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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/microsome assay was performed for the given test chemical.
Author:
Gomes-Carneiro et al.
Year:
1998
Bibliographic source:
Mutation Research

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 / cell type:
S. typhimurium, other: TA102, TA100, TA98 and TA97a
Details on mammalian cell type (if applicable):
Not applicable
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
Lyophilized rat liver S9 fraction induced by Aroclor 1254
Test concentrations with justification for top dose:
0, 250, 500, 750, 1000, 1250, 1500, 2000, 2500 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Ethanol
- Justification for choice of solvent/vehicle: The chemical was soluble in Ethanol
Controls
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
Remarks:
Ethanol
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
sodium azide
benzo(a)pyrene
mitomycin C
other: NPD (1 µg/plate). 2AF (10 µg/plate), 2AA (0.5 or 1 µg/plate)
Details on test system and experimental conditions:
NUMBER OF REPLICATIONS:
- Number of cultures per concentration - triplicate
- Number of independent experiments - each experiment was repeated at least once

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): approximately 1.2x10^9 bacteria per milliliter
- Test substance added - in agar (plate incorporation)

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: No data
- Exposure duration/duration of treatment: 72 h
- Harvest time after the end of treatment (sampling/recovery times): No data
Evaluation criteria:
A dose related increase in the number of revertants was noted whether it be twofold over background or not
Statistics:
means ±SD

Results and discussion

Test results
Species / strain:
S. typhimurium, other: TA102, TA100, TA98 and TA97a
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
True negative controls validity:
not specified
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING/SCREENING STUDIES (if applicable): A preliminary test carried out with TA100 strain without and with addition of S9 mixture. The upper limit of the dose interval tested was either the highest non-toxic dose or the lowest toxic dose determined in this preliminary assay.
Remarks on result:
other: No mutagenic potential

Any other information on results incl. tables

Table - Mutagenicity testing of the test chemical in the Salmonella/microsome assay TA100, TA98, TA97a and TA102 tester strains

Monoterpene

Dose (µg/plate)

Number of revertants (Mean±SD)

TA 100

TA 98

TA 97a

TA 102

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

Test chemical

2500

-

-

-

-

-

142*

-

-

2000

98±6*

157±12

-

51±18

146±8

180±4

-

514±129*

1500

149±26

180±12

37±2

57±13

170±13

140±11

617±140

722±21

1250

164±10

185±14

34±5

58±4

168±6

177±7

605±83

706±4

1000

183±13

179±13

38±1

60±6

166±12

190±40

692±31

777±31

750

196±10

177±26

47±6

59±8

174±19

180±10

642±23

698±30

500

199±16

190±19

41±7

59±2

163±26

210±9

753±41

758±26

250

193±12

-

43±2

-

168±17

-

686±19

794±32

0

176±14

196±18

48±5

54±6

160±22

185±22

660±91

776±36

PC

898±51

1003±66

204±34

210±34

998±52

1048±92

4102±317

1952±86

(–) Dose not tested.

(*) Toxicity apparent as an alteration of the background lawn.

(/+) Mutant counts of individual plates.

Values are the means±SD of three plates of one (out of two) representative experiment.

Applicant's summary and conclusion

Conclusions:
Test chemical did not induce mutation in the Salmonella typhimurium strains TA102, TA100, TA98 and TA97a 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:

Salmonella reverse mutation assay was performed to determine the mutagenic nature of the given test chemical on a tester strains of Salmonella typhimurium TA102, TA100, TA98 and TA97 with and without metabolic activation system extracted from Lyophilized rat liver induced by Aroclor 1254 at dose concentrations of 0, 250, 500, 750, 1000, 1250, 1500, 2000, 2500 µg/plate. The Salmonella mutagenicity test was performed by the plate incorporation method. Briefly, 2 ml of top-agar was mixed with 100µl of an overnight grown culture of S. typhimurium, 100µl of the test substance (diluted in ethanol analytical grade, Merck, KGaA), the negative control, or the positive control (PC) and 500µl of the phosphate buffer or the S9 mixture. Ethanol served as the negative (solvent) controls, while the positive control substances were: SA (0.5µg/plate), NPD (1µg/plate), MC (0.5µg/plate), 4-NQNO (1µg/plate), 2AF (10µg/plate), 2AA (0.5 or 1µg/plate), and B-(α)-P (50µg/plate). SA and MC were dissolved in distilled water and dimethyl sulfoxide (DMSO) was used as vehicle for the other positive controls. Plates were incubated at 37°C for 72 h in the dark and then scored for revertant his+ bacteria colonies. Every determination was made in triplicate and each experiment was repeated at least once in order to check the reproducibility of the results. A preliminary test carried out with TA100 strain without and with addition of S9 mixture. The upper limit of the dose interval tested was either the highest non-toxic dose or the lowest toxic dose determined in this preliminary assay. The given test chemical was non toxic at highest doses of 2000–2500µg/plate. No mutagenic effect was observed with the four bacterial tester strains, in the absence as well as in the presence of extrinsic metabolic activation. Hence, the given test chemical is not likely to classify as a gene mutant in vitro.