Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information
The registered substance was negative in two Ames tests, in a chromosome aberration test in CHO cells and in a gene mutation test (HPRT) in MLA L5178Y tk+/- cells.
Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From April 19 to June 15, 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study following OECD guideline 476 with minor deviations: 3 test concentrations were based on one replicate only as flask contents were lost during expression period and in experiment 1, no concentration gave 10-20% relative survival.
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
yes
Remarks:
3 test concentrations were based on one replicate only as flask contents were lost during expression period and in experiment 1, no concentration gave 10-20% relative survival.
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes
Type of assay:
mammalian cell gene mutation assay
Target gene:
Thymidine kinase, TK +/- locus
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
- Source of cells: Dr Donald Clive, Burroughs Wellcome Co. Cells
- Type and identity of media: RPMI 1640 medium
RPMI A: Penicillin (100 units/mL), streptomycin (100 μg/mL), amphotericin B (2.5 μg/mL) and pluronic acid (0.5 mg/mL)
RPMI 10: Horse serum (heat inactivated, 10% v/v), penicillin (100 units/mL), streptomycin (100 μg/mL), amphotericin B (2.5 μg/mL) and pluronic acid (0.5 mg/mL)
RPMI 20: Horse serum (heat inactivated, 20% v/v), penicillin (100 units/mL), streptomycin (100 μg/mL) and amphotericin B (2.5 μg/mL)
- Properly maintained: Yes
- Periodically checked for Mycoplasma contamination: Yes
- Periodically checked for karyotype stability: No data
- Periodically "cleansed" against high spontaneous background: Yes; each batch was purged of TK- mutants
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
2% S9 fraction of Aroclor 1254-induced male Sprague-Dawley rats
Test concentrations with justification for top dose:
Range-finder experiment: 0, 12.5, 25, 50, 100, 200 and 400 μg/mL (with and without S-9)
Main study:
- Experiment 1: without S-9: 0, 5, 10, 15, 20, 25, 30, 35, 40, 45 and 50 μg/mL; with S-9: 0, 10, 20, 30, 40, 50, 60, 70, 80, 90 and 100 μg/mL
- Experiment 2: without S-9: 0, 10, 20, 25, 30, 35, 37.5, 40, 42.5, 45 and 50 μg/mL; with S-9: 0, 10, 20, 40, 45, 50, 55, 60, 65, 70 and 75 μg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Dimethyl sulphoxide (DMSO)
- Justification for choice of solvent/vehicle: Test material was soluble in anhydrous analytical grade DMSO at concentrations up to at least 269.29 mg/mL
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
with metabolic activation Migrated to IUCLID6: 2 or 3 µg/mL
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
without metabolic activation Migrated to IUCLID6: 0.10 or 0.15 µg/mL
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 3 hours, 37 ± 1 ºC
- Expression time (cells in growth medium): 7 days, 37 ± 1 ºC in a humidified incubator gassed with 5 % v/v CO2 in air
- Selection time (if incubation with a selection agent): 12 days, 37 ± 1 ºC in a humidified incubator gassed with 5 % v/v CO2 in air

SELECTION AGENT (mutation assays): 6-thioguanine (6TG)

NUMBER OF REPLICATIONS: Duplicates (single cultures only used for positive control treatments and range-finding experiment)

NUMBER OF CELLS EVALUATED: 20000 cells/well plated for survival, viability and 6TG resistance

DETERMINATION OF CYTOTOXICITY
- Method: Cloning efficiency

OTHER: Cell viability was identified by eye using background illumination and counted; cell densities were determined using a coulter counter
Evaluation criteria:
For valid data, the test article was considered to induce forward mutation at the hprt locus in mouse lymphoma L5178Y cells if:
1. The mutant frequency at one or more concentrations was significantly greater than that of the negative control (p ≤ 0.05)
2. There was a significant concentration-relationship as indicated by the linear trend analysis (p ≤ 0.05)
3. The effects described above were reproducible.

Results that only partially satisfied the assessment criteria described above were considered on a case-by-case basis.
Statistics:
The experimental data was analysed using UKEMS recommended statistical guidelines
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
The highest concentrations to give >10% Relative Survival were 25 µg/mL in the absence of S-9 and 50 µg/mL in the presence of S-9, which gave 66% and 61% relative survival, respectively.
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No
- Effects of osmolality: No
- Evaporation from medium: No data
- Water solubility: No data; solubility limit in the culture medium: 84.1 to 168.3 μg/mL
- Precipitation: Yes; upon addition of the test article to the cultures, precipitate was observed at the highest four concentrations tested in the absence and presence of S-9 (50 to 400 µg/mL).

RANGE-FINDING/SCREENING STUDIES: Six concentrations were tested in the absence and presence of S-9 ranging from 12.5 to 400 µg/mL (limited by solubility in culture medium).
- Precipitation was observed precipitate was observed at the highest four concentrations tested in the absence and presence of S 9 (50 to 400 µg/mL).
- The highest concentrations to give >10% Relative Survival were 25 µg/mL in the absence of S-9 and 50 µg/mL in the presence of S-9, which gave 66% and 61% Relative Survival, respectively.
- See table 1 for more details

COMPARISON WITH HISTORICAL CONTROL DATA: Yes
Remarks on result:
other: strain/cell type: TK+/- (3.7.2C) cells
Remarks:
Migrated from field 'Test system'.

Table 1: RS values - range-finder experiment

Treatment (µg/mL)

-S-9 %RS

+S-9 %RS

0

100

100

12.5

100

112

25

66

92

50 P

0

61

100 P

NP

0

% RS: Percentage Relative Survival

P: Precipitation observed at time of treatment

NP: Not plated for viability due to excessive toxicity

Table 2: Summary of mutation data

Experiment 1: (3 hour treatment in the absence and presence of S-9)

Treatment

(µg/mL)

-S-9

Treatment

(µg/mL)

+S-9

 

%RS

MF§

 

%RS

MF§

0

 

100

6.51

 

0

 

100

4.96

 

10

 

97

4.91

NS

10

 

92

3.37

NS

15

 

90

3.61

NS

20

 

78

3.89

NS

20

 

91

5.68

NS

30

 

72

5.39

NS

30

 

64

7.11

NS

40

 

52

5.15

NS

35

 

55

4.84

NS

50

P

27

2.51

NS

40

 

32

2.73

NS

60

P

32!

4.55

NS

45

 

7

4.44

NS

70

P

4

5.67

NS

Linear trend

NS

Linear trend

NS

NQO

 

 

 

 

B[a]P

 

 

 

 

0.1

 

70

17.73

 

2

 

85

17.87

 

0.15

 

64

62.76

 

3

 

26

47.67

 

Experiment 2: (3 hour treatment in the absence and presence of S-9)

Treatment

(µg/mL)

-S-9

Treatment

(µg/mL)

+S-9

 

%RS

MF§

 

%RS

MF§

0

 

100

0.74

 

0

 

100

6.83!

 

10

 

117

1.21

NS

20

 

106

1.59

NS

20

 

100

1.74

NS

40

 

89

2.56

NS

30

 

67

4.70

*

50

 

71

3.32

NS

35

 

54

0.68

NS

55

 

77

2.23

NS

37.5

 

36

1.58

NS

60

 

60

1.07!

NS

40

 

23

1.01

NS

65

P

65

0.70

NS

42.5

 

11

4.17

NS

70

P

25

2.03

NS

 

 

 

 

 

75

P

10

1.32

NS

Linear trend

NS

Linear trend

NS

NQO

 

 

 

 

B[a]P

 

 

 

 

0.1

 

70

21.29

 

2

 

45

24.82

 

0.15

 

55

22.80

 

3

 

47

27.60

 

§: 6‑TG resistant mutants/106 viable cells 7 days after treatment

%RS: Percent relative survival adjusted by post treatment cell counts

NS: Not significant

*: Comparison of each treatment with control: Dunnett's test (one-sided), significant at 5% level

!: Based on one replicate only as flask contents lost during expression period

P: Precipitation observed at time of treatment

Conclusions:
Interpretation of results: negative

The test item is not considered as mutagenic in L5178Y cells and should not be classified as mutagen according to Directive 67/548/EEC and CLP Regulation (EC) n° 1272/2008.

