Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In an Ames test performed according to OECD Guideline 471 and in compliance with GLP, a structurally related substance (whose main constituents are beta-phellandrene, d-limonene and l-limonene) was considered to be non mutagenic.

In an in vitro micronucleus test performed according to OECD Guideline 487 and in compliance with GLP, the registered substance did not induce micronuclei.

In an in vitro HPRT test performed according to OECD Guideline 476 and in compliance with GLP, the registered substance was considered to be non mutagenic.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
supporting study
Justification for type of information:
The main constituents of dipentene multiconstituent present at concentration above 10% are d-limonène, l-limonène and beta-phellandrene. It has the same main constituents as REACTION MASS OF BETA-PHELLANDRENE AND D-LIMONENE AND L-LIMONENE. Therefore, data on dipentene multiconstituent can be used for extrapolation to REACTION MASS OF BETA-PHELLANDRENE AND D-LIMONENE AND L-LIMONENE. See read-across justification document in section 13.
Reason / purpose:
read-across source
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
See table 7.6.1/4
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
See table 7.6.1/4
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
See table 7.6.1/5
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
See table 7.6.1/5
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
See table 7.6.1/6
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
See table 7.6.1/6
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
See table 7.6.1/7
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
See table 7.6.1/7
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
See table 7.6.1/8
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
See table 7.6.1/8
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS: not applicable

RANGE-FINDING/SCREENING STUDIES: A first preliminary test was performed testing six dose-levels of the test item (one plate/dose-level) with the TA 98, TA 100 and TA 102 strains, with and without S9 mix. This preliminary test was invalidated. Indeed, the test item used in this test came from a small aliquot taken from one of the flask and stored at +4°C, protected from light and humidity and under nitrogen gas. This conditioning in an inert atmosphere was performed using a layer of parafilm to seal the vessel (as described in CIT’s SOPs). But there were some evidences that this layer of parafilm got deteriorated (most probably by the test item’s emanation) contaminating the aliquot of test item. Because of this contamination, the results of this first preliminary test were invalidated and the study was started all over again using one supplied flask per treatment and avoiding the use of parafilm.
The second preliminary test was performed testing six dose-levels of the test item (two plates/dose-level) with the TA 98, TA 100 and TA 102 strains, with and without S9 mix. In this second preliminary test, the results of the vehicle control obtained with the TA 102 strain with S9 mix were found non valid (inconsistent with the historical data range). No precipitate was observed in the Petri plates when scoring the revertants at any of the tested dose-levels and in any strains. Without S9 mix, a strong toxicity (observation of the decrease in the number of revertant colonies and of a thinning of the bacterial lawn) was observed at dose-levels ≥ 100 µg/plate with the TA 98, TA 100 and TA 102 strains. With S9 mix, a moderate to strong toxicity was observed at dose-levels ≥ 500 µg/plate with the TA 98 and TA 100 strains. Therefore since the test item was toxic in the preliminary test, the choice of the highest dose-level to be tested in the main test was based on the level of toxicity, according to the criteria specified in the international guidelines.

COMPARISON WITH HISTORICAL CONTROL DATA: The number of revertants for the vehicle and positive controls was similar to the historical data.
ADDITIONAL INFORMATION ON CYTOTOXICITY: A strong toxicity was observed at dose-levels  185.2 µg/plate in the TA 1537 and TA 98 strains and at dose-levels  555.6 µg/plate in the TA 1535 strain in the second experiment with S9 mix (see table 7.6.1/9). The results of the vehicle control obtained with the TA 100 and TA 102 strains being inconsistent with the historical data range, therefore the data were invalidated. Since too many toxic dose-levels were obtained leading to less than five analysable dose-levels (which is a requirement of the international guidelines), the values were non-retained. The treatment of the second experiment with S9 mix according to the pre-incubation method was repeated using lower ranges of dose-levels.
Remarks on result:
other: strain/cell type:
Remarks:
Migrated from field 'Test system'.

Table 7.6.1/4: Results for the strainTA1535

Dipentene concentration plate (µg/plate)

Without metabolic activation

With metabolic activation

First experiment (direct plate incorporation)

Second experiment (direct plate incorporation)

First experiment (direct plate incorporation)

Second experiment (pre-incubation)

Ratio mean revertant treated/revertant solvent

Cytotoxicity*

Ratio mean revertant treated/revertant solvent

Cytotoxicity*

Ratio mean revertant treated/revertant solvent

Cytotoxicity*

Ratio mean revertant treated/revertant solvent

Cytotoxicity*

0

-

No

-

No

-

No

-

No

0.8

0.9

No

0.9

No

-

-

-

-

2.3

1.1

No

1.0

No

1.2

No

1.6

No

6.9

0.9

No

0.9

No

0.7

No

1.2

No

20.6

0.9

No

0.9

No

0.8

No

1.5

No

61.7

0.7

No

0.9

St

0.7

No

1.1

No

185.2

0.4

St

0.2

St

0.6

No

1.2

Mt

555.6

-

-

-

-

0.9

No

0.5

St

Postive controls

 

 

 

 

 

 

 

 

NaN3 (1µg)

28.6

No

17.1

No

-

-

-

 

2AM (2µg)

-

-

-

-

13.6

No

16.5

 

*: based on the thining of the bacterial lawn, Mt: Moderate toxicity; St: Strong toxicity

Table 7.6.1/5: Results for the strainTA1537

Dipentene concentration plate (µg/plate)

Without metabolic activation

With metabolic activation

First experiment (direct plate incorporation)

Second experiment (direct plate incorporation)

