Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

An Ames test (OECD 471) is available on 1,10 decanediyl bis methacrylate. In this study, the test item did not induce gene mutations by base pair changes of frameshifts in the genome of the strains used.

No in vitro study on mammalian cells are available on 1,10 decanediyl bis methacrylate. However data are available on an analogue substance of 1,10 decanediyl bis methacrylate : 1,10-decanediol diacrylate. On this analogue substance, HPRT test (OECD 476) and in vitro micronucleus test (OECD 487) showed negative results in presence and in absence of metabolic activation.

Bacterial reverse mutation assay (Sokolowski 2010):

The study was performed to investigate the potential of the test item to induce gene mutations according to the plate incorporation test (experiment 1) and the pre-incubation test (experiment 2) using the Salmonella typhimurium strains TA1535, TA1537, TA98, TA100 and TA102.

The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate. The test item was tested at the following concentrations: 33, 100, 333, 1000, 2500 and 500 µg/plate.

No toxic effects, evident as a reduction in the number of revertants, occurred in the tes groups with and without metabolic activation.

The plates incubated with the test item showed normal background growth up to 5000 µg/plate with and without S9 mix in all strains used.

No substantial increase in revertant colony numbers of any of the five tester srains was observed following treatment with the test item at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.

Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies.

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes of frameshifts in the genome of the strains used. The test item is considered to be non-mutagenic in ths Salmonella typhimurium reverse mutation assay.

In vitro mammalian cell micronucleus test (Brient 2013) / read-across :

The objective of this study was to evaluate the potential of 1,10-decanediol diacrylate to induce an increase in the frequency of micronucleated cellsin the mouse cell line L5178YTK+/-. This study conducted in compliance with OECD Guideline No. 487 and the principles of Good Laboratory Practices.

After a preliminary toxicity test, the test item was tested in two independent experiments, with and without a metabolic activation system, the S9 mix, prepared from a liver microsomal fraction (S9 fraction) of rats induced with Aroclor 1254, as follows:

-First experiment: 3 h treatment + 24 h recovery (without and with S9 mix),

-Second experiment :24 h treatment + 20 h recovery (without S9 mix), and3 h treatment + 24 h recovery (with S9 mix).

Each treatment was coupled to an assessment of cytotoxicity at the same dose-levels. Cytotoxicity was evaluated by determining the PD (Population Doubling) of cells and quality of the cells on the slides has also been taken into account.

For each main experiment (with or without S9 mix), micronuclei were analyzed for three dose-levels of the test item, for the vehicle and the positive controls, in 1000 mononucleated cells per culture (total of 2000 mononucleated cells per concentration). 

The test item was dissolved in dimethylsulfoxide (DMSO).

 Experiments without S9 mix: Following the first experiment,a severe toxicity was induced at the highest tested dose-level of 20 µg/mL, as shown by a 100% decrease in the PD. The immediately lower dose-level of 10 µg/mL induced a slight but acceptable toxicity, as shown by a 37% decrease in the PD. Following the second experiment,a severe toxicity was induced at the highest tested dose-level of 40 µg/mL, as shown by a 100% decrease in the PD. The immediately lower dose-level of 20 µg/mL induced no toxicity, as shown by no noteworthy decrease in the PD.

In the first experiment, a statistically significant increase in the frequency of micronucleated cells was noted at the highest dose-level of 10 µg/mL. However, no dose-response relationship was noted, and only one replicate of the two cultures used for this dose-level showed a frequency of micronucleated cells above the corresponding vehicle control historical data range. These results were thus considered to be equivocal, and the second experiment without S9 mix was performed following a long treatment period. During the second experiment, no statistically significant increase in the frequency of micronucleated cells was noted. Consequently, the increase observed during the first experiment was not reproduced, and was thus not considered to be biologically relevant.

Experiments with S9 mix: Following the first experiment,a marked toxicity was induced at the highest tested dose-level of 80 µg/mL, as shown by a 77% decrease in the PD. The immediately lower dose-level of 40 µg/mL induced a slight but acceptable toxicity, as shown by a 38% decrease in the PD. Following the second experiment, a slight toxicity was induced at the highest tested dose-level of 80 µg/mL, as shown by a 28% decrease in the PD.

In the first experiment, a dose-response relationship was noted, but no statistically significant increase in the frequency of micronucleated cells was observed. In the second experiment performed in the same experimental conditions, some increases in the frequency of micronucleated cells were noted at both higher doses (40 and 80 µg/mL). However, these increases were not statistically significant, and the corresponding frequencies of micronucleated cells remained within the historical data of the vehicle control. Consequently, these increases did not meet the criteria for a positive response and were thus considered as non-biologically relevant.

Under the experimental conditions of the study, the test item did not induce any chromosome damage, or damage to the cell division apparatus, in cultured mammalian somatic cells, using L5178Y TK+/- mouse lymphoma cells, in the absence or in the presence of a rat metabolising system.

 

In vitro mammalian cell gene mutation assay (Massip 2013) / Read-across :

1,10-decanediol diacrylate was assayed for the ability to induce mutation at the hypoxanthine-guanine phosphoribosyl transferase (hprt) locus (6-thioguanine [6TG] resistance) in mouse lymphoma cells using a fluctuation protocol. The study consisted of a cytotoxicity Range-Finder Experiment followed by two independent experiments, each conducted in the absence and presence of metabolic activation by an Aroclor 1254-induced rat liver post-mitochondrial fraction (S-9). The test article was formulated in anhydrous analytical grade dimethyl sulphoxide (DMSO).

A 3 hour treatment incubation period was used for all experiments.

