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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
30 December 1988 to 13 October 1999
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1999
Report Date:
1999

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Principles of method if other than guideline:
The study protocol states that it was written to comply with the standardised guideline OECD 471.
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Appearance: Light yellow liquid, Clear Liquid
- Stability: There was no apparent change in the physical state of the test material during administration
- Storage conditions of test material: Room temperature, protected from exposure to light

Method

Target gene:
Histidine requirement in the Salmonella typhimurium strains (Histidine operon).
Tryptophan requirement in the Escherichia coli strain (Tryptophan operon).
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell type (if applicable):
- Properly maintained: Yes. Overnight cultures were prepared by inoculating from the appropriate master plate or from the appropriate frozen permanent stock into a vessel containing 50 mL of culture medium. To assure that cultures were harvested in late log phase, the length of incubation was controlled and monitored. Following inoculation, each flask was placed in a resting shaker/incubator at room temperature. The shaker/incubator was programmed to begin shaking at approximately 125 rpm at 37 ± 2 °C approximately 12 hours before the anticipated time of harvest. Each culture was monitored spectrophotometrically for turbidity and was harvested at a percent transmittance yielding a titre of approximately 10^9 cells per millilitre. The actual titres were determined by viable count assays on nutrient agar plates.
Additional strain / cell type characteristics:
not applicable
Species / strain / cell type:
E. coli WP2 uvr A
Details on mammalian cell type (if applicable):
- Properly maintained: Yes. Overnight cultures were prepared by inoculating from the appropriate master plate or from the appropriate frozen permanent stock into a vessel containing 50 mL of culture medium. To assure that cultures were harvested in late log phase, the length of incubation was controlled and monitored. Following inoculation, each flask was placed in a resting shaker/incubator at room temperature. The shaker/incubator was programmed to begin shaking at approximately 125 rpm at 37 ± 2 °C approximately 12 hours before the anticipated time of harvest. Each culture was monitored spectrophotometrically for turbidity and was harvested at a percent transmittance yielding a titre of approximately 10^9 cells per millilitre. The actual titres were determined by viable count assays on nutrient agar plates.
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254-induced rat liver S9
Test concentrations with justification for top dose:
- Mutagenicity assay (Experiment B1): 25, 75, 200, 600, 1800 and 5000 µg/plate
Vehicle / solvent:
- Dimethyl sulfoxide (DMSO)
- Justification for choice of solvent/vehicle: Based on solubility of the test material, permitting preparation of a soluble or workable stock concentration up to 500 mg/L.
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
methylmethanesulfonate
other: 2-Aminoanthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)
On the day of its use, minimal top agar, containing 0.8 % agar (w/v) and 0.5 % NaCl (w/v), was melted and supplemented with L-histidine, D-biotin and L-tryptophan solution to a final concentration of 50 µM each. Top agar not used with S9 or Sham mix was supplemented with 25 mL of water for each 100 mL of minimal top agar. Water was sterile, deionised water produced by the Milli-Q Reagent Water System. Bottom agar was Vogel-Bonner minimal medium E containing 1.5 % (w/v) agar. Nutrient bottom agar was Vogel-Bonner minimal medium E containing 1.5 % (w/v) agar and supplemented with 2.5 % (w/v) Oxoid Nutrient Broth No. 2 (dry powder). Nutrient Broth was Vogel-Bonner salt solution supplemented with 2.5 % (w/v) Oxoid Nutrient Broth No. 2 (dry powder).
Test material dilutions were prepared immediately before use. 0.5 mL of S9 or Sham mix, 100 µL of tester strain and 50 µL of vehicle, test or positive control material were added to 2.0 mL of molten selective top agar at 45 ± 2 °C. After vortexing, the mixture was overlaid onto the surface of 25 mL of minimal bottom agar.

DURATION
- Exposure duration: After the overlay had solidified, the plates were inverted and incubated for approximately 48 to 72 hours at 37 ± 2 °C. Plates that were not counted immediately following the incubation period were stored at 2 to 8 °C until colony counting could be conducted.

NUMBER OF REPLICATIONS: Plated in triplicate

DETERMINATION OF CYTOTOXICITY
- Method: The condition of the bacterial background lawn was evaluated for evidence of test material toxicity by using a dissecting microscope. Precipitate was evaluated by visual examination without magnification. Toxicity and degree of precipitation were scored relative to the vehicle control plate.

OTHER: Revertant colonies for a given tester strain and activation condition, except for positive controls, were counted either entirely by automated colony counter or entirely by hand unless the assay was the preliminary toxicity assay or the plate exhibited toxicity. Plates with sufficient test material precipitate to interfere with automated colony counting were counted manually.
Evaluation criteria:
EVALUATION CRITERIA
For each replicate plating, the mean and standard deviation of the number of revertants per plate were calculated. For the test material to be evaluated positive, it must cause a dose-related increase in the mean revertants per plate of at least one tester strain with a minimum of two increasing concentrations of test material. Data sets for strains TA1535 and TA1537 were judged positive if the increase in mean revertants at the peak of the dose response is equal to or greater than three times the mean vehicle control value. Data sets for strains TA98, TA100 and WP2uvrA were judged positive if the increase in mean revertants at the peak of the dose response is equal to or greater than two times the mean vehicle control value.

CRITERIA FOR A VALID TEST
The following criteria must be met for the mutagenicity assay to be considered valid.
All Salmonella and E. coli tester strain cultures must demonstrate the appropriate characteristics.
All cultures must demonstrate the characteristic mean number of spontaneous revertants in the vehicle controls.
To ensure that appropriate numbers of bacteria are plated, tester strain culture titres must be greater than or equal to 0.3 x 10^9 cells/mL.
The mean of each positive control must exhibit at least a three-fold increase in the number of revertants over the mean value of the respective vehicle control.
A minimum of three non-toxic dose levels are required to evaluate assay data. A dose level is considered toxic if one or both of the following criteria are met: (1) A >50 % reduction in the mean number of revertants per plate as compared to the mean vehicle control value. This reduction must be accompanied by an abrupt dose-dependent drop in the revertant count. (2) A reduction in the background lawn.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
No precipitate was observed but toxicity was generally observed at 5000 µg per plate. In Experiment B1, no positive responses were observed with any of the tester strains in the presence and absence of S9 activation. However, a non-dose responsive increase was observed with tester strain TA 1537 (1.7-fold, maximum increase) in the presence of S9 activation. This strain/activation condition was retested in Experiment B2 to clarify the response observed. In Experiment B2, no positive response was observed with tester strain TA1537 in the presence of S9 activation.
All criteria for a valid study were met. The results indicate that under the conditions of this study the test material did not cause a positive response with any of the tester strains in the presence and absence of Aroclor-induced rat liver S9. The study was concluded to be negative without conducting a full independent repeat assay because no unique metabolism requirements were known about the test material and because the equivocal response was retested to resolve the nature of the response.

Any other information on results incl. tables

Table 1: Summary of Experiment B1

± S9 Mix

Concentration

(µg/plate)

Mean Revertants / plate

Base-pair Substitution Type

Frameshift Type

TA100

TA1535

WP2uvrA

TA98

TA1537

-

Solvent

25

75

200

600

1800

5000

105

125

112

106

102

39

0

9

8

10

13

12

8

0

14

16

13

10

11

10

10

15

17

17

16

22

23

3

3

4

5

5

8

3

0

+

Solvent

25

75

200

600

1800

5000

119

121

126

122

131

151

30

13

13

13

13

14

8

1

16

11

17

13

14

12

12

17

19

23

23

23

28

8

7

6

8

5

5

12

5

Positive Controls

-

Name

SA

SA

MMS

2 NF

9AA

Concentration (µg/plate)

1.0

1.0

1000

1.0

75

Mean no. colonies/plate

514

421

166

241

423

+

Name

2AA

2AA

2AA

2AA

2AA

Concentration (µg/plate)

1.0

1.0

10

1.0

1.0

Mean no. colonies/plate

623

70

533

429

62

MMS = Methyl methanesulfonate

9AA = 9-aminoacridine

2AA = 2-aminoanthracene

SA = Sodium azide

2NF = 2-Nitrofluorene

Table 2: Summary of Experiment B2

± S9 Mix

Concentration

(µg/plate)

Mean number of colonies/plate in strain TA1537

+

Solvent

25

75

200

600

1000

1800

5000

7

6

7

7

7

4

8

3

Positive Controls

+

Name

2AA

Concentration (µg/plate)

1

Mean no. colonies/plate

62

2AA = 2-aminoanthracene

Applicant's summary and conclusion

Conclusions:
Under the conditions of this study, the test material was concluded to be negative with regard to genotoxicity in the Bacterial Gene Mutation Assay.
Executive summary:

The mutagenic activity of the test material was evaluated in a bacterial reverse mutation assay conducted using a protocol written to comply with the standardised guideline OECD 471 under GLP conditions.

S. typhimurium tester strains TA98, TA100, TA1535 and TA1537 and E.coli tester strain WP2uvrA were exposed to the test material in the presence and absence of Aroclor-induced rat liver S9 using the plate incorporation method. Following a preliminary toxicity assay, the mutagenicity assay (Experiment B1) was used to evaluate the mutagenic potential of the test material at concentrations of 0, 25, 75, 200, 600, 1800 and 5000 µg/plate in DMSO. Appropriate vehicle and positive controls for each tester strain were evaluated concurrently. All dose levels of test material, vehicle controls and positive controls were plated in triplicate.

No precipitate was observed but toxicity was generally observed at 5000 µg per plate. In Experiment B1, no positive responses were observed with any of the tester strains in the presence and absence of S9 activation. However, a non-dose responsive increase was observed with tester strain TA 1537 (1.7-fold, maximum increase) in the presence of S9 activation. This strain/activation condition was retested in Experiment B2 to clarify the response observed. In Experiment B2, no positive response was observed with tester strain TA1537 in the presence of S9 activation.

All criteria for a valid study were met. The results indicate that under the conditions of this study the test material did not cause a positive response with any of the tester strains in the presence and absence of Aroclor-induced rat liver S9. The study was concluded to be negative without conducting a full independent repeat assay because no unique metabolism requirements were known about the test material and because the equivocal response was retested to resolve the nature of the response.

Under the conditions of this study, the test material was concluded to be negative with regard to genotoxicity in the Bacterial Gene Mutation Assay.