Executive summary:

In an in vitro mammalian cell gene mutation test performed according to OECD guideline 476, in compliance with GLP, mouse lymphoma L5178Y TK+/- (3.7.2C) cells were exposed to the test item in DMSO at concentrations of 0, 12.5, 25, 50, 100, 200 and 400 μg/mL in RPMI 1640 medium with and without 2% S-9 metabolic activation for a preliminary cytotoxicity test.

In the main test, two experiments were performed at the following concentrations:

- Experiment 1: without S-9: 0, 5, 10, 15, 20, 25, 30, 35, 40, 45 and 50 μg/mL; with S-9: 0, 10, 20, 30, 40, 50, 60, 70, 80, 90 and 100 μg/mL

- Experiment 2: without S-9: 0, 10, 20, 25, 30, 35, 37.5, 40, 42.5, 45 and 50 μg/mL; with S-9: 0, 10, 20, 40, 45, 50, 55, 60, 65, 70 and 75 μg/mL.

Mutant frequencies in negative control cultures fell within normal ranges and clear increases in mutation were induced by the positive control chemicals 4-nitroquinoline 1-oxide (without S-9) and benzo(a)pyrene (with S-9). In the absence and presence of S-9 in Experiment 1 and in the presence of S-9 in Experiment 2, there were no significant increases in mutant frequency at any concentration analysed. For Experiment 2 in the absence of S-9, significant increase in mutant frequency was noted at a single intermediate concentration.

As the response is not concentration related, there is no linear trend and the response is not well reproduced, this increase was not considered as biologically relevant. Therefore, the test item is not considered as mutagenic in L5178Y cells and should not be classified as mutagen according to Directive 67/548/EEC and CLP Regulation (EC) n° 1272/2008.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From May 11 to December 22, 1987
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
GLP study performed according to OECD guideline 473 with minor deviations: no data about purity and certificate of analysis of the test substance; evaluation criteria not reported; no information regarding mycoplasma contamination and karyotype stability check
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
yes
Remarks:
no data about purity and certificate of analysis of the test substance; evaluation criteria not reported; no information regarding mycoplasma contamination and karyotype stability check
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test
Target gene:
Not applicable
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
- Source of cell lines: Huntingdon Research Centre
- Type and identity of media: Hams F12 Medium (Flow Laboratories, UK) with 5% foetal calf serum, 2mM L-glutamine and 50 µg/mL gentamicin
- Culture conditions: Cells were cultured in test media in a humidified atmosphere of 5% CO2 in air at 37 °C.
- Properly maintained: Yes; normally subcultured every 2 to 3 days. For long-term preservation, stocks of the cells were stored frozen in culture medium containing 10% v/v DMSO in sealed ampoules in liquid nitrogen. Sterilisation was achieved either by autoclaving at 15 Ib/sq.in. for 20 minutes or by filtration through a 0.22 µm pore size membrane.
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
10% S9 fraction of rat liver supplemented with co-factors
Test concentrations with justification for top dose:
Cytotoxicity test: 1.0, 2.1, 4.6, 9.9, 21.0, 46 or 100 µg/mL
Cytogenetic test:
- Without S9: 0.63, 2.0, 6.3 or 20 µg/mL
- With S9: 1.46, 4.6, 14.6 or 46 µg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: DMSO had been identified as solvent of choice based on a previous bacterial mutation study (PI870203). Based on the solubility and precipitation of the test substance in DMSO, 100 µg/mL was selected as the highest tested dose.
Untreated negative controls:
yes
Remarks:
only medium
Negative solvent / vehicle controls:
yes
Remarks:
DMSO (1% v/v)
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
with metabolic activation Migrated to IUCLID6: 20 µg/mL
Untreated negative controls:
yes
Remarks:
only medium
Negative solvent / vehicle controls:
yes
Remarks:
DMSO (1% v/v)
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
without metabolic activation Migrated to IUCLID6: 25 µg/mL
Details on test system and experimental conditions:
METHOD OF APPLICATION: In medium

