4,4'-[(isopropylidene)bis(p-phenyleneoxy)]diphthalic dianhydride

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2002

Report date:

2002

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

4,4'-[(isopropylidene)bis(p-phenyleneoxy)]diphthalic dianhydride

EC Number:

253-781-7

EC Name:

4,4'-[(isopropylidene)bis(p-phenyleneoxy)]diphthalic dianhydride

Cas Number:

38103-06-9

Molecular formula:

C31H20O8

IUPAC Name:

5,5'-[propane-2,2-diylbis(4,1-phenyleneoxy)]bis(2-benzofuran-1,3-dione)

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 / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Species / strain 2

Species / strain / cell type:

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 / solvent:

Dimethyl sulfoxide (DMSO; CAS No. 67-68-5); from Fisher Scientific

Controls

Controls 1

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Remarks:

All strains; With and without activation

Controls 2

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

Controls 3

Positive controls:

yes

Remarks:

Without S9 activation

Positive control substance:

sodium azide

Remarks:

TA100, TA1535; 1.0 µg/plate

Controls 4

Positive controls:

yes

Remarks:

Without S9 activation

Positive control substance:

methylmethanesulfonate

Remarks:

WP2 uvrA; 1,000 µg/plate

Controls 5

Positive controls:

yes

Remarks:

Without S9 activation

Positive control substance:

9-aminoacridine

Remarks:

TA1537; 75 µg/plate

Controls 6

Positive controls:

yes

Remarks:

Without S9 activation

Positive control substance:

2-nitrofluorene

Remarks:

TA98; 1.0 µg/plate

Details on test system and experimental 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

Test results 1

Key result

Species / strain:

S. typhimurium, other: TA 1535, TA1537, TA98, TA100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

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:

Under the conditions of this study, the test material did not cause a positive response in either the presence or absence of Aroclor induced rat liver S9 and is therefore non-mutagenic.

Executive summary:

The study was conducted in S. typhimurium TA 1535, TA 1537, TA 98 and TA 100 and E. coli WP2 uvr A in accordance with OECD 471 under GLP conditions (BioReliance, 2002).

Bacteria were exposed to the test material at concentrations up to 5000 µg/plate in DMSO both with and without S9 metabolic activation.

Under the conditions of this study, the test material did not cause a positive response in either the presence or absence of Aroclor induced rat liver S9 and is therefore non-mutagenic.