First experiment (direct plate incorporation)

Second experiment (pre-incubation)

Ratio mean revertant treated/revertant solvent

Cytotoxicity*

Ratio mean revertant treated/revertant solvent

Cytotoxicity*

Ratio mean revertant treated/revertant solvent

Cytotoxicity*

Ratio mean revertant treated/revertant solvent

Cytotoxicity*

0

-

No

-

No

-

No

-

No

0.3

-

-

0.3

No

-

-

-

 

0.8

1.0

No

0.7

No

-

-

1.2

-

2.3

1.1

No

1.1

No

-

-

0.9

No

6.9

0.7

No

0.8

No

1.3

No

1.5

No

20.6

0.7

Mt

0.5

Mt

1.3

No

1.4

No

61.7

0.0

St

1.7

St

1.5

No

1.1

No

185.2

0.0

St

-

-

1.7

No

0.7

Mt

555.6

-

-

-

-

0.9

No

-

-

1666.7

 

 

 

 

1.0

Mt

-

-

Postive controls

 

 

 

 

 

 

 

 

9AA (50µg)

18.1

No

32.9

No

-

-

-

-

2AM (2µg)

-

-

-

-

8.7

No

17.1

No

*: based on the thining of the bacterial lawn, Mt: Moderate toxicity; St: Strong toxicity

Table 7.6.1/6:Results for the strainTA98

Dipentene concentration plate (µg/plate)

Without metabolic activation

With metabolic activation

First experiment (direct plate incorporation)

Second experiment (direct plate incorporation)

First experiment (direct plate incorporation)

Second experiment (pre-incubation)

Ratio mean revertant treated/revertant solvent

Cytotoxicity*

Ratio mean revertant treated/revertant solvent

Cytotoxicity*

Ratio mean revertant treated/revertant solvent

Cytotoxicity*

Ratio mean revertant treated/revertant solvent

Cytotoxicity*

0

-

No

-

No

-

No

-

No

0.8

2.1

No

-

-

-

-

-

-

2.3

1.8

No

0.9

No

-

-

0.9

No

6.9

0.9

No

0.7

No

0.9

No

0.6

No

20.6

0.7

No

0.6

No

1.0

No

0.7

No

61.7

0.9

Mt

0.9

No

1.1

No

0.7

Mt

185.2

0.9

Mt

0.2

Mt

1.7

No

0.0

St

555.6

-

-

0.4

St

0.8

Mt

0.0

St

1666.7

-

-

-

-

0.8

Mt

-

-

Postive controls

 

 

 

 

 

 

 

 

2NF (0.5µg)

7.8

No

4.1

No

-

-

-

 

2AM (2µg)

-

-

-

-

26.4

No

45.6

N o

*: based on the thining of the bacterial lawn, Mt: Moderate toxicity; St: Strong toxicity

Table 7.6.1/7: Results for the strainTA100

Dipentene concentration plate (µg/plate)

Without metabolic activation

With metabolic activation

First experiment (direct plate incorporation)

Second experiment (direct plate incorporation)

First experiment (direct plate incorporation)

Second experiment (pre-incubation)

Ratio mean revertant treated/revertant solvent

Cytotoxicity*

Ratio mean revertant treated/revertant solvent

Cytotoxicity*

Ratio mean revertant treated/revertant solvent

Cytotoxicity*

Ratio mean revertant treated/revertant solvent

Cytotoxicity*

0

-

No

-

No

-

No

-

No

0.8

1.1

No

-

-

-

-

-

-

2.3

1.4

No

0.9

No

1.4

-

1.1

No

6.9

1.0

No

1.1

No

1.5

No

1.1

No

20.6

1.1

No

0.9

No

1.1

No

1.3

No

61.7

0.8

No

0.7

Mt

1.4

No

1.1

No

185.2

0.3

Mt

0.4

St

1.1

No

1.0

Mt

555.6

-

-

0.6

St

1.1

No

0.3

St

Postive controls

 

 

 

 

 

 

 

 

NaN3 (1µg)

4.7

No

7.7

No

-

-

-

 

BAP (5µg)

-

-

-

-

8.6

No

5.6

N o

*: based on the thining of the bacterial lawn, Mt: Moderate toxicity; St: Strong toxicity

Table 7.6.1/8:Results for the strainTA102

Dipentene concentration plate (µg/plate)

Without metabolic activation

With metabolic activation

First experiment (direct plate incorporation)

Second experiment (direct plate incorporation)

First experiment (direct plate incorporation)

Second experiment (pre-incubation)

Ratio mean revertant treated/revertant solvent

Cytotoxicity*

Ratio mean revertant treated/revertant solvent

Cytotoxicity*

Ratio mean revertant treated/revertant solvent

Cytotoxicity*

Ratio mean revertant treated/revertant solvent

Cytotoxicity*

0

-

No

-

No

-

No

-

No

0.8

1.4

No

-

-

-

-

-

-

2.3

1.8

No

-

-

-

-

0.8

No

6.9

1.9

No

1.0

No

-

-

1.1

No

20.6

1.3

No

1.0

No

1.0

No

1.1

No

61.7

1.2

No

0.9

No

1.5

No

1.0

No

185.2

1.6

No

0.7

Mt

1.3

No

1.0

No

555.6

-

-

0.6

Mt

1.6

No

0.6

Mt

1666.7

-

-

0.4

Mt

1.0

No

-

-

5000

-

-

-

-

1.4

No

-

-

Postive controls

 

 

 

 

 

 

 

 

MMC (0.5µg)

7.6

No

5.2

No

-

-

-

 

