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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Read-across to DOT bis-(ethylmaleate) (CAS 68109-88-6)

The test material was concluded to be non-mutagenic under the conditions of the test.

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:
17 May 2012 to 03 July 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose for cross-reference:
other: read-across target
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
Qualifier:
according to guideline
Guideline:
other: guidelines published by the Japanese Regulatory Authorities, including METI, MHLW and MAFF.
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: USA, EPA (TSCA) OPPTS harmonised guidelines.
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay
Target gene:
Histidine requirement in the Salmonella typhimurium strains.
Tryptophan requirement in the Escherichia coli strain.
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):
- Type and identity of media: Stock cultures were prepared in Oxoid nutrient broth.
- Properly maintained: yes. Stored at approximately -196 °C in a liquid nitrogen freezer. Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory).
Additional strain / cell type characteristics:
other: S. typhimurium: all strains possess rfa- and uvrB-; TA98 and TA100 also possess the R-factor plasmid pKM101. E. coli strain possesses the uvrA- mutation.
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
Preliminary Toxicity Test
0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate

Mutation Test - Experiments 1 and 2
0, 50, 150, 500, 1500 and 5000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: acetone
- Justification for choice of solvent/vehicle: the test material was insoluble in dimethyl sulphoxide at 50 mg/mL; it was soluble in dimethyl formamide and acetonitrile at 50 mg/mL, acetone at 100 mg/mL and tetrahydrofuran at 200 mg/mL. Acetone was selected as the most appropriate vehicle.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
N-ethyl-N-nitro-N-nitrosoguanidine
benzo(a)pyrene
other: 2-aminoanthracene
Details on test system and experimental conditions:
- EXPERIMENT 1
METHOD OF APPLICATION: in agar (plate incorporation)
2 mL molten top agar (0.6 % agar, 0.5 % NaCl with 5 mL of 1.0 mM histidine and 1.0mM biotin for Salmonella typhimurium or 1.0 mM tryptophan solution for E. coli), 0.1 mL of culture of the appropriate strain, 0.1 mL of the appropriate test material solution or the vehicle or positive control substance and 0.5 mL S9-mix (for the plates with metabolic activation) or 0.5 mL phosphate buffer (for the plates without metabolic activation) were thoroughly mixed and the mix was immediately poured onto the surface of Vogel-Bonner Minimal agar plates.

DURATION
- Exposure duration: 48 hours at 37 °C

NUMBER OF REPLICATIONS: The tests were performed in triplicate


- EXPERIMENT 2
METHOD OF APPLICATION: pre-incubation
0.1 mL of the appropriate bacterial culture was dispensed into a test tube followed by 0.5 mL of S9 mix or phosphate buffer and 0.05 mL of the vehicle or test material formulation and incubated for 20 minutes at 37 °C with shaking at approximately 130 rpm prior to the addition of 2 mL of molten, trace histidine or tryptophan supplemented top agar. The contents of the tube were then mixed and equally distributed on the surface of Vogel-Bonner Minimal agar plates.

DURATION
- Exposure duration: 48 hours at 37 °C

NUMBER OF REPLICATIONS: The tests were performed in triplicate


DETERMINATION OF CYTOTOXICITY
- Method: Cytotoxicity was assessed as a reduction in the number of revertant colonies and/or a clearing of the background lawn of bacterial growth.
Evaluation criteria:
The mutagenicity study was considered valid if the mean colony counts of the control plates were within acceptable ranges and if the results of the positive controls met the criteria for a positive response. Furthermore, all tester strains must have demonstrated the required characteristics as determined by their respective strain checks. There should be a minimum of four non-toxic test material dose levels and no evidence of excessive contamination.

A test material was considered to be positive if the increase in the mean number of revertant colonies on the test plates was concentration-related or if a reproducible two-fold or more increase was observed compared to that of negative control plates.
A test material was considered to be negative in the bacterial gene mutation test if it produced neither a dose-related increase in the mean number of revertant colonies nor a reproducible positive response at any of the test points.
Key result
Species / strain:
S. typhimurium, other: TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
PRE-STUDY CHECKS
The amino acid supplemented top agar and S9-mix used in both experiments was shown to be sterile. The culture density for each bacterial strain was also checked and considered acceptable.

PRELIMINARY TOXICITY TEST
The test material was non-toxic to the strains of bacteria used (TA 100 and WP2uvrA). The test material formulation and S9-mix used in the experiment were both shown to be sterile.

MUTATION TEST
The test material caused no visible reduction in the growth of the bacterial background lawn at any dose level and was therefore tested up to the maximum recommended dose level of 5000 µg/plate. A precipitate (creamy and particulate in appearance) was noted at and above 1500 µg/plate, this observation did not prevent the scoring of revertant colonies.

No toxicologically significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation or exposure method. Small statistically significant increases in WP2uvrA revertant colony frequency were observed in the presence of S9-mix at 50 and 5000 µg/plate in Experiment 2. These increases were considered to be of no biological relevance because there was no evidence of a dose-response relationship or reproducibility. Furthermore, the individual revertant counts at 50 and 5000 µg/plate were within the in-house historical untreated/vehicle control range for the tester strain and the fold increase was only 1.3 times the concurrent vehicle control.

Results for the negative controls were considered to be acceptable. Furthermore, all of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies thus confirming the activity of the S9-mix and the sensitivity of the bacterial strains.

Table 1: Experiment 1 - Mean Number of Revertants

Dose (µg/plate)

 

TA100

TA1535

WP2uvrA

TA98

TA1537

-S9 Mix

+S9 Mix

-S9 Mix

+S9 Mix

-S9 Mix

+S9 Mix

-S9 Mix

+S9 Mix

-S9 Mix

+S9 Mix

0

Mean

115

118

24

12

38

51

35

36

13

12

SD

13.3

14.6

4.5

0.0

4.6

6.4

5.3

6.1

4.4

3.5

50

Mean

103

103

20

9

42

47

32

31

9

12

SD

6.1

8.1

1.0

2.0

1.7

6.2

7.5

2.0

5.9

1.2

150

Mean

112

113

22

10

43

45

24

27

8

12

SD

0.6

2.1

1.7

4.2

4.6

5.5

1.2

6.7

1.0

0.6

500

Mean

99

99

22

12

31

36

35

28

13

8

SD

4.5

4.5

2.6

1.0

5.2

8.5

3.8

10.3

6.0

5.0

1500

Mean

108 P

107 P

23 P

11 P

42 P

41 P

30 P

30 P

12 P

8 P

SD

1.7

4.0

3.5

1.7

5.6

10.5

10.5

4.0

8.0

6.4

5000

Mean

96 P

75 P

22 P

11 P

35 P

41 P

30 P

34 P

10 P

11 P

SD

8.1

6.2

1.5

2.0

3.6

6.1

3.2

2.5

1.2

4.5

Positive Control

Substance (µg/plate)

ENNG

3

2AA

1

ENNG

5

2AA

2

ENNG

2

2AA

10

4NQO

0.2

BP

5

9AA

80

2AA

2

Mean

409

1513

141

288

550

427

137

195

561

278

SD

82.4

77.9

9.9

24.1

28.1

19.4

12.7

3.1

202.6

3.1

 

Table 2: Experiment 2 - Mean Number of Revertants

Dose (µg/plate)

 

TA100

TA1535

WP2uvrA

TA98

TA1537

-S9 Mix

+S9 Mix

-S9 Mix

+S9 Mix

-S9 Mix

+S9 Mix

-S9 Mix

+S9 Mix

-S9 Mix

+S9 Mix

0

Mean

110

99

22

11

40

39

19

31

12

10

SD

10.6

20.1

4.9

3.8

9.0

6.7

4.0

10.2

4.0

5.2

50

Mean

97

106

23

12

45

50*

19

32

8

12

SD

2.9

10.1

0.6

3.1

0.0

4.6

4.0

7.0

4.6

7.2

150

Mean

100

112

20

10

35

46

30

31

13

8

SD

12.9

11.6

4.6

1.2

4.0

4.2

7.6

6.8

5.5

1.0

500

Mean

108

103

21

15

43

48

16

28

10

11

SD

20.3

5.0

2.0

4.6

11.6

7.0

4.6

4.5

4.0

3.2

1500

Mean

104 P

105 P

14 P

12 P

33 P

46 P

20 P

20 P

12 P

14 P

SD

3.8

11.7

6.6

0.0

2.1

2.1

2.3

1.2

0.0

2.1

5000

Mean

103 P

91 P

20 P

9 P

32 P

52 P*

15 P

24 P

17 P

8 P

SD

14.2

0.6

3.6

2.6

3.0

4.0

8.5

7.8

1.7

3.1

Positive Control

Substance (µg/plate)

ENNG

3

2AA

1

ENNG

5

2AA

2

ENNG

2

2AA

10

4NQO

0.2

BP

5

9AA

80

2AA

2

Mean

466

1474

180

280

596

341

129

669

463

236

SD

24.9

187.6

48.6

29.3

47.4

31.9

17.1

41.4

87.2

19.7

ENNG = N-ethyl-N'-nitro-N-nitrosoguanidine

4NQO = 4 -nitroquinoline-1 -oxide

9AA = 9 -aminoacridine

2AA = 2 -aminoanthracene

BP = benzo(a)pyrene

P = precipitate

* = p ≤ 0.05

SD = standard deviation

Conclusions:
Under the conditions of this study, the test material was not mutagenic to S. typhimurium strains TA1535, TA1537, TA98 and TA100 and in the E. coli strain WP2uvrA in both the absence and presence of metabolic activation.
Executive summary:

The mutagenicity of the test material was assessed in a bacterial reverse mutation assay (Ames Test) in accordance with standardised guidelines OECD guideline 471 and EU Method B.13/14. During the study, Salmonella typhimurium strains TA1535, TA1537, TA98, TA100 and Escherichia coli strain WP2uvrA were treated with the test material using both the plate incorporation and pre-incubation methods at five dose levels, in triplicate, both with and without metabolic activation. The dose range for the study was determined in a preliminary toxicity assay and was 50 to 5000 µg/plate. Under the conditions of the study, the vehicle control plates gave counts of revertant colonies generally within the normal range. All positive controls used in the test induced marked increases in the frequency of revertant colonies, both with and without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

The test material caused no visible reduction in the growth of the bacterial background lawn at any dose level and was, therefore, tested up to the maximum recommended dose level of 5000 µg/plate. A test material precipitate (creamy and particulate in appearance) was noted at and above 1500 µg/plate, though this observation did not prevent the scoring of revertant colonies. No toxicologically significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation or exposure method. Small, statistically significant increases in WP2uvrA revertant colony frequency were observed in the presence of S9-mix at 50 and 5000 µg/plate in Experiment 2. These increases were considered to be of no biological relevance because there was no evidence of a dose-response relationship or reproducibility. Furthermore, the individual revertant counts at 50 and 5000 µg/plate were within the in-house historical untreated/vehicle control range for the tester strain and the fold increase was only 1.3 times the concurrent vehicle control.

Therefore, the test material was concluded to be non-mutagenic under the conditions of the test.

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study conducted on read-across material
Justification for type of information:
Read-across to DOT bis-(ethylmaleate) (CAS 68109-88-6), see attached justification.
Reason / purpose for cross-reference:
read-across source
Key result
Species / strain:
S. typhimurium, other: TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Thompson 2012 (Read-Across Study performed on structurally similar substance DOT bis-(ethylmatelate) (CAS 68109-88-6)

The mutagenicity of the test material was assessed in a bacterial reverse mutation assay (Ames Test) in accordance with standardised guidelines OECD guideline 471 and EU Method B.13/14. During the study, Salmonella typhimurium strains TA1535, TA1537, TA98, TA100 and Escherichia coli strain WP2uvrA were treated with the test material using both the plate incorporation and pre-incubation methods at five dose levels, in triplicate, both with and without metabolic activation. The dose range for the study was determined in a preliminary toxicity assay and was 50 to 5000 µg/plate. Under the conditions of the study, the vehicle control plates gave counts of revertant colonies generally within the normal range. All positive controls used in the test induced marked increases in the frequency of revertant colonies, both with and without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

The test material caused no visible reduction in the growth of the bacterial background lawn at any dose level and was, therefore, tested up to the maximum recommended dose level of 5000 µg/plate. A test material precipitate (creamy and particulate in appearance) was noted at and above 1500 µg/plate, though this observation did not prevent the scoring of revertant colonies. No toxicologically significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation or exposure method. Small, statistically significant increases in WP2uvrA revertant colony frequency were observed in the presence of S9-mix at 50 and 5000 µg/plate in Experiment 2. These increases were considered to be of no biological relevance because there was no evidence of a dose-response relationship or reproducibility. Furthermore, the individual revertant counts at 50 and 5000 µg/plate were within the in-house historical untreated/vehicle control range for the tester strain and the fold increase was only 1.3 times the concurrent vehicle control.

Therefore, the test material was concluded to be non-mutagenic under the conditions of the test.

Reverse Mutation: Read-across data (mixture DOTE: MOTE. 70:30) (CAS No 15571-58-1 and CAS 27107-89-7)

Read-across from DOTE can be justified by structural analogy (Dioctyltin and two thiogylocolate ligands).

- Anonymous (1979): An Ames test was carried out with a mixture of 70% dioctyltin bis(2-ethylhexylmercaptoacetate) and 30% mono-octyltin tris(2-ethylhexylmercaptoacetate). The relatively old test was considered less reliable, because the test was not conducted using an E. coli strain and no positive controls for strains TA98, TA100, and TA1537 were included in the test with metabolic activation. No mutagenic activity was observed.

-Anonymous (1978) Two studies:

The mutagenic potential of the test material was investigated in two bacterial reverse mutation assays.

Under the conditions of the first study, no mutagenic effects were seen for all strains tested in the presence of a  metabolic activation system. The test material was found to be toxic to the strain TA1537 at 10 and 20 µL/plate and to the strains TA1538 and D4 at 10 µL/plate.

Under the conditions of the second study the test material was positive in strain TA100 without S9.

- Anonmymous (1983): An Ames test was caried out with the test material. The test was considered less reliable due to the fact that the test was not conducted using E. coli strain WP2 uvrA. Positive controls for the strains TA98, TA1537, and TA1538 were not included in the test with metabolic activation. The test material precipitated in the soft agar at concentrations of 8100 and 24,300 µg/0.1 mL.  

In the tests conducted without microsomal activation, there was a slight increase in the number of back-mutant colonies of strain TA100 in the 2700 µg/0.1 mL concentration and above.  Additionally, a growth-inhibiting effect was reported in the tests without activation for strains TA1535, TA1537, and TA1538 at a  concentration of 24,300 µg/0.1 mL. In the first assay with microsomal activation, there was a slight increase in the number of back-mutant  colonies of strain TA1537 at 300 and 2700 µg/0.1 mL. The study authors attributed this to fluctuation in the rate of spontaneously occurring back-mutants.

Negative and positive controls produced the expected mutant colony counts.

Under the experimental conditions, the test material displayed weak mutagenic activity.

Justification for classification or non-classification

In accordance with the criteria for classification as defined in Annex I, Regulation (EC) No 1272/2008, the substance does not require classification with respect to genetic toxicity.