Ethylenebis(oxyethylene) bis[3-(5-tert-butyl-4-hydroxy-m-tolyl)propionate]

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The substance was not mutagenic in the Ames test in Salmonella typhimurium TA 98, TA 100, TA 1535, TA 1537, TA 1538 and E. coli WP2 uvrA, tested both in the absence and presence of S9 mix. No induction of gene mutations in the in vitro mammalian cell gene mutation in V79 cells was observed with and without metabolic activation. No indication of a cell transformation potential was observed in the Balb 3T3 assay performed without an exogenous metabolic system. Overall, the substance is considered to be non genotoxic.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

18.9.1981 to 2.12.1981

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

- 2-Aminoanthracene was the only compound used to test the efficacy of the S9 mix; no independent repeat

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Target gene:

his/trp

Species / strain / cell type:

other: S. typhimurium TA 1535, TA 1537, TA 1538, TA 98, TA 100 and E. coli WP2 uvr A

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced rat liver S9

Test concentrations with justification for top dose:

5, 10, 50, 100, 500, 1000, and 5000 µg/0.1 mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO.
- Justification for choice of solvent/vehicle: Test article was soluble at >= 5% in DMSO.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other:

Remarks:

See Table 1.

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: Incubated for about 48 hours at 37 ºC in darkness.

NUMBER OF REPLICATIONS: 3 petri dishes/strain/group

Evaluation criteria:

When the colonies had been counted, the arithmetic mean was calculated. The test substance is generally considered to be non-mutagenic 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. No statistical analysis was performed.

Species / strain:

other: S. typhimurium TA 1535, TA 1537, TA 1538, TA 98, TA 100 and E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: At the concentrations of 500 µg/0.1 mL and above the substance precipitated in soft agar.

In the experiments performed without and with microsomal activation, comparison of the number of histidine- or tryptophan-prototrophic mutants in the controls and after treatment with TK 12627 revealed no marked differences. The slight increase in the number of back-mutants observed in the experiment without activation on strain TA 1537 at the concentrations of 10 and 5000 µg/0.1 mL is attributed to fluctuations in the rate of spontaneously occurring back-mutants.

EXPERIMENTAL RESULTS

	TA 98		TA 100		TA 1535		TA 1537		TA 1538		WP2uvrA
Dose (µg/0.1 ml)	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
solvent control	33	41	174	156	19	19	6	10	21	40	21	26
5	32	64	144	146	16	11	7	7	17	35	23	25
10	29	62	161	147	12	11	12	8	24	41	20	26
50	31	45	167	142	17	12	6	10	19	40	17	27
100	33	42	152	153	15	7	9	8	23	35	17	24
500	24	52	152	156	12	12	7	13	21	33	20	28
1000	26	47	163	152	15	9	7	8	20	39	15	34
5000	15	38	157	153	12	18	12	8	22	26	13	24
positive controls:
solvent control	40	62	174	152	17	14	10	11	17	37	18	21
concentration A	249	160	2496	221	464	152	67	70	350	96	409	874
concentration B	394	277	593	277	42	431	606	157	707	160	1346	992

Conclusions:

In the experiments performed without and with microsomal activation, comparison of the number of histidine- or tryptophan-prototrophic mutants in the controls and after treatment revealed no marked differences.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2012

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

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/ß-naphthoflavone induced rat liver S9

Test concentrations with justification for top dose:

Experiment 1, without S9: (79.4), 158.8, 317.5, 635.0, 1270,0, 2540.0 µg/ml
Experiment 1, with S9: 79.4, 158.8, 317.5, 635.0, 1270,0, (2540.0) µg/ml
Experiment 2, with and without S9: 79.4, 158.8, 317.5, 635.0, 1270,0, (2540.0) µg/ml
numbers in parantheses: these cultures were discontinued.

Vehicle / solvent:

DMSO; the final concentration of DMSO in the culture medium was 0.5 % v/v.
- Justification for choice of solvent/vehicle: The solvent was selected based on solubility properties and tolerance by the cell cultures.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

ethylmethanesulphonate

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): 7-10 days
- Fixation time (start of exposure up to fixation or harvest of cells): 17 - 22 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 relevance and statistical significance was considered together.

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH and osmolarity: There was no shift of the pH and osmolarity of the medium even at the maximum concentration of the test item.
- Precipitation: Turbidity was noted in the absence of metabolic activation at 158.8 and 317.5 μg/mL in experiment I and at 158.8, 317.5, and 635.0 μg/mL in experiment II. As in the preexperiment such a broad range of turbidity may be based on denatured protein rather than test item precipitation. Precipitation of the test item was noted at 635 μg/mL and above in the first experiment with and without metabolic activation. In the second experiment precipitation was noted at 635 μg/mL and above with and at 1270 μg/mL and above without metabolic activation.

RANGE-FINDING/SCREENING STUDIES:
The range finding pre-experiment was performed using a concentration range of 39.7 to 3907.7 μg/mL to evaluate toxicity in the presence (4 hours treatment) and absence (4 hours and 24 hours treatment) of metabolic activation. No relevant cytotoxic effect occurred up to the maximum concentration with and without metabolic activation following 4 and 24 hours treatment. The test medium was checked for precipitation or phase separation at the end of each treatment period (4 or 24 hours) prior to removal of the test item. Precipitation occurred at 635.0 μg/mL and above with and without metabolic activation following 4 hours treatment. Following 24 hours treatment precipitation was noted at 2540 μg/mL and above. In the
absence of metabolic activation turbidity was observed from 39.7 to 317.5 μg/mL following 4 hours treatment and from 317.5 to 1270 μg/mL after 24 hours treatment. Such a broad range of turbidity indicates that the turbidity is most likely not based on test item particles but rather denatured protein. Based on the occurrence of precipitation in the pre-experiment the individual concentrations were selected.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
No relevant cytotoxic effect indicated by a relative cloning efficiency I or cell density below 50% in both parallel cultures was noted in both main experiments with and without metabolic activation following 4 and 24 hours treatment.

Summary of Results

	concentration (µg/ml)	T/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	100	100	3	1	100	100	100	17.7	1
positive control (EMS)	150		-	113	76.5	98.2	88.7	29.6	112.2	84.1	113.1	113.1	6.4
test item	79.4	T	-	66.1	culture was discontinued#				80.1	culture was discontinued#
test item	158.8	T	-	60.5	64.5	97.4	4.5	1.5	63.3	66.2	112.8	2.5	0.1
test item	317.5	T	-	21.5	28.5	88.4	15.2	5.1	29.4	34	109.8	25.1	1.4
test item	635	P	-	60.9	42.5	99.5	7.3	2.4	63.9	55.5	98.3	32.7	1.8
test item	1270	P	-	77.3	57.2	93.4	13.8	4.6	78.7	71.1	96.5	6.9	0.4
test item	2540	P	-	78.3	50.7	97.6	9.6	3.2	82.9	70.3	89	13.8	0.8
solvent control (DMSO)			+	100	100	100	37.9	1	100	100	100	11.5	1
positive control (DMBA)	1.1		+	86.4	120.2	66	1014.8	26.8	82.2	109	93.3	626.1	54.4
test item	79.4		+	100.8	102	74.6	62.8	1.7	103.3	96	118.7	7.8	0.7
test item	158.8		+	105.3	114.3	107.9	30	0.8	110.3	100.4	96.2	21.7	1.9
test item	317.5		+	107.2	105.5	111.6	32.6	0.9	106.7	100.8	88.5	33	2.9
test item	635	P	+	108.3	108.6	86.2	11.9	0.3	109.5	111.4	102.2	27.3	2.4
test item	1270	P	+	78.9	112.8	112.6	34.3	0.9	99.2	100	87.8	19.9	1.7
test item	2540	P	+	86.7	culture was discontinued##				100	culture was discontinued##
Experiment II / 24h treatment				culture I					culture II
solvent control (DMSO)			-	100	100	100	16.1	1	100	100	100	30.2	1
positive control (EMS)	150		-	102.7	113.5	90.4	394.8	24.5	97.9	91.4	81.9	496.7	16.5
test item	79.4		-	104.6	113.5	85.7	24.1	1.5	101.9	67.2	134.8	17.5	0.6
test item	158.8	T	-	102.7	71.8	99.3	31.1	1.9	99.1	68.5	140.2	18.5	0.6
test item	317.5	T	-	104.5	56.5	98	20.7	1.3	100.5	59.4	105.2	15.1	0.5
test item	635	T	-	100.4	72.7	100.3	20.4	1.3	98.1	50.9	111.5	12.1	0.4
test item	1270	P	-	102.4	95.9	97	14.2	0.9	97.6	107.2	117.1	36.2	1.2
test item	2540	P	-	100	culture was discontinued##				96.9	culture was discontinued##
Experiment II / 4h treatment
solvent control (DMSO)			+	100	100	100	14.5	1	100	100	100	31.5	1
positive control (DMBA)	1.1		+	80.6	118.3	75.4	757.6	52.3	69.4	47.3	81.3	606.4	19.2
test item	79.4		+	115.6	126.7	93.7	17.6	1.2	96.5	79.6	81.9	26.2	0.8
test item	158.8		+	112.6	130.1	73.2	31.4	2.2	92.9	68.6	81.3	27	0.9
test item	317.5		+	108.4	130.9	82.6	31	2.1	93.2	118.9	107.7	31.4	1
test item	635	P	+	108.1	142.3	89.4	21.7	1.5	91.3	125	87.3	20	0.6
test item	1270	P	+	109	117.6	87	36.3	2.5	94.3	85.9	91.6	38.2	1.2
test item	2540	P	+	112.3	culture was discontinued##				91.4	culture was discontinued##

