Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Based on the results of an in vitro bacterial mutagenicity assay according to OECD 471, the test item is considered to be non-mutagenic (UN GHS) no category) (reference 7.6.1 -1).

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:
06 October 2016 - 22 November 2016
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:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Version / remarks:
1993
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
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Metabolic activation system:
Rat liver S9 induced by ß-Naphthoflavone/phenobarbital
Test concentrations with justification for top dose:
1st series: 5, 18, 50, 158, 500, 1580, 5000 µg/plate (with and without S9 mix)
2nd series: 50, 158, 500, 1580, 5000 µg/plate (with and without S9 mix)
top dose: maximum recommended concentration (according to OECD 471)
Vehicle / solvent:
- Solvent used: DMSO
- Justification for choice of solvent: solubitlity properties of the test item, non toxicity to bacteria
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
sodium azide
other: 2-aminoanthracene 2-10 µg/plate, daunomycin 1 µg/plate
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 2-3 days

NUMBER OF REPLICATIONS: 3 replicates for test item concentrations and positive controls, 6 replicates for solvent controls

DETERMINATION OF CYTOTOXICITY
- Method: counting numbers of revertants

OTHER:
-S9 concentration: 1st series 10%, 2nd series 30%

Evaluation criteria:
A test material was to be defined as negative or non-mutagenic in this assay if
• the assay was to be considered valid, and
• "no" or "weak increases" occurred in the test series performed ("weak increases" randomly occur due to experimental variation)
For valid data, the test material was considered to be positive or mutagenic if:
• a dose dependent (over at least two test material concentrations) increase in the number of revertants was induced, the maximal effect was a "clear increase", and the effects were reproduced at similar concentration levels in the same test system, or
• "clear increases" occurred at least at one test material concentration, higher concentrations showed strong precipitation or cytotoxicity, and the effects were reproduced at the same concentration level in the same test system.
Statistics:
Not performed as not mandatory for this test system.
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:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
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:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
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:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
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:
not examined
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:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No precipitation occurred.

Table 2: Summary of Experiment 1

Metabolic Activation

Test material

Concentr. (µg/plate)

Revertant Colony Counts (Mean ± SD)

TA 98

TA 100

TA 1535

TA 1537

WP2 uvrA

Without Activation

DMSO

 

32 ± 4

124 ± 17

29 ± 4

27 ± 7

39 ± 7

Test item

5

25± 3

112±19

29±6

22±6

34±6

15.8

29±6

122±16

28±7

30±10

32±3

50

29±8

122±14

20±0

28±7

29±2

158

27±7

124±4

27±5

31±1

26±6

500

30±7

112±15

28±1

26±2

32±3

1580

26±11

122±16

32±8

24±3

30±1

5000

21±1

120±8

30±5

29±5

33±5

DAUN

1

393±50

 

 

 

 

NaN3

2

 

1597±59

914±82

 

 

9-AA

50

 

 

 

1812±272

 

NQO

2

 

 

 

 

2128± 269

With Activation

DMSO

 

34±3

144±20

26±6

27±2

36±7

Test item

5

32± 13

140±2

26±1

33±3

38±1

15.8

35±9

144±17

29±4

26±3

39±7

50

26±5

143±2

21±6

23±6

31±1

158

34±3

153±27

22±6

28±3

41±6

500

33±11

131±17

30±2

27±4

33±4

1580

28±10

142±25

26±3

29±5

37±7

5000

25±2

140±22

23±1

23±3

30±2

2-AA

2

426± 24

1286±371

 

 

 

2-AA

5

 

 

265±33

412±48

 

2-AA

10

 

 

 

 

445±161

 

Table 3: Summary of Experiment 2

Metabolic Activation

Test material

Concentr. (µg/plate)

Revertants per plate (Mean ± SD)

TA 98

TA 100

TA 1535

TA 1537

WP2 uvrA

Without Activation

DMSO

 

36 ± 8

123 ± 8

31 ± 2

26 ± 7

26 ± 6

Test item

50

37± 10

115± 8

25±6

29± 2

29± 9

158

42± 8

119± 6

31± 1

28± 8

21± 4

500

37± 4

107± 19

27± 3

23± 6

26± 2

1580

39± 5

115± 7

22± 9

25±8

28± 4

5000

33± 5

111± 9

22± 8

27± 5

28± 6

DAUN

1

196± 41

 

 

 

 

NaN3

2

 

