Registration Dossier

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 16 july 2003 to 22 dec 2003
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study, OECD TG 471 compliant.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2003
Report Date:
2003

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent

Method

Species / strain
Species / strain / cell type:
other: TA98, TA100, TA102, TA1535 and TA1537
Metabolic activation:
with and without
Metabolic activation system:
S9 fraction was purchased from Moltox (Molecular Toxicology, INC, Boone, NC 28607, USA) and obtained from the liver of rats treated with Aroclor 1254 (500 mg/kg) by the intraperitoneal route.
Test concentrations with justification for top dose:
Doses were 312.5, 625, 1250, 2500 and 5000 μg/plate, for the TA 98, TA 100, TA 1535 and TA 1537 strains with and without S9 mix in the first experiment as well as for all the tester strains with and without S9 mix in the second experiment,
Doses were 156.3, 312.5, 625, 1250 and 2500 μg/plate, for the TA 102 strain in the first experiment with and without S9 mix.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: generally used in genotoxicity tests.
Controls
Untreated negative controls:
yes
Remarks:
vehicle (DMSO)
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: see details on test system
Details on test system and experimental conditions:
POSITIVE CONTROLS:
Without S9 mix
. sodium azide (NAN3) 1 µg/plate for TA 1535 – TA 100
. 9-Aminoacridine (9AA) 50 µg/plate for TA 1537
. 2-Nitrofluorene (2NF) 0.5 µg/plate for TA 98
. Mitomycin C (MMC) 0.5 µg/plate for TA 102
With S9 mix
. 2-Anthramine (2AM) 2 µg/plate for TA 1535 – TA 1537 – TA 98 – TA 100
. 2-Anthramine (2AM) 10 µg/plate for TA 102

METHOD OF APPLICATION: in agar (plate incorporation) for both experiments, and preincubation for the second test with S9 mix.

DURATION
- Preincubation period: The preincubation method was performed as follows: test item solution (0.1 mL), S9 mix (0.5 mL) and the bacterial suspension (0.1 mL) were incubated for 60 minutes at 37°C, under shaking, before adding the overlay agar and pouring onto the surface of a minimum agar plate.
- Exposure duration: 48 to 72 hours

The plate incorporation method was performed as follow: test item solution (0.1 mL), S9 mix when required or phophate buffer pH 7.4 (0.5 mL) and bacterial suspension (0.1 mL) were mixed with 2 mL of overlay agar and maintained at 45°C. After rapid homogenization, the mixture was overlaid onto a Petri plate containing minimum medium.
NUMBER OF REPLICATES: 3
Evaluation criteria:
- in TA 98, TA 100 and TA 102: a test article is considered as positive if it produce at least a 2-fold increase in the mean revertant per plate of at least one of
these tester strains over the mean revertants per plate of the appropriate vehicle control. This increase in the number of revertant must be
accompanied by a dose response with increasing concentrations of the test article
- in TA 1535 and TA 1537: idem, but a 3-fold increase is required

Results and discussion

Test results
Species / strain:
other: TA98, TA100, TA102, TA1535 and TA1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 5000 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Tables of results: mean of number of revertants per strain

 

Conc. (µg/plate)

TA98

TA100

TA102

+ S9

-S9

+ S9

-S9

+ S9

-S9

0*

35

32

95

105

614

424

312.5

39

20

142

133

588

312

625

30

38

107

127

652

357

1250

23

31

75

167

654

288

2500

26

25

96

142

535

276

5000

16

24

30

77

68

121

Positive control

1411

189

968

551

2165

1808

 

Conc. (µg/plate)

TA1535

TA1537

+ S9

-S9

+ S9

-S9

0*

16

13

9

5

312.5

13

17

18

4

625

11

12

9

9

1250

16

16

7

8

2500

9

16

8

9

5000

3

11

0

3

Positive control

157

486

92

474

*DMSO

Applicant's summary and conclusion

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

Negative with and without metabolic activation.
Slightly cytotoxic at 5000 µg/plate.
Executive summary:

In a study (CIT, 2003), the potential of the test item GUETOL to induce reverse mutation in Salmonella typhimurium was evaluated according to the international guidelines (OECD 471, Commission Directive No. B13/14) and in compliance with the Principles of Good Laboratory Practice Regulations.

 

A preliminary toxicity test was performed to define the dose-levels of GUETOL to be used for the mutagenicity study. The test item was then tested in two independent experiments, with and without a metabolic activation system, the S9 mix, prepared from a liver microsomal fraction (S9 fraction) of rats induced with Aroclor 1254.

Both experiments were performed according to the direct plate incorporation method except for the second test with S9 mix, which was performed according to the preincubation method (60 minutes,37°C).

Five strains of bacteria Salmonella typhimurium: TA 1535, TA 1537, TA 98, TA 100 and TA 102 were used. Each strain was exposed to five dose-levels of the test item (three plates/dose-level). After 48 to 72 hours of incubation at37°C, the revertant colonies were scored.

The evaluation of the toxicity was performed on the basis of the observation of the decrease in the number of revertant colonies and/or a thinning of the bacterial lawn.

The test item GUETOL was dissolved in dimethylsulfoxide (DMSO).

 

Since the test item was toxic in the preliminary test, the choice of the highest dose-level was based on the level of toxicity, according to the criteria specified in the international guidelines.

The selected treatment-levels were:

312.5, 625, 1250, 2500 and 5000μg/plate, for the TA 98, TA 100, TA 1535 and TA 1537 strains with and without S9 mix in the first experiment as well as for all the tester strains with and without S9 mix in the second experiment,

156.3, 312.5, 625, 1250 and 2500μg/plate, for the TA 102 strain in the first experiment with and without S9 mix.

 

No precipitate was observed in the Petri plates when scoring the revertants at all dose-levels.

A moderate to marked toxicity was noted with S9 mix in the preincubation method, in all the tester strains at 5000μg/plate as well as in the TA 102 strain without S9 mix at 5000μg/plate.

The test item did not induce any noteworthy increase in the number of revertants, both with and without S9 mix, in any of the five strains.

 

Under these experimental conditions, the test item GUETOL did not show mutagenic activity in the bacterial reverse mutation test with Salmonella typhimurium.