Reaction mass of Terpenes and terpenoids, turpentine-oil, alpha-pinene fraction, dimers and Terpenes and terpenoids, turpentine-oil, alpha-pinene fraction, trimers

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The mutagenic activity of individual smoke components, alpha pinene included, was assessed using the Ames test. The information is included in an extended study published by Florin et al. 1980 (Screening of tobacco smoke constituents for mutagenicity using the Ames' test).The 4 bacterial strains used (TA 1535, TA 1537, TA 98 and TA 100) were histidine-requiring mutants of S. typhimurium with and without metabolic activation using liver fraction (S-9). The substance was tested at 3 µmol/plate. In case of toxicity the test was repeated at lower concentrations. Substances giving uncertain results were tested at 4 concentration levels: 0.03, 0.3, 3 and 30 3 µmol/plate. Alpha-pinene did not show mutagenic effects. The substance precipitated at 30 umol/plate.

A Peripheral blood micronucleus teststudy test was conducted as part of a NTP 13 week inhalation study on B6C3F1 female and male mice.

At the end of the treatment period, blood sample 13-week exposure period a blood sample was obtained from male and female mice. Sample collection time was typically between 0 (in the case of continuous exposures) and 24 hours (in the case of single daily exposures). 1,000 to 10,000 mature erythrocytes (normochromatic erythrocytes or NCEs) were scored per animal for presence of micronuclei. The percent PCE (immature erythrocytes) was determined in the blood as a measure of chemical-induced toxicity to the bone marrow. All data are analyzed separately for male and female mice.The results of the study showed no genetic toxicity under the test conditions.

Link to relevant study records

Reference

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

15 sept 1997 - 2 October 1997

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

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

Specific details on test material used for the study:

Test Material: Zonarez A-25
Batch: PC7150
Description: yellow extremely viscous liquid
Date received: 11 August 1997
Storage: Room temperature under nitrogen

Target gene:

TA 100 TA 1535 TA 102 TA 1537 TA98

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

- Supplier: University of California at Berkely
- Date supplier: August 1995
- Storing condition: -196 °C in a Statebourne liquid nitrogen freezer
- Type and identity of media:
- Properly maintained: yes/no
- Periodically checked for Mycoplasma contamination: yes/no
- Periodically checked for karyotype stability: yes/no
- Periodically "cleansed" against high spontaneous background: yes/no

Metabolic activation:

with and without

Test concentrations with justification for top dose:

0, 50, 150, 500, 1500, 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: tetrahydrofuran
- Justification for choice of solvent/vehicle: Not reported

Negative solvent / vehicle controls:

yes

Remarks:

tetrahydrofuran

True negative controls:

yes

Remarks:

2-aminoanthracene, 1,8-dihydroxyantharaquinone

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

N-ethyl-N-nitro-N-nitrosoguanidine

mitomycin C

Details on test system and experimental conditions:

METHOD OF APPLICATION: sterile plates of Vogel-Bonner Minimal agar

DURATION
- Preincubation period: Not reported
- Exposure duration: 48 hr
- Temperature: 37°C
- Expression time (cells in growth medium): Not reported

NUMBER OF REPLICATIONS: Frequency of revertant colonies was assessed using a Domino colony counter

DETERMINATION OF CYTOTOXICITY: Not reported

Evaluation criteria:

For a substance to be considered positive in this test system, it should have induced a dose-related and statistically significant increase in the relevant count in one or more strains of bacteria in the presence and/or absence of S9 in both experiments at sub-toxic dose levels. To be considered negative, the number of revertants at each dose level should be less than twofold that of the vehicle frequency.

Statistics:

All data was analysed using the statistical methods recommended by the UKEMS with Dunnett's method of linear regression used to calculate the result.

Key result

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

No toxicity was exhibited to any of the strains of Salmonella used. A precipitate was observed at 5000 µg/plate. This did not prevent the scoring of revertant colonies.

Remarks on result:

other: strain/cell type: male Sprague-Dawley

Remarks:

Migrated from field 'Test system'.

Mean number of revertant colonies for the toxicity assay
	DOSE (µg/plate)
Strain	0	50	150	500	1500	5000
TA100	122	125	118	127	119	199P
P=precipitate

Conclusions:

Interpretation of results (migrated information):
negative

The test material Zonarez A-25 was considered to be non-mutagenic under the condition of this test.

Executive summary:

A Reverse Mutation Assay "Ames Test" using Salmonella S. typhimurium was conducted to assess the mutagenicity of the test material Zonarez A-25. The test material was tested up to the maximum recommended dose of 5000 µg/plate.

Significant increases in the frequency of revertant colonies of bacteria were recorded at any dose level either with or without metabolic activation. All the positive control chemicals used in the test induced significantly the frequency of revertant colonies and activity of the S9 fraction was shown to be satisfactory.

Results showed no toxicity to any of the strains of Salmonella used up to 5000 µg/plate. A precipitate was observed at this dose level, but it did not prevent the scoring of relevant colonies.

The test item was non-mutagenic under the conditions of the test.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vitro:

Five studies are available on Terpenes and Terpenoids, turpentine oil, alpha-pinene fraction oligomers and Alpha pinene.

A Reverse Mutation Assay "Ames Test" using Salmonella typhimurium strains TA 1535, TA 1537, TA 98 and TA 100 was conducted using the test material Zonarez A-25 (Safepharm Laboratories Limited, 1997). The test material was tested up to the maximum recommended dose of 5000µg/plate. Results showed no toxicity to any of the strains of Salmonella. A precipitate was observed at this dose level, but it did not prevent the scoring of relevant colonies. The test item was non-mutagenic under the conditions of the test.

The mutagenic activity of individual smoke components was studied by Florin et al. 1980, using the Ames test. Alpha pinene was tested in 4 bacterial strains (TA 1535, TA 1537, TA 98 and TA 100) with and without metabolic activation (liver fraction S-9). The substance was tested at 3 µmol/plate (precipitation occurred at 30 umol/plate). Alpha-pinene did not show mutagenic effects.

A second Ames test on Alpha pinene was conducted by NTP (2005a) using Salmonella typhimurium TA 100 and TA 98 and E. coli pKM101 strains with or without S9 mixThe results showed no mutagenic effects.

In addition, a Peripheral blood micronucleus test study test was conducted as part of a NTP 13 week inhalation study on B6C3F1 female and male mice using Alpha pinene. At the end of the treatment period, a blood sample was obtained from male and female mice. 1,000 to 10,000 mature erythrocytes (normochromatic erythrocytes or NCEs) were scored per animal for presence of micronuclei. The percent PCE (immature erythrocytes) was determined in the blood as a measure of chemical-induced toxicity to the bone marrow. All data are analysed separately for male and female mice. The results of the study showed no genetic toxicity under the test conditions.

Finally, a study on food flavouring ingredients was conducted using an unscheduled DNA synthesis assay ( Heck J. D. et al., 1989). Hepatocytes were isolated from adult male Fischer or Sprague-Dawley rats. Per dose level 75 or 150 cells were analysed. The result of the screening study showed that the test item at 10000 µg did not display mutagenic effects.

Justification for classification or non-classification

Based on the available studies and in accordance to Directive 67/548/EEC and EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008, classification is not necessary for genetic toxicity.