2AM (10µg)

-

-

-

-

3.8

No

5.5

N o

*: based on the thining of the bacterial lawn, Mt: Moderate toxicity; St: Strong toxicity

Conclusions:
Test item dipentene multiconstitent did not show any mutagenic activity in the bacterial reverse mutation test with Salmonella typhimurium. Therefore, dipentene multiconstituent is not considered as mutagenic in this bacterial system according to CLP Regulation (EC) No 1272/2008 and Directive 67/548/EEC.
Executive summary:

In a reverse gene mutation assay in bacteria (No. 36799 MMO), performed according to the OECD No. 471 and EC No. B13/14 guidelines, dipentene multiconstiuent (purity of 48.4%) diluted in Dimethylsulfoxide (DMSO) was tested in S. typhimurium TA1535, TA1537, TA100, TA98 and TA102 in the presence and the absence of mammalian metabolic activation (S9) using the direct plate incorporation or the preincubation method.The dipentene content (48.4%) was not taken into account. As a consequence, no correction factor was applied to express the concentrations and dose-levels of test item.

Due to the volatile characteristic of the test item and in order to limit the oxidation of the test item, all the Petri dishes were placed in a sealed jar. One jar was used for each tested dose-level, one jar was used for the vehicle control and another jar for the positive controls. 

Six known mutagens, dissolved in dimethylsulfoxide (except for Mitomycin C which was dissolved in distilled water), were used to check the sensitivity of the test system. The positive controls induced the appropriate responses in the corresponding strains. The number of revertants in the vehicle controls was consistent with the historical data of the testing facility, and the number of revertants in the positive controls was higher than that of the vehicle controls (at least 2-fold increase for the TA 98, TA 100 and TA 102 strains and at least 3-fold increase for the TA 1535 and TA 1537 strains) and was consistent with the historical data of the testing facility. Therefore the study was considered valid.

During the preliminary test, the decrease in the number of revertant colonies and/or thinning of the bacterial lawn showed that test item dipentene multiconstituent was cytotoxic for bacteria. Thus, the choice of the highest dose-level to be tested in the main test was based on the level of toxicity, according to the criteria specified in the international guidelines. During the main test, no induced revertant over background was observed in any strains of S. typhimurium whereas the cytotoxic dose-level was reached.

Therefore, dipentene multiconstituent did not show any mutagenic activity in the bacterial reverse mutation test using Salmonella typhimurium. Dipentene multiconstituent is not considered as mutagenic in this bacterial system according to CLP Regulation (EC) No 1272/2008 and Directive 67/548/EEC.

 

This study is considered as acceptable as it satisfied the criteria of Guideline OECD No. 471.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
16 February to 30 March 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Well conducted and well described study in accordance with GLP and OECD Guideline 476 without any deviation.
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Qualifier:
according to
Guideline:
EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. certificate)
Remarks:
28 October 2016
Type of assay:
other: mammalian cell gene mutation assay
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Les Dérivés Résiniques et Terpéniques / FAB-15052015
- Description: Colourless to slightly yellow liquid
- Expiration date of the lot/batch: 14 May 2016
- Purity test date: 01 June 2015

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Approximately 4 °C and under nitrogen, in the dark until 19 January 2016 where it was stored at room temperature in the dark, under nitrogen until 22 January 2016 and then back to approximately 4 °C, in the dark under nitrogen, thereafter
Target gene:
hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus of the V79 cell line
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: V79 cell stocks were obtained from Harlan CCR in 2010 and originated from Labor für Mutagenitätsprüfungen (LMP); Technical University; 64287 Darmstadt, Germany.
- Suitability of cells: The V79 cell line has been used successfully in in vitro experiments for many years. The high proliferation rate (doubling time 12 - 16 h in stock cultures) and a good cloning efficiency of untreated cells (as a rule more than 50 %) make it an appropriate cell line to use for this study type.
- Modal number of chromosomes: Stable karyotype with a modal chromosome number of 22 (Howard-Flanders, 1981).

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: Cells were grown in Eagles Minimal Essential (MEM) (supplemented with sodium bicarbonate, L-glutamine, penicillin/streptomycin, amphotericin B, HEPES buffer and 10% fetal bovine serum (FBS)) at approximately 37 °C with 5% CO2 in humidified air.
- Properly maintained: Yes
- Periodically checked for Mycoplasma contamination: Yes
- Periodically checked for karyotype stability: Yes
- Periodically 'cleansed' against high spontaneous background: Yes
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9 fraction (2%); S9 fraction was prepared from liver homogenates of male Sprague Dawley rats treated with phenobarbital and β-naphthoflavone
Test concentrations with justification for top dose:
Preliminary Cytotoxicity Test: 9.77, 19.53, 39.06, 78.13, 156.25, 312.5, 625, 1250 and 2500 μg/mL with or without S9

Mutagenicity Test – Main Experiment
4 h without S9: 0, 2, 4, 8, 10, 12, 16 and 20 μg/mL
4 h with S9 (2%): 0, 10, 20, 40, 50, 60, 70 and 80 μg/mL
Justification: The maximum dose level selected for the main mutagenicity experiment was based on the toxicity seen in the preliminary cytotoxicity test and was 20 μg/mL in the absence of S9 and 80 μg/mL in the presence of S9.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Acetone
- Test substance preparation:
The test item is a multiconstituent substance and the maximum dose concentration was initially set at 5000 μg/mL. However, due to the necessity of using acetone as the solvent, the maximum dose level that could be achieved was 2500 μg/mL. The test item was tested with no purity correction applied to the formulations at the request of the Sponsor.
The solubility of the test item was investigated as part of the in vitro Micronucleus Study in Human Lymphocytes, Envigo Study Number TG04KK on the same test item. The test item was insoluble in culture medium at 50 mg/mL and insoluble in dimethyl sulphoxide at 250 mg/mL and 500 μg/mL. The test item was soluble in acetone at 500 mg/mL which allowed a maximum achievable dose of 2500 μg/mL, when dosed at 0.5%.
The test item was formulated within two hours of it being applied to the test system; it is assumed that the formulation was stable for this duration.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
acetone
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
without metabolic activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
acetone
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: Dimethyl benzanthracene
Remarks:
with metabolic activation
Details on test system and experimental conditions:
METHOD OF APPLICATION: Eagles Minimal Essential (MEM) (supplemented with sodium bicarbonate, L-glutamine, penicillin/streptomycin, amphotericin B, HEPES buffer and 10% fetal bovine serum (FBS))
- Cell density at seeding: Cells were seeded at 1 x 10^7 cells/225 cm2 flask approximately 24 hours being exposed to the test or control items.

DURATION
- Exposure duration: 4 h
- Expression time (cells in growth medium): 7 days
- Selection time (if incubation with a selection agent): 7 days
- All incubations were performed at 37 °C in a humidified atmosphere of 5 % CO2 in air.

SELECTION AGENT (mutation assays): 6-Thioguanine (6-TG) at 11 μg/mL

NUMBER OF REPLICATIONS:
- Preliminary toxicity test: Single culture/dose
- Main test: 2 cultures/dose

NUMBER OF CELLS EVALUATED: 200 cells plated/flask for cloning efficiency and 2 x 10^5 cells plated/flask for mutant frequencies.

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency

- OTHER:
- Fixation and staining of all flasks/petri dishes was achieved by aspirating off the media, washing with phosphate buffered saline, fixing for 5 minutes with methanol and finally staining with a 10% Giemsa solution for 5 minutes.
- The cloning efficiency (CE), % control, mutant plate counts, mutant frequency/10^6 (MF10^-6) and mutant frequency/10^6 survival rate (MFSV) were calculated.
Evaluation criteria:
Providing that all of the acceptability criteria are fulfilled, a test item can be considered to be clearly positive if, in any of the experimental conditions examined:
i) At least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control.
ii) The increase is considered to be concentration-related.
iii) The results are outside the range of the historical negative control data for the test item concentrations.
When all these criteria are met, the test chemical is then considered able to induce gene mutations in cultured mammalian cells in this test system.
Providing that all of the acceptability criteria are fulfilled, a test item can be considered to be clearly negative if, in all of the experimental conditions examined:
i) None of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control.
ii) There is no concentration related increase.
iii) The results for the test item concentrations are within the range of the historical negative control data.
The test chemical is then considered unable to induce gene mutations in cultured mammalian cells in this test system.
There is no requirement for verification of a clearly positive or negative response.
In case the response is neither clearly negative nor clearly positive as described above or in order to assist in establishing the biological relevance of a result, the data should be evaluated by expert judgment and/or further investigations. Performing a repeat experiment possibly using modified experimental conditions (e.g. concentration spacing, S9 concentration, and exposure time) may be useful.
Statistics:
When there is no indication of any increases in mutant frequency at any dose level then statistical analysis may not be necessary. In all other circumstances comparisons will be made between the appropriate vehicle control value and each individual dose level, using Student’s t-test. Other statistical analysis may be used if they are considered to be appropriate.
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No significant change in pH when the test item was dosed into media
- Effects of osmolality: Osmolality did not increase by more than 50 mOsm at the dose levels investigated
- Water solubility: None
- Precipitation: Yes

PRELIMINARY CYTOTOXICITY TEST:
- A cloudy precipitate of the test item was observed at the end of exposure at and above 9.77 μg/mL and greasy/oily precipitate was observed at and above 39.06 μg/mL in the 4 h exposure group in the absence of S9. In the presence of S9, precipitate (cloudy and greasy/oily) was observed at the end of exposure at and above 78.13 μg/mL.
- At 4 h exposure in the absence of S9: extreme toxicity with only one surviving dose level (9.77 μg/mL) which demonstrated a 62% reduction in cloning efficiency.
- At 4 h exposure group in the presence of S9: a very sharp toxicity curve between 39.06 μg/mL, where there was no marked toxicity and 78.13 μg/mL where there was a 97% reduction in cloning efficiency.
- Maximum dose level selected for the main mutagenicity experiment was based on the toxicity seen in the preliminary cytotoxicity test and was 20 μg/mL in the absence of S9 and 80 μg/mL in the presence of S9.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation
- Positive historical control data: Ethyl methane sulphonate: 32-370 (226.52 ± 77.63); Dimethyl benzanthracene: 78-1057 (319.07 ± 157.07)
- Negative (solvent/vehicle) historical control data: 4 h without S9: 4-31 (13.92 ± 7.04); 4 h with S9: 4-63 (20.62 ± 13.64)

