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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP-Guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material : J-37
- Substance type: red solid
- Physical state: solid
- Analytical purity: 99.3%
- Lot no.: 91116
- Expiration date of the lot/batch: 22 Jan. 2010
- Storage condition of test material: room temperature (17.6 -20.8 °C) in desiccators
- Handling Instructions: wear cloths, a mask, gloves and goggles. Hygroscopic, control of the temperature - humidity should be necessary to weight.

Method

Target gene:
Histidine operon for S. typhimurium strains and trytophan operon for E.coli strains.
Species / strainopen allclose all
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain characteristics:
other: TA 98 & 100: rfa, uvrB & pKM101. TA1535 & 1537: rfa and uvrB
Metabolic activation:
with and without
Metabolic activation system:
S9-mix (Liver fraction from Phenbarbital (30 mg/kg) and 5,6- benzoflavone (80 mg/kg) induced male Sprague-Dawlet rats.
Species / strain:
E. coli WP2 uvr A
Additional strain characteristics:
other: uvrA and pKM101
Metabolic activation:
with and without
Metabolic activation system:
S9-mix (Liver fraction from Phenbarbital (30 mg/kg) and 5,6- benzoflavone (80 mg/kg) induced male Sprague-Dawlet rats.
Test concentrations with justification for top dose:
Dose range finding study:
0, 8.19, 20.5, 51.2, 128, 320, 800, 2000 and 5000 µg/plate

Main study:
- S9: 0, 1.6, 3.2, 6.4, 12.8, 25.6 and 51.2 µg/plate
+S9: 0, 62.5, 125, 250, 500, 1000 and 2000 µg/plate
Vehicle:
- Vehicle(s)/solvent(s) used: DMSO
Controls
Negative controls:
no
Solvent controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: - S9: TA 98 = 2-NF: 5.0 µg/ plate. - S9: TA 100 & 1535 = SA: 1.5 µg/ plate. - S9: TA 1537 = 9-AA: 80.0 µg/ plate. - S9: WP2uvrA = AF2: 0.005 µg/ plate.+ S9: TA 98, TA 100, TA 1535 & TA 1537 = 2-AA: 1.0, 2.0, 3.0 & 3.0 µg/ plate, respectively.
Details on test system and conditions:
METHOD OF APPLICATION: in agar (plate incorporation).

DURATION
- Preincubation period: 20 min
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: a dense or clear bacterial lawn compared to negative controls was judged as the evidence of bacterial toxicity.

OTHER: following cultivation, the number of revertants colonies was automatically counted by a colony counter (ProtoCOL, SINBIOSIS, UK). When automatic counting was not considered to be accurate by preciptation, deposition or growth inhibition, the number of revertant colonies was counted manual counting. The precipitation and deposition were observed with the naked eye.
Evaluation criteria:
The results of the test substance were considered to be positive when the following conditions were met:
- The number of revertnats colonies in the test substance groups was increased at least twice as compared to the negative control group at one or more doses per plate in at least one strain.
- The number of revertant colonies was increased dose dependently.
Statistics:
Statistical analysis was not performed. Individual plate counts and mean values of revertants colonies were presented.

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
yes
Remarks:
The growth inhibition was evident at 5.12 µg/plate in TA98 and TA100 strains, 64.0 µg/plate in TA1535 strain and 128 µg/plate in the TA1537 strain without metabolic activation.
Vehicle controls valid:
yes
Negative controls valid:
no
Positive controls valid:
yes
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Species / strain:
E. coli WP2 uvr A pKM 101
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
yes
Remarks:
The background lawn could not be observed more than 800 µg/plate in WP2uvrA (pKM101) strain without metabolic activation and 2000 µg/plate in any strains with metabolic activation due to precipitation or deposition.
Vehicle controls valid:
yes
Negative controls valid:
no
Positive controls valid:
yes
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Yes


RANGE-FINDING/SCREENING STUDIES:
The growth inhibition was evident more than 5.12 µg/plate in TA98, TA100 strains and 128 µg/plate in TA1535, TA1537 strains without metabolic activation. The background lawn could not be observed more than 800 µg/plate in WP2uvrA (pKM101) strain without metabolic activation and 2000 µg/plate in any strains with metabolic activation due to precipitation and deposition.
The deposition of the test substance was evident at 800 and 2000 µg/plate in any strains without metabolic activation, respectively. The precipitation of the test substance was evident at 5000 µg/plate in any strain without and with metabolic activation. However, the precipitation and deposition did not interfere with the colony counting.

COMPARISON WITH HISTORICAL CONTROL DATA:
The mean number of revertant colonies for the negative and positive control in any strains of main study was within the range of the historical control data.

Any other information on results incl. tables

The mean number of revertant colonies was less than twice compared to the negative control values at all dose levels of the test substance in the absence and presence of metabolic activation, without dose-related increase.

In the positive control group, the number of revertant colonies was markedly increased as compared to the negative control.

No growth inhibition was evident at any dose levels in WP2uvrA (pKM101) stains without metabolic activation and any strains with metabolic activation at which deposition was not evident.

The deposition of the test substance was evident greater than 400 and 1000 µg/ plate in WP2uvrA (pKM101) stains without metabolic activation and any strains with metabolic activation respectively. However, the deposition did not interfere with the colony counting. 

 

Table 1. The number of revertnats colonies per plate (1st and 2nd main study).

1st experiment

number of revertants: mean value of negative control

number of revertants: mean value of positive control

max. number of revertants: mean value of test material [µg/plate]

without S9-mix

 

 

TA 1535

9 ± 4

256 ± 6

11 ± 2 [ 51.2 ]

TA 100

83 ± 12

572 ± 8 

87 ± 3 [1.60]

TA 1537

6 ± 2

489 ± 25

7 ± 1 [51.2]

TA 98

17 ± 5

472 ± 19

14 ± 4 [3.20]

WP2 uvrA

123 ± 18

 1, 183 ± 93

132 ± 10  [1.60]

with S9-mix

 

 

 

TA 1535

11 ± 1 

185 ± 14

10 ± 3  [62.5]

TA 100

92 ± 2 

580 ± 89

125 ± 19  [500]

TA 1537

9 ± 3

209 ± 35

18 ± 3 [1000]

TA 98

32 ± 5

246 ± 20

54 ± 2 [62.5]

WP2 uvrA

169 ± 17

529 ± 32

206 ± 9 [250]

2nd experiment

number of revertants: mean value of negative control

number of revertants: mean value of positive control

number of revertants: mean value of test material [µg/plate]

without S9-mix

 

 

TA 1535

12 ± 0

273 ± 20

14 ± 8 [3.20]

TA 100

80 ± 2

354 ± 28

76 ± 5 [51.2]

TA 1537

6 ± 1

284 ± 52

8 ± 2 [25.6]

TA 98

12 ± 0

443 ± 19

13 ± 5 [51.2]

WP2 uvrA

79 ± 3

1, 015 ± 204

111 ± 8 [ 12.8]

with S9-mix

 

 

 

TA 1535

8 ± 1

116 ± 6

14 ± 3 [500]

TA 100

78 ± 4

442 ± 42

128 ± 8 [250]

TA 1537

12 ± 4

219 ± 11

15 ± 3 [62.5]

TA 98

27 ± 3

181 ± 8

38 ± 7 [62.5]

WP2 uvrA

119 ± 1

449 ± 23

175 ± 4 [62.5]

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative