1H-Isoindol-1-one, 2,3-dihydro-3,3-bis(4-hydroxyphenyl)-2-phenyl-

Genetic toxicity: in vitro

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 471 (1998); GLP

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2004

Report date:

2004

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes

Remarks:

OECD GLP, US FDA GLP (21 CFR 58); US EPA GLP (40 CFR 160 and 792) UK GLP and Japanese GLP Standard

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

455-890-7

EC Name:

-

Cas Number:

6607-41-6

Molecular formula:

C26H19NO3

IUPAC Name:

455-890-7

Details on test material:

PPP-BP (CAS# 6607-41-6); Purity 99.85%

Method

Species / strain

Species / strain / cell type:

other: Salmonella typhimurium tester strains TA98, TA100, TA1535 and TA1537 and Escherichia coli tester strain WP2 uvrA

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced rat liver S9

Test concentrations with justification for top dose:

10, 33, 67, 100, 333, 667, 1000, 3333 and 5000 microg per plate (Preliminary toxicity assay)
50, 150, 500, 1500 and 5000 microg per plate (Initial and independent repeat mutagenicity assays)

Vehicle / solvent:

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

Controls

Controls 1

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Remarks:

All strains With S9 activation

Positive control substance:

other: 2-aminoanthracene (CAS No. 613-13-8)

Remarks:

1.0 microg/plate all Salmonella strains; 10 microg/plate WP2 uvrA

Controls 2

Positive controls:

yes

Remarks:

TA98 Without S9 activation

Positive control substance:

2-nitrofluorene

Remarks:

1.0 microg/plate

Controls 3

Positive controls:

yes

Remarks:

TA100, TA1535 Without S9 activation

Positive control substance:

sodium azide

Remarks:

1.0 microg/plate

Controls 4

Positive controls:

yes

Remarks:

TA1537 Without S9 activation

Positive control substance:

9-aminoacridine

Remarks:

75 microg/plate

Controls 5

Positive controls:

yes

Remarks:

WP2 uvrA Without S9 activation

Positive control substance:

methylmethanesulfonate

Remarks:

1,000 microg/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. A vehicle control and nine 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: The test system was exposed to the test article via the plate incorporation method. 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 supplemented 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 microL of tester strain and 100 microL of vehicle or test article dilution were added to 2.0 mL of molten selective top agar at 45+/-2°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 microL 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°C. Plates that were not counted immediately following the incubation period were stored at 2 8°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:

other: S.typhimurium TA98, TA100, TA1535 and TA1537 & E. coli WP2 uvrA

Metabolic activation:

with and without

Genotoxicity:

other: No

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

In the preliminary toxicity assay, the doses tested were 10, 33, 67, 100, 333, 667, 1000, 3333 and 5000 microg/plate. Precipitate was observed beginning at 3333 microg/plate. No appreciable toxicity was observed. Based on the preliminary toxicity findings the doses tested for the mutagenicity assay were 50, 150, 500, 1500 and 5000 microg/plate.

In the Initial Mutagenicity Assay (Experiment B1), no positive mutagenic responses were observed with any of the tester strains in either the presence or absence of S9 activation. Precipitate was observed beginning at 1500 microg/plate. Although decreases in revertant counts were observed with tester strain WP2 uvrA in the presence and absence of S9 activation, no reductions in the background lawns were observed.

For the Independent Repeat Mutagenicity Assay (Experiment B2), no positive mutagenic responses were observed with any of the tester strains in either the presence or absence of S9 activation. Precipitate was observed beginning at 1500 or at 5000 microg/plate. Although decreases in revertant counts were observed with tester strain TA1535 in the presence of S9 activation and tester strain WP2 uvrA in the absence of S9 activation, no reductions in the background lawns were observed.

