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Genetic toxicity in vitro

Description of key information

B-TEGME was not mutagenic in several bacterial and mammalian test systems in vitro covering different genetic endpoints:

Negative in two Ames tests: OECD 471, 2007 and 1989

Negative in the HPRT test: OECD 476, 2010

Negative in the chromosome aberration assay: OECD 473, 2010 and 1991

In conclusion there were no indications of genotoxicity.

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:
2007-10-12 to 2007-11-09
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
21 July 1997
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Staatliches Gewerbeaufsichtsamt Hildesheim, Germany
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
- Substance type: Borated Glycol Ether
- Physical state:Clear colourless liquid
- Analytical purity: 100%
- Isomers composition: Not applicable
- Purity test date: Not provided
- Lot/batch No.: DEG4131078
- Expiration date of the lot/batch: 2009-06-05
- Stability under test conditions: Room temperature (20°C)
Target gene:
Defective excision repair gene in Salmonella thyphymirium strains (uvrB) and Escherichia coli strain (uvrA).
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells:
TA 98: University of California, Berkeley Divison of Biochemistry & Molecular Biology, CA 94720 USA
E. coli WP2 (uvrA), TA 1537, TA 1535 and TA 100: TRINOVA BIOCHEM GMBH, Kerkrader Stra e 10, D-35394 Giessen
- Suitability of cells: cells selected according to guidelines
Metabolic activation:
with and without
Metabolic activation system:
S9 (CCR; batch Nos. 250507 and 200707)
Test concentrations with justification for top dose:
1st study (plate incorporation method): 5.0, 1.6, 0.5, 0.16 and 0.05 mg/plate (factor root 10)
2nd study (preincubation method): 5.0, 2.3, 1.1, 0.5 and 0.23 mg/plate (factor root 5)
Vehicle / solvent:
distilled water
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
distilled water
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
sodium azide
benzo(a)pyrene
other: 2-amino-anthracen, ICR 191, 4-nitro-o-phenylendiamine, nitrofurantoine
Details on test system and experimental conditions:
METHOD OF APPLICATION:
- plate incorporation:
2.0 ml top agar with 0.5 mM Histidin/Biotin (or L-Tryptophan for E.coli) for plates without S9
1.5 ml top agar with 0.5 mM Histidin/Biotin (or L-Tryptophan for E.coli) for plates with S9
0.5 ml S9 mix for plates with metabolic activation
0.1 ml bacteria (overnight culture)

- preincubation:
0.35 ml test/reference item solution and double distilled water
1.75 ml buffer solution or S9-mix
0.35 ml bacteria (overnight culture)
After preincubation for 20 min. At 33-37°C: 0.7 ml of the precincubated solution and 1.5 ml top agar with 0.5 nM Histidin/Biotin or L-Tryptophan (for E.coli) solution

DURATION
- Preincubation period: 20 minutes (33-37°c)
- Exposure duration: 48 hours (36.2-37.7°C)

NUMBER OF REPLICATIONS: 3 per concentration level and control (Titer control replicates were prepared 2-fold)
Evaluation criteria:
The rate of induced revertant His+ colonies was derermined.
In addition, genotypes were evaluated. Plates of the mutagenicity test were inspected for present and reduced background lawn after an incubation time of 48h. Plates with colonies which correspond not with the typical shape and colour of Salmonella thyphimuium or Escherichia coli were regarded as contaminated and were not included in calculations.
Statistics:
Arithmetic mean values and standard deviations were calculated based on colonies per plate of three replicates. For evaluation of the results, the induction rate of the mean values was calculated (revertant colonies test item/revertant colonies control). If there is a concentration effect relationship over at least 3 concentrations and/or induction rate equal to or greater than 2, then the test item is classified as mutagenic.
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- There were no confounding factors

RANGE-FINDING/SCREENING STUDIES: No

COMPARISON WITH HISTORICAL CONTROL DATA: No

ADDITIONAL INFORMATION ON CYTOTOXICITY: No
Remarks on result:
other: plate incorporation & preincubation

Table 1: Mutagenic and cytogenic effects of the test item in the Ames test

Strain

S9

Tested concentration range (mg/plate)

Lowest mutagenic concentration

Lowest cytotoxic concentration (mg/plate)

1st study

2nd study

(mg/plate)

1st study

2nd

study

TA 1537

-

0.05 – 0.5

0.23 -5.0

none

not cytotoxic
up to 5.0

not cytotoxic 
up to 5.0

+

TA 98

-

+

TA 100

-

+

TA 1535

-

+

E coli
WP2 (uvrA)

-

+

Conclusions:
The test item is regarded to be not mutagenic under the test conditions.
Executive summary:

The mutagenic effect of B-TEGME was determined in a bacterial reverse mutation test according to OECD No. 471 in two independent study parts (plate incorporation and preincubation method). Test systems were the Salmonnella thyphimurium strains TA 1537, TA 98, TA100 and TA 1535 (uvrB) and Escherichia coli WP2 (uvrA) strains with (+) and without (-) the metabolic system S9 (from male Wistar rats), respectively. Positive and negative conrols were included in each study. B-TEGME was dissolved in double distilled water and applied once at the start of the experiments with the concentration ranges: 0.05 – 0.5 mg/plate in the 1st experiment and 0.23 -5.0 mg/plate in the second experiment. The exposure duration was 48h.

Mutagenic and cytotoxic effects were absent up to the limit dose of 5 mg/plate.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2010/01/14 - 2010/05/28
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to
Guideline:
other: Commission Regulation (EC) No. 440/2008 and the United Kingdom Environmental Mutagen Society (Cole et al, 1990). The technique used is a plate assay using tissue culture flasks and 6-thioguanine (6­TG) as the selective agent.
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
With the exception noted below the work described was performed in compliance with UK GLP standards (Schedule 1, Good Laboratory Practice Regulations 1999 (SI 1999/3106 as amended by SI 2004/0994)). These Regulations are in accordance with GLP standards.
Type of assay:
mammalian cell gene mutation assay
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Lot/batch No.of test material: DEG4129165
- Purity : 89.8 %
- Date received: 03 December 2009
- Expiration date of the lot/batch: 31 October 2011

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature in the dark

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: specific gravity was determined and used to calculate the appropriate dose volume for the required dose level

FORM AS APPLIED IN THE TEST: test material was used as supplied
Target gene:
To assess the potential mutagenicity of the test material the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus of Chinese hamster ovary (CHO) cells was addressed within the assay.
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Remarks:
CHO-K1
Details on mammalian cell type (if applicable):
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination:yes
- Periodically checked for karyotype stability: no
- Periodically "cleansed" against high spontaneous background: yes

Cell Line
The Chinese hamster ovary (CHO-K1) cell line was obtained from ECACC, Salisbury, Wiltshire.

Cell Culture
The stocks of cells were stored in liquid nitrogen at approximately -196°C. Cells were routinely cultured in Ham's F12 medium, supplemented with 5% foetal bovine serum and antibiotics (Penicillin/Streptomycin at 100 units/100 µg per ml) at 37°C with 5% CO2 in air.

