1,4-Methanonaphthalen-5(1H)-one, 9-(dichloromethylene)-2,3,4,6,7,8-hexahydro-, oxime, (5E)-

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: other: bacterial reverse mutation test, gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2010 -01-27 till 2010-02-22

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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)

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:

N-[(3E)-11-(dichloromethylidene)tricyclo[6.2.1.0²,⁷]undec-2(7)-en-3-ylidene]hydroxylamine

EC Number:

700-700-2

Cas Number:

1369492-55-6

Molecular formula:

C12H13Cl2NO

IUPAC Name:

N-[(3E)-11-(dichloromethylidene)tricyclo[6.2.1.0²,⁷]undec-2(7)-en-3-ylidene]hydroxylamine

Details on test material:

- Physical state: solid
- Stability under test conditions: not reported
- Storage condition of test material: At room temperature at about 20 °C

Method

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

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:

sodium azide

methylmethanesulfonate

other: 4-nitro-o-phenylene-diamine; 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 pKM 101, WP2 pKM101

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

minor toxic effect in strain TA98

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 and on the incubated agar plates from 1000 - 5000 µg/plate. 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.

In both experiments, no toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), were observed with the exception of strain TA 98 where a minor toxic effect was observed at 5000 µg/plate with S9 mix in experiment I.

Any other information on results incl. tables

         Summary of Results Pre-Experiment/Experiment I

 

 

Metabolic Activation	Test Group	Dose Level (per plate)		Revertant Colony Counts (Mean ±SD)
				TA 1535	TA 1537	TA 98	TA 100	WP2 pKM101	S uvrA pKM101
Without Activation	DMSO			13 ± 1	12 ± 3	25 ± 4	117 ± 5	191 ± 5	363 ± 8
	Untreated			10 ± 4	11 ± 5	31 ± 3	128 ± 19	202 ± 6	372 ± 13
	Test Substance	3 µg		9 ± 2	11 ± 3	25 ± 4	117 ± 7	215 ± 35	327 ± 17
		10 µg		10 ± 2	12 ± 3	27 ± 4	114 ± 7	200 ± 36	322 ± 14
		33 µg		12 ± 3	11 ± 2	25 ± 4	120 ± 21	195 ± 14	334 ± 34
		100 µg		11 ± 4	9 ± 1	25 ± 8	119 ± 4	194 ± 22	321 ± 35
		333 µg		13 ± 4	14 ± 1	28 ± 7	131 ± 10	203 ± 4	320 ± 23
		1000 µg		12 ± 2^P M	8 ± 3^P M	23 ± 6^P	115 ± 2^P	181 ± 9^P	321 ± 52^P
		2500 µg		11 ± 2^P M	6 ± 1^P M	14 ± 2^P M	107 ± 5^P M	176 ± 17^P	335 ± 23^P M
		5000 µg		8 ± 1^P M	6 ± 2^P M	12 ± 3^P M	104 ± 10^P M	93 ± 14^P M	309 ± 11^P M
	NaN3	10 µg		1620 ± 133			2072 ± 214
	4-NOPD	10 µg				345 ± 18
	4-NOPD	50 µg			75 ± 14
	MMS	3.0 µL						3381 ± 703	2653 ± 127
With Activation	DMSO			20 ± 6	17 ± 2	33 ± 6	108 ± 6	211 ± 30	414 ± 31
	Untreated			15 ± 6	22 ± 1	45 ± 4	122 ± 1	257 ± 11	435 ± 30
	Test Substance	3 µg		18 ± 4	18 ± 5	35 ± 4	115 ± 1	221 ± 39	351 ± 79
		10 µg		17 ± 3	19 ± 2	38 ± 9	113 ± 10	217 ± 2	446 ± 44
		33 µg		15 ± 2	16 ± 3	34 ± 6	115 ± 11	205 ± 22	383 ± 41
		100 µg		19 ± 6	20 ± 1	35 ± 2	117 ± 17	193 ± 15	420 ± 13
		333 µg		13 ± 3	18 ± 4	36 ± 4	102 ± 9	212 ± 54	373 ± 23
		1000 µg		15 ± 2^P M	13 ± 5^P M	35 ± 2^P	106 ± 10^P	157 ± 14^P	396 ± 9^P M
		2500 µg		11 ± 1^P M	10 ± 1^P M	27 ± 1^P M	96 ± 13^P M	133 ± 11^P M	408 ± 5^P M
		5000 µg		14 ± 2^P M	8 ± 2^P M	13 ± 2^P M	102 ± 4^P M	106 ± 11^P M	382 ± 12^P M
	2-AA	2.5 µg		249 ± 6	207 ± 37	1719 ± 236	1585 ± 66
	2-AA	10.0 µg						1261 ± 18	1556 ± 6

