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

Genetic toxicity: in vitro

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Test guideline 3

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

Test material

Test material information

Constituent 1

Reference substance name:

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

EC Number:

275-965-6

EC Name:

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

Cas Number:

71735-74-5

Molecular formula:

C11H23O4PS2

IUPAC Name:

ethyl 3-{[bis(propan-2-yloxy)(sulfanylidene)-λ⁵-phosphanyl]sulfanyl}propanoate

Details on test material:

- Physical state: liquid
- Analytical purity: 98.4 g/100g (31P-NMR spectroscopy)
- Storage condition of test material: At room temperature (15 - 25 °C) in the original container away from direct sunlight.

Method

Target gene:

HPRT (hypoxanthine-guanine phosphoribosyl transferase)

Species / strain

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.

Controls

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.

Results and discussion

Test results

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'.

Any other information on results incl. tables

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

Applicant's summary and conclusion

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.