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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:
from 26-03-1999 to 12-04-1999
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GPL study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1999
Report Date:
1999

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OTS 798.5265 (The Salmonella typhimurium Bacterial Reverse Mutation Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent

Method

Target gene:
not applicable
Species / strain
Species / strain / cell type:
other: Salmonella typhimurium TA1535, TA1537, TA98, TA100, TA102
Details on mammalian cell type (if applicable):
stock of Salmonella tester strains were obtained from Dr. BN.Ames, University of California. Permanent stocks are kept at -80°C, and overnight 
subcultures of these stocks are prepared for each day's work.
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9 - from induce - phenobarbital and betanaphthoflavone - Sprague Dawley rats - liver
Test concentrations with justification for top dose:
Pre-Test: 50, 158, 500, 1580, 5000 µg/plate
main test: 0, 313, 625, 1250, 2500, 5000 µg/plate
Vehicle / solvent:
DMSO
- Justification for choice of solvent/vehicle: no
- Vehicle controls tested: medium with DMSO alone
- volume of vehicle/solvent in the medium: 100 µL/plate
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other:
Remarks:
see below
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation); preincubation 
DURATION
- Preincubation period: 30 min at 37°C
- Exposure duration: 72h at 37°C
NUMBER OF CELLS EVALUATED: not applicable
OTHER: SCORING METHOD: Artek colony counter
Evaluation criteria:
for a mutagenic test substance, two-fold (or more) increase in the mean revertant numbers must be observed at two  
consecutive dose levels or at the highest practicable dose level only. 
In addition, there must be evidence of a dose-response relationship showing increasing numbers of mutant colonies with increasing dose-levels. 
The effect must be reproduced in an independant experiment.
Statistics:
regression line method (includes the solvent control data but not the untreated control data)
Parameters given:
- individual plate counts
- mean number of revertant colonies
- standard error of the mean
- titre of bacterial cultures

Results and discussion

Test results
Species / strain:
other: TA 1535, TA 1537, TA 98, TA 100 and TA 102 
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
see below
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
Positive controls validity:
valid
Additional information on results:
The test substance was found to be soluble in DMSO at a concentration of 100 mg/mL. Since 100µL of the TS solution are used in the preparation 
of each plate, this permitted the maximum concentration of 5000 µg/plate to be used in the toxicity test.
RANGE-FINDING/SCREENING STUDIES:  
The test substance was assayed at a maximum dose-level of 5000 µg/plate and at 4 lower dose-levels spaced at approximately half-log intervals:  
1580, 500, 158 and 50 µg/plate. No sign of toxicity were observed at the dose-levels tested in any tester strain in the absence or presence of S9 
metabolism
COMPARISON WITH HISTORICAL CONTROL DATA: no
ADDITIONAL INFORMATION ON CYTOTOXICITY: In the plate incorporation assay, slight toxicity was observed with TA1537 strain both in the 
presence and absence of S9 metabolism at the highest dose level. In the preincubation assay, with all tester strains, severe toxicity was observed 
in the absence of S9 mix at the highest dose level, while marked toxicity was observed in the presence of S9 mix. Moderate toxicity was observed  
at the dose level of 2500 µg/plate with all tester strain (S9 +/-).
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

RESULTS IN THE PLATE INCORPORATION METHOD:

* In TA1535

Dose (µg/plate)                

Mean number of revertants/plate

Without S9        

With S9

untreated 

18

15

0 (DMSO)                

18

18

313

16

16

625

14

15

1250

16

13

2500

15

14

5000

17

12

Sodium azide                

476

-

2-Aminoanthracene        

-

113


* In TA1537

Dose (µg/plate)                

Mean number of revertants/plate

Without S9        

With S9

untreated 

16

24

0 (DMSO)                

16

24

313

13

22

625

18

21

1250

15

22

2500

14

20

5000

9

14

9-Aminoacridine     

101

-

2-Aminoanthracene        

-

95


* In TA102

Dose (µg/plate)                

Mean number of revertants/plate

Without S9        

With S9

untreated 

434

476

0 (DMSO)                

442

502

313

450

478

625

443

494

1250

443

535

2500

467

535

5000

453

503

Cumene hydroperoxide

1029

-

2-Aminoanthracene        

-

1712


* In TA98

Dose (µg/plate)                

Mean number of revertants/plate

Without S9        

With S9

untreated 

31

41

0 (DMSO)                

31

40

313

29

39

625

30

38

1250

31

38

2500

29

38

5000

31

37

2-Nitrofluorene       

220

-

2-Aminoanthracene        

-

981


* In TA100

Dose (µg/plate)                

Mean number of revertants/plate

Without S9        

With S9

untreated 

134

138

0 (DMSO)                

121

131

313

117

132

625

129

136

1250

116

133

2500

121

127

5000

117

125

Sodium azide                

707

-

2-Aminoanthracene        

-

1239


RESULTS IN THE PREINCUBATION METHOD:

* In TA1535

Dose (µg/plate)                

Mean number of revertants/plate

Without S9        

With S9

untreated 

17

16

0 (DMSO)                

18

16

313

19

17

625

20

16

1250

17

15

2500

15

13

5000

-

13

Sodium azide                

479

-

2-Aminoanthracene        

-

96


* In TA1537

Dose (µg/plate)                

Mean number of revertants/plate

Without S9        

With S9

untreated 

17

23

0 (DMSO)                

25

24

313

17

23

625

16

26

1250

15

25

2500

9

15

5000

-

6

9-Aminoacridine     

109

-

2-Aminoanthracene        

-

88


* In TA 102

Dose (µg/plate)                

Mean number of revertants/plate

Without S9        

With S9

untreated 

340

421

0 (DMSO)                

302

405

313

301

397

625

309

413

1250

288

403

2500

238

355

5000

-

270

Cumene hydroperoxide

1013

-

2-Aminoanthracene        

-

1586


* In TA98

Dose (µg/plate)                

Mean number of revertants/plate

Without S9        

With S9

untreated 

33

43

0 (DMSO)                

30

40

313

31

39

625

30

39

1250

29

43

2500

19

39

5000

-

28

2-Nitrofluorene       

204

-

2-Aminoanthracene        

-

970


* In TA100

Dose (µg/plate)                

Mean number of revertants/plate

Without S9        

With S9

untreated 

126

141

0 (DMSO)                

115

135

313

114

133

625

118

130

1250

116

133

2500

86

131

5000

-

68

Sodium azide                

732

-

2-Aminoanthracene        

-

1191



Applicant's summary and conclusion

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

Paramethoxyphenol ecaille does not induce reverse mutation in Salmonella typhimurium under the reported experimental conditions.
Executive summary:

Two experiments were performed, one using a plate incorporation method, the other using a pre incubation method. In both experiments the test substance was assayed at the dose levels of 5000, 2500, 1250, 625 and 313 µg/plate. In the plate incorporation assay slight toxicity, indicated by a reduction in revertant numbers, was observed with tester strain TA1537 both in the absence or presence of S9 metabolism at the highest dose level. In the pre incubation assay, with all tester strains, severe toxicity, as indicated by microcolony formation, was observed at the highest dose level in the absence of S9 metabolism. Marked toxicity, as indicated by thinning of the background lawn and reduction in revertant numbers, was observed at the highest dose level in the presence of S9 metabolism. The test substance did not induce two-fold increases in the number of revertant colonies at any dose level, in any tester strain, in the plate incorporation or pre incubation assay, in the absence or presence of S9 metabolism. The sterility of the S9 mix and the test substance solutions was confirmed by the absence of colonies on additional agar plates spread separately with these solutions. Marked increases in revertant numbers were obtained in these tests following treatment with the positive control substances, indicating that the assay system was functioning correctly.