methyl-bis[2-(4-methylalicyclyl)propyl]dihydro-heteropolycyle

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:

April 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

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

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Test material form:

liquid

Method

Species / strain

Species / strain / cell type:

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

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:

PRE-EXPERIMENT / EXPERIMENT I:
3, 10, 33, 100, 333, 1000, 2500 and 5000 µg/plate

EXPERIMENT II:
- Without S9 mix: 33, 100, 333, 1000, 2500 and 5000 µg/plate
- With S9 mix: 3, 10, 33, 100, 333, 1000, 2500 and 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Acetone
- Justification for choice of solvent/vehicle: better solubility properties, relative nontoxic to 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 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 (strains TA 98, TA 100, and TA 102) or thrice (strains TA 1535 and TA 1537) 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

Key result

Species / strain:

other: TA 1535, TA 1537, TA 98, TA 100, TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

with S9 mix

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS:
- Effects of pH: none
- Precipitation: Precipitation of the test item in the overlay agar on the incubated agar plates was observed from 2500 to 5000 µg/plate in the presence of metabolic activation in both experiments. The undissolved particles had no influence on the data recording.

COMPARISON WITH HISTORICAL CONTROL DATA: performed

Any other information on results incl. tables

CYTOTOXICITY

The plates incubated with the test item showed normal background growth up to 5000 µg/plate with and without S9 mix in all strains used. Toxic effects, evident as a reduction in the number of revertants (below the induction factor of 0.5), were observed at the following concentrations (µg/plate):

Strain	Experiment I		Experiment II
	Without S9	With S9	Without S9	With S9
TA 1535	no	5000	no	no
TA 1537	no	5000	no	5000
TA 98	no	5000	no	no
TA 100	no	5000	no	no
TA 102	no	5000	no	2500-5000

RESULTS

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with test item at any concentration 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.

Appropriate reference mutagens were used as positive controls. They showed a distinct increase in induced revertant colonies.

SUMMARY EXPERIMENT I

 

Metabolic Activation	Test Group	Dose per plate	Revertant Colony Counts (Mean ±SD)
			TA 1535	TA 1537	TA 98	TA 100	TA 102
Without	Acetone		15 ± 7	11 ± 3	31 ± 10	126 ± 7	458 ± 29
	Untreated		12 ± 3	10 ± 3	34 ± 1	128 ± 13	416 ± 19
	test item	3 µg	15 ± 6	12 ± 3	27 ± 4	130 ± 12	417 ± 30
	test item	10 µg	15 ± 6	10 ± 2	29 ± 5	127 ± 10	443 ± 5
	test item	33 µg	15 ± 7	11 ± 2	34 ± 11	131 ± 6	461 ± 15
	test item	100 µg	14 ± 5	13 ± 4	26 ± 9	137 ± 12	476 ± 26
	test item	333 µg	15 ± 4	10 ± 4	31 ± 10	130 ± 9	411 ± 6
	test item	1000 µg	15 ± 8	11 ± 2	26 ± 5	130 ± 20	421 ± 32
	test item	2500 µg	16 ± 5	10 ± 2	27 ± 5	142 ± 11	430 ± 49
	test item	5000 µg	13 ± 5	8 ± 4	26 ± 1	141 ± 12	389 ± 15
	NaN3	10 µg	1972 ± 38			2206 ± 29
	4-NOPD	10 µg			292 ± 25
	4-NOPD	50 µg		74 ± 2
	MMS	2.0 µL					4996 ± 449
With	Acetone		19 ± 7	17 ± 4	43 ± 2	162 ± 24	583 ± 13
	Untreated		18 ± 3	17 ± 5	36 ± 6	171 ± 12	564 ± 7
	test item	3 µg	15 ± 4	20 ± 3	38 ± 4	152 ± 14	551 ± 55
	test item	10 µg	17 ± 2	21 ± 4	43 ± 6	165 ± 20	610 ± 35
	test item	33 µg	16 ± 5	17 ± 1	38 ± 6	175 ± 5	592 ± 39
	test item	100 µg	16 ± 5	18 ± 3	40 ± 3	182 ± 15	626 ± 24
	test item	333 µg	13 ± 1	18 ± 4	46 ± 8	155 ± 12	576 ± 27
	test item	1000 µg	19 ± 2	22 ± 1	39 ± 9	165 ± 21	528 ± 28
	test item	2500 µg	23 ± 3^P	8 ± 3^P M	38 ± 5^P	165 ± 5^P	177 ± 1^P
	test item	5000 µg	4 ± 2^P M	3 ± 1^P M	2 ± 1^P M	52 ± 6^P M	28 ± 9^P M
	2-AA	2.5 µg	285 ± 21	190 ± 28	1384 ± 35	1850 ± 50
	2-AA	10.0 µg					2101 ± 78

