Registration Dossier

Diss Factsheets

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

No mutagenic potential was observed in a bacterial reverse mutation assay conducted with the test substance. A mouse lymphoma assay was also negative. The test substance did not show any clastogenic activity in a chromosome aberration assay.

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:
1988-02-12 to 1988-02-28
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study without detailed documentation
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
yes
Remarks:
Only 4 tester strains were used.
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
S. typhimurium, other: TA 98, 100, 1535, 1537
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Aroclor induced rat liver (S9 mix)
Test concentrations with justification for top dose:
DRF: 5, 50, 500, 5000 µg/plate with or without metabolic activation
Main test: 50, 150, 500, 1500, 5000 µg/plate with or without metabolic activation
Vehicle / solvent:
- Vehicle used: DMSO
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 µg/plate for strains TA 1535 and TA 1537; 0.5 µ/plate for strain TA 98; 1 µg/plate for strain TA 100
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
2-nitrofluorene
Remarks:
1 µg/plate for strain TA 98, without S9 mix
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
80 µg/plate for strain TA 1537, without S9 mix
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
N-ethyl-N-nitro-N-nitrosoguanidine
Remarks:
Without S9 mix; 5 µg/plate for strain TA 1535; 3 µg/plate for strain TA 100.
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)
- Cell density at seeding: approximately 2 x 10^9 organisms per mL which was assessed photometrically.

DURATION
- Exposure duration: 72 h

NUMBER OF REPLICATIONS: 2

DETERMINATION OF CYTOTOXICITY
- Method: reduction in revertant colony counts or by the absence of a complete background bacterial lawn.
Rationale for test conditions:
According to the guideline
Evaluation criteria:
The mean number of revertant colonies for all treatment groups is compared with those obtained for negative and positive control groups. The effect of metabolic activation is assessed by comparing the results obtained both in the presence and absence of the liver microsomal fraction for each treatment group. A Compound is deemed to provide evidence of mutagenic potential if a statistically significant dose-related increase in the number of revertant colonies of at least twice the concurrent solvent control is obtained in two separate experiments.
Key result
Species / strain:
S. typhimurium TA 1538
Remarks:
Please refer to study entry of YKG26 for further information
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 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 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 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 1537
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
Additional information on results:
Concentration of the test substance resulting in precipitation: 1500 μg/plate

Table 1: Summary of the Results, Test 1

Strain

Dose Level (µg/plate)

S9 mix

Mean revertant colony count

SD

Individual revertant colony count

TA 1535

5000

-

7

0.6

7P,8P,7P

1500

-

10

1.2

11P,9P,11P

500

-

13

2.9

15,15,10

150

-

10

1.0

11,9,10

50

-

16

1.5

14,16,17

0

-

12

2.1

10,11,14

Solvent

-

14

2.3

15,11,15

5000

+

7

1.5

6P,7P,9P

1500

+

9

3.1

12P,10P,6P

500

+

10

1.2

11,11,9

150

+

12

6.1

9,19,8

50

+

10

4.0

6,14,11

0

+

10

1.5

12,9,10

Solvent

+

12

4.2

9,11,17

TA 1537

5000

-

7

2.6

6P,5P,10P

1500

-

8

1.2

7P,9P,9P

500

-

14

2.1

16,12,15

150

-

8

1.5

10,7,8

50

-

8

2.1

6,9,10

0

-

14

1.5

16,14,13

Solvent

-

16

3.5

18,12,18

5000

+

8

1.5

6P,9P,8P

1500

+

9

2.0

9P,11P,7P

500

+

11

1.5

9,11,12

150

+

14

5.1

8,18,15

50

+

11

3.1

8,12,14

0

+

10

1.2

11,11,9

Solvent

+

11

1.5

11,10,13

TA 98

5000

-

20

2.0

20P,22P,18P

1500

-

30

5.7

35P,32P,24P

500

-

27

12.5

23,41,17

150

-

28

3.1

25,31,29

50

-

32

7.5

33,39,24

0

-

35

5.8

38,28,38

Solvent

-

30

4.0

34,31,26

5000

+

11

1.0

12P,10P,11P

1500

+

19

5.3

13P,21P,23P

500

+

21

6.1

22,14,26

150

+

21

5.3

25,23,15

50

+

18

2.6

20,19,15

0

+

19

1.0

18,20,19

Solvent

+

19

1.2

20,20,18

TA 100

5000

-

81

2.5

81P,78P,83P

1500

-

104

15.7

97P,122P,93P

500

-

117

13.1

103,129,119

150

-

122

16.5

141,113,112

50

-

119

8.6

111,128,117

0

-

120

4.5

120,116,125

Solvent

-

113

10.0

103,114,123

5000

+

91

11.5

79P,92P,102P

1500

+

103

4.6

98P,104P,107P

500

+

135

6.1

140,128,136

150

+

129

8.5

132,119,135

50

+

125

15.8

139,129,108

0

+

132

9.1

128,142,125

Solvent

+

121

3.1

122,124,118

Positive Control

TA 1535

ENNG

5

-

771

38.4

740,759,814

TA 1537

9-AA

80

-

X

-

X, X, X,

TA 98

NF

1

-

107

11.4

110,116,94

TA 100

ENNG

3

-

378

25.0

361,367,407

TA 1535

2-AA

2

+

100

10.6

96,92,112

TA 1537

2-AA

2

+

74

9.1

81,64,78

TA 98

2-AA

0.5

+

135

17.1

133,119,153

TA 100

2-AA

1

+

348

32.1

351,378,314

P Precipitation

SD Standard deviation

X Too many colonies for accurate counting

ENNG N-ethyl-N'-nitro-N-nitrosoguanidine

9 AC 9-aminoacridine

2-AA 2-aminoanthracene

NF 2-nitrofluorene

Table 2: Summary of the Results, Test 2

Strain

Dose Level (µg/plate)

S9 mix

Mean revertant colony count

SD

Individual revertant colony count

TA 1535

5000

_

7

0.6

7P,8P,7P

1500

-

10

3.0

10P,7P,13P

500

-

11

1.0

12,11,10

150

-

9

3.5

13,7,7

50

-

13

2.6

10,15,14

0

-

15

1.5

16,15,13

Solvent

-

14

2.5

17,14,12

5000

+

5

1.5

5P,7P,4P

1500

+

11

2.0

9P,11P,13P

500

10

7

2.1

5,8,9

150

+

14

4.2

15,9,17

50

+

7

2.5

7,4,9

0

+

13

3.6

9,16,14

Solvent

+

14

4.6

18,15,9

TA 1537

5000

-

6

2.1

5P,4P,8P

1500

-

9

1.5

9P,10P,7P

500

-

12

1.5

14,11,12

150

-

9

2.5

7,12,9

50

-

9

3.2

8,13,7

0

-

12

3.6

16,11,9

Solvent

-

14

0.6

14,13,14

5000

+

6

1.0

7P,6P,5P

1500

+

10

0.0

10P,10P,10P

500

+

11

2.6

16,11,9

150

+

10

2.1

12,8,9

50

+

11

0.6

11,11,10

0

+

13

2.0

11,13,15

Solvent

+

15

1.5

13,15,16

TA 98

5000

-

24

6.0

30P,25P,18P

1500

-

23

1.7

24P,24P,21P

500

-

34

4.0

34,30,38

150

-

29

8.3

36,20,32

50

-

31

7.0

32,24,38

0

-

37

4.4

32,39,40

Solvent

-

36

8.0

28,37,44

5000

+

13

1.5

15P,13P,12P

1500

+

17

3.1

20P,16P,14P

500

+

22

6.1

15,23,27

150

+

20

5.3

26,18,16

50

+

17

2.5

17,20,15

0

+

19

2.1

17,21,18

Solvent

+

20

1.5

19,20,22

TA 100

5000

-

89

13.9

85P,77P,104P

1500

-

99

6.7

95P,107P,96P

500

-

135

17.0

118,134,152

150

-

133

13.5

133,120,147

50

-

138

2.1

137,140,136

0

-

142

9.1

146,132,149

Solvent

-

127

18.8

117,149,116

5000

+

107

9.0

116P,98P,108P

1500

+

109

19.1

102P,95P,131P

500

+

139

7.8

141,130,145

150

+

137

3.5

139,133,139

50

+

133

7.8

137,138,124

0

+

148

10.1

153,136,154

Solvent

+

142

13.3

157,134,134

Positive Control

TA 1535

ENNG

5

-

231

70.6

158,235,299

TA 1537

9-AA

80

-

2348

55.5

2292,2403,2350

TA 98

NF

1

-

127

4.7

131,129,122

TA 100

ENNG

3

-

357

12.1

368,344,358

TA 1535

2-AA

2

+

124

9.5

131,127,113

TA 1537

2-AA

2

+

76

7.1

77,68,82

TA 98

2-AA

0.5

+

129

7.0

122,136,129

TA 100

2-AA

1

+

432

48.6

377,450,469

P Precipitation

SD Standard deviation

X Too many colonies for accurate counting

ENNG N-ethyl-N'-nitro-N-nitrosoguanidine

9 AC 9-aminoacridine

2-AA 2-aminoanthracene

NF 2-nitrofluorene

Conclusions:
The test substance did not show any mutagenic effects in any of the tester strains.
Executive summary:

In a reverse mutation assay in bacteria according to EU method B.13/14 (Huntingdon, 1988), four strains (TA98, TA100, TA1535, TA1537) of S. typhimurium were exposed to the test substance dissolved in DMSO at concentrations of 50 to 5000 µg/plate in the presence and absence of metabolic activation. The test substance was tested up to a limit concentration of 5000 µg/plate. Precipitation was observed at concentrations of 1500 µg/plate and above. There was no evidence or a concentration related positive response of induced mutant colonies over background. Thus, the test substance is not regarded as mutagenic.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1988-06-15 to 1988-07-28
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Remarks:
K1-BH4
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: BIBRA
- Suitability of cells: The cell line has been frequently used in this test system.
- Methods for maintenance in cell culture: The cells were routinely grown and subcultured in Hams F12 medium (Imperial) supplemented with 5 % foetal calf serum (Gibco) at 37 °C in a humid atmosphere containing 5 % carbon dioxide in 175 cm² plastic tissue culture flasks (Nunc).
- Modal number of chromosomes: 20

MEDIA USED
- Type and identity of media including CO2 concentration: Hams F12 medium (Imperial) 5 %
- Properly maintained: Yes
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254 – induced rat liver (S9 mix)
Test concentrations with justification for top dose:
4, 20 and 40 μg/mL,
At 40 µg/mL, no cytotoxicity was observed in a preliminary toxicity test. However, all final concentrations greater than 40 µg/mL caused precipitate formation in aqueous tissue culture medium. 40 µg/mL is, therefore, considered to be the maximum achievable concentration.
Vehicle / solvent:
- Vehicle used: DMSO
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
0.4 µg/L Mitomycin C (without S9 mix)
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
20 µg/L Cyclophosphamide (with S9 mix)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium
- Cell density at seeding: 8 x 10^4 cells/mL

DURATION
- Exposure duration: 21 hours (without S9 mix), 4 hours (with S9 mix)
- Fixation time (start of exposure up to fixation or harvest of cells): 21 hours

NUMBER OF REPLICATIONS: 2

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth, mitotic index

OTHER EXAMINATIONS:
- Determination of polyploidy: Yes
- Determination of endoreplication: Yes
Evaluation criteria:
The test article was considered to induce a positive response when the percentage of cells with aberrations are increased in a dose-responding manner with one or more concentrations being statistically elevated relative to the solvent control group (p<0.05).
Statistics:
Fisher’s exact test.
Key result
Species / strain:
Chinese hamster Ovary (CHO)
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
Additional information on results:
Concentrations of above 40 µg/mL led to precipitation of the test substance.

Table 1: Summary of the Results

Substance

Conc., µg/mL

No of cells examined

No of Aberrations/100 Cells

Aberrations

No of Aberrant Cells

Exc. Gaps

Inc. gaps

BWF

BF

I

SM

R

A

GT

P

CHR

EXC. Gaps

Mean, %

Inc Gaps

%, Mean

Without S9 mix

DMSO

(Solvent Control)

10 µL/mL

100

0

0

 

 

 

 

 

 

 

 

 

0

0.25

0

0.25

100

0

0

 

 

 

 

 

 

 

 

 

0

0

100

1

1

 

 

 

 

 

1

 

 

 

1

1

100

0

0

 

 

 

 

 

 

 

 

 

0

0

Test Item

4

100

0

0

 

 

 

 

 

 

 

 

 

0

0.0

0

0.0

100

0

0

 

 

 

 

 

 

 

 

 

0

0

20

100

1

1

 

 

 

1

 

 

 

 

 

1

0.5

1

0.5

100

0

0

 

 

 

 

 

 

 

 

 

0

0

40

100

1

1

1

 

 

 

 

 

 

 

 

1

0.5

1

0.5

100

0

0

 

 

 

 

 

 

 

 

 

0

0

Mitomycin C

(pos. Control)

0.4

100

27

29

9

 

6

1

2

4

4

1

2

18

18.7*

19

19.8*

87

28.7

29.9

4

1

9

4

3

1

2

1

1

17

18

With S9 mix

DMSO

(Solvent Control)

10 µL/mL

100

6

6

1

2

1

1

 

1

 

 

 

6

4.25

6

4.25

100

4

4

2

1

 

 

 

1

 

 

 

3

3

100

7

7

1

 

3

 

 

3

 

 

 

5

5

100

3

3

1

 

 

2

 

 

 

 

 

3

3

Test Item

4

100

3

3

 

 

 

1

 

1

1

 

 

3

3.0

3

3.0

100

3

3

 

 

 

 

 

3

 

 

 

3

3

20

100

2

2

 

 

 

2

 

 

 

 

 

2

3.0

2

3.0

100

4

4

 

 

 

4

 

 

 

 

 

4

4

40

100

4

4

 

 

 

 

2

2

 

 

 

4

3.5

4

3.5

100

4

4

2

 

1

1

 

 

 

 

 

3

3

Cyclophosphamide

(pos. Control)

20

78

97.4

107.7

14

2

10

24

 

224

 

 

8

43

54.4*

44

55.3

36

113.9

116.7

5

1

7

7

1

20

2

 

1

19

19

Statistical analysis used was Fisher's test * p < 0.001, Otherwise p > 0.05

BWF Chromatid break with fragment, A Acentric fragment, BF Chromatid break without fragment, GT Greater than 10 aberrations, I Interchange, P Pulverised cell, SM Single minute, CHR Chromatid gap, R Ring

Conclusions:
The test substance has shown no evidence of clastogenic activity in this in vitro cytogenetic test system.
Executive summary:

In a mammalian cell cytogenetics assay (chromosome aberration) according to OECD guideline 473 (Huntingdon, 1989), CHO cell cultures were exposed to the test substance dissolved in DMSO at concentrations of 4, 20 and 40 µg/mL with and without metabolic activation ( Aroclor 1254 – induced rat liver). In a preliminary study the highest dose used did not produce any reduction in mitotic index. The highest dose used (40 µg/mL) was the maximum achievable concentration in the test system. The test substance was tested up to cytotoxic concentrations.

In both the presence and absence of metabolic activation, the test substance caused no statistically significant increase in the proportion of metaphase figures containing chromosomal aberrations at any dose level when compared with the solvent control. Both positive control compounds caused large, statistically highly significant increases in chromosomal damage, demonstrating the sensitivity of this test system and the efficacy of the S9 mix. It is concluded that the test substance has shown no evidence of clastogenic activity in this in vitro cytogenetic test system.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1991-01-23 to 1991-03-06
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Principles of method if other than guideline:
Based on the methods of Clive and Spector (1975) and Amacher et al. (1979, 1980)
GLP compliance:
yes
Type of assay:
in vitro mammalian cell gene mutation tests using the thymidine kinase gene
Target gene:
thymidine kinase
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
CELLS USED
- Doubling time: ca. 12 h
- Methods for maintenance in cell culture: Cells were stored in polypropylene ampules in a freezing mixture consisting of heat-inactivated horse serum (Imperial Laboratories) containing 10 % dimethylsulphoxide DMSO), at -196 °C.

