Registration Dossier

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017-12-20 to 2018-03-15
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:
2018

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1997
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
2008
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent

Method

Species / strain
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Metabolic activation system:
induced rat liver S9 mix
Test concentrations with justification for top dose:
Both trials (plate incorporation and pre-incubation) were performed at concentrations of 0.05, 0.16, 0.5, 1.6 and 5 mg/plate.
Vehicle:
- Vehicle(s)/solvent(s) used: water
- Justification for choice of solvent/vehicle: recommended by guideline
Controls
Negative controls:
no
Solvent controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
mitomycin C
other: 2-Aminoanthracene: 4 µg/plate, all strains with S9
Details on test system and conditions:
METHOD OF APPLICATION: in agar (plate incorporation); preincubation
- Cell density at seeding: 10^8 viable cells per plate

DURATION
- Preincubation period:
30 min
- Exposure duration:
48 hrs +/- 30 min (plate incorporation); 66 hrs (pre-incubation)

NUMBER OF REPLICATIONS:
3

DETERMINATION OF CYTOTOXICITY
- Method: background lawn
Rationale for test conditions:
According to guideline and based on preliminary dose-range finding study.
Evaluation criteria:
Evaluation:
A substance is considered positive if the following criteria apply:
There should be a dose related increase over the range tested and/or a reproducible increase in the mean revertants per plate of at least one tester strain over a minimum of two increasing doses of the test item either in the presence or absence of the metabolic activation system.
The test will be judged positive if the increase in mean revertants at the peak of the dose response is equal to or greater than 2 times the mean vehicle control value in Salmonella typhimurium strains TA98, TA100 and TA102 or equal to or greater than 3 times the mean vehicle control value in tester strains TA1535 and TA1537.

The mutation test is considered acceptable as it met the following criteria:
- All tester strains confirmed to their genetic characteristics.
- The positive controls showed increase in revertant colony numbers of at least twice or thrice the concurrent vehicle control levels with the appropriate bacterial strain.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no
Vehicle controls valid:
yes
Negative controls valid:
not examined
Positive controls valid:
yes
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no
Vehicle controls valid:
yes
Negative controls valid:
not examined
Positive controls valid:
yes
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no
Vehicle controls valid:
yes
Negative controls valid:
not examined
Positive controls valid:
yes
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no
Vehicle controls valid:
yes
Negative controls valid:
not examined
Positive controls valid:
yes
Key result
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no
Vehicle controls valid:
yes
Negative controls valid:
not examined
Positive controls valid:
yes
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH:
none
- Effects of osmolality: none
- Evaporation from medium: no
- Water solubility: well soluble
- Precipitation: not observed

RANGE-FINDING/SCREENING STUDIES:
yes

Any other information on results incl. tables

Table 1 SUMMARY OF COLONY COUNTS OF REVERTANTS OF TRIAL-I: Plate Incorporation Method

 

Treatment

Test Concentration  (mg/plate)

No. of Revertants (Mean of 3 Plates)

With S9

 