In Experiment 1, thirteen concentrations, ranging from 2.5 to 150 µg/mL in the absence of S-9 and from 25 to 400 µg/mL in the presence of S-9, were tested. Seven days after treatment, the highest concentrations analysed to determine viability and 6TG resistance were 40mg/µL in the absence of S-9 and 330 µg/mL in the presence of S-9, limited by toxicity, which gave 17% and 12% RS, respectively.

In Experiment 2 twelve concentrations, ranging from 5 to 80 µg/mL in the absence of S-9 and from 50 to 400 µg/mL in the presence of S-9, were tested.The highest concentrations analysed to determine viability and 6TG resistance were 30 µg/mL in the absence of S-9 and 270 µg/mL in the presence of S-9, which gave 14% and 12% RS, respectively.

In Experiments 1 and 2 no statistically significant increases in MF were observed following treatment with1,10-decanediol diacrylate at any concentration tested in the absence and presence of S-9 and there were no significant linear trends.

It is concluded that 1,10-decanediol diacrylate did not induce mutation at the hprt locus of L5178Y mouse lymphoma cells when tested up to toxic concentrations in two independent experiments, in the absence and presence of a rat liver metabolising system (S-9)



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:
January - February 2001
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reference:
Composition 1
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1997
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Test material information:
Composition 1
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Additional strain characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
rat liver
Test concentrations with justification for top dose:
In the pre-experiment the concentration range of the test item was 3-5000 µg/plate. No relevant toxic effects were observed. 5000 µg/plate was chosen as maximal concentration.
The following concentrations were tested: 33, 100, 333, 1000, 2500 and 5000 µg/plate.
Vehicle:
DMSO
Negative controls:
yes
Solvent controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
methylmethanesulfonate
other: 4-nitro-o-phenylene-diamine, without S9, TA1537, TA98. 2-aminoanthracene : with S9, all strains
Details on test system and conditions:
METHOD OF APPLICATION: in agar (plate incorporation) = experiment 1; preincubation = experiment 2

Pre-experiment for toxicity :
To evaluate the toxicity of the test item a pre-experiment was performed with strains TA98 and TA100. 8 concentrations were tested for toxicity and mutation induction with each 3 plates. The experimental conditions in the pre-experiment were the same as described for the experiment 1 (plate incorporation test).
Toxicity of the test item can be evident as a reduction in the number of spontaneous revertants of a clearing of the bacterial background lawn.
Evaluable plates (> 0 colonies) at five concentrations or more in all strains used.

For each strain and dose level, including the controls, three plates were used.

In the preincubation assay, test solution, S9 mix and bacterial suspension were mixed and incubated at 37°C for 60 minutes. After pre-incubation agar was added to each tube. The mixture was poured on minimal agar plates. After solidification the plates were incubated upside down for at least 48 hours at 37 °C in the dark.

Acceptability of the assay :
-regular background growth in the negative and solvent control
-the spontaneous reversion rates in the negative and solvent control are in the arnge of the historical control data
-the psitive control substances should produce a significant increase in mutant colony frequencies
Evaluation criteria:
A test item is considered positive if either a dose related increase in the number of revertants of a biologically relevant increase for at least one test concentration is induced.
A test item producing neither a dose related increase in the number of revertants nor a biologically relevant positive response at any dose of the test points is considered non-mutagenic in this system.
A biologically relevant response is described as : A test item is considered mutagenic if the number of reversions is at least twice the spontaneous reversion rate instrains TA98, TA 100 and TA 102, or trice in strains TA1535, TA1537. Also a dose-dependent increase in the number of revertants is regarded as an indication of possibly existing mutagenic potential of the test item regardless whether the highest dose induced the criteria described above or not.
Statistics:
no
Key result
Species / strain:
other: TA98, TA100, TA102, TA1535, TA1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no, but tested up to limit concentrations
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Additional information on results:
No precipitation of the test item occurred up to the highest investigated dose.
No toxic effects, evident as a reduction in the number of revertants, occurred in the tes groups with and without metabolic activation.
The plates incubated with the test item showed normal background growth up to 5000 µg/plate with and without S9 mix in all strains used.
No substantial increase in revertant colony numbers of any of the five tester srains was observed following treatment with the test item at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.
Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies.

Ames tables

Pre-experiment

Test group

Concentration per plate (µg)

Revertants per plate (mean of 3 plates)

TA98

TA98

TA 100

TA 100

- S9

+ S9

- S9

+ S9

Negative control

-

28

38

166

138

Solvent control

-

20

32

166

155

4-NOPD

10

223

-

-

-

Sodium azide

10

-

-

690

-

2-AA

2.5

-

223

-

742

Test item

3

22

29

143

126

Test item

10

29

28

139

132

Test item

33

30

29

143

128

Test item

100

27

32

143

126

Test item

333

27

30

133

118

Test item

1000

18

3

 

132

130

Test item

2500

24

23

131

133

Test item

5000

24

22

124

130

 

Experiment 1

TA1535 without S9

Concentration µg/plate

Plate 1

Plate 2

Plate 3

Mean revertant/ plate

s.d

factor

Negative control

8

7

11

9

2.1

 

Solvent control

8

13

8

10

2.9

1.0

Positive control

615

664

664

648

28.3

67.0

33

13

10

7

10

3.0

1.0

100

9

9

10

9

0.6

1.0

333

10

7

8

8

1.5

0.9

1000

6

11

5

7

3.2

0.8

2500

9

5

9

8

2.3

0.8

5000

6

10

8

8

2.0

0.8

 

TA1535 with S9

Concentration µg/plate

Plate 1

Plate 2

Plate 3

Mean revertant/ plate

s.d

factor

Negative control

8

8

11

9

1.7

 

