Terpene

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In vitro gene mutation study in bacteria according to OECD guideline 471 and GLP: Negative

In vitro mammalian cell gene mutation tests using the thymidine kinase gene according to OECD guideline 490 and GLP: Positive (with metabolic activation)

In vitro mammalian cell micronucleus test according to OECD guideline 487 and GLP: Negative

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Additional strain / cell type characteristics:

DNA polymerase A deficient

Metabolic activation:

with and without

Key result

Species / strain:

E. coli WP2 uvr A

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

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

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

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

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

Additional information on results:

Toxicity was observed at the highest or higher dose levels with TA98, TA1537 and TA100 tester strains both in the absence and presence of S9 metabolism and with TA1535 tester strain only in its absence. No toxicity was observed with WP2 uvrA tester strain.
As no relevant increase in revertant numbers was observed at any concentration tested, aMain Assay II was performed using the pre-incubation method for all treatments. The dose-range was slightly modified to take into account the toxicity results ofMain Assay I.

Conclusions:

It is concluded that the test item does not induce reverse mutation in Salmonella typhimurium or Escherichia coli in the absence or presence of S9 metabolism, under the reported experimental conditions.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Qualifier:

according to guideline

Guideline:

OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)

GLP compliance:

yes

Type of assay:

in vitro mammalian cell gene mutation tests using the thymidine kinase gene

Target gene:

assaying for the induction of 5-trifluorothymidine resistant mutants in mouse lymphoma L5178Y cells

Species / strain / cell type:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Test concentrations with justification for top dose:

a preliminary cytotoxicity assay was performed both in the absence and presence of S9 metabolic activation, using the maximum dose level of 5 µL/mL and a wide range of lower dose levels: 2.50, 1.25, 0.625, 0.313, 0.156, 0.0781, 0.0391 and 0.0195 µL/mL.

Vehicle / solvent:

Ethanol

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Conclusions:

It is concluded that the substance induces mutation at the TK locus of L5178Y mouse lymphoma cells in vitro in the presence of S9 metabolic activation, under the reported experimental conditions

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

GLP compliance:

yes

Type of assay:

in vitro mammalian cell transformation assay

Target gene:

Lymphocytes in whole blood cultures are stimulated to divide by exposure to phytohaemagglutinin (PHA).

Species / strain / cell type:

lymphocytes:

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Test concentrations with justification for top dose:

For Main Assay 1, based on the preliminary cytotoxicity results obtained, dose levels of 0.160, 0.123, 0.0947, 0.0728, 0.0560, 0.0431, 0.0331, 0.0255, 0.0196 and 0.0151 µL/mL were used for all treatment series.
Since in the presence of S9 metabolic activation no adequate cytotoxicity was observed at any dose level to select the maximum concentration for scoring micronuclei, treatment was repeated inMain Assay 2 by using a different dose range and space interval. Dose levels of 0.123, 0.112, 0.102, 0.0924, 0.0840, 0.0764, 0.0694, 0.0631, 0.0574, 0.0522 and 0.0474 µL/mL were employed.

Vehicle / solvent:

ethanol

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Positive controls:

yes

Positive control substance:

cyclophosphamide

Key result

Species / strain:

lymphocytes: lymphocytes

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

Conclusions:

it is concluded that TERPENIC BASE does not induce micronucleic in human lymphocytes after in vitro treatment, under the reported experimental conditions.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

An in vivo mammalian alkaline comet assay (Annex VIII, Section 8.4., column 2; test method: OECD TG 489) in rats via the oral route is currently being conducted. This section will be updated as soon as the study is finalized.

Link to relevant study records

Reference

Endpoint:

in vivo mammalian cell study: DNA damage and/or repair

Remarks:

In vivo mammalian alkaline comet assay

Data waiving:

other justification

Justification for data waiving:

other:

Justification for type of information:

JUSTIFICATION FOR DATA WAIVING
Based on an ECHA decision on a testing proposal (decision number TPE-D-2114576945-30-01/F), an in vivo mammalian alkaline comet assay (Annex VIII, Section 8.4., column 2; test method: OECD TG 489) in rats, oral route, in liver, glandular stomach and duodenum tissues has been requested by ECHA. The study is currently planned and will be conducted in 2023.

The OECD TG 489 study will follow the study design specifications made by ECHA:
According to OECD TG 489 (paragraph 31), the comet assay can be performed in either sex, however if there is data demonstrating relevant differences between males and females then a study in both sexes is encouraged. The test is conducted in male rats as there are no data on differences in sensitivity between sexes. Furthermore, the oral route is chosen as it is considered the anticipated route of human exposure. In line with OECD TG 489, the test is performed by analysing tissues from liver as primary site of xenobiotic metabolism, glandular stomach and duodenum as sites of contact. According to the specifications of the study design made by ECHA, a preliminary range-finding study should be considered if there are no suitable data available from other relevant studies. Thus, a dose-range finding study will be conducted to identify the maximum tolerated dose (MTD) for the main test.

Due to the high workload of the laboratories, the deadline of 30 November 2022 specified in ECHA decision TPE-D-2114576945-30-01/F cannot be met. The draft report is planned to be available in 2023 (see laboratory statement attached). This section will be updated with the results of the in vivo mammalian alkaline comet assay presumably end of 2023.

Qualifier:

according to guideline

Guideline:

OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)

GLP compliance:

yes

Type of assay:

mammalian comet assay

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Route of administration:

oral: gavage

Duration of treatment / exposure:

2 treatments at approximately 24 hour intervals

Post exposure period:

2-6 hours after the final administration

No. of animals per sex per dose:

5 males per treated group
3 male animals for positive control group

both sexes should be considered if any existing toxicity, metabolism or exposure data indicate a toxicologically meaningful sex difference.

Tissues and cell types examined:

Liver (other tissues/organs can be selected depending on the proposed use for exposure to the test substance)

Details of tissue and slide preparation:

150 cells analysed per tissue (50 cells scored per each of three replicate slides)

Evaluation criteria:

number of heavily damaged cells (hedgehogs, clouds, ghost cells)
% tail intensity and tail moment

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008

An in vivo mammalian alkaline comet assay according to OECD TG 489 has been initiated. The dossier will be updated as soon as the final report is available. The test substance will be classified or not classified for toxicity to genetic toxicity according to Regulation (EC) No 1272/2008, as amended for the eighteenth time in Regulation (EU) 2022/692, depending on the outcome of the study.