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Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Gene mutation (Bacterial reverse mutation assay / Ames test): S. typhimurium TA 98, TA 100, TA 1535, TA 1537 and Escherichia coli WP2 uvrA: negative with and without metabolic activation (OECD 471)

Mammalian micronucleus (micronucleus test): negative with and without metabolic activation (OECD 487)

Mammalian mutagenicity (HPRT): negative with and without metabolic activation (OECD 476)

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:
5 Oct 2017 - 7 Dec 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
21 July 1997
GLP compliance:
yes (incl. QA statement)
Remarks:
Országos Gyógyszerészeti és Élelmezés-egészégügyi Intézet
Type of assay:
bacterial reverse mutation assay
Target gene:
his, trp operon
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: Trinova Biochem GmbH, Rathenau Str. 2, D-35394 Giessen, Germany
- Methods for maintenance in cell culture if applicable: The viability of each testing culture was determined by plating 0.1 mL of the 10E05, 10E06, 10E07 and 10E08 dilutions of cultures on nutrient agar plates.

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: ready-to-use minimal glucose agar (MGA) plates
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: not reported
- Periodically checked for karyotype stability: not reported
- Periodically 'cleansed' against high spontaneous background: not reported
Additional strain / cell type characteristics:
not applicable
Species / strain / cell type:
E. coli WP2 uvr A
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells:
Trinova Biochem GmbH, Rathenau Str. 2, D-35394 Giessen, Germany
- Methods for maintenance in cell culture if applicable:
The viability of each testing culture was determined by plating 0.1 mL of the 10E05, 10E06, 10E07 and 10E08 dilutions of cultures on nutrient agar plates.

MEDIA USED
- Type and identity of media including CO2 concentration if applicable:
ready-to-use minimal glucose agar (MGA) plates
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: not reported
- Periodically checked for karyotype stability: not reported
- Periodically 'cleansed' against high spontaneous background: not reported
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
post mitochondrial supernatant (S9) prepared from livers of Phenobarbital/β-naphthoflavone-induced rats
Test concentrations with justification for top dose:
5000, 1600, 500, 160, 50 or 16 μg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: ultrapure water (ASTM Type 1) and dimethyl sulfoxide (DMSO)
- Justification for choice of solvent/vehicle: In the preliminary Solubility and Concentration Range Finding Tests ultrapure water (ASTM Type 1) was found as appropriate vehicle for preparing the test item solutions. This vehicle is compatible with the survival of the bacteria and the S9 activity. DMSO also was used depending on the solubility of the test item and the solubility of positive control reference items.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
2-acetylaminofluorene
9-aminoacridine
sodium azide
methylmethanesulfonate
other: 4-Nitro-1,2-phenylenediamine, NPD, 4 μg/plate, -S9, TA98
Details on test system and experimental conditions:
METHOD OF APPLICATION: plate incorporation test for the initial mutation test and a pre-incubation test for the confirmatory mutation test

DURATION
- Preincubation period: 11-13 h
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: Conditions were investigated in triplicate.

DETERMINATION OF CYTOTOXICITY
- Method: colony and background lawn development
Evaluation criteria:
A test item is considered mutagenic if:
- a dose–related increase in the number of revertants occurs and/or;
- a reproducible biologically relevant positive response for at least one of the dose groups occurs in at least one strain with or without metabolic activation.

An increase is considered biologically relevant if:
- in strain Salmonella typhimurium TA100 the number of reversions is at least twice as high as the reversion rate of the vehicle control,
- in strain Salmonella typhimurium TA98, TA1535, TA1537 and Escherichia coli WP2 uvrA the number of reversions is at least three times higher than the reversion rate of the vehicle control.

A test item is considered non-mutagenic if it produces neither a dose-related increase in the number of revertants nor a reproducible biologically.

The tests (Initial and Confirmatory Mutation Tests) are considered valid if:
- All of the Salmonella tester strains demonstrate the presence of the deep rough mutation (rfa) and the deletion in the uvrB gene.
- The Salmonella typhimurium TA98 and TA100 tester strains demonstrate the presence of the pKM101 plasmid R-factor.
- The Escherichia coli WP2 uvrA culture demonstrate the deletion in the uvrA gene.
- The bacterial cultures demonstrate the characteristic mean number of spontaneous revertants in the vehicle controls.
- The tester strain culture titers are in the 109 cells/mL order.
- The batch of S9 used in this study shows the appropriate biological activity.
- The reference mutagens show the expected increase (at least a 3.0-fold increase) in induced revertant colonies over the mean value of the respective vehicle control.
- There are at least five analyzable concentrations (at each tester strain) (a minimum of three non-toxic dose levels is required to evaluate assay data).
Statistics:
Not performed.
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:
not examined
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:
not examined
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:
not examined
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:
not examined
Positive controls validity:
valid
Key result
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 examined
Positive controls validity:
valid

Table 1: Summary of the Initial Mutation Test

Concentrations (µg/plate)

Salmonella typhimurium tester strains

Escherichia coli

TA98

TA100

TA1535

TA1537

WP2uvrA

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

Mean values of revertants per plate and mutation rate(MR)

Mean

MR

Mean

MR

Mean

MR

Mean

MR

Mean

MR

Mean

MR

Mean

MR

Mean

MR

Mean

MR

Mean

MR

UntreatedControl

26.3

0.99

30.0

1.23

99.3

1.07

105.3

0.90

13.7

1.11

11.3

0.97

9.7

0.97

11.3

1.06

47.0

1.09

54.7

1.12

DMSOControl

23.7

1.00

21.0

1.00

94.0

1.00

9.0

1.00

6.3

1.00

11.3

1.00

48.3

1.00

UltrapureWaterControl

26.7

1.00

24.3

1.00

92.7

1.00

117.3

1.00

12.3

1.00

11.7

1.00

10.0

1.00

10.7

1.00

43.0

1.00

49.0

1.00

5000

30.3

1.14

29.0

1.19

89.0

0.96

106.3

0.91

11.7

0.95

10.3

0.89

8.0

0.80

11.0

1.03

53.0

1.23

56.7

1.16

1600

29.0

1.09

27.0

1.11

93.0

1.00

105.0

0.89

12.0

0.97

9.7

0.83

8.3

0.83

8.0

0.75

47.3

1.10

56.7

1.16

500

21.0

0.79

26.3

1.08

86.7

0.94

113.0

0.96

11.7

0.95

9.7

0.83

8.0

0.80

9.7

0.91

43.3

1.01

55.3

1.13

160

20.0

0.75

24.0

0.99

99.7

1.08

104.3

0.89

9.7

0.78

13.3

1.14

8.7

0.87

12.0

1.13

46.3

1.08

49.3

1.01

50

21.7

0.81

24.0

0.99

95.7

1.03

122.0

1.04

12.7

1.03

9.3

0.80

9.0

0.90

10.0

0.94

48.7

1.13

55.7

1.14

16

27.7

1.04

28.0

1.15

91.7

0.99

111.3

0.95

11.0

0.89

9.3

0.80

10.0

1.00

9.7

0.91

43.7

1.02

53.7

1.10

NPD

(4µg)

323.3

13.66

SAZ

(2µg)

1744.0

18.82

1458.7

118.27

9AA

(50µg)

622.7

98.32

MMS

(2µL)

1085.3

25.24

2AA

(2µg)

2666.7

126.98

3162.7

33.65

178.0

19.78

183.3

16.18

2AA(50µg)

241.0

4.99

Conclusions:
No mutagenic effect was observed for the test substance tested up to cytotoxic concentration in any of the test strains without and with metabolic activation. The test substance is non-mutagenic in test strains used.
Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017-12-12 to 2018-05-02
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
July 29, 2016
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
May 30, 2008
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
Bayrisches Landesamt für Gesundheit und Lebensmittelsicherheit
Type of assay:
in vitro mammalian cell gene mutation test using the Hprt and xprt genes
Target gene:
hypoxanthine phosphoribosyltransferase (HPRT)
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Test concentrations with justification for top dose:
500, 600, 700, 800, 1000, 1500 and 2000 µg/mL, ±S9
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: cell culture medium (MEM)
- Justification for choice of solvent/vehicle: The solvent was selected based on the pre-experiment solubility.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
no
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
other: 7,12-dimethylbenz(a)anthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium
- Cell density at seeding: 2 - 5 x 10E6

DURATION
- Exposure duration: 4 h
- Expression time (cells in growth medium): 7 to 9 days
- Selection time (if incubation with a selection agent): 9 - 11 days

SELECTION AGENT (mutation assays): 6-thioguanine

NUMBER OF REPLICATIONS: 2

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: For determination of the relative survival (RS) two 25 cm2 flasks were seeded with approx. 200 cells in complete culture medium for each treatment group at the end of the expression period. After incubation for an appropriate time (6 - 7 days) colonies were fixed with methanol, stained with Giemsa and counted.

At the end of the expression period, for selection the mutants, about 4 x 10E5 cells for each treatment group were seeded in cell culture petri dishes with selective medium containing 11 µg/mL 6-thioguanine (TG) for further incubation. The cloning efficiencies (CE) were determined in parallel to the selection of mutants. For each treatment group two 25 cm2 flasks were seeded with approx. 200 cells in complete culture medium to determine the cloning efficiencies after additional 6 - 8 days. After incubation for an appropriate time (9 - 11 days for mutant frequency and 6 - 8 days for CE) colonies were fixed with methanol, stained with Giemsa and counted.

NUMBER OF CELLS EVALUATED: about 4 x 10E5 cells for mutant frequency; 200 cells for cloning efficiencies


DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: Cytotoxicity was evaluated by relative survival (RS). The cloning efficiency (CE) of cells plated immediately after treatment was adjusted by any loss of cells during treatment as compared with adjusted cloning efficiency in negative / solvent controls (assigned a survival of 100%).
Evaluation criteria:
A mutation assay was considered acceptable if it met the following criteria:
- Negative and/or solvent controls fell within the performing laboratories historical control data range: 7.8-39.7 mutants/10E6 cells (without metabolic activation) and 9.2 - 38.8 mutants/10E6 cells (with metabolic activation).
- The absolute cloning efficiency: ([number of positive cultures x 100] / total number of seeded cultures) of the negative and /or solvent controls was > 50%
- The positive controls (EMS and DMBA) induced a statistically significant increase compared with the concurrent negative control and were compatible with the laboratory historical data base.
- Two experimental conditions (e.g. without and with metabolic activation) were tested unless one results in a positive response

A test chemical was considered to be clearly negative if, in all experimental conditions examined
- none of the test concentrations exhibited a statistically significant increase compared with the concurrent negative control,
- there was no concentration-related increase when evaluated with an appropriate trend-test
- all results were inside the distribution of the historical negative control data

A test chemical was considered to be clearly positive if, in any of the experimental conditions examined
- at least one of the test concentrations exhibited a statistically significant increase compared with the concurrent negative control, and
- the increase was concentration-related when evaluated with an appropriate trend test, and
- any of the results were outside the distribution of the historical negative control data.
- if there was by chance a low spontaneous mutation rate in the corresponding negative and solvent controls a concentration related increase of the mutations within their range has to be discussed.
Statistics:
The non-parametric Mann-Whitney test was applied to the mutation data to prove the dose groups for any significant difference in mutant frequency compared to the negative controls.
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
not applicable
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
not applicable
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: The pH-value detected with the test item was within the physiological range (pH 7.0 ± 0.4).
- Precipitation: No precipitation of the test item was noted in any of the experiments.

RANGE-FINDING/SCREENING STUDIES: The toxicity of the test item was determined in a pre-experiment. Eight concentrations (250, 500, 600, 700, 800, 1000, 1500, 2000 µg/mL) were tested without and with metabolic activation for the 4 h exposure assay. There was no cytotoxicity observed in the pre-experiment.

HISTORICAL CONTROL DATA
- Negative (solvent/vehicle) historical control data: 7.8 - 39.7 mutants per 10E6 cells, -S9; 9.2 - 38.8 mutants per 10E6 cells, +S9

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: In the experiment without metabolic activation the relative survival was 90.27% for the highest concentration (2000 µg/mL) evaluated. The highest biologically relevant concentration evaluated with metabolic activation was 2000 µg/mL with a relative survival of 85.47%.
Remarks on result:
other: At 700 µg/mL and 2000 µg/mL, there was a slight increased mutant frequency outside the historical control data. As there was no evidence for a dose-response relationship, both effects were not considered biologically relevant.

Table 1:  Main Experiment - Toxicity, without metabolic activation

Dose 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

11577000

156

182

169.0

84.50

97.83

100.00

NC2

10000000

11815000

149

157

153.0

76.50

90.38

1

500

10000000

12359000

143

149

146.0

73.00

90.22

95.87

2

600

10000000

12461000

149

148

148.5

74.25

92.52

98.32

3

700

10000000

12869000

119

119

119.0

59.50

76.57

81.37

4

800

10000000

13124000

138

142

140.0

70.00

91.87

97.62

5

1000

10000000

12053000

121

111

116.0

58.00

69.91

74.29

6

1500

10000000

11951000

137

127

132.0

66.00

78.88

83.82

7

2000

10000000

11441000

140

157

148.5

74.25

84.95

90.27

EMS

300

10000000

11645000

100

104

102.0

51.00

59.39

63.11

NC: negative control

EMS: Ethylmethanesulfonate

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:  Main Experiment - Mutagenicity, without metabolic activation

 

CE in non-selective medium

CE in selective medium

 

Dose Group

Concen-tration

[µg/mL]

Number of colonies per flaska

CEb              [%]

Number of colonies per flaskc

CEd              [%]

Mutant Frequency per 106cellse

I

II

mean

I

II

III

IV

V

mean

SD

NC1

0

161

176

168.5

84.25

15

12

9

14

13

12.6

2.1

0.00315

37.4

NC2

164

183

173.5

86.75

9

10

14

9

6

9.6

2.6

0.00240

27.7

3

700

166

163

164.5

82.25

15

14

15

9

14

13.4

2.2

0.00335

40.7

4

800

163

189

176.0

88.00

6

8

5

6

6

6.2

1.0

0.00155

17.6

5

1000

156

170

163.0

81.50

12

9

6

11

14

10.4

2.7

0.00260

31.9

6

1500

170

162

166.0

83.00

6

15

14

4

11

10.0

4.3

0.00250

30.1

7

2000

167

167

167.0

83.50

13

14

10

15

18

14.0

2.6

0.00350

41.9

EMS

300

171

161

166.0

83.00

93

61

87

89

74

80.8

11.8

0.02020

243.4

 NC: negative control

EMS: Ethylmethanesulfonate

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

e: mutant frequency (per 10E6cells): MF = [CE of mutant colonies in selective medium / CE in non-selective medium) x 10E6]

Table 3:  Main Experiment - Toxicity, with metabolic activation

Dose 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

13838000

165

192

178.5

89.25

123.50

100.00

NC2

10000000

13838000

148

152

150.0

75.00

103.79

1

500

10000000

12750000

147

164

155.5

77.75

99.13

87.23

2

600

10000000

12189000

189

173

181.0

90.50

110.31

97.07

3

700

10000000

14093000

171

156

163.5

81.75

115.21

101.38

4

800

10000000

13515000

178

183

180.5

90.25

121.97

107.33

5

1000

10000000

12529000

179

175

177.0

88.50

110.88

97.57

6

1500

10000000

13294000

201

205

203.0

101.50

134.93

118.73

7

2000

10000000

13260000

150

143

146.5

73.25

97.13

85.47

DMBA

1

10000000

12869000

117

106

111.5

55.75

71.74

63.13

 NC: negative control

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:  Main Experiment - Mutagenicity, with metabolic activation

 

CE in non-selective medium

CE in selective medium

 

Dose Group

Concen-tration

[µg/mL]

Number of colonies per flaska

CEb              [%]

Number of colonies per flaskc

CEd              [%]

Mutant Frequency per 106cellse

I

II

mean

I

II

III

IV

V

mean

SD

NC1

0

168

171

169.5

84.75

11

8

15

10

9

10.6

2.4

0.00265

31.3

NC2

169

167

168.0

84.00

13

11

11

18

12

13.0

2.6

0.00325

38.7

3

700

164

172

168.0

84.00

6

7

8

15

7

8.6

3.3

0.00215

25.6

4

800

185

186

185.5

92.75

13

12

17

10

13

13.0

2.3

0.00325

35.0

5

1000

154

142

148.0

74.00

11

17

18

15

16

15.4

2.4

0.00385

52.0

6

1500

147

198

172.5

86.25

12

7

14

7

6

9.2

3.2

0.00230

26.7

7

2000

155

168

161.5

80.75

9

11

7

6

7

8.0

1.8

0.00200

24.8

DMBA

1

140

164

152.0

76.00

151

136

151

137

155

146.0

7.9

0.03650

480.3

 NC: negative control

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:: umber of cells plated: 400000 cells/petri dish

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

e: mutant frequency (per 10E6cells): MF = [CE of mutant colonies in selective medium / CE in non-selective medium) x 10E6]

Table 5:  Statistical Analysis –Main Experiment, without metabolic activation

Statistical significance at the 5% level (p < 0.05) was evaluated by means of the non-parametric Mann-Whitney test.

 

Dose Group

Concentration

[µg/mL]

Mutant Frequency

p-value

Statistical Significance

NC1

0

37.4

/

/

NC2

27.7

/

/

3

700

40.7

0.0360

+

4

800

17.6

0.0013

 +*

5

1000

31.9

0.9999

-

6

1500

30.1

0.9999

-

7

2000

41.9

0.0523

-

EMS

300

243.4

0.0007

+

NC:     Negative control

S:       Solvent Control

EMS:   Ethylmethanesulfonate

+:        significant

-:         not significant

+*:       significantly decreased compared to the negative control, therefore not relevant for interpretation of results

 

 

Table 6: Statistical Analysis–Main Experiment, with metabolic activation

Statistical significance at the 5% level (p < 0.05) was evaluated by means of the non-parametric Mann-Whitney test.

Dose Group

Concentration

[µg/mL]

Mutant Frequency

p-value

Statistical Significance

NC1

0

31.3

/

/

NC2

38.7

/

/

3

700

25.6

0.0693

-

4

800

35.0

0.9257

-

5

1000

52.0

0.0073

+

6

1500

26.7

0.1951

-

7

2000

24.8

0.0360

 +*

DMBA

1

480.3

0.0003

+

 

NC:     Negative control

S:       Solvent Control

DMBA:7,12-dimethylbenz(a)anthracene

+:        significant

-:         not significant

+*:       significantly decreased compared to the negative control, therefore not relevant for interpretation of resul

Conclusions:
In conclusion, in the described mutagenicity test under the experimental conditions reported, the test item butylmonoglycol sulphate, Na-salt is considered to be non-mutagenic at the HPRT locus using V79 cells of the Chinese Hamster.
Endpoint:
in vitro cytogenicity / micronucleus study
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017-11-06 to 2018-04-26
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)
Version / remarks:
29 July, 2016
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
Bayrisches Landesamt für Gesundheit und Lebensmittelsicherheit
Type of assay:
in vitro mammalian cell micronucleus test
Species / strain / cell type:
lymphocytes: human
Details on mammalian cell type (if applicable):
CELLS USED
- Sex, age and number of blood donors if applicable: Human peripheral blood lymphocytes from healthy and non-smoking donors with no known recent exposure to genotoxic chemicals and radiation were used to examine the ability of chemicals to induce cytogenetic damage and thus to identify potential carcinogens or mutagens in vitro.
- Whether whole blood or separated lymphocytes were used if applicable: whole