MEDIA USED
- Type and identity of media including CO2 concentration: RPMI 1640, 5 % CO2
- Properly maintained: Yes
- Periodically 'cleansed' against high spontaneous background: Yes, mutants were eliminated from the cultures by a 24 hour incubation in the presence of methotrexate (0.3 µg/mL), thymidine (9 µg/mL), hypoxanthine (15 µ/mL) and glycine (22.5 µg/mL) followed by 24 hours incubation in similar medium without methotrexate, two days prior to storage at -196 °C.
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254 induced rat liver (S9 mix)
Test concentrations with justification for top dose:
Preliminary toxicity test; 0.5, 1, 5, 10, 15, 30, 40, 50 µg/mL
Mutation tests: -S-9 mix
Test 1 1, 2.5, 5, 10, 15, 30, 50 µg/mL
Test 2 1, 2.5, 5, 10, 15, 30, 50 µg/mL
Mutation tests: +S-9 mix
Test 1 0.1, 0.5, 1, 2.5, 5, 10, 15 µg/mL
Test 2 0.1, 0.5, 1, 2.5, 5, 10, 15 µg/mL
Vehicle / solvent:
- Vehicle used: DMSO
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
without S9 mix, 500 µg/mL
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 20-methylcholanthrene
Remarks:
with S9 mix, 2.5 µg/mL
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 3 hours
- Expression time (cells in growth medium): 48 hours
- Selection time: 48 hours
- Fixation time: Analysis was performed after 12 days incubation following
selection.

SELECTION AGENT (mutation assays): not indicated

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth
Evaluation criteria:
The criteria which must be satisfied before a positive response may be claimed are:
1. The induction of at least a two-fold increase in mutant frequency relative to the concurrent control by the test agent.
2. The demonsfration of a statistically significant response.
3. Evidence of a dose-related response.
4. The response must be reproducible.
5. The data are acceptable according to the criteria detaiied in Arlett et al. (1989), in 'Statistica Evaluation of Mutagenicity Test Data', pp. 66 - 101, Cambridge University Press.
Statistics:
The general method of statistical analysis was by analysis of variance of the mutant frequencies.
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
other: Treatment with 1, 15, 30 and 50 μg/mL in tests 1 and 2 produced cell survival levels of 91 - 31 % and 93 - 26 % respectively.
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
other: Treatment with 0.5, 5, 10 and 15 μg/mL in test 1 and 0.5, 1, 2.5 and 5 μg/mL in test 2 produced cell survival levels of 86 - 31 % and 95 - 23 % in tests 1 and 2 respectively.
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
In the absence of S9-mix treatment with 1 - 50 µg/mL in tests 1 and 2 resulted in relative growth in suspension of 111 - 39 % and 99 - 35 % respectively.
In the presence of S9-mix treatment with 0.1 - 15 µg/mL in tests 1 and 2 resulted in relative growth in suspension of 110 - 31 % and 112 - 5 % respectively.

In the preliminary test:

Treatment with concentrations of 0.5 to 50 μg/mL in the presence and absence of metabolic activation produced relative growth in suspension of 106 to 2 % and 108 to 17 %, respectively.

In the main test:

with metabolic activation:

Treatment with 0.5, 5, 10 and 15 μg/mL in test 1 and 0.5, 1, 2.5, and 5 μg/mL in test 2 produced cell survival levels of 86 - 31 % and 95 - 23 % in tests 1 and 2, respectively.

without metabolic activation:

Treatment with 1, 15, 30 and 50 μg/mL in tests 1 and 2 produced cell survival levels of 91 - 31 % and 93 - 26 %, respectively.

Statistically significant increases in mutant frequency were observed in both tests in the absence of S9 mix. In test 2 the response was dose-related. Although this data does not fulfill all the criteria for a positive response the detection of a dose-related increase may be indicative of mutagenic potential.

In the presence of S9 mix statistically significant increases in mutant frequency were observed in test 1. However, these increases were small and considered to be not biologically significant.

Therefore, the evidence for mutagenic potential of the test substance in the absence of S9 mix is equivocal in this in vitro test system and there is no evidence for mutagenic potential in the presence of S9 mix using this in vitro test system.

Table 1: Summary of the Results, Test 1

Test Item Con. (µg/mL)

No. of colonies on non-selective plates

Survival (% control)

Mean

% survival

No.of colonies on selective plates

Mutant frequency (x 10^-6)+

Mean mutant frequency

(x 10^-6)

1

2

3

Total

Viability (%)

1

2

3

Total

Without S9 mix

0 (DMSO control)

132

139

140

411

100

100

100

49

54

38

141

69

85

131

141

118

390

40

52

37

129

66

96

121

102

319

49

69

C

118

111

122

130

109

361

46

53

69

168

93

1.0

126

115

118

359

97

96

91

60

58

45

171

95

91

106

121

117

344

93

86

43

61

44

148

86

15.0

71

85

73

228

62

31

33

53

46

56

455

136

133*

79

98

105

282

76

34

62

75

46

183

130

30.0

97

63

85

245

66

26

33

47

28

60

135

110

122*

105

104

129

38

91

39

74

93

60

227

134

50.0

98

100

81

279

75

29

31

60

47

68

175

125

132*

105

84

106

295

80

32

70

66

67

203

138

EMS, 500

73

81

74

228

62

36

31

189

220

218

627

550

706***

46

36

63

145

39

25

196

218

2100

624

861

With S9 mix

0 (DMSO control)

121

119

130

370

100

100

100

64

58

59

181

98

106

113

122

122

357

61

50

58

169

95

129

127

122

378

54

73

73

200

106

117

111

105

333

64

70

72

206

124

0.5

102

107

101

310

86

78

86

61

47

53

161

104

92

111

107

103

321

89

94

40

37

50

127

79

5.0

102

95

113

310

86

54

46

41

41

52

134

86

108

84

76

86

246

68

37

52

54

53

159

129

10.0

134

130

139

403

112

43

38

91

73

80

244

121

132

106

130

93

329

91

33

73

82

79

234

142

15.0

146

128

137

411

1144

33

31

84

89

99

272

132

137*

106

98

100

304

84

28

82

64

72

215

141

20-Methylcholanthrene, 2.5

48

50

49

147

41

20

19

165

172

163

500

680

640***

56

48

C

104(156)

43

17

141

170

C

311 (467)

599

+ Where replicate determinations of mutant frequency from a single culture were made, the mean mutant frequency for that culture is shown

C Contaminated. Figure in brackets is adjustment made for contaminated plates and is used in all calculations

* Significantly different from control P < 0.05

*** Significantly different from control P < 0.001

Table 2: Summary of the Results, Test 2

Test Item Con. (µg/mL)

No. of colonies on non-selective plates

Survival (% control)

Mean

% survival

No.of colonies on selective plates

Mutant frequency (x 10^-6)+

Mean mutant frequency (x 10^-6)

1

2

3

Total

Viability (%)

1

2

3

Total

Without S9 mix

0 (DMSO control)

109

107

C

216(324)

100

100

100

53

42

42

137

85

93

109

109

102

320

50

53

59

162

101

107

104

110

321

33

38

59

130

81

100

111

C

211(317)

56

53

C

109(164)

103

1.0

100

118

99

317

99

97

93

29

47

59

135

85

85

87

89

112

288

90

89

47

38

36

121

84

15.0

97

85

93

375

86

30

33

62

35

43

140

 

103

105

105

78

288

90

35

47

447

54

148

102

30.0

102

99

75

376

86

25

27

59

70

77

206

103

138**

95

96

C

191(287)

90

29

C

53

68

121(182)

149

50.0

85

78

72

235

73

22

26

41

50

62

153

127

155**

71

73

91

235

73

29

68

67

77

212

130

EMS, 500

112

105

99

316

99

100

77

259

240

260

759

182

527***

81

77

73

231

72

53

216

219

228

663

480

With S9 mix

0 (DMSO control)

148

136

136

410

100

100

100

96

C

C

96(288)

140

113

134

133

C

267(401)

82

69

64

215

107

143

130

C

273(410)

82

70

66

218

106

126

111

115

352

59

58

C

117(176)

100

0.5

108

122

115

345

88

95

95

47

56

C

103(155)

