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

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames test (bacterial reverse mutation in vitro): Negative

A key study conducted according to guideline OECD 471 and under GLP is available. The study is considered to be relevant, reliable (Klimisch 1) and adequate for the purposes of risk assessment, classification and labeling.

Chromosome aberration: Negative

A key study conducted according to guideline OECD 473 and under GLP is available. The study is considered to be relevant, reliable (Klimisch 1) and adequate for the purposes of risk assessment, classification and labeling.

Cell mutation assay: Negative

A key study conducted according to guideline OECD 476 and under GLP is available. The study is considered to be relevant, reliable (Klimisch 1) and adequate for the purposes of risk assessment, classification and labeling.

Link to relevant study records

Reference

Reference 1

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

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

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

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.

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.

         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

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.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

According to OECD guideline 473 Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive, done to a valid guideline and the study was conducted under GLP conditions.

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

lymphocytes: human

Details on mammalian cell type (if applicable):

- Type and identity of media: Dulbeccos's modified Eagle's medium/Ham's F12 medium
- Properly maintained: yes

Metabolic activation:

with and without

Metabolic activation system:

rat liver S9

Test concentrations with justification for top dose:

With metabolic activation:
Experiment I: 5.5, 9.6, 16.8 µg/mL
Experiment II: 5.5, 9.6, 16.8 µg/mL

Without metabolic activation:
Experiment I: 9.6, 16.8, 29.3, 157.3 µg/mL
Experiment II: 29.3, 51.3, 89.9, 157.3 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: solubility and relatively low cytotoxicity in accordance to the OECD Guideline 473

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

cyclophosphamide

Details on test system and experimental conditions:

Two independent experiments were performed. In Experiment I the exposure period was 4 hours with and without metabolic activation. In Experiment II the exposure period was 4 hours with S9 mix and 22 hours without S9 mix. The chromosomes were prepared 22 hours after start of treatment with the test item. Evaluation of two cultures per dose group.
METHOD OF APPLICATION: in culture medium

DURATION
- Exposure duration: 4 hours (+/- S9 mix) and 22 hours (- S9 mix)
- Fixation time (start of exposure up to fixation or harvest of cells): 22 hours


SPINDLE INHIBITOR (cytogenetic assays): Colcemid
STAIN (for cytogenetic assays): Giemsa


NUMBER OF REPLICATIONS: 2


NUMBER OF CELLS EVALUATED: 100 per culture


DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

Evaluation criteria:

Evaluation of the cultures was performed (according to standard protocol of the "Arbeitsgruppe der Industrie, Cytogenetik") using NIKON microscopes with 100x oil immersion objectives. Breaks, fragments, deletions, exchanges, and chromosome disintegrations were recorded as structural chromosome aberrations. Gaps were recorded as well but not included in the calculation of the aberration rates. 100 well spread metaphases per culture were scored for cytogenetic damage on coded slides.
Only metaphases with characteristic chromosome numbers of 46 ± 1 were included in the analysis. To describe a cytotoxic effect the mitotic index (% cells in mitosis) was determined.

Statistics:

Statistical significance was confirmed by means of the Fisher´s exact test (p < 0.05).

Species / strain:

lymphocytes: human

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

The test item, dissolved in DMSO, was assessed for its potential to induce chromosomal aberrations in human lymphocytes in vitro in the absence and presence of metabolic activation by S9 mix.

Two independent experiments were performed. In Experiment I, the exposure period was 4 hours with and without S9 mix. In Experiment II, the exposure periods were 4 hours with S9 mix and 22 hours without S9 mix. The chromosomes were prepared 22 hours after start of treatment with the test item.

In each experimental group two parallel cultures were analysed. 100 metaphases per culture were scored for structural chromosomal aberrations. 1000 cells per culture were counted for determination of mitotic index.

The highest treatment concentration in this study, 2581.0 µg/mL (approx. 10 mM) was chosen with regard to the molecular weight of the test item and with respect to the OECD Guideline for in vitro mammalian cytogenetic tests.

In Experiment I precipitation of the test item in the culture medium was observed at 29.3 µg/mL and above in the absence of S9 mix and at 16.8 µg/mL in the presence of S9 mix at the end of treatment. In Experiment II precipitation of the test item in the culture medium was observed at 51.3 µg/mL and above in the absence of S9 mix and at 9.6 µg/mL in the presence of S9 mix at the end of treatment. No relevant influence or decrease in the osmolarity or pH value was observed (Experiment I: solvent control: 386 mOsm, pH 7.4 versus 357 mOsm and pH 7.5 at 2581.0 µg/mL; Experiment II: solvent control: 367 mOsm, pH 7.3 versus 355 mOsm and pH 7.3 at 2581.0 µg/mL).

In Experiment I in the absence and presence of S9 mix and in Experiment II in the presence of S9 mix, no cytotoxicity was observed up to the highest applied concentration. However, the mitotic index was markedly decreased to 63.5 % of control at the highest evaluated concentration in the absence of S9 mix. In Experiment II in the absence of S9 mix cytotoxicity was observed at the highest evaluated concentration (40.9 % of control).

In both experiments no statistically significant and biologically relevant increase was observed at the concentrations evaluated. The aberration rates of the cells after treatment with the test item (0.0 – 2.5 % aberrant cells, excluding gaps) were close to the solvent control values (0.5 – 2.0 % aberrant cells, excluding gaps) and within the range of the laboratory historical solvent control data.

No evidence of an increase in polyploid metaphases was noticed after treatment with the test item as compared to the control cultures.

In the experiments, either EMS (825 µg/mL) or CPA (7.5 or 15.0 µg/mL) were used as positive controls and showed distinct increases in cells with structural chromosome aberrations.

Summary of results of the chromosomal aberration study

Exp.	Preparation	Test item	Mitotic indices	Aberrant cells
	interval	concentration	in %	in %
		in µg/mL	of control	incl. gaps*	excl. gaps*	carrying exchanges
Exposure period 4 hrs without S9 mix
I	22 hrs	Solvent control^1	100.0	2.0	2.0	0.0
		Positive control^2	99.7	12.0	12.0^S	2.5
		9.6	100.7	2.5	2.5	0.0
		16.8	101.7	1.0	1.0	0.0
		29.3^P	84.4	0.0	0.0	0.0
		157.3^P	63.5	2.5	2.5	0.0
Exposure period 22 hrs without S9 mix
II	22 hrs	Solvent control^1	100.0	0.5	0.5	0.0
		Positive control^2	48.5	13.5	13.5^S	2.0
		29.3	97.6	2.0	2.0	0.0
		51.3^P	94.8	1.5	1.5	0.0
		89.9^P	103.0	2.0	1.5	0.0
		157.3^P	40.9	3.0	2.5	0.5

*      Including cells carrying exchanges

^P      Precipitation occurred at the end of treatment

^S      Aberration frequency statistically significant higher than corresponding control values

¹       DMSO      0.5 % (v/v)

²         EMS     825.0 µg/mL

 

Summary of results of the chromosomal aberration study (continued)

Exp.	Preparation	Test item	Mitotic indices	Aberrant cells
	interval	concentration	in %	in %
		in µg/mL	of control	incl. gaps*	excl. gaps*	carrying exchanges
Exposure period 4 hrs with S9 mix
I	22 hrs	Solvent control^1	100.0	1.5	1.0	0.5
		Positive control^2	38.8	12.0	11.0^S	2.0
		5.5	99.7	1.0	1.0	0.0
		9.6	90.4	1.0	1.0	0.0
		16.8^P	89.9	1.5	1.5	0.0
II	22 hrs	Solvent control^1	100.0	2.5	1.5	0.0
		Positive control^3	69.2	9.0	9.0^S	1.0
		5.5	109.9	0.5	0.5	0.0
		9.6^P	79.1	1.0	1.0	0.0
		16.8^P	89.3	1.0	0.5	0.0

