Propanoic acid, 3-[[bis(2-methylpropoxy)phosphinothioyl]thio]-2-methyl-

Genetic toxicity: in vitro

Administrative data

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:

October 27, 1999- October 27, 1999

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2000

Report date:

2000

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

(1997)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

434-070-2

EC Name:

-

Cas Number:

268567-32-4

Molecular formula:

C12 H25 O4 P S2

IUPAC Name:

3-{[bis(2-methylpropoxy)(sulfanylidene)-λ⁵-phosphanyl]sulfanyl}-2-methylpropanoic acid

Test material form:

liquid: viscous

Method

Target gene:

All Salmonella Typhimurium strains are histidine auxotrophic mutants. The E. coli strain is a tryptophan auxotrophic mutant.

Species / strain

Species / strain 1

Species / strain / cell type:

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

Additional strain / cell type characteristics:

other: TA1537: his C 3076; rfa-; uvrB-; TA 98: his D 3052; rfa-; uvrB-; TAl535: his G 46; rfa-; uvrB-, TA 100: hisG46;rfa-;uvrB-. The uvrB derivative has a reduction in the activity of an excision repair system.

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Additional strain / cell type characteristics:

other: The uvrA derivative is deficient in the DNA repair process (excision repair damage).

Metabolic activation:

with and without

Metabolic activation system:

Mixture of co-factors with S9 fraction of liver from Wistar rats (HanIbm) induced with phenobarbital and beta-naphthoflavone.

Test concentrations with justification for top dose:

A preliminary toxicity test was carried out with concentrations of 3, 10, 33, 100, 333, 1000, 2500, 5000 µg/plate suspended in dimethylsulfoxide. From the preliminary test, following concentrations: 0, 33, 100, 333, 1000, 2500, 5000 µg/plate, were used for assessment of mutagenicity, since only minor toxic effects were observed in one of the strains at the maximal concentration and 5000 µg/plate were chosen as maximal concentration. These concentrations were designated as Experiment I of the main mutagenicity test (Plate Incorporation Test).
Since results of Experiment I were negative a second mutagenicity test, designated as Experiment II, was undertaken with following concentrations:
0, 33, 100, 333, 1000, 2500, 5000 µg/plate (Pre-Incubation Test).

Vehicle / solvent:

- Vehicle: DMSO
The solvent was chosen because of its solubility properties and its relative non-toxicity to the bacteria
No precipitation of the test item occurred up to the highest investigated dose.

Controls

Controls 1

Untreated negative controls:

yes

Remarks:

concurrent untreated

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Remarks:

(without metabolic activation)

Positive control substance:

sodium azide

Remarks:

TA 1535, TA 100; 10 µg/plate

Controls 2

Untreated negative controls:

yes

Remarks:

concurrent untreated

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Remarks:

(without metabolic activation)

Positive control substance:

other: 4-nitro-o-phenylene-diamine

Remarks:

TA 98, 10 µg/plate; TA 1537, 50 µg/plate

Controls 3

Untreated negative controls:

yes

Remarks:

concurrent untreated

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Remarks:

(without metabolic activation)

Positive control substance:

methylmethanesulfonate

Remarks:

E. coli WP2 uvrA, 5 µL/plate

Controls 4

Untreated negative controls:

yes

Remarks:

concurrent untreated

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Remarks:

(with metabolic activation)

Positive control substance:

other: 2-aminoanthracene

Remarks:

All S. typhimurium, 2.5 µg/plate; E. coli WP2 uvrA, 10 µg/plate

Details on test system and experimental conditions:

The study consisted of a toxicity pre-screen test with and without metabolic activation (plate incorporation, reported as experiment I). Only minor toxic effects were observed. Experiment II was performed as pre-incubation test with and without metabolic activation.

METHOD OF APPLICATION:
Experiment I: plate incorporation test; Experiment II: preincubation test
In each experiment 0.1 mL of the test substance or the vehicle, 0.1 mL of a bacterial culture (in nutrient broth), 0.5 ml S9 mix (with metabolic activation) or S9 mix substitution buffer (without metabolic activation) in 2.0 mL of soft agar was used. In the pre-incubation assay soft agar was added after an incubation period of 1 h at 37 °C. The plates were incubated for about 48 hours at 37 °C in darkness. Each concentration and the controls were tested in triplicate.

DURATION
- Preincubation period: 1 h (Experiment II)
- Expression time: 48 h (Experiment I + II)

DETERMINATION OF CYTOTOXICITY
- Toxicity was assessed as clearing of the bacterial background lawn and/or as reduction of spontaneous revertants.

Evaluation criteria:

A test item was considered positive if either a dose related increase or a biologically relevant increase for at least one test concentration in the number of revertants was induced.
A test item was considered mutagenic if in the S. Typhimurium strains TA 98, TA 100, and E. coli WP2 uvrA the number of reversions was at least twice as high and in the strains TA 1535 and TA 1537 at least three times higher as compared to the spontaneous reversion rate.
Also, a dose-dependent increase in the number of revertants was regarded as an indication of possibly existing mutagenic potential of the test item regardless whether the highest dose induced the above described enhancement factors or not.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

A decrease in the number of revertants was seen at concentrations of at least 2500 µg/plate (Experiment II, without S9 mix) and 1000 µg/plate (Experiment II, with S9 mix).

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

A decrease in the number of revertants was seen at concentrations of at least 2500 µg/plate (Experiment II, with and without S9 mix) and 5000 µg/plate (Experiment I, with S9 mix).

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

After treatment with CG 37-1586 neither the number of histidine-prototrophic mutants nor the number of tryptophane-prototrophic mutants were increased in comparison to the negative control in any of the experiments. Plates incubated with test article showed normal background growth in either concentration with and without metabolic activation.

Any other information on results incl. tables

Summary of Results

without S9 mix

Concentration	TA 1535		TA 1537		TA 98		TA 100		WP2 uvrA
µg/plate	I	II	I	II	I	II	I	II	I	II
Negative control	20	12	10	25	15	20	98	81	44	46
Solvent control	12	12	14	23	15	19	93	88	40	55
Positive control	1297	615	69	89	440	270	Il49	879	820	273
33	14	8	15	26	12	18	93	82	34	54
100	15	6	15	27	12	17	93	87	37	53
333	16	8	13	16	12	17	101	91	36	54
1000	17	8	13	18	12	15	94	87	30	48
2500	18	3	13	10	10	16	92	86	35	34
5000	6	3	8	3	12	11	66	79	38	35

with S9 mix

Concentration	TA 1535		TA 1537		TA 98		TA 100		WP2 uvrA
µg/plate	I	II	I	II	I	II	I	II	I	II
Negative control	13	11	7	25	16	23	115	92	49	52
Solvent control	9	10	11	18	15	16	101	94	49	44
Positive control	105	80	56	81	448	694	896	683	204	182
33	9	8	12	22	11	19	98	92	46	71
100	8	7	12	27	16	12	95	87	45	54
333	12	6	9	28	18	16	97	91	45	42
1000	13	3	10	13	10	12	106	93	37	41
2500	10	I	10	4	9	10	93	83	39	36
5000	8	4	4	2	7	8	80	78	30	35

I Plate incorporation test

II Preincubation test

Applicant's summary and conclusion

Conclusions:

Under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used. Therefore, the test item is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.