Without S9

Salmonella typhimurium

Salmonella typhimurium

TA

98

TA

100

TA 102

TA 1535

TA 1537

TA 98

TA 100

TA

102

TA 1535

TA 1537

Vehicle Control

100 µL of Distilled water

Mean

25.3

102.3

255.3

20.3

8.3

23.0

110.0

256.7

20.7

8.7

±SD

2.5

6.1

7.8

3.5

2.5

3.0

10.6

5.9

2.1

2.3

Lawn Intensity

4+

4+

4+

4+

4+

4+

4+

4+

4+

4+

Test item 

0.05

Mean

19.7

97.0

257.3

18.7

7.3

21.7

98.3

253.3

19.7

6.3

±SD

3.5

8.9

8.5

2.5

1.5

2.5

6.5

5.7

0.6

3.2

Fold Increase

0.8

0.9

1.0

0.9

0.9

0.9

0.9

1.0

1.0

0.7

Lawn Intensity

4+

4+

4+

4+

4+

4+

4+

4+

4+

4+

0.16

Mean

23.0

97.0

252.7

20.7

9.3

19.0

96.3

254.7

15.7

9.3

±SD

3.6

6.0

5.0

1.5

2.3

3.6

5.1

4.0

1.5

2.1

Fold Increase

0.9

0.9

1.0

1.0

1.1

0.8

0.9

1.0

0.8

1.1

Lawn Intensity

4+

4+

4+

4+

4+

4+

4+

4+

4+

4+

0.5

Mean

20.7

106.0

254.0

20.0

7.7

18.0

97.7

258.7

17.3

6.3

±SD

5.0

6.1

7.5

2.6

1.5

2.6

5.7

5.5

2.5

2.1

Fold Increase

0.8

1.0

1.0

1.0

0.9

0.8

0.9

1.0

0.8

0.7

Lawn Intensity

4+

4+

4+

4+

4+

4+

4+

4+

4+

4+

1.6

Mean

22.7

106.0

256.0

20.0

9.0

20.3

99.0

255.0

19.7

8.3

±SD

4.2

7.8

2.0

3.6

3.6

3.1

7.5

4.6

4.0

2.5

Fold Increase

0.9

1.0

1.0

1.0

1.1

0.9

0.9

1.0

1.0

1.0

Lawn Intensity

4+

4+

4+

4+

4+

4+

4+

4+

4+

4+

5

Mean

21.3

107.3

253.3

19.7

7.7

19.7

104.7

257.3

18.3

8.3

±SD

2.5

4.2

6.1

1.5

3.1

2.5

11.6

7.5

2.5

3.2

Fold Increase

0.8

1.0

1.0

1.0

0.9

0.9

1.0

1.0

0.9

1.0

Lawn Intensity

4+

4+

4+

4+

4+

4+

4+

4+

4+

4+

Positive Control

100 µL of respective Positive Control

Mean

375.7

406.0

602.3

146.0

119.3

354.3

392.0

597.0

142.0

115.7

±SD

14.3

6.6

10.1

8.9

3.5

14.5

9.5

7.0

7.5

6.0

Fold Increase

14.8

4.0

2.4

7.2

14.3

15.4

3.6

2.3

6.9

13.3

Lawn Intensity

4+

4+

4+

4+

4+

4+

4+

4+

4+

4+

 

 

Table 2SUMMARY OF COLONY COUNTS OF REVERTANTS OF TRIAL-II: Preincubation Method

 

Treatment

Test Concentration  (mg/plate)

No. of Revertants (Mean of 3 Plates)

With S9

 

