O,O-diethyl phosphorochloridothioate

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:

2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study was conducted in compliance with current guidelines.

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Cross-reference 2

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

O,O-diethyl phosphorochloridothioate

EC Number:

219-755-4

EC Name:

O,O-diethyl phosphorochloridothioate

Cas Number:

2524-04-1

Molecular formula:

C4H10ClO2PS

IUPAC Name:

O,O-diethyl chlorophosphonothioate

Details on test material:

- Name of test material (as cited in study report): EP-2, CAS No 2524-04-1, Phosphorochloridothioic acid,O,O-diethyl ester
- Physical state: Pale yellow liquid
- Analytical purity: > 99 +/- 0.3%
- Lot/batch No.: 2010021503
- Storage condition of test material: at approximately 4°C in the dark

Method

Target gene:

Type of mutations indicated: frame shift mutations and base-pair substitutions

Species / strain

Species / strain 1

Species / strain / cell type:

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

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

S9 mix from the livers of male rats

Test concentrations with justification for top dose:

preliminary toxicity test: 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/plate
Main mutation experiment 1 (plate incorporation method): 5, 15, 50, 150, 500, 1500 and 5000 μg/plate
Main mutation experiment 2 (pre-incubation method): 0.5 - 5000 μg/plate

Vehicle / solvent:

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

- Justification for choice of solvent/vehicle:
The test item was immiscible in dimethyl sulphoxide at 50 mg/ml but was fully miscible in acetone at the same concentration in solubility checks performed. Acetone was therefore selected as the vehicle. Following solubility information provided by the sponsor, sterile distilled water was not evaluated as a potential vehicle in this test system.

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: without S9: N-ethyl-N-nitro-N-nitrosoguanidine, 9-Aminoacridine, 4-Nitroquinoline-1-oxide; with S9: 2-Aminoanthracene, Benzo(a)pyrene

Details on test system and experimental conditions:

METHOD OF APPLICATION: Experiment 1 was performed as plate incorporation assay and experiment 2 was performed as preincubation assay

DURATION
- Preincubation period: 20 minutes (preincubation assay only)
- Exposure duration: 48 hours

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth

Evaluation criteria:

There are several criteria for determining a positive result. Any, one, or all of the following may be used to determine the overall result of the study:
1. A dose-related increase in mutant frequency over the dose range tested (DeSerres and Shelby (1979)).
2. A reproducible increase at one or more concentrations.
3 Biological relevance against in-house historical control ranges.
4. Statistical analysis of data as determined by UKEMS (Mahon et al (1989)).
5. Fold increase greater than two times the concurrent solvent control for any tester strain (especially if accompanied by an out-of-historical range response).

A test item will be considered non-mutagenic (negative) in the test system if the above criteria are not met.

Statistics:

No data

Results and discussion

Test results

Test results 1

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

plate incorporation assay: from 500 µg/plate onwards, pre-incubation assay: from 150 µg/plate onwards

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 2

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

plate incorporation assay: from 500 µg/plate onwards, pre-incubation assay: from 150 µg/plate onwards

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES:
The test item was cytotoxic initially at and above 500 μg/plate to the strains of bacteria used (TA100 and WP2uvrA). The test item formulation and S9-mix used in this experiment were both shown to be sterile.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
In the first experiment (plate incorporation), the test item caused a visible reduction in the growth of the bacterial background lawn in all of the tester strains (with and without metabolic activation) initially at 500 μg/plate. However, in the second experiment (pre-incubation method) the test item caused a visible reduction in the growth of the bacterial background lawn to all of the tester strains (with and without metabolic activation) initially at 150 μg/plate.

No test item precipitate was observed on the plates at any of the doses tested in either the presence or the absence of S9 mix.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Preliminary toxicity test: Numbers of revertant colonies for the toxicity assay:  

S9 mix	Strain	Dose (µg/plate)
		0	0.15	0.5	1.5	5	15	50	150	500	1500	5000
-	TA100	122	127	115	105	104	120	124	116	118 S	60 V	46 V
+	TA100	110	99	101	93	107	113	86	101	104	88 S	77 S
-	WP2uvrA	48	61	44	43	46	43	58	40	43	28 S	29 S
+	WP2uvrA	43	63	66	40	60	69	43	37	55	61	39 S

S            Sparse bacterial background lawn

V            Very weak bacterial background lawn

Experiment 1: Plate incorporation assay – without S9 mix

EP-2 concentration [µg/plate]	Mean number of revertants per plate
	Base-pair substitution type			Frameshift type
	TA100	TA1535	WP2uvrA	TA98	TA1537
0	100	17	54	23	16
5	100	19	47	25	15
15	97	16	49	26	13
50	102	17	48	27	10
150	106	14	51	22	14
500	101 S	14 S	42	21 S	9
1500	68 V	13 S	42 S	18 S	6 S
5000	0 T	7 V	31 S	8 S	2 V
Positive control (name)	ENNG	ENNG	ENNG	4NQO	9AA
Concentration [µg/plate]	3	5	2	0.2	80
Colonies/plate	310	123	327	142	2648

S            Sparse bacterial background lawn

V            Very weak bacterial background lawn

T            Toxic; no bacterial background lawn

ENNG    N-ethyl-N-nitro-N-nitrosoguanidine

4NQO    4-Nitroquinoline-1-oxide

9AA      9-Aminoacridine

 

 

Experiment 1: Plate incorporation assay – with S9 mix

EP-2 concentration [µg/plate]	Mean number of revertants per plate
	Base-pair substitution type			Frameshift type
	TA100	TA1535	WP2uvrA	TA98	TA1537
0	96	19	53	26	13
5	94	20	50	26	13
15	99	20	50	31	14
50	103	22	53	25	15
150	98	19	54	26	13
500	93 S	19	48	23	13
1500	78 S	13 S	46	25	11
5000	50 V	11 S	46 S	21	12 S
Positive control (name)	2AA	2AA	2AA	BP	2AA
Concentration [µg/plate]	1	2	10	5	2
Colonies/plate	1105	50	229	169	335

S            Sparse bacterial background lawn

V            Very weak bacterial background lawn

T            Toxic; no bacterial background lawn

2AA      9-Aminoanthracene

BP          Benzo(a)pyrene

 

 

Experiment 2: Plate incorporation assay – without S9 mix

EP-2 concentration [µg/plate]	Mean number of revertants per plate
	Base-pair substitution type			Frameshift type
	TA100	TA1535	WP2uvrA	TA98	TA1537
0	119	15	30	21	12
0.5	128	13	30	17	11
1.5	125	15	29	19	10
5	124	14	28	20	12
15	106	14	27	20	13
50	102	11	31	18	11
150	53 S	4 V	23 S	0 V	0 V
500	0 T	0 T	0 T	0 T	0 T
Positive control (name)	ENNG	ENNG	ENNG	4NQO	9AA
Concentration [µg/plate]	3	5	2	0.2	80
Colonies/plate	497	162	283	116	2530

S            Sparse bacterial background lawn

V            Very weak bacterial background lawn

T            Toxic; no bacterial background lawn

ENNG    N-ethyl-N-nitro-N-nitrosoguanidine

4NQO    4-Nitroquinoline-1-oxide

9AA      9-Aminoacridine

 

Experiment 2: Plate incorporation assay – with S9 mix

EP-2 concentration [µg/plate]	Mean number of revertants per plate
	Base-pair substitution type			Frameshift type
	TA100	TA1535	WP2uvrA	TA98	TA1537
0	111	20	50	25	11
0.5	111	N/T	N/T	N/T	N/T
1.5	114	N/T	N/T	N/T	N/T
5	111	15	49	24	12
15	113	15	51	23	12
50	105	16	49	27	10
150	107 S	16	46	26	9
500	0 V	13 S	30 S	18	5 S
1500	N/T	0 V	17 V	11 S	0 T
5000	N/T	0 T	13 V	12 S	0 T
Positive control (name)	2AA	2AA	2AA	BP	2AA
Concentration [µg/plate]	1	2	10	5	2
Colonies/plate	801	366	341	186	343

S            Sparse bacterial background lawn

V            Very weak bacterial background lawn

T            Toxic; no bacterial background lawn

N/T        Not tested at this dose level

2AA      9-Aminoanthracene

BP          Benzo(a)pyrene

Results of the negative controls (spontaneous mutation rates):

Mean number of revertants per plate
Base-pair substitution type			Frameshift type
TA100	TA1535	WP2uvrA	TA98	TA1537
Experiment 1:
97	23	46	22	13
Experiment 2:
--	17	49	24	20
111*	15*	33*	26*	11*

*            experiment performed at a later date, due to the toxicity in the original pre-incubation test

 

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative with metabolic activation
negative without metabolic activation

EP-2 is considered to be non-mutagenic both in the presence and in the absence of metabolic activation, but was found to be cytotoxic at 500 µg/plate and 150 µg/plate onwards in the plate incorporation assay and pre-incubation assay, respectively.

Executive summary:

The study was conducted in order to identify possible mutagic activities of EP-2 and was performed according to OECD guideline 471 and EU method B13/14.

In a preliminary toxicity test, Salmonella typhimurium strains TA98, TA100, TA1535 and TA1537 and Escherichia coli strain WP2uvrA were treated at EP-2 concentrations in the range of 0.15 - 5000 µg/plate to determine the dose range to be tested.

The same bacterial strains were treated with EP-2 in the presence and in the absence of metabolic activation (S9 -mix) both in the plate incorporation and in the pre-incubation assay with test concentrationsin the range of 5 - 5000 µg/plate and 0.5 - 5000 µg/plate, respectively. The tester strains without treatment or treated at the vehicle (acetone) alone served as negative controls and appropriate positive controls (with and withot S9 -mix) were examined in the same assays.

Results revealed counts of revertants in the normal range for vehicle control plates. All of the positive control chemicals used in the assays induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

In the first experiment (plate incorporation assay), EP-2 caused a visible reduction in the growth of the bacterial background lawn in all of the tester strains, both in the presence and absence of S9 -mix, initially at 500 µg/plate. In the second experiment (pre-incubation assay), EP-2 caused a visible reduction in the growth of the bacterial background lawn to all of the tester strains, with and without metabolic activation, initially at 150 µg/plate. Thus, EP-2 is cytotoxic at concentrations of 500 µg/plate and 150 µg/plate onwards in the plate incorporation assay and the pre-incubation assay, respectively.

No test item precipitate was observed on the plates at any concentration tested either in the presence or in the absence of metabolic activation.

No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of EP-2 either with or without metabolic activation or exposure method used. It is therefore concluded that EP-2 is non-mutagenic under the conditions of this test.