Registration Dossier

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2015-05-06 to 2015-06-03
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP and guideline compliant study.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report Date:
2015

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
Qualifier:
according to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
BASF SE, Experimental Toxicology and Ecology, 67056 Ludwigshafen, Germany
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Substance type: organic
- Physical state: metallic green-violet solid, melt (highly viscous)
- Analytical purity: 91.6 %
- Expiration date of the lot/batch: 23 January 2019
- Storage condition of test material: at room temperature

Method

Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9 mix (phenobarbital and β-naphthoflavone induced rat liver)
Test concentrations with justification for top dose:
1st experiment: 0; 33; 100; 333; 1000; 3050 and 6100 μg/plate (SPT)
2nd experiment: 0; 0.1; 0.33; 1.0; 3.3; 10 and 33 μg/plate (SPT)
3rd experiment (PIT):
0; 0.1; 0.33; 1.0; 3.3; 10 and 33 μg/plate (TA 1535, TA 98 and E.coli WP2 uvrA)
0; 0.03; 0.1; 0.33; 1.0; 3.3 and 10 μg/plate (TA 100 and TA 1537)
Vehicle / solvent:
- Vehicle used: water
- Justification for choice of vehicle: Good solubility of the test substance in water.
Controlsopen allclose all
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Remarks:
MNNG
Positive control substance:
other: N-methyl-N'-nitro-N-nitrosoguanidine
Remarks:
without S9 mix (TA1535, TA100)
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Remarks:
NOPD
Positive control substance:
other: 4-nitro-o-phenylenediamine
Remarks:
without S9 mix (TA98)
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Remarks:
AAC
Positive control substance:
9-aminoacridine
Remarks:
without S9 mix (TA1537)
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Remarks:
NQO
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
without S9 mix (E. coli WP2 uvrA)
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Remarks:
2-AA
Positive control substance:
other: 2-aminoanthracene
Remarks:
with S9 mix
Details on test system and experimental conditions:
METHOD OF APPLICATION: 1st and 2nd experiment: in agar (plate incorporation); 3rd experiment: preincubation

DURATION
- Preincubation period: 20 min
- Exposure duration: 48 - 72 hours

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency; relative total growth
Evaluation criteria:
Generally, the experiment was considered valid if the following criteria were met:
• The number of revertant colonies in the negative controls was within the range of the historical negative control data for each tester strain.
• The sterility controls revealed no indication of bacterial contamination.
• The positive control substances both with and without S9 mix induced a distinct increase in the number of revertant colonies within the range of the historical positive control data or above.
• Fresh bacterial culture containing approximately 10^9 cells per mL were used.

The test substance was considered positive in this assay if the following criteria were met:
• A dose-related and reproducible increase in the number of revertant colonies, i.e. at least doubling (bacteria strains with high spontaneous mutation rate, like TA 98, TA 100 and E.coli WP2 uvrA) or tripling (bacteria strains with low spontaneous mutation rate, like TA 1535 and TA 1537) of the spontaneous mutation rate in at least one tester strain either without S9 mix or after adding a metabolizing system.

A test substance was generally considered non-mutagenic in this test if:
• The number of revertants for all tester strains were within the historical negative control data range under all experimental conditions in at least two experiments carried out independently of each other.
Statistics:
Not applicable.

Results and discussion

Test results
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Test substance precipitation was found in the standard pate test from about 3050 μg/plate onward with and without S9 mix.

COMPARISON WITH HISTORICAL CONTROL DATA: In this study with and without S9 mix, the number of revertant colonies in the negative controls was within or nearby the range of the historical negative control data for each tester strain. In addition, the positive control substances both with and without S9 mix induced a significant increase in the number of revertant colonies within or nearby the range of the historical positive control data.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
In experiment 1 a strong bacteriotoxic effect (reduced his- or trp- background growth, decrease in the number of his+ or trp+ revertants) was observed in the standard plate test and in the preincubation assay depending on the strain and test conditions from about 10 μg/plate onward.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Table 1 Results

Experiment 1: plate incorporation

 

 

 

 

 

Dose (µg/plate)

Mean number of revertant colonies/3 replicate plates with different strains of Salmonella typhimurium and E. coli

 

TA1535

TA1537

TA98

TA100

WP2 uvrA

 

 

 

 

 

 

 

Results with S9

 

 

 

 

Spontaneous Reversion

9.7

9.3

29.3

109.3

28.0

Positive control

323.0

240.7

2351.3

2818.7

149.0

33

9.3

5.7

18.7

43.7

17.7

100

0.0

0.0

0.0

0.0

15.3

333

0.0

0.0

0.0

0.0

0.0

1000

0.0

0.0

0.0

0.0

0.0

3050

0.0

0.0

0.0

0.0

0.0

6100

0.0

0.0

0.0

0.0

0.0

 

 

 

 

 

 

 

Results without S9

 

 

 

 

Spontaneous Reversion

12.0

5.7

24.0

87.7

28.3

Positive control

5311.0

1403.7

443.3

3192.3

1495.0

33

6.3

0.0

9.0

0.0

15.0

100

0.0

0.0

0.0

0.0

5.7

333

0.0

0.0

0.0

0.0

0.0

1000

0.0

0.0

0.0

0.0

0.0

3050

0.0

0.0

0.0

0.0

0.0

6100

0.0

0.0

0.0

0.0

0.0

 

 

 

 

 

 

Experiment 2: plate incorporation

 

 

 

 

 

Dose (µg/plate)

Mean number of revertant colonies/3 replicate plates with different strains of Salmonella typhimurium and E. coli

 

TA1535

TA1537

TA98

TA100

WP2 uvrA

 

 

 

 

 

 

 

Results with S9

 

 

 

 

Spontaneous Reversion

14.3

9.3

23.3

112.7

28.3

Positive control

367.0

231.0

2168.3

2805.0

112.0

0.1

10.7

10.7

30.7

120.0

20.3

0.33

10.0

10.0

26.7

112.7

26.3

1.0

9.7

8.7

29.7

109.7

25.0

3.0

6.3

9.7

29.3

106.3

31.7

10

12.0

9.3

34.0

117.7

23.3

33

4.3

4.7

18.3

12.0

23.3

 

 

 

 

 

 

 

Results without S9

 

 

 

 

Spontaneous Reversion

11.0

10.7

20.7

105.3

29.3

Positive control

5067.7

1107.3

511.0

4515.0

1520.3

0.1

9.7

6.7

18.7

91.3

22.3

0.33

10.0

8.0

24.3

102.3

27.0

1.0

12.7

7.0

15.3

94.7

28.3

3.0

15.0

8.3

18.0

120.3

21.3

10

9.0

9.0

19.7

66.0

25.3

33

4.3

0.0

3.3

0.0

20.7

33

4.3

0.0

3.3

0.0

20.7

 

 

 

 

 

 

Experiment 3: preincubation

Dose (µg/plate)

Mean number of revertant colonies/3 replicate plates with different strains of Salmonella typhimurium and E. coli

 

TA1535

TA1537

TA98

TA100

WP2 uvrA

 

 

 

Results with S9

Spontaneous Reversion

9.3

6.0

26.0

101.3

21.3

Positive control

230.0

139.7

1530.0

1755.0

83.0

0.03

-

5.7

-

98.3

-

0.1

7.0

5.7

26.3

91.7

21.0

0.33

11.7

5.7

21.0

89.0

21.7

1.0

10.0

8.0

29.0

86.7

23.0

3.3

9.7

7.3

22.3

97.3

22.0

10

9.0

0.0

21.0

57.7

20.0

33

2.7

-

9.0

-

7.0

 

 

 

Results without S9

Spontaneous Reversion

7.7

4.7

18.7

90.0

18.0

Positive control

1612.0

549.0

429.7

1125.3

338.0

0.03

-

4.7

-

92.7

-

0.1

9.7

6.0

16.7

90.7

25.3

0.33

8.3

6.0

18.0

83.7

22.3

1.0

6.7

6.3

16.3

109.3

22.3

3.3

7.7

4.0

19.0

98.7

25.0

10

7.0

0.0

10.3

38.3

18.0

33

2.3

-

0.3

-

10.0

Applicant's summary and conclusion