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Toxicological information

Genetic toxicity: in vitro

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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:
2001
Reliability:
1 (reliable without restriction)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2001
Report Date:
2001

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
Type:
Constituent
Test material form:
liquid

Method

Species / strain
Species / strain / cell type:
other: Salmonella typhimurium strains TA98, TA100, TA1535, TA1537, and Escherichia coli WP2 uvrA
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254-induced rat liver S9
Test concentrations with justification for top dose:
0, 75, 200, 600, 1800, 5000 ug/plate
Vehicle / solvent:
Acetone
Controls
Untreated negative controls:
yes
Remarks:
Acetone
Negative solvent / vehicle controls:
yes
Remarks:
Acetone
Positive controls:
yes
Positive control substance:
other: 1) all Salmonella strains and WP2 urvA with S9 : 2-aminoanthracene 2) TA98 without S9 : 2-nitrofluorene 3) TA100 and TA1535 without S9 : sodium azide 4) TA1537 without S9 : 9-amino-acridine 5) WP2 urvA without S9 : methyl methanesulfonate.
Details on test system and experimental conditions:
NUMBER OF REPLICATIONS: Samples were run in triplicate, with and without metabolic activation.METHOD OF APPLICATION- 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. For the preparation of media and reagents, all references to water imply sterile, deionized water produced by the Milli-Q Reagent Water System. Bottom agar was Vogel-Bonner minimal medium E (Vogel and Bonner, 1956) 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). Each plate was labeled with a code system that identified the test article, test phase, dose level, tester strain, and activation, as described in detail in BioReliance's Standard Operating.- Procedures. Test article dilutions were prepared immediately before use. One-half (0.5) mL of S9 or Sham mix, 100 µL of tester strain and 50 µL of vehicle or test article dilution 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. When plating the positive controls, the test article aliquot was replaced by a 50 µL aliquot of appropriate positive control. After the overlay had solidified, the plates were inverted and incubated for approximately 48 to 72hours at 37±2°C. Plates that were not counted immediately following the incubation period were stored at 2-8°C until colony counting could be conducted. The condition of the bacterial background lawn was evaluated for evidence of test article toxicity by using a dissecting microscope. Precipitate was evaluated by visual examination without magnification. 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 article precipitate to interfere with automated colony counting were counted manually.
Evaluation criteria:
For the test article to be evaluated positive, it must cause a dose-related increase in the mean revertants per plate of at least one tester strain over a minimum of two increasing concentrations of test article. Data sets for tester 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 tester strains TA98, TA100 and WP2 uvrA 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.All criteria for a valid test were met. To meet this criteria, all Salmonella tester strain cultures must demonstrate the presence of the deep rough mutation (rfa) and the deletion in the uvrB gene. Cultures of tester strains TA98 and TA100 must demonstrate the presence of the pKM101 plasmid R-factor. All WP2 uvrA cultures must demonstrate the deletion in the uvrA gene. All cultures must demonstrate the characteristic mean number of spontaneous revertants in the vehicle controls as follows (inclusive): TA98, 10-50; TA100, 80-240; TA1535, 5-45; TA1537, 3-21; WP2uvrA, 10-60. To ensure that appropriate numbers of bacteria are plated, tester strain culture titers must be greater than or equal to 0.3E09 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.
Statistics:
For each replicate plating, the mean and standard deviation of the number of revertants per plate were calculated and are reported.

Results and discussion

Test results
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
other: With metabolic activation: > 5000 ug/plate Without metabolic activation: > 5000 ug/plate
Remarks on result:
other:
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

For a summary of results: See Table 5.5.2 (Summary of Results) in document attached to Chapter 1.11 Additional Remarks.

Dose (mg/plate)

TA98

TA100

TA1535

TA1537

WP2uvrA

Liver Microsomes: None

Vehicle

17±3

96±11

13±2

5±3

17±3

75

16±3

99±10

9±4

5±0

15±3

200

18±2

105±16

9±3

5±3

14±2

600

15±3

108±15

11±1

5±1

15±2

1800

15±3

89±18

14±4

6±1

11±2

5000

11±3

95±13

11±2

6±1

13±2

Positive

91±13

332±10

195±7

448±50

77±10

Dose (mg/plate)

TA98

TA100

TA1535

TA1537

WP2uvrA

Liver Microsomes: Rat Liver S9

Vehicle

18±4

90±4

8±4

7±1

11±2

75

16±6

88±10

10±1

6±3

11±2

200

16±5

97±7

10±3

6±1

14±2

600

20±1

92±11

10±3

5±3

11±1

1800

15±3

85±7

11±2

5±3

12±1

5000

15±3

84±9

10±2

4±2

10±1

Positive

279±134

356±19

50±8

32±11

86±8

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):negativeUsed for data submission
Executive summary:

Used for data submission