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Mutagenicity Test – Main Experiment
- A cloudy precipitate of the test item was seen at and above 70 μg/mL and greasy/oily precipitate at 80 μg/mL at the end of the exposure period in the presence of S9. No precipitate was observed at the end of the exposure in the absence of S9.
- Toxicity was similar to that observed in the Preliminary Toxicity Test. In the 4 h exposure group in the absence of S9 there were no viable cells remaining at 20 μg/mL after the exposure period and therefore this dose level was not plated for cloning efficiency or maintained during the expression period for mutant frequency plating. In the presence of S9 all test item dose levels were plated for mutant frequency.
- There was a dose related reduction in in the Day 0 cloning efficiency demonstrated in both the absence and presence of S9. The 4 h exposure group in the absence of S9 demonstrated a steep toxicity curve with a reduction in cloning efficiency of 26% and 71% at 12 μg/mL and 16 μg/mL, respectively. Although, optimum toxicity has not quite been achieved it is considered that due to the steepness of the toxicity curve the toxicity is satisfactory and the test item has been adequately tested. The higher dose level of 20 μg/mL was too toxic for plating. In the presence of S9, optimum toxicity was achieved at 80 μg/mL with a reduction in cloning efficiency of 82% compared to the vehicle control. There was no marked reduction in the Day 7 cloning efficiencies of either exposure groups indicating full recovery during the expression period from the toxicity observed at the end of exposure to the test item.

MUTAGENICITY RESULTS:
- There were no dose related increases in mutation frequency per survivor or any dose level exceeded the current historical control range for a vehicle control.

See attached document

Conclusions:
The test item is considered to be non-mutagenic to V79 cells at the HPRT locus in the presence or absence of metabolic activation.
Executive summary:

In an in vitro mammalian cell gene mutation test performed according to guideline OECD 476 and in compliance with GLP, Chinese hamster (V79) cells were exposed to test item at the concentrations below.

Preliminary Cytotoxicity Test: 9.77, 19.53, 39.06, 78.13, 156.25, 312.5, 625, 1250 and 2500 μg/mL with and without S9

Mutagenicity Test – Main Experiment

4 h without S9: 0, 2, 4, 8, 10, 12, 16 and 20 μg/mL

4 h with S9 (2%): 0, 10, 20, 40,50, 60, 70 and 80 μg/mL

 

Vehicle and positive control groups were also included in each mutation test. Metabolic activation system used in this test was 2%; S9 fraction was prepared from liver homogenates of male Sprague Dawley rats treated with phenobarbital and β-naphthoflavone.

 

The vehicle (acetone) controls gave mutant frequencies within the range expected of V79 cells at the HPRT locus. The positive control treatments, both in the presence and absence of metabolic activation, gave significant increases in the mutant frequency indicating the satisfactory performance of the test and of the metabolizing system.

 

The maximum dose level selected for the main mutagenicity experiment was based on the toxicity seen in the preliminary cytotoxicity test and was 20 μg/mL in the absence of S9 and 80 μg/mL in the presence of S9.

 

The test item demonstrated no significant increases in mutant frequency at any dose level, either with or without metabolic activation.

 

Therefore, the test item is considered to be non-mutagenic to V79 cells at the HPRT locus in the presence or absence of metabolic activation.

Endpoint:
in vitro cytogenicity / micronucleus study
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
29 January to 05 April 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Well conducted and well described study in accordance with GLP and OECD Guideline 487 without any deviation.
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
Qualifier:
according to
Guideline:
OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. certificate)
Remarks:
28 October 2016
Type of assay:
in vitro mammalian cell micronucleus test
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Les Dérivés Résiniques et Terpéniques / FAB-15052015
- Description: Colourless to slightly yellow liquid
- Expiration date of the lot/batch: 14 May 2016
- Purity test date: 01 June 2015

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Approximately 4 °C and under nitrogen, in the dark until 19 January 2016 where it was stored at room temperature in the dark, under nitrogen until 22 January 2016 and then back to approximately 4 °C, in the dark under nitrogen, thereafter.
Target gene:
Not applicable
Species / strain / cell type:
lymphocytes: human
Details on mammalian cell type (if applicable):
not applicable
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
2 % S9 (final concentration); S9 fraction prepared from liver homogenates of male rats induced with Phenobarbital/β-naphthoflavone
Test concentrations with justification for top dose:
Preliminary Toxicity Test: 0, 9.77, 19.53, 39.06, 78.13, 156.25, 312.5, 625, 1250 and 2500 μg/mL (4 h exposure to the test item without or with S9-mix (2%); 24 h continuous exposure to the test item without S9-mix)

Main Experiment:
0, 16, 24, 32, 64, 128, 160 and 256 μg/mL (4 h exposure to the test item without S9-mix)
0, 16, 24, 32, 64, 128, 160 and 256 μg/mL (4 h exposure to the test item with S9-mix (2%))
0, 24, 32, 40, 64, 80, 100 and 128 μg/mL (24 h continuous exposure to the test item without S9-mix)

Justification: The selection of the maximum dose level for the Main Experiment was based on toxicity for the 24 h exposure group, and limited by the onset of precipitation in the 4 h exposure groups.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Acetone
- Test item preparation: The test item was immiscible in MEM at 50 mg/mL and DMSO at 250 and 500 mg/mL, but was miscible in acetone at 500 mg/mL in solubility checks performed in-house. Prior to each experiment, the test item was accurately weighed, dissolved in acetone and serial dilutions prepared. Due to the sensitivity of human lymphocytes to acetone, the formulations were prepared and dosed in 50 μL aliquots. The test item could not be formulated at 1000 mg/mL due to technical reasons, therefore, the maximum practical concentration was limited to 2500 μg/mL.
The test item was formulated within two hours of it being applied to the test system; it is assumed that the test item formulation was stable for this duration.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
acetone
True negative controls:
no
Positive controls:
yes
Positive control substance:
mitomycin C
other: Demecolcine
Remarks:
without S9 mix
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
acetone
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
with S9 mix
Details on test system and experimental conditions:
TEST SYSTEM:
For each experiment, sufficient whole blood was drawn from the peripheral circulation of a non-smoking volunteer (18-35) who had been previously screened for suitability. The volunteer had not knowingly been exposed to high levels of radiation or hazardous chemicals and had not knowingly recently suffered from a viral infection. Based on over 20 years in-house data for cell cycle times for lymphocytes using BrdU (bromodeoxyuridine) incorporation to assess the number of first, second and third division metaphase cells to calculate the average generation time (AGT) for human lymphocytes it is considered to be approximately 16 h. Therefore using this average the in-house exposure time for the experiments for 1.5 x AGT is 24 h.
The details of the donors used were:
Preliminary Toxicity Test: male, aged 29 years; Main Experiment: male, aged 32 years

