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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The mutagenicity of the test item was determined in a study according to OECD guideline 471. The results indicate that the test item under the experimental conditions described, was not mutagenic to Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100, and to the Escherichia coli strain WP2 uvrA in the presence and absence of a metabolizing system.

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:
January - April 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
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
Target gene:
histidine, tryptophan
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:
Phenobarbital/β-naphthoflavone induced rat liver S9
Test concentrations with justification for top dose:
Strain TA 100: 0.3, 1, 3,10, 33, 100, 333, and 1000 μg/plate
all others: 33, 100, 333, 1000, 2500, and 5000 μg/plate
Based on the observed toxicity in the pretest 1000 μg/plate were chosen as maximal concentration for strain TA 100, 5000 μg/plate for the remaining strains.
Vehicle / solvent:
DMSO
The solvent was chosen because of its solubility properties and its relative nontoxicity to the bacteria.
Untreated negative controls:
yes
Remarks:
untreated
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
TA 1535, TA 100 (Without metabolic activation)
Untreated negative controls:
yes
Remarks:
untreated
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 4-nitro-o-phenylene-diamine, 4-NOPD
Remarks:
TA 1537, TA 98 (Without metabolic activation)
Untreated negative controls:
yes
Remarks:
untreated
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
WP2 uvrA (Without metabolic activation)
Untreated negative controls:
yes
Remarks:
untreated
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
TA 1535, TA 1537, TA 98, TA 100, WP2 uvrA (With metabolic activation)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation) in Experiment I and pre-incubation in Experiment II

DURATION
- Preincubation period: 60 min at 37 °C
- Exposure duration: 48 h at 37 °C

SELECTION AGENT: L-Histidin for S. typhimurium, Tryptophan for E. coli

NUMBER OF REPLICATIONS: 3

Titer of the incubated culture: 10^8 - 10^9 cells/mL.

DETERMINATION OF CYTOTOXICITY
- Method: Background lawn

ACCEPTANCE CRITERIA:
The Salmonella typhimurium and Escherichia coli reverse mutation assay is considered acceptable if it meets the following criteria:
- regular background growth in the negative and solvent control
- the spontaneous reversion rates in the negative and solvent control are in the range of our historical data
- the positive control substances should produce an increase above the threshold of twice (strains TA 98, TA 100, and WP2 uvrA) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control
- a minimum of five analysable dose levels should be present with at least three dose levels showing no signs of toxic effects, evident as a reduction in the number of revertants below the indication factor of 0.5.
Evaluation criteria:
A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100, and WP2 uvrA) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control is observed.
A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.
An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.
A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.
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
Remarks:
at 100 μg/plate
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
Remarks:
at 333 μg/plate
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
Remarks:
at 100 μg/plate
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:
PRE-EXPERIMENT:
3; 10; 33; 100; 333; 1000; 2500; and 5000 μg/plate
Since toxic effects were observed six concentrations were tested in experiment II. Based on the observed toxicity 1000 μg/plate were chosen as maximal concentration for strain TA 100, 5000 μg/plate for the remaining strains.

HISTORICAL CONTROL DATA
see table at "Any other information"
In experiment I the number of colonies did not quite reach the lower limit of our historical control data in the negative control of strain WP2 uvrA with metabolic activation. Since this deviation is rather small, this effect is judged to be based upon statistical fluctuations and has no detrimental impact on the outcome of the study.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: reduced background lawn
- TA 1537: with metabolic activation at 333 μg/plate and without metabolic activation at 100 μg/plate cytotoxic effects
- TA 98: with metabolic activation at 1000 μg/plate and without metabolic activation at 333 μg/plate cytotoxic effects
- TA 100: with metabolic activation at 1000 μg/plate and without metabolic activation at 100 μg/plate cytotoxic effects

Mean number of revertants with S9 mix

Test substance

Dose/Plate (µg)

TA 1535

TA 1537

TA 98

TA 100

WP2 uvrA

DMSO

-

11

18

28

135

35

Test substance

0.3

 

 

 

150

 

1

 

 

 

124

 

3

13

16

37

161

 

10

14

15

32

141

 

33

12

15

28

137

40

100

10

8

35

134

39

333

9

8R

25

69

40

1000

1

0R

14R

5R

40

2500

0

0R

1R

 

24

5000

0

0R

0R

 

27

Positive control

10

 

 

 

 

369

2.5

425

157

3959

5070

 

Positive Control: 2 –Aminoanthracene (2 -AA)

R: reduced background growth

Mean number of revertants without S9 mix

Test substance

Dose/Plate (µg)

TA 1535

TA 1537

TA 98

TA 100

WP2 uvrA

DMSO

-

15

13

26

156

30

Test substance

0.3

 

 

 

140

 

1

 

 

 

187

 

3

11

10

27

145

 

10

11

10

24

154

 

33

8

8

20

142

32

100

7

5R

17

48

30

333

8

7R

20R

48

31

1000

6

7R

15R

43

32

2500

3

4R

14R

 

30

5000

1

2R

7R

 

28

NaN3

10

1151

-

-

2171

-

4-NOPD

10

-

-

317

-

-

4-NOPD

50

-

68

-

-

-

MMS

2.0

-

-

-

-

843

R: reduced background growth

NaN3: sodium azide

4-NOPD: 4-nitro-o-phenylene-diamine

MMS: methyl methane sulfonate

Historical control data

Strain

 

Without S9 mix

With S9 mix

Mean

SD

Min

Max

Mean

SD

Min

Max

TA 1535

Solvent control

12

2.5

6

25

12

2.5

7

26

Untreated control

12

3.1

6

28

12

2.9

7

26

Positive control

1130

143.1

334

1816

388

58.2

176

668

TA1537

Solvent control

10

2.2

6

19

13

3.5

7

30

Untreated control

11

2.7

5

21

14

4.0

7

31

Positive control

82

12.7

43

157

191

60.8

83

434

TA 98

Solvent control

25

4.4

13

43

34

6.2

15

58

Untreated control

27

4.9

12

43

37

6.5

11

57

Positive control

378

73.7

211

627

3949

771.8

360

6586

TA 100

Solvent control

156

26.0

78

209

148

32.3

73

208

Untreated control

176

23.6

79

217

172

25.4

85

218

Positive control

1966

293.2

498

2767

3798

830.4

536

6076

WP2 uvr A

Solvent control

41

5.6

27

63

50

6.8

28

72

Untreated control

42

5.8

30

63

52

6.8

36

88

Positive control

798

362.7

319

4732

378

112.6

167

1265

Conclusions:
The mutagenicity of the test item was determined in a study according to OECD guideline 471. The results indicate that the test item under the experimental conditions described, was not mutagenic to Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100, and to the Escherichia coli strain WP2 uvrA in the presence and absence of a metabolizing system.
Executive summary:

This study was performed to investigate the potential of the test item to induce gene mutations according to the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and the Escherichia coli strain WP2 uvrA. The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate.

The test item was tested at the following concentrations:

Pre-Experiment/Experiment I: 3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate

Experiment II:

Strains TA 1535, TA 1537, and TA 98: 3, 10; 33; 100; 333; 1000; 2500 and 5000 µg/plate

Strain TA 100: 0.3; 1; 3;10; 33; 100; 333; and 1000 µg/plate

Strain WP2 uvrA: 33; 100; 333; 1000; 2500 and 5000 µg/plate

The test item precipitated in the overlay agar in the test tubes from 1000 to 5000 µg/plate with S9 mix in both experiments. Precipitation of the test item in the overlay agar on the incubated agar plates was observed in experiment I at 5000 µg/plate with S9 mix. The undissolved particles had no influence on the data recording. In experiment II no precipitation of the test item was observed in the overlay agar on the incubated agar plates. In experiment I the plates incubated with the test item showed normal background growth in all strains with and without S9 mix up to 5000 µg/plate. In experiment II reduced background growth was observed in strains TA 1537, TA98, and TA 100 with and without S9 mix.

Toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in all strains. No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with test item at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance. Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies.

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

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

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

OECD 471

This study was performed to investigate the potential of the test item to induce gene mutations according to the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and the Escherichia coli strain WP2 uvrA. The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate.

The test item was tested at the following concentrations:

Pre-Experiment/Experiment I: 3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate

Experiment II:

Strains TA 1535, TA 1537, and TA 98: 3, 10; 33; 100; 333; 1000; 2500 and 5000 µg/plate

Strain TA 100: 0.3; 1; 3;10; 33; 100; 333; and 1000 µg/plate

Strain WP2 uvrA: 33; 100; 333; 1000; 2500 and 5000 µg/plate

The test item precipitated in the overlay agar in the test tubes from 1000 to 5000 µg/plate with S9 mix in both experiments. Precipitation of the test item in the overlay agar on the incubated agar plates was observed in experiment I at 5000 µg/plate with S9 mix. The undissolved particles had no influence on the data recording. In experiment II no precipitation of the test item was observed in the overlay agar on the incubated agar plates. In experiment I the plates incubated with the test item showed normal background growth in all strains with and without S9 mix up to 5000 µg/plate. In experiment II reduced background growth was observed in strains TA 1537, TA98, and TA 100 with and without S9 mix.

Toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in all strains. No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with test item at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance. Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies.

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

Justification for classification or non-classification

The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Based on available data on genetic toxicity, the test item is not classified and labelled as mutagen according to Regulation (EC) No 1272/2008 (CLP), as amended for the tenth time in Regulation (EU) No 2017/776.