p-mentha-1,3-diene

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In vitro bacteria reverse mutation: Key study: Test method similar to the OECD Guideline 471. The test item alpha terpinene was found non-mutagenic in the Bacterial Reverse Mutation Test up to the highest tested concentration of 5000 µg/plate with four Salmonella typhimurium strains (TA100, TA98, TA97a and TA1535) in the presence or absence of the metabolic activation system.

In vitro mammalian chromosomal aberration/sister chromatid exchange: Weight of evidence and read-across approach: reliable experimental studies are available from the analogue substance d-limonene as well as tea tree oil (a mixture of some of the components, including 7.9% alpha terpinene) and thus a weight of evidence and read across approach was applied to fulfill this information requirement for alpha terpinene. All studies elicited a negative result and thus, based on weight of evidence and read-across approach, alpha terpinene is considered to be negative for cytogenicity to mammalian cells.

In vitro gene mutation in mammalian cells: Weight of evidence and read-across approach: two reliable experimental studies are available from the analogue substance d-limonene and thus a weight of evidence and read across approach was applied to fulfill this information requirement for alpha terpinene. Both studies were negative and thus, based on weight of evidence and read-across approach, alpha terpinene is considered to be non-mutagenic to mammalian cells.

Other studies: gamma terpinene was determined non-mutagenic in an unscheduled DNA synthesis assay in rat hepatocytes.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

Study performed similarly to OECD guideline 473 with minor deviations: no data on number of replicates; no data on karyotype stability and incubation temperature; only 2-h exposure with test substance with S9

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

yes

Remarks:

no data on number of replicates; no data on karyotype stability and incubation temperature; only 2-h exposure with test substance with S9

GLP compliance:

no

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

No data

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

- Source: Litton Bionetics Inc.
- Type and identity of media: McCoys 5A medium supplemented with antibiotics and 10% fetal calf serum
- Properly maintained: Yes; cells for experiments were thawed and grown in the medium at 37 °C using 5% CO2
- Periodically checked for Mycoplasma contamination: Yes

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9 fraction of livers from Aroclor 1254-induced male Sprague-Dawley rats (20 µL/mL)

Test concentrations with justification for top dose:

- Without S9: 0, 10, 30 or 100 µg/mL
- With S9: 0, 50, 150 or 500 µg/mL
The doses selected for the aberration trials were based on data from the SCE trials. Ten doses were selected which, generally, covered a narrower range than used in the SCE assay. The three highest doses with sufficient metaphase cells were scored for aberrations.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

without metabolic activation (5 µg/mL)

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

with metabolic activation (50 µg/mL)

Details on test system and experimental conditions:

METHOD OF APPLICATION: In medium

DURATION
- Exposure duration: 2 (+S9) or 8 (-S9) hours
- Fixation time (start of exposure up to fixation or harvest of cells): 10.5 (-S9) or 12 (+S9) hours

SPINDLE INHIBITOR (cytogenetic assays): Colcemid
STAIN (for cytogenetic assays): Giemsa

NUMBER OF CELLS EVALUATED: 100 cells/dose in vehicle control and treatment groups; 50 cells/dose in positive control groups. Cells were scored for simple (chromatid gaps and breaks, fragments, deletions, chromosome gaps and breaks, and double minutes), complex (interstitial deletions, triradials, quadriradials, rings and dicentrics) and other pulverized, polyploids, and endoreduplications) aberrations.

DETERMINATION OF CYTOTOXICITY
- Based on SCE trials (visual estimate of the confluency of each flask)

Evaluation criteria:

- If a trial had a positive trend and no significant doses, or if there was no trend and only one significant dose, the trial was judged equivocal;
- If a trial had significant trend and one significant dose it was judged weak positive;
- If the trial had two significant doses it was judged positive, whether or not a positive trend was obtained.
- If only one dose was significant and the increase over the control was P <0.0005 the trial was denoted weak positive*

Statistics:

- Data were evaluated for both trend and dose point increase over the solvent control.
- A binomial sampling assumption was used to evaluate an absolute increase in aberrations over the solvent control. Dose points with P values adjusted by Dunnett's method were considered significant if < 0.05, whereas a trend of P < 0.003 was significant.

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

cytotoxic above 100 (-S9) and 500 (+S9) µg/mL

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

ADDITIONAL INFORMATION ON CYTOTOXICITY: Cytotoxic above 100 (-S9) and 500 (+S9) µg/mL

Table 1: Results obtained in trial 1 (without S9)

 

Dose (µg/mL)	Cells	Percent cells with aberrations
		Total	Simple	Complex
0.0000	100	4.00	4.00	0.00
10.0000	100	2.00	2.00	0.00
30.0000	100	2.00	2.00	0.00
100.0000	100	6.00	6.00	0.00
Positive control - MMC
5.0000	50.	50.00	42.00	18.00
Trend statistic = 0.75E+00
Trend probability = 0.23E+00

 

Table 2: Results obtained in trial 1 (with S9)

 

Dose (µg/mL)	Cells	Percent cells with aberrations
		Total	Simple	Complex
0	100	4	3	1
50	100	0	0	0
150	100	4	4	0
500	100	5	5	0
Positive control - CPA
50	50	40	26	18
Trend statistic = 0.88E+00
Trend probability = 0.19E+00

Conclusions:

Under the test conditions, d-limonene is not considered as cytogenetic in CHO cells according to the CLP Regulation (EC) N° (1272-2008).

Executive summary:

In an in vitro mammalian chromosome aberration test performed similarly to OECD guideline 473, Chinese hamster Ovary (CHO) cells were exposed to d-limonene in McCoys 5A medium with and without metabolic activation [S9 fraction of livers from Aroclor 1254-induced male Sprague-Dawley rats (20 µL/mL)] at the following concentrations: Without S9: 0, 10, 30 and 100 µg/mL, and with S9: 0, 50, 150 and 500 µg/mL. Positive controls (mitomycin C at 5 µg/mL without S9 and cyclophosphamide at 50 µg/mL with S9) induced the appropriate response. Chromosome aberrations were not induced in treatment groups over background at any tested concentrations in the presence or absence of activation system. Under the test conditions, d-limonene is not considered as cytogenetic in CHO cells according to the criteria of the CLP Regulation (EC) N° (1272-2008).

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

abstract

Remarks:

Study performed similarly to OECD Guideline 476 with deviations: no data on test material purity, source and concentration units; no data on negative/positive controls; evaluation criteria not reported

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

yes

Remarks:

no data on test material purity, source and concentration units; no data on negative/positive controls; evaluation criteria not reported

GLP compliance:

not specified

Type of assay:

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

Target gene:

Thymidine kinase, TK +/- locus

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media: Fischer's medium containing 10% horse serum, antibiotics, glutamine, sodium pyruvate and Pluronic F68

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9 fraction of induced rat liver supplemented with cofactors (CORE)

Test concentrations with justification for top dose:

Up to 100 µg/mL

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

not specified

True negative controls:

not specified

Positive controls:

not specified

Positive control substance:

not specified

Remarks:

no data

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not applicable

Untreated negative controls validity:

not applicable

Positive controls validity:

not applicable

Remarks on result:

other: strain/cell type: -3.7.2C heterozygote

Conclusions:

Under the test conditions, d-limonene is not considered as mutagenic in L5178Y cells according to the criteria of the CLP Regulation (EC) N° (1272-2008).

