Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The test item was not mutagenic in the bacterial reverse mutation assay.

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:
1990-10-02 to 1990-10-11
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1983
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay
Test material information:
Composition 1
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: D 271



Target gene:
The Salmonella typhimurium histidine (his) reversion system measures his- → his+ reversions. The Salmonella typhimurium strains are constructed to differentiate between base pair (TA1535, TA100) and frameshift (TA1537, TA98) mutations.
Species / strain:
other: S. typhimurium TA 98, TA 100, TA 1535, TA 1537, TA 1538
Metabolic activation:
with and without
Metabolic activation system:
rat liver S9 mix, Aroclor 1254 induced
Test concentrations with justification for top dose:
The test compound was tested at doses of 4 to 10000 microgram/plate and proved to be toxic to the bacterial strains at doses of 2500 microgram/ plate. Thinning of the bacterial lawn and a reduction in the number of colonies have been observed at this dose.
Concentrations: 0, 4, 20, 100, 500, 2500, 5000, 10000 µg/plate.
In the second experiment, 5000 µg/plate was chosen as the highest dose.
Vehicle:
- Vehicle/solvent used: DMSO
- Justification for choice of solvent/vehicle: soluble in DMSO
Negative controls:
no
Solvent controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
without metabolic activation
Negative controls:
no
Solvent controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
without metabolic activation
Negative controls:
no
Solvent controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
2-nitrofluorene
Remarks:
without metabolic activation
Negative controls:
no
Solvent controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
with metabolic activation
Negative controls:
no
Solvent controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2- aminoanthracene
Remarks:
with metabolic activation
Details on test system and conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 h

SELECTION AGENT: histidine

NUMBER OF REPLICATIONS: 3

NUMBER OF CELLS EVALUATED: all revertant colonies were counted


DETERMINATION OF CYTOTOXICITY
- Method: relative total growth


Rationale for test conditions:
standard test conditions for this assay
Evaluation criteria:
A test article is classified mutagenic if either of the following conditions under a) and b) is achieved:
a) a test article produces at least a 2-fold increase in the mean number of revertants per plate of at least one of the tester strains over the mean number of revertants per plate of the appropriate vehicle control at complete bacterial background lawn
b) a test article induces a dose-related increase in the mean number of revertants per plate of at least one of the tester strains over the mean number of revertants per plate of the appropriate vehicle control in at least two to three concentrations of the test article at complete bacterial background lawn.
The test results must be reproducible.
Statistics:
Statistical analysis is not neccessary as only the number of colonies has to be compared to the controls.
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
yes
Remarks:
cytotoxicity observed at doses of 2500 µg/plate onwards
Vehicle controls valid:
yes
Negative controls valid:
not applicable
Positive controls valid:
yes
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
yes
Remarks:
Cytotoxicity was observed in the preliminary experiment at doses of 2500 µg/plate and above.
Vehicle controls valid:
yes
Negative controls valid:
not applicable
Positive controls valid:
yes
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
yes
Remarks:
cytotoxicity observed at doses of 2500 µg/plate onwards
Vehicle controls valid:
yes
Negative controls valid:
not applicable
Positive controls valid:
yes
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
yes
Remarks:
cytotoxicity observed at doses of 2500 µg/plate onwards
Vehicle controls valid:
yes
Negative controls valid:
not applicable
Positive controls valid:
yes
Key result
Species / strain:
S. typhimurium TA 1538
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
yes
Remarks:
cytotoxicity observed at doses of 2500 µg/plate onwards
Vehicle controls valid:
yes
Negative controls valid:
not applicable
Positive controls valid:
yes

Table 1: Number of revertants per plate (mean of three plates), Experiment 1

strain

TA 100

strain

TA 1535

Strain

TA 1537

strain

TA 1538

strain

TA 98

conc. [µg] per plate

-MA

+MA

-MA

+MA

-MA

+MA

-MA

+MA

-MA

+MA

0

162

178

8

8

12

15

23

22

28

34

4

168

180

9

17

15

16

16

18

26

33

20

176

180

11

9

12

16

20

18

34

34

100

196

198

11

11

15

14

18

18

29

34

500

201

204

10

8

13

14

16

20

25

29

2500

168

177

13

13

11

17

17

19

27

35

10000

147

134

5

10

12

10

17

17

33

25

Sodium azide

1 µg

639

 

