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Genetic toxicity in vitro

Description of key information

In an in vitro bacterial reverse mutation assay according to OECD Guideline 471 (AMES test) with and without metabolic activation (S9 mix).(BASF Colors&Effects, 2017), three out of five tested strains (TA98, TA 100 and TA1537) showed a biological relevant increase of mutant colonies after incubation.

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:
2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1997-07-21
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Batch No. of test material: 002-151203
- Expiration date of the batch: 2020-04-16

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature

OTHER SPECIFICS: Solid / orange
Target gene:
his / trp
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Remarks:
uvrA
Metabolic activation:
with and without
Metabolic activation system:
phenobarbital and β-naphthoflavone induced rat liver S9 mix
Test concentrations with justification for top dose:
1st Experiment
Doses: 0, 33, 100, 333, 1000, 2500, and 5000 μg/plate

2nd Experiment
Doses: 0, 3.3, 10, 33, 100, 333, and 1000 μg/plate

In agreement with the recommendations of current guidelines 5 mg/plate or 5 μL/plate were generally selected as maximum test dose at least in the 1st Experiment. However, this maximum dose was tested even in the case of relatively insoluble test compounds to detect possible mutagenic impurities. Furthermore, doses > 5 mg/plate or > 5 μL/plate might also be tested in repeat experiments for further clarification/substantiation.
Vehicle / solvent:
- Solvent used: DMSO
- Justification for choice of solvent/vehicle: Due to the insolubility of the test substance in water, DMSO was used as vehicle, which had been demonstrated to be suitable in bacterial reverse mutation tests and for which historical control data are available.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene (2-AA)
Remarks:
With S9 mix: 2.5 μg/plate, TA 1535, TA 100, TA 1537, TA 98; 60 μg/plate, Escherichia coli WP2 uvrA
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)
Remarks:
Without S9 mix: 5 μg/plate, TA 1535, TA 100
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 4-nitro-o-phenylenediamine (NOPD)
Remarks:
Without S9 mix: 10 μg/plate, TA 98
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
Without S9 mix: 100 μg/plate, TA 1537
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
Without S9 mix: 5 μg/plate, E. coli WP2 uvrA
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 - 72 hours at 37 °C
NUMBER OF REPLICATIONS: in triplicate
NUMBER OF CELLS EVALUATED: all revertants / colonies counted
DETERMINATION OF CYTOTOXICITY
Toxicity detected by a
- decrease in the number of revertants (factor ≤ 0.6)
- clearing or diminution of the background lawn (= reduced his- or trp- background growth)
was recorded for all test groups both with and without S9 mix in all experiments and indicated in the tables. Single values with a factor ≤ 0.6 were not detected as toxicity in low dose groups.
Evaluation criteria:
Acceptance 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 10E9 cells per mL were used.


Assessment criteria
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 range of the historical negative control data under all experimental conditions in at least two experiments carried out independently of each other.
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
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:
positive
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
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:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
A weak bacteriotoxic effect (decrease in number of his+ revertants) was observed only using the tester strain TA 1537 without S9 mix at 5000 μg/plate in the 1st Experiment
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:
positive
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
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:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Test substance precipitation was found from about 1000 μg/plate onward with and without S9 mix.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
No bacteriotoxic effect (reduced his- or trp- background growth, decrease in the number of his+ or trp+ revertants) was observed in the standard plate test up to the highest required concentration.

MUTAGENICITY

According to the results of the present study the test substance led to an evident and dose dependent increase in the number of his+ revertants with the tester strains TA 100, TA 1537, and TA 98, all with and without S9 mix. The increase of revertants was reproducible in two experiments carried out independently of each other. Based on the recent assessment criteria the test substance has to be considered positive.

Tests without S9 mix (see Table 1):

- TA 1535: No relevant increase in the number of his+ revertants.

-TA 100: 1st Exp.: Increase in the number of his+ revertants at concentrations of 100, 333, 1000, 2500, and 5000 μg/plate (factors 3.2, 6.9, 12.5, 13.5, and 8.4, respectively).

2nd Exp.: Increase in the number of his+ revertants at concentrations of 100, 333, and 1000 μg/plate (factors 3.0, 7.0, and 15.5, respectively).

-TA 1537: 1st Exp.: Increase in the number of his+ revertants at concentrations of 100, 333, 1000, 2500, and 5000 μg/plate (factors 3.2, 8.1, 13.4,

13.9, and 3.1, respectively). 2nd Exp.: Increase in the number of his+ revertants at concentrations of 100, 333, and 1000 μg/plate (factors 5.3, 11.1, and 26.0, respectively).

-TA 98: 1st Exp.: Increase in the number of his+ revertants at concentrations of 100, 333, 1000, 2500, and 5000 μg/plate (factors 2.9, 5.6, 11.0, 13.0, and 20.7, respectively). 2nd Exp.: Increase in the number of his+ revertants at concentrations of 100, 333, and 1000 μg/plate (factors 2.3, 6.9, and 16.1, respectively).

-E. coli WP2 uvrA: No relevant increase in the number of trp+ revertants.

Tests with S9 mix (see Table 2):

- TA 1535: 1st Exp.: Slight increase in the number of his+ revertants at a concentration of 2500 μg/plate (factor 2.5) 2nd Exp.: No relevant increase in the number of his+ revertants.

- TA 100: 1st Exp.: Increase in the number of his+ revertants at concentrations of 100, 333, 1000, 2500, and 5000 μg/plate (factors 2.8, 4.9, 11.4, 10.7, and 10.3, respectively). 2nd Exp.: Increase in the number of his+ revertants at concentrations of 100, 333, and 1000 μg/plate (factors 2.5, 4.5, and 11.7, respectively).

- TA 1537: 1st Exp.: Increase in the number of his+ revertants at concentrations of 333, 1000, 2500, and 5000 μg/plate (factors 5.6, 13.3, 12.2, and 4.5, respectively). 2nd Exp.: Increase in the number of his+ revertants at concentrations of 333 and 1000 μg/plate (factors 4.7 and 15.7, respectively).

- TA 98: 1st Exp.: Increase in the number of his+ revertants at concentrations of 333, 1000, 2500, and 5000 μg/plate (factors 2.3, 5.7, 15.5, and 16.3, respectively). 2nd Exp.: Increase in the number of his+ revertants at concentrations of 333 and 1000 μg/plate (factors 2.8 and 7.9, respectively).

- E. coli WP2 uvrA: No relevant increase in the number of trp+ revertants.

Table 1: SPT – without metabolic activation

Strain

Test group

Dose

(µg/plate)

Mean revertants

per plate

Standard

deviation

Factor

TA 1535

DMSO

-

15.7

3.8

-

 

Test item

33

18.7

5.7

1.2

 

 

100

14.3

3.2

0.9

 

 

333

13.0

4.0

0.8

 

 

1000

16.0

3.0

P 1.0

 

 

2500

20.3

2.1

P 1.3

 

 

5000

16.7

2.1

P 1.1

 

MNNG

5.0

4606.0

133.4

294.0

TA 100

DMSO

-

106.3

4.0

-

 

Test item

33

174.7

10.7

1.6

 

 

100

337.0

15.1

3.2

 

 

333

734.0

40.7

6.9

 

 

1000

1329.7

57.6

P 12.5

 

 

2500

1430.7

282.9

P 13.5

 

 

5000

889.3

86.9

P 8.4

 

MNNG

5.0

4396.3

126.1

41.3

TA1537

DMSO

-

10.0

4.6

-

 

Test item

33

18.3

2.3

1.8

 

 

100

32.3

11.7

3.2

 

 

333

81.0

3.5

8.1

 

 

1000

133.7

10.5

P 13.4

 

 

2500

138.7

20.1

P 13.9

 

 

5000

30.7

7.1

P 3.1

 

AAC

100

922.3

88.3

92.2

TA 98

DMSO

-

24.7

5.8

-

 

Test item

33

36.7

4.9

1.5

 

 

100

71.7

6.1

2.9

 

 

333

137.0

20.3

5.6

 

 

1000

270.7

26.8

P 11.0

 

 

2500

320.0

122.0

P 13.0

 

 

5000

510.3

48.7

P 20.7

 

NOPD

10

980.0

21.5

39.7

E. coli

DMSO

-

27.0

9.5

-

 

Test item

33

30.7

4.5

1.1

 

 

100

30.0

2.6

1.1

 

 

333

26.7

7.0

1.0

 

 

1000

21.7

3.5

P 0.8

 

 

2500

25.0

3.0

P 0.9

 

 

5000

18.7

2.9

P 0.7

 

4-NQO

5.0

1082.3

6.1

40.1

P Precipitation


 

Table 2: SPT – with metabolic activation

Strain

Test group

Dose

(µg/plate)

Mean revertants

per plate

Standard

deviation

Factor

TA 1535

DMSO

-

11.0

1.7

-

 

Test item

33

20.3

2.1

1.8

 

 