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: Complete culture medium was RPMI 1640 medium supplemented with: 15% fetal bovine serum (FBS); 100 U/100 µg/mL penicillin/streptomycin solution; and 2.4 µg/mL phytohemagglutinin (PHA). Treatment medium for the short-term exposure was complete culture medium without FBS. After treatment medium or treatment medium for the long-term exposure was complete culture medium with 15% FBS and 6 µg/mL cytochalasin B.
Additional strain / cell type characteristics:
not applicable
Cytokinesis block (if used):
cytochalasin B
Metabolic activation:
with and without
Metabolic activation system:
phenobarbital/β-naphtholflavone induced rat liver S9-mix
Test concentrations with justification for top dose:
Experiment I with short-term exposure (4 h): 1000, 1500 and 2000 µg/mL, ±S9
Experiment II with long-term exposure (44 h): 1000, 1500 and 2000 µg/mL, -S9

The selection of the concentrations was based on data from the pre-experiment.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: cell culture medium (RPMI)
- Justification for choice of solvent/vehicle: The test material was soluble in cell culture medium and the solvent was compatible with the survival of the cells and the S9 activity.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
no
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
methylmethanesulfonate
other: Colchicine
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 44 to 48 h
- Exposure duration: 4 h, ± S9 or 44 h, -S9
- Selection time (if incubation with a selection agent): 40 h for Experiment I and 43 for Experiment II
- Fixation time (start of exposure up to fixation or harvest of cells): 44 h

SPINDLE INHIBITOR (cytogenetic assays): Cytochalasin B

STAIN (for cytogenetic assays): acridine orange solution

NUMBER OF REPLICATIONS: 2

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: At the end of the cultivation, the complete culture medium was removed. Subsequently, the cells were treated with cold hypotonic solution (0.075 M KCl) for some minutes at room temperature and immediately centrifuged. The pellet was resuspended with a solution consisted of fixation solution + NaCl 0.9% (1+1) and centrifuged. After that the cells were fixed with methanol + glacial acetic acid (3+1). The cells were resuspended gently and the suspension was dropped onto clean glass slides. Consecutively, the cells were dried on a heating plate. The cells were stained with acridine orange solution.

NUMBER OF CELLS EVALUATED: 2000 binucleated cells (if possible) per concentration (1000 binucleated cells per slide)

CRITERIA FOR MICRONUCLEUS IDENTIFICATION: All slides, including those of positive and negative controls were independently coded before microscopic analysis. For each dose group at least 2000 binucleated cells (if possible) per concentration (1000 binucleated cells per slide) were analysed for micronuclei according to the criteria of Fenech, i.e. clearly surrounded by a nuclear membrane, having an area of less than one-third of that of the main nucleus, being located within the cytoplasm of the cell and not linked to the main nucleus via nucleoplasmic bridges. Mononucleated and multinucleated cells and cells with more than six micronuclei were not considered.

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: Cytokinesis block proliferation index (CBPI) was determined from 500 cells. The CBPI was used to calculate the % cytostasis, which indicates the inhibition of cell growth of treated cultures in comparison to control cultures.
Evaluation criteria:
A mutation assay was considered acceptable if it met the following criteria:
- The concurrent negative control was considered acceptable for addition to the laboratory historical negative control database.
- Concurrent positive controls induced responses that were compatible with those generated in the laboratory’s historical positive control data base and produced a statistically significant increase compared with the concurrent negative control.
- Cell proliferation criteria in the negative control were fulfilled.
- All experimental conditions were tested unless one resulted in positive results.
- Adequate number of cells and concentrations were analysable.

A test item was considered to be clearly positive if, in any of the experimental conditions examined:
- at least one of the test concentrations exhibited a statistically significant increase compared with the concurrent negative control
- the increase was concentration-related in at least one experimental condition when evaluated with an appropriate trend test
- any of the results were outside the distribution of the historical negative control data (e.g. Poisson-based 95% control limits).

When all of these criteria were met, the test item was considered able to induce chromosome breaks and/or gain or loss in this test system.
A test item was considered to be clearly negative if in all experimental conditions examined none of the criteria mentioned above were met.
Statistics:
The nonparametric χ² Test was performed to verify the results in both experiments.
Key result
Species / strain:
lymphocytes: human
Remarks:
Experiment I and II
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
not applicable
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No precipitate of the test item was noted in any concentration group evaluated in the main experiments.

RANGE-FINDING/SCREENING STUDIES: No precipitate of the test item was noted in the preliminary finding. The highest dose group evaluated in the pre-experiment was 2000 µg/mL.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Negative (solvent/vehicle) historical control data: Experiment I and II, -S9: micronucleated cell frequency 0.28% – 1.21%; Experiment I, +S9: micronucleated cell frequency 0.25% – 1.33%

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: In experiment I and II with and without metabolic activation no increase of the cytostasis above 30% was noted.

Table 1:  Summary: Experiment I and II, without metabolic activation

Dose Group

Concentration [µg/mL]

Number of cells evaluated

Cytostasis

[%]

Relative Cell Growth
[%]

Micro-nucleated
Cells Frequency
[%]

Historical Control Limits Negative Control

P

Statistical Significant Increasea

Exp. I

4 h treatment, 44 h fixation interval

C

0

2000

0

100

0.50

0.28% - 1.21%

/

/

4

1000

2000

1

99

0.55

-

-

5

1500

2000

 0*

127

0.35

-

-

6

2000

2000

 0*

119

0.45

-

-

MMS

50

2000

30

70

4.05

-

+

Colc

0.8

2200

32

68

1.30

-

+

 

 

Exp. II

44 h treatment, 44 h fixation interval

C

0

2000

0

100

0.65

0.28% - 1.21%

/

/

5

1000

2000

2

98

0.75

-

-

6

1500

2000

1

99

0.50

-

-

7

2000

2000

 0*

101

0.45

-

-

MMS

50

1708

27

73

3.61

-

+

Colc

0.02

2000

49

51

1.45

-

+

 

 

Table 2:  Summary: Experiment I, with metabolic activation

Dose Group

Concentration [µg/mL]

Number
 of cells evaluated

Cytostasis

[%]

Relative Cell Growth
[%]

Micro-

Nucleated
Cells
Frequency
[%]

Historical Control Limits Negative Control

P

Statistical Significant Increasea

Exp. I

4 h treatment,

44 h fixation interval

C

0

2000

0

100

0.65

0.25% - 1.33%

/

/

4

1000

2000

5

95

0.35

-

-

5

1500

2000

0

100

0.60

-

-

6

2000

2000

11

89

1.00

-

-

CPA

12.5

1755

41

59

2.56

-

+

C: Negative Control (Culture medium)

P: Precipitation (+: precipitation, -: no precipitation)

a: statistically significant increase compared to negative control (c² test , p<0.05).

+: significant; -: not significant

MMS: Methylmethanesulfonate, Positive Control (without metabolic activation) [50 µg/mL and 50 µg/mL]

Colc.: Colchicine, Positive Control (without metabolic activation) [0.02 µg/mL and 0.8 µg/mL]

CPA: Cyclophosphamide, Positive Control (with metabolic activation) [12.5 µg/mL]

Relative Cell Growth: 100 x ((CBPITest conc– 1) / (CBPIcontrol -1))

Cytostasis [%] = 100- Relative Cell Growth [%]

*: the cytostasis is defined 0, when the relative cell growth exceeds 100%

Conclusions:
In conclusion, it can be stated that during the study described and under the experimental conditions reported, the test item butylmonoglycol sulphate, Na-salt did not induce structural and/or numerical chromosomal damage in human lymphocytes. Therefore, butylmonoglycol sulphate, Na-salt is considered to be non-mutagenic with respect to clastogenicity and/or aneugenicity in the in vitro Mammalian Cell Micronucleus Test.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Mode of Action Analysis / Human Relevance Framework

not applicable

Additional information

Gene mutation

The test item butylmonoglycol sulphate, Na-salt was tested with regard to a potential mutagenic activity using the Bacterial Reverse Mutation Assay (OECD 471 and in compliance with GLP). The experiments were carried out using Salmonella typhimurium (Salmonella typhimurium TA98, TA100, TA1535 and TA1537), and Escherichia coli (Escherichia coli WP2 uvrA) in the presence and absence of a post mitochondrial supernatant (S9) prepared from livers of phenobarbital/β-naphthoflavone-induced rats (Toxi-Coop, 2017). The following concentrations of the test item were prepared and investigated in the initial and confirmatory mutation tests: 5000; 1600; 500; 160; 50 and 16 μg/plate.

 

No precipitation of the test item was observed on the plates in the examined bacterial strains at any examined concentration level (±S9 Mix) throughout the study. The test item did not show inhibitory, cytotoxic effects in the performed experiments. The colony and background lawn development was not affected in any case; the obtained revertant colony number decreases (compared to the revertant colony numbers of the vehicle control) remained within the biological variability range of the applied test system. The reported data of this mutagenicity assay showed that under the experimental conditions applied, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

In conclusion, the test item butylmonoglycol sulphate, Na-salt has no mutagenic activity on the applied bacterium tester strains under the test conditions used in this study.

 

Mammalian mutagenicity

The test item butylmonoglycol sulphate, Na-salt was assessed for its potential to induce mutations at the HPRT locus using Chinese hamster V79 cells in an OECD 476 study conducted in compliance with GLP. Test concentration evaluated were 500, 600, 700, 800, 1000, 1500 and 2000 µg/mL,±S9 for a 4-exposure period (Eurofins, 2018a). Due to the non-toxic effects at the lower concentrations (500 and 600 µg/mL), only the five highest concentrations were investigated further for determination of mutagenicity.

No precipitation of the test item was noted in the experiments. No growth inhibition evidenced by the relative survival (RS) was observed in the experiment without and with metabolic activation. In the experiments no biologically relevant increase of mutants was found after treatment with the test item (without and with metabolic activation). In conclusion, in the described mutagenicity test under the experimental conditions reported, the test item butylmonoglycol sulphate, Na-salt is considered to be non-mutagenic at the HPRT locus using V79 cells of the Chinese Hamster.

  

Mammalian cytogenicity

In order to investigate a possible potential of butylmonoglycol sulphate, Na-salt to induce micronuclei in human lymphocytes an in vitro micronucleus assay was carried out in an OECD 487 study conducted in compliance with GLP. In the main experiment without and with metabolic activation 2000 µg/mL test item was selected as the highest concentration (Eurofins, 2018b). The following concentrations were evaluated for micronuclei frequencies:

 

Experiment I with short-term exposure (4 h):1000, 1500 and 2000 µg/mL, ±S9

Experiment IIwith long-term exposure (44 h):1000, 1500 and 2000 µg/mL, -S9

 

No precipitate of the test item was noted in any concentration group evaluated in the main experiments. In experimentI and II with and without metabolic activation no increase of the cytostasis above 30% was noted. In experiment I and II with and without metabolic activation no biologically relevant increase of the micronucleus frequency was noted after treatment with the test item.

The nonparametric χ² Test was performed to verify the results in both experiments. No statistically significant enhancement (p<0.05) of cells with micronuclei was noted in the concentration groups of the test item evaluated in experiment I and II.

The χ² Test for trend was performed to test whether there is a concentration-related increase in the micronucleated cells frequency in the experimental conditions. No statistically significant increase in the frequency of micronucleated cells was observed in experiment I and II without metabolic activation. In experiment I with metabolic activation, evaluation of the data with the χ² Test for trend showed a statistically significant concentration-related increase in the micronucleated cells frequency. There were no statistically significant increases in the micronuclei frequencies of the concentration groups when compared to the corresponding negative control and all values were within the historical negative control limits. Therefore, the statistically significant concentration-related increase obtained by the χ² Test for trend was considered not to be biologically relevant.

In conclusion, it can be stated that during the study described and under the experimental conditions reported, the test item Butylmonoglycol sulphate, Na-salt did not induce structural and/or numerical chromosomal damage in human lymphocytes. Therefore, butylmonoglycol sulphate, Na-salt is considered to be non-mutagenic with respect to clastogenicity and/or aneugenicity in the in vitro Mammalian Cell Micronucleus Test.

Justification for classification or non-classification

The available data on genetic toxicity of butylmonoglycol sulphate, Na-salt do not meet the criteria for classification according to Regulation (EC) No 1272/2008, and are therefore conclusive but not sufficient for classification.