Reaction mass of (2R*,4R*,4aR*,9bS*)-2,4-dimethyl-4,4a,5,9b-tetrahydroindeno[1,2-d][1,3]dioxine and (2R*,4R*,4aS*,9bR*)-2,4-dimethyl-4,4a,5,9b-tetrahydroindeno[1,2-d][1,3]dioxine

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

Study period:

22 Feb - 23 Mar 2001

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP Guideline study.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2001

Report date:

2001

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted 21 July 1997

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Ministerium für Umwelt und Verkehr, Baden-Württemberg, Stuttgart, Germany

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

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

EC Number:

944-528-8

Molecular formula:

C13H16O2

IUPAC Name:

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

Method

Target gene:

his operon (for S. typhimurium strains)

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:

First experiment:
5, 15, 50, 150 and 500 µg/plate without metabolic activation (TA1537)
15, 50, 150, 500 and 1500 µg/plate without metabolic activation (TA98, TA100 and TA 102)
50, 150, 500, 1500 and 5000 µg/plate without metabolic activation (TA1535)
50, 150, 500, 1500 and 5000 µg/plate with metabolic activation (all strains)

Second experiment:
15, 50, 150, 500, 1500 and 5000 µg/plate without metabolic activation (TA1535)
15, 50, 150, 500 and 1500 µg/plate without metabolic activation (TA102)
50, 150, 500, 1500 and 5000 µg/plate without metabolic activation (TA98, TA100)
500, 1000, 1500, 2000 and 3000 µg/plate without metabolic activation (TA1535)
15, 50, 150, 500 and 1500 µg/plate with metabolic activation (TA100, TA102, TA1537)
50, 150, 500, 1500 and 5000 µg/plate with metabolic activation (TA98, TA1535)

Vehicle / solvent:

- Vehicle/solvent used: DMSO

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

other: sodium azide (NaN3), 2-nitrofluorene (2-NF), 9-aminoacridine (9-AA), 2-aminoanthracene (2-AA)

Remarks:

+S9: 2-AA (0.8 µg/plate TA98, TA100; 0.9 µg/plate TA102, TA1535; 1.7 µg/plate, TA1537); -S9: NaN3 (0.7 µg/plate, TA100, TA1535); 2-NF (2.5 µg/plate, TA98); 9-AA (50 µg/plate, TA1537); Mitomycin C (0.15 µg/plate, TA102)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 to 72 h

NUMBER OF REPLICATIONS: triplicates each in two independent experiments

DETERMINATION OF CYTOTOXICITY
- Method: reduction in the number of revertant colonies and/or diminution of the background lawn

Statistics:

X²-test was used to estimate the statistical significance of the difference between the mean number of revertants in the negative controls and the plates at each dosage level. Mean values and standard deviation were calculated.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 5000 µg/plate (+S9)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 5000 µg/plate (-/+S9)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 5000 µg/plate (-/+S9)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 1500 µg/plate (-S9) and at 5000 µg/plate (+S9)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No precipitation occurred up to the highest investigated dose.

RANGE-FINDING/SCREENING STUDIES:
An initial toxicity test was conducted. No further details were given in the study report.

COMPARISON WITH HISTORICAL CONTROL DATA:
The negative and positive control data were between the minimum and maximum value of the historical control data of the laboratory.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
In the absence of S9 mix the test substance was bacteriotoxic towards the strains TA102 at 1500 µg/plate and towards the strains TA1537 and TA100 at 5000 µg/plate. In the presence of S9 mix the test substance was bacteriotoxic towards the strains TA1535, TA1537, TA100, and TA102 at 5000 µg/plate.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

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
NC	34 ± 6	99 ± 8	216 ± 26	19 ± 3	10 ± 3
Solvent control (DMSO)	30 ± 3	86 ± 2	235 ± 11	17 ± 3	10 ± 0
5	-	-	-	-	8 ± 2
15	33 ± 6	86 ± 5	216 ± 22	-	9 ± 2
50	33 ± 8	94 ± 14	240 ± 19	19 ± 2	8 ± 3
150	33 ± 7	98 ± 6	211 ± 19	24 ± 4	6 ± 3
500	36 ± 5	100 ± 2	253 ± 7	29 ± 1	7 ± 3
1500	32 ± 4	106 ± 6	172 ± 6 (T)	34 ± 8	-
5000	-	-	-	25 ± 5	-
NaN3 (0.7)	-	503 ± 58	-	885 ± 104	-
2-NF (2.5)	812 ± 39	-	-	-	-
9-AA (50)	-	-	-	-	272 ± 43
Mitomycin C (0.15)	-	-	723 ± 53	-	-
S9-Mix	With
Test item (µg/plate)	TA 98	TA 100	TA 102	TA 1535	TA 1537
NC	32 ± 8	97 ± 10	174 ± 29	7 ± 3	10 ± 4
Solvent control (DMSO)	25 ± 2	91 ± 7	208 ± 3	9 ± 2	14 ± 2
50	22 ± 3	86 ± 2	204 ± 29	10 ± 2	11 ± 4
150	25 ± 4	86 ± 3	229 ± 7	9 ± 2	13 ± 4
500	25 ± 3	83 ± 4	239 ± 12	6 ± 3	13 ± 3
1500	24 ± 1	81 ± 11	187 ± 27	6 ± 1	9 ± 4
5000	25 ± 7 (T)	58 ± 4 (T)	38 ± 10 (T)	6 ± 1	3 ± 3 (T)
2-AA (0.8)	926 ± 31	627 ± 90	-	-	-
2-AA (0.9)	-	-	308 ± 62	135 ± 20	-
2-AA (1.7)	-	-	-	-	362 ± 58
NC = Negative Control T = bacteriotoxic 2-NF: 2-nitrofluorene; 9-AA: 9-aminoacridine; 2-AA: 2-aminoanthracene; NaN3: sodium azide

 

 

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
NC	18 ± 4	201 ± 16	328 ± 28	22 ± 4	8 ± 2
Solvent control (DMSO)	21 ± 3	158 ± 6	300 ± 13	24 ± 1	6 ± 2
15	-	-	282 ± 34	-	7 ± 2
50	19 ± 3	138 ± 12	273 ± 14	-	8 ± 4
150	18 ± 2	177 ± 10	282 ± 12	-	7 ± 3
500	17 ± 4	166 ± 29	250 ± 18	35 ± 7	7 ± 5
1000	-	-	-	38 ± 9	-
1500	16 ± 2	130 ± 28	199 ± 12 (T)	38 ± 3	7 ± 3
2000	-	-	-	33 ± 15	-
3000	-	-	-	41 ± 3	-
5000	16 ± 2	79 ± 23 (T)	-	-	0 ± 0 (T)
NaN3 (0.7)	-	471 ± 91	-	841 ± 127	-
2-NF (2.5)	607 ± 67	-	-	-	-
9-AA (50)	-	-	-	-	381 ± 54
Mitomycin C (0.15)	-	-	967 ± 67	-	-
S9-Mix	With
Test item (µg/plate)	TA 98	TA 100	TA 102	TA 1535	TA 1537
NC	23 ± 5	171 ± 23	228 ± 38	12 ± 3	9 ± 3
Solvent control (DMSO)	22 ± 4	174 ± 6	272 ± 16	13 ± 3	11 ± 2
15	-	170 ± 12	223 ± 12	-	12 ± 5
50	22 ± 3	179 ± 14	263 ± 15	11 ± 1	13 ± 4
150	20 ± 6	180 ± 13	227 ± 17	14 ± 4	14 ± 4
500	21 ± 5	144 ± 23	252 ± 19	13 ± 5	10 ± 2
1500	24 ± 3	140 ± 12	209 ± 45	13 ± 3	12 ± 4
5000	20 ± 4	-	-	6 ± 4 (T)	-
2-AA (0.8)	420 ± 19	647 ± 113	-	-	-
2-AA (0.9)	-	-	477 ± 19	308 ± 51	-
2-AA (1.7)	-	-	-	-	321 ± 21
NC = Negative Control T = bacteriotoxic 2-NF: 2-nitrofluorene; 9-AA: 9-aminoacridine; 2-AA: 2-aminoanthracene; NaN3: sodium azide

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

Under the conditions of the Ames test the test substance was not mutagenic in any of the five strains (TA 1535, TA 1537, TA 98, TA 100 and TA 102) tested with and without metabolic activation.