METHOD OF APPLICATION:
Cell culture: Cells (whole blood cultures) were grown in Eagle's minimal essential medium with HEPES buffer (MEM), supplemented “in-house” with L-glutamine, penicillin/streptomycin, amphotericin B and 10% fetal bovine serum (FBS), at approximately 37 °C with 5% CO2 in humidified air. The lymphocytes of fresh heparinized whole blood were stimulated to divide by the addition of phytohaemagglutinin (PHA).

DURATION
- Exposure duration: 4 h (± S9) and 24 h continuous exposure (-S9) in preliminary toxicity test; 4 h (± S9) and 24 h continuous exposure (-S9) in main experiment
- At the end of the exposure period, the cell cultures were washed and then incubated for a further 24 h in the presence of Cytochalasin B.

SPINDLE INHIBITOR (cytogenetic assays): Prior to the mitosis (after exposure of the test substance) the chemical cytochalasin B (4.5 μg/mL) was added to the cultures.

STAIN (for cytogenetic assays): 5 % Giemsa for 5 minutes

NUMBER OF REPLICATIONS:
- Preliminary toxicity test: Single culture for test item and vehicle control
- Main test: Duplicate cultures per dose for test item, vehicle and positive controls

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED:
- At the end of the Cytochalasin B treatment period the cells were centrifuged, the culture medium was drawn off and discarded, and the cells resuspended in MEM. The cells were then treated with a mild hypotonic solution (0.0375 KCl) before being fixed with fresh methanol/glacial acetic acid (19:1 v/v). The fixative was changed at least three times and the cells stored at approximately 4 ºC prior to slide making. The lymphocytes were re-suspended in several mL of fresh fixative before centrifugation and re-suspension in a small amount of fixative. Several drops of this suspension were dropped onto clean, wet microscope slides and left to air dry. When the slides were dry they were stained in 5% Giemsa for 5 minutes, rinsed, dried and a cover slip applied using mounting medium.

NUMBER OF CELLS EVALUATED:
- Cytokinesis Block Proliferation Index (CBPI): A minimum of approximately 500 cells per culture were scored for the incidence of mononucleate, binucleate and multinucleate cells and the CBPI value expressed as a percentage of the vehicle controls. The CBPI indicates the number of cell cycles per cell during the period of exposure to Cytochalasin B.
- Scoring of Micronuclei:
The micronucleus frequency in 2000 binucleated cells was analyzed per concentration (1000 binucleated cells per culture, two cultures per concentration) except in the 4 h exposure group without S9-mix at 64 μg/mL where only 332 and 714 cells were assessed for micronuclei from the A and B cultures, respectively. This change to the number of cells assessed was necessitated by toxicity limiting the number of binucleate cells available for assessment, despite the apparent optimum toxicity of the dose level.
Cells with 1, 2 or more micronuclei were recorded as such but the primary analysis was on the combined data. Experiments with human lymphocytes have established a range of micronucleus frequencies acceptable for control cultures in normal volunteer donors.

CRITERIA FOR MICRONUCLEUS IDENTIFICATION:
- The criteria for identifying micronuclei were that they were round or oval in shape, non-refractile, not linked to the main nuclei and with a diameter that was approximately less than a third of the mean diameter of the main nuclei. Binucleate cells were selected for scoring if they had two nuclei of similar size with intact nuclear membranes situated in the same cytoplasmic boundary. The two nuclei could be attached by a fine nucleoplasmic bridge which was approximately no greater than one quarter of the nuclear diameter.

DETERMINATION OF CYTOTOXICITY
- Method: Cytotoxicity of test item in the lymphocyte cultures was determined using the cytokinesis-block proliferation index (CBPI index).
% Cytostasis = 100-100{(CBPIT – 1)/(CBPIC –1)}
CBPI = [(No. mononucleate cells) + (2 x No. binucleate cells) + (3 x No. multinucleate cells)] / [Total number of cells]
T = test substance treatment culture
C = vehicle control culture
Evaluation criteria:
Providing that all of the acceptability criteria are fulfilled, a test item is considered to be clearly negative if, in most/all of the experimental conditions examined:
1. None of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control.
2. There is no dose-related increase.
3. The results in all evaluated dose groups should be within the range of the laboratory historical control data.
Providing that all of the acceptability criteria are fulfilled, a test item may be considered to be clearly positive, if in any of the experimental conditions examined, there is one or more of the following applicable:
1. At least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control.
2. There is an increase which can be considered to be dose-related.
3. The results are substantially outside the range of the laboratory historical negative control data.
When all the criteria are met, the test item is considered able to induce chromosome breaks and/or gain or loss in this test system.
There is no requirement for verification of a clear positive or negative response.
In case the response is neither clearly negative nor clearly positive as described above or in order to assist in establishing the biological relevance of a result, the data should be evaluated by expert judgement and/or further investigations. The Study Director may make a judgement based on experience and the biological relevance of the data and any justification for acceptance of the data will be included in the report.
Statistics:
The frequency of binucleate cells with micronuclei was compared, where necessary, with the concurrent vehicle control value using the Chi-squared Test on observed numbers of cells with micronuclei. Other statistical analyses may be used if appropriate (Hoffman et al., 2003). A toxicologically significant response was recorded when the p value calculated from the statistical analysis of the frequency of binucleate cells with micronuclei was less than 0.05 and there was a dose-related increase in the frequency of binucleate cells with micronuclei which was reproducible.
Key result
Species / strain:
lymphocytes: human
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH and osmolality: There was no significant change in pH when the test item was dosed into media and the osmolality did not increase by more than 50 mOsm at the dose levels tested.
- Water solubility: None
- Precipitation: Yes