*      Including cells carrying exchanges

^P      Precipitation occurred at the end of treatment

^S      Aberration frequency statistically significant higher than corresponding control values

¹       DMSO      0.5 % (v/v)

²       CPA        15.0 µg/mL

³       CPA          7.5 µg/mL

Conclusions:

In conclusion, it can be stated that under the experimental conditions reported, the test item did not induce structural chromosomal aberrations in human lymphocytes in vitro. Therefore, CA4920 is considered to be non-clastogenic in this chromosome aberration test, when tested up to cytotoxic and/or precipitating concentrations.

Executive summary:

This in vitro assay was performed to assess the potential of the test substance to induce structural chromosomal aberrations in the absence and presence of an exogenous metabolic activation system (liver S9 mix from phenobarbital/b-naphthoflavone treated male rats).

In each experimental group two parallel cultures were analysed. Per culture 100 metaphases were scored for structural chromosomal aberrations.

 

The highest applied concentration in this study (2581.0 µg/mL of the test item, approx. 10 mM) was chosen with regard to the molecular weight of the test item and with respect to the current OECD Guideline 473.

Dose selection of the cytogenetic experiments was performed considering the toxicity data and test item precipitation and in accordance with OECD Guideline 473. 

 

In Experiment I in the absence and presence of S9 mix and in Experiment II in the presence of S9 mix, no marked cytotoxicity was observed up to the highest applied concentration. In Experiment II in the absence of S9 mix cytotoxicity was observed at the highest evaluated concentration. 

 

In both independent experiments, neither a statistically significant nor a biologically relevant increase in the number of cells carrying structural chromosomal aberrations was observed after treatment with the test item.

 

No evidence of an increase in polyploid metaphases was noticed after treatment with the test item as compared to the control cultures.

 

Appropriate mutagens were used as positive controls. They induced statistically significant increases (p < 0.05) in cells with structural chromosome aberrations.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

from 2011-01-19 to 2011-03-21 (experimental phase)

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

from February 1998

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Version / remarks:

from 1998

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

from May 30, 2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Target gene:

Thymidine Kinase Locus

Species / strain / cell type:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

Pre-Experiment:
without metabolic activation: 20.3, 40.6, 81.2, 162.5, 325.0, 650.0, 1300.0, 2600.0 µg/mL
with metabolic activation: 20.3, 40.6, 81.2, 162.5, 325.0, 650.0, 1300.0, 2600.0 µg/mL

Experiment I:
without S9 mix: 2.5, 5.0, 10.0, 20.0, 40.0, 60.0 (not continued) µg/mL
wtih S9 mix: 10.0, 20.0, 40.0, 80.0, 120.0, 160.0 (not continued) µg/mL

Experiment II:
without S9 mix: 2.5, 5.0, 10.0, 20.0, 30.0, 40.0 (not continued) µg/mL
wtih S9 mix: 10.0, 20.0, 40.0, 80.0, 100.0, 120.0 (not continued) µg/mL

Experiment III:
wtih S9 mix: 40.0, 50.0, 60.0 (culture II continued only), 70.0 (not continued) , 80.0 (not continued) , 90.0 (not continued) µg/mL

Vehicle / solvent:

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

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: without metaboilc activation: Methyl Methane Sulfonate (MMS); with metabolic activation: Cyclophosphamide (CPA)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4 hours
- Expression time (cells in growth medium): 48 hours
- Selection time (if incubation with a selection agent): 10-15 days

SELECTION AGENT (mutation assays): RPMI 1640 (complete culture medium) by addition of 5 µg/mL TFT

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: 10^6

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency

OTHER EXAMINATIONS:
- Determination of polyploidy: not done
- Determination of endoreplication: not done

Evaluation criteria:

A mutation assay is considered acceptable if it meets the following criteria:
1. All plates, from either the cloning efficiency 2 or the TFT resistance-testing portion of the experiment are analysable.
2. The absolute cloning efficiency 2 at the time of mutant selection (CE) of the negative/vehicle controls is 65 – 120%.
3. The total suspension growth of the negative/vehicle control calculated by the day 1 fold-increase in cell number multiplied by the day 2 fold-increase in cell number is 8 – 32.
4. The range of the negative/vehicle control mutant frequency is in the range of 50 – 170 × 10^-6.
5. The positive controls (MMS and CPA) should yield an absolute increase in total MF that is an increase above spontaneous background MF (an induced MF [IMF]) of at least 300 × 10^-6 cells. At least 40 % of the induced mutation frequency (IMF) should be reflected in the small colony MF. Alternatively, the positive controls should induce at least 150 small colonies.
6. The upper limit of cytotoxicity observed in the positive control culture should be the same as for the experimental cultures (i.e. the relative total growth – RTG- should be greater than 10 % of the concurrent selective control group).
7. The highest concentration of the test item should be 10 mM or 5000 µg/mL, unless limited by toxicity or solubility of the test item. If toxicity occurred, the highest concentration should lower the cloning efficiency 1 or the relative total growth to 10 to 20 % of survival. If precipitation is noted, the highest analysed concentration should be the lowest concentration where precipitation is observed by the naked eye.

Statistics:

A linear regression (least squares) was performed to assess a possible dose dependent increase of mutant frequencies using statistics software. The number of mutant colonies obtained for the groups treated with the test item was compared to the solvent control groups. A trend is judged as significant whenever the p-value (probability value) is below 0.05. However, both, biological relevance and statistical significance were considered
together.

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Experiment I: Relevant cytotoxic effectsat 40 µg/mL without S9 mix, at 80.0 µg/mL with S9 mix. Experiment II: cytotoxicity observed at 30.0 µg/mL without S9 mix,at 80.0 µg/mL with S9 mix. Experiment III: strong cytotoxic effects at 40 µg/mL and above.

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: The test medium was checked for precipitation visible to the naked eye at the end of the treatment period just before the test item was removed. Precipitation was noted in the pre-experiment at 162.5 µg/mL and above without S9 mix and at 81.3 µg/mL with S9 mix. In experiment I precipitation was observed at 160.0 µg/mL with S9 mix. In experiment II precipitation occurred at 100 µg/mL and above with S9 mix. In experiment III precipitation was noted at 60 µg/mL and above.

RANGE-FINDING/SCREENING STUDIES: The pre-experiment was performed in the presence and absence of metabolic activation with a treatment time of 4 hours. Test item concentrations between 20.3 µg/mL and 2600 µg/mL were used. The highest concentration in the pre-experiment was chosen with regard to the molecular weight (258.1 g/mol) and the purity (99.2 % w/w) of the test item. The stock solution formed a clear solution in DMSO. Cytotoxic effects leading to RSG values below 50% were observed at 40.6 µg/mL in the absence of metabolic activation and at 162.5 µg/mL in the presence of metabolic activation. The test medium was checked for precipitation at the end of the treatment period (4 hours) before the test item was removed. Precipitation occurred at 162.5 µg/mL and above without metabolic activation and at 81.3 µg/mL with metabolic activation. Both, pH value and osmolarity were determined in the pre-experiment at the maximum concentration of the test item and at the solvent control without metabolic activation. No relevant increase of the osmolarity or pH value was observed (solvent control: 365 mOsm, pH 7.38 versus 332 mOsm and pH 7.37 at 2600 µg/mL).

COMPARISON WITH HISTORICAL CONTROL DATA: MMS (19.5 µg/mL) and CPA (3.0 and 4.5 µg/mL) were used as positive controls and showed a distinct increase in induced total mutant colonies at acceptable levels of toxicity with at least one of the concentrations of the controls. The relative total growth of the MMS control in the second culture of the second experiment without metabolic activation was just 7.9 but the mean value of both parallel cultures (13.5 and 7.9, equal to a mean of 10.7) was acceptable.

ADDITIONAL INFORMATION ON CYTOTOXICITY: The recommended cytotoxic range of approximately 10-20% RTG or relative cloning efficiency (survival) was covered in both parallel cultures of the first experiment with and without metabolic activation. In the second experiment this range was solely covered in both parallel cultures without metabolic activation. In the presence of metabolic activation severe cytotoxic effects occurred at 80.0 µg/mL in both parallel cultures. Based on the steep gradient of toxicity virtually no cytotoxicity was noted at the next lower concentration of 40.0 µg/mL. Therefore, a supplementary experiment was performed with very narrowly spaced concentrations within the cytotoxic range to investigate the cytotoxic region of the steep toxicity gradient.
The data generated at 120 µg/mL with metabolic activation in experiment I in both cultures and at 100 µg/mL in experiment II, culture I and at 50.0 in experiment III, culture I are not considered valid since the relative total growth (RTG) fell below 1%.

Increase of mutation frequency

A single isolated increase of the mutation frequency exceeding the threshold occurred in the second culture of the second experiment without metabolic activation at 20.0 µg/mL. However, this increase was not reproduced in the parallel cultures performed under identical experimental conditions. Furthermore, the increase of the mutation frequency above the threshold was not dose dependent as indicated by the lacking statistical significance and was consequently judged as biologically irrelevant artefact.

Viablitiy

The viability exceeded the upper limit of 120% in the first culture of experiment III. The data are acceptable however, since the cloning efficiency of the parallel culture remained within the acceptable range.

Summary of Results, Experiment I, II, and III

	Conc. µg/mL	S9 mix	Relative cloning efficiency	Relative total growth	Mutant colonies/10^6 cells	threshold	Relative cloning efficiency	Relative total growth	Mutant colonies/10^6 cells	threshold
Experiment I/4h treatment			Culture I				Culture II
Solv.control with DMSO		-	100	100	91	217	100	100	97	223
Pos.control with MMS	19.5	-	98.4	31.5	266	217	107.9	32.1	345	223
Test item	2.5	-	105.1	131.4	75	217	136.3	139	108	223
Test item	5	-	142.8	83.1	108	217	200	96.4	128	223
Test item	10	-	108.7	86.2	109	217	144.2	115.4	103	223
Test item	20	-	89.6	85.7	87	217	150	82.2	136	223
Test item	40	-	11.3	3.5	132	217	11.8	6.3	92	223
Test item	60	-	10.1	Culture was not continued #			3.9	Culture was not continued #
Solv.control with DMSO		+	100	100	76	202	100	100	99	225
Pos.control with CPA	3	+	65	57.9	198	202	98.4	64.5	387	225
Pos. Control with CPA	4.5	+	44.8	30.8	340	202	66.7	42.5	379	225
Test item	10	+	91.1	101.5	61	202	105.1	171.1	73	225
Test item	20	+	94.4	69.1	97	202	88.1	104.3	97	225
Test item	40	+	68	39.7	70	202	84.2	89.9	85	225
Test item	80	+	39.4	20.9	68	202	59.6	28.2	82	225
Test item	120	+	3.2 *	0.8 *	123 *	202 *	2.6 *	0.7 *	189 *	225 *
Test item	160 (p)	+	0.3	Culture was not continued #			0.6	Culture was not continued #
Experiment II/ 4h treatment			Culture I				Culture II
Solv.control with DMSO		-	100	100	82	208	100	100	87	213
Pos.control with MMS	19.5	-	37.3	13.5	739	208	123.8	7.9	695	213
Test item	2.5	-	67.3	121.1	115	208	52.4	70	118	213
Test item	5	-	130.7	144.5	130	208	88.1	78.8	166	213
Test item	10	-	78.9	125.1	102	208	100	48.1	158	213
Test item	20	-	92.4	141.8	61	208	74.8	33	248	213
Test item	30	-	25.8	53.5	72	208	20.5	16.2	117	213
Test item	40	-	3.6	Culture was not continued #			8.8	Culture was not continued #
Solv.control with DMSO		+	100	100	116	242	100	100	80	206
Pos.control with CPA	3	+	39.9	32.8	381	242	58.8	41.9	242	206
Pos. Control with CPA	4.5	+	62.4	40.9	372	242	56.3	47.4	267	206
Test item	10	+	51.5	60.7	141	242	84.8	85.8	92	206
Test item	20	+	104.5	48.9	239	242	90.4	75.6	91	206
Test item	40	+	85.3	52.1	118	242	77.4	66.9	82	206
Test item	80	+	14.4	5.1	178	242	3.9	3.4	87	206
Test item	100 (p)	+	3.3 *	0.8 *	173 *	242 *	0.6	Culture was not continued #
Test item	120 (p)	+	1.6	Culture was not continued #			0	Culture was not continued #
Experiment III/ 4h treatment			Culture I				Culture II
Solv.control with DMSO		+	100	100	55	181	100	100	84	210
Pos.control with CPA	3	+	69	49.6	142	181	69.6	59.7	179	210
Pos. Control with CPA	4.5	+	38.3	24.2	233	181	43.4	30.5	338	210
Test item	40	+	21.4	11.9	80	181	13.6	17.8	156	210
Test item	50	+	2.6 *	0.9 *	232 *	181 *	19.5	11.1	119	210
Test item	60	+	0	Culture was not continued #			2.4	1.4	170	210
Test item	70	+	0	Culture was not continued #			10.6	Culture was not continued #
Test item	80	+	0	Culture was not continued #			3.3	Culture was not continued #
Test item	90	+	0	Culture was not continued #			0	Culture was not continued #

 threshold = number of mutant colonies per 10^6cells of each solvent control plus 126

p precipitation

# culture was not continued due to exceedingly severe cytotoxic effects

* values are judged as invalid, since the acceptance criteria and the exception criteria of the IWGT are not met (RTG < 1%)

Conclusions:

In conclusion it can be stated that during the mutagenicity test described and under the experimental conditions reported the test item did not induce mutations in the mouse lymphoma thymidine kinase locus assay using the cell line L5178Y in the absence and presence of metabolic activation. The study is regarded to be adequate and reliable.

Executive summary:

A valid GLP study was performed to investigate the potential of the test item to induce mutations at the mouse lymphoma thymidine kinase locus using the cell line L5178Y. The method followed was that described in OECD TG 476.

The assay was performed in three independent experiments, using two parallel cultures each. Experiment I, and II were performed with and without liver microsomal activation and a treatment period of 4 hours. The supplementary experiment III was solely performed with metabolic activation (4 hours treatment) to cover the cytotoxic range of approximately 10- 20% RTG that was not covered in the second experiment with metabolic activation.

The concentration range of the main experiments was limited by the solubility of the test item in aqueous media and by cytotoxic effects. Relevant cytotoxic effects occurred at 40 µg/mL in the first experiment without metabolic activation and at 80.0 µg/mL in the presence of metabolic activation. In the second experiment cytotoxicity was observed at 30.0 µg/mL without metabolic activation and at 80.0 µg/mL with metabolic activation. In the supplementary experiment III strong cytotoxic effects occurred at 40.0 µg/mL and above.

No substantial and reproducible dose dependent increase in mutant colony numbers was observed up to the maximum concentrations tested with and without metabolic activation. Appropriate reference mutagens were used as positive controls and showed a distinct increase in induced mutant colonies, indicating that the tests were sensitive and valid.

