Ethyl 3-[[bis(1-methylethoxy)phosphinothioyl]thio]propionate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames test: negative (BASF, 2012) HPRT test: negative (Harlan, 2012)

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

September 13, 2012 - October 18, 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: 12.5, 25.0, 50.0, 100.0, (200.0), (400.0) µg/ml
Experiment 1, with S9: (12.5), 25.0, 50.0, 100.0, 200.0, 400.0 µg/ml
Experiment 2, without S9: 12.5, 25.0, 50.0, 100.0, (200.0), (400.0) µg/ml
Experiment 2, with S9: (12.5), 25.0, 50.0, 100.0, 200.0, 400.0 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.

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): 8 days
- Fixation time (start of exposure up to fixation or harvest of cells): 17days

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.

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

cytotoxicity occurred at 50.0 μg/mL and above without and at 400 μg/mL with metabolic activation

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Phase separation was observed at 100 μg/mL and above without and at 200 μg/mL and above with metabolic activation.

RANGE-FINDING/SCREENING STUDIES:
In the pre-test Relevant cytotoxic effects were observed at 200.6 μg/mL, and 802.5 μg/mL, and above after 4 hours treatment without metabolic activation and at 3210 μg/mL with metabolic activation. Following 24 hours treatment cytotoxic effects occurred at 100.3 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 observed at 200.6 μg/mL and above in the presence and absence of metabolic activation following 4 and 24 hours treatment. Based on the results of the pre-experiment, the individual concentrations of the main experiments were selected. A series of concentrations spaced by a factor of 2 was applied.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Relevant cytotoxic effects indicated by a relative cloning efficiency I and/or a relative cell density below 50% were observed in the first experiment at 50.0 μg/mL and above without and at 400 μg/mL with metabolic activation. In the second experiment cytotoxic effects as described above occurred at 50.0 μg/mL and above without and at 400 μg/mL with metabolic activation.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Summary of Results

	concentration (µg/ml)	PS	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	10.9	1	100	100	100	11.5	1
positive control (EMS)	150		-	95.5	123.2	91.9	64.1	5.9	101.7	79.8	80.2	74.2	6.5
test item	12.5		-	97.1	161.2	75.4	10.1	0.9	104.6	92	65.6	29.2	2.5
test item	25		-	90	73.6	96.6	10.5	1	92.5	62.8	91.9	4.3	0.4
test item	50		-	14.3	14.1	83.9	6.3	0.6	12.7	10.8	87.3	11	1
test item	100	PS	-	39.3	20.2	89.8	5	0.5	28.9	15.7	99.2	9.3	0.8
test item	200	PS	-	0	culture was discontinued#				0	culture was discontinued#
test item	400	PS	-	0	culture was discontinued#				0	culture was discontinued#
solvent control (DMSO)			+	100	100	100	15.5	1	100	100	100	12.5	1
positive control (DMBA)	1.1		+	92.7	79.9	94.2	559	36.1	87.1	94.2	120.4	367.4	29.4
test item	12.5		+	101.2	culture was discontinued##				101	culture was discontinued##
test item	25		+	96.8	88.7	104.6	10.7	0.7	95.1	119.6	115.4	17.4	1.4
test item	50		+	94.6	112.7	101.8	14.7	1	92.7	113	99.2	15	1.2
test item	100		+	94.9	90.9	96.6	10.8	0.7	94.4	105.6	77.4	35.4	2.8
test item	200	PS	+	94.2	95.1	103	20.3	1.3	92.6	102.4	109.4	7.4	0.6
test item	400	PS	+	36.5	26	93.7	12.4	0.8	28.1	41	104.2	10.1	0.8
Experiment II / 24h treatment				culture I					culture II
solvent control (DMSO)			-	100	100	100	24.4	1	100	100	100	30.6	1
positive control (EMS)	150		-	78.6	50.7	106.4	275.6	11.3	78.1	65.6	99.8	314.4	10.3
test item	12.5		-	92.6	94.8	98	29.4	1.2	95.5	97.2	106.2	34.5	1.1
test item	25		-	92.3	63	100.5	17.7	0.7	98.4	110.1	95	26.1	0.9
test item	50		-	46.2	17.5	94.2	22.3	0.9	44.2	14.4	103.5	15	0.5
test item	100	PS	-	32.9	11.2	89	9.4	0.4	31.1	11	74.3	33.1	1.1
test item	200	PS	-	0	culture was discontinued#				0	culture was discontinued#
test item	400	PS	-	0	culture was discontinued#				0	culture was discontinued#
Experiment II / 4h treatment
solvent control (DMSO)			+	100	100	100	28.7	1	100	100	100	14.9	1
positive control (DMBA)	1.1		+	105.1	69.6	94.6	686	23.9	87.1	74.2	71.2	851.2	57.2
test item	12.5		+	112.4	culture was discontinued##				96.1	culture was discontinued##
test item	25		+	103.8	97.5	132.7	20.4	0.7	142.6	86.8	124	18.4	1.2
test item	50		+	98.5	93.8	114.2	28.2	1	105.2	95.8	103.4	19.8	1.3
test item	100		+	98.5	95.2	112	23.2	0.8	94.9	95.4	102.1	33.5	2.2
test item	200	PS	+	113.5	95.2	120.5	13	0.5	100	95	122.5	30.1	2
test item	400	PS	+	39.3	59.4	105.6	27.4	1	35.8	10.4	93.1	51.1	3.4

PS = Phase Separation visible at the end of treatment

# culture was not continued due to exceedingly severe cytotoxic effects

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

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 assay was performed in two independent experiments, using two parallel cultures each. 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 test item was dissolved in DMSO. The tested concentrations ranged from 12.5 to 400 µg/ml. Phase separation was observed at 100 μg/mL and above without and at 200 μg/mL and above with metabolic activation. Relevant cytotoxic effects indicated by a relative cloning efficiency I and/or a relative cell density below 50% were observed in the first experiment at 50.0 μg/mL and above without and at 400 μg/mL with metabolic activation. In the second experiment cytotoxic effects as described above occurred at 50.0 μg/mL and above without and at 400 μg/mL with metabolic activation. The recommended cytotoxic range of approximately 10-20% relative cloning efficiency I or relative cell density was covered without metabolic activation. In the presence of metabolic activation phase separation was limiting.

No relevant and reproducible increase in mutant colony numbers/10⁶ cells was observed in the main experiments up to the maximum concentration. An isolated increase of the induction factor exceeding the induction factor of three times the mutation frequency of the corresponding solvent control was observed in the second culture of the second experiment with metabolic activation at 400 μg/mL with metabolic activation. At this data point the absolute value of the mutation frequency (50.5 mutant colonies per 10⁶ cells) also exceeded the historical range of solvent controls (3.4 - 36.6 mutant colonies/10⁶ cells). However, the effect was not reproduced in the parallel culture performed under identical experimental conditions and was therefore, judged as biologically irrelevant artefact based on phase separation. A linear regression analysis (least squares) was performed to assess a possible dose dependent increase of mutant frequency. A significant dose dependent trend of the mutation frequency indicated by a probability value of <0.05 was determined in the first experiment at culture I without metabolic activation and in the second culture of experiment II with metabolic activation. However, the trend noted in the first culture of the first experiment without metabolic activation was reciprocal, going down versus increasing concentrations and thus, irrelevant. The trend noted in the second culture of the second experiment with metabolic activation was based on an irreproducible phase separation artefact as discussed above and consequently, irrelevant as well. 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.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

27.2.2012 - 15.3.2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Qualifier:

according to guideline

Guideline:

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

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

GLP compliance:

yes (incl. QA statement)

Remarks:

BASF SE, Experimental Toxicology and Ecology, 67056 Ludwigshafen, Germany

Type of assay:

bacterial reverse mutation assay

Target gene:

his, trp

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

liver S9 mix from induced rats

Test concentrations with justification for top dose:

33 μg - 5 500 μg/plate (SPT)
33 μg - 5 500 μg/plate (PIT)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Due to the limited solubility of the test substance in ultrapure water, DMSO was used as vehicle, which had been demonstrated to be suitable in bacterial reverse mutation tests and for which historical control data are available.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: see "details on test system"

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation); preincubation

DURATION
- Preincubation period: 20 min
- Exposure duration: 48 - 72 h

NUMBER OF REPLICATIONS: three

DETERMINATION OF CYTOTOXICITY
• decrease in the number of revertants
• clearing or diminution of the background lawn (= reduced his- or trp- background growth)
• reduction in the titer

POSITIVE CONTROLS
With S9 mix
• 2-aminoanthracene (2-AA) (SIGMA, A-1381) 1)
- 2.5 μg/plate, dissolved in DMSO
- strains: TA 1535, TA 100, TA 1537, TA 98
- 60 μg/plate, dissolved in DMSO
- strain: Escherichia coli WP2 uvrA
Without S9 mix
• N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) (FLUKA, 68051) 1)
- 5 μg/plate, dissolved in DMSO
- strains: TA 1535, TA 100
• 4-nitro-o-phenylenediamine (NOPD) (SIGMA, N-9504) 1)
- 10 μg/plate, dissolved in DMSO
- strain: TA 98
• 9-aminoacridine (AAC) (SIGMA, A-1135) 1)
- 100 μg/plate, dissolved in DMSO
- strain: TA 1537
• 4-nitroquinoline-N-oxide (4-NQO) (SIGMA, N-8141) 1)
- 5 μg/plate, dissolved in DMSO
- strain: E. coli WP2 uvrA

Evaluation criteria:

The test substance is considered positive in this assay if the following criteria are met:
• A dose-related and reproducible increase in the number of revertant colonies, i.e. about doubling of the spontaneous mutation rate in at least one tester strain either without S9 mix or after adding a metabolizing system.

A test substance is generally considered non-mutagenic in this test if:
• The number of revertants for all tester strains were within the historical negative control range under all experimental conditions in at least two experiments carried out independently of each other.

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No test substance precipitation was found with and without S9 mix.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
A weak bacteriotoxic effect (slight decrease in the number of his+ revertants, slight reduction in the titer) was occasionally observed in the standard plate test and in the preincubation assay depending on the strain and test conditions from about 2 750 μg/plate onward only with metabolic activation.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

EXPERIMENTAL RESULTS

Standard Plate Test:

	TA 1535		TA 100		TA 1537		TA 98		WP2 uvra
Dose (µg/plate)	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
DMSO	12	14	71	86	6	8	21	29	47	54
33	11	14	78	88	5	7	21	23	45	53
100	12	13	67	83	6	7	20	26	48	51
333	12	13	69	80	7	7	20	26	51	54
1000	13	15	76	92	5	7	22	25	49	52
2750	13	14	74	72	6	6	22	20	49	54
5500	9	13	61	84	5	5	17	18	43	54
positive control*	633	165	694	998	448	113	606	795	614	252

Preincubation Test:

	TA 1535		TA 100		TA 1537		TA 98		WP2 uvra
Dose (µg/plate)	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
DMSO	13	12	72	76	7	7	19	25	43	41
33	13	12	72	74	7	8	19	25	43	40
100	12	15	77	75	7	8	20	24	41	44
333	12	12	79	74	7	7	17	21	47	43
1000	12	13	76	81	6	8	19	23	44	40
2750	11	13	76	82	7	6	18	18	43	37
5500	10	13	72	74	6	6	16	21	43	36
positive control*	556	146	884	803	354	122	595	634	575	258

* see "details on test system and conditions"

Conclusions:

Under the experimental conditions chosen here, it is concluded that the test article is not a mutagenic test substance in the bacterial reverse mutation test in the absence and the presence of metabolic activation.

Executive summary:

The test article's potential to cause point mutations in bacteria was assessed in a reverse mutation assay according to OECD guideline 471 and in compliance with GLP. The test article was tested independently in a standard plate test and by the preincubation method at concentrations ranging from 33 - 5500 µg/plate with S. typhimurium TA 1535, 100, 1537, 98 and E. coli WP2 uvrA. A weak bacteriotoxic effect (slight decrease in the number of his+ revertants, slight reduction in the titer) was occasionally observed in the standard plate test and in the preincubation assay depending on the strain and test conditions from about 2 750 μg/plate onward only

with metabolic activation.According to the results of the present study, the test substance did not lead to a relevant increase in the number of revertant colonies either without S9 mix or after adding a metabolizing system. The results of the negative as well as the positive controls performed in parallel corroborated the validity of this study, since the values fulfilled the acceptance criteria of this study. In this study with and without S9 mix, the number of revertant colonies in the negative controls was within the range of the historical negative control data for each tester strain. In addition, the positive control substances both with and without S9 mix induced a significant increase in the number of revertant colonies within the range of the historical positive control data or above. Thus, under the experimental conditions chosen here, it is concluded that the test substance is not mutagenic in the bacterial reverse mutation test in the absence and the presence of metabolic activation.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

MNT (structural analogue): negative (RCC, 2000)

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

(1997)

Deviations:

no

Remarks:

Deviations from the protocol were stated (example given: relative humidity under which the experiment was conducted ranged between 18-70 % and not between 30-70 % as described in the protocol) which however, did not affect the validity of the experiment.

GLP compliance:

yes (incl. QA statement)

Type of assay:

micronucleus assay

Species:

mouse

Strain:

NMRI

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: RCC Ltd. Biotechnology & Animal Breeding Division CH-4414 Füllinsdorf
- Age at study initiation: 8 - 10 weeks
- Weight at study initiation: 33 +/- 2.7 g (males), 28.4 +/- 1.8 g (females)
- Assigned to test groups randomly: yes
- Housing: individually in Makrolon type-I cages
- Diet (ad libitum): pelleted standard diet, ALTROMIN 1324
- Fasting period before study: 18 h
- Water (ad libitum): tap water
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 +/- 4
- Humidity (%): 18 - 70
- Photoperiod (hrs dark / hrs light): 12 / 12

Route of administration:

oral: gavage

Vehicle:

- Vehicle: corn oil
- Concentration of test material in vehicle: In a pretest 2000 mg/kg bw, 1800 mg/kg bw in main study
- Application volume: 10 ml/kg bw

Duration of treatment / exposure:

After single oral administration the following dose levels of the test item were investigated:
24 h preparation interval: 450, 900 and 1800 mg/kg bw
48 h preparation interval: 1800 mg/kg bw

Frequency of treatment:

Single oral administration

Post exposure period:

MAIN STUDY:
450, 900 and 1800 mg/kg bw: 24 h
1800 mg/kg bw: 48 h

Dose / conc.:

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

Remarks:

observation period 24 h

Dose / conc.:

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

Remarks:

observation period 24 h

Dose / conc.:

1 800 mg/kg bw/day (actual dose received)

Remarks:

observation period 24 h and 48 h

No. of animals per sex per dose:

Six animals per sex per dose (5 per sex were evaluated)

Control animals:

yes, concurrent no treatment

Positive control(s):

- Positive Control: 40 mg/kg bw Cyclophosphamide
- Volume administered: 10 ml/kg bw
- Route of administration: single oral application

Tissues and cell types examined:

Bone marrow was harvested from the femurs. 2000 PCE per animal were used to determine the incidence of micronucleated PCE. Normochromatic erythrocytes (NCE) as well as polychromatic erythrocytes (PCE) were counted in the same sample for determination of the PCE/NCE-ratio.

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
In a pre-test with 2000 mg/kg bw a female died 24 h after application of CG 37-1586 per gavage. In a second pre-test with 4 animals (2 males, 2 females) no mortality was observed within 48 h after administration of 1800 mg/kg bw test article. Therefore, 1800 mg/kg bw CG 37-1586 was selected as maximum tolerated dose level.

DETAILS OF SLIDE PREPARATION:
Bone marrow was harvested from the shafts of femurs. Preparations were air-dried and stained in May-Grünwald/Giemsa's. Cover slips were mounted in Eukitt.

Evaluation criteria:

In this report, a test article was classified as mutagenic if it induces either a dose-related increase in the number of micronucleated polychromatic erythrocytes or a statistically significant and biologically relevant positive response.

A test item producing neither a dose-related increase in the number of micronucleated polychromatic erythrocytes nor a statistically significant and biologically relevant positive response is considered non-mutagenic.

Statistics:

The significance of difference was assessed by the non-parametric Mann-Whitney test.

Sex:

male/female

Genotoxicity:

negative

Remarks:

PCEs with micronuclei 0.03, 0.05, 0.035 % after 24 h at 450, 900 and 1800 mg/kg bw, respectively versus 0.05 % in control. After 48 h at 1800 mg/kg bw the percentage was 0.015 %. Positive control (24 h) showed 1.67 % micronucleated PCE.

Toxicity:

yes

Remarks:

The number of NCE increased slightly at the dose level of 1800 mg/kg bw (2027 NCE/2000 PCE after 24 h; 2117 NCE/2000 PCE after 48 h) in comparison to vehicle control (1732 NCE/2000 PCE).

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

In a pre-test with 2000 mg/kg bw a female died 24 h after application of CG 37-1586 per gavage. In a second pre-test with 4 animals (2 males, 2 females) no mortality was observed within 48 h after administration of 1800 mg/kg bw test article. Therefore, 1800 mg/kg bw CG 37-1586 was selected as maximum tolerated dose level. However, animals at this dose level showed clinical signs of toxicity (reduction of spontaneous activity, eyelid closure, apathy, abdominal position). During main study one female died in the highest dose level within 24 h. It was replaced with a satellite animal so that the overall number of animals was sufficient.

No statistically significant increase in the frequency of micronuclei at any preparation interval and dose level after administration of the test item was seen. The mean values of micronuclei observed after treatment with CG 37-1586 were below or as high as the value of the vehicle control group and within the historical control data range.

The percentage of micronucleated cells in the high dose group at the 48 h sampling time was below the historical data range. However, the authors stated that such values have been obtained in previous studies and do not affect the validity of the study.

 

The positive control (cyclophosphamide) showed a statistically significant increase of induced micronucleus frequency.

Additional information

Point Mutation in Bacteria

The test material was tested in an Ames test following OECD test guideline 471 and under GLP quality control. According to the results of the present study, the test substance did not lead to a relevant increase in the number of revertant colonies either without S9 mix or after adding a metabolizing system in two experiments carried out independently of each other (standard plate test and preincubation assay). The number of revertant colonies in the negative controls was within the range of the historical negative control data for each tester strain. In addition, the positive control substances both with and without S9 mix induced a significant increase in the number of revertant colonies within the range of the historical positive control data or above. Thus, the test is considered valid and it is concluded that the test article is not mutagenic in the bacterial reverse mutation test in the absence and the presence of metabolic activation.

Point Mutation in Mammalian Cells

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 assay was performed in two independent experiments, using two parallel cultures each. The first main experiment was performed with and without liver microsomal activation and a treatment period of 4 hours. The second experiment was performed with a treatment time of 4 hours with and 24 hours without metabolic activation. The maximum concentration of the pre-experiment was 3210 μg/mL or approximately 10 mM according to the current OECD guideline 476. The test item was dissolved in DMSO. The concentration range of the main experiments was limited by cytotoxic effects and phase separation of the test item. The test item was dissolved in DMSO. The tested concentrations ranged from 12.5 to 400 µg/ml. Phase separation was observed at 100 μg/mL and above without and at 200 μg/mL and above with metabolic activation. Relevant cytotoxic effects indicated by a relative cloning efficiency I and/or a relative cell density below 50% were observed in the first experiment at 50.0 μg/mL and above without and at 400 μg/mL with metabolic activation. In the second experiment cytotoxic effects as described above occurred at 50.0 μg/mL and above without and at 400 μg/mL with metabolic activation. In the presence of metabolic activation phase separation was limiting. No substantial and reproducible dose dependent increase of the mutation frequency was observed up to the maximum concentration with and without metabolic activation. 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. Therefore, the test article is considered to be non-mutagenic in this HPRT assay.

Chromosomal Damage

This information is taken from a structural analogue (see attached document for read across justification). No statistically significant increase in the frequency of micronuclei at any preparation interval and dose level after administration of the test item was seen. The mean values of micronuclei observed after treatment with the test item were below or as high as the value of the vehicle control group and within the historical control data range. The percentage of micronucleated cells in the high dose group at the 48 h sampling time was below the historical data range. However, the authors stated that such values have been obtained in previous studies and do not affect the validity of the study. The positive control (cyclophosphamide) showed a statistically significant increase of induced micronucleus frequency.

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.