Indeno[4,3a-b]furan, decahydro-2,2,7,7,8,9,9-heptamethyl-

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:

22 April 2003 to 22 May 2003

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2003

Report date:

2003

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

449-360-4

EC Name:

-

Cas Number:

647828-16-8

Molecular formula:

C18H32O

IUPAC Name:

decahydro-2,2,6,6,7,8,8-heptamethyl-2H-Indeno[4,5-b] furan

Test material form:

liquid

Method

Target gene:

Histidine for Salmonella
Tryptophan for E.Coli

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:

Phenobarbitone and beta­naphthoflavone induced rat liver (S9-mix)

Test concentrations with justification for top dose:

Preliminary Toxicity Test: 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate.
Main experiment 1 and 2: 50, 150, 500, 1500, 5000 µg/plate.

Vehicle / solvent:

Dimethyl sulphoxide

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

other: 2-Aminoanthracene

Remarks:

With S9 mix

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

without S9 mix

Details on test system and experimental conditions:

METHOD OF APPLICATION:
In agar (plate incorporation)

DURATION:
- Pre-incubation period for bacterial strains: 10 hours
- Exposure duration: 48 - 72 hours
- Expression time: Not applicable
- Selection time: Not applicable

NUMBER OF REPLICATIONS:
Triplicate plating

DETERMINATION OF CYTOTOXICITY:
- Method: plates were assessed for numbers of revertant colonies and examined for effects on the growth of the bacterial background lawn.

Evaluation criteria:

Acceptance Criteria:
- All tester strain cultures should exhibit a characteristic number of spontaneous revertants per plate in the vehicle and untreated controls.
- The appropriate characteristics for each tester strain should be confirmed, e.g., rfa cell-wall mutation and pKM101 plasmid R-factor etc.
- All tester strain cultures should be in the approximate range of 1 to 9.9 X 109 bacteria per mL.
- Each mean positive control value should be at least 2 times the respective vehicle control value for each strain, thus demonstrating both the intrinsic sensitivity of the tester strains to mutagenic exposure and the integrity of the S9-mix.
- There should be a minimum of 4 non-toxic test substance dose levels.
- There should not be an excessive loss of plates due to contamination.

Evaluation Criteria:
- When the test substance has induced a reproducible, dose-related and statistically (Dunnett's method of linear regression) significant increase in the revertant count in at least 1 strain of bacteria, it is considered to be a positive result.

Statistics:

The mean and standard deviation were calculated. Dunnetts linear regression analysis was performed.

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:

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

Vehicle controls validity:

valid

Remarks:

The number of revertant colonies per plate for the concurrent vehicle control was inside the historical control range of the laboratory.

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Remarks:

The positive control induced a number of revertant colonies per plate that demonstrated the effective performance of the assay.

Test results 2

Key result

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

Remarks:

The number of revertant colonies per plate for the concurrent vehicle control was inside the historical control range of the laboratory.

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Remarks:

The positive control induced a number of revertant colonies per plate that demonstrated the effective performance of the assay.

Test results 3

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

Tested up to maximum recommended dose of 5000 µg/plate

Vehicle controls validity:

valid

Remarks:

The number of revertant colonies per plate for the concurrent vehicle control was inside the historical control range of the laboratory.

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Remarks:

The positive control induced a number of revertant colonies per plate that demonstrated the effective performance of the assay.

Test results 4

Key result

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

Remarks:

The number of revertant colonies per plate for the concurrent vehicle control was inside the historical control range of the laboratory.

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Remarks:

The positive control induced a number of revertant colonies per plate that demonstrated the effective performance of the assay.

Test results 5

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

Tested up to maximum recommended dose of 5000 µg/plate

Vehicle controls validity:

valid

Remarks:

The number of revertant colonies per plate for the concurrent vehicle control was inside the historical control range of the laboratory.

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Remarks:

The positive control induced a number of revertant colonies per plate that demonstrated the effective performance of the assay.

Additional information on results:

Preliminary Toxicity Test
- The test material was non-toxic to the strains of bacteria used (TA100 and WP2uvrA). The test material formulation and S9-mix used in this experiment were both shown to be effectively sterile.

