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Genetic toxicity: in vitro

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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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study in compliance with the GLP

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2000
Report Date:
2000

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
OECD Guideline 472 (Genetic Toxicology: Escherichia coli, Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
28 february 2000
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: liquid
Details on test material:
- Name of test material (as cited in study report): TEPA

Method

Target gene:
S. typhimurium: histidine gene
E. coli: tryptophan gene
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
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
Controlsopen allclose all
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
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
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
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
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 independant 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 resultsopen allclose all
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
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
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
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
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 sudy (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.