1-(N,N-bis(2-ethylhexyl)aminomethyl)-1,2,4-triazole

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The test substance was not mutagenic in an Ames test performed with strains Salmonella typhimurium TA 98, TA 100, TA 102, TA 1535 and TA 1537 with and without S9 mix. No genotoxic potential was evident in an HPRT study. Furthermore, the test article did not induce chromosomal aberrations in vitro (in human lymphocytes).

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

May - June 1985

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

The only difference to guideline was that the repeat experiment was not performed by pre-incubation method but rather as a second plate-incorporation test. Study was performed under GLP-like quality control.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted 1997

Deviations:

no

Remarks:

The only difference to guideline was that the repeat experiment was not performed by pre-incubation method but rather as a second plate-incorporation test. Study was performed under GLP-like quality control.

GLP compliance:

no

Remarks:

but performed under GLP like quality assurance with QAU statement included.

Type of assay:

bacterial reverse mutation assay

Target gene:

his

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

S9 fraction of liver from rats induced with Aroclor 1254

Test concentrations with justification for top dose:

0.08 - 5000 µg/plate in the toxicity test.
20, 78, 313, 1250 and 5000 µg/plate in the mutagenicity test.

Vehicle / solvent:

Acetone

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: TA 98: daunorubicin-HCl; TA 100: 4-nitroquinoline-N-oxide; TA 102: mitomycin-C; TA 1535: sodium azide; TA 1537: 9(5)-aminoacridine hydrochloride monohydrate

Remarks:

without S9 mix

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: TA 98, TA 100, TA 1537, TA 102: 2-aminoanthracene; TA 1535: cyclophosphamide

Remarks:

with S9 mix

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

- Exposure duration: 48 h

NUMBER OF REPLICATIONS: without and with the addition of microsomal activation mixture three Petri dishes were prepared per strain and per group (i.e. per concentration or per control group). In order to confirm the results the experiments were repeated.

DETERMINATION OF CYTOTOXICITY
- Method: other: reduction in the colony count

Evaluation criteria:

The test substance is generally considered to be nonmutagenic if the colony count in relation to the negative control is not doubled at any concentration.

Statistics:

When the colonies had been counted, the arithmetic mean was calculated.

Key result

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES:
A preliminary toxicity test was carried out with the concentrations ranging from 0.08 to 5000 µg/0.1 ml. Thereafter, the concentration of 5000 µg/0.1 ml was used as the highest in the mutagenicity test.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
A growth-inhibiting effect of the test item occurred in the experiments without microsomal activation at the upper concentrations (cytotoxicity started to occur at 313 or 1250 µg/plate, depending on test strain). In the experiments with microsomal activation this effect was less pronounced (cytotoxicity started to occur at 1250 and/or 5000 µg/plate, depending on test strain).

Experiment I

	TA 98		TA 100		TA 102		TA 1535		TA 1537
Dose (µg/plate)	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
solvent control	22	37	185	204	230	263	16	18	10	15
20	25	36	176	191	186	264	15	19	4	11
78	28	41	166	251	178	257	14	18	7	11
313	23	44	171	192	71	225	8	15	6	16
1250	16	26	95	145	17	90	0	13	0	0
5000	0	0	0	0	0	0	0	4	0	0
solvent control	20	45	171	170	210	275	13	19	9	10
positive control A	264	1148	665	608	887	907	470	353	684	98
positive control B	668		1215		1255		387		1597

Experiment II

	TA 98		TA 100		TA 102		TA 1535		TA 1537
Dose (µg/plate)	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
solvent control	24	47	158	171	322	298	13	17	8	20
20	31	47	143	153	298	344	12	13	11	15
78	28	40	183	179	276	326	14	12	8	11
313	20	34	158	165	215	263	9	12	10	14
1250	5	29	92	172	0	52	0	3	0	1
5000	0	0	0	0	0	0	0	0	0	0
solvent control	20	45	160	157	256	332	12	16	7	15
positive control A	442	1451	670	1489	797	2170	246	300	558	262
positive control B	649		1286		992		528		1520

Positive Controls

Without S9 mix:

TA 98: daunorubicin-HCl, A: 5 and B: 10 µg/0.1 mL phosphate buffer

TA 100: 4-nitroquinoline-N-oxide, A: 0.125 and B: 0.25 µg/0.1 mL phosphate buffer

TA 102: mitomycin-C, A: 0.5 and B: 1.0 µg/0.1 mL bidistilled water

TA 1535: sodium azide, A: 2.5 and B: 5.0 µg/0.1 mL bidistilled water

TA 1537: 9(5)- aminoacridine hydrochloride monohydrate, A: 50 and B: 100 µg/0.1 mL DMSO

With S9-Mix:

TA 98, TA 100, TA 1537: 2-aminoanthracene, 5 µg/0.1 mL DMSO

TA 102: 2-aminoanthracene, 20 µg/0.1 mL DMSO

TA 1535: cyclophosphamide, 250 µg/0.1 mL phosphate buffer

Conclusions:

Interpretation of results: Negative with and without metabolic activation

Based on the presented results and under the conditions employed, the test article did not induce point mutations in presence or absence of a metabolic activation system and is therefore regarded as not mutagenic in the Ames test.

Executive summary:

In order to investigate the test article's potential to cause point mutation in bacteria, an AMES test similar in design to the OECD guideline No. 471 was carried out with the tester strains Salmonella typhimurium TA 98, TA 100, TA 102, TA 1535 and TA 1537. The test article was applied by the plate incorporation method at concentrations of 20, 78, 313, 1250 and 5000 µg/0.1 ml either with or without a metabolic activation system (rat liver S9 mix). The experiment was performed in triplicates and repeated once for confirmation. Positive controls were performed in parallel to check the tester strains sensitivity. In none of the experiments did treatment with the test substance lead to an increase in the incidence of histidine-prototrophic mutants in comparison with the controls. A growth-inhibiting effect of the test item occurred in the experiments without microsomal activation at the upper concentrations (cytotoxicity started to occur at 313 or 1250 µg/plate, depending on test strain). In the experiments with microsomal activation this effect was less pronounced (cytotoxicity started to occur at 1250 and/or 5000 µg/plate, depending on test strain). In conclusion, no evidence of the induction of point mutations by the test substance or by its metabolites formed as a result of microsomal activation was detectable in the strains of S. typhimurium used in these experiments. Therefore, the substance is considered as not mutagenic in this assay.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

January 03, 2013 - March 15, 2013

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)

Qualifier:

according to guideline

Guideline:

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

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

GLP compliance:

yes (incl. QA statement)

Remarks:

Harlan Cytotest Cell Research GmbH, In den Leppsteinswiesen 19, 64380 Rossdorf, Germany

Type of assay:

mammalian cell gene mutation assay

Target gene:

HPRT (hypoxanthine-guanine phosphoribosyl transferase)

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

- Type and identity of media: MEM (minimal essential medium) containing Hank’s salts supplemented with 10% foetal bovine serum (FBS), neomycin (5 µg/mL) and amphotericin B (1%).
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically checked for spontaneous mutant frequency: yes

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/ß-naphthoflavone induced rat liver S9

Test concentrations with justification for top dose:

Experiment I:
4 hours, without S9 mix: (0.1), 0.3, 0.5, (1.0), 2.0, 4.0, 6.0, (8.0) µg/ml
4 hours, with S9 mix: 13.0, 26.0, (52.0), (104.0), 156.0, 208.0 µg/ml
Experiment II:
24 hours, without S9 mix: (0.2), 0.4, 0.8, 1.6, 3.3, 6.5, (13.0), (19.5) µg/ml
4 hours, with S9 mix: (3.3), 6.5, 13.0, 26.0, 52.0, 104.0, (156.0), (208.0) µg/ml
numbers in parantheses: these cultures were discontinued.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

ethylmethanesulphonate

Remarks:

without S9: EMS, 0.15 mg/ml; with S9: DMBA, 1.1 µg/ml

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4 h (with and without S9), 24h (without S9)
- Expression time (cells in growth medium): 7 days
- Selection time (if incubation with a selection agent): 8 days
- Fixation time (start of exposure up to fixation or harvest of cells): 18 - 20 days

SELECTION AGENT (mutation assays): 11 μg/mL 6-thioguanine
STAIN (for cytogenetic assays): 10% methylene blue in 0.01% KOH solution

NUMBER OF REPLICATIONS: two independent cultures were used

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency, cell density

Evaluation criteria:

A test item is classified as positive if it induces either a concentration-related increase of the mutant frequency or a reproducible and positive response at one of the test points.
A test item producing neither a concentration-related increase of the mutant frequency nor a reproducible positive response at any of the test points is considered non-mutagenic in this system.
A positive response is described as follows:
A test item is classified as mutagenic if it reproducibly induces a mutation frequency that is three times above the spontaneous mutation frequency at least at one of the concentrations in the experiment.
The test item is classified as mutagenic if there is a reproducible concentration-related increase of the mutation frequency. Such evaluation may be considered also in the case that a threefold increase of the mutant frequency is not observed.
However, in a case by case evaluation this decision depends on the level of the corresponding solvent control data. If there is by chance a low spontaneous mutation rate within the laboratory´s historical control data range, a concentration-related increase of the mutations within this range has to be discussed. The variability of the mutation rates of solvent controls within all experiments of this study was also taken into consideration.

Statistics:

A linear regression (least squares) was performed to assess a possible dose dependent increase of mutant frequencies. The number of mutant colonies obtained for the groups treated with the test item were 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 and statistical significance was considered together.

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

for details see below

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No precipitation or phase separation, visible to the unaided eye, was noted up to the maximum evaluated concentrations with and without metabolic activation.

RANGE-FINDING/SCREENING STUDIES:
In the repeated pre-experiment no relevant toxic effect occurred up to the maximum concentration in the absence of metabolic activation following 4 hours treatment. In the presence of metabolic activation a complete inhibition of the cell growth was observed at 206.3 µg/mL and above (4 hours treatment). After 24 hours treatment the cell growth was completely inhibited at 26.0 µg/mL and above. The test medium was checked for precipitation or phase separation at the end of each treatment period (4 or 24 hours) prior to removal to the test item. Phase separation was solely noted in the presence of metabolic activation at 412.5 µg/mL and above. There was no relevant shift of pH and osmolarity of the medium even at the maximum concentration of the test item. Based on the results of the pre-experiment, the individual concentrations of the main experiments were selected.

COMPARISON WITH HISTORICAL CONTROL DATA:
The mutation frequency did not exceed the historical range of solvent controls.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Relevant cytotoxic effects indicated by a relative cloning efficiency I or cell density below 50% in both parallel cultures occurred in the first experiment at 6.0 µg/mL without metabolic activation. In the second experiment with and without metabolic activation no relevant cytotoxic effects were noted up to the maximum analysable concentration of 104 µg/mL with and 6.5 µg/mL without metabolic activation. Exceedingly severe cytotoxicity precluded analysis at the next higher concentrations of 156.0 µg/mL with and 13.0 µg/mL without metabolic activation. As the concentrations were spaced by a factor of 2 or lower, the data are acceptable.

Summary of Results

	concentration (µg/ml)	P	S9 Mix	relative cloning efficiency I (%)	relative cell density (%)	relative cloning efficiency II (%)	mutant colonies / 10^6cells	induction factor	relative cloning efficiency I (%)	relative cell density (%)	relative cloning efficiency II (%)	mutant colonies / 10^6cells	induction factor
Experiment I / 4h treatment				culture I					culture II
solvent control (DMSO)			-	100.0	100.0	100.0	6.4	1.0	100.0	100.0	100.0	22.8	1.0
positive control (EMS)	150.0		-	91.7	80.5	104.3	79.1	12.4	93.3	88.6	94.3	93.4	4.1
test item	0.1		-	97.4	culture was not continued#				101.2	culture was not continued#
test item	0.3		-	99.6	96.8	94.7	12.9	2.0	102.2	109.0	90.9	9.8	0.4
test item	0.5		-	98.2	90.9	89.9	6.4	1.0	102.9	114.9	80.4	23.1	1.0
test item	1.0		-	96.0	culture was not continued##				104.9	0.9
test item	2.0		-	99.2	85.1	98.1	7.5	1.2	99.6	76.1	71.3	20.0	0.9
test item	4.0		-	68.4	80.0	77.4	22.3	3.5	58.0	91.9	53.4	5.6	0.2
test item	6.0		-	13.6	34.1	59.4	37.9	6.0	12.4	38.4	45.8	10.3	0.5
test item	8.0		-	3.6	1.2	culture was not continued###			3.1	3.6	culture was not continued###
Experiment I / 4h treatment				culture I					culture II
solvent control (DMSO)			+	100.0	100.0	100.0	11.7	1.0	100.0	100.0	100.0	21.7	1.0
positive control (DMBA)	1.1		+	52.7	70.7	72.6	1262.2	107.8	54.7	65.2	78.9	1471.0	67.9
test item	13.0		+	102.7	126.6	92.4	10.0	0.9	95.4	137.2	108.4	16.3	0.8
test item	26.0		+	105.3	113.5	89.0	19.2	1.6	97.4	110.5	91.1	22.4	1.0
test item	52.0		+	101.4	culture was not continued##				92.2	culture was not continued##
test item	104.0		+	94.7	culture was not continued##				85.0	culture was not continued##
test item	156.0		+	59.7	54.9	58.0	28.5	2.4	69.8	71.0	65.5	30.0	1.4
test item	208.0		+	83.7	121.4	72.0	19.3	1.6	83.2	117.1	95.7	16.4	0.8
Experiment II / 24h treatment				culture I					culture II
solvent control (DMSO)			-	100.0	100.0	100.0	6.4	1.0	100.0	100.0	100.0	8.6	1.0
positive control (EMS)	150.0		-	95.4	99.6	100.8	284.1	44.5	99.4	86.1	94.4	192.3	22.4
test item	0.2		-	102.3	culture was not continued#				101.8	culture was not continued#
test item	0.4		-	98.5	94.6	95.4	9.2	1.4	100.1	88.1	93.7	5.8	0.7
test item	0.8		-	99.5	102.3	94.1	5.4	0.8	99.9	82.1	91.0	7.8	0.9
test item	1.6		-	96.6	105.7	91.2	10.6	1.7	97.2	81.3	92.3	17.2	2.0
test item	3.3		-	96.5	111.6	90.4	10.7	1.7	96.4	77.0	88.2	7.6	0.9
test item	6.5		-	87.8	80.4	87.8	13.5	2.1	86.0	51.2	89.3	12.5	1.5
test item	13.0		-	0.0	culture was not continued###				0.0	culture was not continued###
test item	19.5		-	0.0	culture was not continued###				0.0	culture was not continued###
Experiment II / 4h treatment				culture I					culture II
solvent control (DMSO)			+	100.0	100.0	100.0	5.7	1.0	100.0	100.0	100.0	7.6	1.0
positive control (DMBA)	1.1		+	62.9	64.5	98.6	506.9	88.3	66.2	49.8	94.2	608.7	79.8
test item	3.3		+	98.6	culture was not continued#				97.3	culture was not continued#
test item	6.5		+	98.6	90.0	98.3	10.3	1.8	99.0	95.2	98.3	8.5	1.1
test item	13.0		+	96.5	107.7	94.6	5.5	1.0	98.7	96.2	96.5	10.7	1.4
test item	26.0		+	95.5	111.1	94.1	9.5	1.6	100.0	81.9	97.4	8.2	1.1
test item	52.0		+	97.8	101.9	90.1	6.0	1.1	96.3	80.4	98.2	6.6	0.9
test item	104.0		+	94.5	93.5	96.8	6.3	1.1	94.7	74.9	110.7	8.8	1.2
test item	156.0		+	0.0	culture was not continued###				0.0	culture was not continued###
test item	208.0	PS	+	0.0	culture was not continued###				0.0	culture was not continued###

# culture was not continued as a minimum of only 4 concentrations is required

## culture was not continued due to microbial contamination

### culture was not continued due to exceedingly severe cytotoxic effects

PS phase separation visible at the end of treatment

Conclusions:

Interpretation of results: negative with and without metabolic activation.

In conclusion it can be stated that under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells and is therefore considered to be non-mutagenic in this HPRT assay.

Executive summary:

A GLP-compliant mammalian cell mutagenicity test according to OECD guideline 476 was performed to investigate the potential of the test article to induce gene mutations at the HPRT locus in V79 cells of the Chinese hamster. The study was performed in two independent experiments, using identical experimental procedures. In the first experiment the treatment period was 4 hours with and without metabolic activation. The second experiment was performed with a treatment time of 4 hours with and 24 hours without metabolic activation. The concentration range of the main experiments (0.1 – 208.0 µg/ml) was limited by cytotoxic effects. The test item was dissolved in DMSO.

No precipitation or phase separation, visible to the unaided eye, was noted up to the maximum evaluated concentrations with and without metabolic activation. Relevant cytotoxic effects indicated by a relative cloning efficiency I or cell density below 50% in both parallel cultures occurred in the first experiment at 6.0 µg/mL without metabolic activation. In the second experiment with and without metabolic activation no relevant cytotoxic effects were noted up to the maximum analysable concentration of 104 µg/mL with and 6.5 µg/mL without metabolic activation. Exceedingly severe cytotoxicity precluded analysis at the next higher concentrations of 156.0 µg/mL with and 13.0 µg/mL without metabolic activation. No relevant and reproducible increase in mutant colony numbers/10⁶ cells was observed in the main experiments up to the maximum concentration. The mutation frequency did not exceed the historical range of solvent controls. The induction factor exceeded the threshold of three times the corresponding solvent control in the first culture of experiment I without metabolic activation at 4.0 and 6.0 µg/mL. These effects however, were based on the rather low solvent control of 6.4 mutant colonies/10⁶ cells. The actual values of the mutation frequency remained within the historical range of solvent controls. Furthermore, the increase was not reproduced in the parallel culture under identical experimental conditions. In both experiments of this study (with and without S9 mix) the range of the solvent controls was from 5.7 up to 22.8 mutants per 10⁶ cells; the range of the groups treated with the test item was from 5.4 up to 37.9 mutants per 10⁶ cells. EMS (150 µg/mL) and DMBA (1.1 µg/mL) were used as positive controls and showed a distinct increase in induced mutant colonies. In conclusion it can be stated that under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells and is therefore considered to be non-mutagenic in this HPRT assay.

Reference 3

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

October - November 2005

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

adopted July 21, 1997

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

published June 8, 2000

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

lymphocytes: human

Details on mammalian cell type (if applicable):

- Blood was collected from healthy adult, non-smoking male volunteers.
- Cultured in the presence of phytohaemagglutinin (stimulant).
- Culture medium consisted of RPM11640 medium, supplemented with 20% (v/v) heat-inactivated (56°C; 30 min) foetal calf serum, L-glutamine (2 mM), penicillin/streptomycin (50 U/ml and 50 µg/ml respectively) and 30 U/ml heparin.

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9 mix, derived from phenobarbital and beta-naphthoflavone induced rat liver

Test concentrations with justification for top dose:

Dose range finding test
- 3 h exposure time, with and without S9 mix: 1, 3, 10, 33 and 100 µg test item /mL culture medium
- 24 h and 48 h exposure time, without S9-mix: 1, 3, 10, 33, 100 and 333 µg test item /mL culture medium

First cytogenetic assay
Experiment 1:
- 3 h exposure time, 24 h fixation time, with and without S9-mix: 30, 40, 50, 60, 80 and 100 µg test item /mL culture medium (no concentrations were scored)
Experiment 1A:
- 3 h exposure time, 24 h fixation time, without S9-mix: 10, 20, 30, 40 and 50 µg test item /mL culture medium (scored concentrations: 20, 30 and 40 µg test item /mL)
- 3 h exposure time, 24 h fixation time, with S9-mix: 30, 50, 60, 65, 70, 75, 80 and 85 µg test item /mL culture medium (scored concentrations: 60, 70 and 80 µg test item /mL)

Second cytogenetic assay
Experiment 2:
- 24 h exposure time, 24 h fixation time, without S9-mix: 5, 10, 15, 20, 25 and 30 µg test item /mL culture medium (no concentrations were scored)
- 48 h exposure time, 48 h fixation time, without S9-mix: 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10 µg test item /mL culture medium (scored concentrations: 1, 3 and 6 µg test item /mL)
- 3 h exposure time, 48 h fixation time, with S9-mix: 30, 60, 65, 70, 75, 80 and 100 µg test item /mL culture medium (scored concentrations: 60, 80 and 100 µg test item /mL)
Experiment 2A:
- 24 h exposure time, 24 h fixation time, without S9-mix : 1, 3, 5, 7,10, 15 and 20 µg test item /mL culture medium (scored concentrations: 3, 7 and 15 µg test item/mL)

Vehicle / solvent:

Ethanol

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

Without metabolic activation (-S9-mix) Migrated to IUCLID6: 0.5 µg/ml (3 h exposure), 0.2 µg/ml (24 h exposure), 0.1 µg/ml (48 h exposure).

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

With metabolic activation (-S9-mix)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Culturing period before exposure: 48 h
- Exposure duration: 3h, 24h, 48h
- Fixation time (start of exposure up to fixation or harvest of cells): 24 h, 48 h

SPINDLE INHIBITOR: Colchicine 0.5 µg/mL medium (metaphase-arresting chemical)

STAIN: 5% (v/v) Giemsa (Merck) solution in tap water.

NUMBER OF CELLS EVALUATED: 100 metaphase chromosome spreads per culture. In case the number of aberrant cells, gaps excluded, was >25 in 50 metaphases, no more metaphases were examined.

NUMBER OF REPLICATIONS: Double cultures per concentration

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

OTHER EXAMINATIONS:
- Determination of polyploidy: Yes
- Determination of endoreplication: Yes

Evaluation criteria:

The number of cells with aberrations and the number of aberrations were calculated. A test substance was considered positive (clastogenic) in the chromosome aberration test if:
a) It induced a dose-related statistically significant (Chi-square test, one-sided, p < 0.05) increase in the number of cells with chromosome aberrations.
b) A statistically significant and biologically relevant increase in the frequencies of the number of cells with chromosome aberrations was observed in the absence of a clear dose-response relationship.

A test substance was considered negative (not clastogenic) in the chromosome aberration test if none of the tested concentrations induced a statistically significant (Chi-square test, one-sided, p < 0.05) increase in the number of cells with chromosome aberrations.

Statistics:

Chi-square test, one-sided, p < 0.05

Key result

Species / strain:

lymphocytes: human

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: At a concentration of 100 µg/ml the test article precipitated in the culture medium.

RANGE-FINDING/SCREENING STUDIES:
In the dose range finding study, at the 3 h exposure time, blood cultures were treated with 1, 3, 10, 33 and 100 µg/ml culture medium with and without S9-mix. At the 24 h and 48 h continuous exposure time blood cultures were treated with 1, 3, 10, 33, 100 and 333 µg/ml culture medium without S9-mix. The test substance was tested beyond the limit of solubility to obtain adequate toxicity data. After 3 hour exposure, a reduction in mitotic index below 50% was observed at the concentration of 100 µg/ml. At longer exposure (without S9-mix), this reduction was evident at 33 µg/ml and 10 µg/ml (24 and 48 hours, respectively).

COMPARISON WITH HISTORICAL CONTROL DATA:
The number of cells with chromosome aberrations found in the solvent control cultures was within the laboratory historical control data range. The number of polyploid cells and cells with endoreduplicated chromosomes found in the solvent control cultures was within the laboratory historical control data range.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Cytotoxicity (as indicated by mitotic index of 50% and below) was observed at concentrations of approximately 40 µg/ml and higher after 3h exposure without S9-mix. With S9-mix, this effect was observed at higher concentrations (starting at around 80 µg/ml). After 24 and 48 exposures without S9-mix, a mitotic index of 50% and below was evident at concentrations from around 10 µg/ml and higher.

OTHER REMARKS:
- The test item did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations.
- No effects on the number of polyploid cells and cells with endoreduplicated chromosomes were observed.
- The highest concentration analysed induced an inhibition of the mitotic index of 66%.
- The positive control chemicals produced a statistically significant increase in the frequency of aberrant cells. It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.

Experimental Result

							Number of cells with aberrations
Experiment	Exposure Time	Fixation Time	Dose (µg/ml)	Mitotic Index	S9-Mix	number of metaphases	(+ gaps)	(- gaps)
1A	3 h	24 h	vehicle	100	-	200	1	1
	3 h	24 h	20	98	-	200	5	4
	3 h	24 h	30	84	-	200	4	3
	3 h	24 h	40	47	-	200	6	6
	3 h	24 h	MMC-C	73	-	100	51^***	51^***
	3 h	24 h	vehicle	100	+	200	1	1
	3 h	24 h	60	101	+	200	4	4
	3 h	24 h	70	71	+	200	8^*	8^*
	3 h	24 h	80	53	+	200	9^*	7^*
	3 h	24 h	CP	65	+	100	53^***	53^***
2A	24 h	24 h	vehicle	100	-	200	2	2
	24 h	24 h	3	101	-	200	6	6
	24 h	24 h	7	72	-	200	5	3
	24 h	24 h	15	34	-	200	5	3
	24 h	24 h	MMC-C	63	-	100	65^***	60^***
2	48 h	48 h	vehicle	100	-	200	1	1
	48 h	48 h	1	95	-	200	2	1
	48 h	48 h	3	73	-	200	0	0
	48 h	48 h	6	45	-	200	3	2
	48 h	48 h	MMC-C	81	-	100	57^***	57^***
	3 h	48 h	vehicle	100	+	200	1	0
	3 h	48 h	60	96	+	200	3	3
	3 h	48 h	80	88	+	200	2	2
	3 h	48 h	100	57	+	200	5	5
	3 h	48 h	CP	-^a)	+	100	58^***	48^***

^a): CP was fixed after 24 hours. Therefore, the mitotic index could not be calculated as percentage of control

Significantly different from control group (Chi-square test), * P < 0.05, ** P < 0.01 or *** P < 0.001

CP: cyclophosphamide; MMC-C: mitomycin C

Interpretation of results:

In the absence of S9-mix the test substance did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations in two independently repeated experiments. In the first cytogenetic assay, in the presence of S9-mix the test article induced a statistically significant increase in the number of cells with chromosome aberrations at the highest tested concentration of 70 and 80 µg/ml, both when gaps were included and excluded. Since the type of aberrations observed were only breaks and gaps, the increase was not dose related and moreover the number of cells with chromosome aberrations was well within the historical control data range (calculated for 200 cells), the increase was considered not to be biologically relevant.

In the second experiment, in the presence of S9-mix, the test substance did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations.

Conclusions:

Interpretation of results: negative with and without metabolic activation

It is concluded that this test is valid and that the test substance is not clastogenic in human lymphocytes under the experimental conditions described in this report.

Executive summary:

The ability of the test substance to induce chromosome aberrations in cultured peripheral human lymphocytes was assessed in a GLP-compliant cytogenetic assay according to OECD guideline 473. The possible clastogenicity of the test article was tested in two independent experiments. In the first cytogenetic assay, the test substance was tested up to 40 and 80 µg/ml for a 3 h exposure time with a 24 h fixation time in the absence and presence of S9-mix, respectively. Appropriate toxicity was reached at these dose levels. In the second cytogenetic assay, the test substance was tested up to 15 µg/ml for a 24 h continuous exposure time with a 24 h fixation time and up to 6 µg/ml for a 48 h continuous exposure time with a 48 h fixation time in the absence of S9-mix. In the presence of S9-mix the test substance was tested up to 100 µg/ml for a 3 h exposure time with a 48 h fixation time. Appropriate toxicity was reached at these dose levels. Positive control chemicals, mitomycin C and cyclophosphamide, both produced a statistically significant increase in the incidence of cells with chromosome aberrations, indicating that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly. In the absence of S9-mix the test item did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations in two independently repeated experiments. In the first cytogenetic assay, in the presence of S9-mix the test item induced a statistically significant increase in the number of cells with chromosome aberrations at the highest tested concentration of 70 and 80 µg/ml, both when gaps were included and excluded. Since the type of aberrations observed were only breaks and gaps, the increase was not dose related and moreover the number of cells with chromosome aberrations was well within our historical control data range (calculated for 200 cells), the increase was considered not to be biologically relevant. In the second experiment, in the presence of S9-mix, the test article did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations. No effects on the number of polyploid cells and cells with endoreduplicated chromosomes were observed both in the absence and presence of S9-mix. Therefore it can be concluded that the test article does not disturb mitotic processes and cell cycle progression and does not induce numerical chromosome aberrations under the experimental conditions described in this report. Finally, it is concluded that this test is valid and that the test article is not clastogenic in human lymphocytes under the experimental conditions described in this report.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

The test article did not show any signs of genotoxicity in two in vivo micronucleus tests in hamsters.

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

May - August 1985

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

not applicable

Principles of method if other than guideline:

Meets the principles of OECD guideline 474 with the following restriction: only 1000 immature erythrocytes per animal were counted.

GLP compliance:

no

Remarks:

but GLP like quality assurance

Type of assay:

mammalian erythrocyte micronucleus test

Species:

hamster, Chinese

Strain:

other: random outbred strain

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: CIBA-GEIGY Tierfarm, Sisseln.
- Age at study initiation: female 6-10 weeks, male 4-9 weeks
- Weight at study initiation: female 21-32 g, male 21-38 g (tolerability test), female 20-31 g, male 21-34 g (mutagenicity test)
- Water: ad libitum
- Diet: standard diet, NAFAG No.924

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22-23°C
- Humidity (%): 46-50%
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

Vehicle used: aqueous solution of sodium carboxymethylcellulose (0.5%)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: 5000 mg/kg test item in 20 mL/kg 0.5% aqueous solution of sodium carboxymethylcellulose.