Mutation Test
- Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory). The S9-mix used in both experiments of the main test was shown to be sterile.
- Results for the negative controls (spontaneous mutation rates) are presented in Table 1 and were considered to be acceptable. These data are for concurrent untreated control plates performed on the same day as the Mutation Test.
- The individual plate counts, the mean number of revertant colonies and the standard deviations for the test material, vehicle and positive controls both with and without metabolic activation, are presented in Table 2.
- A history profile of vehicle and positive control values is presented in Table 3.
- The test material caused no visible reduction in the growth of the bacterial background lawn at any dose level. The test material was, therefore, tested up to the maximum recommended dose level of 5000 µg/plate. No test material precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix.
- No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, at any dose level either with or without metabolic activation.
- All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies thus confirming the activity of the S9-mix and the sensitivity of the bacterial strains.

Any other information on results incl. tables

Preliminary Toxicity Test:

The test substance was non-toxic to the strains of bacteria used (TA100 and WP2uvrA^-). The test substance formulation and S9-mix used in this experiment were both shown to be effectively sterile.

The number of revertant colonies for the toxicity assay were:

With (+) or without (-) S9-mix	Strain	Dose (µg/plate)
		0	0.15	0.5	1.5	5	15	50	150	500	1500	5000
-	TA100	74	56	61	73	62	67	66	63	61	70	93
+	TA100	89	110	92	118	105	67	82	89	102	96	101
-	WP2uvrA-	15	14	15	19	12	26	17	13	15	14	15
+	WP2uvrA-	13	11	15	9	16	14	9	14	18	14	24

   

Mutation Test:

Table 1. Spontaneous Mutation Rates (Concurrent Negative Control).

Experiment 1

Number of revertants (mean number of colonies per plate)
Base-pair Substitution Type						Frameshift Type
TA100		TA1535		WPAuvrA		TA98		TA1537
106	(91)	13	(11)	18	(20)	24	(18)	8	(7)
86		11		19		12		9
80		9		22		17		4

 

Experiment 2

Number of revertants (mean number of colonies per plate)
Base-pair Substitution Type						Frameshift Type
TA100		TA1535		WPAuvrA		TA98		TA1537
81	(82)	32	(27)	26	(26)	23	(19)	5	(8)
81		21		24		17		9
83		29		28		18		10

 

Table 2a: Range-finding test – Without Metabolic Activation: Mean value and SD

	TA100	TA1535	WP2uvrA-	TA98	TA1537
0	70 (7.5)	13 (1.2)	20 (5.5)	23 (5.0)	8 (5.1)
50	70 (6.7)	16 (0.6)	16 (2.9)	19 (3.8)	7 (0.6)
150	56 (6.7)	14 (4.0)	20 (3.8)	21 (5.8)	8 (4.2)
500	52 (9.5)	12 (1.2)	16 (4.0)	18 (5.8)	9 (0.6)
1500	55 (4.4)	13 (4.4)	19 (1.2)	31 (2.1)	7 (4.6)
5000	66 (5.8)	14 (5.8)	22 (3.8)	23 (2.0)	12 (4.6)
Positive control	ENNG	ENNG	ENNG	4NQO	9AA
	365 (112.8)	258 (9.6)	762 (43.8)	277 (29.5)	1263 (131.8)

 

Table 2a: Range-finding test – With Metabolic Activation: Mean value and SD

	TA100	TA1535	WP2uvrA-	TA98	TA1537
0	96 (6.4)	15 (1.5)	32 (0.6)	26 (3.1)	15 (2.5)
50	97 (7.2)	13 (2.9)	24 (0.6)	32 (5.7)	11 (2.9)
150	79 (0.6)	11 (0.6)	24 (3.0)	32 (2.5)	7 (1.5)
500	85 (4.7)	12 (6.1)	27 (5.3)	24 (5.8)	12 (1.2)
1500	88 (2.1)	16 (3.1)	24 (5.6)	29 (0.0)	5 (0.6)
5000	74 (17.5)	14 (4.0)	26 (5.5)	22 (0.0)	9 (4.6)
Positive control	2AA	2AA	2AA	BP	2AA
	1749 (242)	221 (7.8)	637 (172.8)	326 (22.6)	341 (14.7)

 

Table 2c: Main test – Without Metabolic Activation: Mean value and SD

	TA100	TA1535	WP2uvrA-	TA98	TA1537
0	108 (11.1)	15 (1.5)	14 (3.5)	17 (1.0)	8 (2.5)
50	86 (7.8)	12 (3.5)	17 (4.5)	15 (4.0)	8 (0.6)
150	88 (11.0)	12 (4.5)	13 (2.3)	22 (5.6)	6 (1.7)
500	96 (18.8)	10 (0.6)	14 (2.0)	16 (3.2)	7 (1.0)
1500	81 (5.0)	10 (1.7)	13 (5.6)	19 (8.0)	7 (1.2)
5000	80 (13.4)	15 (4.2)	18 (3.0)	28 (8.4)	7 (2.5)
Positive control	ENNG	ENNG	ENNG	4NQO	9AA
	361 (80.9)	176 (32.1)	717 (37.4)	109 (8.4)	759 (20.2)

 

Table 2d: Main test – With Metabolic Activation: Mean value and SD

	TA100	TA1535	WP2uvrA-	TA98	TA1537
0	106 (12.9)	13 (6.1)	19 (2.3)	20 (2.5)	10 (0.0)
50	86 (2.1)	8 (3.1)	22 (1.5)	21 (4.2)	7 (2.5)
150	95 (11.6)	7 (2.3)	17 (3.6)	29 (6.6)	4 (0.6)
500	87 (8.0)	14 (9.6)	14 (1.0)	26 (2.6)	7 (1.7)
1500	74 (10.1)	16 (10.2)	12 (1.7)	24 (3.0)	4 (1.0)
5000	85 (16.5)	12 (1.7)	21 (3.1)	22 (4.9)	10 (3.6)
Positive control	2AA	2AA	2AA	BP	2AA
	1363 (49.7)	239 (22.7)	362 (4.6)	202 (16.3)	288 (48.8)

ENNG: N-ethyl-N’-nitro-N-nitrosoguanidine; 4NQO: 4-Nitroquinoline-1-oxide; 9AA: 9-Aminoacridine

 

Table 3a: Historical Control Data: Combined vehicle and untreated control: 2001

	TA100		TA1535		WP2uvrA-		TA98		TA1537
	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
Mean	106	116	16	15	22	26	23	32	11	13
SD	23	23	4	4	5	6	7	8	4	4
Min	58	62	8	7	11	13	10	13	2	4
Max	178	178	37	37	41	52	56	58	23	29
N	817	658	790	621	656	501	814	662	793	630

 

Table 3b: Historical Control Data: Combined vehicle and untreated control: 2002

	TA100		TA1535		WP2uvrA-		TA98		TA1537
	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
Mean	91	100	20	17	23	27	22	35	12	17
SD	16.6	18.0	5.5	4.3	4.8	5.3	5.2	6.8	4	4.7
Min	61	68	8	8	10	14	11	13	4	5
Max	160	162	38	37	47	45	44	66	29	38
N	939	742	912	718	685	521	946	749	918	718

 

Table 3c: Historical Control Data: Positive control: 2001

	TA100		TA1535		WP2uvrA-		TA98		TA1537
	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
Mean	505	1582	431	336	685	756	140	303	1415	428
SD	190	541	412	110	323	243	43	96	713	146
Min	259	454	98	113	248	242	72	136	197	139
Max	1829	2738	2039	990	1960	1340	362	679	3616	792
N	165	163	163	161	156	155	166	164	162	160

 

Table 3d: Historical Control Data: Positive control: 2002

	TA100		TA1535		WP2uvrA-		TA98		TA1537
	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
Mean	460	1880	347	310	621	866	136	239	1953	453
SD	118.2	594.3	204.0	119.7	235.5	350.5	38.2	74.7	803.1	145.9
Min	235	499	80	91	185	210	66	91	486	140
Max	952	3397	1385	810	1295	3406	323	507	4622	1365
N	190	190	188	186	169	168	192	192	188	184

 

Applicant's summary and conclusion

Conclusions:

The test substance was considered to be non-mutagenic under the conditions of this Bacterial Reverse Mutation Test (Ames) that was performed in accordance with OECD TG 471.

Executive summary:

A Bacterial Reverse Mutation Test (Ames) was performed in accordance with OECD TG 471 and under GLP conditions to determine the mutagenic potential of the test substance. Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA were treated with the test substance using the Ames plate incorporation method at five dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolising system (S9-mix). The dose range of the main test was determined in a preliminary toxicity assay using TA100 and WP2uvrA- and was determined to be 50 to 5000 µg/plate. Both main tests were performed at 50, 150, 500, 1500, and 5000 µg/plate. The vehicle (dimethyl sulphoxide) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with and without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated. The test substance caused no visible reduction in the growth of the bacterial background lawn at any dose level. The test substance was, therefore, tested up to the maximum recommended dose level of 5000 µg/plate. No test substance precipitate was observed on the plates at any of the doses tested in either the presence or absence of the S9-mix. No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test substance, either with or without metabolic activation. Based on the observed results, the test substance was considered to be non-mutagenic under the conditions of this test.