Solvent control

15

15

9

13

3.5

1.0

Positive control

73

86

64

74

11.1

5.7

33

8

11

17

12

4.6

0.9

100

8

8

12

9

2.3

0.7

333

7

12

10

10

2.5

0.7

1000

11

11

12

11

0.6

0.9

2500

10

7

5

7

2.5

0.6

5000

6

9

7

7

1.5

0.6

 

TA1537 without S9

Concentration µg/plate

Plate 1

Plate 2

Plate 3

Mean revertant/ plate

s.d

factor

Negative control

6

5

6

6

0.6

 

Solvent control

10

7

4

7

3.0

1.0

Positive control

52

51

52

52

0.6

7.4

33

8

5

7

7

1.5

1.0

100

8

5

4

6

2.1

0.8

333

5

8

5

6

1.7

0.9

1000

5

7

8

7

1.5

1.0

2500

6

4

4

5

1.2

0.7

5000

4

5

5

5

0.6

0.7

 

TA1537 with S9

Concentration µg/plate

Plate 1

Plate 2

Plate 3

Mean revertant/ plate

s.d

factor

Negative control

16

11

7

11

4.5

 

Solvent control

5

7

5

6

1.2

1.0

Positive control

107

96

84

96

11.5

16.9

33

12

7

7

9

2.9

1.5

100

8

8

6

7

1.2

1.3

333

6

6

6

6

0

1.1

1000

8

7

4

6

2.1

1.1

2500

5

5

5

5

0

0.9

5000

8

4

4

5

2.3

0.9

 

 

TA98 without S9

Concentration µg/plate

Plate 1

Plate 2

Plate 3

Mean revertant/ plate

s.d

factor

Negative control

27

27

31

28

2.3

 

Solvent control

23

20

18

20

2.5

1.0

Positive control

241

212

217

223

15.5

11.0

33

29

32

30

30

1.5

1.5

100

28

29

25

27

2.1

1.3

333

24

29

27

27

2.5

1.3

1000

17

18

19

18

1.0

0.9

2500

25

22

26

24

2.1

1.2

5000

23

24

26

24

1.5

1.2

 

TA98 with S9

Concentration µg/plate

Plate 1

Plate 2

Plate 3

Mean revertant/ plate

s.d

factor

Negative control

38

39

38

38

0.6

 

Solvent control

34

32

29

32

2.5

1.0

Positive control

379

430

402

404

25.5

12.7

33

29

27

30

29

1.5

0.9

100

31

31

34

32

1.7

1.0

333

29

32

28

30

2.1

0.9

1000

21

23

25

23

2.0

0.7

2500

22

23

24

23

1.0

0.7

5000

19

23

24

22

2.6

0.7

 

TA100 without S9

Concentration µg/plate

Plate 1

Plate 2

Plate 3

Mean revertant/ plate

s.d

factor

Negative control

156

161

180

166

12.7

 

Solvent control

166

160

171

166

5.5

1.0

Positive control

721

702

648

690

37.9

4.2

33

143

148

138

143

5.0

0.9

100

143

142

145

143

1.5

0.9

333

132

135

131

133

2.1

0.8

1000

139

128

130

132

5.9

0.8

2500

130

133

129

131

2.1

0.8

5000

125

121

127

124

3.1

0.8

 

TA100 with S9

Concentration µg/plate

Plate 1

Plate 2

Plate 3

Mean revertant/ plate

s.d

factor

Negative control

147

132

135

138

7.9

 

Solvent control

158

157

149

155

4.9

1.0

Positive control

713

757

756

742

25.1

4.8

33

130

129

124

128

3.2

0.8

100

127

121

131

126

5.0

0.8

333

122

118

115

118

3.5

0.8

1000

125

129

135

130

5.0

0.8

2500

132

129

139

133

5.1

0.9

5000

132

128

131

130

2.1

0.8

 

TA102 without S9

Concentration µg/plate

Plate 1

Plate 2

Plate 3

Mean revertant/ plate

s.d

factor

Negative control

207

194

194

198

7.5

 

Solvent control

195

182

201

193

9.7

1.0

Positive control

973

915

1026

971

55.5

5.0

33

233

198

205

212

18.5

1.1

100

229

195

178

201

26.0

1.0

333

210

210

183

201

15.6

1.0

1000

190

190

177

186

7.5

1.0

2500

193

205

177

192

14.0

1.0

5000

217

203

185

202

16.0

1.0

 

TA102 with S9

Concentration µg/plate

Plate 1

Plate 2

Plate 3

Mean revertant/ plate

s.d

factor

Negative control

192

149

149

163

24.8

 

Solvent control

161

194

238

198

38.6

1.0

Positive control

749

785

806

780

28.8

3.9

33

184

198

237

206

27.5

1.0

100

196

196

167

186

16.7

0.9

333

147

165

181

164

17.0

0.8

1000

172

182

198

184

13.1

0.9

2500

156

139

117

137

19.6

0.7

5000

116

107

117

113

5.5

0.6

 

 

Experiment 2

TA1535 without S9

Concentration µg/plate

Plate 1

Plate 2

Plate 3

Mean revertant/ plate

s.d

factor

Negative control

9

10

8

9

1.0

 

Solvent control

13

11

9

11

2.0

1.0

Positive control

738

754

677

723

40.6

65.7

33

13

11

7

10

3.1

0.9

100

14

10

13

12

2.1

1.1

333

10

10

11

10

0.6

0.9

1000

8

10

10

9

1.2

0.8

2500

10

7

7

8

1.7

0.7

5000

6

8

8

7

1.2

0.7

 

TA1535 with S9

Concentration µg/plate

Plate 1

Plate 2

Plate 3

Mean revertant/ plate

s.d

factor

Negative control

10

13

13

12

1.7

 

Solvent control

13

10

9

11

2.1

1.0

Positive control

120

128

119

122

4.9

11.5

33

10

11

11

11

0.6

1.0

100

13

9

8

10

2.6

0.9

333

8

7

9

8

1.0

0.8

1000

5

13

6

8

4.4

0.8

2500

11

12

8

10

2.1

1.0

5000

13

9

10

11

2.1

1.0

 

TA1537 without S9

Concentration µg/plate

Plate 1

Plate 2

Plate 3

Mean revertant/ plate

s.d

factor

Negative control

7

5

7

6

1.2

 

Solvent control

5

7

6

6

1.0

1.0

Positive control

47

49

65

54

9.9

8.9

33

6

6

8

7

1.2

1.1

100

6

6

6

6

0.0

1.0

333

5

7

7

6

1.2

1.1

1000

8

6

6

7

1.2

1.1

2500

4

7

5

5

1.5

0.9

5000

5

5

4

5

0.6

0.8

 

TA1537 with S9

Concentration µg/plate

Plate 1

Plate 2

Plate 3

Mean revertant/ plate

s.d

factor

Negative control

12

11

11

11

0.6

 

Solvent control

12

8

9

10

2.1

1.0

Positive control

57

55

55

56

1.2

5.8

33

7

9

10

9

1.5

0.9

100

12

9

12

11

1.7

1.1

333

9

8

13

10

2.6

1.0

1000

9

11

10

10

1.0

1.0

2500

9

0

10

6

5.5

0.7

5000

11

11

11

11

0.0

1.1

 

 

TA98 without S9

Concentration µg/plate

Plate 1

Plate 2

Plate 3

Mean revertant/ plate

s.d

factor

Negative control

25

24

32

27

4.4

 

Solvent control

31

21

23

25

5.3

1.0

Positive control

220

203

234

219

15.5

8.8

33

34

26

23

28

5.7

1.1

100

30

30

21

27

5.2

1.1

333

19

25

30

25

5.5

1.0

1000

21

30

23

25

4.7

1.0

2500

27

27

21

25

3.5

1.0

5000

19

24

26

23

3.6

0.9

 

TA98 with S9

Concentration µg/plate

Plate 1

Plate 2

Plate 3

Mean revertant/ plate

s.d

factor

Negative control

30

44

40

38

7.2

 

Solvent control

45

44

34

41

6.1

1.0

Positive control

586

591

548

575

23.5

14.0

33

52

38

46

45

7.0

1.1

100

47

47

42

45

2.9

1.1

333

44

38

46

43

4.2

1.0

1000

35

33

36

35

1.5

0.8

2500

40

30

32

34

5.3

0.8

5000

37

38

34

36

2.1

0.9

 

TA100 without S9

Concentration µg/plate

Plate 1

Plate 2

Plate 3

Mean revertant/ plate

s.d

factor

Negative control

97

112

129

113

16.0

 

Solvent control

129

107

100

112

15.1

1.0

Positive control

832

902

856

863

35.6

7.7

33

99

100

106

102

3.8

0.9

100

101

107

113

107

6.0

1.0

333

91

102

100

98

5.9

0.9

1000

116

109

101

109

7.5

1.0

2500

90

97

104

97

7.0

0.9

5000

62

84

66

71

11.7

0.6

 

TA100 with S9

Concentration µg/plate

Plate 1

Plate 2

Plate 3

Mean revertant/ plate

s.d

factor

Negative control

81

78

113

91

19.4

 

Solvent control

132

125

131

129

3.8

1.0

Positive control

691

711

698

700

10.1

5.4

33

119

111

124

118

6.6

0.9

100

104

104

114

107

5.8

0.8

333

98

83

84

88

8.4

0.7

1000

83

100

95

93

8.7

0.7

2500

100

82

84

89

9.9

0.7

5000

89

90

78

86

6.7

0.7

 

TA102 without S9

Concentration µg/plate

Plate 1

Plate 2

Plate 3

Mean revertant/ plate

s.d

factor

Negative control

210

220

212

214

5.3

 

Solvent control

199

157

189

182

21.9

1.0

Positive control

1040

984

1090

1038

53.0

5.7

33

212

214

183

203

17.3

1.1

100

228

214

207

216

10.7

1.2

333

205

215

198

206

8.5

1.1

1000

184

196

182

187

7.6

1.0

2500

196

199

166

187

18.2

1.0

5000

174

156

163

164

9.1

0.9

 

TA102 with S9

Concentration µg/plate

Plate 1

Plate 2

Plate 3

Mean revertant/ plate

s.d

factor

Negative control

275

191

191

219

48.5

 

Solvent control

191

201

229

207

19.7

1.0

Positive control

805

723

822

783

52.9

3.8

33

218

234

214

222

10.6

1.1

100

262

251

217

243

23.5

1.2

333

230

224

218

224

6.0

1.1

1000

219

215

202

212

8.9

1.0

2500

225

214

218

219

5.6

1.1

5000

218

228

223

223

5.0

1.1

 

 

 

 

Conclusions:
In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes of frameshifts in the genome of the strains used.
The test item is considered to be non-mutagenic in ths Salmonella typhimurium reverse mutation assay.
Executive summary:

The study was performed to investigate the potential of the test item to induce gene mutations according to the plate incorporation test (experiment 1) and the pre-incubation test (experiment 2) sing the Salmonella typhimurium strains TA1535, TA1537, TA98, TA100 and TA102.