 

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

 

Study Name: 1389001	Study Code: Harlan CCR 1389001
Experiment: 1389001 HV2 Pre	Date Plated: 16/02/2011
Assay Conditions:	Date Counted: 22/02/2011

 

Metabolic Activation	Test Group	Dose Level (per plate)		Revertant Colony Counts (Mean ±SD)
				TA 1535	TA 1537	TA 98	TA 100	WP2 pKM101	WP2 uvrA pKM101
Without Activation	DMSO			12 ± 2	11 ± 4	23 ± 3	122 ± 6	178 ± 4	343 ± 13
	Untreated			12 ± 8	12 ± 5	33 ± 8	136 ± 3	206 ± 19	363 ± 35
	Test Substance	3 µg		12 ± 5	9 ± 1	25 ± 4	112 ± 4	192 ± 13	347 ± 29
		10 µg		10 ± 2	12 ± 3	20 ± 4	118 ± 16	177 ± 11	320 ± 12
		33 µg		15 ± 6	12 ± 2	25 ± 2	115 ± 12	168 ± 4	274 ± 21
		100 µg		15 ± 2	8 ± 3	22 ± 5	109 ± 9	173 ± 10	275 ± 28
		333 µg		14 ± 5	11 ± 4	22 ± 5	113 ± 23	179 ± 25	335 ± 14
		1000 µg		10 ± 3^P M	12 ± 1^P M	22 ± 5^P M	108 ± 8^P	179 ± 1^P	308 ± 23^P
		2500 µg		13 ± 2^P M	13 ± 2^P M	23 ± 4^P M	112 ± 8^P M	124 ± 9^P M	312 ± 40^P M
		5000 µg		10 ± 2^P M	12 ± 4^P M	17 ± 3^P M	112 ± 8^P M	106 ± 15^P M	244 ± 4^P M
	NaN3	10 µg		2047 ± 55			2262 ± 79
	4-NOPD	10 µg				344 ± 25
	4-NOPD	50 µg			63 ± 4
	MMS	3.0 µL						2911 ± 21	2536 ± 47
With Activation	DMSO			19 ± 3	18 ± 2	37 ± 10	145 ± 8	207 ± 9	396 ± 19
	Untreated			23 ± 7	27 ± 4	44 ± 8	163 ± 16	263 ± 15	501 ± 26
	Test Substance	3 µg		21 ± 6	21 ± 6	38 ± 6	140 ± 10	205 ± 14	339 ± 20
		10 µg		21 ± 3	17 ± 2	37 ± 3	153 ± 12	202 ± 15	382 ± 73
		33 µg		20 ± 6	17 ± 4	33 ± 8	141 ± 8	172 ± 11	359 ± 60
		100 µg		16 ± 4	16 ± 6	36 ± 10	144 ± 3	173 ± 23	365 ± 38
		333 µg		14 ± 2	21 ± 3	40 ± 6	147 ± 27	194 ± 33	368 ± 57
		1000 µg		14 ± 0^P M	14 ± 2^P M	26 ± 3^P M	143 ± 13^P	176 ± 2^P	319 ± 30^P
		2500 µg		10 ± 4^P M	11 ± 3^P M	31 ± 1^P M	113 ± 6^P M	146 ± 8^P M	303 ± 40^P M
		5000 µg		11 ± 3^P M	9 ± 3^P M	29 ± 1^P M	116 ± 6^P M	128 ± 6^P M	292 ± 21^P M
	2-AA	2.5 µg		307 ± 15	209 ± 26	1323 ± 97	1540 ± 56
	2-AA	10.0 µg						1721 ± 77	1322 ± 74

 

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

Applicant's summary and conclusion

Conclusions:

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 theSalmonella typhimurium strains TA 1535, TA 1537, TA 98, and TA 100, and the Escherichia coli strains WP2uvrApKM101 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.

 

In both experiments, no toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), were observed with the exception of strain TA 98 where a minor toxic effect was observed at 5000 µg/plate with S9 mix in experiment I.

 

No substantial increase in revertant colony numbers of any of the five 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.

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.