100

8.3

2.1

0.8

 

 

333

17.3

2.1

1.6

 

 

1000

24.0

6.9

P 2.2

 

 

2500

27.3

6.1

P 2.5

 

 

5000

21.7

1.5

P 2.0

 

2-AA

2.5

311.0

33.0

28.3

TA 100

DMSO

-

110.0

6.2

-

 

Test item

33

179.0

27.6

1.6

 

 

100

303.7

22.2

2.8

 

 

333

538.3

34.3

4.9

 

 

1000

1252.3

86.9

P 11.4

 

 

2500

1173.3

153.1

P 10.7

 

 

5000

1133.3

84.0

P 10.3

 

2-AA

2.5

2296.7

272.2

20.9

TA1537

DMSO

-

12.0

3.0

-

 

Test item

33

21.0

4.0

1.8

 

 

100

22.0

7.0

1.8

 

 

333

66.7

9.1

5.6

 

 

1000

159.0

6.2

P 13.3

 

 

2500

146.0

16.4

P 12.2

 

 

5000

53.7

11.2

P 4.5

 

2-AA

2.5

217.7

21.7

18.1

TA 98

DMSO

-

28.0

2.0

-

 

Test item

33

31.0

5.6

1.1

 

 

100

42.0

8.7

1.5

 

 

333

63.7

11.7

2.3

 

 

1000

159.3

16.0

P 5.7

 

 

2500

435.0

36.9

P 15.5

 

 

5000

457.3

32.1

P 16.3

 

2-AA

2.5

1729.3

409.1

61.8

E. coli

DMSO

-

24.0

6.2

-

 

Test item

33

35.7

12.1

1.5

 

 

100

39.0

7.9

1.6

 

 

333

38.0

2.0

1.6

 

 

1000

25.0

3.5

P 1.0

 

 

2500

18.7

1.2

P 0.8

 

 

5000

15.3

4.9

P 0.6

 

2-AA

60

204.0

2.6

8.5

P Precipitation

Conclusions:
Thus, under the experimental conditions chosen here, it is concluded that the test item is a potent mutagenic test substance in the bacterial reverse mutation test in the absence and the presence of metabolic activation.
Endpoint conclusion
Endpoint conclusion:
adverse effect observed (positive)

Additional information

In an in vitro bacterial reverse mutation assay according to OECD Guideline 471 (AMES test) (BASF Colors&Effects, 2017), the test substance was tested for its mutagenic potential based on the ability to induce point mutations in selected loci of several bacterial strains (TA 1535, TA 100, TA 1537, TA 98 and E. coli WP2 uvrA). In a stadard plate test with and without metabolic activation (liver S9 mix from induced rats), the bacterial strains were exposed to doses of the test substance up to 5000 µg/plate. Precipitation of the test substance was found from about 1000 μg/plate onward with and without S9 mix. No bacteriotoxic effect was observed under all test conditions. Adequate negative and positive controls were valid. A relevant increase in the number of his+ or trp+ revertants (factor ≥ 2: TA 100, TA 98 and E.coli WP2 uvrA or factor ≥ 3: TA 1535 and TA 1537) was not observed using the tester strains TA 1535 and E. coli WP2 uvrA with or without metabolic activation. A relevant, reproducible and dose dependent increase in the number of his+ revertants exceeding a factor of 2 compared to the concurrent vehicle control was observed with TA 100 and TA 98 with and without metabolic activation in the standard plate. In TA 100, with and without S9 mix, an increase at concentrations of 100 up to 5000 μg/plate was observed. In TA 98 without S9 mix an increase at concentrations of 100 up to 5000 μg/plate and with S9 mix an increase at concentrations of 333, 1000, 2500 and 5000 μg/plate was observed. Using tester strains TA 1537, a relevant, reproducible and dose depending increase of his+ revertants exceeding a factor of 3 compared to the concurrent vehicle control was observed with and without adding metabolic activation. Wihout S9 mix, an increase at concentrations of 100 up to 5000 μg/plate and with S9 mix, an increase at concentrations of 333, 1000, 2500 and 5000 μg/plate was observed.

Thus, under the experimental conditions of this study, the test substance is mutagenic in the Salmonella typhimurium/Escherichia coli reverse mutation assay in the absence and the presence of metabolic activation.

Justification for classification or non-classification

The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008.

Bacterial in vitro results with the test substance were positive. However, for a conclusive justification for classification or non-classification under Regulation (EC) No 1272/2008, as amended for the thenth time in Regulation (EU) No 2017/776, further studies in mammalian cells are necessary.