DURATION
- Exposure duration: 6 hours with S9 and 18 hours without S9
- Fixation time (start of exposure up to fixation or harvest of cells): 18-24 hours

SPINDLE INHIBITOR (cytogenetic assays): Colchicine (0.5 µg/mL)
STAIN (for cytogenetic assays): 5% Giemsa

NUMBER OF REPLICATIONS: Duplicates

NUMBER OF CELLS EVALUATED: 1000 cells/dose for toxicity test, 200 cells/dose (100 cells/replicate) for cytogenicity experiments

DETERMINATION OF CYTOTOXICITY
- Method: Mitotic index; cells were exposed to Chandanol at concentrations of 1.0, 2.1, 4.6, 9.9, 21, 46 or 100 µg/mL in the absence and presence of S9 and the number of dividing nuclei per 1000 nuclei per dose were examined microscopically to calculate the mitotic index.
Evaluation criteria:
No data
Statistics:
Criteria for scoring chromosome aberrations followed the recommendations of the UKEMS (1983).
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at ≥ 21 µg/mL (without S9); at 100 µg/mL (with S9)
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Solubility or precipitation: Based on the solubility and precipitation of the test substance in DMSO, 100 µg/mL was selected as the highest tested dose in the cytogenetic test.

RANGE-FINDING/SCREENING STUDIES: Cells were exposed to Chandanol at concentrations of 1.0, 2.1, 4.6, 9.9, 21, 46 or 100 µg/mL in the absence and presence of S9 and the number of dividing nuclei per 1000 nuclei per dose were examined microscopically to calculate the mitotic index. Cytotoxicity was observed at or above 21 µg/mL without S9 and at 100 µg/mL with S9.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Table 1: Mitotic indices of chandanol with and without metabolic activation

 

Concentration (µg/mL)

Without metabolic activation

With metabolic activation

 Mitotic index 

% of control

 Mitotic index 

% of control

 Control 

 14 

-

 15 

-

 100.0 

 toxic 

 

 toxic 

 

 46.0 

 toxic 

 

 7 

47

 21.0 

 14* 

100

 12 

80

 9.9 

 14 

100

 10 

67

 4.6 

 8 

57

 13 

87

 2.1 

 12 

86

 21 

140

 1.0 

 14 

100

 14 

93

* number of cells greatly reduced; Mitotic index = no. of mitoses per 1000 cells scored

 

Table 2: Cytogenetics assay of Chandanol with CHO cells in the absence of exogenous metabolism (replicates A and B combined)

 

Dose
(µg/mL)

Number of divisions

Normal

Chromatid

Isochromatid

Chromatid exchange

Chromosome exchange

Fragments

M.A.

Aberrant divisions

Frequency of aberrations

Gap

Break

Gap

Break

Control DMSO
(1% v/v)

200

186

8

2

_

1

_

_

2 pr+1

_

7

7 (3)

200

183

6

1

_

2

1

_

8 pr

_

9

9 (6)

0.63

200

182

7

3

2

1

1

_

3 pr+2

_

9

10 (6)

2

200

185

6

2

_

2

_

1

4 pr

_

8

8 (5)

6.3

200

184

5

2

_

6

_

2

3 pr

_

8

9 (7)

20

200

177

9

_

2

3

_

 

8 pr+2

_

12

12 (8)

MMS (25 µg/mL)

197

122

40

25

9

13

7

2

8 pr+1

_

38

53 (33)

 

Table 3: Cytogenetics assay of Chandanol with CHO cells in the presence of exogenous metabolism (replicates A and B combined)

 

Dose
(µg/mL)

Number of divisions

Normal

Chromatid

Isochromatid

Chromatid exchange

Chromosome exchange

Fragments

M.A.