PRELIMINARY TOXICITY TEST:
- A precipitate of the test item was observed in the parallel blood-free cultures at the end of the exposure at and above 156.25 μg/mL in the 4 h exposure group without metabolic activation (S9), at and above 312.5 μg/mL in the 4 h exposure with metabolic activation in the 24 h continuous exposure groups.
- Hemolysis was observed following exposure to the test item at all of the dose levels in the absence of metabolic activation. In the 4-h exposure group with metabolic activation, hemolysis was observed at and above 19.53 μg/mL. Hemolysis is an indication of a toxic response to the erythrocytes and not indicative of any genotoxic response to the lymphocytes.
- Microscopic assessment of the slides prepared from the exposed cultures showed that binucleate cells were present at up to 2500 μg/mL in the 4 h exposure groups. Maximum exposure of the test item to the cells seemed to be at 312.5 μg/mL where cell quality was at its worse but improved with increasing concentration. The maximum dose with adequate numbers of binucleate cells present in the 24 h continuous exposure was 156.25 μg/mL.
The selection of the maximum dose level for the Main Experiment was based on toxicity for the 24 h exposure group, and limited by the onset of precipitation in the 4 h exposure groups.

NUMBER OF CELLS WITH MICRONUCLEI
Mean % binucleate cells with micronuclei:
4 h exposure without S9: 0.95, 0.40, 0.80, 0.45 and 1.01 at 0, 16, 24, 32 and 64 μg/mL
4 h exposure with S9: 0.40, 0.15, 0.65, 0.75 and 1.15 at 0, 16, 24, 32 and 64 μg/mL
24 h exposure without S9: 0.50, 0.45, 0.75, 0.60 and 0.70 at 0, 24, 32, 40 and 64 μg/mL

HISTORICAL CONTROL DATA (with ranges, means and standard deviation
- Vehicle historical control data:
4 h exposure without S9: 0-1.30 (0.38 ± 0.24)
4 h exposure with S9: 0.05-0.90 (0.40 ± 0.20)
24 h exposure without S9: 0.10-0.75 (0.40 ± 0.18)
- Positive control historical control data:
4 h exposure without S9 (Mitomycin C): 1.75-10.10 (5.03 ± 2.34)
4 h exposure with S9 (Cyclophosphamide): 1.55-6.60 (3.83 ± 1.42)
24 h exposure without S9 (Demecolcine): 2.25-7.20 (3.72 ± 1.03)

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Micronucleus Test – Main Experiment
- The qualitative assessment of the slides determined that the toxicity was marginally increased to that observed in the Preliminary Toxicity Test and that there were binucleate cells suitable for scoring at 64 μg/mL in all three exposure groups. Precipitate of test item was noted at 128 μg/mL in all three exposure groups.
Hemolysis was observed at all of the dose levels tested in all three exposure groups.
In the 4 h exposure groups, the qualitative observations confirmed a dose-related inhibition of CBPI and that 52% and 50% cytostasis was achieved at 64 μg/mL in the absence and presence of S9-mix, respectively. This indicated that optimum toxicity had been achieved for these dose groups (as specified in OECD Guideline 487). In the 24 h continuous exposure group, a dose-related inhibition of CBPI was observed and 67% cytostasis was achieved at 64 μg/mL. Whilst this figure exceeded the optimum toxicity level, it was selected for micronucleus analysis as no toxicity was observed at lower dose levels but excessive toxicity was observed above it. Therefore, the maximum dose level selected for analysis of binucleate cells was limited by toxicity in all three exposure groups.

See attached document

Conclusions:
The test item was considered to be non-clastogenic and non-aneugenic to human lymphocytes in vitro.
Executive summary:

In an in vitro micronucleus test performed according to OECD Guideline 487 and in compliance with GLP, cultured peripheral human lymphocytes were exposed to the test item in the presence and absence of a metabolic activation system. Three exposure conditions in a single experiment were used for the study using a 4 h exposure in the presence and absence of a standard metabolizing system (S9 at a 2% final concentration) and a 24 h exposure in the absence of metabolic activation. At the end of the exposure period, the cell cultures were washed and then incubated for a further 24 h in the presence of Cytochalasin B.

Preliminary Toxicity Test: 0, 9.77, 19.53, 39.06, 78.13, 156.25, 312.5, 625, 1250 and 2500 μg/mL (4 h exposure to the test item without or with S9-mix (2%); 24 h continuous exposure to the test item without S9-mix)

Main Experiment:

0, 16, 24, 32, 64, 128, 160 and 256 μg/mL (4 h exposure to the test item without S9-mix)

0, 16, 24, 32, 64, 128, 160 and 256 μg/mL (4 h exposure to the test item with S9-mix (2%))

0, 24, 32, 40, 64, 80, 100 and 128 μg/mL (24 h continuous exposure to the test item without S9-mix)

All vehicle (acetone) controls had frequencies of cells with micronuclei within the range expected for normal human lymphocytes. The positive control items induced statistically significant increases in the frequency of cells with micronuclei. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

The test item was toxic but did not induce any statistically significant increases in the frequency of cells with micronuclei, in any of the exposure groups, using a dose range that included a dose level that induced approximately 50% reduction in Cytokinesis Block Proliferation Index (CBPI).