Cell Cleansing
Cell stocks spontaneously mutate at a low but significant rate. Before the stocks of cells were frozen down they were cleansed of HPRT- mutants by culturing in HAT medium for 4 days. This is Ham's F12 growth medium supplemented with Hypoxanthine (13.6 µg/ml, 100 µM), Aminopterin (0.0178 µg/ml, 0.4 µM) and Thymidine (3.85 µg/ml, 16 µM). After 4 days in medium containing HAT, the cells were passaged into HAT-free medium and grown for 4 to 7 days. Bulk frozen stocks of HAT cleansed cells were frozen down, with fresh cultures being recovered from frozen before each experiment.
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
PB/betaNF S9 was prepared from the livers of male Sprague Dawley CD strain rats. These had received three daily oral doses of a mixture of phenobarbitone (80 mg/kg) and beta-naphthoflavone (100 mg/kg), prior to S9 prepreparation on the fourth day.
Test concentrations with justification for top dose:
The molecular weight of the test material was calculated to be approximately 500.4, therefore the maximum dose level was 5000 µg/ml. The purity of the test material was 89.8% and was accounted for in the formulations. There was no significant change in pH when the test material was dosed into media. However the osmolality did increase by 50 mOsm at the top dose and was considered to be at the limit of acceptability therefore the maximum dose was limited to 4000 µg/ml. Further details for osmalality and pH are given in table 1.

Preliminary Cytotoxicity Test
Doses of 15.63 to 4000 µg/ml were used in the preliminary cytotoxicity test.

Mutagenicity Test - Experiment 1
The dose levels of the controls and the test material are given table below:
Group Final concentration of B-TEGME (µg/ml)
4(20)-hour without S9 0*, 125*, 250*, 500*, 1000*, 2000*, 4000*, EMS 500* and 750*
4(20)-hour with S9 0*, 125*, 250*, 500*, 1000*, 2000*, 4000*, DMBA 0.5* and 1.0*

Mutagenicity Test - Experiment 2
The dose levels of the controls and the test material are given below:
Group Final concentration of B-TEGME (µg/ml)
4(20)-hour without S9 0*, 125*, 250*, 500*, 1000*, 2000*, 4000*, EMS 200*
4(20)-hour with S9 0*, 125*, 250*, 500*, 1000*, 2000*, 4000*, DMBA 0.5* and 1.0*

EMS = Ethylmethanesulphonate
* = Dose levels plated for mutant frequency
DMBA = Dimethyl benzanthracene

No analysis was conducted to determine the homogeneity, concentration or stability of the test material formulation. This is an exception with regard to GLP and has been reflected in the GLP compliance statement.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Hams F12 cell culture media
- Justification for choice of solvent/vehicle: The test material formed a solution with the solvent suitable for dosing.
No analysis was conducted to determine the homogeneity, concentration or stability of the test material formulation. This is an exception with regard to GLP and has been reflected in the GLP compliance statement.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
Hams F12
True negative controls:
no
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
Remarks:
With metabolic activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
Hams F12
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
Without metabolic activation
Details on test system and experimental conditions:
Preliminary Cytotoxicity Test
A preliminary cytotoxicity test was performed on cell cultures using a 4-hour exposure time with and without metabolic activation and a continuous exposure of 24 hours without metabolic activation. The cells were plated out at 3 x 10^6 cells/75 cm2 flask for the 4-hour exposure groups and at 1.5 x 10^6 cells/75 cm2 flask for the 24-hour exposure group approximately 20 hours before dosing. On dosing, the growth media was removed and replaced with serum free media (Ham's F12) for the 4-hour exposure group and with Ham’s F12 with 1% serum for the 24-hour exposure group. One flask per dose level was treated with and without S9 metabolic activation, 9 dose levels using halving dilutions and vehicle controls were dosed. The dose range of test material used was 15.63 to 4000 µg/ml. Exposure was for 4 hours or 24 hours at 37°C, after which the cultures were washed twice with phosphate buffered saline (PBS) before being trypsinised. Cells from each flask were suspended in growth medium; a sample was removed from each dose group and counted using a Coulter counter. For each culture, 200 cells were plated out into three 25 cm2 flasks with 5 ml of growth medium and incubated for 6 to 7 days at 37°C ± 2°C in an incubator with a humidified atmosphere of 5% CO2 in air. The cells were then fixed and stained and total numbers of colonies in each flask counted to give cloning efficiencies.
Results from the preliminary cytotoxicity test were used to select the test material dose levels for the mutagenicity experiments.

Mutagenicity Test
Several days before starting each experiment, a fresh stock of cells was removed from the liquid nitrogen freezer and grown up to provide sufficient cells for use in the test. Cells were seeded at 3 x 10^6/75 cm2 flask for the 4 hour exposure groups or 1.5 x 10^6/75 cm2 flask for the 24 hour exposure group and allowed to attach overnight before being exposed to the test or control materials. Duplicate cultures were set up, both in the presence and absence of metabolic activation, with six dose levels of test material, and vehicle and positive controls. Treatment was either for 4 hours in serum free media (Ham's F12) or for 24 hours in Ham’s F12 with 1% serum at 37°C in an incubator with a humidified atmosphere of 5% CO2 in air. The dose range of test material was 125 to 4000 µg/ml both in the presence and absence of S9 metabolic activation and in both Experiment 1 and Experiment 2.
At the end of the treatment period the flasks were washed twice with PBS, trypsinised and the cells suspended in growth medium. A sample of each dose group cell suspension was counted using a Coulter counter. Cultures were plated out at 2 x 10^6 cells/flask in a 225 cm2 flask to allow growth and expression of induced mutants, and in triplicate in 25 cm2 flasks at 200 cells/flask for an estimate of cytotoxicity. Cells were grown in growth media and incubated at 37°C ± 1.5°C in an incubator with a humidified atmosphere of 5% CO2 in air.
Cytotoxicity flasks were incubated for 7 days then fixed with methanol and stained with Giemsa. Colonies were manually counted and recorded to estimate cytotoxicity.

During the 7 Day expression period the cultures were sub-cultured and maintained at 2 x 10^6 cells/225 cm2 flask on days 3 or 4 to maintain logarithmic growth. At the end of the expression period the cell monolayers were trypsinised, cell suspensions counted using a Coulter counter and plated out as follows:
i) In triplicate at 200 cells/25 cm2 flask in 5 ml of growth medium to determine cloning efficiency. Flasks were incubated for 6 to 7 days, fixed with methanol and stained with Giemsa. Colonies were manually counted, counts were recorded for each culture and the percentage cloning efficiency for each dose group calculated.
ii) At 2 x 10^5 cells/75 cm2 flask (5 replicates per group) in Ham's F12 growth media (5% serum), supplemented with 10 µg/ml 6-Thioguanine (6-TG), to determine mutant frequency. The flasks were incubated for 14 days at 37°C in an incubator with a humidified atmosphere of 5% CO2 in air, then fixed with methanol and stained with giemsa. Mutant colonies were manually counted and recorded for each flask.
The percentage cloning efficiency and mutation frequency per survivor were calculated for each dose group.
Fixation and staining of all flasks was achieved by aspirating off the media, washing with phosphate buffered saline, fixing for 5 minutes with methanol and finally staining with a 10% Giemsa solution for 5 minutes.

