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Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
From 15 May to 14 December 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2012

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
N° 2011/40
Type of assay:
other: mammalian cell gene mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent

Method

Target gene:
Thymidine Kinase locus (Chromosome 11)
Species / strain
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
- Type and identity of media: RPMI 1640 medium supplemented by heat inactivated horse serum at 10%, v/v
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: no data
- Periodically "cleansed" against high spontaneous background: yes
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9 fraction was purchased from Moltox (Molecular Toxicology, INC, Boone, NC 28607, USA) and obtained from the liver of rats treated with Aroclor 1254 (500 mg/kg) by the intraperitoneal route.
Test concentrations with justification for top dose:
Preliminary study: (cytotoxicity assessment)
10, 100, 500, 1000, 2500 and 5000 μg/mL.

Main study (mutagenicity experiments)
Without S9 mix:
first experiment (3-hour treatment): 6.25, 12.5, 25, 50, 75, 100 and 200 μg/mL
second experiment (24-hour treatment): 3.13, 6.25, 12.5, 25, 50 and 100 μg/mL
With S9 mix (3h treatment):
first experiment: 12.5, 25, 50, 100, 200, 300 and 400 μg/mL
second experiment: 6.25, 12.5, 25, 50, 100 and 200 μg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: water for injections: batch Nos. 2F0284 and 2F0703 (CDM Lavoisier, Paris, France).
- Justification for choice of solvent/vehicle: no data
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
water
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
methylmethanesulfonate
Remarks:
-S9 mix: MMS at 25 μg/mL (3-hour treatment) or 5 μg/mL (24-hour treatment) / +S9 mix: CPA at 3 μg/mL. Positive controls were diluted in water for injections.
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: not applicable
- Exposure duration: 3h (first experiment without S9 mix, both experiments with S9 mix) or 24h (second experiment without S9 mix)
- Expression time (cells in growth medium): for 2 days after the end of the treatment period
- Selection time (if incubation with a selection agent): 2 days after the end of the treatment period (at the end of the expression time). Cell were incubated with TFT for 11-12 days
- Fixation time (start of exposure up to fixation or harvest of cells): not applicable

SELECTION AGENT (mutation assays): TFT (trifluorothymidine): 4 μg TFT/mL culture medium
SPINDLE INHIBITOR (cytogenetic assays): not applicable
STAIN (for cytogenetic assays): not applicable

NUMBER OF REPLICATIONS: 2 independent experiments, 2 parallel cultures/dose-level

NUMBER OF CELLS EVALUATED: not applicable.

DETERMINATION OF CYTOTOXICITY
- Method: Adjusted relative total growth (Adj. RTG); to define the number of viable cells (CE2: Cloning Efficiency at the end of the expression period), an average of 1.6 cells/well were seeded in two 96-well plates/culture (four plates/dose-level). After at least 7 days of incubation, in a 37°C, 5% CO2 humidified incubator, the clones were counted.

Adj. RTG (Adjusted Relative Total Growth) = (Adjusted RSG x RCE2) / 100

With RSG= Relative suspension growth calculated as follows: RSG = SG treated / SG vehicle control x 100
Suspension Growth (SG) for the 3-hour treatment = Daily growth on day 1 x daily growth on day 2
Suspension Growth (SG) for the 24-hour treatment = Cell growth during the 24-hour treatment x daily growth on day 1 x daily growth on day 2
Adjusted RSG = Cell count factor x (SG treated/SG vehicle control) x 100
with Cell count factor = treated post-treatment cell count / vehicle control post-treatment cell count

RCE2 = (CE2 treated/ CE2 vehicle control) x 100
CE2 = - Ln [empty wells / total wells] / number of cells per wells (ca. 1.6)

OTHER EXAMINATIONS:
For scoring of colonies in mutant plates, the following parameters were considered:
. well containing mutant colony (small or large),
. well not containing mutant colony,
. when both small and large colonies are present in the same well both mutant colonies were counted (one small and one large).
size of small colonies: < 25% of the diameter of the well (sign of inter-gene mutations: chromosomal rearrangement, translocation)
size of large colonies: > 25% of the diameter of the well (sign of intra-gene mutations: point mutations, base deletions)
Evaluation criteria:
Acceptance criteria:
Criteria for the vehicle controls:
. the Cloning Efficiency (CE2) of the vehicle controls at the end of the expression time should be between 0.65 and 1.2,
. the mutation frequency of the vehicle controls should fall within the normal range of 50 x 10-6 - 170 x 10-6,
. the suspension growth of the vehicle controls should be between 8 and 32 for the 3-hour treatment period, and between 32 and 180 for the 24-hour treatment period.
Criteria for the positive controls:
. the increase above the vehicle control mutation frequency (IMF) should be at least 300 x 10-6, the increase in the small colony mutation frequency accounting for at least 40%,
. or the increase in the small colony mutation frequency should be at least 150 x 10-6 above that seen in the concurrent vehicle control.
In addition, the upper limit of cytotoxicity observed in the positive control culture should have an Adj. RTG greater than 10%.

Evalution criteria:
a positive result, which should fulfill the following criteria:
. at least at one dose-level the mutation frequency minus the mutation frequency of the vehicle control (IMF) equals or exceeds the Global Evaluation Factor (GEF) of 126 x 10-6,
. a dose-response relationship is demonstrated by a statistical analysis.

Noteworthy increases in the mutation frequency observed only at high-levels of cytotoxicity (Adj. RTG lower than 10%), but with no evidence of mutagenicity at dose-levels with Adj. RTG between 10 and 20%, are not considered as positive results.

A test item may be considered as non-mutagenic when there is no culture showing an Adj. RTG value between 10 and 20% if (Moore et al., 2002):
. there is at least one negative data point between 20 and 25% Adj. RTG and no evidence of mutagenicity in a series of data points between 100 and 20% Adj. RTG,
. there is no evidence of mutagenicity in a series of data points between 100 and 25% and there is also a negative data point between 10 and 1% Adj. RTG.
Statistics:
A linear regression was performed to assess the linear trend between the mutation frequency and the dose. This statistical analysis was performed using SAS Enterprise Guide software version 4.2 (SAS version 9.2, SAS Institute Inc; Appendix 4). Only individual mutation frequencies obtained from cultures showing an Adj. RTG ≥ 10% were used in this analysis.

Results and discussion

Test results
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
see Tables 7.6.1/1 to 7.6.1/4
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
see Tables 7.6.1/1 to 7.6.1/4
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: at 5000 µg/mL, the pH was approximately 7.4 (7.1 for the vehicle control)
- Effects of osmolality: at 5000 µg/mL, the osmolality was equal to 331 mOsm/kg H2O (298 mOsm/kg H2O for the vehicle control).
- Evaporation from medium: no data
- Water solubility: no data
- Precipitation: no data but a priori the test substance is freely soluble in water

RANGE-FINDING/SCREENING STUDIES:To assess the cytotoxicity of the test item, six dose-levels (one culture/dose-level) were tested both with and without metabolic activation.
Following the 3-hour treatment without S9 mix, a severe toxicity was induced at dose-levels ≥ 100 μg/mL, as shown by a 82-100% decrease in the Adjusted Relative Total Growth. Following the 24-hour treatment without S9 mix, a moderate to severe toxicity was induced at dose-levels ≥ 10 μg/mL, as shown by a 41-100% decrease in Adj. RTG. Following the 3-hour treatment with S9 mix, a moderate to severe toxicity was induced at dose-levels ≥ 100 μg/mL, as shown by a 45-100% decrease in Adj. RTG.

COMPARISON WITH HISTORICAL CONTROL DATA: With one exception which is not considered to have a biological impact on the validity of the
study, the Cloning Efficiencies (CE2), the Suspension Growths (SG) and the mutation frequencies of the vehicle controls were as specified in the acceptance criteria. Moreover, the induced mutation frequencies obtained for the positive controls met the acceptance criteria specified in the study plan. The study was therefore considered as valid.

ADDITIONAL INFORMATION ON CYTOTOXICITY: Without S9 mix, following the 3-hour treatment, a slight to severe toxicity was induced at dose-levels
≥ 25 μg/mL, as shown by a 35-100% decrease in Adj. RTG. Following the 24-hour treatment, a moderate to severe toxicity was induced at dose-levels ≥ 3.13 μg/mL, as shown by a 43-100% decrease in Adj. RTG.
With S9 mix, in the first experiment, a slight to severe toxicity was induced at dose-levels ≥ 50 μg/mL, as shown by a 37-100% decrease in Adj. RTG.
In the second experiment, a slight to severe toxicity was induced at dose-levels ≥ 25 μg/mL, as shown by a 33-95% decrease in Adj. RTG.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Table 7.6.1/1: mutagenicity results of first experiment without S9 mix, 3-hour treatment:

Doses (µg/mL)

Individual values

Mean values of C1 and C2

Cultures

Adj RTG %

MF x 10-6

Adj RTG %

MF x 10-6

Large colony (x 10-6)

Small colony

0

C1

100

95

100

110

47

62

C2

100

126

6.25

C1

67

99

84

95

42

50

C2

100

91

12.5

C1

84

103

83

109

59

47

C2

83

114

25

C1

54

81

65

85

30

53

C2

75

90

50

C1

20

106

22

131

44

83

C2

23

156

75

C1

2

189

2

190

66

119

C2

2

192

MMS (25 µg/mL)

