4,4a,5,9b-tetrahydroindeno[1,2-d]-1,3-dioxin

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:

13 Aug - 17 Nov 1999

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1999

Report date:

1999

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Version / remarks:

Commission Directive 92/69/EEC

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

The Department of Health of the Government of the United Kingdom

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

4,4a,5,9b-tetrahydroindeno[1,2-d]-1,3-dioxin

EC Number:

241-997-4

EC Name:

4,4a,5,9b-tetrahydroindeno[1,2-d]-1,3-dioxin

Cas Number:

18096-62-3

Molecular formula:

C11H12O2

IUPAC Name:

2H,4H,4aH,5H,9bH-indeno[1,2-d][1,3]dioxine

Method

Target gene:

his operon

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Metabolic activation system:

co-factor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with Aroclor 1254

Test concentrations with justification for top dose:

Experiment 1 and 2: 50, 150, 500, 1500 and 5000 µg/plate with and without metabolic activation

Vehicle / solvent:

- Vehicle/solvent used: DMSO

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

benzo(a)pyrene

mitomycin C

other: 2-aminoanthracene (2AA); 1,8-dihydroxyanthraquinone (Danthron)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: approx. 48 h

NUMBER OF REPLICATIONS: triplicates each in two independent experiments

DETERMINATION OF CYTOTOXICITY
- Method: Inspection of the bacterial background lawn

Evaluation criteria:

The test material may be considered to be positive in this test system if the following criteria are met:
The test material should have induced a reproducible, dose-related and statistically (Dunnett´s method of linear regression) significant increase in the revertant count in at least one strain of bacteria.

Statistics:

Mean values and standard deviation were calculated.

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 4

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 5

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No test material precipitate was observed on the plates at any of the doses tested in either the presence or absence of metabolic activation.

RANGE-FINDING/SCREENING STUDIES:
In order to select appropriate dose levels for the main study, a preliminary test was carried out in S. typhimurium strain TA 100 to determine the toxicity of the test substance. The dose range for the test substance used was 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate wiht and without metabolic activation. The test substance was non-toxic to strain TA 100 at the tested concentrations.

Any other information on results incl. tables

Table 1: Test Results of Experiment 1

Experiment 1
S9-Mix	Without
Test item (µg/plate)	TA 98	TA 100	TA 102	TA 1535	TA 1537
Solvent control (DMSO)	30 ± 4	124 ± 18	299 ± 20	28 ± 7	18 ± 4
50	30 ± 5	123 ± 7	286 ± 4	20 ± 6	11 ± 0
150	26 ± 8	122 ± 10	301 ± 14	23 ± 6	15 ± 3
500	31 ± 7	145 ± 13	297 ± 27	27 ± 9	19 ± 3
1500	25 ± 4	110 ± 14	287 ± 26	30 ± 6	16 ± 1
5000	20 ± 6	128 ± 5	307 ± 35	33 ± 2	9 ± 1
ENNG (3)	-	539 ± 64	-	-	-
ENNG (5)	-	-	-	222 ± 39	-
9-AA (80)	-	-	-	-	784 ± 251
4NQO (0.2)	160 ± 2	-	-	-	-
MMC (0.5)	-	-	751 ± 185	-	-
S9-Mix	With
Test item (µg/plate)	TA 98	TA 100	TA 102	TA 1535	TA 1537
Solvent control (DMSO)	44 ± 6	143 ± 21	330 ± 24	24 ± 4	22 ± 2
50	50 ± 17	129 ± 16	313 ± 26	13 ± 5	22 ± 1
150	48 ± 11	138 ± 8	321 ± 9	18 ± 7	23 ± 3
500	56 ± 14	144 ± 5	331 ± 22	20 ± 1	24 ± 1
1500	37 ± 5	131 ± 17	316 ± 19	16 ± 5	22 ± 3
5000	38 ± 5	127 ± 3	289 ± 12	17 ± 5	20 ± 1
2-AA (1)	-	2057 ± 428	-	-	-
2-AA (2)	-	-	-	274 ± 43	814 ± 11
DAN (10)	-	-	813 ± 53	-	-
BP (5)	814 ± 11	-	-	-	-
EENG: N-ethyl-N-nitro-N-nitrosoguanidin 4NQO: 4-Nitroquinoline-1-oxide 9-AA: 9-Aminoacridine MMC: Mitomycin C 2-AA: 2-Aminoanthracene BP: Benzo(a)pyrene DAN: 1,8-Dihydroxyanthraquinone

Table 2: Test Results of Experiment 2

Experiment 2
S9-Mix	Without
Test item (µg/plate)	TA 98	TA 100	TA 102	TA 1535	TA 1537
Solvent control (DMSO)	27 ± 4	119 ± 11	352 ± 4	25 ± 2	16 ± 4
50	29 ± 3	123 ± 4	338 ± 5	20 ± 3	16 ± 5
150	29 ± 7	137 ± 15	331 ± 15	25 ± 7	17 ± 3
500	23 ± 6	135 ± 6	350 ± 12	23 ± 1	14 ± 2
1500	27 ± 7	121 ± 7	323 ± 56	24 ± 2	11 ± 3
5000	22 ± 8	124 ± 16	324 ± 17	32 ± 3	15 ± 5
ENNG (3)	-	481 ± 25	-	-	-
ENNG (5)	-	-	-	167 ± 15	-
9-AA (80)	-	-	-	-	926 ± 324
4NQO (0.2)	168 ± 14	-	-	-	-
MMC (0.5)	-	-	955 ± 75	-	-
S9-Mix	With
Test item (µg/plate)	TA 98	TA 100	TA 102	TA 1535	TA 1537
Solvent control (DMSO)	39 ± 2	127 ± 15	311 ± 20	15 ± 7	20 ± 1
50	34 ± 3	112 ± 11	308 ± 17	14 ± 1	19 ± 5
150	40 ± 8	157 ± 18	318 ± 25	18 ± 1	22 ± 3
500	31 ± 4	132 ± 29	317 ± 3	16 ± 3	17 ± 4
1500	29 ± 7	119 ± 11	317 ± 3	17 ± 4	14 ± 2
5000	38 ± 7	138 ± 19	269 ± 18	14 ± 1	12 ± 1
2-AA (1)	-	1484 ± 94	-	-	-
2-AA (2)	-	-	-	279 ± 41	551 ± 35
DAN (10)	-	-	875 ± 204	-	-
BP (5)	257 ± 6	-	-	-	-
EENG: N-ethyl-N-nitro-N-nitrosoguanidin 4NQO: 4-Nitroquinoline-1-oxide 9-AA: 9-Aminoacridine MMC: Mitomycin C 2-AA: 2-Aminoanthracene BP: Benzo(a)pyrene DAN: 1,8-Dihydroxyanthraquinone

Applicant's summary and conclusion

Conclusions:

Under the conditions of the Ames Assay the substance was not mutagenic in any of the five strains (TA 100, TA 1535, TA 102, TA 98 and TA 1537) tested with and without metabolic activation up to 5000 µg/plate.