ASSAY ACCEPTANCE CRITERIA
An assay will normally be considered acceptable for the evaluation of the test results only if all the following criteria are satisfied. The with and without metabolic activation portions of mutation assays are usually performed concurrently, but each portion is, in fact, an independent assay with its own positive and negative controls. Activation or non-activation assays will be repeated independently, as needed, to satisfy the acceptance criteria.

i) The average absolute cloning efficiency of negative controls should be between 70 and 115% with allowances being made for errors in cell counts and dilutions during cloning and assay variables. Assays in the 50 to 70% range may be accepted but will be dependent on the scientific judgement of the Study Director. All assays below 50% cloning efficiency will be unacceptable.
ii) The background (spontaneous) mutant frequency of the vehicle controls are generally in the range of 0 to 25 x 10^-6. The background values for the with and without-activation segments of a test may vary even though the same stock populations of cells may be used for concurrent assays. Assays with backgrounds greater than 35 x 10^-6 will not be used for the evaluation of a test material.
iii) Assays will only be acceptable without positive control data (loss due to contamination or technical error) if the test material clearly shows mutagenic activity. Negative or equivocal mutagenic responses by the test material must have a positive control mutant frequency that is markedly elevated over the concurrent negative control.
iv) Test materials with little or no mutagenic activity, should include an acceptable assay where concentrations of the test material have reduced the clonal survival to approximately 10 to 15% of the average of the negative controls, reached the maximum recommended dose (10 mM or 5 mg/ml) or twice the solubility limit of the test material in culture medium. Where a test material is excessively toxic, with a steep response curve, a concentration that is at least 75% of the toxic dose level should be used. There is no maximum toxicity requirement for test materials that are clearly mutagenic.
v) Mutant frequencies are normally derived from sets of five dishes for mutant colony count and three dishes for viable colony counts. To allow for contamination losses it is acceptable to score a minimum of four mutant selection dishes and two viability dishes.
vi) Five dose levels of test material, in duplicate, in each assay will normally be assessed for mutant frequency. A minimum of four analysed duplicate dose levels is considered necessary in order to accept a single assay for evaluation of the test material.
Evaluation criteria:
Extended information are given above in the section 'Details on test system and conditions'.
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
non-mutagenic
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:
Preliminary Cytotoxicity Test
Doses of 15.63 to 4000 µg/ml were used in the preliminary cytotoxicity test. The results of the individual flask counts and their analysis are presented in Table 1. It can be seen that there was no consistent dose-related reduction in the cloning efficiency (CE) either in the presence or absence of S9.

Mutagenicity Test - Experiment 1
The Day 0 and Day 7 cloning efficiencies are presented in Tables 2 and 3. All of the vehicle controls had acceptable levels for cloning efficiencies. It can be seen that there was no marked toxicity with the test material when compared to the vehicle controls. The toxicity observed was similar to that seen in the preliminary cytotoxicity test.
All of the vehicle control cultures had mutant frequencies within the expected range. The positive control materials induced significant increases in mutant frequency. The metabolic activation system was therefore shown to be functional and the test method itself was operating as expected. The mutation frequency counts and mean mutation frequency per survivor values are presented in Tables 2 and 3. There were no increases in mutation frequency per survivor which exceeded the vehicle control value by 20 x 10^-6 with or without the presence of S9.

Mutagenicity Test - Experiment 2
The Day 0 and Day 7 cloning efficiencies are presented in Tables 4 and 5. All of the vehicle controls had acceptable levels for cloning efficiencies. It can be seen that, as in Experiment 1, there was no marked toxicity with the test material when compared to the vehicle controls, and the toxicity observed was similar to that seen in the preliminary cytotoxicity test. All of the vehicle control cultures had mutant frequencies within the expected range. The positive control materials induced significant increases in mutant frequency. The metabolic activation system was therefore shown to be functional and the test method itself was operating as expected. The mutation frequency counts and mean mutation frequency per survivor per values are presented in Tables 4 and 5. There were no increases in mutation frequency per survivor which exceeded the vehicle control value by 20 x 10^-6 with or without the presence of S9.

Table 1: Preliminary Cytotoxicity Results

 

4-hour -S9

4-hour +S9

24-hour -S9

Dose

(µg/ml)

 

Count

 

Mean

 

% CE

%

Control

 

Count

 

Mean

 

% CE

% Control

 

Count

 

Mean

 

% CE

%

Control

0

142

146

156

148.0

74.0

100

142

146

118

135.3

67.7

100

168

170

164

167.3

83.7

100

15.63

132

134

140

135.3

67.7

91

110

111

126

115.7

57.8

85

167

161

160

162.7

81.3

97

31.25

130

126

132

129.3

64.7

87

104

102

116

107.3

53.7

79

169

170

166

168.3

84.2

101

62.5

136

138

125

133.0

66.5

90

113

120

135

122.7

61.3

91

155

157

141

151.0

75.5

90

125

148

135

119

134.0

67.0

91

117

134

128

126.3

63.2

93

166

151

154

157.0

78.5

94

250

139

151

132

140.7

70.3

95

100

112

125

112.3

56.2

83

127

157

128

137.3

68.7

82

500

109

130

148

129.0

64.5

87

101

108

103

104.0

52.0

77

136

142

142

140.0

70.0

84

1000

138

120

130

129.3

64.7

87

117

123

120

120.0

60.0

89

168

155

151

158.0

79.0

94

2000

121

123

128

124.0

62.0

84

121

118

118

119.0

59.5

88

144

155

151

150.0

75.0

90

4000

145

138

135

139.3

69.7

94

140

150

129

139.7

69.8

103

156

170

156

160.7

80.3

96

CE = cloning efficiency

Table 2: Experiment 1 - Without Metabolic Activation (S9)

 

 

Day 0 Viability

Day 7 Viability

Day 7 Mutant

 

Dose (µg/ml)

 

Colonies/flask (200 cells plated/flask)

 % CE

% Control

Mean % Control

Colonies/flask (200cells plated /flask)

 

% CE

 

% Control

 

Mean % Control

Colonies /flask (2x105cells plated/flask )

 

MF

MFS 

10-6

Group SD

Group MFS 10-6

 

0

A

160

162

166

81.3

100.0

100

156

173

192

86.8

100.0

100

3

4

3

2

5

17

19.6

1.33

16

B

143

155

161

76.5

100.0

208

185

207

100.0

100.0

5

2

3

2

1

13

13.0

 

125

A

141

144

161

74.3

91.4

93

192

182

202

96.0

110.6

103

3

5

4

2

3

17

17.7

0.92

17

B

150

149

139

73.0

95.4

187

198

189

95.7

95.7

3

4

3

3

2

15

15.7

 

250

A

131

169

162

77.0

94.7

102

166

182

172

86.7

99.8

96

1

3

0

3

5

12

13.8

1.78

15

B

164

166

171

83.5

109.2

160

198

191

91.5

91.5

5

4

3

1

1

14

15.3

 

500

A

132

137

141

68.3

84.0

92

187

178

194

93.2

107.3

88

3

6

8

8

5

30

32.2

2.41

25

B

149

159

150

76.3

99.8

142

142

132

69.3

69.3

4

3

1

3

2

13

18.8

 

1000

A

162

144

149

75.8

93.2

105

127

121

131

63.2

72.7

72

1

1

1

0

1

4

6.3

0.63

9

B

181

183

175

89.8

117.4

154

152

121

71.2

71.2

1

2

2

2

1

8

11.2

 

2000

A

161

122

163

74.3

91.4

99

157

154

136

74.5

85.8

84

3

3

2

4

2

14

18.8

1.32

14

B

169

155

163

81.2

106.1

186

181

122

81.5

81.5

1

1

0

4

2

8

9.8

 

4000

A

135

133

132

66.7

82.0

91

196

176

199

95.2

109.6

101

6

2

2

4

5

19

20.0

1.57

17

B

164

150

143

76.2

99.6

195

193

172

93.3

93.3

3

4

2

4

1

14

15.0

EMS

500

A

71

54

60

30.8

37.9

42

139

137

161

72.8

83.9

77

26

30

28

23

27

134

184.0

5.76

156

B

57

78

79

35.7

46.6

160

117

139

69.3

69.3

14

19

18

14

24

89

128.4

EMS

750

A

39

33

36

18.0

22.1

29

107

115

112

55.7

64.1

62

29

40

34

37

38

178

319.8

7.93

253

B

47

61

55

27.2

35.5

112

116

126

59.0

59.0

24

18

21

22

25

110

186.4

EMS = Ethylmethanesulphonate

CE = Cloning efficiency

MF = Mutant frequency

MFS = Mutant frequency per survivor

SD = Standard deviation

Table 3: Experiment 1 - With Metabolic Activation (S9) 