In conclusion it can be stated that during the mutagenicity test described and under the experimental conditions reported, the test item did not induce mutations in the mouse lymphoma thymidine kinase locus assay using the cell line L5178Y in the absence and presence of metabolic activation. Therefore, the test item is considered to be non mutagenic in this mouse lymphoma assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

Erythrocyte micronucleus test: Negative

A key study conducted according to guideline OECD 474 and under GLP is available. The study is considered to be relevant, reliable (Klimisch 1) and adequate for the purposes of risk assessment, classification and labeling.

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

The in vivo micronucleus study was conducted solely to comply with non-EU national registration requirement, and has been provided here in accordance with REACH, Article 22(1)e.

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 09 January 2014 to 11 June 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Qualifier:

according to guideline

Guideline:

EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5395 (In Vivo Mammalian Cytogenetics Tests: Erythrocyte Micronucleus Assay)

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian erythrocyte micronucleus test

Species:

rat

Strain:

Wistar

Details on species / strain selection:

The rat is an animal that has been used for many years as a suitable experimental animal in cytogenetic investigations. There are many data available from such investigations, which may be helpful in the interpretation of results from the micronucleus test. In addition, the rat is an experimental animal in many physiological, pharmacological and toxicological studies.

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Laboratories B.V. Postbus 6174 5960 AD Horst / The Netherlands
- Age at study initiation: 6 - 7 weeks
- Weight at study initiation: 203.8 g to 238.5 g
- Assigned to test groups randomly: yes
- Fasting period before study:
- Housing:group housing in Makrolon Type II (pre-test) / III (main study), with wire mesh top
- Diet (e.g. ad libitum): Pelleted standard diet, ad libitum
- Water (e.g. ad libitum): Tap water, ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2°C
- Humidity (%): 45 - 65 % (for a short period 7 h the relative humidity was decreased to 23%)
- Air changes (per hr): 15/hour
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

Name: 1% CMC
Batch no.: BCBD7651V
Expiry Date: January 2017
Suspended in: sterile water
Batch no.: 134618061
Expiry Date: October 2016

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
On the day of the experiment, the test substance was suspended in 1% CMC. Grinding of the test substance in a mortar was used to formulate the test substance. An ultraturrax was used to formulate the test substance for the high dose level.

Duration of treatment / exposure:

All animals received a single standard volume once orally

Frequency of treatment:

once orally

Dose / conc.:

500 mg/kg bw/day (nominal)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Dose / conc.:

2 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

14 males

Control animals:

yes, concurrent vehicle

Positive control(s):

Name: Cyclophosphamide,
CPA Batch: A0302605
Expiry Date: October 2014
Dissolved in: sterile water
Batch no.: 134618061
Expiry Date: October 2016
Dosing: 20 mg/kg b.w.
Route and frequency of administration: orally, once
Volume administered: 10 mL/kg b.w.

Tissues and cell types examined:

Polychromatic erythrocytes (PCE) in the bone marrow

Details of tissue and slide preparation:

The femora were removed, the epiphyses were cut off and the marrow was flushed out with fetal calf serum using a syringe. The nucleated cells were separated from the erythrocytes using the method of Romagna (8). The cell suspensions were passed through a column consisting of α-Cellulose and Cellulose. The columns will then be washed with Hank´s buffered saline. The cell suspension was centrifuged at 1500 rpm (390 × g) for 10 minutes and the supernatant was discarded. A small drop of the re-suspended cell pellet was spread on a slide. The smear was air-dried and then stained with May-Grünwald /Giemsa. Cover slips were mounted with EUKITT. At least one slide was made from each bone marrow sample.

Evaluation criteria:

Evaluation of the slides was performed using NIKON microscopes with 100× oil immersion objectives. Per animal 2000 PCEs were analysed for micronuclei. To describe a cytotoxic effect the ratio between polychromatic and normochromatic erythrocytes was determined from the same slide and expressed in polychromatic erythrocytes per 2000 erythrocytes. The analysis was performed with coded slides. Immature and mature erythrocytes were identified by their pale and blue to green colour, respectively. Micronuclei are distinguished by being small nuclei separate from and additional to the main nuclei of the cells. A test substance is classified as mutagenic if it induces either a dose-related increase or a clear increase in the number of micronucleated polychromatic erythrocytes in a single dose group.

