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Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2018-02-12 to 2018-10-29
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report date:
2018

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
Adopted July 29, 2016
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
in vitro mammalian cell gene mutation test using the Hprt and xprt genes

Test material

Constituent 1
Chemical structure
Reference substance name:
Sodium 2-(1-carboxylatoethoxy)-1-methyl-2-oxoethyl isooctadecanoate
EC Number:
266-533-8
EC Name:
Sodium 2-(1-carboxylatoethoxy)-1-methyl-2-oxoethyl isooctadecanoate
Cas Number:
66988-04-3
Molecular formula:
C24H44O6.Na
IUPAC Name:
sodium 2-(1-carboxylatoethoxy)-1-methyl-2-oxoethyl isooctadecanoate
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch no. of test material: Pationic ISL (Lot: 1731200025)
- Expiration date of the lot/batch: 12 months after opening

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature
- Solubility and stability of the test substance in the solvent/vehicle: All test item solutions were freshly prepared immediately prior to use.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: A solubility test was performed with different solvents and vehicles. Based on the results of the solubility test, treatment medium (MEM + 0% FBS) was used as solvent. The solvent was compatible with the survival of the cells and the S9 activity. The pH-value detected with the test item was within the physiological range (pH 7.0 ± 0.4). Osmolality of the highest test item concentration was 324 mOsmol/kg.

Method

Target gene:
Hprt
Species / strain
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: ATCC (CCL-93)
- Suitability of cells: These cells are characterised by their high proliferation rate and their high cloning efficiency of untreated cells (usually more than 50%). These features of the cells are necessary for the appropriate performance of the study.
- Cell cycle length, doubling time or proliferation index: 12 - 14 h doubling time
- Methods for maintenance in cell culture: Freshly thawed cells from stock cultures were maintained in plastic culture flasks in minimal essential medium (MEM) and cultured at a humidified atmosphere of 5% CO2 and at 37 °C.


MEDIA USED
- Type and identity of media including CO2 concentration if applicable: MEM supplemented with 10% (v/v) fetal bovine serum (FBS), 100 U/100 µg/mL penicillin/streptomycin, 2 mM L-glutamine, 25 mM HEPES and 2.5 µg/mL amphotericin B; cultured at a humidified atmosphere of 5% CO2 and at 37 °C
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically 'cleansed' against high spontaneous background: yes
Metabolic activation:
with and without
Metabolic activation system:
S9 liver
Test concentrations with justification for top dose:
Cytotoxicity of the test item was determined in a pre-experiment. In the pre-experiment it was observed that cytotoxic effects occurred without metabolic activation at concentrations of 10 µg/mL or higher and with metabolic activation at concentrations of 100 µg/mL or higher. Based on these data the concentrations for the main experiment were selected.
The test item was tested for cytotoxicity at the following concentrations:
* without metabolic activation: 2, 4, 6, 8, 10, 14, 16 and 20 µg/mL
* with metabolic activation: 25, 50, 55, 60, 65, 70, 75, 80, 85 and 90 µg/mL
According to OECD test guideline 476, more than 4 concentrations may be particularly important for evaluating mutagenicity when using single cultures. Therefore, the following 5 concentrations were selected on the basis of cytotoxicity for evaluating mutagenicity.
* without metabolic activation: 4, 6, 8, 10 and 14 µg/mL
* with metabolic activation: 25, 55, 65, 75 and 85 µg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: treatment medium (MEM + 0 % FBS)
- Justification for choice of solvent/vehicle: A solubility test was performed with different solvents and vehicles. Based on the results of the solubility test, the treatment medium was used as solvent. The solvent was compatible with the survival of the cells and the S9 activity.
Controls
Untreated negative controls:
yes
Remarks:
Treatment medium
Negative solvent / vehicle controls:
no
Remarks:
Not required as treatment medium was used as vehicle for test item.
True negative controls:
no
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
ethylmethanesulphonate
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium
- Cell density at seeding: Approximately 10–20E+06 cells per each concentration, negative and positive controls, were seeded in complete cell culture medium (MEM supplemented with 10 % FBS) in a 175 cm³ culture flask.