 

 

Day 0 Viability

Day 7 Viability

Day 7 Mutant

 

Dose (µg/ml)

 

Colonies/flask

(200 cells plated/flask)

 

% CE

 

%

Control

 

Mean % Control

Colonies/flask (200 cells plated

/flask)

 

% CE

 

%

Control

 

Mean % Control

 

Colonies /flask (2x105cells plated/flask )

 

MF

MFS  

10-6

 

Group SD

Group MFS 10-6

 

0

A

173

175

171

86.5

100.0

100

194

192

180

94.3

100.0

100

3

3

2

1

4

13

13.8

1.23

13

B

162

188

199

91.5

100.0

159

156

151

77.7

100.0

0

1

2

3

3

9

11.6

 

125

A

150

160

164

79.0

91.3

96

152

154

160

77.7

82.3

93

2

3

4

4

3

16

20.6

1.10

18

B

185

185

184

92.3

100.9

167

162

157

81.0

104.3

2

4

4

1

2

13

16.0

 

250

A

170

163

178

85.2

98.5

99

144

163

146

75.5

80.0

93

2

3

4

2

3

14

18.5

0.97

17

B

172

180

195

91.2

99.6

167

166

157

81.7

105.2

3

2

1

4

2

12

14.7

 

500

A

191

154

178

87.2

100.8

98

156

157

166

79.8

84.6

93

4

2

5

3

4

18

22.5

1.10

20

B

177

151

191

86.5

94.5

164

150

158

78.7

101.3

4

2

2

3

2

13

16.5

 

1000

A

186

197

198

96.8

111.9

105

148

141

153

73.7

78.1

85

0

2

3

4

4

13

17.6

1.49

14

B

180

181

177

89.7

98.0

158

131

141

71.7

92.3

0

2

3

1

1

7

9.8

 

2000

A

186

138

147

78.5

90.8

89

196

187

164

91.2

96.6

96

2

2

3

3

2

12

13.2

1.57

17

B

179

148

150

79.5

86.9

153

140

153

74.3

95.7

2

6

3

4

0

15

20.2

 

4000

A

185

151

168

84.0

97.1

97

164

169

191

87.3

92.6

92

5

3

5

2

3

18

20.6

1.81

13

B

158

174

195

87.8

96.0

135

151

141

71.2

91.6

0

1

2

0

1

4

5.6

DMBA

0.5

A

153

159

163

79.2

91.5

87

151

156

140

74.5

79.0

76

45

35

43

36

38

197

264.4

5.07

321

B

145

151

155

75.2

82.1

115

112

114

56.8

73.2

51

41

37

40

46

215

378.3

DMBA

1

A

113

105

94

52.0

60.1

54

89

91

90

45.0

47.7

49

45

45

40

36

44

210

466.7

8.06

583

B

78

90

92

43.3

47.4

77

74

86

39.5

50.9

58

62

53

52

51

276

698.7

DMBA = Dimethyl benzanthracene

CE = Cloning efficiency

MF = Mutant frequency

MFS = Mutant frequency per survivor

SD = Standard deviation

Table 4: Experiment 2 - Without Metabolic Activation (S9) 

 

 

Day 0 Viability

Day 7 Viability

Day 7 Mutant

 

Dose (µg/ml)

 

 

Colonies/flask (200 cells plated/flask)

 

%CE

 

%

Control

 

Mean % Control

 

Colonies/flask (200 cells plated /flask)

 

% CE

 

%

Control

 

Mean % Control

 

Colonies /flask

(2x105cells plated/flask)

 

MF

 

MFS  10 -6

 

GroupSD

 

Group MFS 10-6

 

0

A

149

159

168

79.3

100.0

100

149

171

174

82.3

100.0

100

0

0

0

0

1

1

1.2

0.32

1

B

149

153

161

77.2

100.0

148

169

144

76.8

100.0

0

0

0

0

0

0

0.0

 

125

A

146

152

171

78.2

98.5

98

171

179

146

82.7

100.4

100

0

0

0

0

0

0

0.0

0.00

0

B

155

144

148

74.5

96.5

143

145

174

77.0

100.2

0

0

0

0

0

0

0.0

 

250

A

163

149

140

75.3

95.0

96

139

147

142

71.3

86.6

90

0

0

0

1

0

1

1.4

0.32

1

B

143

150

152

74.2

96.1

139

161

132

72.0

93.7

0

0

0

0

0

0

0.0

 

500

A

142

161

158

76.8

96.8

96

152

151

141

74.0

89.9

90

2

0

0

0

0

2

2.7

0.67

2

B

148

156

137

73.5

95.2

129

147

139

69.2

90.0

1

0

0

0

0

1

1.4

 

1000

A

143

159

155

76.2

96.0

93

159

160

174

82.2

99.8

100

0

0

1

0

0

1

1.2

0.84

4

B

138

143

139

70.0

90.7

159

162

139

76.7

99.8

0

2

1

0

2

5

6.5

 

2000

A

137

159

141

72.8

91.8

90

168

172

147

81.2

98.6

94

0

0

0

0

1

1

1.2

0.53

4

B

127

133

150

68.3

88.6

141

121

149

68.5

89.2

1

1

1

1

0

4

5.8

 

4000

A

147

132

121

66.7

84.0

89

158

172

164

82.3

100.0

102

0

0

1

1

1

3

3.6

0.48

2

B

153

144

136

72.2

93.5

171

159

148

79.7

103.7

0

0

0

0

0

0

0.0

EMS

200

A

59

56

40

25.8

32.6

25

98

87

96

46.8

56.9

60

18

12

9

12

14

65

138.8

3.69

130

B

35

26

23

14.0

18.1

102

96

92

48.3

62.9

18

11

9

14

7

59

122.1

EMS

300

A

26

34

23

13.8

17.4

14

86

88

79

42.2

51.2

53

19

28

30

18

25

120

284.6

4.67

279

B

12

21

15

8.0

10.4

79

82

94

42.5

55.3

27

20

27

25

17

116

272.9

EMS =Ethylmethanesulphonate

CE = Cloning efficiency

MF = Mutant frequency

MFS = Mutant frequency per survivor

SD = Standard deviation

Table 5: Experiment 2 - With Metabolic Activation (S9)

 

 

Day 0 Viability

Day 7 Viability

Day 7 Mutant

 

Dose (µg/ml)

 

 Colonies/flask (200 cells plated/flask)

 

% CE

 

%

Control

 

Mean % Control

 

Colonies/flask (200cells plated/flask)

 

% CE

 

%

Control

 

Mean % Control

 Colonies /flask (2x105cells plated/flask)

 

MF

 

MFS  10-6

 

Group SD

 

Group MFS 10-6

 

0

A

161

154

151

77.7

100.0

100

170

168

161

83.2

100.0

100

0

0

2

1

1

4

4.8

0.79

5

B

195

190

188

95.5

100.0

149

159

166

79.0

100.0

1

2

0

0

1

4

5.1

 

125

A

152

144

143

73.2

94.2

85

170

181

163

85.7

103.0

98

0

0

2

1

2

5

5.8

0.82

6

B

122

164

147

72.2

75.6

141

149

155

74.2

93.9

1

2

1

0

1

5

6.7

 

250

A

116

143

138

66.2

85.2

84

176

149

178

83.8

100.8

97

0

0

1

0

0

1

1.2

0.32

1

B

149

164

166

79.8

83.6

143

154

149

74.3

94.1

0

0

0

0

0

0

0.0

 

