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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Reverse gene mutation assay: Negative (Non-mutagenic); OECD 471; M. Sarada., 2018

Link to relevant study records
Reference
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
06 April - 08 April 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
The study was conducted under GLP in accordance with the international guideline.
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1997
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
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 Mix
Test concentrations with justification for top dose:
Doses selected for the mutagenicity assay was based on data generated in preliminary study (Doses of 1.2, 4.9,20, 20, 78, 313, 1250 and 5000 μg per plate) where growth inhibition was observed at 313 μg per plate and more in all TA strains with and without metabolic activation at 1250 μg per plate in E. coli WP2 uvr A. Precipitation was also observed at 1250 μg per plate with and without metabolic activation. Therefore the doses for the main test were as follows;
0, 10, 20, 39, 78, 156 & 313 μg per plate for salmonella strains and 39, 78, 156, 313, 625 & 1250 μg per plate for WP2 uvrA.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: guideline recommended
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
2-acetylaminofluorene
sodium azide
benzo(a)pyrene
other: 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide For TA100, TA98 & Wp2uvrA and 2-Methoxy-6-chloro-9-[3-(2-chloroethyl)aminopropylamino]acridine.2HCl for TA1537
Details on test system and experimental conditions:
NUMBER OF REPLICATIONS:
- Number of cultures per concentration (single, duplicate, triplicate): Triplicate
- Number of independent experiments: Three ; preliminary test and two main experiment

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): n/a
- Test substance added in medium; For tests without metabolic activation, 0.5 mL of 0.1 M Na-phosphate buffer (pH 7.4) and 0.1 mL of each fresh bacterial culture were added to each tube containing 0.1 mL of the test solution or or the negative control solution. For the test with metabolic activation, 0.5 mL of the S9 mix was added to each tube instead of 0.1 M Na-phosphate buffer. This was followed by pre-incubation at at 37°C for the 20 mins, then 2.0mL of top agar were added to the mixture, and the contents of each tube were pourers over the surface of the minimal glucose agar plate. And 0.1 mL of positive control solution was carried out equally.
For sterility test, 0.1 mL of test solution of the maximum concentration and 0.5 mL of S9 mix were added to each tube followed by 2.0 mL agar then transferred to surface of minimal glucose agar plate.
These preparation took place over UV absorbent filter.
As top agar, 0.5 mM biotin-0.5 mM L-histidine solution and 0.5 mM l-tryptophan solution were added to the safe agar solution (0.6% agar, 0.5% NaCl) by volume of 1/10 for TA and E.Coli strains respectively.
All plates wear incubated at at 37°C for 48 hrs, the number of revertant colonies were counted. Afterwards, growth inhibition of the test strains was checked using a stereoscopic microscope.

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: 20 mins
- Exposure duration/duration of treatment: 48 hours at 37°C
- Harvest time after the end of treatment (sampling/recovery times): not stated
Rationale for test conditions:
The study was based on the in vitro (pre-incubation) technique described by Ames et al (1975), Maron and Ames (1983), in which mutagenic effects are determined by exposing mutant strains of Salmonella typhimurium to various concentrations of the test item. These strains have a deleted excision repair mechanism which makes them more sensitive to various mutagens and they will not grow on media which does not contain histidine. When large numbers of these organisms are exposed to a mutagen, reverse mutation to the original histidine independent form takes place. These are readily detectable due to their ability to grow on a histidine deficient medium. Using these strains of Salmonella typhimurium revertants may be produced after exposure to a chemical mutagen, which have arisen as a result of a base-pair substitution in the genetic material (miscoding) or as a frameshift mutation in which genetic material is either added or deleted. Additionally, a mutant strain of Escherichia coli (WP2uvrA) which requires tryptophan and can be reverse mutated by base substitution to tryptophan independence is used to complement the Salmonella strains.

Evaluation criteria:
In the two main tests, if the number of revertant colonies on the test plates increased significantly in comparison with that on the control plates (based on twice as many as that of the negative control), and dose-response and reproducibility were also observed, the test substance was to be judged positive. The results of each concentrations were demonstrated with the mean and the standard deviation.
Statistics:
Not stated
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:
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:
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:
cytotoxicity
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:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
MTEST-SPECIFIC CONFOUNDING FACTORS: 
- Data on pH:Not specified
- Data on osmolality:Not specified
- Possibility of evaporation from medium:Not specified
- Water solubility:Not specified
- Precipitation and time of the determination: Precipitation was observed at 625 μg per plate with and without metabolic activation.
- Definition of acceptable cells for analysis: All bacterial strains must have demonstrated the required characteristics as determined by their respective strain checks according to Ames et al (1975), Maron and Ames (1983) and Mortelmans and Zeiger (2000).
- Other confounding effects: no



RANGE-FINDING/SCREENING STUDIES (if applicable): Was conducted,
growth inhibition was observed at ≥ 313 μg per plate in all TA strains with and without metabolic activation and at 1250 μg per plate in E. coli WP2 uvr A. Precipitation was observed in control groups at ≥ 1250 μg per plate with and without metabolic activation.