472

 

 

 

 

 

 

 

9-Aminoacridine

50 µg

 

 

 

 

133

 

 

 

 

 

2-Nitrofluorene

2.5 µg

 

 

 

 

 

 

597

 

527

 

2-Aminoanthracene

0.5 µg (TA 100, TA1538, TA98)

1µg (TA1535, TA1537)

 

992

 

191

 

133

 

820

 

855

Benzo[a]pyrene 10 µg

 

1661

 

32

 

109

 

229

 

724

 

 

Table 2: Number of revertants per plate (mean of three plates), Experiment 2

strain

TA 100

strain

TA 1535

Strain

TA 1537

strain

TA 1538

strain

TA 98

conc. [µg]

-MA

+MA

-MA

+MA

-MA

+MA

-MA

+MA

-MA

+MA

0

141

153

12

13

7

8

16

18

21

23

4

154

155

12

11

5

6

14

15

25

25

20

140

153

11

9

7

9

19

16

24

29

100

162

164

9

16

7

9

14

17

21

34

500

147

149

8

11

9

8

16

16

23

27

2500

161

145

8

8

7

7

17

15

25

32

10000

134

152

13

12

8

7

17

11

25

28

Sodium azide

1 µg

705

 

555

 

 

 

 

 

 

 

9-Aminoacridine

50 µg

 

 

 

 

621

 

 

 

 

 

2-Nitrofluorene

2.5 µg

 

 

 

 

 

 

1332

 

835

 

2-Aminoanthracene

0.5 µg (TA 100, TA1538, TA98)

1µg (TA1535, TA1537)

 

1094

 

184

 

115

 

973

 

1161

Benzo[a]pyrene 10 µg

 

1396

 

32

 

118

 

197

 

604

Conclusions:
The test substance was not mutagenic in this bacterial reverse mutation assay.
Executive summary:

The test item was tested for mutagenicity with the strains TA 100, TA 1535, TA 1537, TA 1538, TA 98 of Salmonella typhimurium. The mutagenicity studies were conducted in the absence and in the presence of a metabolizing system derived from rat liver homogenate (S9 mix, Aroclor 1254 induced). A dose range of 6 different doses from 4 microgram/plate to 10000 microgram/plate was used. Control plates without mutagen showed that the number of spontaneous revertant colonies was similiar to that described in the literature. All the positive control compounds gave the expected increase in the number of revertant colonies. The test compound proved to be toxic to the bacterial strains at 2500 microgram/plate. In the absence of the metabolic activation system the test compound did not show a dose dependent increase in the number of revertants in any of the bacterial strains. Also in the presence of a metabolic activation system, treatment of the cells with the test item did not result in relevant increases in the number of revertant colonies. Summarizing, it can be stated that the test item is not mutagenic in these bacterial test systems either with or without exogenous metabolic activation at the dose levels investigated.

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

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Ames test:

The test item was tested for mutagenicity with the strains TA 100, TA 1535, TA 1537, TA 1538, TA 98 of Salmonella typhimurium.

The mutagenicity studies were conducted in the absence and in the presence of a metabolizing system derived from rat liver homogenate (S9 mix, Aroclor 1254 induced). A dose range of 6 different doses from 4 microgram/plate to 10000 microgram/plate was used. Control plates without mutagen showed that the number of spontaneous revertant colonies was similiar to that described in the literature. All the positive control compounds gave the expected increase in the number of revertant colonies. The test compound proved to be toxic to the bacterial strains at 2500 microgram/plate. In the absence of the metabolic activation system the test compound did not show a dose dependent increase in the number of revertants in any of the bacterial strains. Also in the presence of a metabolic activation system, treatment of the cells with the test item did not result in relevant increases in the number of revertant colonies. Summarizing, it can be stated that the test item is not mutagenic in these bacterial test systems either with or without exogenous metabolic activation at the dose levels investigated.

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008

The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. As a result the substance is not considered to be classified for genetic toxicity under Regulation (EC) No 1272/2008, as amended for the tenth time in Regulation (EU) No 2017/776.