  

SUMMARY EXPERIMENT II

 

Metabolic Activation	Test Group	Dose per plate	Revertant Colony Counts (Mean ±SD)
			TA 1535	TA 1537	TA 98	TA 100	TA 102
Without	Acetone		12 ± 3	9 ± 1	32 ± 7	127 ± 17	389 ± 6
	Untreated		16 ± 4	9 ± 1	33 ± 3	131 ± 8	384 ± 14
	test item	33 µg	14 ± 1	10 ± 1	33 ± 3	121 ± 6	412 ± 14
	test item	100 µg	12 ± 5	9 ± 2	28 ± 4	120 ± 21	435 ± 19
	test item	333 µg	15 ± 1	10 ± 3	29 ± 4	128 ± 9	412 ± 18
	test item	1000 µg	12 ± 4	12 ± 3	26 ± 2	129 ± 8	364 ± 36
	test item	2500 µg	12 ± 5	8 ± 2	33 ± 6	117 ± 12	372 ± 31
	test item	5000 µg	15 ± 2	7 ± 2	28 ± 5	129 ± 14	388 ± 20
	NaN3	10 µg	2113 ± 40			2345 ± 48
	4-NOPD	10 µg			343 ± 17
	4-NOPD	50 µg		75 ± 1
	MMS	2.0 µL					4245 ± 164
With	Acetone		18 ± 5	15 ± 1	44 ± 2	149 ± 17	512 ± 10
	Untreated		16 ± 3	20 ± 7	46 ± 7	159 ± 7	509 ± 29
	test item	3 µg	15 ± 3	17 ± 3	43 ± 7	134 ± 7	450 ± 11
	test item	10 µg	15 ± 2	17 ± 2	46 ± 4	144 ± 22	446 ± 48
	test item	33 µg	17 ± 4	16 ± 1	45 ± 4	153 ± 3	506 ± 55
	test item	100 µg	17 ± 6	18 ± 5	41 ± 3	157 ± 22	498 ± 93
	test item	333 µg	16 ± 3	18 ± 7	51 ± 5	125 ± 12	434 ± 49
	test item	1000 µg	16 ± 5	16 ± 6	43 ± 3	157 ± 14	435 ± 50
	test item	2500 µg	20 ± 7^P	16 ± 5^P	37 ± 4^P	139 ± 8^P	160 ± 24^P
	test item	5000 µg	13 ± 4^P M	7 ± 3^P M	25 ± 4^P M	115 ± 11^P M	116 ± 10^P M
	2-AA	2.5 µg	251 ± 12	102 ± 9	1069 ± 17	1045 ± 150
	2-AA	10.0 µg					2439 ± 35

 

Key to Positive Controls

NaN3          sodium azide

2-AA           2-aminoanthracene

MMS          methyl methane sulfonate

4-NOPD      4-nitro-o-phenylene-diamine

 

Key to Plate Postfix Codes

P                Precipitate

M               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.

Executive summary:

SUMMARY

This study was performed to investigate the potential of the test item to induce gene mutations according to the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and TA 102.

The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate. The test item was tested at the following concentrations:

Pre-Experiment / Experiment I: 

3, 10, 33, 100, 333, 1000, 2500 and 5000 µg/plate

Experiment II:

- without S9 -mix: 33, 100, 333, 1000, 2500 and 5000 µg/plate

- with S9 -mix: 3, 10, 33, 100, 333, 1000, 2500 and 5000 µg/plate

Precipitation of the test item in the overlay agar on the incubated agar plates was observed from 2500 to 5000 μg/plate in the presence of metabolic activation in both experiments. The undissolved particles had no influence on the data recording.

The plates incubated with the test item showed normal background growth up to 5000 µg/plate with and without S9 mix in all strains used.

No toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in the test groups without metabolic activation. With metabolic activation toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred.

No substantial increase in revertant colony numbers of any of the fivetester strains was observed following treatment with the test item 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.

Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies.