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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:
6 to 27 April 1995
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline and GLP-compliant study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1995
Report Date:
1995

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Test material form:
solid: crystalline
Details on test material:
- Name of test material (as cited in study report): BEPD
- Physical state: colourless solid
- Analytical purity: 99.5%
- Batch number: HRC 28.3.95/9
- Storage conditions: at room temperature
- Stability: assumed stable for 6 months from receipt at HRC on 28 March 1995

Method

Target gene:
Reversion to histidine-independence
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:
Liver post-mitochondrial supernatant (S9 fraction) from male Sprague-Dawley rats induced with Aroclor 1254 prior to sacrifice. Before use, the S9 fraction was supplemented with appropriate co-factors.
Test concentrations with justification for top dose:
0 (ethanol), 50, 150, 500, 1500, 5000 µg/plate (-S9)
0 (ethanol), 50, 150, 500, 1500, 5000 µg/plate (+S9)
Vehicle / solvent:
Ethanol
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
ethanol
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: The positive control compounds used were 2-aminoanthracene (+S9); N-ethyl-N'-nitro-N-nitrosoguanidine, 2-nitrofluorene and 9-aminoacridine (-S9)
Details on test system and experimental conditions:
Triplicate cultures of each bacterial strain were exposed to the test material dissolved in ethanol in the absence and presence of S9-fraction. The plate incorporation method was used for both the initial and confirmatory assays. Plates were incubated for 48 hours. From each triplicate plate, the number of revertant colonies was determined (using a Seescan automatic colony counter); mean revertants were also calculated for each exposure condition. Cytotoxicty was also observed.
Evaluation criteria:
The laboratory's criterion for a positive response in this assay is shown below:
At least a 2-fold increase in the mean number of revertants per plate in at least one tester strain compared to the solvent control in two separate experiments, accompanied by a concentration-response relationship.
Statistics:
Not applicable

Results and discussion

Test results
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
In the preliminary cytotoxicity assessments, BEPD was not toxic towards the tester strains, therefore, 5000 µg/plate was therefore chosen as the top dose level in the mutation tests.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

BEPD toxicity was observed at 5000 µg/plate with TA98 in the absence of S9 -fraction in both mutation tests. No substantial increases in revertant colony numbers were recorded for any of the tester strains following treatment with BEPD at any dose level, in the presence or absence of S9 -fraction, in either mutation test.

Ames test - summary of findings

Mean number of revertant colonies

Initial experiment

Exposure (µg/plate)

+S9

-S9

TA 1535

TA 1537

TA 98

TA 100

TA 1535

TA 1537

TA 98

TA 100

ethanol

19

15

28

127

14

13

23

122

0

17

14

28

141

14

14

26

124

50

15

10

32

126

17

14

25

123

150

18

13

29

109

14

12

29

112

500

8

11

28

113

10

10

26

109

1500

15

10

21

88

12

11

22

119

5000

11

9

27

117

9

7

-

113

Confirmatory experiment

Exposure (µg/plate)

+S9

-S9

TA 1535

TA 1537

TA 98

TA 100

TA 1535

TA 1537

TA 98

TA 100

ethanol

13

11

26

118

13

10

25

121

0

15

11

24

118

11

15

21

120

50

13

9

25

118

9

8

22

96

150

11

9

21

125

7

9

20

102

500

10

9

24

105

10

13

23

109

1500

11

12

26

107

9

7

23

109

5000

10

8

19

86

9

12

-

95

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

BEPD was not mutagenic under the conditions of this study.
Executive summary:

The mutagenicity of BEPD was investigated in an Ames test (plate incorporated method) using Salmonella typhimurium strains TA 1535, TA 1537, TA 98 and TA 100. Triplicate plates were exposed to the test substance (dissolved in ethanol) at 0, 50, 150, 500, 1500 and 5000 µg/plate in the presence and absence of exogenous metabolic activation system (Aroclor 1254 -induced male Sprague-Dawley rat liver S9 fraction). The assay was repeated using the same concentrations of BEPD. No evidence for mutagenicity was seen in the absence or presence of a metabolic activation system. Results were confirmed in an independently repeated assay. Appropriate positive control compounds confirmed the sensitivity of the assay.