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Toxicological information

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
Study period:
2000-11-10 to 2001-02-08
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2001
Report date:
2001

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1997
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Methyl phenylacetate
EC Number:
202-940-9
EC Name:
Methyl phenylacetate
Cas Number:
101-41-7
Molecular formula:
C9H10O2
IUPAC Name:
methyl phenylacetate

Method

Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535
Species / strain / cell type:
S. typhimurium TA 98
Species / strain / cell type:
S. typhimurium, other: TA97a
Species / strain / cell type:
S. typhimurium TA 100
Species / strain / cell type:
S. typhimurium TA 102
Metabolic activation:
with and without
Metabolic activation system:
Induced with Phenobarbital intraperitoneally and 13-Naphtoflavone orally
Test concentrations with justification for top dose:
0.05 - 0.16 - 0.5 - 1.6 - 5 mg/plate
Vehicle / solvent:
DMSO
Controlsopen allclose all
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: ICR 191 acridine mutagen dihydrochloride
Remarks:
TA97a (without metabolic activation)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 4-Nitro-1,2-phenylenediamine
Remarks:
TA98 (without metabolic activation)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: Nitrofurantoine
Remarks:
TA100 (without metabolic activation)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
TA1535 (wihtout metabolic activation)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-Aminoanthraceen
Remarks:
TA 97a, TA 98, TA 100, TA 1535 (with metabolic activation)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: Cumene Hydroperoxide
Remarks:
TA102 (without metabolic activation)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: Danthron
Remarks:
TA102 (with metabolic activation)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 h at 37 °C (Plates of the mutagenicity test)
24 h (Plates for confirmation of the genotypes)

SELECTION AGENT: L-Histidin

NUMBER OF REPLICATIONS: 3

Titer of the overnight culture: > 1*10^8 cells/mL.

DETERMINATION OF CYTOTOXICITY
- Method: Background lawn

OTHER EXAMINATIONS:
- Other: Genotypes were evaluated for each study.

Evaluation criteria:
Since a reduced background lawn is regarded to be a cytotoxic effect, plates with reduced background lawn were not included into evaluation procedures.
Arithmetic mean values and standard deviations were calculated from colonies per plate of three replicates. For evaluation of the results the induction rate of the mean values was calculated :
Induction rate: revertant colonies of test item/revertant colonies of the corresponding control
The test item is to be interpretated mutagenic if there is a concentration effect relationship and the induction rate is >=2. The data presented in the tables are computer generated and rounded for presentation. Thus manual calculation of results based an the data in this report may yield minor deviations from these figures.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium, other: TA97a
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
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:
not examined
Positive controls validity:
valid
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:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
ADDITIONAL INFORMATION ON CYTOTOXICITY: TA97a showed at a concentration of 5 mg/plate with and without metabolic activation cytotoxic effects. The other strains showed no cytotoxic effects.

Any other information on results incl. tables

 

Concentration
(mg/plate)

S9

TA97a

TA98

TA100

TA102

TA 1535

 

5

-

n.a.

22

109

93

37

 

1.6

-

334

24

157

307

39

 

0.5

-

311

33

162

294

32

 

0.16

-

355

21

155

357

29

 

0.05

-

407

31

131

307

27

2nd test

5

-

173

31

77

258

20

 

1.6

-

325

29

152

463

26

 

0.5

-

422

32

125

486

19

 

0.16

-

388

32

142

484

19

 

0.05

-

415

34

158

554

19

 

5

+

n.a.

13

94

298

21

 

1.6

+

292

22

100

300

13

 

0.5

+

341

21

98

287

14

 

0.16

+

329

32

80

251

14

 

0.05

+

349

23

79

241

12

2nd test

5

+

n.a.

34

102

439

11

 

1.6

+

306

45

131

539

11

 

0.5

+

326

36

118

647

10

 

0.16

+

340

45

129

639

16

 

0.05

+

434

38

107

708

14

ICR

-

>3.1 (1st test),
>3.3 (2nd test)

 

 

 

 

2-AA

+

>3.8 (1st test),
>3.6 (2nd test)

>45.3 (1st test),
>32.1 (2nd test)

>11.8 (1st test),
>9.3 (2nd test)

 

9.8 (1st test),
6.3 (2nd test)

 

4-nitro-1,2-phenylenediamine

-

 

6.0 (1st test),
3.6 (2nd test)

 

 

 

 

Nitrofurantoine

-

 

 

3.5 (1st test),
2.4 (2nd test)

 

 

 

Cumene hydroperoxide

-

 

 

 

>3.7 (1st test),
>2.8 (2nd test)

 

Danthron

+

 

 

 

>2.7 (1st test),
>2.2 (2nd test)

 

Sodium azide

-

 

 

 

 

9.0 (1st test),
9.9 (2nd test)

Applicant's summary and conclusion

Conclusions:
In this study the test item was found to have no mutagenic effects an Salmonella typhimurium strains TA 97 a, TA 98, TA 100, TA 102 and TA 1535 with (+) and without (-) the metabolic activation system S9 from male Wistar rats at concentrations up to 5 mg/plate.
Executive summary:

The mutagenic effects of the test item were determined in a reverse mutation assay according to the principles of OECD Guideline No. 471 and EEC Directive 2000/32/EEC Method B. 13/14. Test systems were the Salmonelle typhimurium strains TA 97a, TA 98, TA 100, TA 102 and TA 1535 with (+) and without (-) the metabolic activation system S9 (from male Wistar rats) each. Positive and negative controls were included in each study. Duration of each study was 48 h. The test item was dissolved in DMSO and applied once at test initiation with the concentrations 0.05 - 0.16 - 0.5 - 1.6 - 5 mg/plate. The test substance showed no mutagenic effects with and without metabolic activation. Cytotoxic effects were observed only for TA97a in the highest concentration of 5 mg/plate.