Statistics:

Statistical methods (nonparametric Mann-Whitney test) were used as an aid in evaluating the results.

Sex:

male

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

valid

Remarks:

used as negative control

Negative controls validity:

not applicable

Positive controls validity:

valid

Preliminary toxicity assay: In a pre-experiment 4 animals (2 males, 2 females) received orally a single dose of 2000 mg/kg b.w.test item suspended in1% CMC. The volume administered was 10 mL/kg b.w.. The animals treated with 2000 mg/kg b.w. expressed signs of toxicity, reduced activity. On the basis of these data 2000 mg/kg b.w., the maximum OECD Guideline recommended dose for this assay was considered suitable. No substantial gender specific differences in toxicity were observed, thus, the main study was performed using male animals only, as permitted by the Guideline.

Conclusions:

The test substance did not induce micronuclei as determined by the micronucleus test in the bone marrow cells of the rat. Therefore, the substance is considered to be non-mutagenic in this bone marrow micronucleus assay.

Executive summary:

This study was performed in order to investigate the potential of the test item to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the rat. The test item was formulated in 1% carboxymethylcellulose (CMC), which was also used as vehicle control. A correction factor of 1.02 was applied. The volume administered orally was 10 mL/kg body weight (b.w.). At 24 h and 48 h after a single administration of the test item, the bone marrow cells were collected for micronuclei analysis. Seven males per test group (except the negative and positive control groups with five males only) were evaluated for the occurrence of micronuclei. Per animal 2000 PCEs were scored for micronuclei. To describe a cytotoxic effect due to the treatment with the test substance the ratio between polychromatic and normochromatic erythrocytes was determined per slide and reported as the number of PCEs per 2000 erythrocytes. The following dose levels of the test substance were investigated: 

24 h preparation interval: 500, 1000, and 2000 mg/kg b.w.
48 h preparation interval: 2000 mg/kg b.w. 

The highest dose was estimated to be a suitable maximum tolerated dose based on a pre-experiment. After treatment with the test substance the number of PCEs per 2000 erythrocytes was not decreased as compared to the mean value of PCEs per 2000 erythrocytes of the vehicle control, thus indicating that the test itm did not exert any significant cytotoxic effects in the bone marrow. In comparison to the corresponding vehicle controls there was no biologically relevant or statistically significant enhancement in the frequency of the detected micronuclei at any preparation interval after administration of the test substance with any dose level used. For all treatment groups the mean values of micronuclei observed after treatment with the test item were very well within the historical vehicle control range. Additionally, no dose dependency was observed. A dose of 20 mg/kg b.w. cyclophosphamide administered orally was used as the positive control, which showed a substantial increase of induced micronucleus frequency. The volume of the positive control administered was 10 mL/kg b.w.. In conclusion, it can be stated that under the experimental conditions reported, the test substancedid not induce micronuclei as determined by the micronucleus test with bone marrow cells ofthe rat. Therefore, the substance is considered to be non-mutagenic in this bone marrow micronucleus assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

The in vivo micronucleus study was conducted solely to comply with non-EU national registration requirements.

Justification for classification or non-classification

Three in vitro mutagenicity studies conducted according to OECD guidelines 471, 473, 476 and one in vivo mutagenicity study conducted according to OECD guideline 474 were all found to give negative results. As a result, and in accordance with Directive 2001/59/EC, Annex VI, 4.2.2.3, the substance is not considered to be classified as mutagenic.

Three in vitro mutagenicity studies conducted according to OECD guidelines 471, 473, 476 and one in vivo mutagenicity study conducted according to OECD guideline 474 were all found to give negative results. As a result, and in accordance with Regulation (EC) No. 1272/2008, Annex I, Part 3, 3.5.2, the substance is not considered to be classified as mutagenic.