500

A

132

135

146

68.8

88.6

80

148

174

139

76.8

92.4

93

0

0

1

1

1

3

3.9

0.70

4

B

137

139

135

68.5

71.7

145

142

161

74.7

94.5

0

2

0

1

0

3

4.0

 

1000

A

184

151

149

80.7

103.9

90

176

178

156

85.0

102.2

101

0

0

0

3

1

4

4.7

1.06

4

B

150

130

156

72.7

76.1

141

163

165

78.2

98.9

1

0

2

0

0

3

3.8

 

2000

A

155

162

153

78.3

100.9

89

171

178

196

90.8

109.2

98

2

1

2

0

0

5

5.5

0.82

6

B

149

132

162

73.8

77.3

151

132

129

68.7

86.9

0

2

1

1

1

5

7.3

 

4000

A

134

152

156

73.7

94.8

91

145

165

178

81.3

97.8

99

0

0

1

0

0

1

1.2

0.84

4

B

160

168

167

82.5

86.4

141

171

162

79.0

100.0

2

1

2

0

0

5

6.3

DMBA

0.5

A

110

111

129

58.3

75.1

67

115

116

111

57.0

68.5

69

25

18

17

24

22

106

186.0

2.77

188

B

127

107

106

56.7

59.3

104

101

121

54.3

68.8

18

23

19

21

22

103

189.6

DMBA

1

A

118

112

112

57.0

73.4

69

108

101

119

54.7

65.7

72

24

24

20

17

24

109

199.4

12.29

267

B

109

142

116

61.2

64.0

116

122

129

61.2

77.4

32

34

56

46

37

205

335.1

DMBA = Dimethyl benzanthracene

CE = Cloning efficiency

MF = Mutant frequency

MFS = Mutant frequency per survivor

SD = Standard deviation

Conclusions:
The test material did not induce significant or dose-related increases in mutant frequency per survivor in either the presence or absence of metabolic activation in the two experiments. The test material was therefore considered to be non-mutagenic to CHO cells at the HPRT locus under the conditions of this test.
Executive summary:

The study was conducted to assess the potential mutagenicity of the test material on the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus of Chinese hamster ovary (CHO) cells. The protocol used was designed to comply with the OECD Guidelines for Testing of Chemicals No. 476 'In Vitro Mammalian Cell Gene Mutation Tests', Commission Regulation (EC) No. 440/2008 and the United Kingdom Environmental Mutagen Society (Cole et al, 1990). The technique used was the plate assay using tissue culture flasks and 6-thioguanine (6­TG) as the selective agent.

Chinese hamster ovary (CHO) CHO-Kl cells were treated with the test material at six dose levels, in duplicate, together with vehicle (solvent) and positive controls. Four treatment conditions were used for the study, i.e. In Experiment 1, a 4-hour exposure in the presence of an induced rat liver homogenate metabolising system (S9), at a 2% final concentration and a 4-hour exposure in the absence of metabolic activation (S9). In Experiment 2, the 4-hour exposure with addition of S9 was repeated (using a 1% final S9 concentration), whilst in the absence of metabolic activation the exposure time was increased to 24 hours.

The dose range of test material was selected based on the results of a preliminary cytotoxicity test and was 125 to 4000 µg/ml in both the presence and absence of metabolic activation. The maximum dose was limited to 4000 µg/ml as a result of an increase in osmolality of 50 mOsm at the maximum recommended dose of 5000 µg/ml, which was considered to be at the limit of acceptability.

The vehicle (solvent) controls gave mutant frequencies within the range expected of CHO cells at the HPRT locus.

The positive control treatments, both in the presence and absence of metabolic activation, gave significant increases in the mutant frequency indicating the satisfactory performance of the test and of the metabolising system.

The test material demonstrated no significant increases in mutant frequency at any dose level, either with or without metabolic activation, in either the first or second experiment.

The test material was considered to be non-mutagenic to CHO cells under the conditions of the test.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2010/01/28 - 2010/04/28
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
21 July 1997
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
other: is acceptable to the Japanese New Chemical Substance Law (METI)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
UK GLP standards (Schedule 1, Good Laboratory Practice Regulations 1999 (SI 1999/3106 as amended by SI 2004/0994)). These Regulations are in accordance with GLP standards published as OECD Principles on Good Laboratory Practice.
Type of assay:
other: in vitro mammalian chromosome aberration assay
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Substance type: Borated Glycol Ether
- Lot/batch No.of test material: DEG4129165
- Purity : 89.8 %
- Date received: 03 December 2009
- Expiration date of the lot/batch: 31 October 2011

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature in the dark

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: specific gravity was determined and used to calculate the appropriate dose volume for the required dose level

FORM AS APPLIED IN THE TEST: test material was used as supplied
Target gene:
Not applicable.
Species / strain / cell type:
lymphocytes: human
Details on mammalian cell type (if applicable):
For each experiment, sufficient whole blood was drawn from the peripheral circulation of a volunteer who had been previously screened for suitability. The volunteer had not been exposed to high levels of radiation or hazardous chemicals and had not knowingly recently suffered from a viral infection
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
phenobarbitone and beta-naphthoflavone induced rat liver, S9
Test concentrations with justification for top dose:
Cell Growth Inhibition test (µg/mL)
4h exposure (after 20 hour cell harvest) without S9: 0, 15.63, 31.25, 62.5, 125, 250, 500, 1000, 2000, 4000
4(20)-hour with S9 0, 15.63, 31.25, 62.5, 125, 250, 500, 1000, 2000, 4000
24-hour without S9 0, 15.63, 31.25, 62.5, 125, 250, 500, 1000, 2000, 4000

Experiment 1: (µg/mL)
4(20)-hour without S9 0*, 125, 250, 500, 1000*, 2000*, 4000*, MMC 0.4*
4(20)-hour with S9 0*, 125, 250, 500, 1000*, 2000*, 4000*, CP 5*
(* Dose levels selected for metaphase analysis)


Experiment 2: (µg/mL)
24-hour without S9 0*, 125, 250*, 500*, 1000*, 2000*, 4000, MMC 0.4*
4(20)-hour with S9 0*, 125, 250, 500, 1000*, 2000*, 4000*, CP 5*
(* Dose levels selected for metaphase analysis)

CP: cyclophosphoamide
MMC: mitomycin C
Vehicle / solvent:
MEM tissue culture medium was selected as the solvent because the test material was soluble in it at the required concentrations. A correction for the purity of the test material was made when the test material formulations were prepared.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
MEM tissue culture medium
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
In the presence of S9
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
MEM tissue culture medium
True negative controls:
no
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
In the absence of S9
Details on test system and experimental conditions:
METHOD OF APPLICATION:
in medium

DURATION
- Preincubation period: Approximately 48 hrs
- Exposure duration:
Experiment 1: 4 hrs with and without S9
Experiment 2: 24 hrs without S9, 4 hrs with S9

- Expression time (cells in growth medium): 20 hrs for 4 hrs exposure
- Selection time (if incubation with a selection agent): Not applicable
- Fixation time (start of exposure up to fixation or harvest of cells): 24 hrs

SPINDLE INHIBITOR: Demecolcine

STAIN: When the slides were dry they were stained in 5% Giemsa for 5 minutes, rinsed, dried and coverslipped using mounting medium.

NUMBER OF REPLICATIONS: Duplicate cultures

NUMBER OF CELLS EVALUATED: 100/culture

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index - A total of 2000 lymphocyte cell nuclei were counted and the number of cells in metaphase recorded and expressed as the mitotic index and as a percentage of the vehicle control value.

