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Genetic toxicity: in vitro

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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.
Cross-referenceopen allclose all
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010

Materials and methods

Test guideline
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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
impurity
Test material form:
liquid
Details on test material:
Batch No. : 094678
Purity : 92.8%
Name of test material (as cited in study report): 2-ethyl-4-(2,2,3-trimethyl-3-cyclopenten-1-yl)-2-buten-1-ol
Physical state: liquid
Storage Conditions: +2°C to +8°C, under nitrogen and protected from light
Date of manufacture: 25 November 2009
Expiry Date: 24 November 2011

Method

Target gene:
Thymidine kinase, TK +/- locus
Species / strain
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
Controlsopen allclose all
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

Results and discussion

Test results
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'.

Any other information on results incl. tables

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

Applicant's summary and conclusion

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.