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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:
1982
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Meets generally accepted scientific standards with GLP and is described in sufficient detail
Cross-referenceopen allclose all
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1982
Report Date:
1982

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
other: Microbiological Associates Protocol
Deviations:
no
Remarks:
Not specified in the report.
Principles of method if other than guideline:
none
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): Zoldine ZE (BIOBANTM CS-1246 Antimicrobial)
- Physical state: Orange Liquid
- Analytical purity: 96.84% active ingredient (Oxazolidine E)
- Lot/batch No.: 1

Method

Target gene:
Histedine operon
Species / strain
Species / strain:
other: Salmonella typhimurium strains TA98, TA100, TA1535, TA1537, and TA1538
Details on mammalian cell lines (if applicable):
not applicable
Additional strain characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S-9 liver homogenates prepared from Aroclor 1254-induced male Sprague-Dawley rats and were stored at approximately -70 °C or below.
Test concentrations with justification for top dose:
Toxicity Assay: 10,000, 3,125, 977, 305, 95, 30, 9.4, 2.9 and 0 ug/plate

Mutagenesis Assay:
In the absence & presence of metabolic activation (S9): 600, 300, 150, 30, 6.0 and 0 ug/plate
Vehicle:
- Vehicle(s)/solvent(s) used: water ( distilled water)
Controls
Negative controls:
yes
Solvent controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
other: All strains with activation = 2-aminoanthracene, TA98 without activation = 2-nitrofluorene, TA100 without activation = 1,3-propane sultone, TA1535(-S9) = 1,3-propane sultone, TA1537 (-S9) = 9-aminoacriaminoacridine, TA1538(-S9)=2-nitrofluorene
Details on test system and conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Preincubation period: No pre-incubation, plate incorporation method utilized.
- Exposure duration: 48 hours

NUMBER OF REPLICATIONS: Triplicate


DETERMINATION OF CYTOTOXICITY
- Method: Bacterial background lawn evaluation

Evaluation criteria:
Concentrations of the test chemical which are overtly toxic are easily visualized as showing no bacterial growth on the plate (i.e. absence of background lawn). Lower levels of toxicity may be seen as a thin or sparse bacterial lawn, a reduction in the number of revertants, or the appearance of microcolonies (overgrown background lawn). Positive and negative controls were run concurrently with the test chemical, and appropriate responses for these controls are prerequesites for evaluating the response of the bacteria to the test chemical.

A test chemical is considered a bacterial mutagen if both the mean number of revertant colonies observed is at least three times higher than the mean of the negative (solvent) control and at the same time it produces a dose response relationship over several concentrations. If a chemical produces reproducible reversion rates in excess of 3x over background, but no definitive dose response relationship, it is considered to be a presumptive bacterial mutagen. If a chemical produces reproducible reversion rates greater than 2x but less than 3x over the negative controls, the results are considered to be equivocal or inconclusive. Test substances failing to meet the above criteria are considered non-mutagenic in this system.

The test material must cause at least a doubling in the average revertants per plate or at least one tester strain. The increase must be accompanied by a dose response to increasing concentrations of the test article, and in the cases where the increase is less than three-fold, the response must be reproducible.
Statistics:
(Only average and standard deviation calculated)

Results and discussion

Test results
Species / strain:
other: TA98, TA100, TA1535, TA1537 and TA1538
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
other: Results indicated that the maximum dose to be tested, which would cause a detectable reduction in the number of revertants per plate in the mutagenicity assay, would be 600 ug/plate. Slight toxicity was noted at 305 uL/plate, and the viable counts and re
Vehicle controls valid:
yes
Negative 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:
Preliminary Toxicity Assay
Results indicated that the maximum dose to be tested, which would cause a detectable reduction in the number of revertants per plate in the mutagenicity assay, would be 600 ug/plate. Slight toxicity was noted at 305 uL/plate, and the viable counts and revertants on plates dosed with 977 uL/plate were drastically reduced.

Mutagenicity Assay
CS-1246 was tested at 0, 6, 30, 150, 300, and 600 ug/plate in the five Salmonella tester strains with and without metabolic activation. The test material was negative in all tester strains with and without activation, and is considered negative for mutagenicity in this system.

Any other information on results incl. tables

Table for Bacterial Assay for Gene Mutations in the Absence of S-9

Revertants Per Plate

 

 

TA1535

TA1537

TA1538

TA98

TA100

Vehicle Control

38

7

11

19

183

Test Article (ug/plate)

6.0

36

6

5

22

188

30

37

6

7

17

163

150

33

8

8

26

185

300

29

10

5

29

193

600

1

3

0

2

22

Positive Control

1693

332

1949

1152

1316

Table forBacterial Assay for Gene Mutations in the Presence of S-9

Revertants Per Plate

 

 

TA1535

TA1537

TA1538

TA98

TA100

Vehicle Control

11

9

19

32

158

Test Article (ug/plate)

6.0

9

13

19

31

143

30

16

9

20

28

145

150

8

9

15

32

158

300

15

12

16

36

197

600

4

5

3

14

54

Positive Control

205

254

326

337

700

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

The test material was negative in all tester strains with and without activation, and is considered negative for mutagenicity in this system.
Executive summary:

The Ames mutagenicity test was conducted in the Salmonella/mammalian-microsome assay using five tester strains TA98, TA100, TA1535, TA1537 and TA1538 ; both with and without metabolic actavation by rat liver microsomes.

The test material was prepared in water. Top agar for selection of histidine revertants was prepared with Difco bacto agar and NaCl. The top agar was supplemented with histidine and D-biotin for viable cell count determination plates. The Salmonella tester strains were were received from Dr. Ames, of UC Berkeley.

Toxicity Assay
Eight serial half-log dilutions of the test article were plated with a diluted TA100 culture on non-selective agar, and with an undiluted TA100 culture on selective agar.
 For viability determinations, equal numbers of cells were seeded on each plate in the presence of the solvent and the test article. The percent survival of an appropriately-diluted TA100 culture on non-selective agar was determined by comparing the number of colonies on the solvent control with those on the plates containing the test article. Toxicity on the revertant plates was detectable by a decrease in the number of revertant colonies per plate, or by a thinning or disappearace of the background bacterial lawn.

Mutagenicity Assay
The test article was solubilized and serially diluted immediately before its use.
 Five doses of the test article were first plated with all five tester strains with metabolic activation (Aroclor 1254-induced rat liver homogenate), after which they were plated on all tester strains without metabolic activation. All positive controls, solvent controls, negative controls, and test material doses were plated in triplicate. Without activation, 50uL of tester strain and 50 uL of solvent or test material were added to 2.5 mL of molten top agar. In activated systems, 0.5 mL of S-9 mix was added in addition. After mixing, the agar mixtures were poured over the minimal bottom agar. Plates were inverted and incubated for 48 hours at 37C. Positive controls were:
All strains with activation = 2-aminoanthracene
TA98 without activation = 2-nitrofluorene
TA100 without activation = 1,3-propane sultone
TA1535 without activation = 1,3-propane sultone
TA1537 without activation = 9-aminoacridine
TA1538 without activation = 2-nitrofluorene

Revertant colonies were counted either entirely by an automated colony counter or entirely by hand. Plates with precipitate were counted manually. The condition of the background bacterial lawn was evaluated macroscopically and microscopically for evidence of test material toxicity. All plating was done in triplicate. For each triplicate set, an average and standard deviation were calculated.

Criteria for Positive Result
The test material must cause at least a doubling in the average revertants per plate or at least one tester strain.
 The increase must be accompanied by a dose response to increasing concentrations of the test article, and in the cases where the increase is less than three-fold, the response must be reproducible.

The test material was tested at 0, 6, 30, 150, 300, and 600 ug/plate in the five Salmonella tester strains with and without metabolic activation. The test material was negative in all tester strains with and without activation, and is considered negative for mutagenicity in this system. The test material was negative in all tester strains with and without activation, and is considered negative for mutagenicity in this system.

The test material was negative in all tester strains with and without activation, and is considered negative for mutagenicity in this system.