STUDY RESULTS
- Concurrent vehicle negative and positive control data: Both with historical control range


For all test methods and criteria for data analysis and interpretation:
- Concentration-response relationship where possible: No increase in revertant were observed.
- Statistical analysis; not stated

- Any other criteria: not stated


Ames test:
- Signs of toxicity: Toxicity were observed beginning at 650 μg per plate in all test conditions
- Individual plate counts: yes
Remarks on result:
other: Not Mutagenic

Table 1. Preliminary Test Results

A term of test

April 10 - 13th2018

With or without S9 mix

Dose ug/plate

No. Revertant colonies/plste

Base-pair substitution type

Frame-shift type

TA100

TA1535

WP2uvrA

TA98

TA1537

-S9

0

106

14

28

31

8

1.2

106

15

24

30

7

4.9

118

16

21

18

9

20

112

16

24

21

11

78

130

12

22

26

8

313

104*

8*

29

25*

7*

1250+

108*

15*

25*

24*

8*

5000+

118*

13*

25*

19*

4*

+S9

0

123

13

29

36

16

1.2

126

8

29

35

16

4.9

112

11

36

35

13

20

117

13

27

27

17

78

98

14

24

38

18

313

136*

10*

28

24*

14*

1250+

102*

12*

27*

23*

15*

5000+

109*

6*

20*

24*

10*

Positive control -S9

Name

AF-2

NaN3

AF-2

AF-2

ICR-191

Dosemg/plate

0.01

0.5

0.01

0.1

1.0

No. Colonies/plate

503

420

151

561

1375

Positive control +S9

Name

B[a]P

2AA

2AA

B[a]P

B[a]P

Dosemg/plate

5.0

2.0

10.0

5.0

5.0

No. Colonies/plate

682

218

325

212

59

 

 Table 2. The 1st Main Test Results

A term of test

April 17 - 20th2018

With or without S9 mix

Dose ug/plate

No. Revertant colonies/plste

Base-pair substitution type

Frame-shift type

TA100

TA1535

WP2uvrA

TA98

TA1537

-S9

0

109±7.0

12±1.7

30±3.5

20±2.0

9±1.0

10

95±10.4

9±1.0

NT

19±1.5

9±1.7

20

93±8.3

9±1.0

NT

19±1.5

9±0.6

39

90±6.1

12±2.6

27±2.1

18±2.3

10±1.7

78

92±3.8

11±1.5

28±1.2

20±1.5

8±1.5

156

91±6.7*

11±1.0*

24±2.1

20±3.6*

7±0.6*

313

91±6.7*

9±2.3*

32±2.1

20±4.4*

9±1.5*

 

625+

NT

NT

23±3.5*

NT

NT

 

1250+

NT

NT

23±4.6*

NT

NT

+S9

0

106±5.3

13±2.1

32±2.6

27±2.5

16±2.6

10

110±3.2

13±3.0

NT

30±15.0

14±1.0

20

100±2.1

10±2.1

NT

25±3.1

15±2.3

39

95±3.2

10±2.1

31±2.9

26±3.2

16±2.3

78

101±3.6

12±1.5

32±1.5

30±3.5

16±2.6

156

93±4.0*

11±3.2

37±2.5

30±4.2*

14±1.7*

313

88±6.6*

8±1.7*

25±3.1

24±5.2*

13±0.6*

625

NT

NT

31±2.6*

NT

NT

1250+

NT

NT

30±3.2*

NT

NT

Positive control -S9

Name

AF-2

NaN3

AF-2

AF-2

ICR-191

Dosemg/plate

0.01

0.5

0.01

0.1

1.0

No. Colonies/plate

535±26.1

383±3.8

147±7.4

523±14.4

1305±92.9

Positive control +S9

Name

B[a]P

2AA

2AA

B[a]P

B[a]P

Dosemg/plate

5.0

2.0

10.0

5.0

5.0

No. Colonies/plate

728±26.6

286±15.5

345±34.4

175±9.3

63±70

 

 