Without S9

Salmonella typhimurium

Salmonella typhimurium

TA

98

TA

100

TA 102

TA 1535

TA 1537

TA

98

TA 100

TA 102

TA 1535

TA 1537

Vehicle Control

100 µL of Distilled water

Mean

27.0

106.0

269.0

22.7

9.3

22.0

98.0

269.3

19.0

7.3

±SD

4.4

5.6

12.5

1.5

3.5

4.6

8.7

11.5

3.6

2.5

Lawn Intensity

4+

4+

4+

4+

4+

4+

4+

4+

4+

4+

1-(carboxylatomethyl)methylpyridinium

0.05

Mean

25.3

98.3

260.3

19.3

9.3

20.3

102.3

253.0

19.0

7.3

±SD

3.1

5.7

9.7

3.8

3.8

3.1

8.5

7.0

3.0

2.1

Fold Increase

0.9

0.9

1.0

0.9

1.0

0.9

1.0

0.9

1.0

1.0

Lawn Intensity

4+

4+

4+

4+

4+

4+

4+

4+

4+

4+

0.16

Mean

22.7

105.3

263.0

20.7

10.7

20.7

104.0

269.7

17.0

6.0

±SD

3.8

8.6

3.6

2.5

1.5

1.5

3.6

11.7

2.0

1.7

Fold Increase

0.8

1.0

1.0

0.9

1.1

0.9

1.1

1.0

0.9

0.8

Lawn Intensity

4+

4+

4+

4+

4+

4+

4+

4+

4+

4+

0.5

Mean

23.0

97.7

262.0

20.7

9.7

20.0

97.0

259.7

19.0

6.0

±SD

1.7

4.2

9.8

4.5

1.5

3.6

5.6

5.9

3.0

2.0

Fold Increase

0.9

0.9

1.0

0.9

1.0

0.9

1.0

1.0

1.0

0.8

Lawn Intensity

4+

4+

4+

4+

4+

4+

4+

4+

4+

4+

1.6

Mean

25.7

98.0

266.3

18.7

7.0

20.7

95.3

255.0

16.3

5.7

±SD

2.5

8.5

11.8

4.5

2.0

1.5

6.8

8.2

0.6

0.6

Fold Increase

1.0

0.9

1.0

0.8

0.8

0.9

1.0

0.9

0.9

0.8

Lawn Intensity

4+

4+

4+

4+

4+

4+

4+

4+

4+

4+

5

Mean

23.3

101.7

262.7

18.0

6.0

21.3

99.0

269.7

16.0

5.7

±SD

2.5

4.0

10.7

2.0

2.0

3.1

4.4

4.5

2.6

1.5

Fold Increase

0.9

1.0

1.0

0.8

0.6

1.0

1.0

1.0

0.8

0.8

Lawn Intensity

4+

4+

4+

4+

4+

4+

4+

4+

4+

4+

Positive Control

100 µL of respective Positive Control

Mean

373.3

396.0

605.0

144.3

118.3

348.0

395.7

592.0

139.3

118.3

±SD

7.5

8.2

7.5

12.1

6.7

6.2

7.5

15.9

9.3

5.0

Fold Increase

13.8

3.7

2.2

6.4

12.7

15.8

4.0

2.2

7.3

16.1

Lawn Intensity

4+

4+

4+

4+

4+

4+

4+

4+

4+

4+

 

 

Positive controls:

With S9:     

For Salmonella typhimurium TA98, TA100, TA102, TA1535 and TA1537 = 4 µg/plate of 2-Aminoanthracene

Without S9:

For TA98: 2 µg/plate of 2-Nitrofluorene

For TA100 and TA1535: 1 µg/plate of Sodium azide.

For TA102: 0.5 µg/plate of Mitomycin C

For TA1537: 50 µg/plate of 9-Aminoacridine

 

Applicant's summary and conclusion

Conclusions:
Based on the results obtained from this study, it is concluded that the test item is non-mutagenic in the Bacterial Reverse Mutation Test up to the highest tested concentration of 5 mg/plate under the test conditions chosen.
Executive summary:

The test item was evaluated for mutagenicity in a Bacterial Reverse Mutation Test according to OECD guideline for testing of chemicals No. 471, “Bacterial Reverse Mutation Test”, adopted on 21st July 1997.

The test concentrations used in the mutation assay were selected based on the results obtained in the pre-tests. The mutagenic potential of the test item was investigated in two independent experiments, in plate incorporation test (trial 1) and pre-incubation test (trial 2). Each assay was conducted with and without metabolic activation (± S9 mix). Both trials were performed at concentrations of 0.05, 0.16, 0.5, 1.6 and 5 mg/plate. All concentrations, including the controls, were tested in triplicate. Vehicle control (distilled water) and appropriate positive controls (2-nitrofluorene, sodium azide and 9-Aminoacridine, Mitomycin C for trials without metabolic activation and 2-Aminoanthracene for trials with metabolic activation) were tested simultaneously.

The tester strains used in the mutation assay were Salmonella typhimurium TA98, TA100, TA 102, TA1535 and TA1537.

In the performed trials all of the validity criteria regarding the investigated strains, negative (vehicle) and positive controls, S9 activity and number of investigated analyzable concentration levels were fulfilled. Based on the experimental results obtained, the mean numbers of revertant colonies at the tested concentrations were comparable to those of the vehicle control, in both trials in the presence and absence of metabolic activation. There was no substantial increase in number of revertant colonies at any of the tested concentrations in both trials. In the performed experiments an inhibitory effect of the test item (decreased number of revertant colony numbers and/or affected background lawn development) was not observed in any case.

The number of revertant colonies in the positive controls resulted in 2.2 to 16.1 fold increase under identical conditions.