbital and 5,6-benzoflavone
Test concentrations with justification for top dose:
Dose range finding study (all strains): 4.88, 19.5, 78.1, 313, 1250 and 5000 μg/plate (top dose was selected according to the test guideline).
First and second (main) experiment (without S9): TA98 and TA1535: 9.77, 19.5, 39.1, 78.1, 156 and 313 μg/plate; TA100 and TA1537: 2.44, 4.88, 9.77, 19.5, 39.1 and 78.1 μg/plate; WP2uvrA: 39.1, 78.1, 156, 313, 625 and 1250 μg/plate
First and second (main) experiment (with S9): TA100 and TA1537: 9.77, 19.5, 39.1, 78.1, 156 and 313 μg/plate; TA98 and TA1535: 39.1, 78.1, 156, 313, 625 and 1250 μg/plate; WP2uvrA: 156, 313, 625, 1250, 2500 and 5000 μg/plate
(top doses were selected at the lowest dose level at which growth inhibition was confirmed)
Vehicle / solvent:
- Vehicle used: dimethyl sulfoxide
- Justification for choice of vehicle: because the test substance is unstable in water, a preliminiary solubility test was performed. The test substance was dissolved in DMSO. The vehicle is selected according to OECD guideline 471.
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
2-nitrofluorene
sodium azide
benzo(a)pyrene
other: 2-aminoanthracene
Remarks:
See table 1 in ''Any other information on materials and methods incl. tables''
Details on test system and experimental conditions:
METHOD OF APPLICATION: in glucose agar
- Cell density at seeding: 10^9 cells/mL

DURATION
- Preincubation period: no
- Exposure duration: 48 hours

NUMBER OF REPLICATIONS: 3 plates/dose

DETERMINATION OF CYTOTOXICITY
- Method: colonies were counted by a colony counter (ProtoCOL3, Syniosis, UK). To confirm the presence or absence of growth inhibition, the background lawn was observed using a stereoscopic microscope (45-fold magnification, SZ61, Olympus, Japan).

OTHER:
- Evaluation of microbial contamination: the presence or absence of colonies formed by microbial contamination in the plates was evaluated by placing 100 μL of the high dose formulation, 500 μL of 0.1 mol/L sodium phosphate buffer (pH 7.4) and 500 μL of S9 mix in tubes and incubating these in a shaking water bath (37°C, 90 rpm) for 20 minutes, followed by a 48 hour incubation period.

ACCEPTABILITY CRITERIA
- The mean number of revertant colonies for the negative and positive controls should be within the range of the historical control data or the mean number of revertant colonies in the positive control groups should be increased at least twice as compared to the negative control group.
- No plate should show any evidence of contamination.
Evaluation criteria:
The results of the study were considered to be positive when the number of revertant colonies in any strain at one or more doses was increased at least two times when compared to the negative control group. There should be dose dependency or reproducibility as dose increases.

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:
not applicable
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:
not applicable
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:
not applicable
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:
not applicable
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:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation (dose range finding study): at 5000 μg/plate (without S9) and from 1250 μg/plate (with S9)
- Precipitation (main studies): only with S9: at dose levels > 625 μg/plate for TA98, TA1535 and WP2uvrA

GROWTH INHIBITION:
Main studies (without S9): at 313 μg/plate for TA98; above 39.1 μg/plate for TA100 and TA1537; above 156 μg/plate for TA1535; at 1250 μg/plate for WP2uvrA.
Main studies (with S9): above 625 μg/plate for TA98 and TA1535; at 313 μg/plate for TA100 and TA1537 and above 2500 μg/plate for WP2uvrA.

HISTORICAL DATA: the mean number of revertant colonies for the negative and positive control groups was within the range of the historical control data (Table 5) and the number of revertant colonies of the positive control groups was markedly increased at least twice when compared to the negative control group. Also, there was no contamination.

Any other information on results incl. tables

Table 2 Historical negative control values of revertant colonies

 

TA1535

TA1537

TA98

TA100

WP2uvrA

S9-mix

-

+

-

+

-

+

-

+

-

+

Range

4.7-15.5

3.9-14.9

3.3-11.7

8.4-21.4

9.6-26.1

14.3-37.6

60.5-110.9

64.9-125.1

73.9-167.6

87.3-198.2

Mean

10.1

9.4

7.5

14.9

17.9

25.9

85.7

95.0

120.8

142.7

SD

2.1

1.9

1.4

2.6

3.0

4.0

10.2

12.0

17.3

18.7

n

125

125

125

125

125

125

125

125

125

125

Table 3 Historical positive control values of revertant colonies

 

TA1535

TA1537

TA98

TA100

WP2uvrA

S9-mix

-

+

-

+

-

+

-

+

-

+

Range

353.6-582.6

67.0-165.7

237.0-638.9

77.9-189.5

392.3-762.9

84.6-258.5

440.0-711.4

149.8-314.3

209.1-1163.3

304.5-610.5

Mean

468.1

116.3

437.9

133.7

577.6

171.5

575.7

232.1

686.2

457.5

SD

47.4

19.2

128.5

18.6

87.4

32.3

55.0

32.1

160.7

59.3

n

119

125

119

52

119

52

119

52

23

125

Applicant's summary and conclusion

Conclusions:
Based on the results of an Ames test, performed according to OECD guideline 471, STABiO D-376N was concluded not to be mutagenic.
Executive summary:

An Ames test was performed, according to OECD guideline 471 and GLP principles, to assess the mutagenic potential of STABiO D-376N. Four Salmonella typhimurium strains (TA98, TA100, TA1535 and TA1537) and one Escherichia coli strain

(WP2uvrA) were used in the presence and absence of a metabolic activation system (S9). In the dose range finding test, the test substance was tested up to a dose level of 5000 μg/plate. In the two main tests, dose levels were tested up to the lowest dose level where precipitation occurred in the range finding test. The mean number of revertant colonies for the negative and positive controls were within the historical data range. In the positive control group, the mean number of revertant colonies was markedly increased more than twice when compared to that of the negative control group. In the main tests, the mean number of revertant colonies was less than twice when compared to the negative control group at all dose levels of the test substance in the presence and absence of metabolic activation. Based on the results of this study, STABiO is considered not to be mutagenic in the Salmonella typhimurium reverse assay and the Escherichia coli reverse assay.