90

90

-

-

-

-

-

-

-

-

-

-

-

5.0

127

103

105

335

85

83

85

19

42

39

100

60

73

145

111

119

375

95

87

56

52

53

161

86

10.0

127

124

116

367

93

65

61

72

58

77

207

113

118

111

100

108

319

81

57

50

79

C

129(194)

122

15.0

73

52

93

218

55

16

23

39

36

38

113

104

91

93

117

108

318

81

30

39

29

56

124

78

20-Methylcholanthrene, 2.5

41

56

56

153

39

19

19

148

C

133

281(422)

552

570***

47

54

C

101(152)

39

18

143

154

C

297(446)

587

+ Where replicate determinations of mutant frequency from a single culture were made, the mean mutant frequency for that culture is shown

C Contaminated. Figure in brackets is adjustment made for contaminated plates and is used in all calculations

* Significantly different from control P < 0.05

** Significantly different from control P<O.Ol

*** Significantly different from control P < 0.001

Conclusions:
Under the present test conditions, the test substance tested up to cytotoxic concentrations in the absence and presence of metabolic activation in two independent experiments, was negative with respect to the mutant frequency in the L5178Y TK +/- mammalian cell mutagenicity test with S9 mix. The evidence for mutagenic potential of the test substance in the absence of S9 mix is equivocal in this in vitro test system.
Executive summary:

In a mammalian cell gene mutation assay (TK) according to OECD guideline 476 (Huntingdon, 1991), mouse lymphoma L5178Y cells cultured in vitro were exposed to the test substance dissolved in DMSO at concentrations of 0.5, 1, 5, 10, 15, 30 and 50 µg/mL with and without metabolic activation (S9 mix).

In the absence of S9-mix treatment with 1 - 50 µg/mL in tests 1 and 2 resulted in relative growth in suspension of 111 - 39 % and 99 - 35 %, respectively. In the presence of S9-mix treatment with 0.1 - 15 µg/mL in tests 1 and 2 resulted in relative growth in suspension of 110 - 31 % and 112 - 5 %, respectively.

Statistically significant increases in mutant frequency were observed in both tests in the absence of S9 mix. In test 2 the response was dose-related. Although this data does not fulfil all the criteria for a positive response the detection of a dose-related increase may be indicative of mutagenic potential.

In the presence of S9 mix statistically significant increases in mutant frequency were observed in test 1. However, these increases were small and considered to be not biologically significant.

Therefore, the evidence for mutagenic potential of the test substance in the absence of S9 mix is equivocal in this in vitro test system and there is no evidence for mutagenic potential in the presence of S9 mix using this in vitro test system.

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

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

3 key studies are available to cover genetic toxicity. In accordance with Article 25(3) of the REACH Regulation the robust study summaries submitted 12 years previously, are used as supporting information for the purpose of the registration.

Bacterial reverse mutation

Key study

In a reverse mutation assay in bacteria according to EU method B.13/14 (Huntingdon, 1988), strains (TA98, TA100, TA1535, TA1537) of S. typhimurium were exposed to the test substance dissolved in DMSO at concentrations of 50 to 5000 µg/plate in the presence and absence of metabolic activation. The test substance was tested up to a limit concentration of 5000 µg/plate. There was no evidence or a concentration related positive response of induced mutant colonies over background. Thus, the test substance is not regarded as mutagenic.

Supporting study

The results of the key study were supplemented by a bacterial reverse mutation assay (Ames) in the S. typhimurium tester strain TA1538 (Huntingdon, 1990). The bacteria were exposed to the test substance dissolved in DMSO at concentrations of 50 to 5000 µg/plate in the presence and absence of metabolic activation. The test substance was tested up to a limit concentration of 5000 µg/plate. There was no evidence or a concentration related positive response of induced mutant colonies over background. Thus, the test substance is not regarded as mutagenic.

In a reverse mutation assay in bacteria according to EU method B.13/14 (Mitsubishi, ECHA RSS, 2017), strains (TA98, TA100, TA1535, TA1537, E. coli WP uvrA) of S. typhimurium and E. coli were exposed to the test substance dissolved in DMSO at concentrations of 313 to 5000 µg/plate in the presence and absence of metabolic activation. The test substance was tested up to a limit concentration of 5000 µg/plate. There was no evidence or a concentration related positive response of induced mutant colonies over background. Thus, the test substance is not regarded as mutagenic.