DURATION
- Preincubation period: 24 h after seeding
- Exposure duration: 4 h
- Expression time (cells in growth medium): 7–9 days after treatment
- Selection time (if incubation with a selection agent): 9–11 days for mutant frequency and 6–8 days for cloning efficiency

SELECTION AGENT (mutation assays): 11 µg/mL 6-thioguanine (TG)

NUMBER OF REPLICATIONS: 2 for cytotoxicity; 2–5 for mutagenicity

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: Colonies were fixed with methanol, stained with Giemsa and counted.

NUMBER OF CELLS EVALUATED: 200 cells for cloning efficiency and 400000 cells for mutagenicity

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency
- Any supplementary information relevant to cytotoxicity: The relative survival (indicator of cytotoxicity) was calculated based on the cloning efficiency of the cells plated immediately after treatment adjusted by any loss of cells during treatment.
Evaluation criteria:
A test chemical is considered to be clearly negative if, in all experimental conditions examined,
- none of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
- there is no concentration-related increase when evaluated with an appropriate trend-test, and
- all results are inside the distribution of the historical negative control data.
A test chemical is considered to be clearly positive if, in any of the experimental conditions examined,
- at least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
- the increase is concentration-related when evaluated with an appropriate trend test, and
- any of the results are outside the distribution of the historical negative control data.
- if there is by chance a low spontaneous mutation rate in the corresponding negative control a concentration-related increase of the mutations within their range has to be discussed.
According to the OECD guideline, the biological relevance is considered first for the interpretation of results.
Statistics:
The non-parametric Mann-Whitney test was applied to the mutation data to determine any significant difference in mutant frequency for the concentration groups when compared to the negative control. Mutant frequencies of the negative control were used as reference. Additionally, the chi-squared test for trend was conducted to determine a statistically significant concentration-response relationship.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
At the highest tested concentration for mutagenicity (75 µg/mL)
Vehicle controls validity:
not examined
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
At the highest tested concentration for mutagenicity (14 µg/mL)
Vehicle controls validity:
not examined
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING/SCREENING STUDIES: In the first pre-experiment eight concentrations [250, 500, 600, 700, 800, 1000, 1500 and 2000 µg/mL] were tested. Due to very high toxicity, the pre-experiment had to be repeated with lower concentrations (1–250 µg/mL), which then allowed selection of the concentrations to be tested in the main experiments.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95 %): See Table 5 in “any other information on materials and methods”.

Any other information on results incl. tables

Cytotoxicity: A biologically relevant growth inhibition (i.e. reduction of relative survival below 70%) was observed after the treatment with the test item in the experiment with and without metabolic activation.

In the experiment without metabolic activation the relative survival was 10% for the highest concentration (14 µg/mL) evaluated for mutagenicity.

The highest biologically relevant concentration evaluated in the mutagenicity experiment with metabolic activation was 75 µg/mL with a relative survival of 15%. At concentration of 60 µg/mL the relative survival value was decreased (RS = 7%) but was considered to be an outlier in comparison to the next lower (55 µg/mL, RS = 77%) and the next higher (65 µg/mL; RS = 76%) tested concentrations. As the next lower and higher concentrations showed no cytotoxicity, this decrease was regarded as not biologically relevant and potentially caused by technical reasons.

Mutagenicity: In the experiments no biologically relevant increase of mutants (i.e. above the upper limit of the historical control data and no statistically relevant increase) was found after treatment with the test item (without and with metabolic activation). In the experiment without metabolic activation one mutant value (at concentration 14 µg/mL with 41.5 mutants/106cells) was found to be slightly above the upper limit of the historical control data (40.2 mutants/106cells). However, at this concentration, the relative survival is 10% and this positive result, according to the OECD guideline 476, should be interpreted with caution. Since this was only a slight increase and no statistically significant increase was determined, this effect was considered as not biologically relevant. All the other mutant values were within the historical control data range of the test facility.

Table 1: Cytotoxicity, without metabolic activation

Group

Concen-tration

[µg/mL]

Number of cells at the

Number of colonies per flaska

CEb [%]

Adjusted CEc [%]

Relative Survival (RS)d

[%]

beginning of treatment

end of treatment

I

II

mean

NC1

0

10000000

9996000

161

156

159

79

79

100

NC2

10000000

10693000

137

167

152

76

81

1

2

10000000

10540000

192

189

191

95

100

125

2

4

10000000

10812000

162

175

169

84

91

114

3

6

10000000

10523000

169

143

156

78

82

102

4

8

10000000

8823000

127

125

126

63

56

69

5

10

10000000

6817000

63

53

58

29

20

25

6

14

20000000

6358000

46

51

49

24

8

10

7

16

20000000

4148000

70

59

65

32

7

8

8

20

20000000

2754000

77

81

79

40

5

7

EMS

300

10000000

11577000

163

170

167

83

96

120

NC: negative control

CE: cloning efficiency

EMS: ethyl methanesulfonate

a: number of cells plated: 200 cells/flask

b: cloning efficiency: CE [%] = [(number of colonies / number of cells plated) x 100]

c: adjusted CE [%] = [CE x (number of cells at the end of treatment / number of cells at the beginning of treatment)]

d: relative survival: RS [%] = [(adjusted CE in treated culture / adjusted CE in the negative control) x 100]


Table 2: Mutagenicity, without metabolic activation

 

CE in non-selective medium

CE in selective medium

 

Group

Concen-tration

[µg/mL]

Number of colonies per flaska

CEb [%]

Number of colonies per flaskc

CEb[%]

Mutant Frequency per 106cellsd

I

II

mean

I

II

III

IV

V

mean

SD

NC1

0

168

163

166

83

13

8

11

12

8

10.4

2.1

0.0026

31.4

NC2

150

160

155

78

8

13

13

11

14

11.8

2.1

0.0030

38.1

2

4

150

165

158

79

9

7

11

8

8

8.6

1.4

0.0022

27.3

3

6

143

161

152

76

11

14

6

16

8

11.0

3.7

0.0028

36.2

4

8

171

150

161

80

7

13

10

9

6

9.0

2.4

0.0023

28.0

5

10

161

175

168

84

9

7

8

4

6

6.8

1.7

0.0017

20.2

6

14

138

146

142

71

11

10

11

13

14

11.8

1.5

0.0030

41.5

EMS

300

155

165

160

80

60

75

76

76

77

72.8

6.4

0.0182

227.5

NC: negative control

CE: cloning efficiency

EMS: ethyl methanesulfonate

a: number of cells plated: 200 cells/flask

b: cloning efficiency:CE [%] = [(number of colonies / number of cells plated) x 100]

c: number of cells plated: 400000 cells/petri dish

d: mutant frequency (per 106cells): MF = [CE of mutant colonies in selective medium / CE in non-selective medium) x 106]


 Table 3: Cytotoxicity, with metabolic activation

Group

Concen-tration

[µg/mL]

Number of cells at the

Number of colonies per flaska

CEb [%]

Adjusted CEc [%]

Relative Survival (RS)d

[%]

beginning of treatment

 end of treatment

I

 II

 mean

NC1

0

10000000

13991000

113

132

123

61

86

100

NC2

10000000

13566000

148

148

148

74

100

1

25

10000000

12563000

153

141

147

74

92

99

2

50

10000000

13549000

108

114

111

56

75

81

3

55

10000000

13566000

109

103

106

53

72

77

4

60

10000000

4471000

28

29

29

14

6

7

5

65

10000000

13634000

114

93

104

52

71

76

6

70

10000000

8755000

35

38

37

18

16

17

7

75

20000000

12070000

31

63

47

24

14

15

8

80

20000000

9520000

31

41

36

18

9

9

9

85

20000000

7344000

37

37

37

19

7

7

10

90

20000000

7242000

14

21

18

9

3

3

DMBA

1.0

10000000

14484000

142

145

144

72

104

112

DMBA

1.5

10000000

14484000

146

142

144

72

104

112

NC: negative control

CE: cloning efficiency

DMBA: 7,12-dimethylbenz(a)anthracene

a: number of cells plated: 200 cells/flask

b: cloning efficiency: CE [%] = [(number of colonies / number of cells plated) x 100]

c: adjusted CE [%] = [CE x (number of cells at the end of treatment / number of cells at the beginning of treatment)]

d: relative survival: RS [%] = [(adjusted CE in treated culture / adjusted CE in the negative control) x 100]

 

Table 4: Mutagenicity, with metabolic activation

 

CE in non-selective medium

CE in selective medium

 

Group

Concen-tration

[µg/mL]

Number of colonies per flaska

CEb[%]

Number of colonies per flaskc

CEb

[%]

Mutant Frequency per 106cellsd

I

II

mean

I

II

III

IV

V

mean

SD

NC1

0

155

137

146

73

9

7

9

13

3

8.2

3.2

0.0021

28.1

NC2

146

140

143

72

13

7

10

11

7

9.6

2.3

0.0024

33.6

1

25

143

128

136

68

11

7

13

14

5

10.0

3.5

0.0025

36.9

3

55

156

178

167

84

13

15

16

10

9

12.6

2.7

0.0032

37.7

5

65

167

181

174

87

16

20

11

12

6

13.0

4.7

0.0033

37.4

7

75

167

163

165

83

15

12

8

14

8

11.4

2.9

0.0029

34.5

DMBA

1.0

167

144

156

78

89

110

96

103

110

101.6

8.2

0.0254

326.7

DMBA

1.5

158

147

153

76

121

100

114

121

116

114.4

7.7

0.0286

375.1

NC: negative control

CE: cloning efficiency

DMBA: 7,12-dimethylbenz(a)anthracene

a: number of cells plated: 200 cells/flask

b: cloning efficiency: CE [%] = [(number of colonies / number of cells plated) x 100]

c: number of cells plated: 400000 cells/petri dish

d: mutant frequency (per 106cells): MF = [CE of mutant colonies in selective medium / CE in non-selective medium) x 106] 

Table 5: Historical Laboratory Control Data (January 2015 – April 2018)

 

Negative Control

Positive Control

 

-S9

+S9

EMS

DMBA

Mean

24

27

290

414

Min

5

8

186

117

Max

43

49

631

788

SD

7.92

8.60

71.86

170.31

RSD [%]

32.51

32.12

24.74

41.15

n =

64

70

63

62

LCL

8.5

9.6

146.7

73.2

UCL

40.2

44.0

434.2

754.4

NC: negative control

PC: positive controls (-S9 EMS; +S9 DMBA)

S9: metabolic activation

Mean: mean of mutants/106cells

Min.: minimum of mutants/106cells

Max.: maximum of mutants/106cells

SD: standard deviation

RSD: relative standard deviation

n: number of control values

LCL: lower control limit

UCL: upper control limit

Applicant's summary and conclusion

Conclusions:
In an in vitro cell gene mutagenicity test, the test item sodium isostearoyl lactylate is considered to be non-mutagenic in the HPRT locus using V79 cells of the Chinese hamster.
Executive summary:

The test item sodium isostearoyl lactylate was assessed for its potential to induce mutations at the HPRT locus using V79 cells of the Chinese hamster. The selection of the concentrations was based on data from the pre-experiments. The main experiments with and without metabolic activation were performed as a 4 h short-term exposure assay. Cytotoxicity was tested in the main experiment at the ranges of 2–20 µg/mL without metabolic activation and 25–90 µg/mL with metabolic activation. The mutagenicity of the test item was investigated at the following concentrations: without metabolic activation: 4, 6, 8, 10 and 14 µg/mL and with metabolic activation: 25, 55, 65 and 75 µg/mL No precipitation of the test item was noted in the experiments.

Biologically relevant growth inhibition (i.e. relative survival < 70 %) was observed in the experiment with and without metabolic activation. In the experiment without metabolic activation the relative survival was 10 % for the highest concentration (14 µg/mL) evaluated for mutagenicity. The highest biologically relevant concentration evaluated for mutagenicity with metabolic activation was 75 µg/mL with a relative survival of 15 %.

In the experiments no biologically relevant increase of mutants (i.e. above the upper limit of the historical control data) was found after treatment with the test item (without and with metabolic activation) and no concentration-response relationship was observed. DMBA and EMS were used as positive controls and showed distinct and biologically relevant effects in mutation frequency.

Thus, in this in vitro cell gene mutagenicity test, the test item sodium isostearoyl lactylate is considered to be non-mutagenic in the HPRT locus using V79 cells of the Chinese hamster.

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