Registration Dossier

Diss Factsheets

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Link to relevant study records
Reference
Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
from 2010-04-26 to 2010-08-23
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was performed according to the OECD guideline (No. 471) and is in compliance to the GLP.
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to other study
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay
Target gene:
Each strain derived from S. typhimurium LT2 contains one mutation in the histine operon, resulting in a requirement for histidine.
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell type (if applicable):
Not applicable
Additional strain / cell type characteristics:
other: additional mutations in rfa and uvrB genes. For the strains TA98 and TA100 presence of an additional plasmid pKM101 in order to enhance their sensitivity of detection of some mutagens. See Table 1
Species / strain / cell type:
S. typhimurium TA 102
Details on mammalian cell type (if applicable):
Not applicable
Additional strain / cell type characteristics:
other: mutated in rfa gene and presence of an additional plasmid pKM101 in order to enhance the sensitivity of detection of some mutagens. See Table 1
Metabolic activation:
with and without
Metabolic activation system:
S9 fraction was purchased from Moltox (Molecular Toxicology, INC, Boone, NC 28607, USA) and obtained from the liver of rats treated with Aroclor 1254 (500 mg/kg) by the intraperitoneal route.
Test concentrations with justification for top dose:
Preliminary test: 10, 100, 500, 1000, 2500, 5000 µg/plate with and without S9 mix.
Mutagenicity experiments: first experiment = 156.3 to 5000 µg/plate without and with S9 mix for all strains. Second experiment = 156.3 to 5000 µg/plate without S9 mix for all strains; 156.3 to 5000 µg/plate with S9 mix for TA1535, TA1537, TA98 and TA102; 78.1 to 2500 µg/plate with S9 mix for TA 100. See details in table 2.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Ethanol, batch Nos. V8F771168F and V0F701170F (Carlo Erba, Val de Reuil, France).
- Justification for choice of solvent/vehicle: During the solubility assay, the test item was found freely soluble in the vehicle (Ethanol) at 200 mg/mL. Consequently, with a treatment volume of 25 µL/plate, the highest dose-level to be tested in the preliminary test was 5000 µg/plate, which is the highest dose-level recommended in the international guidelines. Successive dilutions of the stock preparation were also performed in Ethanol and used with a treatment volume of 25 µL/plate. Therefore the dose-levels to be tested in the preliminary test were 10, 100, 500, 1000, 2500 and 5000 µg/plate.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
Ethanol
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: sodium azide, 9-Aminoacridine, 2-Nitrofluorene, Mitomycin C for without S9 mix efficacy control. 2-Anthramine for with S9 mix efficacy control. See details in Table 3
Remarks:
no remarks
Details on test system and experimental conditions:
METHOD OF APPLICATION:
direct plate incorporation: test item solution (0.025 mL), S9 mix when required or phosphate buffer pH 7.4 (0.5 mL) and bacterial suspension (0.1 mL) were mixed with 2 mL of overlay agar (containing traces of the relevant aminoacid and biotin and maintained at 45°C). After rapid homogenization, the mixture was overlaid onto a Petri plate containing minimum medium.
or preincubation method: test item solution (0.025 mL), S9 mix (0.5 mL) and the bacterial suspension (0.1 mL) were incubated for 60 minutes at 37°C, under shaking, before adding the overlay agar and pouring onto the surface of a minimum agar plate.
DURATION
- Preincubation period: 60 min at 37°C
- Exposure duration: 48 or 72 hours
SELECTION AGENT (mutation assays): not applicable
SPINDLE INHIBITOR (cytogenetic assays): not applicable
STAIN (for cytogenetic assays): not applicable
NUMBER OF REPLICATIONS: 3 plates/dose/strain. For the mutagenicity experiments, two independent experiments were performed.
NUMBER OF CELLS EVALUATED: not applicable
DETERMINATION OF CYTOTOXICITY
- Method: observation of the decrease in the number of revertant colonies and/or thinning of the bacterial lawn
OTHER EXAMINATIONS:
- Determination of polyploidy: not required
- Determination of endoreplication: not required

OTHER: All the Petri dishes obtained were placed in a sealed jar using one jar for each dose-level tested, one jar for the vehicle control and another jar for the positive controls. The jars were then incubated at 37°C. The precaution of using jars in this study was due to the volatile characteristic of the test item and to limit the oxidation of the test item.
The revertants were scored with an automatic counter (Cardinal counter, Perceptive Instruments, Suffolk CB9 7 BN, UK). Manual counting was used as needed.


Evaluation criteria:
A reproducible 2-fold increase (for the TA 98, TA 100 and TA 102 strains) or 3-fold increase (for the TA 1535 and TA 1537 strains) in the number of revertants compared with the vehicle controls, in any strain at any dose-level and/or evidence of a dose-relationship was considered as a positive result. Reference to historical data, or other considerations of biological relevance may also be taken into account in the evaluation of the data obtained.
Statistics:
no data
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
See tables 4; 5; 6; 7
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Remarks:
See tables 4; 5; 6; 7
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
Remarks:
See tables 4; 5; 6; 7
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
See tables 4; 5; 6; 7
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
Remarks:
See tables 4; 5; 6; 7
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Remarks:
See tables 4; 5; 6; 7
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 100
Metabolic activation:
without
Genotoxicity:
negative
Remarks:
See tables 4; 6
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Remarks:
See tables 4; 6
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with
Genotoxicity:
negative
Remarks:
See tables 5; 7
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
only according to the pre-incubation method. See tables 5; 7
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
See tables 4; 5; 6; 7
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Remarks:
See tables 4; 5; 6; 7
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS: not applicable
RANGE-FINDING/SCREENING STUDIES: A first preliminary test was performed testing six dose-levels of the test item (one plate/dose-level) with the TA 98, TA 100 and TA 102 strains, with and without S9 mix. This preliminary test was invalidated. Indeed, the test item used in this test came from a small aliquot taken from one of the flask and stored at +4°C, protected from light and humidity and under nitrogen gas. This conditioning in an inert atmosphere was performed using a layer of parafilm to seal the vessel (as described in CIT’s SOPs). But there were some evidences that this layer of parafilm got deteriorated (most probably by the test item’s emanation) contaminating the aliquot of test item. Because of this contamination, the results of this first preliminary test were invalidated and the study was started all over again using one supplied flask per treatment and avoiding the use of parafilm.
The second preliminary test was performed testing six dose-levels of the test item (two plates/dose-level) with the TA 98, TA 100 and TA 102 strains, with and without S9 mix. No precipitate was observed in the Petri plates when scoring the revertants at any of the tested dose-levels. A strong toxicity (observation of a strong thinning of the bacterial lawn) was observed at dose levels 5000 µg/plate with the TA 98 strain without S9 mix and with the TA 100 strain with S9 mix. No noteworthy toxicity was noted in the other tested strains up to the highest dose level of 5000 µg/plate. Since the test item was freely soluble and only toxic in the preliminary test at the highest dose level recommended in the international guidelines, the highest dose-level to be used in the main mutagenicity experiments was 5000 µg/plate, according to the criteria specified in the international guidelines.
COMPARISON WITH HISTORICAL CONTROL DATA: The numbers of revertants for the vehicle and positive controls were as specified in the acceptance criteria. The study was therefore considered valid.
ADDITIONAL INFORMATION ON CYTOTOXICITY: In the first experiment without S9 mix, a moderate toxicity was noted at dose-levels 1250 µg/plate and above in the TA 100 strain and a moderate to strong toxicity was noted at 5000 µg/plate in the TA 1537 and TA 98 strains (see table 4).
In the second experiment without S9 mix, a moderate toxicity was noted at dose-level 1250 µg/plate and above in the TA 100 strain and at dose-levels 5000 µg/plate in TA98 strain, and a moderate to strong toxicity was noted at dose level 2500 µg/plate and above in the TA1537 strain (see table 6).
In the first experiment with S9 mix (direct plate incorporation), a moderate toxicity was noted at 5000 µg/plate in the TA 98 strain (see table 5).
In the second experiment with S9 mix (preincubation method), a moderate to strong toxicity was noted at dose-level 625 µg/plate and above in the TA 100 strain and from 1250 µg/plate in the TA1537 strain, and a moderate toxicity was noted at dose-level 2500 µg/plate and above in the TA 1535 strain and at 5000 µg/plate in the TA 98 and TA 102 strains (see table 7).
Remarks on result:
other: strain/cell type:
Remarks:
Migrated from field 'Test system'.

Table 4: Number of revertants per plate in the absence of metabolic activation in the first test (direct plate incorporation method)

Dimethyloctadiene Concentration
(µg/plate)

TA 1535

TA1537

TA 98

TA 100

TA 102

Mean$

Standard deviation

Mean$

Standard deviation

Mean$

Standard deviation

Mean$

Standard deviation

Mean$

Standard deviation

0*

23

7

10

3

34

4

142

46

295

9

156.3

16

3

6

1

34

22

108

15

346

41

312.5

19

4

15

8

39

7

123

32

310

29

625

14

5

9

3

51

13

99

45

343

65

1250

23

10

10

2

41

6

127Mt

68

219

20

2500

22

6

5

1

46

24

165Mt

51

243

37

5000

19

2

9St

4

32St

12

128Mt

12

346

42

Positive control**

530

98

377

92

155

3

507

61

1877

210

$: Mean of triplicate

*Solvent control = negative control: ethanol

**Mutagens positive controls:

- NaN3(1 µg/plate) in TA1535 and TA100 strains

- 9AA (50 µg/plate) in TA1537 strain

- 2NF (0.5 µg/plate) in TA 98 strain

- MMC ( 0.5 µg/plate) in TA 102 strain

Mt : Moderate cytotoxicity

St : Strong cytotoxicity

 

 

 

Table 5:Number of revertants per plate in the presence of metabolic activation (S9 mix) in the first test (direct plate incorporation method) 

Dimethyloctadiene Concentration
(µg/plate)

TA 1535

TA1537

TA 98

TA 100

TA 102

Mean$

Standard deviation

Mean$

Standard deviation

Mean$

Standard deviation

Mean$

Standard deviation

Mean$

Standard deviation

0*

22

3

12

2

41

16

150

58

400

85

156.3

24

5

12

2

28

13

123

20

471

21

312.5

25

4

10

6

49

16

159

48

507

172

625

21

9

10

3

54

5

146

22

468

195

1250

18

7

17

7

40

8

163

33

672

56

2500

19

3

9

2

50

6

132

12

614

96

5000

23

6

8

5

55Mt

13

138

17

596

142

Positive control**

256

69

85

17

577

53

1560

308

958

30

$: Mean of triplicate

*Solvent control = negative control: ethanol

**Mutagens positive controls:

- 2AM (2µg/plate) in TA1535, TA1537, TA98

- 2AM (10µg/plate) in TA102 strain

- BAP (5µg/plate) in TA100 strain

Mt : Moderate cytotoxicity

St : Strong cytotoxicity

 

 

Table 6:Number of revertants per plate in the absence of metabolic activation in the second test (direct plate incorporation method)

Dimethyloctadiene Concentration
(µg/plate)

TA 1535

TA1537

TA 98

TA 100

TA 102

Mean

Standard deviation

Mean

Standard deviation

Mean

Standard deviation

Mean

Standard deviation

Mean

Standard deviation

0*

24

8

10

6

28

2

95

18

452

100

156.3

21

3

7

3

19

2

67

8

463

14

312.5

15

4

9

2

17

12

69

10

474

50

625

16

5

11

5

20

0

71

20

512

19

1250

20

6

8

4

15

5

76Mt

5

512

42

2500

28

17

8Mt

5

19

8

73Mt

15

487

31

5000

23

5

8St

5

18Mt

6

76Mt

35

455

23

Positive control**

745

103

242

55

116

10

815

20

1999

125

$: Mean of triplicate

*Solvent control = negative control: ethanol

- NaN3(1 µg/plate) in TA1535 and TA100 strains

- 9AA (50 µg/plate) in TA1537 strain

- 2NF (0.5 µg/plate) in TA 98 strain

- MMC ( 0.5 µg/plate) in TA 102 strain

Mt : Moderate cytotoxicity

St : Strong cytotoxicity

 

 

Table 7: Number of revertants per plate in the presence of metabolic activation (S9 mix) in the second test (pre-incubation method)

 

TFE Concentration
(µg/plate)

TA 1535

TA1537

TA 98

TA 100

TA 102

Mean

Standard deviation

Mean

Standard deviation

Mean

Standard deviation

Mean

Standard deviation

Mean

Standard deviation

0*

23

2

9

3

37

9

130

19

476

84

78.1

-

-

-

-

-

-

117

3

-

-

156.3

29

6

9

2

29

4

120

16

437

159

312.5

24

7

11

2

38

3

95

11

350

57

625

26

5

10

7

32

3

88Mt

3

408

56

1250

19

8

9Mt

2

31

5

69St

19

412

96

2500

17Mt

4

8St

3

31

5

82St

23

503

25

5000

21Mt

10

9St

2

35Mt

4

-

-

352Mt

75

Positive control**

139

17

159

36

1497

169

390

34

3418

605

$: Mean of triplicate

*Solvent control = negative control: ethanol

**Mutagens positive controls:

- 2AM (2µg/plate) in TA1535, TA1537, TA98 strains

- 2AM (10µg/plate) in TA102 strain

- BAP (5 µg/plate) in TA100 strain

Mt : Moderate cytotoxicity

St : Strong cytotoxicity

Conclusions:
Interpretation of results (migrated information):
negative with or without metabolic activation

Under the test conditions, the test item dimethyloctadiene did not show mutagenic activity in the bacterial reverse mutation test with Salmonella typhimurium strains. Dimethyloctadiene is not considered as mutagenic in this bacterial system according to the criteria of the CLP Regulation (EC) n° 1272/2008 and according to the Annex VI of Directive 67/548/EEC.
Executive summary:

In a reverse gene mutation assay in bacteria performed according to the OECD No.471 and EC No.B13/14 guidelines, dimethyloctadiene (purity of 91.1%) diluted in ethanol was tested in S. typhimurium TA1535, TA1537, TA100, TA98 and TA102 in the presence and the absence of mammalian metabolic activation (S9) using the direct plate incorporation or the preincubation method. All the concentrations and dose-levels were expressed as active item, taking into account the purity of the test item (91.1%).

Due to the volatile characteristic of the test item and in order to limit the oxidation of the test item, all the Petri dishes were placed in a sealed jar. One jar was used for each tested dose-level, one jar was used for the vehicle control and another jar for the positive controls. 

Six known mutagens, dissolved in dimethylsulfoxide (except for Mitomycin C which was dissolved in distilled water), were used to check the sensitivity of the test system. The positive controls induced the appropriate responses in the corresponding strains. The number of revertants in the vehicle controls was consistent with the historical data of the testing facility, and the number of revertants in the positive controls was higher than that of the vehicle controls (at least 2-fold increase for the TA 98, TA 100 and TA 102 strains and at least 3-fold increase for the TA 1535 and TA 1537 strains) and was consistent with the historical data of the testing facility. Therefore the study was considered valid.

During the preliminary test, a strong thinning of the bacterial lawn was observed at the highest recommended dose level (5000 µg/plate) in TA98 without metabolic activation and in TA100 with metabolic activation. Since the test item was freely soluble and only toxic in the preliminary test at the highest dose‑level recommended in the international guidelines, the highest dose-level to be used in the main mutagenicity experiments was 5000 µg/plate.

During the main tests, no induced revertant over background was observed in any strains of S. typhimurium whereas the cytotoxic dose-level was reached or the test was conducted up to the highest recommended dose-level (5000 µg/plate).

 

Therefore, under the test conditions, dimethyloctadiene did not show any mutagenic activity in the bacterial reverse mutation test using Salmonella typhymurium strains. Dimethytloctadiene is not considered as mutagenic according to the criteria of the CLP Regulation (EC) n° 1272/2008 and according to the Annex VI of Directive 67/548/EEC.

Additional information

A study was identified as a key study (Sarlang, 2010). In a GLP reverse gene mutation assay in bacteria, performed according to the OECD No.471 and EC No.B13/14 guidelines, Dimethyloctadiene was tested in S. typhimurium TA1535, TA1537, TA100, TA98 and TA102 in the presence and the absence of mammalian metabolic activation (S9) using the direct plate incorporation or the preincubation method. During the main tests, no induced revertant over background was observed in any strains whereas the cytotoxic dose-level was reached or the test was conducted up to the highest recommended dose-level (5000 µg/plate). Therefore, under the test conditions, dimethyloctadiene did not show any mutagenic activity in the bacterial reverse mutation test using Salmonella typhimurium strains.


Short description of key information:
Bacterial Reverse Mutation assay (Ames test, OECD 471): negative (Kr. 1)

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

In a GLP reverse gene mutation assay in bacteria, performed according to the OECD No.471 and EC No.B13/14 guidelines, dimethyloctadiene was negative in S. typhimurium TA1535, TA1537, TA100, TA98 and TA102 with and without metabolic activation therefore dimethyloctadiene does not need to be classified as mutagenic according to the criteria of the CLP Regulation (EC) n° 1272 /2008 and according to the annex VI of Directive 67/548/EEC.

Categories Display