(1R,4S,4aR,8aS)-9-(dichloromethylidene)octahydro-1,4-methanonaphthalene-5,8-dione

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:

2011 -01-26 till 2011-01-22

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline conforming study under GLP without deviations

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Report date:

2011

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)

Version / remarks:

from May 30, 2008

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

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:

(1R,4S,4aR,8aS)-9-(dichloromethylidene)octahydro-1,4-methanonaphthalene-5,8-dione

EC Number:

700-729-0

Cas Number:

1369500-14-0

Molecular formula:

C12H12Cl2O2

IUPAC Name:

(1R,4S,4aR,8aS)-9-(dichloromethylidene)octahydro-1,4-methanonaphthalene-5,8-dione

Details on test material:

- Physical state: solid, brown
- Storage condition of test material: at room temperature < 30° C, light protected

Method

Species / strain

Species / strain / cell type:

other: TA 1535, TA 1537, TA 98, TA 100, WP2 uvrA pKM101, WP2 pKM101

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/ß-Naphthoflavone induced rat liver S9

Test concentrations with justification for top dose:

3, 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate / pre-experiment/experiment I; experiment II

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The solvent was chosen because of its solubilisation properties and its relative non-toxicity to the bacteria.

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: sodium azide; 4-nitro-o-phenylene-diamine; methyl methane sulfonate, 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium; in agar; plate incorporation; preincubation;


DURATION
- Preincubation period: 1 hour
- Exposure duration: 72 hours


NUMBER OF REPLICATIONS: 3 plates


DETERMINATION OF CYTOTOXICITY
A reduction in the number of spontaneous revertants (below the induction factor of 0.5) or a clearing of the bacterial background lawn.

Evaluation criteria:

A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice the colony count of the corresponding solvent control is observed.

A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.

An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.

A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.

Statistics:

According to the OECD guideline 471, a statistical analysis of the data is not mandatory.

Results and discussion

Test results

Species / strain:

other: TA 1535, TA 1537, TA 98, TA 100, WP2 uvrA pKM101, WP2 pKM101

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation:
Precipitation of the test item was observed in the overlay agar in the test tubes from 1000 - 5000 µg/plate. Precipitation was also observed on the incubated agar plates without S9 mix from 333 - 5000 µg/plate in experiment I and at 2500 and 5000 µg/plate in experiment II, and with S9 mix from 1000 - 5000 µg/plate in both experiments. The undissolved particles had no influence on the data recording.
- Other confounding effects:
COMPARISON WITH HISTORICAL CONTROL DATA: performed
ADDITIONAL INFORMATION ON CYTOTOXICITY:
The plates incubated with the test item showed normal background growth up to 5000 µg/plate with and without metabolic activation in both independent experiments.

Toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), were observed at higher concentrations in strain WP2 pKM101 without S9 mix and in strains TA1535, TA98, WP2 pKM101, and WP2 uvrA pKM101 with S9 mix in experiment I. In experiment II, toxic effects were observed without S9 mix in all strains and with S9 mix in strains TA 1537, TA 97, and WP2 pKM101.

Remarks on result:

other: other: reverse mutation assay

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

 Summary of Results Pre-Experiment/Experiment

 

Metabolic Activation	Test Group	Dose Level (per plate)		Revertant Colony Counts (Mean ±SD)
				TA1535	TA1537	TA98	TA100	WP2 pKM101	WP2 uvrA pKM101
Without Activation	DMSO			15 ± 4	9 ± 3	25 ± 2	138 ± 4	170 ± 5	340 ± 10
	Untreated			19 ± 3	10 ± 4	30 ± 5	156 ± 2	224 ± 10	383 ± 34
	Test substance	3 µg		15 ± 4	8 ± 1	23 ± 5	142 ± 20	187 ± 43	417 ± 18
		10 µg		15 ± 5	8 ± 1	25 ± 4	117 ± 13	180 ± 25	334 ± 25
		33 µg		13 ± 4	8 ± 1	26 ± 3	132 ± 21	147 ± 13	322 ± 18
		100 µg		13 ± 2	7 ± 2	20 ± 2	134 ± 18	151 ± 22	301 ± 20
		333 µg		19 ± 2^P	9 ± 1^P	28 ± 6^P	143 ± 12^P	162 ± 21^P	397 ± 25^P
		1000 µg		13 ± 3^P	7 ± 1^P	25 ± 8^P	158 ± 3^P	101 ± 1^P	347 ± 43^P
		2500 µg		9 ± 1^P M	6 ± 1^P	21 ± 5^P M	106 ± 1^P	100 ± 2^P	259 ± 7^P
		5000 µg		8 ± 2^P M	5 ± 1^P M	22 ± 4^P M	86 ± 10^P M	42 ± 9^P M	258 ± 25^P M
	NaN3	10 µg		2129 ± 83			2025 ± 101
	4-NOPD	10 µg				277 ± 20
	4-NOPD	50 µg			76 ± 3
	MMS	3.0 µL						2531 ± 197	3035 ± 329
With Activation	DMSO			18 ± 2	13 ± 3	32 ± 5	126 ± 9	174 ± 6	404 ± 24
	Untreated			24 ± 5	22 ± 2	33 ± 10	145 ± 6	241 ± 29	497 ± 4
	Test substance	3 µg		20 ± 4	14 ± 7	32 ± 5	124 ± 19	199 ± 23	395 ± 51
		10 µg		20 ± 2	11 ± 4	34 ± 7	134 ± 14	180 ± 35	353 ± 42
		33 µg		14 ± 5	13 ± 2	33 ± 9	113 ± 7	188 ± 20	344 ± 36
		100 µg		16 ± 1	13 ± 3	29 ± 5	132 ± 26	137 ± 4	316 ± 35
		333 µg		16 ± 7	18 ± 2	29 ± 4	147 ± 8	151 ± 3	344 ± 19
		1000 µg		8 ± 2^P M	8 ± 2^P M	30 ± 6^P	121 ± 11^P	113 ± 12^P	294 ± 32^P
		2500 µg		8 ± 2^P M	8 ± 2^P M	13 ± 2^P M	117 ± 3^P	105 ± 12^P	233 ± 9^P
		5000 µg		8 ± 1^P M	7 ± 3^P M	12 ± 2^P M	96 ± 8^P M	41 ± 8^P M	168 ± 34^P
	2-AA	2.5 µg		392 ± 36	269 ± 40	1731 ± 173	1984 ± 352
	2-AA	10.0 µg						1412 ± 60	1443 ± 27

 

Key to Positive Controls		Key to Plate Postfix Codes
NaN3 2-AA 4-NOPD MMS	sodium azide 2-aminoanthracene 4-nitro-o-phenylene-diamine methyl methane sulfonate	P M	Precipitate Manual count

 Summary of Results Experiment II

 

Metabolic Activation	Test Group	Dose Level (per plate)		Revertant Colony Counts (Mean ±SD)
				TA1535	TA1537	TA98	TA100	WP2 pKM101	WP2 uvrA pKM101
Without Activation	DMSO			16 ± 1	10 ± 3	22 ± 6	124 ± 10	183 ± 21	365 ± 20
	Untreated			20 ± 1	13 ± 2	30 ± 1	160 ± 5	202 ± 40	372 ± 18
	Test substance	3 µg		16 ± 4	11 ± 4	21 ± 1	130 ± 5	199 ± 5	331 ± 30
		10 µg		14 ± 2	11 ± 4	24 ± 3	115 ± 15	185 ± 13	312 ± 10
		33 µg		17 ± 3	10 ± 2	24 ± 2	104 ± 3	178 ± 13	324 ± 25
		100 µg		17 ± 2	13 ± 1	23 ± 4	111 ± 11	177 ± 7	336 ± 19
		333 µg		15 ± 2	14 ± 1	21 ± 1	89 ± 12	173 ± 19	334 ± 21
		1000 µg		10 ± 2	12 ± 4	23 ± 2	78 ± 22	110 ± 4	288 ± 50
		2500 µg		8 ± 1^P	7 ± 2^P	21 ± 2^P	54 ± 8^P	93 ± 4^P	195 ± 11^P
		5000 µg		2 ± 0^P M	1 ± 1^P M	5 ± 2^P M	21 ± 5^P M	50 ± 6^P M	149 ± 17^P M
	NaN3	10 µg		1735 ± 114			2196 ± 108
	4-NOPD	10 µg				374 ± 14
	4-NOPD	50 µg			74 ± 9
	MMS	3.0 µL						3224 ± 165	2523 ± 147
With Activation	DMSO			15 ± 7	21 ± 1	34 ± 6	133 ± 22	200 ± 6	397 ± 11
	Untreated			20 ± 6	26 ± 5	44 ± 7	154 ± 12	249 ± 39	412 ± 47
	Test substance	3 µg		17 ± 7	22 ± 2	37 ± 6	136 ± 10	196 ± 27	373 ± 18
		10 µg		16 ± 6	19 ± 3	29 ± 7	130 ± 7	186 ± 38	379 ± 9
		33 µg		11 ± 3	19 ± 7	31 ± 5	129 ± 18	219 ± 22	368 ± 18
		100 µg		14 ± 1	19 ± 4	36 ± 1	116 ± 12	221 ± 17	369 ± 15
		333 µg		18 ± 2	19 ± 3	33 ± 9	125 ± 15	198 ± 26	358 ± 19
		1000 µg		15 ± 2^P	15 ± 5^P	35 ± 2^P	104 ± 13^P	122 ± 4^P	332 ± 26^P
		2500 µg		12 ± 2^P M	11 ± 4^P M	22 ± 2^P M	84 ± 12^P M	77 ± 6^P M	233 ± 14^P M
		5000 µg		9 ± 3^P M	6 ± 2^P M	14 ± 1^P M	85 ± 6^P M	65 ± 11^P M	201 ± 22^P M
	2-AA	2.5 µg		296 ± 16	199 ± 1	1447 ± 136	2103 ± 137
	2-AA	10.0 µg						1459 ± 66	1664 ± 73

 

Key to Positive Controls		Key to Plate Postfix Codes
NaN3 2-AA 4-NOPD MMS	sodium azide 2-aminoanthracene 4-nitro-o-phenylene-diamine methyl methane sulfonate	P M	Precipitate Manual count

Toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), were observed at the following concentrations (μg/plate):

Strain	Experiment I		Experiment II
	without S9 mix	with S9 mix	without S9 mix	with S9 mix
TA1535	/	2500	5000	/
TA1537	/	/	5000	5000
TA98	/	2500, 5000	5000	5000
TA100	/	/	2500, 5000	/
WP2 pKM101	5000	5000	5000	2500, 5000
WP2 uvrA pKM101	/	5000	5000	/

/ = no toxic effects observed

 

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used. Therefore, the test substance is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.

Executive summary:

This study was performed to investigate the potential of the test substance to induce gene mutations in the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using the Salmonella typhimurium strains TA1535, TA1537, TA98, and TA100, and the Escherichia coli strains WP2 uvrA pKM101 and WP2 pKM101.

The plates incubated with the test item showed normal background growth up to 5000 μg/plate with and without metabolic activation in both independent experiments.

Toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5),

were observed at higher concentrations in strain WP2 pKM101 without S9 mix and in strains TA1535, TA98, WP2 pKM101, and WP2 uvrA pKM101 with S9 mix in experiment I. In experiment II, toxic effects were observed without S9 mix in all strains and with S9 mix in strains TA 1537, TA97, and WP2 pKM101.

No substantial increase in revertant colony numbers of any of the six tester strains was observed following treatment with the test substance at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.