 Table 3. The 2nd Main Test Results

A term of test

April 19 - 23th2018

With or without S9 mix

Dose ug/plate

No. Revertant colonies/plste

Base-pair substitution type

Frame-shift type

TA100

TA1535

WP2uvrA

TA98

TA1537

+S9

0

117±11.0

13±1.7

29±4.2

27±1.0

18±3.2

10

131±8.1

9± 2.1

NT

27±2.9

16±2.3

20

126±8.2

14±2.9

NT

28±6.2

15±0.6

39

116±8.5

10±2.9

34±1.7

23±1.7

14±1.2

78

131±7.4

10±1.5

27±6.0

27±1.7

17±4.7

156

126±11.8*

9±1.7

24±1.2

21±1.5*

18±4.5*

313

119±11.8*

10±3.1*

26±4.6

24±2.9*

17±1.7*

625+

NT

NT

29±4.0*

NT

NT

1250+

NT

NT

25±3.5*

NT

NT

-S9

0

113±12.3

12±1.0

29±4.2

27±1.0

18±3.2

10

113±13.3

10± 1.2

NT

27±2.9

16±2.3

20

109±11.5

14±1.7

NT

28±6.2

15±0.6

39

117±2.1

11±0.6

34±1.7

23±1.7

14±1.2

78

117±2.6

10±0.6

27±6.0

27±1.7

17±4.7

156

116±12.6*

12±2.0*

24±1.2

21±1.5*

18±4.5*

Please refer to 'Attached background material', where figures are presented in support of the lack of signficant increase in revertant colonies.

Conclusions:
Based on the condition of the study, the test item did not induce mutagenicity in bacterial reverse mutation assay with or without metabolic activation.
Executive summary:

OECD 471 ( 2018): The test item, was tested to evaluate its mutagenic potential using the preincubation method by measuring its ability to induce reverse mutations at selected loci of several strains of Salmonella typhimurium and at the tryptophan locus of Escherichia coli strain WP2 uvrA in the presence and absence of an exogenous metabolic activation system. Dimethyl sulfoxide (DMSO) was used as the vehicle.

Doses selected for the mutagenicity assay were 1.2, 4.9,20, 20, 78 1250 and 5000 μg per plate, based on data generated in preliminary study where growth inhibition was observed at ≥ 313 μg per plate in all TA strains with and without metabolic activation at 1250 μg per plate in E. coli WP2 uvr A. Precipitation was also observed at 1250 μg per plate with and without metabolic activation. All criteria for a valid study were met as described in the protocol.

The test item in DMSO, at concentrations of 2.018, 6.224 and 6.304 mg/mL, was stable at room temperature under the condition of the study. No contaminant colonies were observed on the sterility plates for the vehicle control, the test substance dilutions or the S9 and Sham mixes.

Precipitate was observed at 625 μg per plate and toxicity was observed beginning at 625 μg per plate with all conditions. No dose dependent increases were in all tester strain with or without metabolic activation. The test item was negative for mutagenicity in bacterial reverse mutation assay with or without metabolic activation.

Under the conditions of this study, test item did not meet the criteria for classification for mutagenicity in accordance with Globally Harmonized Classification System or the Regulation (EC) No. 1272/2008; relating to the Classification, Labelling and Packaging of Substances and Mixtures.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Mode of Action Analysis / Human Relevance Framework

The test item is not mutagenic and therefore no mode of action is required.

Additional information

OECD 471 ( 2018): The test item was tested to evaluate its mutagenic potential using the preincubation method by measuring its ability to induce reverse mutations at selected loci of several strains of Salmonella typhimurium and at the tryptophan locus of Escherichia coli strain WP2 uvrA in the presence and absence of an exogenous metabolic activation system. Dimethyl sulfoxide (DMSO) was used as the vehicle.

Doses selected for the mutagenicity assay were 1.2, 4.9, 20, 78 1250 and 5000 μg per plate, based on data generated in preliminary study where growth inhibition was observed at 313 μg per plate and more in all TA strains with and without metabolic activation at 1250 μg per plate in E. coli WP2 uvrA. Precipitation was also observed at 1250 μg per plate with and without metabolic activation.  All criteria for a valid study were met as described in the protocol.

The test item in DMSO, at concentrations of 2.018, 6.224 and 6.304 mg/mL, was stable at room temperature under the condition of the study. No contaminant colonies were observed on the sterility plates for the vehicle control, the test substance dilutions or the S9 and Sham mixes.

Precipitate was observed at 625 μg per plate and toxicity was observed beginning at 625 μg per plate with all conditions. No dose dependent increases were in all tester strain with or without metabolic activation. The test item was negative for mutagenicity in bacterial reverse mutation assay with or without metabolic activation.

Justification for classification or non-classification

The test item did not meet the criteria for classification for mutagenicity in accordance with Globally Harmonized Classification System or the Regulation (EC) No. 1272/2008; relating to the Classification, Labelling and Packaging of Substances and Mixtures.