Executive summary:

In an in vitro mammalian cell gene mutation test performed similarly to OECD Guideline 476, mouse lymphoma L5178Y TK+/- (-3.7.2C heterozygote) cells were exposed to d-limonene up to 100 µg or nL/mL in both the absence and presence of metabolic activation (S9 fraction of induced rat liver supplemented with cofactors). D-Limonene showed no substantial increases in mutant frequency up to the highest concentration tested in either presence or absence of S9 mix. Under the test conditions, d-limonene is not considered as mutagenic in L5178Y cells according to the criteria of the CLP Regulation (EC) N° (1272-2008).

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

Study performed similarly to OECD Guideline 476 with minor deviations: no data on maintenance of cell cultures and absence of mycoplasma

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

yes

Remarks:

no data on maintenance of cell cultures and absence of mycoplasma

Principles of method if other than guideline:

Not applicable

GLP compliance:

not specified

Type of assay:

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

Target gene:

Thymidine kinase, TK +/- locus

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Source of cells: National Toxicology Program's (NTP) chemical repository (Radian Corporation, Austin, USA)
- Type and identity of media: Fischer’s medium used for expression and cloning; horse serum used at 20% v/v for soft agar cloning

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9 fraction of male Fischer 344 rat liver induced with Aroclor-1254

Test concentrations with justification for top dose:

Without S9:
- Trial 1: 0, 10, 20, 30, 40, 50 and 60 nL/mL
- Trial 2: 0, 30, 40, 50, 60, 80 and 100 nL/mL
- Trial 3: 0, 5, 10, 20, 30, 40 and 50 nL/mL
- Trial 4: 0, 5, 10, 20, 30, 40, 50 and 60 nL/mL

With S9:
- Trail 1: 0, 10, 20, 30, 40, 50, 60 and 80 nL/mL
- Trail 2: 0, 10, 20, 30, 40, 50, 60 and 80 nL/mL
- Trail 3: 0, 30, 40, 50, 60, 80 and 100 nL/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: 1% ethanol

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

1% ethanol

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

without metabolic activation(5 nL/mL)

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

1% ethanol

True negative controls:

no

Positive controls:

yes

Positive control substance:

3-methylcholanthrene

Remarks:

with metabolic activation (2.5 µg/mL )

Details on test system and experimental conditions:

METHOD OF APPLICATION: In medium

DURATION
- Exposure duration: 4 hours at 37 °C
- Expression time (cells in growth medium): 48 hours at 37 °C
- Selection time (if incubation with a selection agent): 9-12 days at 37 °C

SELECTION AGENT (mutation assays): Trifluorothymidine (TFT)

NUMBER OF REPLICATIONS: Duplicate (at least)

NUMBER OF CELLS EVALUATED: 6 x 10^6 cells exposed to the test item, 3 x 10^6 cells to select mutant cells; 600 cells to determine cloning efficiency

DETERMINATION OF CYTOTOXICITY
- Method: Relative growth on Days 1 and 2, cloning efficiency and relative total growth

OTHER EXAMINATIONS:
- Colony sizing: Number of small and large mutant colonies were determined by recording TFT colony counts for increments of 0.2 on the colony size discriminator.

OTHER: Colonies were counted on an Artek 880 colony counter fitted with a 10-turn size discriminator.

Evaluation criteria:

Positive (+):
- Significant response for at least one of the three highest dose sets and a significant trend (P ≤ 0.05)

Questionable (?):
- Significant response for one of the three highest dose sets but no significant trend
- Significant trend but no significant dose set

Inconclusive (i):
- Significant response for a dose set other than one of the three highest but no significant trend
- No significant responses or trend, but the relative total growth is greater than 30% and higher toxicity can be attained

No response (=):
- No significant responses or trend, and the relative total growth is greater than 30% under conditions where a 1.5-fold increase in dose cause precipitation or where the 5 mg/mL (or 5 µL/mL) concentration limit is attained.

Negative (-):
- No significant responses or trend, and either the relative total growth is less than 30% or excessive toxicity occurs for a 1.5-fold higher dose.

Statistics:

Consistency among the mutant frequencies was analysed using chi-square test; acceptable cultures must be significant at P ≤ 0.05

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at or above 50 and 60 nL/mL (with and without S9, respectively)

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

Response in trials without S9:
- Trial 1: Inconclusive (i)
- Trial 2: Questionable (?)
- Trial 3 and 4: Negative (-)
- Overall response: Negative (-)

Response in trials with S9:
- Trial 1 and 2: Negative (-)
- Trial 3: Inconclusive (i)
- Overall response: Negative (-)


ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Without S9: Cytotoxicity was observed in one or more replicates tested at concentration of 50 nL/mL or above
- With S9: Cytotoxicity was observed in one or more replicates tested at concentration of 60 nL/mL or above

Table 1: Chemical-induced changes in the large and small classes of mutant colonies

 

Chemical treatment	Trial	Mutant colony count and CE						Mutant frequency				Mutant frequency change
		Treatment			Solvent control			Treatment		Solvent control		Difference
		L	S	CE	L	S	CE	L	S	L	S	L	S
50 nL/mL	WO 2	48	119	98	44	53	85	16	40	17	21	-1	19
50 nL/mL	S9 2	98	143	79	72	109	115	41	60	21	32	20	28

Conclusions:

Under the test conditions, d-limonene was not considered as mutagenic in mouse lymphoma L5178Y cells and does not need to be classified according to the criteria of the CLP Regulation (EC) N° (1272-2008).

Executive summary:

In an in vitro mammalian cell gene mutation test performed similarly to OECD guideline 476, mouse lymphoma L5178Y TK+/- cells were exposed to d-limonene in 1% ethanol in Fischer’s medium with and without metabolic activation (S9 fraction of male Fischer 344 rat liver induced with Aroclor-1254) at the following concentrations:

Without S9:

- Trial 1: 0, 10, 20, 30, 40, 50 and 60 nL/mL

- Trial 2: 0, 30, 40, 50, 60, 80 and 100 nL/mL

- Trial 3: 0, 5, 10, 20, 30, 40 and 50 nL/mL

- Trial 4: 0, 5, 10, 20, 30, 40, 50 and 60 nL/mL

With S9:

- Trial 1: 0, 10, 20, 30, 40, 50, 60 and 80 nL/mL

- Trial 2: 0, 10, 20, 30, 40, 50, 60 and 80 nL/mL

- Trial 3: 0, 30, 40, 50, 60, 80 and 100 nL/mL

 

Positive controls (methyl methanesulphonate at 5 nL/mL without S9 and 3-methylcholanthrene at 2.5 µg/mL with S9) induced the appropriate response. In experiment without S9, mutagenic responses in trials 1, 2, 3 and 4 were inconclusive, questionable, negative and negative, respectively. In experiment with S9, mutagenic responses in trials 1, 2 and 3 were negative, negative and inconclusive, respectively. Overall, d-limonene was not considered as mutagenic in either presence or absence of S9 mix. Cytotoxicity was observed in one or more replicates tested at or above 50 nL/mL.

 

Under the test conditions, d-limonene was not considered as mutagenic in mouse lymphoma L5178Y cells and does not need to be classified according to the criteria of the CLP Regulation (EC) N° (1272-2008).

Reference 4

Endpoint:

in vitro DNA damage and/or repair study

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

Study performed similarly to OECD guideline 479 with minor deviations: no data on number of replicates; no data on karyotype stability

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 479 (Genetic Toxicology: In Vitro Sister Chromatid Exchange Assay in Mammalian Cells)

Deviations:

yes

Remarks:

no data on number of replicates; no data on karyotype stability

GLP compliance:

no

Type of assay:

sister chromatid exchange assay in mammalian cells

Target gene:

No data

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

- Source: Litton Bionetics Inc.
- Type and identity of media: McCoys 5A medium supplemented with antibiotics and 10% fetal calf serum
- Properly maintained: Yes; cells for experiments were thawed and grown in the medium at 37 °C using 5% CO2
- Periodically checked for Mycoplasma contamination: Yes

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9 fraction of livers from Aroclor 1254-induced male Sprague-Dawley rats (20 µL/mL)

Test concentrations with justification for top dose:

Without S9:
- Trial 1: 0, 16.2, 54 or 162 µg/mL
- Trial 2: 0, 30, 50 or 100 µg/mL
- Trial 3: 0, 15, 30 or 50 µg/mL
With S9:
- Trial 1: 0, 16.2, 54 or 162 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

without metabolic activation (0.0015 or 0.01 µg/mL)

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

with metabolic activation (0.4 or 2.5 µg/mL)

Details on test system and experimental conditions:

METHOD OF APPLICATION: In medium

DURATION
- Exposure duration: 2 hours at 37 °C
- Expression time (cells in growth medium): 24 hours in presence of bromodeoxyuridine (BrdUrd): 10^-5 M
- Fixation time (start of exposure up to fixation or harvest of cells): 25-29 hours (standard harvest) or > 29 hours (delayed harvest)

SPINDLE INHIBITOR (cytogenetic assays): Colcemid, 0.1 or 0.4 µg/mL for 2-2.5 h

NUMBER OF CELLS EVALUATED: 50 cells/dose

DETERMINATION OF CYTOTOXICITY
- Method: Visual estimate of the confluency of each flask at the end of the treatment

Evaluation criteria:

- If a trial had a positive trend and no significant doses, or if there was no trend and only one significant dose, the trial was judged equivocal;
- If a trial had significant trend and one significant dose it was judged weak positive;
- If the trial had two significant doses it was judged positive, whether or not a positive trend was obtained.

Statistics:

- Data were evaluated for both trend and dose point increase over the solvent control.
- A trend of P < 0.005 or an individual dose with a 20% increase over the solvent control was considered significant.

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Remarks:

cytotoxic above 162 µg/mL

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

ADDITIONAL INFORMATION ON CYTOTOXICITY: Cytotoxic above 162 µg/mL

Table 1: Results obtained in trial 1 (without S9)

 

Dose (µg/mL)	Total chromosomes	Total SCE	SCE per cell
0	1050	389	7.78
16.2	1050	447	6.94
54	1051	463	9.26
162	1051	457	9.14
Positive control - MMC
0.0015	1048	701	14.02
0.0100	211	341	34.10
Trend statistic: 0.23E+01
Trend probability: 0.96E-02

 

Table 2: Results obtained in trial 2 (without S9)

 

Dose (µg/mL)	Total chromosomes	Total SCE	SCE per cell	Harvest time
0.0000	1049	366	7.32	26.50
30.0000	1049	407	8.14	26.50
50.0000	1048	405	8.10	30.50
100.0000	1041	475	9.50*	30.50
Positive control - MMC
0.0015	1046	476	9.52	26.50
0.0100	210	252	25.20	26.50
Trend statistic: 0.38E+01
Trend probability: 0.81E-04

* significant (20%) increase of SCE per chromosome over the control

 

Table 3: Results obtained in trial 3 (without S9)

 

Dose (µg/mL)	Total chromosomes	Total SCE	SCE per cell	Harvest time
0.0000	1048	345	6.90	26.50
15.0000	1049	343	6.86	26.50
30.0000	1048	349	6.98	26.50
50.0000	1046	406	8.12	30.50
Positive control - MMC
0.0015	1051	516	10.32	26.50
0.0100	209	230	23.00	26.50
Trend statistic: 0.22E+01
Trend probability: 0.15E-01

 

Table 4: Results obtained in trial 1 (with S9)

 

Dose (µg/mL)	Total chromosomes	Total SCE	SCE per cell
0.0000	1047	398	7.96
16.2000	1048	404	8.08
54.0000	1049	399	7.98
162.0000	1045	394	7.88
Positive control - CPA
0.4000	1046	620	12.40
2.5000	210	405	40.50
Trend statistic: -0.17E+00
Trend probability: 0.57E+00

Conclusions:

Under the test conditions, d-limonene is not considered as cytogenetic in CHO cells according to the criteria of the CLP Regulation (EC) N° (1272-2008).

Executive summary:

In an in vitro sister chromatid exchange assay performed similarly to OECD guideline 479, Chinese hamster Ovary (CHO) cells were exposed to d-limonene in McCoys 5A medium with and without metabolic activation [S9 fraction of livers from Aroclor 1254-induced male Sprague-Dawley rats (20 µL/mL)] at the following concentrations: Without S9: trial 1: 0, 16.2, 54 and 162 µg/mL; trial 2: 0, 30, 50 and 100 µg/mL and trial 3: 0, 15, 30 and 50 µg/mL. With S9: trial 1: 0, 16.2, 54 and 162 µg/mL. Clear increases in mean SCE/cell were induced by the positive control chemicals mitomycin C (without S-9) and cyclophosphamide (with S-9). In trial 2 (without S9), a significant linear trend and a significant increase in SCE/cell were observed at a concentration of 100 µg/mL. However, no significant increases and no significant linear trends were observed in trial 1 (with and without S9) and trial 3 (without S9). Under the test conditions, d-limonene is not considered as cytogenetic in CHO cells according to the criteria of the CLP Regulation (EC) N° (1272-2008).

Reference 5

Endpoint:

in vitro DNA damage and/or repair study

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 479 (Genetic Toxicology: In Vitro Sister Chromatid Exchange Assay in Mammalian Cells)

Deviations:

yes

Remarks:

test conducted only without metabolic activation

GLP compliance:

not specified

Type of assay:

sister chromatid exchange assay in mammalian cells

Target gene:

Not applicable

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Remarks:

(CHO K-1)

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: American Type Culture Collection, cloned in testing laboratory

MEDIA USED
- Type and identity of media including CO2 concentration if applicable:
Ham's F12 medium and humidified atmosphere with 5% CO2 at 37ºC.
The medium was supplemented with 10% fetal bovine serum, 50 IU/ml penicillin G, 50 µg/ml streptomycin sulfate and 2.5 µg/ml fungizon. Medium and all antibiotics were obtained from Flow Laboratories, Inc. (U.S.A.).

Metabolic activation:

without

Test concentrations with justification for top dose:

0, 10, 33.3, 100, 333, 1000 µM

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

no

Details on test system and experimental conditions:

METHOD OF APPLICATION: in culture medium. Treatment was done with cells in the log-phase. The cells were exposed to Mitomycin C (MMC) for 21 h, and washed twice with Hanks' balanced salt solution. Treated cells were cultured in the presence or absence of tested material for 1 cell cycle.
- Cell density at seeding (if applicable): CHO K-1 cells were seeded at a density of 0.5-1.0 x 10^6 cells/100-mm dish.

DURATION
- Exposure duration: 1 cell cycle (21 h)

SPINDLE INHIBITOR (cytogenetic assays): The cells were treated with colchicine for 2 h at a final concentration of 50 µg/ml.

STAIN (for cytogenetic assays): modified Giemsa procedure (Sakanishi and Takayama, 1977)

NUMBER OF REPLICATIONS: 3 independent experiments

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: BrdUrd (final concentration 5 µM) was added 2 cell cycles before fixation. After addition of BrdUrd, the cultures were incubated in total darkness and all operations were performed under a red safe light. Preparations were processed using a modified Giemsa procedure (Sakanishi and Takayama, 1977) and harlequin-stained chromosomes in 50 metaphases per culture were analyzed for SCEs.

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): 50 metaphases per culture

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; mitotic indices were determined and given as numbers of mitotic cells per 1000 cells. In addition, the numbers of cells which completed 2 cell cycles (M2) or less than 2 cycles (MI) were determined.

Statistics:

The SCE data were statistically analyzed using Student's t-test.

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 1000 µM with and without MMC treatment

Untreated negative controls validity:

not examined

Positive controls validity:

not examined

Table 1. Effect of components of plant essence on MMC-induced SCEs

Component	MMC treatment (0.15 µM)	Mean frequency of SCEs
		Concentration (µM) of components
		0	10	33.3	100	333	1000
d-(+)-limonene	+	81.7	81.1	83.8	83.7	T
	-	9.0	8.9	9.1	9.0	T

Cells treated with 0.15 µM MMC for 21 h were post-treated with test item at the indicated doses for 21 h.

Each value represents the mean of 3 independent experiments.

T, toxic; -, not tested.

Conclusions:

d-limonene did not induce sister chromatid exchanges in CHO cells tested without metabolic activation.

Executive summary:

D-limonene was tested on sister chromatid exchange assay in cultured Chinese hamster ovary CHO-K1 cells without metabolic activation using an method comparable to OECD guideline 479. 3 independent experiments were conducted with and without initial induction during 1 cell cycle (21 h) of Mitomycin C (MMC) and post treatment during 1 cell cycle (21 h) of tested material at concentrations of 0, 10, 33.3, 100, 333, 1000 µM. The tested material was found toxic at the dose of 333 μM. Under these test conditions, d-limonene did not induce sister chromatid exchanges in CHO cells at any of the doses tested.

Reference 6

Endpoint:

in vitro DNA damage and/or repair study

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

abstract

Remarks:

Data published in a peer reviewed journal. Original study report not available.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 482 (Genetic Toxicology: DNA Damage and Repair, Unscheduled DNA Synthesis in Mammalian Cells In Vitro)

Deviations:

yes

Remarks:

no data on replications

GLP compliance:

not specified

Type of assay:

other: unscheduled DNA synthesis (UDS)

Target gene:

Not applicable

Species / strain / cell type:

hepatocytes: Rat/Fischer and Sprague Dawley adult male

Metabolic activation:

without

Test concentrations with justification for top dose:

Up to 30 μg/ml

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: no data

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium; Livers were perfused in situ with 0.5 mM EDTA in HEPES buffer (pH 7.2) for four minutes. Cultures of rat liver hepatocytes were incubated with the test material for 18-20 hours.

DURATION
- Exposure duration: 18-20 h

NUMBER OF CELLS EVALUATED: 75-150 cells were analyzed for each dose level.

Evaluation criteria:

UDS was measured by electronically counting nuclear grains and subtracting the average number of grains in 3 adjacent nuclear sized cytoplasmic areas.
The test was considered positive if an increase in net nuclear grain count of at least six grains per nucleus above the solvent control and/or an increase in the percent of nuclei with at least 6 net grains to more than 10% above the negative control value.

Key result

Species / strain:

hepatocytes: Rat/Fischer and Sprague Dawley adult male

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Remarks:

the positive induced significant increases in the mean number of net nuclear grain counts compared to the solvent control.

Gamma terpinene did not cause a significant increase in UDS as measured by the mean number of net nuclear grain counts at any dose level.

Conclusions:

Gamma terpinene was not mutagenic based on the results of the rat hepatocyte unscheduled DNA synthesis assay.

Executive summary:

Gamma terpinene was tested on the rat hepatocyte unscheduled DNA synthesis assay following the OECD Guideline 482. Cultures of rat liver hepatocytes were incubated with the test material for 18-20 hours at concentrations of up to 30 μg/ml without metabolic activation. The tested material did not cause a significant increase in the mean number of net nuclear grain counts compared to the control at any dose level. Thus, gamma terpinene is considered to be negative in the unscheduled DNA synthesis assay.

Reference 7

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

Study performed according to OECD guideline 473.

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

yes

Remarks:

only tested without metabolic activation

GLP compliance:

not specified

Remarks:

(no data reported)

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

No data

Species / strain / cell type:

lymphocytes: Human

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: Peripheral venous human blood
- Suitability of cells: Current study was approved by the Ethics Committee of the Universidade Estadual de Maringá, Maringá PR Brazil.
- Sex, age and number of blood donors if applicable: two healthy males and one female aged 25 years, non-smoking, non-alcoholic, not under drug therapy and with no recent history of exposure to mutagens.
- Whether whole blood or separated lymphocytes were used if applicable: yes, separated by centrifugation at 1100 rpm for 5 min.

