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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:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
data from handbook or collection of data
Justification for type of information:
Data is from peer reviewed publication

Data source

Reference
Reference Type:
publication
Title:
Salmonella Mutagenicity Test Results for 250 Chemicals
Author:
Steve Haworth, Timothy Lawlor, Kristien Mortelmans, William Speck, and Errol Zeiger
Year:
1983
Bibliographic source:
Environmental Mutagenesis Volume 5, Supplement 1: 3 -142 (1983)

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Principles of method if other than guideline:
Gene mutation toxicity study was performed to determine the mutagenic nature of the test chemical.
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material: Cineol (eucalyptol)
- IUPAC name: 1,3,3-Trimethyl-2-oxabicyclo[2.2.2]octane
- Molecular formula: C10H18O
- Molecular weight: 154.2512 g/mol

Method

Target gene:
Histidine
Species / strain
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell lines (if applicable):
Not applicable
Additional strain characteristics:
not specified
Cytokinesis block (if used):
No data
Metabolic activation:
with and without
Metabolic activation system:
Liver S-9 fractions were routinely prepared from male Sprague-Dawley rats and male Syrian hamsters that were injected, ip, with Aroclor 1254 (200 mg/ml in corn oil) at 500 mg/kg.
Test concentrations with justification for top dose:
0, 3.3, 10, 33, 100, 333, 1000 or 3333 µg/plate
Vehicle:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The chemical was soluble in DMSO
Controls
Negative controls:
not specified
Solvent controls:
yes
Remarks:
DMSO
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
9-aminoacridine
sodium azide
other: 2-Aminoanthracene (2-AA), 4-Nitro-o-phenylenediamine (NOPD)
Details on test system and conditions:
METHOD OF APPLICATION: preincubation

DURATION
- Preincubation period: 20 mins
- Exposure duration: 48 hrs
- Expression time (cells in growth medium): 48 hrs
- Selection time (if incubation with a selection agent): No data
- Fixation time (start of exposure up to fixation or harvest of cells): No data

SELECTION AGENT (mutation assays): No data
SPINDLE INHIBITOR (cytogenetic assays): No data
STAIN (for cytogenetic assays): No data

NUMBER OF REPLICATIONS: Triplicate

NUMBER OF CELLS EVALUATED: No data

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: No data

OTHER EXAMINATIONS:
- Determination of polyploidy: No data
- Determination of endoreplication: No data
- Other: No data

OTHER: No data
Rationale for test conditions:
No data
Evaluation criteria:
A dose related increase in the number of revertants was noted whether it be twofold over background or not
Statistics:
No data

Results and discussion

Test results
Species / strain:
other: TA1535, TA1537, TA98, and TA100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
not specified
Vehicle controls valid:
yes
Negative controls valid:
not specified
Positive controls valid:
yes
Remarks on result:
other: No mutagenic potential
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No data
- Effects of osmolality: No data
- Evaporation from medium: No data
- Water solubility: No data
- Precipitation: No data
- Other confounding effects: No data

RANGE-FINDING/SCREENING STUDIES: To select the dose range for the mutagenesis assay, the test chemical was checked for toxicity to TA100 up to a concentration of 10 mg/plate or the limit of solubility, both in the presence and absence of S-9 mix. One or more parameters were used as an indication of toxicity: viability on complete medium and reduced numbers of revertant colonies per plate and/or thinning or absence of the bacterial lawn. If toxicity was not apparent in the preliminary toxicity determination, the highest dose tested was 10 mg/plate; otherwise the upper limit of solubility was used. If toxicity was observed, the doses of test chemical were chosen so that the high dose exhibited some degree of toxicity. Occasionally, in the earlier tests, the high dose was greater than 10 mg/plate.

COMPARISON WITH HISTORICAL CONTROL DATA: No data

ADDITIONAL INFORMATION ON CYTOTOXICITY: No data

Any other information on results incl. tables

Table: Mutagenicity of Cineol (eucalyptol)

Dose (µg/plate)

TA100

TA1535

NA

RLI

HLI

NA

RLI

HLI

0

94±7.1

120±8.2

91±5.5

4±1.2

7±1.3

8±3.3

3.3

85±6.0

 

 

 

 

 

10

76±6.0

 

 

 

 

 

33

79±7.9

127±20.4

105±15.0

4± 1.2

6±0.3

6±0.9

100

95±11.2

113± 16.2

100±6.4

4±0.9

8±2.0

8±1.0

333

97 ±3.2

92 ±6.5

88±8.0

4±0.7

4±0.7

4±1.9

1000

 

104±6.4

93±4.0

3±0.6

t

5±1.3

3333

 

t

3±3.0

T

T

T

Pos

773±61.2

2096±89.8

1834±29.2

1260±85.5

157±27.

180±39.4

 

Dose (µg/plate)

TA1537

TA98

NA

RLI

HLI

NA

RLI

HLI

0

4±1.8

10±2.6

7±1.0

19±2.6

27±4.4

24±3.8

3.3

7±1.2

 

 

 

 

 

10

8±3.6

6±1.7

9±0.6

19±2.1

 

 

33

6±1.3

7±1.5

9±2.1

14±2.3

27±3.7

22±1.5

100

4±0.5

5±0.7

8±3.4

15±3.2

23±6.2

19±9.5

333

T

6±2.5

7±1.9

21±0.9

14±2.2

17±4.1

T: complete clearing of background lawn

Applicant's summary and conclusion

Conclusions:
Test chemical did not induce mutation in the Salmonella typhimurium strains TA1535, TA1537, TA98, and TA100 both in the presence and absence of S9 metabolic activation system and hence is not likely to classify as a gene mutant in vitro.
Executive summary:

Gene mutation toxicity study was performed to determine the mutagenic nature of the testchemical . The study was performed by the Preincubation protocol using Salmonellatyphimurium strains TA1535, TA1537, TA98, and TA100 both in the presence and absence of S9 metabolic activation system. Preincubation was carried at 37°C for 20 mins followed by exposure period of 48 hrs at dose levels of 0, 3.3, 10, 33, 100, 333, 1000 or 3333µg/plate. DMSO was used as solvent control and concurrent positive control chemicals were included in the study. A dose related increase in the number of revertants was noted whether it be twofold over background or not. Test chemical did not induce mutation in the Salmonellatyphimurium strains TA1535, TA1537, TA98, and TA100 both in the presence and absence of S9 metabolic activation system and hence is not likely to classify as a gene mutant in vitro.