Triethyl phosphonoacetate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Study conducted in compliance with international standard guidelines under GLP conditions.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2000

Report date:

2000

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:

OECD Guideline 472 (Genetic Toxicology: Escherichia coli, Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

28 february 2000

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Triethyl phosphonoacetate

EC Number:

212-757-6

EC Name:

Triethyl phosphonoacetate

Cas Number:

867-13-0

Molecular formula:

C8H17O5P

IUPAC Name:

ethyl 2-(diethoxyphosphoryl)acetate

Test material form:

liquid

Method

Target gene:

S. typhimurium: histidine gene
E. coli: tryptophan gene

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:

Phenobarbital/beta-Naphthaflavone induced rat liver S9

Test concentrations with justification for top dose:

Preliminary test: 0.15 - 5000 µg/plate (+/- metabolic activation)
Main test: 50 - 5000 µg/plate (+/- metabolic activation)

Vehicle / solvent:

Solvent: Water

Controls

Controls 1

Untreated negative controls:

yes

Remarks:

sterile water

Negative solvent / vehicle controls:

yes

Remarks:

sterile water and DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

for TA100; TA1535; WP2uvrA and TA1537 +S9

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

for TA98 + S9

Controls 3

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

for TA 1537 -S9

Controls 4

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

for TA 98 -S9

Controls 5

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

for TA100, TA1535 and WP2P uvrA -S9

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)
DURATION
- Preincubation period: none
- Exposure duration: 2 days

NUMBER OF REPLICATIONS: Preliminary study on TA100 and WP2 uvrA strains and 2 independent main studies with triplicate for each test item concentration

DETERMINATION OF CYTOTOXICITY
- Method: growth of the bacterial background lawn and number of revertant

Evaluation criteria:

Statistically significant dose-related increase in mean number of revertant colonies and/or two-fold increase in mean number of revertant colonies.

Statistics:

Dunnett's method of linear regression

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

see results tables 7.6.1/1 to 7.6.1/4

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

See results 7.6 1/1 to 7.6 1/4

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 3

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

See results 7.6 1/1 to 7.6 1/4

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 4

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

See results 7.6 1/1 to 7.6 1/4

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 5

Species / strain:

E. coli WP2

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

See results 7.6 1/1 to 7.6 1/4

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS

- Precipitation: no


RANGE-FINDING/SCREENING STUDIES: the test material was non-toxic to the strains of bacteria used in the range finding study (TA100 and WP2 uvrA) up to 5000 µg/plate.

COMPARISON WITH HISTORICAL CONTROL DATA: The results for the vehicle control and the positive control groups are in concordance with historical control data pooled in 1998 and 1999.

Any other information on results incl. tables

Table 7.6.1/1:Number of revertants per plate in the absence of metabolic activation in the first test

Test item Concentration (µg/plate)	TA 100		TA1535		WP2 uvrA		TA 98		TA 1537
	Mean$	SD	Mean$	SD	Mean$	SD	Mean$	SD	Mean$	SD
0	130	6.6	24	7.9	22	3.5	23	5.3	10	2.1
50	114	9.6	28	4.0	22	2.3	22	4.6	10	1.5
150	117	13.8	23	8.7	25	4.0	20	5.5	9	0.6
500	130	1.0	24	2.0	26	4.0	18	3.8	9	5.9
1500	129	7.4	27	8.1	23	1.5	17	2.5	10	1.2
5000	120	2.0	25	6.0	12	3.2	19	7.5	8	1.0
Positive control**	554	35.1	543	36.1	859	59.1	129	12.0	1350	147.1

$: Mean of triplicate

**Mutagens positive controls:

- TA100: ENNGat 3µg/plate

- TA1535: ENNGat 5µg/plate

- WP2 uvrA: ENNG 2 µg/plate

- TA98: 4NQO at 0.2 µg/plate

- TA1537: 9AA at 80 µg/plate

 

 

Table 7.6.1/2: Number of revertants per plate in the presence of metabolic activation (S9 mix) in the first test 

Test item Concentration (µg/plate)	TA 100		TA1535		WP2 uvrA		TA 98		TA 1537
	Mean$	SD	Mean$	SD	Mean$	SD	Mean$	SD	Mean$	SD
0	121	11.3	17	3.5	25	2.6	34	0.6	9	4.0
50	119	1.0	15	2.5	20	3.5	31	6.8	9	4.9
150	117	7.0	16	4.0	26	4.2	21	6.0	10	0.6
500	119	10.4	13	0.6	22	2.1	24	8.0	11	0.6
1500	125	20.7	13	3.2	18	4.6	29	9.7	9	2.0
5000	119	2.5	12	1.5	16	2.6	25	5.8	10	4.5
Positive control**	2635	110.4	275	17.1	828	41.9	338	25.1	652	34.4

$: Mean of triplicate

**Mutagens positive controls:

- TA100, TA1535, WP2 uvrA and TA1537: 2AAat 1;2; 10 and 2 µg/plate respectively

- TA98: BAPat 5µg/plate

 

Table 7.6.1/3: Number of revertants per plate in the absence of metabolic activation in the second test

Test item Concentration (µg/plate)	TA 100		TA1535		WP2 uvrA		TA 98		TA 1537
	Mean$	SD	Mean$	SD	Mean$	SD	Mean$	SD	Mean$	SD
0	103	5.9	31	5.6	28	2.9	25	2.3	11	4.6
50	102	4.0	25	8.1	23	0.6	19	4.5	14	3.8
150	84	7.9	22	7.8	25	5.3	21	2.3	9	3.8
500	95	5.0	24	7.0	21	5.7	15	5.2	10	2.5
1500	105	9.0	23	2.6	18	2.1	21	1.2	6	2.0
5000	110	11.4	22	5.6	19	3.6	18	1.2	12	2.9
Positive control**	666	39.0	325	17.8	603	94.2	104	2.5	663	11.2

$: Mean of triplicate

**Mutagens positive controls:

- TA100: ENNGat 3µg/plate

- TA1535: ENNGat 5µg/plate

- WP2 uvrA: ENNG 2 µg/plate

- TA98: 4NQO at 0.2 µg/plate

- TA1537: 9AA at 80 µg/plate

 

Table 7.6.1/4: Number of revertants per plate in the presence of metabolic activation (S9 mix) in the second test

 

Test item Concentration (µg/plate)	TA 100		TA1535		WP2 uvrA		TA 98		TA 1537
	Mean$	SD	Mean$	SD	Mean$	SD	Mean$	SD	Mean$	SD
0	118	4.7	16	7.0	19	7.9	22	2.0	11	2.1
50	94	20.0	19	0.6	13	3.1	27	2.6	12	1.7
150	103	23.0	12	2.1	15	3.5	24	11.5	8	4.2
500	95	15.2	17	3.8	18	1.2	15	4.2	11	2.0
1500	110	11.5	14	1.0	21	1.5	24	6.1	14	2.3
5000	90	11.5	14	4.6	21	5.0	25	3.0	10	1.2
Positive control**	1304	101.7	519	180.0	516	7.6	135	21.0	606	16.0

$: Mean of triplicate

**Mutagens positive controls:

- TA100, TA1535, WP2 uvrA and TA1537: 2AAat 1;2; 10 and 2 µg/plate respectively

- TA98: BAPat 5µg/plate

Applicant's summary and conclusion

Conclusions:

Interpretation of results:
negative with metabolic activation all strains
negative without metabolic activation all strains

The test substance shows no evidence of mutagenic potential in the presence or absence of metabolic activation.

Executive summary:

In a reverse gene mutation assay in bacteria, performed according to the OECD No.471 guideline and in compliance with the GLP, Triethyl phosphonoacetate diluted in water was tested in S. typhimurium TA1535, TA1537, TA100, TA98 strains and E. coli WP2 uvrA strain in the presence and the absence of mammalian metabolic activation (S9) using the direct plate incorporation method. Five known mutagens (9-aminoacridine, 4-nitroquinoline-N-oxide, N-ethyl-N-nitro-N-nitrosoguanidine, 2-aminoanthraceneand benzoapyrene) were used to check the sensitivity of the test system. They gave appropriate response, therefore the test was considered as valid.

In two independent assays the test item was used at the following concentrations: 0; 50; 150; 500; 1500 and 5000 µg/plate (triplicate). The test item was non cytotoxic up to 5000 µg/plate. Furthermore, the test substance did not induce any increase in the observed numbers of revertant colonies in any test strain either in the presence or absence of an auxiliary metabolising system (S9).

 

Under the test conditions, Triethyl phosphonoaceate did not show any mutagenic activity in the bacterial reverse mutation test using Salmonella typhimurium and E. coli.

 

This study is considered as acceptable as it satisfied the criteria of the OECD Guideline No. 471.