-Scoring of Chromosome Damage: Where possible the first 100 consecutive well-spread metaphases from each culture were counted, where there was approximately 30 to 50% of cells with aberrations, slide evaluation was terminated at 50 cells. If the cell had 44-48 chromosomes, any gaps, breaks or rearrangements were noted according to the simplified system of Savage (1976) recommended in the 1983 UKEMS guidelines for mutagenicity testing. Cells with chromosome aberrations were reviewed as necessary by a senior cytogeneticist prior to decoding the slides.

OTHER EXAMINATIONS:
- Determination of polyploidy: Frequency of polyploid cells
Evaluation criteria:
A positive response was recorded for a particular treatment if the % cells with aberrations, excluding gaps, markedly exceeded that seen in the concurrent control, either with or without a clear dose-relationship. For modest increases in aberration frequency a dose response relationship is generally required and appropriate statistical tests may be applied in order to record a positive response.
Statistics:
The frequency of cells with aberrations excluding gaps and the frequency of polyploid cells was compared, where necessary, with the concurrent vehicle control value using Fisher's Exact test.
Species / strain:
lymphocytes: Human
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Refer to information on results and tables.
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Result tables are given below. Please refer to tables 1 to 4.

Preliminary Toxicity Test (Cell Growth Inhibition Test)
The dose range of the test material for the Preliminary Toxicity Test was 15.63 to 4000 µg/ml. The maximum dose was limited by osmolality issues observed at the maximum recommended 5000 µg/ml concentration. It can be seen that the test material showed some evidence of toxicity in all three exposure groups. No precipitate of the test material was observed in the parallel blood-free cultures at the end of the exposure period, in any of the exposure groups. Microscopic assessment of the slides prepared from the treatment cultures showed that metaphase cells were present up to 4000 µg/ml in the 4(20)-hour treatment, both in the presence and absence of metabolic activation (S9). The maximum dose with metaphases present in the 24-hour continuous exposure was 4000 µg/ml.
Dose selection for Experiment 1 and 2 was based on mild toxicity and the maximum practical dose level (4000 µg/ml).

Chromosome Aberration Test – Experiment 1
The dose levels of the controls and the test material are given in the table below:
Group Final concentration of B-TEGME (µg/ml)
4(20)-hour without S9 0*, 125, 250, 500, 1000*, 2000*, 4000*, MMC 0.4*
4(20)-hour with S9 0*, 125, 250, 500, 1000*, 2000*, 4000*, CP 5*
(* Dose levels selected for metaphase analysis)
The qualitative assessment of the slides determined that there were metaphases suitable for scoring present at the maximum test material dose level of 4000 µg/ml in the presence and absence of metabolic activation (S9). The data show that no growth inhibition was achieved in either the presence or absence of S9.
No precipitate of the test material was observed at the end of the treatment period in the presence or absence of S9.
The maximum dose level selected for metaphase analysis was the maximum practical dose level (4000 µg/ml).
All of the vehicle control cultures had frequencies of cells with chromosome aberrations within the expected range. The positive control materials induced statistically significant increases in the frequency of cells with aberrations. The metabolic activation system was therefore shown to be functional and the test method itself was operating as expected.
The test material did not induce any statistically significant increases in the frequency of cells with aberrations either in the absence or presence of metabolic activation (S9).
The test material did not induce a statistically significant increase in the numbers of polyploid cells at any dose level in either of the exposure groups.

Chromosome Aberration Test - Experiment 2
The dose levels of the controls and the test material are given in the table below:
Group Final concentration of B-TEGME (µg/ml)
24-hour without S9 0*, 125, 250*, 500*, 1000*, 2000*, 4000, MMC 0.4*
4(20)-hour with S9 0*, 125, 250, 500, 1000*, 2000*, 4000*, CP 5*
(* Dose levels selected for metaphase analysis)
The qualitative assessment of the slides determined that there were metaphases suitable for scoring present at the maximum test material dose level of 4000 µg/ml in the absence and presence of S9.
These data show 50% growth inhibition was achieved at 2000 µg/ml in the absence of S9. However, only 7% growth inhibition was achieved at 4000 µg/ml in the presence of S9.
No precipitate of the test material was observed at the end of the treatment period in the presence or absence of S9.
The maximum dose level selected for metaphase analysis in the presence of S9 was the same as Experiment 1, and was the maximum practical dose level of 4000 µg/ml. However the maximum dose level selected in the absence of S9 was limited by toxicity to 2000 µg/ml.
All of the vehicle control cultures had frequencies of cells with chromosome aberrations within the expected range. The positive control materials induced statistically significant increases in the frequency of cells with aberrations. The metabolic activation system was therefore shown to be functional and the test method itself was operating as expected.
The test material did not induce any statistically significant increases in the frequency of cells with chromosome aberrations either in the absence or presence of metabolic activation.
The test material did not induce a significant increase in the numbers of polyploid cells at any dose level in either of the exposure groups.

Table 1: Short Term Treatment Test - Experiment 1 Without Metabolic Activation (S9)

 

Treatment Period (hours)

 

S9

mix

 

Concentration

ȝg/ml

 

 

Number and Percentages of Cells Showing Structural Chromosome Aberrations (%)

 

g

 

Cell Growth Index

 

Number and Percentages of Cells Showing Numerical Aberrations (%)

 

Observed

 

ctb

 

cte

 

csb

 

cse

 

Others

 

Total

Mitotic Index (%)

 

Observed

 

Polyploids

 

Others

 

Total

 

4

 

-

Negative Control (MEM)

0

A

100

2

0

0

0

0

2

0

4.80

100

0

0

0

B

100

0

0

0

0

0

0

0

3.50

100

0

0

0

Total

%

200

(100)

2

(1.0)

0

(0.0)

0

(0.0)

0

(0.0)

0

(0.0)

2

(1.0)

0

(0.0)

 

(100)

200

0

0

0

(0.0)

 

-

 

1000

A

100

0

0

0

0

0

0

2

5.00

100

0

0

0

B

100

1

0

1

0

0

2

0

6.35

100

0

0

0

Total

%

200

(100)

1

(0.5)

0

(0.0)

1

(0.5)

0

(0.0)

0

(0.0)

2

(1.0)

2

(1.0)

 

(137)

200

0

0

0

(0.0)

 

-

 

2000

A

100

0

0

0

0

0

0

1

6.70

100

0

0

0

B

100

0

0

0

0

0

0

2

4.75

100

0

0

0

Total

%

200

(100)

0

(0.0)

0

(0.0)

0

(0.0)

0

(0.0)

0

(0.0)

0

(0.0)

3

(1.5)

 

(138)

200

0

0

0

(0.0)

 

-

 

4000

A

100

0

0

0

0

0

0

1

5.30

100

0

0

0

B

100

0

0

0

0

0

0

0

4.90

100

0

0

0

Total

%

200

(100)

0

(0.0)

0

(0.0)

0

(0.0)

0

(0.0)

0

(0.0)

0

(0.0)

1

(0.5)

 

(123)

200

0

0

0

(0.0)

 

-

Positive Control (MMC)

0.4

A

50a

17

16

2

1

0

29

3

2.05

50

0

0

0

B

50a

16

21

3

0

0

31

5

2.05

50

0

0

0

Total

%

100

(100)

33

(33.0)

37

(37.0)

5

(5.0)

1

(1.0)

0

(0.0)

60***

(60.0)

8

(8.0)

 

(49)

100

0

0

0

(0.0)

MMC = Mitomycin C

*** = p < 0.001

a = Slide evaluation terminated at 50 cells because at least 30% cells with aberrations had been observed.