 

Therefore, the test item was considered to be non-clastogenic and non-aneugenic to human lymphocytes in vitro.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

In a reverse gene mutation assay in bacteria, performed according to guideline OECD 471, a structurally related substance (whose main constituents are d-limonene, l-limonene and beta-phellandrene) diluted in Dimethylsulfoxide (DMSO) was tested in S. typhimurium TA1535, TA1537, TA100, TA98 and TA102 in the presence and the absence of mammalian metabolic activation (S9) using the direct plate incorporation or the preincubation method.

The positive controls induced the appropriate responses in the corresponding strains. The number of revertants in the vehicle controls was consistent with the historical data of the testing facility, and the number of revertants in the positive controls was higher than that of the vehicle controls (at least 2-fold increase for the TA 98, TA 100 and TA 102 strains and at least 3-fold increase for the TA 1535 and TA 1537 strains) and was consistent with the historical data of the testing facility. Therefore the study was considered valid.

During the preliminary test, the decrease in the number of revertant colonies and/or thinning of the bacterial lawn showed that the test item was cytotoxic for bacteria. Thus, the choice of the highest dose-level to be tested in the main test was based on the level of toxicity, according to the criteria specified in the international guidelines. During the main test, no induced revertant over background was observed in any strains of S. typhimurium whereas the cytotoxic dose-level was reached. Therefore, the test item did not induce any mutagenic activity in the bacterial reverse mutation test using Salmonella typhimurium.

 

In an in vitro micronucleus test performed according to guideline OECD 487 and in compliance with GLP, cultured peripheral human lymphocytes were exposed to the registered substance in the presence and absence of a metabolic activation system. Three exposure conditions in a single experiment were used for the study using a 4 h exposure in the presence and absence of a standard metabolizing system (S9 at a 2% final concentration) and a 24 h exposure in the absence of metabolic activation. At the end of the exposure period, the cell cultures were washed and then incubated for a further 24 h in the presence of Cytochalasin B.

Preliminary Toxicity Test: 0, 9.77, 19.53, 39.06, 78.13, 156.25, 312.5, 625, 1250 and 2500 μg/mL (4 h exposure to the test item without or with S9-mix (2%); 24 h continuous exposure to the test item without S9-mix)

Main Experiment:

0, 16, 24, 32, 64, 128, 160 and 256 μg/mL (4 h exposure to the test item without S9-mix)

0, 16, 24, 32, 64, 128, 160 and 256 μg/mL (4 h exposure to the test item with S9-mix (2%))

0, 24, 32, 40, 64, 80, 100 and 128 μg/mL (24 h continuous exposure to the test item without S9-mix)

All vehicle (acetone) controls had frequencies of cells with micronuclei within the range expected for normal human lymphocytes. The positive control items induced statistically significant increases in the frequency of cells with micronuclei. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

The registered substance was toxic but did not induce any statistically significant increases in the frequency of cells with micronuclei, in any of the exposure groups, using a dose range that included a dose level that induced approximately 50% reduction in Cytokinesis Block Proliferation Index (CBPI).

In an in vitro mammalian cell gene mutation test performed according to guideline OECD 476 and in compliance with GLP, Chinese hamster (V79) cells were exposed to the registered substance at the concentrations below.

Preliminary Cytotoxicity Test: 9.77, 19.53, 39.06, 78.13, 156.25, 312.5, 625, 1250 and 2500 μg/mL with and without S9

Mutagenicity Test – Main Experiment

4 h without S9: 0, 2, 4, 8, 10, 12, 16 and 20 μg/mL

4 h with S9 (2%): 0, 10, 20, 40,50, 60, 70 and 80 μg/mL

Vehicle and positive control groups were also included in each mutation test. Metabolic activation system used in this test was 2%; S9 fraction was prepared from liver homogenates of male Sprague Dawley rats treated with phenobarbital and β-naphthoflavone.

The vehicle (acetone) controls gave mutant frequencies within the range expected of V79 cells at the HPRT locus. The positive control treatments, both in the presence and absence of metabolic activation, gave significant increases in the mutant frequency indicating the satisfactory performance of the test and of the metabolizing system.

The maximum dose level selected for the main mutagenicity experiment was based on the toxicity seen in the preliminary cytotoxicity test and was 20 μg/mL in the absence of S9 and 80 μg/mL in the presence of S9.

The registered substance did not induce any significant increases in mutant frequency at any dose level, either with or without metabolic activation.

Justification for classification or non-classification

In an Ames test performed according to OECD Guideline 471 and in compliance with GLP, the read-across substance reaction mass of dl-limonene, alpha-, gamma- terpinenes, terpinolene was considered to be non mutagenic.

In an in vitro micronucleus test performed according to OECD Guideline 487 and in compliance with GLP, the registered substance did not induce micronuclei.

In an in vitro HPRT test performed according to OECD Guideline 476 and in compliance with GLP, the registered substance was considered to be non mutagenic.

According to these results the registered substance can be considered as non genotoxic according to the CLP Regulation (EC) No 1272/2008.