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: RPMI 1640 medium, supplemented with 15% fetal calf serum, 1% L-glutamine 200 mM and 2% phytohemagglutinin.
- Properly maintained: yes

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

without

Test concentrations with justification for top dose:

95 μg/ml, 182 μg/ml and 365 μg/ml.
4 concentrations of TTO (95 μg/ml, 182 μg/ml, 365 μg/ml and 548 μg/ml) were evaluated by the mitotic index (MI). At 548 μg/ml a cytotoxicity of approx. 80% was obtained when compared to the negative control. Thus, the TTO concentrations selected were 95 μg/ml, 182 μg/ml and 365 μg/m which produced cytotoxicity ranging approximately 9–40% when compared to the negative control.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: none

Untreated negative controls:

yes

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

0.1 µg/mL

Details on test system and experimental conditions:

METHOD OF APPLICATION: In medium; The lymphocyte cultures were incubated at 37ºC, 5.0% CO2 for 72 h. At 48 h incubation, TTO and mitomycin C were added to each culture individually.

DURATION
- Preincubation period: 48 h
- Exposure duration: 24 h

SPINDLE INHIBITOR (cytogenetic assays): Colchicine

STAIN (for cytogenetic assays): 5% Giemsa

NUMBER OF REPLICATIONS: 2

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: After 72 h incubation, the cells from the culture were treated with a hypotonic solution (75 mM KCl) for 20 min at 37ºC and fixed with a cold solution of methanol:glacial acetic acid (3:1 v/v). The cells were fixed with two changes of fixatives. Slides were prepared for microscopic analysis by dripping 3–4 drops of the pre-fixed lymphocyte suspension from a distance of 30 cm, dried for 5 days at 22ºC and stained with 5% Giemsa (pH 6.8 Sorensen's buffer).

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): 400 well-spread metaphases (200 metaphases per dose and per replicate)

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: mitotic index
- Any supplementary information relevant to cytotoxicity: level of cytotoxicity was determined by the reduction in mitotic index (MI) when compared to that in negative control, with the highest oil concentration producing 50–60% cytotoxicity. MI was calculated by the number of dividing cells/total number of the cells x 100 (Chandrasekaran et al., 2009).

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: yes

Evaluation criteria:

Results were judged as follows:
negative if the frequency of chromosomal aberrations was <5%; inconclusive if the frequency of chromosomal aberrations was ≥5% but <10%; and positive if the frequency of chromosomal aberrations was ≥10% (Maenosono et al., 2009).

Statistics:

Results were analyzed statistically (Z-test, p <0.05).

Key result

Species / strain:

lymphocytes: human

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not applicable

Untreated negative controls validity:

valid

Positive controls validity:

valid

Table 1. Effect of TTO on the mitotic index (MI) in human lymphocytes.

TTO concentrations(μg/ml)	MI ± SD (%)
0	3.3 ± 0.948
95	2.9 ± 0.360
182	3.0 ± 0.665
365	2.2 ± 0.450
548	0.7 ± 0.776 *
positive control (a)	2.2 ± 0.608

a Positive control: mitomycin C (0.1 µg/ml).

* Significantly different from negative control (Kruskal–Wallis test, p< 0.05).

Table 2. Chromosome analysis of human lymphocytes treated with TTO

Compounds	Concentrations (μg/ml)	Frequency of chromosomal aberrations (%)
		Cog	Ceg	Cag	Cob	Ceb	Cab	Frag	Rear	Endo	Total (a)	Judge (b)
None	0	0	0	0	0	0	0	0	0	0	0	-
Mytomycin C	0.1	0.25	1	1	5.25	2.75	10.5	0	0	0.5	19 *	+
TTO	95	0	0.25	0.5	0	0	0	0	0	0.75	0.75	-
	182	0	0.75	0.5	0.25	0	0.75	0	0	0.75	1.75	-
	365	0	0	0.25	0.25	0	1	0	0	0.25	1.5	-

Cog: chromosome gap; Ceg: centromeric gap; Cag: chromatid gap; Cob: chromosome break; Ceb: centromeric break; Cab: chromatid break; Frag: fragment; Rear: rearrangement; Endo: endorreduplication. Four hundred metaphases were scored for each treatment.

a Cog, Ceg and Cag, according to OECD guideline no. 473 (1997), are classified as achromatic lesions and therefore should not be included in the total chromosomal aberrations.

b According to Maenosono et al. (2009).

* Significantly different from negative control (Z-test, p < 0.05).

Conclusions:

Under the test conditions, the test substance was found negative in a chromosomal aberration test using human blood lymphocytes.

Executive summary:

In an in vitro mammalian chromosomal aberration test performed accordingly with OECD guideline 473, human blood lymphocytes cells were exposed to the test substance tea tree oil (TTO) composed of the following main components as identified by GC/MS and NMR: Terpinen-4-ol (42.8%), γ-terpinene (20.4%), p-cymene (9.6%), α-terpinene (7.9%), 1,8-cineole (3%), α-terpineol (2,8%) and α-pinene (2.4%). Based on the cytotoxicity observed in a mitotic index (MI), the concentrations used in the test were 95, 182 and 365 μg/ml. Untreated culture was used as negative control and mitomycin C at 0.1 µg/mL was used as positive control. None of the tested TTO concentrations caused significant differences in the frequencies of both structural and numerical (polyploidy and endorreduplication) chromosomal aberrations when compared to those of the negative control. Thus, it was concluded that TTO is not genotoxic in in vitro mammalian cells.

Reference 8

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2005

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

Salmonella/microsome assay performed by the plate incorporation procedure as described by Maron and Ames, 1983, and Gomes-Carneiro et al., 1998

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Test material: alpha terpinene
- Source of test material: Sigma Chemical Co. (St Louis, MO, USA)
- Purity: 95% or higher

Target gene:

The histidine-dependent Salmonella typhimurium strains were used.

Species / strain / cell type:

other: Salmonella typhimurium TA100, TA98, TA97a and TA1535

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

Extrinsic metabolic system (rat liver S9 fraction induced by Aroclor 1254

Test concentrations with justification for top dose:

Concentration range 0-5000 µg/plate: 0, 100, 250, 400, 500, 600, 700, 800, 900, 1000, 1250, 1500, 2000, 2500 and 5000 µg/plate

Vehicle / solvent:

- Vehicle / solvent used: ethanol

Negative solvent / vehicle controls:

yes

Remarks:

100 µl

Positive controls:

yes

Positive control substance:

2-acetylaminofluorene

4-nitroquinoline-N-oxide

2-nitrofluorene

sodium azide

other: 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

BACTERIAL STRAINS:
- TA100, TA98, TA97a and TA1535 strains of S. typhimurium
- Source: University of California, Berkeley, USA
- An inoculum (200 µl) of a thawed permanent culture was added to 20 ml of Nutrient Broth No.2 and incubated at 37°C with shaking until a concentration of approximately 1-2x10^9 bacteria per ml was obtained.

METABOLIC ACTIVATION SYSTEM (S9 mixture):
- Lyophilized rat liver S9 fraction induced by Aroclor 1254 was purchased from Moltox (Molecular Toxicology Inc., Boone, NC, USA).
- S9 mixture: 7.0ml of ultrapure water; 10.5ml of 200mM sodium phosphate bu¿er pH7.4; 0.84ml of 100mM NADP solution; 0.105ml of 1M glucose-6-phosphate; 0.42ml of 1.65M KCl + 0.4M MgCl2 salt solution; and 2.1ml of lyophilized S9 fraction reconstituted with water provided by a MilliQ water puri¿cation system.

SALMONELLA/MICROSOME ASSAY:
The following was mixed with 2 ml of top agar which poured onto a minimal glucose plate:
- 100 µl of an overnight grown culture
- 100 µl test substance (diluted in analytical grade ethanol, Vetec)
- the negative (solvent) control or positive control
- 500 µl sodium-phosphate buffer or S9 mix

- The plates were incubated at 37°C for 72-hr in the dark and then scored for revertant his+ bacterial colonies
- Each determination was made in triplicate and at least two independent experiments were carried out

POSITIVE CONTROLS:
- tested in concentration range 0-1500 µg/plate: 0, 1, 5, 10, 25, 50, 75, 100, 200, 250, 300, 400, 500, 600, 700, 750, 800, 900 and 1000 µg/plate

Rationale for test conditions:

The assay is specifically designed to determine the ability of alpha terpinene to produce genetic damage that leads to gene mutations.

Evaluation criteria:

Toxicity was apparent either as a reduction in the number of his+ revertant bacterial colonies and/or as a change in the auxotrophic background growth (i.e. the background lawn). The criteria for a positive mutagenic response was a clear dose-dependent increase in the number of revertants within the non-toxic range.

Statistics:

Values recorded are means ± SD of three plates.

Key result

Species / strain:

other: Salmonella typhimurium TA100, TA98, TA97a and TA1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Addition of the S9-mixture seemed to have reduced toxicity.

Remarks on result:

other: not mutagenic

Table1: First assay testing of alpha terpinene (1-isopropyl-4-methyl-1,3-cyclohexadiene) in the Salmonella/microsome assay
Dose (µg/plate)		Number of revertants (Mean ± SD)
		TA100		TA98		TA97a		TA1535
		-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
a-terpinene	5000	-	-	-	43 ± 36*	-	45 ± 17*	2 ± 3*	6 ± 4*
	2500	-	-	-	65 ± 16*	-	108 ± 22*	0 ± 0*	14 ± 5*
	2000	-	104 ± 28*	13 ± 13*	-	-	-	-	-
	1500	-	-	23 ± 12*	-	-	-	-	-
	1250	-	73 ± 35*	19 ± 14*	-	-	-	-	-
	1000	-	51 ± 35*	9 ± 12*	62 ± 12	-	144 ± 17*	12 ± 1*	15 ± 6*
	750	-	-	35 ± 8*	-	-	-	-	-
	500	-	84 ± 18	20 ± 17	59 ± 2	110 ± 16*	142 ± 5	8 ± 4*	17 ± 2
	400	-	-	-	-	79 ± 2*	-	-	-
	300	-	-	-	-	83 ± 10*	-	-	-
	250	-	-	-	67 ± 4	-	146 ± 19	-	-
	200	-	-	-	-	101 ± 8*	-	-	-
	100	102 ± 8*	116 ± 35*	-	57 ± 4	97 ± 17	185 ± 28	16 ± 2	17 ± 4
	75	128 ± 19	-	-	-	-	-	-	-
	50	118 ± 2	-	-	-	141 ± 6	-	-	-
	35	147 ± 19	-	-	-	-	-	-	-
	20	139 ± 8	-	-	-	-	-	-	-
	10	152 ± 36	-	-	-	-	-	-	-
Negative control (100 µl)		167 ± 10	206	43 ± 12	58 ± 8	150 ± 2	202 ± 28	18 ± 4	27 ± 5
Positive control (1 µg/plate)		SA	2-AA	2-NF	2AA	4-NQNO	2AF	SA	2AA
		804 ± 59	614 ± 97	173 ± 35	430 ± 15	845 ± 17	928 ± 24	506 ± 8	154 ± 4

Negative control: 100 µl ethanol. Positive controls: sodium azide (SA), 2-aminoanthracene (2-AA), 2-nitrofluorene (2-NF), 4-nitroquinoline-N-oxide (4-NQNO), 2-aminofluorene (2-AF). Dose not tested: (-). Toxicity apparent as an alteration of the background lawn: (*). Values are means ± SD of three plates.

Table 2. Second assay testing of alpha terpinene in the Salmonella/microsome assay
Dose (µg/plate)		Number of revertants (Mean±SD)
		TA100		TA98		TA97a		TA1535
		-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
Alpha terpinene	1500	-	-	-	50 ± 2*	-	-	-	-
	750	-	-	-	58 ± 14	-	-	-	19 ± 6*
	500	-	-	34 ± 6*	58 ± 8	-	172 ± 4	0 ± 0*	21 ± 4
	250	-	-	25 ± 5*	58 ± 4	-	220 ± 8	2 ± 3*	16 ± 2
	200	-	-	-	-	107 ± 20*	-	-	-
	100	-	-	36 ± 12	56 ± 8	109 ± 13	267 ± 8	4 ± 3	19 ± 5
	75	149 ± 7	120 ± 3	-	-	138 ± 20	-	-	-
	50	133	130 ± 12	41 ± 6	60 ± 3	123 ± 16	226 ± 14	20 ± 4	26 ± 2
	35	170 ± 15	146 ± 19	-	-	-	-	-	-
	25	-	-	37± 2	-	141 ± 14	194 ± 4	22 ± 5	22 ± 7
	20	162 ± 12	132 ± 39	-	-	-	-	-	-
	10	170 ± 6	140 ± 1	-	-	158 ± 2	-	21 ± 5	-
	5	154 ± 15	145 ± 18	-	-	158 ± 8	-	28 ± 7	-
Negative control (100 µl)		158 ± 26	148 ± 19	37 ± 7	47 ± 1	152 ± 13	190 ± 4	30 ± 4	23 ± 5
Positive control (1 µg/plate)		SA	2-AA	2-NF	2AA	4-NQNO	2AF	SA	2AA
		449 ± 32	774 ± 54	88 ± 8	183 ± 22	752 ± 87	841 ± 24	696 ± 81	182 ± 10

Negative control: 100 µl ethanol. Positive controls: sodium azide (SA), 2-aminoanthracene (2-AA), 2-nitrofluorene (2-NF), 4-nitroquinoline-N-oxide (4-NQNO), 2-aminofluorene (2-AF). Dose not tested: (-). Toxicity apparent as an alteration of the background lawn: (*). Values are means ± SD of three plates.

Conclusions:

Based on the results of this study, alpha terpinene was shown to be not mutagenic in the Ames assay in the four Salmonella typhimurium strains tested, either in the presence or absence of the metabolic activation system.

Executive summary:

The aim of this study performed using the plate incorporation procedure as described by Maron and Ames, 1983, and Gomes-Carneiro et al., 1998, was to investigate the genotoxicity of alpha terpinene when tested at different concentrations in four Salmonella typhimurium strains in the Salmonella/microsome assay, with and without the addition of an extrinsic metabolic activation system.

200 µl of four S. typhimurium strains (TA100, TA98, TA97a and TA1535) were incubated separately with 20 ml Nutrient Broth 2 at 37 °C with shaking until approximately 1-2x10^9 bacteria per ml were obtained. 100 µl of overnight culture, 100 µl alpha- terpinene (diluted in ethanol)/ negative control /positive control and 500 µl sodium phosphate buffer or S9-mix were mixed with 2ml top agar and then poured into a minimal glucose plate. The plates were incubated in the dark at 37°C for 72-h.

The negative control used was ethanol and the positive controls were TA100/-S9 and TA1535/-S9, SA (1 µg/plate); TA100/+S9 and TA1535/-S9, 2AA (1µg/plate); TA98/-S9, 2-NF (1,5lµ/plate); TA98/+S9; 2AA (0,5µg/plate); TA97a/-S9, 4-NQNO (1µg/plate); TA97a/+S9, 2AF (10µg/plate).

Alpha terpinene was tested at several concentrations in the range of 0-5000 µg/plate and the positive controls were tested in the range of 0-1500 µg/plate.

Based on the results of this study, alpha terpinene did not induce any increase in the number of his+ revertant colonies over the negative control values in the four S. typhimurium strains tested, either in the presence or absence of the metabolic activation system. The results also suggest that the addition of the S9-mixture seemed to have induced a reduced toxicity.

Based on the results of this study, it was concluded that alpha terpinene was not mutagenic in the Salmonella/microsome assay.

Reference 9

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH
The analogue substance (R)-p-mentha-1,8-diene (d-limonene) which shares the same functional groups with the substance p-mentha-1,3-diene (alpha terpinene) also has comparable values for the relevant molecular properties.
See attached the reporting format.

Reason / purpose for cross-reference:

read-across source

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Read across from an analogue for which cytotoxic was above 100 (-S9) and 500 (+S9) µg/mL.

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other: Read-across from an analogue determined to be negative in the chromosome aberration assay.

Conclusions:

Based on the read-across approach from the analogue d-limonene, alpha terpinene is not considered as cytogenetic in CHO cells according to the CLP Regulation (EC) N° (1272-2008).

Executive summary:

In an in vitro mammalian chromosome aberration test performed similarly to OECD guideline 473, Chinese hamster Ovary (CHO) cells were exposed to d-limonene in McCoys 5A medium with and without metabolic activation [S9 fraction of livers from Aroclor 1254-induced male Sprague-Dawley rats (20 µL/mL)] at the following concentrations: Without S9: 0, 10, 30 and 100 µg/mL, and with S9: 0, 50, 150 and 500 µg/mL. Positive controls (mitomycin C at 5 µg/mL without S9 and cyclophosphamide at 50 µg/mL with S9) induced the appropriate response. Chromosome aberrations were not induced in treatment groups over background at any tested concentrations in the presence or absence of activation system. Based on these results, the read-across was applied and alpha terpinene is not considered as cytogenetic in CHO cells according to the criteria of the CLP Regulation (EC) N° (1272-2008).

Reference 10

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH
The analogue substance (R)-p-mentha-1,8-diene (d-limonene) which shares the same functional groups with the substance p-mentha-1,3-diene (alpha terpinene) also has comparable values for the relevant molecular properties.
See attached the reporting format.

Reason / purpose for cross-reference:

read-across source

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not applicable

Untreated negative controls validity:

not applicable

Positive controls validity:

not applicable

Remarks on result:

other: read-across from an analogue determined to be not mutagenic in mammalian cell assay.

Conclusions:

Based on the read-across approach from the analogue d-limonene, alpha terpinene is not considered as mutagenic in L5178Y cells according to the criteria of the CLP Regulation (EC) N° (1272-2008).

Executive summary:

In an in vitro mammalian cell gene mutation test performed similarly to OECD Guideline 476, mouse lymphoma L5178Y TK+/- (-3.7.2C heterozygote) cells were exposed to d-limonene up to 100 µg or nL/mL in both the absence and presence of metabolic activation (S9 fraction of induced rat liver supplemented with cofactors). D-Limonene showed no substantial increases in mutant frequency up to the highest concentration tested in either presence or absence of S9 mix. Based on these results, the read-across was applied and alpha terpinene is not considered as mutagenic in L5178Y cells according to the criteria of the CLP Regulation (EC) N° (1272-2008).

Reference 11

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH
The analogue substance (R)-p-mentha-1,8-diene (d-limonene) which shares the same functional groups with the substance p-mentha-1,3-diene (alpha terpinene) also has comparable values for the relevant molecular properties.
See attached the reporting format.

Reason / purpose for cross-reference:

read-across source

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Read across from an analogue for which cytotoxicity was found at or above 50 and 60 nL/mL (with and without S9, respectively).

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other: read-across from an analogue determined to be not mutagenic in mammalian cell assay.

Conclusions:

Based on the read-across approach from the analogue d-limonene, alpha terpinene should not be considered as mutagenic in mouse lymphoma L5178Y cells and does not need to be classified according to the criteria of the CLP Regulation (EC) N° (1272-2008).

Executive summary:

In an in vitro mammalian cell gene mutation test performed similarly to OECD guideline 476, mouse lymphoma L5178Y TK+/- cells were exposed to d-limonene in 1% ethanol in Fischer’s medium with and without metabolic activation (S9 fraction of male Fischer 344 rat liver induced with Aroclor-1254) at the following concentrations:

Without S9:

- Trial 1: 0, 10, 20, 30, 40, 50 and 60 nL/mL

- Trial 2: 0, 30, 40, 50, 60, 80 and 100 nL/mL

- Trial 3: 0, 5, 10, 20, 30, 40 and 50 nL/mL

- Trial 4: 0, 5, 10, 20, 30, 40, 50 and 60 nL/mL

With S9:

- Trial 1: 0, 10, 20, 30, 40, 50, 60 and 80 nL/mL

- Trial 2: 0, 10, 20, 30, 40, 50, 60 and 80 nL/mL

- Trial 3: 0, 30, 40, 50, 60, 80 and 100 nL/mL

 

Positive controls (methyl methanesulphonate at 5 nL/mL without S9 and 3-methylcholanthrene at 2.5 µg/mL with S9) induced the appropriate response. In experiment without S9, mutagenic responses in trials 1, 2, 3 and 4 were inconclusive, questionable, negative and negative, respectively. In experiment with S9, mutagenic responses in trials 1, 2 and 3 were negative, negative and inconclusive, respectively. Overall, d-limonene was not considered as mutagenic in either presence or absence of S9 mix. Cytotoxicity was observed in one or more replicates tested at or above 50 nL/mL.

 

Based on these results, the read-across was applied and alpha terpinene should not be considered as mutagenic in mouse lymphoma L5178Y cells and does not need to be classified according to the criteria of the CLP Regulation (EC) N° (1272-2008).