Aberrant divisions

Frequency of aberrations

Gap

Break

Gap

Break

Control DMSO
(1% v/v)

200

191

4

1

_

_

_

1

3 pr

_

5

5 (3)

200

189

9

1

_

_

_

_

2 pr

_

6

6 (2)

1.46

200

192

7

_

_

_

_

_

1 pr

_

4

4 (1)

4.6

200

186

5

4

1

 

1

_

4 pr+1

_

7

8 (6)

14.6

200

183

11

3

_

1

1

_

2 pr

_

9

9 (4)

46

200

180

12

2

_

_

2

_

3 pr+1

_

10

10 (4)

CP (20 µg/mL)

200

41

53

60

16

25

99

_

40 pr+13

12

77

206 (176)

MMS = Methyl methane sulphonate; CP = Cyclophosphamide

*per 100 divisions to nearest whole numbers;

M.A. Multiple aberrations assigned nominal score of 5

Conclusions:
Interpretation of results (migrated information):
negative with metabolic activation
negative without metabolic activation

The test item did not induce chromosome aberrations in cultured Chinese hamster ovary (CHO) cells with and without metabolic activation.
Executive summary:

In an in vitro chromosome aberration test performed according to OECD guideline 473 and in compliance with GLP, Chinese hamster ovary (CHO) cells were exposed to the test item. In a first experiment, cells were exposed to the test item at concentrations of 1.0, 2.1, 4.6, 9.9, 21, 46 or 100 µg/mL in the absence and presence of S9 (10% S9 fraction of rat liver supplemented with co-factors). From the results of this cytotoxicity test, the test item was tested at 0.63, 2.0, 6.3 or 20 µg/mL without S9 and at 1.46, 4.6, 14.6 or 46 µg/mL with S9. Treatment was for 6 hours with S9 and 18 hours without S9.

 

Positive controls (methylmethanesulfonate at 25 µg/mL without S9 and cyclophosphamide at 20 µg/mL with S9) induced the appropriate response. Chromosome aberrations were not induced over background at any tested concentrations in the absence or presence of metabolic activation.

 

Therefore, the test item did not induce chromosome aberrations in cultured Chinese hamster ovary (CHO) cells with and without metabolic activation.

 

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From June 20 to July 12, 2007
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study conducted similarly to OECD Guideline 471 with minor deviations: no certificate of analysis of test substance; only duplicate cultures used /dose
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
no certificate of analysis of test substance; only duplicate cultures used /dose
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
Not applicable
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell type (if applicable):
not applicable
Additional strain / cell type characteristics:
not specified
Species / strain / cell type:
E. coli WP2 uvr A
Details on mammalian cell type (if applicable):
not applicable
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
10% S9 fraction of male SD rat liver induced with phenobarbital and 5, 6-benzoflavone and supplemented with Cofactor I® (Oriental Yeast Co., Ltd.)
Test concentrations with justification for top dose:
Range-finding test 1 (all 5 strains): 0.305, 1.22, 4.88, 19.5, 78.1, 313, 1250 or 5000 µg/plate with or without S9 mix
Range-finding test 2 (all 4 Salmonella strains): 0.610, 1.22, 2.44, 4.88, 9.77 or 19.5 µg/plate without S9 mix
Main test:
- Without S9 (all 4 Salmonella strains): 0.610, 1.22, 2.44, 4.88, 9.77 or 19.5 µg/plate
- Without S9 (WP2 uvrA) or with S9 (all 5 strains): 2.44, 4.88, 9.77, 19.5, 39.1 or 78.1 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Test substance was soluble and stable in DMSO at 50 mg/mL but insoluble in distilled water
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-Aminoanthracene
Remarks:
with metabolic activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide (TA 100, TA 98 and WP2 uvrA); sodium azide (TA 1535); 2-methoxy-6-chloro-9-[3-(2-chloroethyl)-aminopropylamino]acridine.2HCI (TA 1537)
Remarks:
without metabolic activation
Details on test system and experimental conditions:
METHOD OF APPLICATION: Preincubation

DURATION
- Preincubation period: 20 minutes at 37 ± 0.5 °C
- Exposure duration: 48 hours at 37 ± 0.5 °C

NUMBER OF REPLICATIONS: Duplicates (for test substance and positive control groups) or triplicates (for negative control group)

NUMBER OF CELLS EVALUATED: 10^9 cells/mL
Evaluation criteria:
- Test substance was judged to be positive when the number of revertant colonies increased to twice or more that in the negative control in a concentration-dependent manner and also the reproducibility of the test results was obtained.
- In all other cases, it was judged to be negative.
Statistics:
None
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
see table 1
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Yes; in the range-finding test-1, precipitation was observed at 1250 µg/plate or more without S9 mix and at 5000 µg/plate or more with S9 mix.

RANGE-FINDING/SCREENING STUDIES:

Range-finding test-1:
- Number of revertant colonies in the test substance treatment groups was less than twice that in the solvent control
- Precipitation was observed at 1250 µg/plate or more without S9 mix and at 5000 µg/plate or more with S9 mix.
- Bacterial growth inhibition was observed at 19.5 µg/plate or more in TA100, TA1535, TA98 and TA1537 without S9 mix, at 78.1 µg/plate or more in WP2uvrA without S9 mix and at 78.1 µg/plate or more in all test strains with S9 mix.

Range-finding test-2:
- Number of revertant colonies in the test substance treatment groups was less than twice that in the solvent control in TA100, TA1535, TA98 and TA1537 without S9 mix
- Number of revertant colonies was increased nearly twice in TA1537
- Bacterial growth inhibition was observed at 19.5 µg/plate in all test strains


COMPARISON WITH HISTORICAL CONTROL DATA: Yes; numbers of revertant colonies in the negative control and the positive controls were within the range of the historical data (December, 2006 - May, 2007)

ADDITIONAL INFORMATION ON CYTOTOXICITY: In the range-finding test-1, complete cytotoxicity was observed at 78.1 µg/plate or more in TA100, TA1535 and TA1537 without S9 mix, at 313 µg/plate or more in TA98 without S9 mix and at 313 µg/plate or more in TA100, TA1535 and TA1537 with S9 mix
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Table 1: Results of the main test

 

With or without S9 mix

Test substance dose (µg/plate)

Number of revertant colonies per plate

Base-pair substitution type

Frameshift type

TA100

TA1535

WP2uvrA

TA98

TA1537

 

Mean

 

Mean

 

Mean

 

Mean

 

Mean

Without S9 mix

Negative control

151 

142

10

37

43

22

19

8

10

144

13

49

20

11

130

8

44

14

11

0.61

123

122

13

11

 

 

26

23

11

11

120

8

20

11

1.22

146

136

11

8

 

 

19

20

15

13

126

5

21

11

2.44

138

143

10

10

36

38

28

28

15

12

148

10

40

27

8

4.88

130

139

7

8

41

41

25

28

16

18

148

8

41

31

20

9.77

104

126

14

15

50

42

21

22

12

12

148

15

33

22

12

19.5

78*

66

3*

4

43

37

21*

19

0*

2

53*

5*

31

16*

4*

39.1

-

 

-

 

20*

16

-

 

-

 

12*

78.1

-

 

-

 

20*

22

-

 

-

 

23*

With S9 mix

Negative control

118

116

8

10

40

39

47

41

29

32

112

10

31

41

31

117

12

47

34

37

2.44

152

144

12

9

44

39

31

37

29

35

135

6

34

42

40

4.88

134

128

11

12

47

49

38

32

28

29

122

12

50

26

29

9.77

142

145

10

8

55

48

36

36

23

19

147

5

41

36

15

19.5

141

148

11

12

41

44

35

39

30

29

134

12

47

42

28

39.1

153

147

11

12

42

41

35

35

23

20

141

13

40

34

16

78.1

101*

90

5*

4

24*

27

23*

28

8*

7

79*

3*

29*

32*

5*

Positive control without S9 mix

Chemical

AF-2

NaN3

AF-2

AF-3

ICR-191

Dose (µg/plate)

0.01

0.5

 

0.01

 

0.1

 

0.5

 

Number of revertant colonies/plate

770

756

626

620

502

479

627

628

1682

1720

742

613

455

629

1757

Positive control with S9 mix

Chemical

2AA

2AA

2AA

2AA

2AA

Dose (µg/plate)

1

2

10

0.5

2

Number of revertant colonies/plate

1050

1025

239

236

863

866

498

502

239

215

1000

233

868

505

191

*: Bacterial growth inhibition was observed.

AF-2: 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide

NaN3: Sodium azide

ICR-191: 2-Methoxy-6-chloro-9-[3-(2-chloroethyl)-aminopropylamino ]acridine.2HCI

2AA: 2-Aminoanthracene

Conclusions:
Interpretation of results::
negative with metabolic activation
negative without metabolic activation

The test item is not considered as mutagenic in S. typhimurium strains (TA 1535, TA 1537, TA 100 and TA 98) and Escherichia coli strain (WP2 uvrA) according to the criteria of Directive 67/548/EEC and CLP Regulation (EC) n° 1272/2008.
Executive summary:

In a reverse gene mutation assay in bacteria, performed similarly to the OECD guideline 471 and in compliance with GLP, strains of S. typhimurium (TA 1535, TA 1537, TA 100 and TA 98) and one Escherichia coli strain (WP2 uvrA) were exposed to the test item at concentrations of 0, 0.305, 1.22, 4.88, 19.5, 78.1, 313, 1250 or 5000 µg/plate in both the absence and presence of S9 metabolic activation (10% S9 fraction of male SD rat liver induced with phenobarbital and 5, 6-benzoflavone) according to the pre-incubation method for the first range finding test.

In the second range finding test, all four Salmonella test strains were exposed to the test item at concentrations of 0.610, 1.22, 2.44, 4.88, 9.77 or 19.5 µg/plate without S9 mix.

In the main test, experiment was performed at the following concentration according to the pre-incubation method:

- without S9 (all four Salmonella test strains) at 0.610, 1.22, 2.44, 4.88, 9.77 or 19.5 µg/plate;

- without S9 (WP2 uvrA) or with S9 (all 5 strains) at 2.44, 4.88, 9.77, 19.5, 39.1 or 78.1 µg/plate.

 

In the range finding test, precipitation of the test substance was observed at 1250 µg/plate or more without S9 mix and at 5000 µg/plate with S9 mix. Cytotoxicity was observed at 78.1 µg/plate or more in TA100, TA1535 and TA1537 without S9 mix, at 313 µg/plate or more in TA98 without S9 mix and at 313 µg/plate or more in TA100, TA1535 and TA1537with S9 mix. The positive and negative (vehicle) controls induced the appropriate responses in the corresponding strains. The test item showed no substantial increases in revertant colony numbers over control count obtained with any of the tester strains at any concentrations in either presence or absence of S9 mix.

 

Therefore, the test item is not considered as mutagenic in bacteria according to the criteria of Directive 67/548/EEC and CLP Regulation (EC) n° 1272/2008.

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

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

In two reverse gene mutation assays in bacteria, performed similarly to the OECD guideline 471 and in compliance with GLP, the test item was negative in S. typhimuriumstrains (TA 1535, TA 1537, TA 100 and TA 98) and in Escherichia colistrain (WP2 uvrA-) in presence and absence of metabolic activation, up to limit or cytotoxic concentrations.

The test item was also negative in presence and absence of metabolic activation in a chromosome aberration test performed in CHO cells performed according to OECD guideline 473 and in compliance with GLP.

The test item was also negative in presence and absence of metabolic activation in a gene mutation test (HPRT) in MLA L5178Y tk+/- cells performed according to OECD guideline 476 and in compliance with GLP.


Justification for selection of genetic toxicity endpoint
Several in vitro studies were used to complete this endpoint: two Ames tests, chromosome aberration test in CHO cells and gene mutation test (HPRT) in MLA L5178Y tk+/- cells.


Short description of key information:
The registered substance was negative in two Ames tests, in a chromosome aberration test in CHO cells and in a gene mutation test (HPRT) in MLA L5178Y tk+/- cells.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

As the test item is negative in two Ames tests, in a chromosome aberration test in CHO cells and in a gene mutation test (HPRT) in MLA L5178Y tk+/- cells, it is not classified according to Directive 67/548/EEC and CLP Regulation (EC) n° 1272/2008.