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

## culture was not continued to avoid analysis of too many precipitating concentrations

T Turbidity

P Preparation visible at the end of treatment

Conclusions:

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 tested concentrations ranged from 79.4 to 1270 µg/ml.

Turbidity was noted in the absence of metabolic activation at 158.8 and 317.5 μg/mL in experiment I and at 158.8, 317.5, and 635.0 μg/mL in experiment II. As in the preexperiment such a broad range of turbidity may be based on denatured protein rather than test item precipitation. Precipitation of the test item was noted at 635 μg/mL and above in the first experiment with and without metabolic activation. In the second experiment precipitation was noted at 635 μg/mL and above with and at 1270 μg/mL and above without metabolic activation. No relevant cytotoxic effect indicated by a relative cloning efficiency I or cell density below 50% in both parallel cultures was noted in both main experiments with and without metabolic activation following 4 and 24 hours treatment.

No relevant and reproducible increase in mutant colony numbers/10⁶ cells was observed in the main experiments up to the maximum concentration with and without metabolic activation. In experiment I the induction factor exceeded the threshold of 3.0 at 317.5, 1270, and 2540 μg/mL in the first culture without metabolic activation (induction factors of 5.1, 4.6, and 3.2). These isolated effects were biological irrelevant as they were based on the rather low solvent control of just 3.0 colonies per 10⁶ cells and were not confirmed in the parallel culture II performed under identical experimental conditions or in the second experiment. In the first culture of experiment I the mutant colonies/10⁶ cells exceeded the range of the historical solvent control data at 79.4 μg/mL with metabolic activation. Another increase exceeding the range of the historical solvent control data was observed in the second culture of experiment II at 1270 μg/mL with metabolic activation. However, the threshold of three times the mutation frequency of the corresponding solvent control was not reached or exceeded. A linear regression analysis (least squares) was performed to assess a possible dose dependent increase of mutant frequencies. No significant dose dependent trend of the mutation frequency indicated by a probability value of <0.05 was determined in any of the experimental groups. In both experiments of this study (with and without S9 mix) the range of the solvent controls was from 3.0 up to 37.9 mutants per 10⁶ cells; the range of the groups treated with the test item was from 2.5 up to 62.8 mutants per 10⁶ cells. The highest solvent control of 37.9 colonies per 10⁶ cells slightly exceeded the historical range of solvent controls with metabolic activation (3.4 - 36.6 mutant colonies per 10⁶). The mean value of both parallel cultures (37.9 and 11.5 equal to a mean of 24.7) remained well within the historical range of solvent controls with metabolic activation. 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:

genetic toxicity in vitro

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

no guideline available

Principles of method if other than guideline:

Balb 3T3 cell transformation assay

GLP compliance:

no

Type of assay:

in vitro mammalian cell transformation assay

Target gene:

Morphological detection of changes due to transformations in mammalian cells induced by chemical substances.

Species / strain / cell type:

mammalian cell line, other: Mouse BALB/3T3 cells

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

not applicable

Test concentrations with justification for top dose:

40.0, 20.0, 10.0, 5.0, and 2.5 µg/mL.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

3-methylcholanthrene

Species / strain:

mammalian cell line, other: Mouse BALB/3T3 cells

Metabolic activation:

not applicable

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Under the given experimental conditions no effects were obtained that must be interpreted as suggestive of a transformative property .

Conclusions:

Under the given experimental conditions no effects were obtained that must be interpreted as suggestive of a transformative property.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

No genotoxicity was observed in Chinese hamsters at doses of 750, 1500 and 3000 mg/kg bw in the nucleus anomaly test.

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:

1982

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:

yes

Remarks:

1000 instead of 2000 cells scored per animal

GLP compliance:

no

Type of assay:

micronucleus assay

Species:

hamster, Chinese

Strain:

not specified

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Weight at study initiation: Females weighed 21-28 g and males weighed 21-30 g.
- Housing: Individual caging.
- Diet (e.g. ad libitum): Standard diet (NAFAG No.924) ad libitum.
- Water (e.g. ad libitum): Ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22-23 °C.
- Humidity (%): 54-57%.
- Photoperiod (hrs dark / hrs light): 12 h/12 h.

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: CMC (carboxymethyl cellulose) plus Tween 80.
- Amount of vehicle (if gavage or dermal): 20 mL CMC plus Tween 80/kg body weight.
- Vehicle(s)/solvent(s) used: Sodium CMC (carboxymethyl cellulose) plus Tween 80.
- Concentration of test material in vehicle: 750, 1,500, and 3,000 mg/kg body weight in 20 mL/kg body weight of 0.5% aqueous solution of sodium CMC containing 0.1% Tween 80.

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: No details.

Duration of treatment / exposure:

The preparation was administered orally to groups of 6 female and 6 male animals each. Treatment consisted of daily one gavage administration on 2 consecutive days.

Frequency of treatment:

Once/day.

Post exposure period:

24 h after the second application, the animals were sacrificed by dislocation of the cervical vertebrae.

Dose / conc.:

750 mg/kg bw/day (actual dose received)

Dose / conc.:

1 500 mg/kg bw/day (actual dose received)

Dose / conc.:

3 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

6 Males and 6 females.

Control animals:

yes, concurrent vehicle

Positive control(s):

Cyclophosphamide
- Route of administration: Oral (gavage).
- Doses/concentrations: 128 mg/kg body weight in 20 mL/kg body weight.

Tissues and cell types examined:

Bone marrow was harvested from the shafts of both femurs.

Details of tissue and slide preparation:

DETAILS OF SLIDE PREPARATION:
In a siliconized pipette filled with approx. 0.5 µL rat serum the bone marrow was drawn up. In order to receive a homogeneous suspension the content of pipette was aspirated gently about 3 times. 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. 3 Hours later, the slides were stained in undiluted May-Grünwald solution for 2 minutes then in May-Grünwald solution:water 1:1 for 2 minutes and then in Giemsa's (40%) for 20 minutes. After being rinsed in methanol (55%) for 5-8 seconds and washed off twice in water, they were left immersed in water for approximately 2 minutes. After rinsing with distilled water and air-drying, the slides were cleared in Xylol and mounted in Eukitt.

Evaluation criteria:

The slides of 3 female and 3 male animals each of the negative control group, the positive control group and of the groups treated with various dose were examined. 1000 Bone marrow cells each were scored per animal and the following anomalies were registered:
a) Single Jolly bodies;
b) fragments of nuclei in erythrocytes;
c) micronuclei in erythroblasts;
d) micronuclei in leucopoietic cells; and
e) polyploid cells.

Statistics:

The significance of difference was assessed by chi-square test.

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:

In all dosage groups the percentage of cells displaying anomalies of nuclei did not differ significantly from the negative control. By contrast, the positive control (cyclophosphamide, 128 mg/kg body weight) yielded a marked increase of the percentage of cells with anomalies. Here the mean percentage of anomalies was 9.68, whereas the negative control yielded a percentage of 0.10. The difference is highly significant (p<0.05).

EXPERIMENTAL RESULTS

			percent of cells with anomalies of nuclei
	number of animal	sex of animal	single jolly bodies	frasgments of nuclei in erythrocytes	micronuclei in erythroblasts	micronuclei in leucopoietic cells	polyploid cells	total
Control (0.5% CMC + 0.1% Tween 80)	1	f				0.1		0.1
	2	f					0.1	0.1
	3	f	0.2			0.1		0.3
	4	m						0.0
	5	m						0.0
	6	m	0.1					0.1
Cyclophosphamide (128 mg/kg)	1	f	5.5	0.9	2.1	0.6		9.1
	2	f	10.9	1.7	1.7	0.4		14.7
	3	f	6.0	0.2	2.4	0.1	0.1	8.8
	4	m	5.1	0.6	0.8	0.2		6.7
	5	m	5.7	1.6	1.4	0.3	0.1	9.1
	6	m	8.2	0.9	0.2	0.4		9.7
test article (750 mg/kg)	1	f						0.0
	2	f	0.2					0.2
	3	f	0.2					0.2
	4	m	0.2					0.2
	5	m						0.0
	6	m						0.0
test article (1500 mg/kg)	1	f	0.1					0.1
	2	f	0.1					0.1
	3	f	0.1					0.1
	4	m	0.1					0.1
	5	m						0.0
	6	m	0.1					0.1
test article (3000 mg/kg)	1	f	0.1					0.1
	2	f	0.2		0.1	0.1		0.4
	3	f	0.1			0.1		0.2
	4	m						0.0
	5	m						0.0
	6	m	0.1					0.1