Frequency of treatment:

Single application

Post exposure period:

16, 24 and 48h after application

Dose / conc.:

5 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

In the tolerability test: 2 males and 2 females

In the mutagenicity test: 24 males and 24 females in each treatment group and in the negative control group (8 males and 8 females per sampling time).

8 males and 8 females in the positive control group.

Control animals:

yes, concurrent vehicle

Positive control(s):

cyclophosphamide (64 mg/kg)

Tissues and cell types examined:

Bone marrow; erythrocytes

Details of tissue and slide preparation:

SAMPLING TIME: 16, 24 and 48 h after application

DETAILS OF SLIDE PREPARATION: Bone marrow was harvested from the shafts of both femurs. In a siliconized pipette filled with approx. 0.5 µL rat serum the bone marrow was drawn up. Small drops of the mixture were transferred on the end of a slide, spread out by pulling it behind a polished cover glass and the preparations were air-dried. Three hours later, the slides were stained in undiluted May-Grünwald solution for 2 min then in May-Grünwald solution/water 1/1 and then in Giemsa's, 40% .

METHOD OF ANALYSIS:1000 polychromatic erythrocytes each were scored for the incidence of micronuclei per animal. The ratio of polychromatic to normochromatic erythrocytes was determined for each animal by counting a total of 1000 erythrocytes.

Statistics:

The significance of difference was assessed by X2-test.

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: 5000 mg/kg bw
- Mortality: none

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei: The bone marrow smears from animals treated with the dose of 5000 mg/kg of the test item showed no statistically significant increase (p>0,05) in the number of micronucleated polychromatic erythrocytes compared to the negative control animals at all three sampling times.
- Ratio of PCE/NCE: Ratio was not influenced by the test item.
- The respective "positive control" experiments with cyclophosphamide (64 mg/kg) yielded an average of 4.15% polychromatic erythrocytes with micronuclei. This is significantly different from the controls (0.15%) treated with the vehicle (0.5% CMC) alone.

Conclusions:

Interpretation of results: negative

Under the conditions of this experiment, no evidence of mutagenic effects was obtained in Chinese hamsters treated with the test substance.

Executive summary:

A micronucleus experiment was performed to evaluate any mutagenic effect of the test article on polychromatic erythrocytes in bone marrow cells in vivo. The test article in 0.5% carboxymethyl cellulose was administered orally to groups of 24 female and 24 male animals each in the negative and in the 5000 mg/kg dose group. The positive control group consisted of 8 female and 8 male animals and was treated with Cyclophosphamide (64 mg/kg). Treatment consisted of a single application. 16, 24 and 48h after application 8 female and 8 male animals per group and sampling time were sacrificed by dislocation of the cervical vertebrae. From the bone marrow smears were made. The bone marrow smears from animals treated with the dose of 5000 mg/kg showed no statistically significant increase (p>0,05) in the number of micronucleated polychromatic erythrocytes compared to the negative control animals at all three sampling times. The respective "positive control" experiments with cyclophosphamide yielded an average of 4.15% polychromaticerythrocytes with micronuclei. This is significantly different from the controls (0.15%) treated with the vehicle (0.5% CMC) alone. It is concluded that under the conditions of this experiment, no evidence of mutagenic effects was obtained in Chinese hamsters treated with the test substance.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

IN VITRO

Ames test

The mutagenic potential of the test substance was investigated in an Ames test similar to OECD TG 471 (Ciba-Geigy 1992), performed on Salmonella typhimurium TA 98, TA 100, TA 102, TA 1535 and TA 1537 in the presence and absence of a metabolic activation system (S9 mix). None of the tested concentrations (20 to 5000 µg/plate) led to an increase in the incidence of histidine-prototrophic mutants by comparison with the negative control either with or without metabolic activation. This result was confirmed in a second independent experiment. Owing to a growth-inhibiting effect of the substance a significant reduction in the colony count was observed at concentrations from 313 µg/plate and higher in experiments without metabolic activation and from 1250 µg/plate and higher in experiments with metabolic activation. Based on the results of these experiments and on standard evaluation criteria, it is concluded that the test article and its metabolites did not induce gene mutations in the strains of S. typhimurium and E. coli used.

HPRT Test

A GLP-compliant mammalian cell mutagenicity test according to OECD guideline 476 was performed to investigate the potential of the test article to induce gene mutations at the HPRT locus in V79 cells of the Chinese hamster (Harlan, 2013). The study was performed in two independent experiments, using identical experimental procedures. In the first experiment the treatment period was 4 hours with and without metabolic activation. The second experiment was performed with a treatment time of 4 hours with and 24 hours without metabolic activation. The concentration range of the main experiments (0.1 – 208.0 µg/ml) was limited by cytotoxic effects. The test item was dissolved in DMSO. No precipitation or phase separation, visible to the unaided eye, was noted up to the maximum evaluated concentrations with and without metabolic activation. Relevant cytotoxic effects occurred in the first experiment at 6.0 µg/mL without metabolic activation. In the second experiment with and without metabolic activation no relevant cytotoxic effects were noted up to the maximum analysable concentration of 104 µg/mL with and 6.5 µg/mL without metabolic activation. Exceedingly severe cytotoxicity precluded analysis at the next higher concentrations of 156.0 µg/mL with and 13.0 µg/mL without metabolic activation. No relevant and reproducible increase in mutant colony numbers/10⁶ cells was observed in the main experiments up to the maximum concentration. The mutation frequency did not exceed the historical range of solvent controls. The induction factor exceeded the threshold of three times the corresponding solvent control in the first culture of experiment I without metabolic activation at 4.0 and 6.0 µg/mL. These effects however, were based on the rather low solvent control of 6.4 mutant colonies/10⁶ cells. Furthermore, the increase was not reproduced in the parallel culture under identical experimental conditions. Appropriate reference mutagens (EMS and DMBA), used as positive controls, induced a distinct increase in mutant colonies and thus, showed the sensitivity of the test system and the activity of the metabolic activation system. In conclusion it can be stated that under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells and is therefore considered to be non-mutagenic in this HPRT assay.