 

Table 2: Short Term Treatment Test - Experiment 1 With Metabolic Activation (S9)

 

Treatment Period (hours)

 

S9

mix

 

Concentration

ȝg/ml

 

Number and Percentages of Cells Showing Structural Chromosome Aberrations (%)

 

g

Cell Growth Index

Number and Percentages of Cells Showing Numerical Aberrations (%)

 

Observed

 

ctb

 

cte

 

csb

 

cse

 

Others

 

Total

Mitotic Index (%)

 

Observed

 

Polyploids

 

Others

 

Total

 

4

 

+

Negative Control(MEM)

0

A

100

0

0

0

0

0

0

0

5.90

100

0

0

0

B

100

1

0

1

0

0

2

1

5.30

100

0

0

0

Total

%

200

(100)

1

(0.5)

0

(0.0)

1

(0.5)

0

(0.0)

0

(0.0)

2

(1.0)

1

(0.5)

 

(100)

200

0

0

0

(0.0)

 

+

 

1000

A

100

0

0

1

0

0

1

0

7.40

100

0

0

0

B

100

0

0

0

0

0

0

0

7.00

100

0

0

0

Total

%

200

(100)

0

(0.0)

0

(0.0)

1

(0.5)

0

(0.0)

0

(0.0)

1

(0.5)

0

(0.0)

 

(129)

200

0

0

0

(0.0)

 

+

 

2000

A

100

0

0

0

0

0

0

0

6.90

100

0

0

0

B

100

1

0

1

0

0

2

0

6.30

100

0

0

0

Total

%

200

(100)

1

(0.5)

0

(0.0)

1

(0.5)

0

(0.0)

0

(0.0)

2

(1.0)

0

(0.0)

 

(118)

200

0

0

0

(0.0)

 

+

 

4000

A

100

0

0

0

0

0

0

0

6.25

100

0

0

0

B

100

1

0

0

0

0

1

0

6.55

100

0

0

0

Total

%

200

(100)

1

(0.5)

0

(0.0)

0

(0.0)

0

(0.0)

0

(0.0)

1

(0.5)

0

(0.0)

 

(114)

200

0

0

0

(0.0)

 

+

Positive Control(CP)

5

A

100

11

4

1

0

0

15

9

2.00

100

0

0

0

B

50a

14

6

4

0

0

20

9

1.15

50

0

0

0

Total

%

150

(100)

25

(16.7)

10

(6.7)

5

(3.3)

0

(0.0)

0

(0.0)

35***

(23.3)

18

(12.0)

 

(28)

150

0

0

0

(0.0)

CP = Cyclophosphamide

*** = p < 0.001

a = Slide evaluation terminated at 50 cells because at least 30% cells with aberrations had been observed.

Table 3: Continuous Treatment Test - Experiment 2

 

Treatment Period (hours)

 

S9

mix

 

Concentration mg/ml

 

Number and Percentages of Cells Showing Structural Chromosome Aberrations (%)

 

g

Cell Growth Index

Number and Percentages of Cells Showing Numerical Aberrations (%)

 

Observed

 

ctb

 

cte

 

csb

 

cse

 

Others

 

Total

Mitotic Index (%)

 

Observed

 

Polyploids

 

Others

 

Total

 

24

 

-

Negative Control (MEM)

0

A

100

1

0

0

0

0

1

0

6.60

102

2

0

2

B

100

0

0

0

0

0

0

1

6.25

100

0

0

0

Total

%

200

(100)

1

(0.5)

0

(0.0)

0

(0.0)

0

(0.0)

0

(0.0)

1

(0.5)

1

(0.5)

 

(100)

202

2

0

2

(1.0)

 

-

 

250

A

100

1

0

1

0

0

2

0

5.00

100

0

0

0

B

100

0

0

0

0

0

0

0

5.20

100

0

0

0

Total

%

200

(100)

1

(0.5)

0

(0.0)

1

(0.5)

0

(0.0)

0

(0.0)

2

(1.0)

0

(0.0)

 

(79)

200

0

0

0

(0.0)

 

-

 

500

A

100

1

0

0

0

0

1

0

4.55

100

0

0

0

B

100

0

0

0

0

0

0

0

5.95

100

0

0

0

Total

%

200

(100)

1

(0.5)

0

(0.0)

0

(0.0)

0

(0.0)

0

(0.0)

1

(0.5)

0

(0.0)

 

(82)

200

0

0

0

(0.0)

 

-

 

1000

A

100

0

0

0

0

0

0

0

4.20

100

0

0

0

B

100

0

0

0

0

0

0

0

3.70

100

0

0

0

Total

%

200

(100)

0

(0.0)

0

(0.0)

0

(0.0)

0

(0.0)

0

(0.0)

0

(0.0)

0

(0.0)

 

(61)

200

0

0

0

(0.0)

 

-

 

2000

A

100

0

0

0

0

0

0

0

3.40

100

0

0

0

B

100

0

0

1

0

0

1

1

3.00

100

0

0

0

Total

%

200

(100)

0

(0.0)

0

(0.0)

1

(0.5)

0

(0.0)

0

(0.0)

1

(0.5)

1

(0.5)

 

(50)

200

0

0

0

(0.0)

 

-

Positive Control (MMC)

0.2

A

50a

10

12

0

1

0

16

2

0.85

50

0

0

0

B

50a

6

12

8

1

0

23

5

0.80

50

0

0

0

Total

%

100

(100)

16

(16.0)

24

(24.0)

8

(8.0)

2

(2.0)

0

(0.0)

39***

(39.0)

7

(7.0)

 

(13)

100

0

0

0

(0.0)

MMC = Mitomycin C

*** = p < 0.001

a = Slide evaluation terminated at 50 cells because at least 30% cells with aberrations had been observed.

Table 4: Short Term Treatment Test - Experiment 2 With Metabolic Activation (S9)

 

Treatment Period (hours)

 

S9

mix

 

Concentration mg/ml

 

Number and Percentages of Cells Showing Structural Chromosome Aberrations (%)

 

g

Cell Growth Index

Number and Percentages of Cells Showing Numerical Aberrations (%)

 

Observed

 

ctb

 

cte

 

csb

 

cse

 

Others

 

Total

Mitotic Index (%)

 

Observed

 

Polyploids

 

Others

 

Total

 

4

 

+

Negative Control (MEM)

0

A

100

0

0

0

0

0

0

1

8.75

100

0

0

0

B

100

0

0

0

0

0

0

0

5.55

100

0

0

0

Total

%

200

(100)

0

(0.0)

0

(0.0)

0

(0.0)

0

(0.0)

0

(0.0)

0

(0.0)

1

(0.5)

 

(100)

200

0

0

0

(0.0)

 

+

 

1000

A

100

0

0

1

0

0

1

0

6.70

100

0

0

0

B

100

1

0

0

0

0

1

0

6.60

100

0

0

0

Total

%

200

(100)

1

(0.5)

0

(0.0)

1

(0.5)

0

(0.0)

0

(0.0)

2

(1.0)

0

(0.0)

 

(93)

200

0

0

0

(0.0)

 

+

 

2000

A

100

0

0

0

0

0

0

0

6.95

100

0

0

0

B

100

0

0

0

0

0

0

0

7.20

100

0

0

0

Total

%

200

(100)

0

(0.0)

0

(0.0)

0

(0.0)

0

(0.0)

0

(0.0)

0

(0.0)

0

(0.0)

 

(99)

200

0

0

0

(0.0)

 

+

 

4000

A

100

0

0

0

0

0

0

1

5.55

100

0

0

0

B

100

1

0

0

0

0

1

0

7.80

100

0

0

0

Total

%

200

(100)

1

(0.5)

0

(0.0)

0

(0.0)

0

(0.0)

0

(0.0)

1

(0.5)

1

(0.5)

 

(93)

200

0

0

0

(0.0)

 

+

Positive Control (CP)

5

A

50a

11

3

1

0

0

15

6

2.25

50

0

0

0

B

100

6

8

9

0

0

21

10

1.80

100

0

0

0

Total

%

150

(100)

17

(11.3)

11

(7.3)

10

(6.7)

0

(0.0)

0

(0.0)

36***

(24.0)

16

(10.7)

 

(28)

150

0

0

0

(0.0)

CP = Cyclophosphamide

*** = p < 0.001

a = Slide evaluation terminated at 50 cells because at least 30% cells with aberrations had been observed.

