Phosphorothioic triamide, N-propyl-

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

October - November 2007

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2008

Report date:

2008

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

21 Jul 1997

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

(from the competent authority) Landesamt für Umwelt, Wasserwirtschaft und Gewerbeaufsicht Rheinland-Pfalz

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of N-butylphosphorothioic triamide and N-propylphosphorothioic triamide

EC Number:

700-457-2

Molecular formula:

Unspecified

IUPAC Name:

Reaction mass of N-butylphosphorothioic triamide and N-propylphosphorothioic triamide

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Batch No.of test material: 8712 / 056
- Purity: 99.6 g / 100 g
- Date of production: 01 Oct 2007
- Physical state and appearance: solid, white

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Refrigerator - 20 °C
- Stability under test conditions: The stability of the test substance at room temperature in the vehicle water over a period of 4 hours was verified analytically.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The substance was dissolved in water. To achieve a solution of the test substance in the vehicle, the test substance preparation was treated with ultrasonic waves and was shaken thoroughly. All test substance formulations were prepared immediately before administration.

Method

Target gene:

his, trp

Species / strain

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:

Aroclor induced rat liver S9 fraction

Test concentrations with justification for top dose:

1st Experiment (Standard Plate Test): 0; 20; 100; 500; 2500 and 5000 µg/plate
2nd Experiment (Preincubation Test): 0; 312.5; 625; 1250; 2500 and 5000 µg/plate

In agreement with the recommendations of current guidelines 5 mg/plate or 5 µL/plate are generally selected as maximum test dose at least in the 1st Experiment. However, this maximum dose will be tested even in the case of relatively insoluble test compounds to detect possible mutagenic impurities. Furthermore, doses > 5 mg/plate or > 5 µL/plate might also be tested in repeat experiments for further calculation / substantiation.

Vehicle / solvent:

- Vehicle used: water
- Justification for choice of vehicle: Due to the good solubility of the test substance in water, water was used as vehicle.

Controls

Untreated negative controls:

yes

Remarks:

sterility control

Negative solvent / vehicle controls:

yes

Remarks:

vehicle control

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

other: - 2-aminoanthracene (2-AA): with S9 mix, 2.5 µg/plate dissolved in DMSO (TA 1535, TA 100, TA 1537, TA 98) or 60 µg/plate dissolved in DMSO (E.coli WP2 uvrA)

Details on test system and experimental conditions:

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

DURATION
- Exposure duration: 48 - 72 hours in the dark at 37 °C

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

Rationale for test conditions:

Bacterial reverse mutation assays using amino-acid requiring strains of Salmonella typhimurium and Escherichia coli are commonly employed as initial screening methods for detecting a point mutagenic activity of chemical substances and are used to screen for possible mammalian mutagens and carcinogens.

Evaluation criteria:

- Mutagenicity: Individual plate counts, the mean number of revertant colonies per plate and the standard deviations were given for all dose groups as well as for the positive and negative (vehicle) controls in all experiments. In general, five doses of the test substance are tested with a maximum of 5 mg/plate, and triplicate plating is used for all test groups at least in the 1st Experiment. Dose selection and evaluation as well as the number of plates used in repeat studies or further experiments are based on the findings of the 1st Experiment.
- Titer: The titer is generally determined only in the experimental parts with S9 mix both for the negative controls (vehicle only) and for the two highest doses in all experiments.
- Toxicity: Toxicity detected by a decrease in the number of revertants, clearing or diminution of the background lawn (= reduced his- or trp- background growth), reduction in the titer is recorded for all test groups both with and without S9 mix in all experiments and indicated in the tables.
- Solubility: Precipitation of the test material is recorded and indicated in the tables. As long as precipitation does not interefere with the colony scoring, 5 mg/plate is generally selected and analyzed (in cases of nontoxic compounds) as the maximum dose at least in the 1st Experiment even in the case of relatively insoluble test compounds to detect possible mutagenic impurities. Furthermore, doses > 5 mg/plate might also be tested in repeat experiments for further clarification / substantiation.

Statistics:

Acceptance criteria
Generally, the experiment is considered valid if the following criteria are met:
- The number of revertant colonies in the negative controls was within the range of the historical negative control data for each tester strain.
- The sterility controls revealed no indication of bacterial contamination.
- The positive control substances both with and without S9 mix induced a distinct increase in the number of revertant colonies within the range of the historical positive control data or above.
- The titer of viable bacteria was >= 10^8 / mL.

Assessment criteria
The test chemical 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.

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

in preincubation test only at 5000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with

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:

valid

Positive controls validity:

valid

Test results 3

Species / strain:

S. typhimurium TA 1537

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:

valid

Positive controls validity:

valid

Test results 4

Species / strain:

S. typhimurium TA 98

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:

valid

Positive controls validity:

valid

Test results 5

Species / strain:

S. typhimurium TA 100

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:

valid

Positive controls validity:

valid

Test results 6

Species / strain:

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:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No test substance precipitation was found.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: see tables below
- Negative (solvent/vehicle) historical control data: see tables below

Any other information on results incl. tables

Table 1: Historical Negative Control Data TA 1535

Method	S9 mix	Negative control	Period	No. of plates	Min*	Max*	Mean*	SD
SPT	-	Water	01.00 – 02.07	400	10	24	17	2
SPT	-	DMSO	11.04 – 02.07	400	10	25	17	2
SPT	-	Acetone	01.96 – 11.06	259	11	25	18	2
SPT	1 : 9	Water	01.00 – 01.07	400	12	24	18	2
SPT	1 : 9	DMSO	11.04 – 03.07	400	10	24	17	2
SPT	1 : 9	Acetone	01.96 – 11.06	258	11	25	19	2
PIT	-	Water	04.98 – 02.07	400	10	25	18	2
PIT	-	DMSO	07.03 – 03.07	400	10	25	17	2
PIT	-	Acetone	01.96 – 11.06	216	10	25	18	3
PIT	1 : 9	Water	11.97 – 02.07	400	11	25	18	2
PIT	1 : 9	DMSO	06.03 – 03.07	400	10	24	16	2
PIT	1 : 9	Acetone	01.96 – 11.06	201	10	24	18	2

*: revertants/plate

Table 2: Historical Negative Control Data TA 100

Method	S9 mix	Negative control	Period	No. of plates	Min*	Max*	Mean*	SD
SPT	-	Water	12.99 – 02.07	400	82	150	110	9
SPT	-	DMSO	11.04 – 02.07	400	80	135	109	9
SPT	-	Acetone	01.96 – 11.06	306	91	160	117	14
SPT	1 : 9	Water	12.99 – 01.07	400	88	152	115	2
SPT	1 : 9	DMSO	11.04 – 03.07	400	81	154	111	11
SPT	1 : 9	Acetone	01.96 – 11.06	301	85	160	119	15
PIT	-	Water	04.98 – 02.07	400	90	160	115	13
PIT	-	DMSO	09.03 – 03.07	400	81	153	109	10
PIT	-	Acetone	01.96 – 11.06	223	91	160	116	14
PIT	1 : 9	Water	03.98 – 02.07	400	92	159	120	15
PIT	1 : 9	DMSO	07.03 – 03.07	400	81	157	110	10
PIT	1 : 9	Acetone	05.96 – 11.06	198	96	160	118	15

*: revertants/plate

Table 3: Historical Negative Control Data TA 1537

Method	S9 mix	Negative control	Period	No. of plates	Min*	Max*	Mean*	SD
SPT	-	Water	11.99 – 02.07	400	5	16	10	2
SPT	-	DMSO	10.04 – 03.07	400	5	16	10	2
SPT	-	Acetone	01.96 – 11.06	261	5	18	10	2
SPT	1 : 9	Water	10.99 – 01.07	400	6	17	11	2
SPT	1 : 9	DMSO	09.04 – 03.07	400	5	16	10	2
SPT	1 : 9	Acetone	01.96 – 11.06	261	5	20	11	2
PIT	-	Water	04.98 – 02.07	400	5	16	10	2
PIT	-	DMSO	07.03 – 03.07	400	5	17	10	2
PIT	-	Acetone	01.96 – 11.06	213	6	17	10	2
PIT	1 : 9	Water	11.97 – 02.07	400	6	20	11	2
PIT	1 : 9	DMSO	07.03 – 03.07	400	5	17	10	2
PIT	1 : 9	Acetone	01.96 – 11.06	204	5	20	11	2

*: revertants/plate

Table 4: Historical Negative Control Data TA 98

Method	S9 mix	Negative control	Period	No. of plates	Min*	Max*	Mean*	SD
SPT	-	Water	01.00 – 02.07	400	17	44	29	5
SPT	-	DMSO	10.04 – 03.07	400	15	40	29	4
SPT	-	Acetone	01.96 – 11.06	263	19	50	29	5
SPT	1 : 9	Water	01.00 – 01.07	400	20	49	36	5
SPT	1 : 9	DMSO	10.04 – 03.07	400	21	50	35	5
SPT	1 : 9	Acetone	01.96 – 11.06	262	25	50	38	5
PIT	-	Water	01.98 – 02.07	400	18	44	28	4
PIT	-	DMSO	07.03 – 03.07	400	17	40	29	4
PIT	-	Acetone	01.96 – 11.06	216	19	45	28	5
PIT	1 : 9	Water	11.97 – 02.07	400	17	49	35	2
PIT	1 : 9	DMSO	07.03 – 03.07	400	20	50	33	5
PIT	1 : 9	Acetone	01.96 – 11.06	203	20	50	36	6

*: revertants/plate

Table 5: Historical Negative Control Data E.coli WP2 uvrA

Method	S9 mix	Negative control	Period	No. of plates	Min*	Max*	Mean*	SD
SPT	-	Water	09.99 – 01.07	400	25	55	33	5
SPT	-	DMSO	09.04 – 03.07	400	25	59	34	5
SPT	-	Acetone	04.96 – 11.06	234	25	55	34	5
SPT	1 : 9	Water	09.99 – 01.07	400	25	56	36	6
SPT	1 : 9	DMSO	09.04 – 03.07	400	25	60	38	6
SPT	1 : 9	Acetone	04.96 – 11.06	242	25	59	37	6
PIT	-	Water	10.97 – 02.07	400	25	52	32	5
PIT	-	DMSO	06.03 – 03.07	400	25	54	33	5
PIT	-	Acetone	01.96 – 11.06	191	25	54	32	5
PIT	1 : 9	Water	11.97 – 02.07	400	25	57	37	6
PIT	1 : 9	DMSO	07.03 – 03.07	400	25	56	36	5
PIT	1 : 9	Acetone	01.96 – 11.06	197	25	52	36	6

*: revertants/plate

Table 6: Historical Positive Control Data TA 1535

Method	S9 mix	Positive control µg/plate	Period	No. of plates	Min*	Max*	Mean*	SD
SPT	-	MNNG 5.0	03.05 – 02.07	400	509	1602	752	187
SPT	1 : 9	2-AA 2.5	02.05 – 03.07	400	77	342	132	28
PIT	-	MNNG 5.0	06.04 – 03.07	400	512	1317	708	150
PIT	1 : 9	2-AA 2.5	03.04 – 03.07	400	83	398	130	33

*: revertants/plate

Table 7: Historical Positive Control Data TA 100

Method	S9 mix	Positive control µg/plate	Period	No. of plates	Min*	Max*	Mean*	SD
SPT	-	MNNG 5.0	02.05 – 03.07	400	500	1574	823	201
SPT	1 : 9	2-AA 2.5	02.05 – 03.07	400	521	1878	876	205
PIT	-	MNNG 5.0	06.04 – 03.07	400	520	1302	797	141
PIT	1 : 9	2-AA 2.5	04.04 – 03.07	400	517	1497	829	174

*: revertants/plate

Table 8: Historical Positive Control Data TA 1537

Method	S9 mix	Positive control µg/plate	Period	No. of plates	Min*	Max*	Mean*	SD
SPT	-	AAC 100	02.05 – 03.07	400	212	1172	441	109
SPT	1 : 9	2-AA 2.5	02.05 – 03.07	400	72	200	129	22
PIT	-	AAC 100	05.04 – 03.07	400	221	1028	429	100
PIT	1 : 9	2-AA 2.5	03.04 – 03.07	400	80	198	123	19

*: revertants/plate

Table 9: Historical Positive Control Data TA 98

Method	S9 mix	Positive control µg/plate	Period	No. of plates	Min*	Max*	Mean*	SD
SPT	-	NOPD 10	02.05 – 03.07	400	332	1316	616	145
SPT	1 : 9	2-AA 2.5	02.05 – 03.07	400	502	1414	697	160
PIT	-	NOPD 10	06.04 – 03.07	400	388	1430	630	169
PIT	1 : 9	2-AA 2.5	03.04 – 03.07	400	510	1223	663	120

*: revertants/plate

Table 10: Historical Positive Control Data E.coli WP2 uvrA

Method	S9 mix	Positive control µg/plate	Period	No. of plates	Min*	Max*	Mean*	SD
SPT	-	4-NQO 5.0	01.05 – 03.07	400	500	1587	653	188
SPT	1 : 9	2-AA 60.0	01.05 – 03.07	400	150	383	231	30
PIT	-	4-NQO 5.0	05.04 – 03.07	400	500	1339	593	102
PIT	1 : 9	2-AA 60.0	05.04 – 03.07	400	150	307	226	25

*: revertants/plate

Applicant's summary and conclusion

Conclusions:

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

Executive summary:

The test substance was tested for its mutagenic potential based on the ability to induce point mutations in selected loci of several bacterial strains, i.e. Salmonella typhimurium and Escherichia coli, in a reverse mutation assay.

Strains: TA 1535, TA 100, TA 1537, TA 98 and E.coli WP2 uvrA

Dose Range: 20 µg - 5000 µg/plate (SPT), 312.5 µg - 5000 µg/plate (PIT)

Test Conditions: Standard plate test (SPT) and preincubation test (PIT) both with and without metabolic activation (Aroclor-induced rat liver S9 mix).

Solubility: No precipitation of the test substance was found.

Toxicity: A weak bacteriotoxic effect was occasionally observed depending on the strain and test conditions at 5000 µg/plate.

Mutagenicity: A relevant increase in the number of his+ or trp+ revertants was not observed in the standard plate test or in the preincubation test either without S9 mix or after the addition of a metabolizing system.

According to the results of the present study, the test substance is not mutagenic in the Salmonella typhimurium / Escherichia coli reverse mutation assay under the experimental conditions chosen here.