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Toxicological information

Genetic toxicity: in vitro

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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:
30 April 1996 to 13 August 1996
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study conducted to GLP in accordance with generally accepted scientific principles, possibly with incomplete reporting or methodological deficiencies, which do not affect the quality of the relevant results.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1996
Report Date:
1996

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
Test material form:
other: liquid (unspecified)
Details on test material:
- Appearance: dark liquid
- Storage conditions: room temperature in a clear glass container

Method

Target gene:
Histidine requirement in the Salmonella typhimurium strains.
Tryptophan requirement in the Escherichia coli strain.
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Details on mammalian cell type (if applicable):
- Type and identity of media: Stock cultures were prepared in Oxoid nutrient broth #2.
- Properly maintained: yes, stored frozen. All tester strains were checked for the presence of the appropriate genetic markers on approximately a monthly basis.
Additional strain / cell type characteristics:
other: All strains contain an rfa mutation and all except TA102 contain a uvrB deletion mutation. In addition, strains TA98, TA100 and TA102 contain the plasmid pKM101.
Species / strain / cell type:
E. coli WP2 uvr A
Details on mammalian cell type (if applicable):
- Type and identity of media: Stock cultures were prepared in Oxoid nutrient broth #2.
- Properly maintained: yes, stored frozen. All tester strains were checked for the presence of the appropriate genetic markers on approximately a monthly basis.
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Rat liver homogenate (6 % S9 in standard co-factors)
Test concentrations with justification for top dose:
Preliminary Toxicity Test:
0, 50, 167, 500, 1670 and 5000 µg/plate

Mutation Test:
Experiments 1 and 2: 0, 50, 167, 500, 1670, 5000 and 10 000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: isocetyl alcohol
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
isocetyl alcohol
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
N-ethyl-N-nitro-N-nitrosoguanidine
mitomycin C
other: 2-aminofluorene, 2-anthramine
Details on test system and experimental conditions:
- EXPERIMENT 1
METHOD OF APPLICATION: in agar (direct plate incorporation)
2 mL of molten top agar (supplemented with 0.5 mM histidine or tryptophan/0.5 mM biotin) was combined with 0.1 mL tester strain and 0.1 mL of the appropriate concentration of the test material or solvent in sterile glass tubes. Cultures treated in the presence of metabolic activation also contained 0.5 mL of the S9 mixture. The tubes were vortexed and the mixture was poured onto minimal glucose plates, evenly distributed and allowed to solidify. Within an hour all plates were inverted and incubated in the dark.

DURATION
- Exposure duration: 48 hours at 37 °C.

NUMBER OF REPLICATIONS: The tests were performed in triplicate.


- EXPERIMENT 2
METHOD OF APPLICATION: pre-incubation
In the absence of metabolic activation, 0.5 mL 0.2 M Na₂HPO₄ (pH 7.4), 0.1 mL tester strain and 0.1 mL of the appropriate concentration of the test material or solvent first were combined and incubated, with shaking, at approximately 30 °C for approximately 30 minutes.
In the presence of metabolic activation, 0.5 mL S9 mixture, 0.1 mL tester strain and 0.1 mL of the appropriate concentration of the test material or solvent first were combined and incubated, with shaking, at approximately 30 °C for approximately 30 minutes.
Following the 30-minute pre-incubation period, 2 mL of molten, supplemented top agar was added to each tube. The tubes were vortexed and the mixture was poured onto minimal glucose plates, evenly distributed and allowed to solidify. Within an hour all plates were inverted and incubated in the dark.

DURATION
- Exposure duration: 48 hours at 37 °C.

NUMBER OF REPLICATIONS: The tests were performed in triplicate.


DETERMINATION OF CYTOTOXICITY
- Method: Inhibited growth was characterised by the absence of a confluent bacterial lawn and/or the presence of pindot colonies.
Evaluation criteria:
A positive result is defined as a statistically significant, dose-dependent increase in the number of histidine- or tryptophan-independent revertants with at least one dose level inducing a revertant frequency that is two-fold the solvent control value. Statistical analyses are performed using the program developed by Snee and Irr (1981), with significance established at the 95 % confidence limit. If the test material does not induce a statistically significant, dose-dependent increase in revertant frequency, but does induce a revertant frequency at one dose level that is two-fold the spontaneous control value, the result is considered equivocal. A negative result is defined as the absence of a statistically significant or dose-dependent increase in the number of histidine or tryptophan-independent revertants.
Statistics:
Statistical analyses are performed only when a 50 % increase in revertant frequency, relative to the concurrent negative controls, is observed. As this was not the case for the test material, no analysis took place.

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING/SCREENING STUDIES
Results indicated that the test material was not toxic to any strain at any dose evaluated under either treatment condition. In addition, the test material was found to be incompletely soluble (droplets were observed) at all doses.

BACTERIAL CONTAMINANT EVALUATION
To ensure sterility, standard contamination evaluations were performed with each assay. The solvent, S9 mix and highest dose of the test material were evaluated at the same volumes used in the assay. Both top agars also were plated alone on minimal glucose plates. All plating was done in triplicate. Plates were incubated for 48 hours at 37°C and then scored for bacterial growth.

MUTAGENICITY EXPERIMENTS
Six doses of the test material were evaluated in the event of insolubility at the highest dose levels. Normal growth was observed in all tester strains at all doses evaluated with and without S9, and the test material was found to be incompletely soluble at all doses (droplets were seen), under both treatment conditions.
Revertant frequencies for all doses in all tester strains with and without S9 under both treatment conditions approximated or were less than those observed in the concurrent negative control cultures.
All positive and negative control values were within acceptable ranges.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Table 1: Experiment 1

+/- S9 Mix

Concentration

(µg/plate)

Mean number of colonies/plate

Base-pair Substitution Type

Frameshift Type

TA100

TA1535

TA102

WP2uvrA

TA98

TA1537

-

-

-

-

-

-

-

Solvent

50

167

500

1670

5000

10 000

96

67

96

113

108

100

104

7

7

7

9

6

6

10

292

294

292

275

277

269

290

5

7

3

5

4

5

7

26

30

30

27

27

25

23

10

9

12

11

9

11

12

+

+

+

+

+

+

+

Solvent

50

167

500

1670

5000

10 000

98

119

112

100

101

95

101

10

6

5

5

8

5

9

290

300

309

289

311

297

307

7

6

6

4

5

5

8

35

35

25

30

38

36

31

10

10

12

11

8

10

13

 

 

Positive Controls

 

 

-

Name

SA

SA

MMC

ENNG

2NF

9AA

Concentration (µg/plate)

10

10

2.5

2

5

150

Mean no. colonies/plate

1014

1109

1599

158

422

947

 

 

+

Name

2AM

2AM

2AF

2AM

2AM

2AM

Concentration (µg/plate)

2.5

2.5

30

80

2.5

2.50

Mean no. colonies/plate

1905

124

852

506

2345

489

SA = Sodium azide

2AM = 2-anthramine

9AA = 9-aminoacridine

2NF = 2-nitrofluorene

MMC = Mitomycin C

2AF = 2-aminofluorene

ENNG = N-ethyl-N’-nitro-N-nitrosoguanidine

 

Table 2: Experiment 2

+/- S9 Mix

Concentration

(µg/plate)

Mean number of colonies/plate

Base-pair Substitution Type

Frameshift Type

TA100

TA1535

TA102

WP2uvrA

TA98

TA1537

-

-

-

-

-

-

-

Solvent

50

167

500

1670

5000

10 000

91

83

99

86

94

8

92

10

12

10

9

7

9

8

270

291

252

268

288

272

263

7

6

4

6

6

6

5

29

26

30

28

28

30

29

9

9

8

7

10

11

9

+

+

+

+

+

+

+

Solvent

50

167

500

1670

5000

10 000

97

101

108

95

94

104

97

8

8

8

4

5

7

5

312

332

302

298

315

318

286

8

5

6

5

7

6

9

31

35

35

32

34

35

34

11

14

11

8

9

11

7

 

 

Positive Controls

 

 

-

Name

SA

SA

MMC

ENNG

2NF

9AA

Concentration (µg/plate)

10

10

2.5

2

5

150

Mean no. colonies/plate

958

1023

1343

527

523

1746

 

 

+

Name

2AM

2AM

2AF

2AM

2AM

2AM

Concentration (µg/plate)

2.5

2.5

30

80

2.5

2.5

Mean no. colonies/plate

1533

113

744

508

1821

496

SA = Sodium azide

2AM = 2-anthramine

9AA = 9-aminoacridine

2NF = 2-nitrofluorene

MMC = Mitomycin C

2AF = 2-aminofluorene

ENNG = N-ethyl-N’-nitro-N-nitrosoguanidine

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

Under the conditions of this study, the test material was considered to be non-mutagenic.
Executive summary:

The potential of the test material to cause mutagenic effects in bacteria was assessed in accordance with the standardised guideline OECD 471 under GLP conditions.

Salmonella typhimurium strains TA1535, TA1537, TA98, TA100 and TA102 and Escherichia coli strain WP2uvrA were treated with the test material, using the plate incorporation and pre-incubation methods, at six dose levels, both with and without metabolic activation. The dose levels assessed were 50, 167, 500, 1670, 5000 and 10 000 µg/plate.

The test material caused no visible reduction in the growth of the bacterial background lawn at any dose level. No toxicologically significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation or exposure method.

The vehicle and positive controls were within the normal range.

The test material was considered to be non-mutagenic under the conditions of this test.