Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In an in vitro bacterial reverse mutation assay (Ames) according to OECD guideline 471 (BASF SE, 2004), the test item did not show a mutagenic potential.

In an in vitro mammalian cell forward mutation assay according to EU Method B.17 (BASF SE, 2014), the test item did not show a mutagenic potential.

In an in vitro chromosome aberration assay in human lymphochytes according to OECD guideline 473 (O´Hagan, 2003), the test item did not show a clastogenic potential.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2003-10-13 to 2004-01-08
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:
1997-07-21
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
2000-05-19
Deviations:
no
GLP compliance:
yes (incl. certificate)
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: GR31143161
- Expiration date of the batch: 24.04.2014

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

OTHER SPECIFICS: colourless-yellowish liquid
Target gene:
his
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital and ß-Naphthoflavone induced male rat liver.
Test concentrations with justification for top dose:
Experiment I and II ( except for strains TA 1537 and TA 102 without 89 mix): 33, 1 00, 333, 1000, 2500, and 5000 µg/plate
Experiment II (strains TA 1537 and TA 102 without 89 mix): 0.3, 1, 3, 10, 33, 100, 250, and 500 µg/plate
ln the pre-experiment the concentration range of the test item was 3- 5000 µg/plate. The pre-experiment is reported as part of experiment I since no relevant toxic effects were observed and 5000 µg/plate were chosen as maximal concentration for experiment I.
Vehicle / solvent:
- Solvent used: ethanol
- Justification for choice of solvent: The solvent was chosen because of its solubility properties and its relative nontoxicity to the bacteria.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
without S9 mix, TA1535, TA100, 10 µg/plate
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 4-nitro-o-phenylene-diamine
Remarks:
without S9 mix, TA1537, 50 µg/plate; TA98, 10 µg/plate
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
without S9 mix, TA102, 4 µL/plate
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
with S9 mix, TA1535, TA1537, TA98, TA100, 2.5 µg/plate; TA102, 10 µg/plate
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation); preincubation

DURATION
- Preincubation period: 60 min
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: Reduction in the number of spontaneaus revertants or a clearing of the bacterial background lawn.

Rationale for test conditions:
To establish a dose response effect six dose Ievels with adequately spaced concentrations were tested. The maximum dose Ievel was 5000 µg/plate.
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 TA 102) 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 secend 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 contros such an increase is not considered biologically relevant.
Statistics:
A statistical analysis of the data is not required.
Key result
Species / strain:
S. typhimurium TA 98
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 100
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 102
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
Key result
Species / strain:
S. typhimurium TA 1535
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
Key result
Species / strain:
S. typhimurium TA 1537
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

Table 1: Cytotoxic effects

Strain

Experiment I

Experiment II

 

- S9 mix

+ S9 mix

- S9 mix

+ S9 mix

TA1535

-

-

5000

-

TA1537

2500

5000

100

2500, 5000

TA98

-

-

-

-

TA100

-

-

-

-

TA102

-

-

33 - 500

-

 Table 2: Summary of results, without S9 mix

Concentration [µg/plate]

Revertants/plate [Mean]

TA1535

TA1537

TA98

TA100

TA102

I

II

I

II

I

II

I

II

I

II

Neg. Control

11

19

6

5

25

28

185

17

200

234

Solvent Control

16

22

6

5

25

29

188

183

177

169

Pos. Control #

602

1075

54

73

165

199

557

502

1339

917

0.3

-

-

-

7

-

-

-

-

-

150

1

-

-

-

4

-

-

-

-

-

166

3

-

-

-

6

-

-

-

-

-

172

10

-

-

-

6

-

-

-

-

-

175

33

13

18

6

6

23

20

222

158

152

52

100

11

18

5

8

20

21

225

174

161

32

250

-

-

-

8

-

-

-

-

-

18

333

9

14

4

-

21

17

148

152

159

-

500

-

-

-

4

-

-

-

-

-

0

1000

7

14

3

-

19

14

206

124

152

-

2500

11

16

3

-

19

20

232

154

155

-

5000

9

9

5

-

20

21

208

178

153

-

- Not performed

# Sodium azide (10.0 µg/plate) strains TA 1535 and TA 100; 4-nitro-o-phenylene-diamine strains TA 1537 (50 µg/plate) and TA 98 (10.0 µg/plate), Methylmethanesulfonate (4 1 µL/plate) strain TA 102

Table 3: Summary of results, with S9 mix 

Concentration [µg/plate]

Revertants/plate [Mean]

TA1535

TA1537

TA98

TA100

TA102

I

II

I

II

I

II

I

II

I

II

Neg. Control

11

11

10

14

21

18

179

188

184

216

Solvent Control

19

27

9

13

28

31

202

190

176

134

Pos. Control ##

312

274

265

224

498

472

801

709

1355

1246

33

20

29

10

8

30

29

257

156

156

122

100

18

19

10

12

33

25

211

196

172

108

333

16

17

9

11

32

25

210

167

153

116

1000

12

14

6

6

29

19

198

114

152

98

2500

13

19

6

4

31

26

179

137

162

102

5000

13

18

4

3

30

27

174

142

109

156

## 2-aminoanthracene (2.5 µg/plate) strains TA 1535, TA 1537, TA 98, and TA 100; 2-aminoanthracene (10.0 µg/plate) strain TA 102

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2003-09 to 2004-01-12
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
1997-07-21
Deviations:
yes
Remarks:
mutations at the mouse lymphoma thymidine kinase locus
Qualifier:
according to
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
2000-05-19
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
other: mutations at the mouse lymphoma thymidine kinase locus
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Batch No. of test material: GR31143161
- Expiration date of the batch: 24.04.2014

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

OTHER SPECIFICS: colourless-yellowish liquid
Target gene:
thymidine kinase (TK) locus
Species / strain / cell type:
mouse lymphoma L5178Y cells
Remarks:
heterozygous L5178Y/TK+/-
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: cell bank of RCC Cytotest Cell Research
- Suitability of cells: The L5178Y cell line has successfully been used in in vitro experiments for many years.
- Doubling time: 10 -12 h
- Methods for maintenance in cell culture if applicable: Large stocks of the cleansed L5178Y cell line are stored in liquid nitrogen in the cell bank of RCC Cytotest Cell Research allowing the repeated use of the same cell culture batch in many experiments. Before freezing, each batch was screened for mycoplasma contamination and checked for karyotype stability. Consequently, the parameters of the experiments remain similar because of the reproducible characteristics of the cells. The cells are sub-cultured at least three times a week.
- Modal number of chromosomes: 40 ± 2

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: Thawed stock cultures are propagated in plastic flasks in RPMI 1640 complete culture medium. The cell cultures are incubated at 37 °C in a humidified atmosphere with 4.5 % carbon dioxide and 95.5 % air.
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically 'cleansed' against high spontaneous background: yes
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital/beta-Naphthoflavone induced rat liver (S9 mix)
Test concentrations with justification for top dose:
Experiment I:
without S9 mix 2.5, 5.0, 10.0, 15.0, and 20.0 µg/mL
with S9 mix 5.0, 10.0, 15.0, 20.0, 25.0, and 30.0 µg/mL

Experiment II: 10.0, 20.0, 40.0, 60.0, 80.0, 100.0 µg/mL

According to exceedingly toxic effects, no higher concentrations were investigated.
Vehicle / solvent:
- Solvent used: DMSO, Tween
- Justification for choice of solvent: The solvent was chosen because of its solubility properties and its relative non-toxicity to the cell cultures. In order to increase the solubility of the test item 0.016 % Tween was added to the culture medium. Due to the known toxicity of Tween, the optimal concentration to be applied in the experiments was determined in a separate experiment.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
without S9 mix, 6.5, 13.0 µg/mL
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
with S9 mix, 3.0 µg/mL
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium
- Cell density at seeding: 10E7 cells/flask (80 cm² flasks)

DURATION
- Exposure duration:
Experiment I: 4 h
Experiment II: 24 h
- Expression time: 72 h

SELECTION AGENT (mutation assays): RPMI 1640 (complete culture medium) by addition of 5 pg/mL TFT.

NUMBER OF REPLICATIONS: 2

DETERMINATION OF CYTOTOXICITY
- Method: relative suspension growth




Evaluation criteria:
A test item is classified as positive if it induces either a reproducible concentration-related increase in the mutant frequency or a reproducible positive response for at least one of the test points.
A test item producing neither a reproducible concentration-related increase in the mutant frequency nor a reproducible positive response at any of the test points is considered nonmutagenic in this system.
A significant response is described as follows: The test item is classified as mutagenic if it reproducibly induces a mutation frequency that is at least two times higher than the spontaneous mutation frequency (negative or solvent control) in the experiment.
The test item is classified as mutagenic if there is a reproducible concentration-related increase in the mutation frequency. Such an evaluation may be considered independently of the enhancement factor for induced mutants. However, in the evaluation of the test results the historical variability of the mutation rates in negative and solvent controls and the mutation rates of ail negative and solvent controls of this study are taken into consideration.
Results of test groups are rejected if the relative total growth, the relative suspension growth and/or cloning efficiency 1 is less than 10 % of the solvent control or the cloning efficiency 2 after the expression period is less than 20 %. Whenever a test item is considered mutagenic according to the above mentioned criteria, the ratio of small versus large colonies is used to differentiate point mutations and clastogenic effects. If the increase of the mutation frequency is accompanied by a reproducible and dose dependent shift in the ratio of small versus large colonies clastogenic effects are indicated
Key result
Species / strain:
mouse lymphoma L5178Y cells
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:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH/osmolality: There was no relevant shift in osmolarity and pH-values of the medium even at the maximal concentration of the test item in the pre-experiment.
- Precipitation: not observed


RANGE-FINDING/SCREENING STUDIES: Test item concentrations between 0.47 and 60 µg/mL (with and without S9-mix) were used to evaluate toxicity. Relevant toxic effects were observed at 15.0 µg/mL and above in the absence and at 60.0 µg/mL in the presence of metabolic activation (4 hours treatment). Following puise treatment (24 hours) a toxic effect occurred at 60.0 µg/mL. According to these results the concentrations for experiment I were set up. However, due to exceedingly severe toxic effects, the first experiment had to be terminated prior to the generation of mutagenicity data and had to be repeated in a lower dose range.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data:
4 h treatment:
MMS: without S9 mix, Range: 185 - 1398, Mean 468, S.D. 203
CPA: with S9 mix, historical data of the positive control CPA used in the experiments are not available at present. Both positive contros, 3-MC and CPA, are covered by the OECD guideline. The use of 3-MC was discontinued, because this substance is not available anymore in Europe due to its carcenogenicity

24 h treatment
MMS: without S9 mix, Range 203 - 1792, Mean 705, S.D. 330

- Negative historical control data:
4 h treatment:
without S9 mix, Range 41 - 175, Mean 97, S.D. 30
with S9 mix, Range 43 - 186, Mean 103, S.D. 31

24 h treatment
without S9 mix, Range 41 - 186, Mean 92, S.D. 30

- Solvent historical control data:
4 h treatment:
without S9 mix, Range 4 - 181, Mean 98, S.D. 31
with S9 mix, Range 3 - 202, Mean 106, S.D. 30

24 h treatment
without S9 mix, Range 33 - 192, Mean 97, S.D. 35

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: total suspension growth, relative total growth

Table 1: Summary of Results 

 

Conc. [µg/mL]

S9 mix

Relative Cloning Efficiency

Relative Total Growth

Mutant Colonies/10E6 cells

Induction Factor

Relative Cloning Efficiency

Relative Total Growth

Mutant Colonies/10E6 cells

Induction Factor

4 h Treatment

Culture I

Culture II

Neg. Control

 

-

100.0

100.0

113

1.0

100.0

100.0

71

1.0

Neg. Control *

 

-

100.0

100.0

138

1.0

100.0

100.0

71

1.0

Solvent Control

 

-

100.0

100.0

132

1.0

100.0

100.0

97

1.0

Pos. Control

13.0

-

107.6

30.1

809

7.2

76.0

13.4

898

12.6

Test Item

1.25

-

98.2

Culture was not continued#

105.9

Culture was not continued

2.5

-

100.0

103.2

93

0.7

82.8

93.3

90

0.9

5.0

-

98.2

91.0

89

0.7

82.8

135.1

98

1.0

10.0

-

98.2

74.6

102

0.8

75.4

92.6

65

0.7

15.0

-

75.4

36.9

105

0.8

70.9

86.0

79

0.8

20.0

-

28.0

46.4

90

0.7

44.2

61.0

65

0.7

25.0

-

0.5

Culture was not continued##

6.3

Culture was not continued##

30.0

-

-1.0

Culture was not continued##

2.8

Culture was not continued##

 

Neg. Control

 

+

100.0

100.0

95

1.0

100.0

100.0

87

1.0

Neg. Control *

 

+

100.0

100.0

97

1.0

100.0

100.0

75

1.0

Solvent Control

 

+

100.0

100.0

93

1.0

100.0

100.0

108

1.0

Pos. Control

3.0

+

49.9

42.4

231

2.4

63.3

55.4

285

12.6

Test Item

1.25

+

111.6

Culture was not continued#

76.9

Culture was not continued#

2.5

+

97.6

Culture was not continued#

82.4

Culture was not continued#

5.0

+

65.2

115.6

84

0.9

72.0

90.5

49

0.5

10.0

+

82.0

116.2

84

0.9

67.6

54.1

163

1.5

15.0

+

66.3

78.5

139

1.5

61.7

63.1

164

1.5

20.0

+

91.0

44.5

189

2.0

66.6

77.8

127

1.2

25.0

+

47.5

31.7

169

1.8

41.3

48.4

176

1.6

30.0

+

18.8

21.4

103

1.1

22.0

40.1

107

1.0

24 h Treatment

Culture I

Culture II

Neg. Control

 

-

100.0

100.0

109

1.0

100.0

100.0

164

1.0

Neg. Control *

 

-

100.0

100.0

179

1.0

100.0

100.0

90

1.0

Solvent Control

 

-

100.0

100.0

124

1.0

100.0

100.0

129

0.8

Pos. Control

6.5

-

61.2

69.8

371

3.4

88.7

76.3

204

1.2

Pos. Control

13.0

-

70.9

17.4

943

8.6

29.7

16.8

508

3.1

Test Item

10.0

-

81.6

87.4

124

1.0

40.7

78.9

222

1.7

20.0

-

83.2

71.2

111

0.9

64.5

76.6

206

1.6

40.0

-

32.9

27.4

136

1.1

42.6

28.5

217

1.7

60.0

-

28.9

15.1

113

0.9

39.5

13.6

305

2.4

80.0

-

10.7

4.3

182

1.5

9.6

11.1

189

1.5

100.0

-

0.4

Culture was not continued##

0.3

Culture was not continued##

# culture was not continued since a minimum of four concentrations is required by the guidelines

## not determined, culture not continued due to exceedingly strong toxic effects

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
2002
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study without detailed documentation
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
1997
Deviations:
no
GLP compliance:
yes
Type of assay:
other: Chromosome aberration assay
Specific details on test material used for the study:
The 9cis,11trans and 10trans,12cis CLA isomers are present in approximately equal proportions in the test item, and these two isomers account for approximately 75% of the total fatty acid content of the product. Other CLA isomers are also present and account for approximately 4.4% of the total fatty acid content. The remaining fatty acids are mainly monounsaturated e.g. oleic acid, and saturated e.g. palmitic and stearic acid. The poly- and saturated e.g. palmitic and stearic acid. The poly-unsaturated fatty acids, other than CLA, consist mainly of linoleic acid. The typical fatty acid composition of the test item is given in table 1.
Species / strain / cell type:
lymphocytes:
Remarks:
human peripheral blood lymphocytes
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
Experiment I: 128, 160 and 200 µg/mL
Experiment II:
192, 240 and 300 µg/mL, without S9 mix;
153, 240 and 300 µg/mL, with S9 mix
Vehicle / solvent:
- Vehicle/solvent used: not specified
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
other: 4-nitroquinoline 1-oxide
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium;
- Cell density at seeding: not specified

DURATION
- Exposure duration: 3 h
- Expression time:
Experiment I: 17 h
Experiment II: 20 h after exposure without S9 mix, 17 h after exposure with S9 mix

Evaluation criteria:
The test article was considered positive if the proportion of cells with structural aberrations at one or more concentrations exceeded the normal range in both replicates and a statistically significant increase in the proportion of cells with structural aberrations (excluding gaps) occurred at these doses.
Key result
Species / strain:
lymphocytes:
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
not specified
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation:
Experiment I: 200 µg/plate
Experiment II: not specified
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

In vitro reverse bacterial mutation assay

In an in vitro bacterial reverse mutation assay (Ames) according to OECD guideline 471 (BASF SE, 2004), the potential of the test item to induce gene mutations in the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and TA 102 was investigated (experiment I: plate incorporation; experiment II: pre-incubation). The experiments were performed in triplicates, with or without S9 mix (Phenobarbital/ ß-Naphthoflavone induced male rat liver). No visible reduction of the background growth was observed in experiment I. ln experiment II, reduced background growth was observed at higher concentrations, except in strains TA 1537 and TA 102 reduced background growth was observed atconcentrations of 33 µg/plate and above. Toxic effects, evident as a reduction in the number of revertants, were observed in strain TA 1537 with and without S9 mix in experiment I. ln experiment II, toxic effects were observed in strains TA 1535, TA 1537, and TA 102 without S9 mix and in strain TA 1537 with S9 mix. Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies. Parallel performed negative and solvent controls were valid. No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with the test item at any dose Ievei, 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.

ln 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 basepair changes or frameshifts in the genome of the strains used. Therefore, the test item is considered to be non-mutaganic in this Salmonella typhimurium reverse mutation assay.

These results are supported by further in vitro bacterial reverse mutation assays (Ames) similar or according to OECD guideline 471. Strains of S. typhimurium were exposed to concentrations up to 5000 µg/plate (TA 1535, TA 1537, TA 98, and TA 100) or up to 3930 µg/plate (TA102, calculation error), respectively, with or without S9 mix (O´Hagan, 2003). In another published study (Zeiger, 1983), strains of S. typhimurium TA98, TA100, TA1535, and TA1537 and/or TA97 were tested for mutagenicity. A further study on mutagenicity was performed by BASF SE (BASF SE, 1980). TA1535 and E. coli WP2 uvrA were exposed to concentrations up to 1000 µg/plate, TA98, TA1537, TA1538 to concentrations up to 3333.3 µg/plate ans TA100 up to 10000 µg/plate with or without S9 mix. In any study was an increased revertant count observed under the given experimental conditions .

 

In vitro mammalian cell mutation assay

In an in vitro mammalian cell gene mutation assay according to EU method B.17 (BASF SE, 2004), the potential of the test item to induce mutations at the mouse lymphoma thymidine kinase locus using the cell line L5178Y was determined. The assay was performed in two independent experiments, using two parallel cultures each. The first main experiment was performed with and without liver microsomal activation and a treatment period of 4 h. Due to exceedingly severe toxic effects, the first experiment had to be terminated prior to the generation of mutagenicity data and repeated in a lower dose range. The second experiment was performed solely without metabolic activation with a treatment period of 24 h. Due to the exceedingly high toxicity of positive control, the second experiment was terminated prematurely and the second experiment was repeated under identical conditions. The test item was evaluated at the following concentrations:

Experiment I

without S9 mix: 2.5; 5.0; 10.0; 15.0; and 20.0 µg/mL

with S9 mix: 5.0; 10.0; 15.0; 20.0; 25.0; and 30.0 µg/mL

Experiment Il

without S9 mix: 10.0; 20.0; 40.0; 60.0; and 80.0 µg/mL

In experiment I strong toxic effects occurred at concentrations of 25 µg/mL and above in the absence and at 20 µg/mL and above in the presence of metabolic activation. In the second experiment (solely performed without metabolic activation) strong toxic effects were observed at 40 µg/mL and above after 24 hour treatment with the test item. The cell growth observed at the lowest concentrations was approximately in the range of the negative control. No precipitation of the test item was observed in both main experiments up to the maximum concentration. In experiment I without S9 mix (4 hour treatment interval) no increase in mutant colony numbers was observed up to the highest investigated concentration (20.0 µg/mL).The toxicity range of 10 - 20 % as recommended in the current OECD guideline was not reached at the highest evaluated concentration in both cultures (cloning efficiency: 28.0 and 44.2 %, respectively and relative total growth: 46.4 and 61.0 %, respectively). The next higher concentration (25.0 µg/mL) could not be evaluated due to the extreme toxicity of the test item. However, a negative result in an assay is considered acceptable if a dose spacing of < 2 was used and the highest dose tested showed < 20 % cloning efficiency or relative total growth, or total kill. Therefore, the obtained results are considered to be valid. In experiment I in the presence of metabolic activation (4 hour treatment interval) an isolated and moderate increase in the frequency of mutant colonies reaching the threshold of twice the frequency of mutant colonies occurred at 20.0 µg/mL in culture I. As the number of mutant colonies exceeded our historical data range by only 3 colonies (186 versus 189 mutant colonies/per 10E6 cells) and such an increase was not obtained in culture Il, this effect is not considered to be biologically relevant. In experiment Il in culture Il without metabolic activation (24 hour treatment interval) the mutant colony numbers obtained at 10, 20, 40, and 60 µg/mL exceeded our historical control data range. However, the threshold of twice the frequency of mutant colonies was only exceeded in the second culture at 60.0 µg/mL (induction factor 2.4). Since no such increase was found in the parallel culture, the observed effects in culture Il are considered not to be biologically relevant.

In this study the range of the negative controls was from 71 up to 179 mutant colonies per 10E6 cells; the range of the groups treated with the test item was from 49 up to 305 mutant colonies per 10E6 cells.

 Methylmethanesulfonate (MMS, 13 µg/mL) and cyclophosphamide (CPA, 3.0 µg/mL) were used as positive controls and showed a distinct increase in induced total mutant colonies and an increase of the relative quantity of small versus large colonies. Since the second experiment had to be repeated due to exceedingly high toxicity in the positive control (MMS 13.0 µg/mL), the repeat experiment was performed with an additional positive control concentration (MMS 6.5 µg/mL). In this additional positive control the threshold of twice the mutant colony number of the corresponding negative control was not reached. However, the number of mutant colony numbers at the standard concentration (MMS 13.0 µg/mL) showed a distinct increase in mutant colony numbers (943 and 508/ 10E6 cells, respectively) and remained well within the range of our historical data (203 to 1792 mutant colonies/10E6 cells).

In conclusion, it can be stated that under the experimental conditions reported the test item did not induce mutations in the mouse lymphoma thymidine kinase locus assay using the cell line L5178Y in the absence and presence of metabolic activation.

 

In vitro chromosome aberration assay

The clastogenic potential of the test item was determined in an in vitro chromosome aberration assay in human lymphochytes according to OECD guideline 473 (O´Hagan, 2003). Treatment of lymphocytes with the test item did not induce elevated frequencies of cells with chromosomal aberrations (structural or numerical) compared to concurrent negative control. Under the test conditions reported, the test item did not show a clastogenic potential.

 

Conclusion

The test item did not show a mutagenic potential in bacterial or mammalian cell test systems.

Justification for classification or non-classification

Classification, Labeling, 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. The test item did not show a mutagenic potential. As a result the test 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.