The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate. The test item was tested at the following concentrations: 33, 100, 333, 1000, 2500 and 500 µg/plate.

No toxic effects, evident as a reduction in the number of revertants, occurred in the tes groups with and without metabolic activation.

The plates incubated with the test item showed normal background growth up to 5000 µg/plate with and without S9 mix in all strains used.

No substantial increase in revertant colony numbers of any of the five tester srains was observed following treatment with the test item at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.

Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies.

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes of frameshifts in the genome of the strains used.

The test item is considered to be non-mutagenic in ths Salmonella typhimurium reverse mutation assay.

Endpoint:
in vitro cytogenicity / micronucleus study
Type of information:
experimental study
Adequacy of study:
key study
Study period:
23 April 2013 - 18 June 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reference:
Composition 1
Qualifier:
according to
Guideline:
other: OECD Guideline No. 487 (In vitro mammalian cell micronucleus test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian cell micronucleus test
Test material information:
Composition 1
Target gene:
Not applicable (not a gene mutation assay).
Species / strain:
other: L5178Y TK+/- mouse lymphoma cells
Details on mammalian cell lines (if applicable):
- Type and identity of media: RPMI 1640 medium containing 10% (v/v) heat-inactivated horse serum, L-Glutamine (2 mM), penicillin (100 U/mL), streptomycin (100 µg/mL) and sodium pyruvate (200 µg/mL)
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
Additional strain characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
rat liver S9 mix
Test concentrations with justification for top dose:
With a treatment volume of 1% (v/v) in culture medium, the dose-levels used for treatments, were as follows:
. 0.16, 0.31, 0.63, 1.25, 2.5, 5, 10 and 20 µg/mL in the first experiment without S9 mix,
. 0.31, 0.63, 1.25, 2.5, 5, 10, 20 and 40 µg/mL in the second experiment without S9 mix,
. 0.63, 1.25, 2.5, 5, 10, 20, 40 and 80 µg/mL in both experiments with S9 mix.
Vehicle:
- Vehicle used: the vehicle was dimethylsulfoxide (DMSO), batch No. K42474850 145.
- Justification for choice according to solubility assays performed at CiToxLAB France: highest recommended dose-level of 5000 µg/mL was achievable using a test item solution at 500 mg/mL under a treatment volume of 1% (v/v) in the culture medium.
Negative controls:
no
Solvent controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: mitomycin C, colchicine (without S9 mix); cyclophosphamide (with S9 mix)
Details on test system and conditions:
METHOD OF APPLICATION: in medium

DURATION
-First experiment: 3 h treatment + 24 h recovery (without and with S9 mix),
-Second experiment : 24 h treatment + 20 h recovery (without S9 mix), and 3 h treatment + 24 h recovery (with S9 mix).

NUMBER OF CELLS EVALUATED: 2000 mononucleated cells/dose

DETERMINATION OF CYTOTOXICITY
- Method: population doubling
Evaluation criteria:
A test item was considered to have clastogenic and/or aneugenic potential, if all the following criteria were met:
- a dose-related increase in the frequency of micronucleated cells was observed,
- for at least one dose-level, the frequency of micronucleated cells of each replicate culture was above the corresponding vehicle historical range,
- a statistically significant difference in comparison to the corresponding vehicle control was obtained at one or more dose-levels.

The biological relevance of the results was considered first.
If the criteria of a positive response are only partially met, results will be evaluated on a case by case basis, taking into account other parameters such as reproducibility between experiments. If results remain inconclusive, or when the highest analyzable dose-level does not exhibit about 55% toxicity (in case of toxic items), additional confirmatory experiments may be needed.
Statistics:
no
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
yes
Vehicle controls valid:
yes
Negative controls valid:
not applicable
Positive controls valid:
yes
Additional information on results:
Experiments without S9 mix
Cytotoxicity
Following the first experiment,a severe toxicity was induced at the highest tested dose-level of 20 µg/mL, as shown by a 100% decrease in the PD. The immediately lower dose-level of 10 µg/mL induced a slight but acceptable toxicity, as shown by a 37% decrease in the PD.
Following the second experiment,a severe toxicity was induced at the highest tested dose-level of 40 µg/mL, as shown by a 100% decrease in the PD. The immediately lower dose-level of 20 µg/mL induced no toxicity, as shown by no noteworthy decrease in the PD.
 
Micronucleus analysis
The dose-levels selected for micronucleus analysis were as follows:
. 2.5, 5 and 10 µg/mL for the 3-hour treatment, the higher being too cytotoxic,
. 5, 10 and 20 µg/mL for the 24-hour treatment, the higher being too cytotoxic.
 
In the first experiment, a statistically significant increase in the frequency of micronucleated cells was noted at the dose-level of 10 µg/mL. However, no dose-response relationship was noted, and only one replicate of the two cultures used for this dose-level showed a frequency of micronucleated cells above the corresponding vehicle control historical data range. These results were thus considered to be equivocal, and the second experiment without S9 mix was performed following a long treatment period. During the second experiment, no statistically significant increase in the frequency of micronucleated cells was noted. Consequently, the increase observed during the first experiment was not reproduced, and was thus not considered to be biologically relevant.

Experiments with S9 mix
Cytotoxicity
Following the first experiment,a marked toxicity was induced at the highest tested dose-level of 80 µg/mL, as shown by a 77% decrease in the PD. The immediately lower dose-level of 40 µg/mL induced a slight but acceptable toxicity, as shown by a 38% decrease in the PD.
Following the second experiment, a slight toxicity was induced at the highest tested dose-level of 80 µg/mL, as shown by a 28% decrease in the PD.
 