911± 25

678± 25

 

 

9-AA

50

 

 

 

539± 139

 

NQO

2

 

 

 

 

1173± 58

With Activation

DMSO

 

47± 9

118± 9

28± 3

31± 1

30± 3

Test item

50

42± 7

117± 14

28± 4

29± 6

29±1

158

41±5

124± 16

24± 10

31± 6

35± 4

500

38±7

119± 10

31± 6

30± 5

32± 6

1580

48± 3

134± 15

29± 4

30± 6

32± 3

5000

35± 5

117± 14

31± 4

38±4

37± 7

2-AA

2

226± 4

 

 

 

 

2-AA

5

 

1073± 42

 

 

 

2-AA

10

 

 

390± 36

523± 81

122±1

 

Key to Positive Controls

NaN3                Sodium azide

2-AA                2-Aminoanthracene

9-AA                9-Aminoacridine

DAUN              Daunomycin

NQO                4-Nitroquinoline-N-oxide

Conclusions:
The test item is considered non-mutagenic under the test conditions described.
Executive summary:

The test item was examined for its mutagenic activity in two series of in vitro microbial assays employing Salmonella typhimurium TA 98, TA 100, TA 1535 and TA 1537, and Escherichia coli WP2 uvrA as indicator organisms. The plate incorporation test with and without addition of liver S9 mix from Aroclor 1254-pre-treated rats was used. In this study, two experimental series was performed. In the experiments with S9 mix, 10 and 30% S9 were used in the 1st and 2nd series, respectively. Treatments of all tester strains were performed using test item formulations prepared in anhydrous analytical grade dimethyl sulfoxide (DMSO) in the absence and in the presence of S9 mix, using final concentrations between 5 and 5000 µg/plate, plus vehicle and positive controls. The strain specific positive control test materials, namely daunomycin, sodium azide, 4-nitroquin-olin-N-oxide, and 9-aminoacridine in the absence of S9 mix, yielded the expected mutant frequencies that were greatly in excess of the negative controls. The genotype of the tester strains used was thus confirmed. 2-Aminoanthracene which requires metabolic activation was strongly mutagenic. This indicates that the exogenous metabolizing system used in the present investigation (S9 mix) was functioning. Thus, the acceptance criteria have been met in total and the study is considered valid. Following test item treatments, precipitation of the test material on the agar plates was not occurred. There was no toxicity to the bacteria observed. Under the conditions described, there were no relevant increases in revertant numbers after test item exposure observed in both, the absence and presence of S9 mix. According to the criteria for negative and positive results, the test item was not mutagenic under the described experimental conditions (UN GHS: no category).

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

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

The test item was examined for its mutagenic activity in two series of in vitro microbial assays employing Salmonella typhimurium TA 98, TA 100, TA 1535 and TA 1537, and Escherichia coli WP2 uvrA as indicator organisms. The plate incorporation test with and without addition of liver S9 mix from Aroclor 1254-pre-treated rats was used. In this study, two experimental series was performed. In the experiments with S9 mix, 10 and 30% S9 were used in the 1st and 2nd series, respectively. Treatments of all tester strains were performed using test item formulations prepared in anhydrous analytical grade dimethyl sulfoxide (DMSO) in the absence and in the presence of S9 mix, using final concentrations between 5 and 5000 µg/plate, plus vehicle and positive controls. The strain specific positive control test materials, namely daunomycin, sodium azide, 4-nitroquin-olin-N-oxide, and 9-aminoacridine in the absence of S9 mix, yielded the expected mutant frequencies that were greatly in excess of the negative controls. The genotype of the tester strains used was thus confirmed. 2-Aminoanthracene which requires metabolic activation was strongly mutagenic. This indicates that the exogenous metabolizing system used in the present investigation (S9 mix) was functioning. Thus, the acceptance criteria have been met in total and the study is considered valid. Following test item treatments, precipitation of the test material on the agar plates was not occurred. There was no toxicity to the bacteria observed. Under the conditions described, there were no relevant increases in revertant numbers after test item exposure observed in both, the absence and presence of S9 mix. According to the criteria for negative and positive results, the test item was not mutagenic under the described experimental conditions (UN GHS: no category).

Justification for classification or non-classification

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

The available experimental 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 considered to be classified for genetic toxicity (UN GHS: no category) according to Regulation (EC) No 1272/2008 (CLP), as amended for the tenth time in Regulation (EC) No 2017/776.