Conclusions:
The test material did not induce any statistically significant increases in the frequency of cells with chromosome aberrations in either the absence or presence of a liver enzyme metabolising system in either of two separate experiments. The test material was therefore considered to be non-clastogenic to human lymphocytes in vitro.
Executive summary:

This report describes the results of an in vitro study for the detection of structural chromosomal aberrations in cultured mammalian cells. It supplements microbial systems insofar as it identifies potential mutagens that produce chromosomal aberrations rather than gene mutations (Scott et al, 1990). The method used followed that described in the OECD Guidelines for Testing of Chemicals (1997) No. 473 "Genetic Toxicology: Chromosome Aberration Test" and Method B10 of Commission Regulation (EC) No. 440/2008 of 30 May 2008 and is acceptable to the Japanese New Chemical Substance Law (METI).

Duplicate cultures of human lymphocytes, treated with the test material, were evaluated for chromosome aberrations at up to four dose levels, together with vehicle and positive controls. Four treatment conditions were used for the study, i.e. In Experiment 1, a 4-hour exposure in the presence of an induced rat liver homogenate metabolising system (S9), at a 2% final concentration with cell harvest after a 20-hour expression period and a 4-hour exposure in the absence of metabolic activation (S9) with a 20-hour expression period. In Experiment 2, the 4-hour exposure with addition of S9 was repeated (using a 1% final S9 concentration); whilst in the absence of metabolic activation the exposure time was increased to 24 hours.

All vehicle (solvent) control groups had frequencies of cells with aberrations within the range expected for normal human lymphocytes. All the positive control materials induced statistically significant increases in the frequency of cells with aberrations indicating the satisfactory performance of the test and of the activity of the metabolising system.

The test material did not induce any statistically significant increases in the frequency of cells with aberrations, in either of two separate experiments, using a dose range that included a dose level that induced approximately 50% mitotic inhibition in one of the exposure groups.

In conclusion, the test material was considered to be non-clastogenic to human lymphocytes in vitro.

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

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

The potential mutagenic effect of B-TEGME was determined in two reverse mutation assays according to OECD test guideline No. 471, of which the most recent study according to GLP and current standards was considered as the key study (Clariant, USO117742, 2007), and a previous study (non-GLP) was considered as supporting (BASF, 40M0575/884367, 1989). In both studies, there was no mutagenicity up to the limit dose of 5000 µg/plate and two independent study parts took place (plate incorporation and preincubation method). Test systems were the Salmonnella thyphimurium strains TA 1537, TA 98, TA100 and TA 1535 (uvrB) and Escherichia coli WP2 (uvrA) strains with (+) and without (-) the metabolic system S9 (from male Wistar rats), respectively. Positive and negative controls were included in each study. The test item was dissolved in distilled water and applied once at experimental starting with the concentration ranges given. The duration was 48h. Mutagenic and cytotoxic effects were absent.

A key study for gene mutation potential in mammalian cells of B-TEGME was conducted to assess the potential mutagenicity of the test material on the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus of Chinese hamster ovary (CHO) cells (Shell, 2259-0067, 2010). The protocol used was designed to comply with the OECD Guidelines for Testing of Chemicals No. 476 'In Vitro Mammalian Cell Gene Mutation Tests', Commission Regulation (EC) No. 440/2008 and the United Kingdom Environmental Mutagen Society (Cole et al, 1990). The technique used is a plate assay using tissue culture flasks and 6-thioguanine (6­TG) as the selective agent. Chinese hamster ovary (CHO) CHO-Kl cells were treated with the test material at six dose levels, in duplicate, together with vehicle (solvent) and positive controls. Four treatment conditions were used for the study, i.e. In Experiment 1, a 4-hour exposure in the presence of an induced rat liver homogenate metabolising system (S9), at a 2% final concentration and a 4-hour exposure in the absence of metabolic activation (S9). In Experiment 2, the 4-hour exposure with addition of S9 was repeated (using a 1% final S9 concentration), whilst in the absence of metabolic activation the exposure time was increased to 24 hours.

The dose range of test material was selected based on the results of a preliminary cytotoxicity test and was 125 to 4000 µg/ml in both the presence and absence of metabolic activation. The maximum dose was limited to 4000 µg/ml as a result of an increase in osmolality of 50 mOsm at the maximum recommended dose of 5000 µg/ml, which was considered to be at the limit of acceptability.

The vehicle (solvent) controls gave mutant frequencies within the range expected of CHO cells at the HPRT locus.

The positive control treatments, both in the presence and absence of metabolic activation, gave significant increases in the mutant frequency indicating the satisfactory performance of the test and of the metabolising system.

The test material demonstrated no significant increases in mutant frequency at any dose level, either with or without metabolic activation, in either the first or second experiment. The test material was considered to be non-mutagenic to CHO cells under the conditions of the test.

A key study for chromosome aberration potential of B-TEGME was studied in vitro in human peripheral lymphocytes (Shell, 2259-0066, 2010). This study was conducted according to OECD 473 and GLP standards, and was therefore considered to be relevant, adequate and reliable. The study supplements microbial systems insofar as it identifies potential genotoxins that produce chromosomal aberrations rather than gene mutations. Duplicate cultures of human lymphocytes, treated with the test material, were evaluated for chromosome aberrations at up to four dose levels, together with vehicle and positive controls. Four treatment conditions were used for the study, i.e. In Experiment 1, a 4-hour exposure in the presence of metabolising system S9, at a 2% final concentration with cell harvest after a 20-hour expression period and a 4-hour exposure in the absence of metabolic activation with a 20-hour expression period. In Experiment 2, the 4-hour exposure with addition of S9 was repeated (using a 1% final S9 concentration); whilst in the absence of metabolic activation the exposure time was increased to 24 hours. All vehicle (solvent) control groups had frequencies of cells with aberrations within the range expected for normal human lymphocytes. All the positive control materials induced statistically significant increases in the frequency of cells with aberrations indicating the satisfactory performance of the test and of the activity of the metabolising system. The test material did not induce any statistically significant increases in the frequency of cells with aberrations, in either of two separate experiments, using a dose range that included a dose level that induced approximately 50% mitotic inhibition in one of the exposure groups. In conclusion, the test material was considered to be non-clastogenic to human lymphocytes in vitro.

Chromosome aberration potential was also studied with a brake fluid containing 17% B-TEGME in cultured Chinese Hamster Ovary (CHO) cells (Shell, 92.090, 1991). Although the study was also conducted under GLP and according to OECD test 473, this was considered as a supporting study sine the test material contained less B-TEGME. Cells were treated for either 3 hours in the presence of S9 mix or for 24 hours in the absence of S9 mix. Metaphase cells were analysed, demonstrating that the brake fluid and hence also B-TEGME did not induce structural chromosome aberrations in cultured human lymphocytes both in the presence and in the absence of S9 mix, under the experimental conditions described.

Justification for classification or non-classification

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008, as amended for the tenth time in Regulation (EU) No 2017/776. As a result the substance is not considered to be classified for genotoxicity under Regulation (EC) No. 1272/2008.