Chromosme Aberration test

The ability of the test substance to induce chromosome aberrations in cultured peripheral human lymphocytes was assessed in a GLP-compliant cytogenetic assay according to OECD guideline 473 (Notox, 2005). The possible clastogenicity of the test article was tested in two independent experiments. In the first cytogenetic assay, the test substance was tested up to 40 and 80 µg/ml for a 3 h exposure time with a 24 h fixation time in the absence and presence of S9-mix, respectively. In the second cytogenetic assay, the test substance was tested up to 15 µg/ml for a 24 h continuous exposure time with a 24 h fixation time and up to 6 µg/ml for a 48 h continuous exposure time with a 48 h fixation time in the absence of S9-mix. In the presence of S9-mix the test substance was tested up to 100 µg/ml for a 3 h exposure time with a 48 h fixation time. In the absence of S9-mix the test item did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations in two independently repeated experiments. In the presence of S9-mix, in the first cytogenetic assay, the test item induced a statistically significant increase in the number of cells with chromosome aberrations at the highest tested concentration of 70 and 80 µg/ml, both when gaps were included and excluded. Since the type of aberrations observed were only breaks and gaps, the increase was not dose related and moreover the number of cells with chromosome aberrations was well within the historical control data range (calculated for 200 cells), the increase was considered not to be biologically relevant. This was confirmed in the second experiment performed in the presence of S9-mix, where the test article did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations. No effects on the number of polyploid cells and cells with endoreduplicated chromosomes were observed both in the absence and presence of S9-mix. Therefore it can be concluded that this test is valid and that the test article is not clastogenic in human lymphocytes under the experimental conditions described in this report.

 

In a study performed as an additional repeat experiment of above study, human lymphocytes were exposed to the test substance in the presence of S9-mix (Notox, 2006). The test substance was tested at concentrations up to 80 µg/ml for a 3 h exposure time with a 24 h fixation. The test article did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations. No effects on the number of polyploid cells and cells with endoreduplicated chromosomes were observed. Therefore, the conclusions drawn from the above experiment could be confirmed and the test substance is considered not to be clastogenic in human lymphocytes under the experimental conditions described.

IN VIVO

Micronucleus Test in the hamster

A micronucleus experiment was performed to evaluate any mutagenic effect of the test article on polychromatic erythrocytes in bone marrow cells in vivo (Ciba-Geigy, 1986). The test article was administered to groups of 24 female and 24 male Chinese hamsters each in the negative and in the 5000 mg/kg dose group.8 males and 8 females were sacrificed 16, 24 and 48 hours thereafter. The bone marrow smears from animals treated with the dose of 5000 mg/kg showed no statistically significant increase (p > 0,05) in the number of micronucleated polychromatic erythrocytes compared to the negative control animals at all three sampling times. The respective "positive control" experiments with cyclophosphamide (64 mg/kg) yielded an average of 4.15% polychromaticerythrocytes with micronuclei. This is significantly different from the controls (0.15%) treated with the vehicle (0.5% CMC) alone. It is concluded that under the conditions of this experiment, no evidence of mutagenic effects was obtained in Chinese hamsters treated with the test substance.

This experimental result is confirmed by a second micronucleus experiment (Ciba-Geigy, 1985). In this study, the test article in 0.5% carboxymethyl cellulose was administered orally to groups of 6 female and 6 male Chinese hamsters at one daily dose of 750, 1500 or 3000 mg/kg on each of two consecutive days. The animals were sacrificed 24 h after the second application. Three male animals each of the intermediate and of the high dose group died in the course of the experiment. The bone marrow smears from animals treated with the various doses of the test substance showed no significant difference from the control. The incidence of bone marrow cells with anomalies of nuclei corresponds to the frequency observed in the control group. It is concluded that under the conditions of this experiment, no evidence of mutagenic effects was obtained in Chinese hamsters treated with the test substance.

Justification for selection of genetic toxicity endpoint
Several studies are required for this endpoint, nevertheless, the most recent guideline study was selected.

Justification for classification or non-classification

Classification, Labeling, and Packaging Regulation (EC) No. 1272/2008

The available experimental test data are reliable and suitable for the purpose of classification under Regulation (EC) No.1272/2008. Based on the data, classification for genotoxicity is not warranted under Regulation (EC) No.1272/2008.