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:
2010-05-07 to 2010-05-17
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)
Deviations:
no
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 (as cited in study report): Silica-Zirkonia Filler
- Physical state: White powder
- Batch: IT-253
- Analytical purity of components: 72.4% silicon dioxide (CAS No. 7631-86-9) and 26.0% zirconium dioxide (CAS No. 1314-23-4) and a not stated percentage of disodium oxide (CAS No. 1313-59-3)
- Storage: at room temperature in the dark
- Stability: stable under storage conditions
- Expiry date: 20 Nov 2010
- not soluble in water

Method

Target gene:
S. typhimurium: Histidine operon
E. coli: Tryptophan operon
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 (80 mg/kg bw) and ß-naphthoflavone (100 mg/kg bw) induced rat liver S9-mix
Test concentrations with justification for top dose:
3, 10, 33, 100, 333, 1000, 3330, and 5000 µg/plate with and without 3.6% S9-mix for dose range finding test
3, 10, 33, 100, 333, 1000, 3330, and 5000 µg/plate with and without 5% S9-mix for experiment 1
33, 100, 333, 1000 and 3330 µg/plate with and without 10% S9-mix for experiment 2
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
Controlsopen allclose all
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
0.1 mL DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2 aminoanthracene: 2.5 µg/plate for TA 1535 and TA 1537, 1 µg/plate for TA 98 and TA 100 and 10 µg/plate for WP2 uvrA
Remarks:
with 5% S9-mix
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
0.1 mL DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2 aminoanthracene: 2.5 µg/plate for TA 1535 and TA 100, 1 µg/plate for TA 98, 5 µg/plate for TA 1537 and 10 µg/plate for WP2 uvrA
Remarks:
with 10% S9-mix
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
0.1 mL DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
without S9-mix Migrated to IUCLID6: 5 µg/plate for TA 1535
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
0.1 mL DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
without S9-mix Migrated to IUCLID6: 60 µg/plate for TA 1537
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
0.1 mL DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
2-nitrofluorene
Remarks:
without S9-mix Migrated to IUCLID6: 10 µg/plate for TA 98
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
0.1 mL DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
without S9-mix Migrated to IUCLID6: 650 µg/plate for TA 100
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
0.1 mL DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
without S9-mix Migrated to IUCLID6: 10 µg/plate for WP2 uvrA
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 ± 4 hours

NUMBER OF REPLICATIONS: triplicate

NUMBER OF CELLS EVALUATED: if less than 40 colonies/plate were present they were counted manually, if more than 40 colonies were present, they were counted automatically with a Biocount 4000 Pro-S colony counter.

DETERMINATION OF CYTOTOXICITY
- Method: reduction of the bacterial background lawn, increase in the size of the microcolonies and reduction of the revertant colonies
Evaluation criteria:
A test substance is considered negative (not mutagenic) in the test if:
a) The total number of revertants in tester strain TA1OO is not greater than two (2) times the concurrent control, and the total number of revertants in tester strains TA1535, TA1537, TA98 or WP2uvrA is not greater than three (3) times the concurrent control.
b) The negative response should be reproducible in at least one independently repeated experiment.

A test substance is considered positive (mutagenic) in the test if:
a) The total number of revertants in tester strain TA1OO is greater than two (2) times the concurrent control, or the total number of revertants in tester strains TA1535, TA1537, TA98 or WP2uvrA is greater than three (3) times the concurrent control.
b) In case a repeat experiment is performed when a positive response is observed in one of the tester strains, the positive response should be reproducible in at least one independently repeated experiment.

The preceding criteria were not absolute and other modifying factors might enter into the final evaluation decision.

Results and discussion

Test results
Species / strain:
other: E. coli WP2uvrA and S. typhimurium TA 1535, TA 1537, TA 98 and 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
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: at concentrations of 3330 and 5000 µg/plate

RANGE-FINDING/SCREENING STUDIES: tested in the tester strains TA100 and WP2uvrA with concentrations of 3, 10, 33, 100, 333, 1000, 3330 and 5000 pg/plate in the absence and presence of S9-mix.

COMPARISON WITH HISTORICAL CONTROL DATA: In range of historical control data from the laboratory.

Any other information on results incl. tables

Table 1: Maximum number of revertants from experiment 1

 

Maximum number of revertants

Solvent control

Positive control

Treatment

(at dose level [µg/mL])

Strain

With S9

Without S9

With S9

Without S9

With S9

Without S9

TA 1535

6

5

332

960

10 (100)

9 (333)

TA 1537

3

4

364

268

4 (333)

5 (33)

TA 98

19

13

1027

1131

25 (1000)

18 (100)

TA 100

93

95

1197

603

104 (5000)

101 (33)

WPA2uvrA

20

18

376

1082

23 (1000)

25 (333)

 

Table 2: Maximum number of revertants from experiment 2

 

Maximum number of revertants

Solvent control

Positive control

Treatment

(at dose level [µg/mL])

Strain

With S9

Without S9

With S9

Without S9

With S9

Without S9

TA 1535

6

12

224

942

10 (333)

13 (33)

TA 1537

3

4

283

296

4 (33)

5 (100)

TA 98

25

15

694

1098

27 (3330)

22 (100)

TA 100

64

106

1239

1097

74 (3330)

109 (333)

WPA2uvrA

17

10

331

1045

20 (1000)

20 (333)

 

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative