Registration Dossier

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
1999-06-09
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: OECD guideline compliant GLP compliant

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)
Version / remarks:
as at 1999
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
as at 1999
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Type:
Constituent
Type:
Constituent
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Ro 01-4479/000
- Physical state: colourless coarse crystals
- Analytical purity: 100 % (anhydrous)
- Purity test date: not reported, but Analysis No.: A981 9037
- Lot/batch No.: 805046
- Expiration date of the lot/batch: May 2002
- Stability under test conditions: It is to be expected that no gross degradation is occuring under the specified storage conditions for a period of a few months or when dissolved in the solvent for the test duration.
- Storage condition of test material: room temperature

Method

Target gene:
his G 46: TA1535, TA100
his C 3076: TA1537
his D 3052: TA98
his G 428: TA102



Species / strainopen allclose all
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Metabolic activation system:
S9 extract from phenobarbital/5,6 benzoflavone treated rats
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with and without
Metabolic activation system:
S9 extract from male, phenobarbital/5,6 benzoflavone treated Sprague Dawley rats
Test concentrations with justification for top dose:
0, 50, 158.1, 500, 1581, 5000 µg/plate
Vehicle:
- Vehicle(s)/solvent(s) used: distilled water
Controls
Solvent controls:
yes
Positive controls:
yes
Positive control substance:
other: 2-Nitrofluorene, ICR 191 , Mitomycin C, Sodium azide, 2-Aminoanthracene, see Tables 3 & 4
Details on test system and conditions:
METHOD OF APPLICATION: in medium (exp. 1) as well as preincubation (exp. 2)

DURATION
- Preincubation period: 30 min at 37 °C
- Exposure duration: 2 days
- Selection time (if incubation with a selection agent): 2 days

SELECTION AGENT (mutation assays): histidine-less Vogel-Bonner minimal agar medium

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency

OTHER:
Colony counting: electronically using a DOMINO automatic image analysis system (Perceptive Instruments, Haverhill, Suffolk, England) after having inspected the background lawn for signs of toxicity

Evaluation criteria:
Positive result:
- A reproducible, dose-related increase in the number of his+ revertants.
- The increase should reach at least a doubling of the number of spontaneous revertants for Salmonella fyphimurium strains TA1535 and TA98. For strains TA97, TA100 and TA102 a 1.5 - fold increase over control values might be indicative of a mutagenic effect provided the negative control values fall within the historical control data.
- Biological relevance should always be taken into account.
Negative result:
- Absence of a reproducible increase in the number of his+ revertant colonies.
Statistics:
not used

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:
no
Vehicle controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no
Vehicle controls valid:
yes
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
- Water solubility: water solubility is high
- Precipitation: no precipitation

RANGE-FINDING/SCREENING STUDIES:
No toxic effects were apparent by reduction of background growth and by reduction or absence of revertant colonies in a dose range finder assay
with TA100 (see Table 2).

COMPARISON WITH HISTORICAL CONTROL DATA:
Mutant frequencies of the controls were in the range of the historical control values (values provided in the appendix of the study report)

Any other information on results incl. tables

- Table 1: Number of cells plated in respective experiment

Exp. No.

Strain

Colonies per plate

Cells plated x 10^6

1

TA1535

185 / 211

198

1

TA97

217 / 205

211

1

TA98

182 / 212

197

1

TA100

50 / 30

40

1

TA102

136 / 204

170

2

TA1535

105 / 108

107

2

TA97

130 / 103

117

2

TA98

205 / 219

212

2

TA100

67 / 59

63

2

TA102

124 / 133

129

- Table 2: Dose range finder assay with TA100

Concen- tration µg/plate

Precipitation in standard assay

Precipitation in preincubation assay

Revertants per plate (two plates average)

Toxicity on VB plate

-S9

+S9

-S9

+S9

0

-

-

141

180

growth

growth

50

-

-

143

148

growth

growth

158.1

-

-

133

171

growth

growth

500

-

-

120

156

growth

growth

1581

-

-

134

154

growth

growth

5'000

-

-

118

147

growth

growth

- Table 3: Summary table of experiment 1 (standard test procedure without preincubation)

Strain

TA1535

TA1535

TA97

TA97

TA98

TA98

TA100

TA100

TA102

TA102

Activation

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

Concentration [µg/plate]

 

 

 

 

 

 

 

 

 

0

16 ± 4

13 ± 5

188 ± 4

204 ± 4

22 ± 5

19 ± 3

123 ± 8

145 ± 35

369 ± 19

348 ± 22

50

14 ± 4

10 ± 2

205 ± 20

210 ± 13

19 ± 6

26 ± 9

129 ± 5

146 ± 20

355 ± 10

391 ± 17

158.1

13 ± 5

9 ± 3

192 ± 18

201 ± 8

20 ± 9

20 ± 2

120 ± 14

139 ± 6

370 ± 12

403 ± 10

500

16 ± 4

11 ± 2

212 ± 9

226 ± 10

18 ± 2

22 ± 7

123 ± 7

132 ± 16

362 ± 19

354 ± 29

1581

17 ± 5

5 ± 3

197 ± 3

206 ± 23

20 ± 3

22 ± 5

128 ± 14

130 ± 13

368 ± 11

339 ± 22

5000

20 ± 2

7 ± 6

193 ± 9

206 ± 6

15 ± 1

28 ± 5

134 ± 7

131 ±8

359 ± 12

340 ± 23

Positive controls, Concentration [µg/plate]

 

 

 

 

 

 

 

 

 

 

2-Nitrofluorene, 0.5

 

 

 

 

155 ± 20

 

 

 

 

 

ICR 191, 1

 

 

954 ± 23

 

 

 

 

 

 

 

Mitomycin C, 0.4

 

 

 

 

 

 

 

 

1327 ± 32

 

Sodium azide, 1

831 ± 18

 

 

 

 

 

530 ± 32

 

 

 

2-Aminoanthracene, 4

21 ± 2

292 ± 21

248 ± 19

1970 ± 231

64 ± 13

4077 ± 173

192 ± 9

3859 ± 165

340 ± 2

950 ± 23

- Table 4: Summary table of experiment 2 (with preincubation)

Strain

TA1535

TA1535

TA97

TA97

TA98

TA98

TA100

TA100

TA102

TA102

Activation

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

Concentration [µg/plate]

 

 

 

 

 

 

 

 

 

 

0

12 ± 5

6 ± 2

194 ± 14

235 ± 6

31 ± 5

32 ± 3

145 ± 12

170 ± 16

409 ± 5

409 ± 38

50

10 ± 3

8 ± 6

198 ± 12

224 ± 19

30 ± 2

33 ± 8

135 ± 11

169 ± 10

409 ± 32

430 ± 22

158.1

10 ± 4

5 ± 3

157 ± 12

220 ± 11

30 ± 8

33 ± 7

139 ± 15

154 ± 11

397 ± 32

427 ± 43

500

11 ± 3

7 ± 4

176 ± 21

237 ± 11

35 ± 13

37 ± 7

139 ± 10

158 ± 7

415 ± 15

478 ± 26

1581

19 ± 4

7 ± 5

160 ± 7

213 ± 14

28 ± 3

35 ± 2

143 ± 13

164 ± 13

403 ± 18

408 ± 10

5000

17 ± 6

10 ± 4

170 ± 10

236 ± 31

36 ± 6

30 ± 4

143 ± 15

154 ± 13

397 ± 10

419 ± 14

Positive controls, Concentration [µg/plate]

 

 

 

 

 

 

 

 

 

 

2-Nitrofluorene, 0.5

 

 

 

 

264 ± 16

 

 

 

 

 

ICR 191, 1

 

 

2901 ± 25

 

 

 

 

 

 

 

Mitomycin C, 0.4

 

 

 

 

 

 

 

 

1050 ± 60

 

Sodium azide, 1

970 ± 54

 

 

 

 

 

562 ± 0

 

 

 

Applicant's summary and conclusion

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

The test compound was evaluated for mutagenic activity in the standard plate incorporation and in the preincubation versions of the Ames test according to
OECD 471 using Salmonella typhimurium (TA1535, TA97, TA98, TA100, TA102).
The test was negative for all strains up to the limit concentration of 5000 µg/plate with and without activation.
Executive summary:

The test compound was evaluated for mutagenic activity in the standard plate incorporation and in the preincubation versions of the Ames test according to OECD 471 using Salmonella typhimurium (TA1535, TA97, TA98, TA100, TA102).

The test was negative for all strains up to the limit concentration of 5000 µg/plate with and without activation.

RS-Pantolactone (named Ro 01-4479/000 in the study report) was evaluated for mutagenic activity in the standard plate incorporation and in the preincubation versions of the Ames test according to OECD 471 using Salmonella typhimurium (TA1535, TA97, TA98, TAl 00, TA102) in presence of an exogenous metabolic activation system (S9).

All strains were exposed up to the limit concentration of 5000 µg/plate without signs of cytotoxicity or precipitation. Distilled water was used as vehicle. The activity of the S9-mix and the responsiveness of the tester strains were verified by including appropriate controls into each experiment.

No increase in the number of revertant colonies was apparent for any of the five tester strains after treatment with the test item.

Thus it can be concluded that neither RS-Pantolactone per se, nor any of the metabolites formed is mutagenic in the Ames test under the described experimental conditions.

 

By expert judgement it is concluded that L-Pantolactone will get the same results.