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Toxicological information

Genetic toxicity: in vitro

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Administrative data

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:
May 1980
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: study similar to OECD 471

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1980
Report date:
1980

Materials and methods

Test guideline
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
: only 4 of 5 recommended strains (TA1535, TA1537, TA98, TA100) were used in this study
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Reaction mass of dimethyl adipate and dimethyl glutarate and dimethyl succinate
EC Number:
906-170-0
Molecular formula:
CH3CO2(CH2)nCO2CH3 Where n = 2, 3 and 4
IUPAC Name:
Reaction mass of dimethyl adipate and dimethyl glutarate and dimethyl succinate
Test material form:
other: liquid
Details on test material:
See information in the field "Confidential details on test material"

Method

Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Metabolic activation system:
rat liver S9 mix
Test concentrations with justification for top dose:
0, 100, 500, 1000, 5000, 10000 µg/plate for all bacterial strains with and without S9
Vehicle / solvent:
DMSO
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene (all strains with S9), 9-aminoacridine (TA1537 without S9), N-methyl-N'-nitrosoguanidine (TA1535 and TA100 without S9), 2-nitrofluorene (TA98 without S9)
Details on test system and experimental conditions:
METHOD OF APPLICATION: as impregnation on paper disk (spot test in a closed system)

Experiments without S9 were performed by adding 10E8 bacteria (in 0.1 mL) to 2 mL of standard top agr, mixing immediately, and pouring on a Davis minimal agar plate. Experiments with S9 were performed in the same manner except that 0.5 mL of S9 mix was added before mixing.
A sterile disk saturated with test substance was placed in the center of one of two replicate plates. The plate with the disk was inverted and the other plate was stacked on top of it. Both plates were sealed in a plastic bag and incubated for 48 hours at 37°C.
Evaluation criteria:
A chemical was judged to be mutagenic or nonmutagenic in the assay after considering the various pieces of information provided by the statistical analysis
Statistics:
The data points (number of revertants/plate = x) were transformed prior to analysis using the power transformation y = xE0.2. Two analyses were performed. In the first analysis, the response observed at each concentration was compared to the control by a t-test of significance to determine which dose levels produced significant increases in mutation frequency. In the second analysis, the significance of the dose-response relationship was tested. Linear, quadratic, and higher order dose-response effects were tested in an F-test of significance. In addition, an analysis was conducted to determine whether the dose-response relationship was different in different trials.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Remarks:
up to the top concentration of 10000 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Remarks:
This strain was tested in another assay described in Groot 2019 endpoint of this dossier
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
In an intial cytotoxicity experiment on strain TA1535, DBE was tested at concentrations up to 10000 µg/plate and induced no toxicity.
No indication of mutagenic activity was observed in the spot test. The mutation frequencies were not significantly greater than te solvent control values and significant dose-response effects were not observed.

Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Table 1: Mutagenicity testing in strain TA1535

 

Test concentration (µg/plate)

Number of revertants per plate (duplicate plates)

Trial 1

Trial 2

Without metabolic activation

0 (DMSO)

21

32

24

34

100

37

32

31

33

500

35

38

29

32

1000

40

30

27

25

5000

31

36

32

37

10000

40

37

35

39

Positive control
(MNNG, 4 µg/plate)

1521

1876

700

1070

Without metabolic activation

0 (DMSO)

9

22

15

21

100

15

23

20

18

500

12

20

16

18

1000

14

25

9

14

5000

13

28

25

19

10000

19

25

24

17

Positive control
(2-AA, 10 µg/plate)

294

259

283

266

MNNG: N-methyl-N'-nitro-N-nistrosoguanidine2-AA: 2-aminoanthracene

Table 2: Mutagenicity testing in strain TA1537

 

Test concentration (µg/plate)

Number of revertants per plate (duplicate plates)

Trial 1

Trial 2

Without metabolic activation

0 (DMSO)