Conclusions:

It was concluded that, under the conditions of this experiment, no evidence of mutagenic effects were noted in Chinese hamsters treated with the test substance.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Genetic toxicity in vitro

Gene mutation in bacteria

The substance was not mutagenic in the Ames test in Salmonella typhimurium TA 98, TA 100, TA 1535, TA 1537, TA 1538 and E. coli WP2 uvrA, tested both in the absence and presence of S9 mix prepared from Arochlor-1254-induced rats following the procedure of OECD testing guideline 471. The study was not performed under GLP but contains sufficient documentation to suggest GLP-like characteristics. Incubations were performed in a plate-incorporation design without an independent repeat experiment. Tested concentrations were up to 5000 μg per plate, with precipitation occurring at 500 μg/plate and above. DMSO was used as vehicle. Cytotoxicity was not observed. The validity of the experiment was confirmed by incubation with positive control substances. Treatment with the test item did not cause an increase in the number of reverse mutations.

 

Gene mutation in mammalian cells

In a GLP compliant in vitro mammalian cell gene mutation test (HPRT-Test) according to OECD Guideline 476 the test substance was investigated in two independent experiments, using two parallel cultures each. In the main experiment I cells were exposed for 4 hours to the test item at concentrations of 79.4; 158.8; 317.5; 635.0; 1270.0; 2540.0 μg/mL with and without liver microsomal activation, whereas the second experiment was performed with a treatment time of 4 hours with metabolic activation and a treatment time of 24 hours without metabolic activation at the same concentrations. The test item was dissolved in DMSO. The maximum concentration of the pre-experiment and the main experiments was 2540.0 μg/mL. 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. Precipitation of the test item was noted at 635 μg/mL and above. No relevant cytotoxic effect was found. No relevant and reproducible increase in mutant colony numbers/106cells was observed in the main experiments up to the maximum concentration with and without metabolic activation. According to the results of the study, the test substance did not induce gene mutations in the in vitro mammalian cell gene mutation test under the experimental conditions chosen.

 

Mammalian cell transformation assay

The BALB 3T3 cell transformation assay was performed with 3 -methylcholanthrene as positive control which yielded transformation frequencies of 6.84 and 11.5 at 1.5 and 3μg/ml, respectively. Incubations with test item concentrations of 2.5 to 40μg/ml, vehicle or culture medium resulted in transformation frequencies between 0 and 1.07 which was neither dose-dependent nor outside the historical range. The highest test item concentration was determined in a cytotoxicity experiment and chosen based on a 25% reduction in colony forming ability. The final concentration of the solvent DMSO in the culture medium was 1%. It was not performed under GLP, but contains sufficient documentation to suggest GLP-like characteristics.

Genetic toxicity in vitro

Chromosome aberration in mammals

The micronucleus study was performed prior to the introduction of the OECD testing guideline and therefore contains minor differences in terminology and design. It was not performed under GLP, but contains sufficient documentation to suggest GLP-like characteristics. Only 1000 cells per animal scored, whereas OECD 474 requires 2000 cells per animal. This is considered acceptable because each six animals for 1500 and 3000 mg/kg bw were investigated, whereas a guideline compliant study requires five animals for a limit dose of 2000 mg/kg bw. The substance was applied by gavage in a suspension in 0.5% carboxymethylcellulose containing 0.1% Tween 80. Each one dose of 750, 1500 or 3000 mg/kg bw was given on two consecutive days. Cyclophosphamide given at 128 mg/kg bw served as positive control. Negative control group animals received the vehicle only. Animals were sacrificed 24h after the last treatment and bone marrow cells were prepared and mounted on slides for scoring. The following anomalies were investigated: single jolly bodies, fragments of nuclei in erythrocytes, micronuclei in erythroblasts, micronuclei in leucopoietic cells and polyploid cells. In contrast to the positive control, no increase in the number of anomal nuclei was observed for the test item.

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 classification purposes under Regulation 1272/2008. As a result the substance is not considered to be classified for genotoxicity under Regulation (EC) No. 1272/2008.