Terpenes and Terpenoids, sinpine

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

23 June - 10 September 2004

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2004

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

1

Reference substance name:

Terpenes and Terpenoids, sinpine

EC Number:

272-842-9

EC Name:

Terpenes and Terpenoids, sinpine

Cas Number:

68917-63-5

IUPAC Name:

Reaction mass of 1-methyl-4-(propan-2-ylidene)cyclohex-1-ene and 1-methyl-4-(prop-1-en-2-yl)cyclohex-1-ene and 2,6,6-trimethylbicyclo[7.1.1]hept-2-ene and 1-isopropyl-4-methylcyclohexa-1,4-diene

Test material form:

not specified

Method

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

33; 100; 333; 1000; 2500 and 5000 microg/plate to validate the test, reference mutagens are tested in parallel to the test item.

Vehicle / solvent:

DMSO at 99%

Controls

Controls 1

Untreated negative controls:

yes

Remarks:

DMSO

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Positive controls:

no

Controls 2

Untreated negative controls:

yes

Remarks:

solvent

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

sodium azide

methylmethanesulfonate

other: 4-NOPD

Details on test system and experimental conditions:

The histidine dependent strains are derived from S.typhimurium strain LT2 through a mutation in the histidine locus. Additionally due to "deep rough" muttation they posses a faulty lipopolysaccharide envelope which enables substances to penetrate the cell wall more easily. A further mutation causes an inactivation of the excision repair system. The latter alteration aslso includes a deletion in the nitrate reductase and biotin genes. In the strains TA98, TA 100 and TA 102 teh R-factor plasmid pKM 101 carries unu DC analogous genes that are involved in error-prone repair and the ampicillin reistance marker. The strain TA 102 does not contain the uvrB- mutation and is excision repair proficient. Additionally, TA 102 contains the multicopy plasmid pAQ1 carrying the hisG428 mutation and a tetracycline resistance gene.
Regular checking of the properties of the strains regarding the mebrane permeability, ampicillin-and tetracycline-resistance as well as spontaneous mutation rates is performed in the CRO according to B. Ames et al. and D. MAron and B. Ames. In this way it was ensured that the expiremental conditions set down by Ames were fulfilled.
The bacterial strains TA 1535; 1537; 98; 100 and 102 were obtained from Trinova Biochem GmbH.

The strain cultures were stored as stock cultures in ampoules with nutrient broth + 5% DMSO in liquid nitrogen

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Applicant's summary and conclusion

Conclusions:

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.
Therefore, SinPine P is considered to be non-mutagenic in this Salmonella typhimurium reverse mutation assay.

Executive summary:

This study was performed to investigated the potential of SinPine P to induce gene mutation according to the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using the Salmonella typhimurium strains TA 1535, Ta 1537, Ta 98, TA 100 and TA 102.

The assay was performed in two independent experiemnts both with and without liver microsomal activation. Each concentration, including teh controls, was tested in triplicate. The test item was tested at the following concentrations:

33; 100; 333; 1000: 2500 and 5000 microg/plate

Reduced background growth was observed with and without S9 mix at 333 microg/plate and above in experiment I and at 1000microg/plate and above in experiement II ina ll strains used.

Toxic effects, evident as a reduction in the number of revertants, were observed at higher concentrations with and without metabolic activation in nearly all strains used.

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with SinPine P at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.

Appropiate reference mutagens were used as positive controls and showed a distint increase of induced revertant colonless.