C1

59

351

64

385

134

 

222

C2

69

418

 

Table 7.6.1/2: mutagenicity results of second experiment without S9 mix, 24-hour treatment:

Doses (µg/mL)

Individual values

Mean values of C1 and C2

Cultures

Adj RTG %

MF x 10-6

Adj RTG %

MF x 10-6

Large colony (x 10-6)

Small colony

0

C1

100

118

100

113

39

69

C2

100

108

3.13

C1

46

141

57

153

66

82

C2

68

165

6.25

C1

33

166

50

154

44

105

C2

68

142

12.5

C1

14

205

21

199

58

130

C2

28

193

25

C1

6

320

11

212

57

149

C2

16

104

50

C1

0

227

0

200

50

142

C2

0

174

MMS (5 µg/mL)

C1

35

1058

38

929

182

587

C2

42

800

 

Table 7.6.1/3: mutagenicity results of first experiment with S9 mix, 3-hour treatment:

Doses (µg/mL)

Individual values

Mean values of C1 and C2

Cultures

Adj RTG %

MF x 10-6

Adj RTG %

MF x 10-6

Large colony (x 10-6)

Small colony

0

C1

100

145

100

147

47

95

C2

100

149

12.5

C1

94

135

92

113

49

60

C2

90

92

25

C1

122

147

102

160

47

104

C2

83

172

50

C1

63

177

63

171

52

116

C2

63

165

100

C1

18

180

15

196

58

129

C2

12

213

CPA (3 µg/mL)

C1

54

990

45

1047

238

674

C2

35

1104

 

Table 7.6.1/4: mutagenicity results of second experiment with S9 mix, 3-hour treatment:

Doses (µg/mL)

Individual values

Mean values of C1 and C2

Cultures

Adj RTG %

MF x 10-6

Adj RTG %

MF x 10-6

Large colony (x 10-6)

Small colony

0

C1

100

47

100

63

30

32

C2

100

78

6.25

C1

68

101

83

96

56

36

C2

98

91

12.5

C1

65

75

80

74

39

33

C2

96

74

25

C1

56

109

64

104

41

60

C2

73

98

50

C1

57

84

67

81

23

57

C2

76

79

100

C1

49

91

51

100

43

54

C2

52

110

200

C1

2

131

5

111

54

55

C2

8

92

CPA (3 µg/mL)

C1

31

613

46

598

152

389

C2

61

584

 

Applicant's summary and conclusion

Conclusions:
Under the experimental conditions of this study, the test item, Cocamidopropyl hydroxysultaine, did not show any mutagenic activity in the mouse lymphoma assay, up to 200 μg/mL in the presence of a rat metabolizing system (3-hour treatment) or up to 75 μg/mL (3-hour treatment) or 50 μg/mL (24-hour treatment) in the absence of a rat metabolizing system.
Executive summary:

In an in vitro mammalian cell mutation assay, performed according to OECD 476 and GLP,  cocamidopropyl hydroxysultaine (as an aqueous solution of purity 36.2%) diluted in water for injections was tested in the L5178Y Tk +/- mouse lymphoma cell line in the presence and the absence of mammalian metabolic activation (S9 mix). Prior to the mutagenicity experiments, a preliminary assay was conducted in order to assess the cytotoxicity of the test substance.The substance was tested for its toxicity at dose levels between 0 and 5000 µg/mL as recommended in the OECD guideline.The amount used for the test (preliminary and main assays) was calculated as concentration of Cocamidopropylhydroxysultaine and therefore a correction factor of 2.76 was applied. Two independent experiments were then performed. In Experiment 1, L5178Y TK +/- mouse lymphoma cells (heterozygous at the thymidine kinase locus) were exposed to the test item at eight dose levels, in duplicate, together with vehicle (water) and positive controls (Methylmethane sulphonate (MMS) or Cyclophosphamide (CPA) without and with metabolic activation respectively) using 3-hour exposure groups both in the absence and presence of metabolic activation. In Experiment 2, the cells were exposed to the test item at seven dose levels using a 3hour exposure group in the presence of metabolic activation and a 24hour exposure group in the absence of metabolic activation. The concentration range of test item was selected following the results of the preliminary toxicity test, and in Experiment 1 was 6.25 to 200 µg/ml in the absence of metabolic activation and 12.5 to 400 µg/mL in the presence of metabolic activation. In Experiment 2 the dose range was 3.13 to 400 µg/ml in the absence of metabolic activation, and 6.25 to 200 µg/ml in the presence of metabolic activation. The vehicle controls had acceptable mutant frequency values that were within the normal range for the L5178Y cell line at the TK +/- locus. The positive control items induced marked increases in the mutant frequency indicating the satisfactory performance of the test and of the activity of the metabolising system.