3-chlorophthalic anhydride

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2004

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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)

Version / remarks:

1998

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

3-chlorophthalic anhydride

EC Number:

204-179-8

EC Name:

3-chlorophthalic anhydride

Cas Number:

117-21-5

Molecular formula:

C8H3ClO3

IUPAC Name:

4-chloro-1,3-dihydro-2-benzofuran-1,3-dione

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-induced rat liver S9

Test concentrations with justification for top dose:

- Preliminary assay: 6.7, 10, 33, 67, 100, 333, 667, 1000, 3333 and 5000 µg/plate
- Initial mutagenicity assay: 15, 50, 150, 500, 1500 and 5000 µg/plate
- Repeat mutagenicity assay: 50, 150, 500, 1500 and 5000 µg/plate

Vehicle / solvent:

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

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

methylmethanesulfonate

other: 2-aminoanthracene

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 microL of tester strain and 50 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

Test results 1

Species / strain:

other: S. typhimurium TA 98, TA 100, TA 1535, TA 1537, E. coli WP2 uvrA

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 5000 µg/plate

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

other: S. typhimurium TA 98, TA 100, TA 1535, TA 1537, E. coli WP2 uvrA

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: Yes; beginning at 1500 µg/plate or at 5000 µg/plate

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

All validity criteria were met.

Remarks on result:

other: initial assay

Any other information on results incl. tables

In the preliminary toxicity assay, toxicity was observed beginning at 333 µg/plate.  Precipitate was not observed.  The doses for the initial mutagenicity assay were selected based on the preliminary toxicity findings.

In the Initial Mutagenicity Assay, no positive mutagenic responses were observed with any of the tester strains in either the presence or absence of S9 activation.  Toxicity was observed at 5000 µg/plate.  No precipitate was observed.

For the Independent Repeat Mutagenicity Assay, no positive mutagenic responses were observed with any of the tester strains in either the presence or absence of S9 activation.  The doses for the repeat assay were selected based on the results of the initial mutagenicity assay. 

Toxicity was observed beginning at 1500 or 5000 µg/plate.  No precipitate was observed.

Initial Mutagenicity Assay Mean Number of Revertants Per Plate

Activation:  None

Dose (µg/plate)	TA98	TA100	TA1535	TA1537	WP2 uvrA
Vehicle (DMSO)	11 ± 1	141 ± 17	22 ± 3	5 ± 2	15 ± 2
15	12 ± 4	145 ± 10	21 ± 2	6 ± 4	12 ± 1
50	10± 2	161 ± 13	21 ± 3	7 ± 3	12 ± 2
150	10 ± 2	149 ± 2	24 ± 10	3 ± 1	14 ± 2
500	14 ± 1	140 ± 6	19 ± 2	4 ± 3	13 ± 5
1500	13 ± 2	102 ± 15	13 ± 1	3 ± 0	12 ± 3
5000	0 ± 0	0 ± 0	0 ± 0	0 ± 0	0 ± 0
Positive Control	60 ± 17	532 ± 26	360 ± 19	657 ± 110	102 ± 7

Initial Mutagenicity Assay Mean Number of Revertants Per Plate

Activation:  S9

Dose (µg/plate)	TA98	TA100	TA1535	TA1537	WP2 uvrA
Vehicle (DMSO)	20 ± 2	236 ± 10	14 ± 3	5 ± 1	18 ± 3
15	19 ± 2	220 ± 16	18 ± 2	7 ± 3	13 ± 3
50	21 ± 1	216 ± 4	13 ± 2	5 ± 2	19 ± 5
150	20 ± 3	209 ± 6	17 ± 2	6 ± 2	16 ± 3
500	22 ± 3	192 ± 5	17 ± 3	5 ± 1	13 ± 1
1500	21 ± 6	191 ± 9	15 ± 5	5 ± 1	16 ± 5
5000	14 ± 3	63 ± 10	3 ± 2	2 ± 2	10 ± 1
Positive Control	463 ± 106	1233 ± 395	129 ± 1	75 ± 24	727 ± 95

Independent Repeat Mutagenicity Assay Mean Number of Revertants Per Plate

Activation:  None

Dose (µg/plate)	TA98	TA100	TA1535	TA1537	WP2 uvrA
Vehicle (DMSO)	15 ± 2	208 ± 23	17 ± 2	6 ± 3	14 ± 3
50	13 ± 3	198 ± 5	15 ± 3	3 ± 1	12 ± 4
150	15 ± 6	208 ± 24	17 ± 2	6 ± 3	11 ± 5
500	13 ± 5	169 ± 6	12 ± 2	3 ± 2	11 ± 4
1500	11 ± 3	97 ± 38	10 ± 4	4 ± 2	9 ± 1
5000	0 ± 0	0 ± 0	0 ± 0	0 ± 0	0 ± 0
Positive Control	112 ± 4	801 ± 86	223 ± 18	605 ± 50	76 ± 11

Independent Repeat Mutagenicity Assay Mean Number of Revertants Per Plate

Activation:  S9

Dose (µg/plate)	TA98	TA100	TA1535	TA1537	WP2 uvrA
Vehicle (DMSO)	23 ± 5	233 ± 29	13 ± 3	7 ± 1	15 ± 5
50	28 ± 7	232 ± 6	13 ± 5	8 ± 3	18 ± 1
150	24 ± 4	225 ± 10	14 ± 5	5 ± 2	13 ± 4
500	21 ± 6	233 ± 7	12 ± 1	4 ± 2	16 ± 1
1500	20 ± 1	211 ± 5	7 ± 3	6 ± 0	16 ± 8
5000	9 ± 6	54 ± 10	3 ± 3	1 ± 1	8 ± 3
Positive Control	1025 ± 106	1118 ± 80	138 ± 21	133 ± 14	565 ± 178

Applicant's summary and conclusion

Conclusions:

The test substance was not mutagenic in the presence and absence of metabolic activation.

Executive summary:

Genetic toxicity was determined in an Ames test performed according to OECD 471 and was in compliance with GLP criteria. In the plate incorporation method, the Salmonella typhimurium tester strains TA98, TA100, TA1535 and TA1537 and Escherichia coli tester strain WP2 uvrA were exposed to concentrations of 15, 50, 150, 500, 1500 and 5000 µg/plate in the initial and 50, 150, 500, 1500 and 5000 µg/plate in the repeated mutagenicity assay, in the presence and absence of metabolic activation (aroclor-induced rat liver S9). These concentrations were based on a preliminary toxicity assay. The number of revertant colonies were determined after 48 - 72 hours incubation at 37 ± 2 °C. Under the conditions of this study, no positive response was observed in the presence or absence of a metabolic activation system. Based on these results, the test substance was determined to be not mutagenic in an Ames test.