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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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: OECD Test Guideline 471 (1998)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2002
Report Date:
2002

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
GLP compliance:
yes
Remarks:
OECD GLP, US FDA GLP (21 CFR 58); US EPA GLP (40 CFR 160 and 40 CFR 792) UK GLP and Japanese GLP Standard
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Bisphenol A Dianhydride (BPA-DA; CAS No. 38103-06-9); Lot No. UI0049; Purity: 98.5%; from GE Plastics

Method

Species / strainopen allclose all
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Metabolic activation system:
Aroclor-induced rat liver S9
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
Aroclor-induced rat liver S9
Test concentrations with justification for top dose:
6.7, 10, 33, 67, 100, 333, 667, 1000, 3333 and 5000 µg per plate (Preliminary assay)
100, 333, 1000, 3333 and 5000 µg per plate (Mutagenicity assays)
Vehicle:
Dimethyl sulfoxide (DMSO; CAS No. 67-68-5); from Fisher Scientific
Controlsopen allclose all
Solvent controls:
yes
Remarks:
DMSO
Remarks:
All strains; With and without activation
Positive controls:
yes
Remarks:
With S9 activation
Positive control substance:
other: 2-aminoanthracene
Remarks:
All strains; 1.0 µg/plate all Salmonella strains; 10 µg/plate WP2 uvrA
Positive controls:
yes
Remarks:
Without S9 activation
Positive control substance:
sodium azide
Remarks:
TA100, TA1535; 1.0 µg/plate
Positive controls:
yes
Remarks:
Without S9 activation
Positive control substance:
methylmethanesulfonate
Remarks:
WP2 uvrA; 1,000 µg/plate
Positive controls:
yes
Remarks:
Without S9 activation
Positive control substance:
9-aminoacridine
Remarks:
TA1537; 75 µg/plate
Positive controls:
yes
Remarks:
Without S9 activation
Positive control substance:
2-nitrofluorene
Remarks:
TA98; 1.0 µg/plate
Details on test system and conditions:
Preliminary Toxicity Assay: The preliminary toxicity assay was used to establish the dose range over which the test article would be assayed. Vehicle control and ten dose levels of the test article were plated, one plate per dose, with overnight cultures of TA98, TA100, TA1535, TA1537 and WP2 uvrA on selective minimal agar in the presence and absence of Aroclor induced rat liver S9.
Mutagenicity Assay: The mutagenicity assay (initial and independent repeat assays), was used to evaluate the mutagenic potential of the test article. Five dose levels of test article along with vehicle control and appropriate positive controls were plated with overnight cultures of TA98, TA100, TA1535, TA1537 and WP2 uvrA in the presence and absence of Aroclor induced rat liver S9. All dose levels of test article, vehicle control and positive controls were plated in triplicate.
Plating and Scoring Procedures: On the day of its use, minimal top agar was melted and supplemented. Top agar not used with S9 or Sham mix was supplemented with 25 mL of water for each 100 mL of minimal top agar. For the preparation of media and reagents, all references to water imply sterile, deionized water produced by the Milli Q Reagent Water System. Bottom agar was Vogel Bonner minimal medium E.
Each plate was labeled with a code system that identified the test article, test phase, dose level, tester strain and activation.
One half (0.5) milliliter of S9 or Sham mix, 100 uL of tester strain and 50 uL of vehicle or test article dilution were added to 2.0 mL of molten selective top agar at 45±2 degrees C. After vortexing, the mixture was overlaid onto the surface of 25 mL of minimal bottom agar. When plating the positive controls, the test article aliquot was replaced by a 50 µL aliquot of appropriate positive control. After the overlay had solidified, the plates were inverted and incubated for approximately 48 to 72 hours at 37±2 degrees C. Plates that were not counted immediately following the incubation period were stored at 2 8 degrees C until colony counting could be conducted.
The condition of the bacterial background lawn was evaluated for evidence of test article toxicity by using a dissecting microscope. Precipitate was evaluated by visual examination without magnification.
Revertant colonies for a given tester strain and activation condition, except for positive controls, were counted either entirely by automated colony counter or entirely by hand unless the assay was the preliminary toxicity assay or the plate exhibited toxicity.
Evaluation criteria:
For each replicate plating, the mean and standard deviation of the number of revertants per plate were calculated and are reported.
For the test article to be evaluated positive, it must cause a dose-related increase in the mean revertants per plate of at least one tester strain over a minimum of two increasing concentrations of test article. Data sets for tester strains TA1535 and TA1537 were judged positive if the increase in mean revertants at the peak of the dose response is equal to or greater than 3.0-times the mean vehicle control value. Data sets for tester strains TA98, TA100 and WP2 uvrA were judged positive if the increase in mean revertants at the peak of the dose response is equal to or greater than 2.0-times the mean vehicle control value.

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:
no
Vehicle controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'. Remarks: including Escherichia coli tester strain WP2 uvrA
Additional information on results:
All validity criteria were met

Any other information on results incl. tables

Initial Mutagenicity Assay - Mean Number of Revertants Per Plate

Activation:  None

Dose (µg/plate)

TA98

TA100

TA1535

TA1537

WP2 uvrA

Vehicle (DMSO)

10 ± 1

137 ± 12

12 ± 1  

11 ± 5  

11 ±   3

100

9 ± 2

137 ± 14  

13 ± 4

10 ± 2  

10 ±   1

333

11± 4

133 ± 15  

12 ± 2  

10 ± 2  

11 ±   1

1000

13 ± 1

135 ± 14

8 ± 1  

12 ± 1  

10 ±   5

3333

14 ± 5

125 ± 3

12 ± 0   

9 ± 3   

9 ±   2

5000

15 ± 3

113 ± 15

7 ± 2   

9 ± 1   

9 ±   1

Positive Control

128 ± 12

582 ± 28

211 ± 27  

68 ± 6  

74 ±   3

Initial Mutagenicity Assay - Mean Number of Revertants Per Plate

Activation:  S9

Dose (µg/plate)

TA98

TA100

TA1535

TA1537

WP2 uvrA

Vehicle (DMSO)

18 ± 2

151 ± 17

11 ± 3

11 ± 3

10 ± 1

100

13 ± 2

129 ± 3

12 ± 3

10 ± 2

8 ± 1

333

13 ± 3

118 ± 14

9 ± 1

10 ± 1

11 ± 2

1000

9 ± 2

116 ± 17

10 ± 2

9 ± 3

9 ± 1

3333

13 ± 2

120 ± 9

12 ± 3

7 ± 2

9 ± 1

5000

14 ± 2

117 ± 5

10 ± 4

10 ± 3

12 ± 4

Positive Control

713 ± 51

678 ± 42

185 ± 9

246 ± 19

770 ± 90

Confirmatory Mutagenicity Assay - Mean Number of Revertants Per Plate

Activation:  None

Dose (µg/plate)

TA98

TA100

TA1535

TA1537

WP2 uvrA

Vehicle (DMSO)

15 ± 5 

177 ± 19

15 ± 4

14 ± 1

18 ± 3

100

12 ± 4

187 ± 6

13 ± 2  

19 ± 2  

18 ± 7

333

14 ± 2

202 ± 25

11 ± 1

16 ± 6

14 ± 4

1000

12 ± 1

186 ± 10

13 ± 3

12 ± 3

9 ± 1

3333

15 ± 2

180 ± 34

15 ± 7

9 ± 2

11 ± 5

5000

15 ± 5

148 ± 17

10 ± 3

15 ± 5   

11 ± 2

Positive Control

133 ± 7

636 ± 27

73 ± 9 

83 ± 4 

157 ± 3

Confirmatory Mutagenicity Assay - Mean Number of Revertants Per Plate

Activation:  S9

Dose (µg/plate)

TA98

TA100

TA1535

TA1537

WP2 uvrA

Vehicle (DMSO)

27 ± 7

168 ± 11  

11 ± 1  

11 ± 4  

20 ± 5

100

27 ± 4 

183 ± 15

9 ± 5  

11 ± 1

21 ± 2

333

17 ± 5

181 ± 7

11 ± 7  

10 ± 6  

16 ± 3

1000

14 ± 6

166 ± 16

8 ± 3  

11 ± 4  

14 ± 1

3333

16 ± 6

164 ± 4

11 ± 4

13 ± 4

15 ± 3

5000

15 ± 3

173 ± 24

13 ± 3

10 ± 3  

11 ± 1

Positive Control

920 ± 6

1051 ± 25

235 ± 4

234 ± 34 

791 ± 40

Applicant's summary and conclusion

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

The results of the Bacterial Reverse Mutation Assay with an Independent Repeat Assay indicate that, under the conditions of this study, Bisphenol A Dianhydride (CAS 38103-06-9) did not cause a positive response in either the presence or absence of Aroclor induced rat liver S9 and is therefore non-mutagenic.