Micronucleus analysis
The dose-levels selected for micronucleus analysis were as follows:
. 10, 20 and 40 µg/mL for the first experiment, the higher being too cytotoxic,
. 20, 40 and 80 µg/mL for the second experiment, the latter showing a precipitate at the end of the treatment period.
 
In the first experiment, a dose-response relationship was noted, but no statistically significant increase in the frequency of micronucleated cells was observed. In the second experiment performed in the same experimental conditions, some increases in the frequency of micronucleated cells were noted at 40 and 80 µg/mL. However, these increases were not statistically significant, and the corresponding frequencies of micronucleated cells remained within the historical data of the vehicle control. Consequently, these increases did not meet the criteria for a positive response and were thus considered as non-biologically relevant.
Conclusions:
Under the experimental conditions of the study, 1,10-decanediol diacrylate did not induce any chromosome damage, or damage to the cell division apparatus, in cultured mammalian somatic cells, using L5178Y TK+/- mouse lymphoma cells, in the absence or in the presence of a rat metabolising system.

Executive summary:

The objective of this study was to evaluate the potential of 1,10-decanediol diacrylate to induce an increase in the frequency of micronucleated cellsin the mouse cell line L5178YTK+/-. This study conducted in compliance with OECD Guideline No. 487 and the principles of Good Laboratory Practices.

 

Methods

After a preliminary toxicity test, the test item was tested in two independent experiments, with and without a metabolic activation system, the S9 mix, prepared from a liver microsomal fraction (S9 fraction) of rats induced with Aroclor 1254, as follows:

-First experiment: 3 h treatment + 24 h recovery (without and with S9 mix),

-Second experiment : 24 h treatment + 20 h recovery (without S9 mix), and 3 h treatment + 24 h recovery (with S9 mix).

Each treatment was coupled to an assessment of cytotoxicity at the same dose-levels. Cytotoxicity was evaluated by determining the PD (Population Doubling) of cells and quality of the cells on the slides has also been taken into account.

For each main experiment (with or without S9 mix), micronuclei were analyzed for three dose-levels of the test item, for the vehicle and the positive controls, in 1000 mononucleated cells per culture (total of 2000 mononucleated cells per concentration).

 

The test item was dissolved in dimethylsulfoxide (DMSO).

 

Results

 

The mean PD and mean frequencies of micronucleated cells for the vehicle control cultures were as specified in the acceptance criteria. Positive control cultures showed clear statistically significant increases in the frequency of micronucleated cells. The study was therefore considered to be valid.

 

Since the test item was found to be cytotoxic and poorly soluble in the preliminary test, the selection of the highest dose-level to be used in the main experiments was based on the level of precipitate/emulsion and/or cytotoxicity, according to the criteria specified in the international guidelines.

 

With a treatment volume of 1% (v/v) in culture medium, the dose-levels used for treatments, were as follows:

. 0.16, 0.31, 0.63, 1.25, 2.5, 5, 10 and 20 µg/mL in the first experiment without S9 mix,

. 0.31, 0.63, 1.25, 2.5, 5, 10, 20 and 40 µg/mL in the second experiment without S9 mix,

. 0.63, 1.25, 2.5, 5, 10, 20, 40 and 80 µg/mL in both experiments with S9 mix.

 

A precipitate was observed at the end of the treatments performed with S9 mix at the highest tested dose-level of 80 µg/mL.

Experiments without S9 mix

Cytotoxicity

Following the first experiment,a severe toxicity was induced at the highest tested dose-level of 20 µg/mL, as shown by a 100% decrease in the PD. The immediately lower dose-level of 10 µg/mL induced a slight but acceptable toxicity, as shown by a 37% decrease in the PD.

Following the second experiment,a severe toxicity was induced at the highest tested dose-level of 40 µg/mL, as shown by a 100% decrease in the PD. The immediately lower dose-level of 20 µg/mL induced no toxicity, as shown by no noteworthy decrease in the PD.

 

Micronucleus analysis

The dose-levels selected for micronucleus analysis were as follows:

. 2.5, 5 and 10 µg/mL for the 3-hour treatment, the higher being too cytotoxic,

. 5, 10 and 20 µg/mL for the 24-hour treatment, the higher being too cytotoxic.

 

In the first experiment, a statistically significant increase in the frequency of micronucleated cells was noted at the dose-level of 10 µg/mL. However, no dose-response relationship was noted, and only one replicate of the two cultures used for this dose-level showed a frequency of micronucleated cells above the corresponding vehicle control historical data range. These results were thus considered to be equivocal, and the second experiment without S9 mix was performed following a long treatment period. During the second experiment, no statistically significant increase in the frequency of micronucleated cells was noted. Consequently, the increase observed during the first experiment was not reproduced, and was thus not considered to be biologically relevant.

Experiments with S9 mix

Cytotoxicity

Following the first experiment,a marked toxicity was induced at the highest tested dose-level of 80 µg/mL, as shown by a 77% decrease in the PD. The immediately lower dose-level of 40 µg/mL induced a slight but acceptable toxicity, as shown by a 38% decrease in the PD.

Following the second experiment, a slight toxicity was induced at the highest tested dose-level of 80 µg/mL, as shown by a 28% decrease in the PD.

 

Micronucleus analysis

The dose-levels selected for micronucleus analysis were as follows:

. 10, 20 and 40 µg/mL for the first experiment, the higher being too cytotoxic,

. 20, 40 and 80 µg/mL for the second experiment, the latter showing a precipitate at the end of the treatment period.

 

In the first experiment, a dose-response relationship was noted, but no statistically significant increase in the frequency of micronucleated cells was observed. In the second experiment performed in the same experimental conditions, some increases in the frequency of micronucleated cells were noted at 40 and 80 µg/mL. However, these increases were not statistically significant, and the corresponding frequencies of micronucleated cells remained within the historical data of the vehicle control. Consequently, these increases did not meet the criteria for a positive response and were thus considered as non-biologically relevant.

 

Conclusion

Under the experimental conditions of the study, the test item did not induce any chromosome damage, or damage to the cell division apparatus, in cultured mammalian somatic cells, using L5178Y TK+/- mouse lymphoma cells, in the absence or in the presence of a rat metabolising system.

 

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
17 April 2013 to 20 August 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reference:
Composition 1
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
mammalian cell gene mutation assay
Test material information:
Composition 1
Target gene:
HPRT gene
Species / strain:
mouse lymphoma L5178Y cells
Details on mammalian cell lines (if applicable):
The master stock of L5178Y tk+/- (3.7.2°C) mouse lymphoma cells originated from Dr Donald Clive, Burroughs Wellcome Co. Cells supplied to Covance Laboratories Ltd. were stored as frozen stocks in liquid nitrogen. Each batch of frozen cells was purged of mutants and confirmed to be mycoplasma free. For each experiment, at least one vial was thawed rapidly, the cells diluted in RPMI 10 and incubated in a humidified atmosphere of 5 ± 1% v/v CO2 in air. When the cells were growing well, subcultures were established in an appropriate number of flasks.
Additional strain characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
The mammalian liver post-mitochondrial fraction (S-9) used for metabolic activation were from male Sprague Dawley rats induced with Aroclor 1254
Test concentrations with justification for top dose:
In the cytotoxicity Range-Finder Experiment, six concentrations were tested in the absence and presence of S-9 ranging from 88.13 to 2820 µg/mL (equivalent to 10 mM at the highest concentration tested).

In Experiment 1 thirteen concentrations, ranging from 2.5 to 150 µg/mL in the absence of S-9 and from 25 to 400 µg/mL in the presence of S-9, were tested.
In Experiment 2 twelve concentrations, ranging from 5 to 80 µg/mL in the absence of S-9 and from 50 to 400 µg/mL in the presence of S-9, were tested.

Positive controls
4-nitroquinoline 1-oxide (NQO), stock solution: 0.015 and 0.020 mg/mL and final concentration: 0.15 and 0.20 µg/mL, no metabolic activation
Benzo[a]pyrene (B[a]P), stock solution: 0.200 and 0.300 mg/mL and final concentration: 2.00 and 3.00 µg/mL with metabolic activation
Vehicle:
DMSO diluted 100 fold in the treatment medium
Negative controls:
yes
Remarks:
DMSO diluted 100 fold in the treatment medium
Solvent controls:
no
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
benzo(a)pyrene
Remarks:
For positive control concentrations see test concentration section
Details on test system and conditions:
DURATION
- Preincubation period: Not applicable
- Exposure duration: 3-hour exposure followed by 7-day incubation period
Evaluation criteria:
For valid data, the test article was considered to induce forward mutation at the hprt locus in mouse lymphoma L5178Y cells if:
1. The mutant frequency at one or more concentrations was significantly greater than that of the negative control (p < 0.05).
2. There was a significant concentration relationship as indicated by the linear trend analysis (p < 0.05).
3. The effects described above were reproducible.
The test article was considered positive in this assay if all of the above criteria were met.
The test article was considered negative in this assay if none of the above criteria were met.
Statistics:
Not applicable
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
yes
Vehicle controls valid:
not applicable
Negative controls valid:
yes
Positive controls valid:
yes
Additional information on results:
In the cytotoxicity Range-Finder Experiment, six concentrations were tested in the absence and presence of S-9 ranging from 88.13 to 2820 µg/mL (equivalent to 10 mM at the highest concentration tested). The highest concentration to provide >10% relative survival (RS) in the presence of S-9 was 176.3 µg/mL, which gave 96% RS. Extreme or complete toxicity was observed at the remaining higher concentrations analysed in the presence of S-9 (352.5 to 705 µg/mL). In the absence of S-9, complete toxicity (0% RS) was observed at all concentrations analysed (88.13 to 705 µg/mL).
Following consultation with the Study Monitor, an extended range of closely spaced concentrations was tested in the absence of S-9 in Experiment 1.
In Experiment 1, thirteen concentrations, ranging from 2.5 to 150 µg/mL in the absence of S-9 and from 25 to 400 µg/mL in the presence of S-9, were tested. Seven days after treatment, the highest concentrations analysed to determine viability and 6TG resistance were 40 µg/mL in the absence of S-9 and 330 µg/mL in the presence of S-9, limited by toxicity, which gave 17% and 12% RS, respectively.
In Experiment 2, twelve concentrations, ranging from 5 to 80 µg/mL in the absence of S-9 and from 50 to 400 µg/mL in the presence of S-9, were tested. The highest concentrations analysed to determine viability and 6TG resistance were 30 µg/mL in the absence of S-9 and 270 µg/mL in the presence of S-9, which gave 14% and 12% RS, respectively.
In both Experiments 1 and 2, no statistically significant increases in mutant frequency were observed following treatment with 1,10-decanediol diacrylate at any concentration tested, in the absence or presence of S-9, and there were no significant linear trends. Mutant frequencies in vehicle control cultures fell within acceptable ranges and clear increases in mutation were induced by the positive control chemicals. The study was therefore accepted as valid.

Table 1: %RS Values –Range-Finder Experiment

Treatment

(µg/mL)

-S-9

% RS

+S-9

% RS

0

100

100

88.13

0

122

176.3

0

96

352.5 P

0

3

705.0 P, PP

0

0

1410 P, PP

NP

NP

2820 P, PP

NP

NP

P Precipitation observed at time of treatment

PP Precipitation observed following treatment incubation period

NP Not plated

 

Table 2: Summary of Mutation Data

Experiment 1 (3-hour treatment in the absence and presence of S-9)

Treatment

(mg/mL)

-S-9

Treatment

(mg/mL)

+S-9

%RS

MF§

%RS

MF§

0

100

5.38

0

100

2.79

2.5

103

1.29

NS

50

101

2.39

NS

5

101

2.34

NS

100

77

1.71

NS

10

73

3.45

NS

150

$$, P

51

(4.76)

20

59

2.46

NS

200

P

39

2.45

NS

30

31

2.91

NS

230

P

29

4.18

NS

40

17

4.28

NS

250

P

22

4.00

NS

270

$$, P

18

(6.36)

300

P

10

2.18

NS

330

P

12

1.32

NS

Linear trend

NS

Linear trend

NS

NQO

B[a]P

0.15

63

31.36

2

79

21.52

0.2

43

47.63

3

57

56.34

 

Experiment 2 (3-hour treatment in the absence and presence of S-9)

Treatment

(µg/mL)

-S-9

Treatment

(µg/mL)

+S-9

%RS

MF§

%RS

MF§

0

100

3.31

0

100

2.06

5

78

3.20

NS

50

92

5.28

NS

7.5

76

1.52

NS

75

84

1.55

NS

10

64

1.86

NS

100

65

2.83

NS

20

31

1.88

NS

150

43

2.67

NS

25

18

2.15

NS

200

23

3.36

NS

30

14

3.01

NS

250

P

14

3.23

NS

270

P

12

2.00

NS

Linear trend

NS

Linear trend

NS

NQO

B[a]P

0.15

71

54.02

2

58

23.88

0.2

61

47.44

3

22

53.43

§ 6-TG resistant mutants/106viable cells 7 days after treatment

%RS Percent relative survival adjusted by post treatment cell counts

$$ Treatment excluded from analysis due to excessive heterogeneity for mutation

Data in parentheses indicates marked heterogeneity observed

P Precipitation noted at time of treatment only

NS Not significant

 

Conclusions:
It is concluded that 1,10-decanediol diacrylate did not induce mutation at the hprt locus of L5178Y mouse lymphoma cells when tested up to toxic concentrations in two independent experiments, in the absence and presence of a rat liver metabolising system (S-9).
Executive summary:

1,10-decanediol diacrylate was assayed for the ability to induce mutation at the hypoxanthine-guanine phosphoribosyl transferase (hprt) locus (6-thioguanine [6TG] resistance) in mouse lymphoma cells using a fluctuation protocol. The study consisted of a cytotoxicity Range-Finder Experiment followed by two independent experiments, each conducted in the absence and presence of metabolic activation by an Aroclor 1254-induced rat liver post-mitochondrial fraction (S-9). The test article was formulated in anhydrous analytical grade dimethyl sulphoxide (DMSO).

A 3 hour treatment incubation period was used for all experiments.

In the cytotoxicity Range-Finder Experiment, six concentrations were tested in the absence and presence of S-9, ranging from 88.13 to 2820 µg/mL (equivalent to 10 mM at the highest concentration tested). The highest concentration to provide >10% relative survival (RS) in the presence of S-9 was 176.3 µg/mL, which gave 96% RS. Extreme or complete toxicity was observed at the remaining higher concentrations analysed in the presence of S-9 (352.5 to 705 µg/mL). In the absence of S-9 complete toxicity (0% RS) was observed at all concentrations analysed (88.13 to 705 µg/mL). Following consultation with the Study Monitor, an extended range of closely spaced concentrations was tested in the absence of S-9 in Experiment 1.

In Experiment 1 thirteen concentrations, ranging from 2.5 to 150 µg/mL in the absence of S-9 and from 25 to 400 µg/mL in the presence of S-9, were tested. Seven days after treatment, the highest concentrations analysed to determine viability and 6TG resistance were 40mg/µL in the absence of S-9 and 330 µg/mL in the presence of S-9, limited by toxicity, which gave 17% and 12% RS, respectively.

In Experiment 2 twelve concentrations, ranging from 5 to 80 µg/mL in the absence of S-9 and from 50 to 400 µg/mL in the presence of S-9, were tested.The highest concentrations analysed to determine viability and 6TG resistance were 30 µg/mL in the absence of S-9 and 270 µg/mL in the presence of S-9, which gave 14% and 12% RS, respectively.

Negative (vehicle) and positive control treatments were included in each Mutation Experiment in the absence and presence of S-9. Mutant frequencies (MF) in vehicle control cultures fell within acceptable ranges and clear increases in mutation were induced by the positive control chemicals 4-nitroquinoline 1-oxide (NQO) (without S-9) and benzo(a)pyrene (B[a]P) (with S-9). Therefore the study was accepted as valid.

In Experiments 1 and 2 no statistically significant increases in MF were observed following treatment with1,10-decanediol diacrylate at any concentration tested in the absence and presence of S-9 and there were no significant linear trends.

It is concluded that 1,10-decanediol diacrylate did not induce mutation at the hprt locus of L5178Y mouse lymphoma cells when tested up to toxic concentrations in two independent experiments, in the absence and presence of a rat liver metabolising system (S-9).

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

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Justification for classification or non-classification

Based on the negative results in all three regulatory in vitro genotoxicity tests, no classification for 1,10-decanediyl bis methacrylate is required for genotoxicity according to the Regulation EC n°1272/2008.