Remarks on result:

other: other: All strains tested; Initial Mutagenicity Assay

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Initial Mutagenicity Assay

Mean Number of Revertants Per Plate

Activation:  None

Dose (microg/plate)	TA98	TA100	TA1535	TA1537	WP2uvrA
Vehicle (DSMO)	17 ± 5	115 ± 3	18 ± 7	5 ± 1	17 ± 2
50	15 ± 3	130 ± 14	18 ± 4	4 ± 2	15 ± 1
150	15 ± 1	136 ± 6	17 ± 4	4 ± 4	15 ± 7
500	17 ± 3	124 ± 3	21 ± 1	4 ± 2	14 ± 4
1500	18 ± 2	119 ± 12	19 ± 5	4 ± 2	11 ± 3
5000	16 ± 2	107 ± 7	17 ± 5	4 ± 2	5 ± 1
Positive Control	129 ± 7	598 ± 8	402 ± 9	1200 ± 112	137 ± 8

Initial Mutagenicity Assay

Mean Number of Revertants Per Plate

Activation:  S9

Dose (microg/plate)	TA98	TA100	TA1535	TA1537	WP2uvrA
Vehicle (DSMO)	21 ± 5	126 ± 12	15 ± 3	6 ± 3	19 ± 1
50	23 ± 5	129 ± 7	16 ± 4	5 ± 3	20 ± 1
150	23 ± 4	132 ± 15	13 ± 2	4 ± 3	21 ± 2
500	28 ± 6	132 ± 5	18 ± 4	6 ± 3	18 ± 1
1500	27 ± 1	133 ± 23	13 ± 3	6 ± 1	15 ± 3
5000	24 ± 2	121 ± 11	13 ± 3	4 ± 0	7 ± 2
Positive Control	768 ± 70	743 ± 36	109 ± 15	76 ± 7	683 ± 5

Independent Repeat Mutagenicity Assay

Mean Number of Revertants Per Plate

Activation:  None

Dose (microg/plate)	TA98	TA100	TA1535	TA1537	WP2uvrA
Vehicle (DSMO)	21 ± 2	115 ± 13	23 ± 2	6 ± 2	16 ± 5
50	15 ± 3	118 ± 17	21 ± 1	5 ± 3	15 ± 2
150	17 ± 4	112 ± 12	25 ± 1	10 ± 5	17 ± 2
500	19 ± 2	122 ± 5	25 ± 6	7 ± 2	15 ± 4
1500	14 ± 5	107 ± 13	16 ± 3	6 ± 2	12 ± 3
5000	11 ± 2	85 ± 8	18 ± 7	3 ± 2	6 ± 2
Positive Control	154 ± 17	473 ± 5	406 ± 60	1459 ± 268	120 ± 9

Independent Repeat Mutagenicity Assay

Mean Number of Revertants Per Plate

Activation:  S9

Dose (microg/plate)	TA98	TA100	TA1535	TA1537	WP2uvrA
Vehicle (DSMO)	21 ± 7	118 ± 13	27 ± 7	9 ± 1	21 ± 3
50	26 ± 7	114 ± 5	42 ± 4	8 ± 1	19 ± 4
150	32 ± 9	133 ± 9	28 ± 1	7 ± 2	20 ± 2
500	30 ± 4	139 ± 6	14 ± 4	8 ± 2	23 ± 2
1500	29 ± 8	115 ± 12	18 ± 5	6 ± 1	16 ± 7
5000	18 ± 2	117 ± 11	12 ± 3	5 ± 1	12 ± 3
Positive Control	620 ± 115	805 ± 32	104 ± 16	106 ± 15	614 ± 24

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

Conclusion: The results of the Bacterial Reverse Mutation Assay with an Independent Repeat Assay indicate that, under the conditions of this study, PPP-BP (CAS No. 6607-41-6) did not cause a positive response in either the presence or absence of Aroclor-induced rat liver S9.

Executive summary:

In this in vitro assessment of mutagenic potential, within Salmonella typhimurium tester strains TA98, TA100, TA1535 and TA1537 and Escherichia coli tester strain WP2 uvrA were exposed to the test substance diluted in dimethylsulfoxide. The DMSO was also used as a solvent/vehicle control. Two independent mutation tests were performed, in the presence and absence of liver preparations from Aroclor 1254-induced rats. Based on preliminary range finding results, the highest dose level of 5000 µg/plate was chosen for the subsequent mutation study. Other dose levels used in the mutation assays were 50, 150, 500, 1500 µg/plate. When tested at dose levels up to 5000 µg/plate in dimethylsulfoxide, the test substance was not mutagenic in the bacterial test system.