Chromosome aberration

Key study

In a mammalian cell cytogenetics assay (chromosome aberration) according to OECD guideline 473 (Huntingdon, 1989), CHO cell cultures were exposed to the test substance dissolved in DMSO at concentrations of 4, 20 and 40 µg/mL with and without metabolic activation (Aroclor 1254 – induced rat liver). In a preliminary study the highest dose used did not produce any reduction in mitotic index. The highest dose used (40 µg/mL) was the maximum achievable concentration in the test system. The test substance was tested up to cytotoxic concentrations.

In both the presence and absence of metabolic activation, the test substance caused no statistically significant increase in the proportion of metaphase figures containing chromosomal aberrations at any dose level when compared to the solvent control. Both positive control compounds caused large, statistically highly significant increases in chromosomal damage, demonstrating the sensitivity of this test system and the efficacy of the S9 mix. It is concluded that the test substance has shown no evidence of clastogenic activity in this in vitro cytogenetic test system.

Supporting study

In a mammalian cell cytogenetics assay (chromosome aberration) according to EU method B.10 (Toxicol. Lab. Ltd., ECHA RSS, 2017), CHO cell cultures were exposed to the test substance dissolved in DMSO at concentrations of 12.5 to 125 µg/mL with and without metabolic activation (Aroclor 1254 – induced rat liver). The highest concentration represented the solubility limit for this substance. A dose-related decrease in the mitotic index occurred both with and without S9 mix. At the highest dose level the decrease was over 40 % compared with control values. The test substance was tested up to cytotoxic concentrations.

In both the presence and absence of metabolic activation, the test substance caused no statistically significant increase in the proportion of metaphase figures containing chromosomal aberrations at any dose level when compared to the solvent control. It is concluded that the test substance has shown no evidence of clastogenic activity in this in vitro cytogenetic test system.

In vitro mammalian cell gene mutation

Key study

In a mammalian cell gene mutation assay (TK) according to OECD guideline 476 (Huntingdon, 1991), mouse lymphoma L5178Y cells cultured in vitro were exposed to the test substance dissolved in DMSO at concentrations of 0.5, 1, 5, 10, 15, 30 and 50 µg/mL with and without metabolic activation (S9 mix).

In the absence of S9-mix treatment with 1 - 50 µg/mL in tests 1 and 2 resulted in a relative cell growth in suspension of 111 - 39 % and 99 - 35 %, respectively. In the presence of S9-mix treatment with 0.1 - 15 µg/mL in tests 1 and 2 resulted in relative growth in suspension of 110 - 31 % and 112 - 5 %, respectively.

Statistically significant increases in mutant frequency were observed in both tests in the absence of S9 mix. In test 2 the response was dose-related. Although this data does not fulfil all the criteria for a positive response, the detection of a dose-related increase may be indicative of mutagenic potential.

In the presence of S9 mix statistically significant increases in mutant frequency were observed in test 1. However, these increases were small and considered to be not biologically significant.

Therefore, the evidence for mutagenic potential of the test substance in the absence of S9 mix is equivocal in this in vitro test system and there is no evidence for mutagenic potential in the presence of S9 mix using this in vitro test system.

Supporting study

In a mammalian cell gene mutation assay (TK) according to OECD guideline 476 (Pharmaco-LSR Ltd., ECHA RSS, 2017), mouse lymphoma L5178Y cells cultured in vitro were exposed to the test substance dissolved in DMSO at concentrations of 2.73 to 730 µg/mL with and without metabolic activation (S9-mix).

Although some toxicity was seen in preliminary tests at low concentrations, no dose-response relationship was apparent. Some precipitation of the test substance was observed at concentrations of 175 µg/mL and above. Cultures exposed to the test substance showed no increase in mutant colony numbers or increase in the mutant frequencies (per 100000 survivors) compared to controls. Thus, no evidence for a mutagenic potential was observed for the test substance.

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 according to Regulation (EC) No 1272/2008 (CLP).