11

15

11

8

100

13

15

10

13

500

14

8

18

9

1000

13

7

7

14

5000

6

13

9

14

10000

13

12

12

11

Positive control
(9-AAC, 50 µg/plate)

580

411

383

357

Without metabolic activation

0 (DMSO)

12

11

10

9

100

8

12

10

13

500

11

9

11

9

1000

13

8

15

7

5000

9

10

12

8

10000

12

13

9

15

Positive control
(2-AA, 10 µg/plate)

328

228

176

172

9-AAC: 9-aminoacridine          2-AA: 2-aminoanthracene

Table 3: Mutagenicity testing in strain TA98

 

Test concentration (µg/plate)

Number of revertants per plate (duplicate plates)

Trial 1

Trial 2

Without metabolic activation

0 (DMSO)

31

34

37

32

100

26

22

28

30

500

20

27

30

26

1000

22

18

24

31

5000

26

23

30

31

10000

29

26

29

23

Positive control
(2-NF, 25 µg/plate)

2772

2624

2619

2388

Without metabolic activation

0 (DMSO)

47

41

43

39

100

37

42

45

48

500

39

35

41

48

1000

43

47

58

54

5000

40

36

47

38

10000

39

37

34

54

Positive control
(2-AA, 10 µg/plate)

1200

1363

1166

1114

2-NF: 2-nitrofluorene   2-AA: 2-aminoanthracene

Table 4: Mutagenicity testing in strain TA100

 

Test concentration (µg/plate)

Number of revertants per plate (duplicate plates)

Trial 1

Trial 2

Without metabolic activation

0 (DMSO)

115

112

143

104

100

129

114

141

143

500

130

124

113

129

1000

105

115

112

115

5000

116

90

136

105

10000

100

121

130

98

Positive control
(MNNG, 4 µg/plate)

1908

1989

1430

17995

Without metabolic activation

0 (DMSO)

146

127

143

172

100

120

115

139

114

500

146

117

130

138

1000

132

99

145

176

5000

117

114

126

136

10000

128

118

155

148

Positive control
(2-AA, 5 µg/plate)

1102

820

863

1012

MNNG: N-methyl-N'-nitro-N-nistrosoguanidine2-AA: 2-aminoanthracene

Applicant's summary and conclusion

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

DBE is not mutagenic in the Ames reverse mutation assay (spot test format) with strains TA1535, TA1537, TA98 and TA100 up to the test concentration of 10000 µg/plate.
Executive summary:

The mutagenic potential of DBE has been assessed in the Salmonella typhimurium Ames test according to OECD guideline n° 471 and EC guideline n° B13/14.

Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 were tested in presence and in absence of metabolic activation system from liver fraction of rats (S9 mix). The negative control was DMSO.

DBE was tested in 2 independent experiments performed according to the spot test method in which a sterile disk saturated with DBE was placed in the center of the plate.

Each strain of bacteria was exposed to dose-levels of 0, 100, 500, 1000, 5000 or 10000 µg/plate of the test item (two plates/dose-level). In each experiment, negative and positive controls were included using duplicate plates.

After 48 hours of incubation at 37°C in a sealed plastic bag, the number of revertant colonies per plate was scored in each experiment, for each strain and for each experimental point. The evaluation of the toxicity was performed on the basis of the observation of the decrease in the number of revertant colonies and/or thinning of the bacterial lawn.

No noteworthy toxicity was induced in an initial cytotoxicity experiment on TA1535.

Negative and positive controls responded adequately. The study was therefore considered valid.

The test substance DBE did not induce any noteworthy increase in the number of revertants which could be considered as relevant, either with or without S9 mix, in any of the 4 tester strains.

The test item did not demonstrate any in vitro mutagenic activity in this bacterial test system, therefore DBE is not classified according to Annex VI of the Directive 67/548/CEE and according to EU Regulation 1272/2008 (CLP).

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