Reference 12

Endpoint:

in vitro DNA damage and/or repair study

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH
The analogue substance (R)-p-mentha-1,8-diene (d-limonene) which shares the same functional groups with the substance p-mentha-1,3-diene (alpha terpinene) also has comparable values for the relevant molecular properties.
See attached the reporting format.

Reason / purpose for cross-reference:

read-across source

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Remarks:

Read across from an analogue for which cytotoxic is above 162 µg/mL.

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other: Read-across from an analogue determined to be negative in the SCE assay.

Conclusions:

Based on the read-across approach from the analogue d-limonene, alpha terpinene is not considered as cytogenetic in CHO cells according to the criteria of the CLP Regulation (EC) N° (1272-2008).

Executive summary:

In an in vitro sister chromatid exchange assay performed similarly to OECD guideline 479, Chinese hamster Ovary (CHO) cells were exposed to d-limonene in McCoys 5A medium with and without metabolic activation [S9 fraction of livers from Aroclor 1254-induced male Sprague-Dawley rats (20 µL/mL)] at the following concentrations: Without S9: trial 1: 0, 16.2, 54 and 162 µg/mL; trial 2: 0, 30, 50 and 100 µg/mL and trial 3: 0, 15, 30 and 50 µg/mL. With S9: trial 1: 0, 16.2, 54 and 162 µg/mL. Clear increases in mean SCE/cell were induced by the positive control chemicals mitomycin C (without S-9) and cyclophosphamide (with S-9). In trial 2 (without S9), a significant linear trend and a significant increase in SCE/cell were observed at a concentration of 100 µg/mL. However, no significant increases and no significant linear trends were observed in trial 1 (with and without S9) and trial 3 (without S9). Based on these results, the read-across was applied and alpha terpinene is not considered as cytogenetic in CHO cells according to the criteria of the CLP Regulation (EC) N° (1272-2008).

Reference 13

Endpoint:

in vitro DNA damage and/or repair study

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH
The analogue substance (R)-p-mentha-1,8-diene (d-limonene) which shares the same functional groups with the substance p-mentha-1,3-diene (alpha terpinene) also has comparable values for the relevant molecular properties.
See attached the reporting format.

Reason / purpose for cross-reference:

read-across source

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Read across from an analogue for which cytotoxicity was found at 1000 µM with and without MMC treatment.

Untreated negative controls validity:

not examined

Positive controls validity:

not examined

Remarks on result:

other: Read-across from an analogue determined to be negative in the SCE assay.

Conclusions:

Based on the read-across approach from the analogue d-limonene, alpha terpinene is considered to be negative in the SCE assay in CHO cells without metabolic activation.

Executive summary:

D-limonene was tested on sister chromatid exchange assay in cultured Chinese hamster ovary CHO-K1 cells without metabolic activation using an method comparable to OECD guideline 479. 3 independent experiments were conducted with and without initial induction during 1 cell cycle (21 h) of Mitomycin C (MMC) and post treatment during 1 cell cycle (21 h) of tested material at concentrations of 0, 10, 33.3, 100, 333, 1000 µM. The tested material was found toxic at the dose of 333 μM. Based on these results, the read-across was applied and alpha terpinene is considered to be negative in the SCE assay in CHO cells without metabolic activation.

Reference 14

Endpoint:

in vitro DNA damage and/or repair study

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH
The analogue substance gamma terpinene and the target substance alpha terpinene are two enantiomeric forms of terpinene, therefore they share the same functional groups and also have comparable values for the relevant molecular properties.
See attached the reporting format.

Reason / purpose for cross-reference:

read-across source

Key result

Species / strain:

hepatocytes: Rat/Fischer and Sprague Dawley adult male

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Remarks:

the positive induced significant increases in the mean number of net nuclear grain counts compared to the solvent control.

Remarks on result:

other: read-across from an analogue determined to be negative in the UDS assay.

Conclusions:

Based on the read-across approach from the analogue gamma terpinene, alpha terpinene is not considered mutagenic based on the results of the rat hepatocyte unscheduled DNA synthesis assay.

Executive summary:

Gamma terpinene was tested on the rat hepatocyte unscheduled DNA synthesis assay following the OECD Guideline 482. Cultures of rat liver hepatocytes were incubated with the test material for 18-20 hours at concentrations of up to 30 μg/ml without metabolic activation. The tested material did not cause a significant increase in the mean number of net nuclear grain counts compared to the control at any dose level. Based on these results, the read-across was applied and alpha terpinene is considered to be negative in the unscheduled DNA synthesis assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

Genetic toxicity in vivo: Weight of evidence and read-across approach: reliable experimental in vivo studies are available from the analogue substance d-limonene and thus a weight of evidence and read across approach was applied to strengthen the in vitro information used for alpha terpinene. All studies elicited a negative result and thus, based on weight of evidence and read-across approach, alpha terpinene is considered to not have a potential for in vivo mutagenicity.

Link to relevant study records

Reference

Reference 1

Endpoint:

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

Remarks:

Comet assay.

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)

Version / remarks:

Methodology described in several publications (Sasaki et al., 1997; Sasaki et al., 1999; Sasaki et al., 2000; Tsuda et al., 2000)

GLP compliance:

no

Type of assay:

mammalian comet assay

Species:

mouse

Strain:

other: ddY

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Japan SLC Co. (Shizuoka, Japan)
- Age at study initiation: 8 weeks
- Weight at study initiation: no available
- Fasting period before study: not reported
- Diet (e.g. ad libitum): commercial pellets MF (Oriental Yeast Industries Co., Tokyo, Japan) ad l
ibitum
- Water (e.g. ad libitum): tap water ad libitum
- Acclimation period: 1 week.
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24ºC
- Humidity (%): 55-65%
- Photoperiod (hrs dark / hrs light): 12 h light–dark cycle

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: olive oil
- Amount of vehicle (if gavage or dermal): 10 mL/kg

Duration of treatment / exposure:

3, 8 and 24 hours (sampling times after single dose)

Frequency of treatment:

Once

Post exposure period:

No data

Dose / conc.:

2 000 mg/kg bw (total dose)

Remarks:

(for sampling times of 3, 8 and 24 hours)

No. of animals per sex per dose:

- Treatment groups: 4 males per sampling time
- Vehicle control: 12 males per sampling time
- Untreated control group: 12 males

Control animals:

yes, concurrent no treatment

yes, concurrent vehicle

Positive control(s):

None.

Tissues and cell types examined:

Stomach, colon, liver, kidney, urinary, bladder, lung, brain and bone marrow.

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
A single dose was set at 0.5 X LD50 (with a limit of 2000 mg/kg) with the purpose of using a dose at which gross necrosis was not observed in order to avoid false-positive results of the comet assay due to cytotoxicity.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
A preliminary range-finding test was conducted using 4-5 male mice/dose to determine the LD50 value.
Animals were observed for pharmacotoxic signs and were macroscopically necropsied 3, 8 and 24 hours after treatment.

METHOD OF ANALYSIS:
Stomach, colon, liver, kidney, urinary bladder, lung, brain and bone marrow were isolated and the prepared slides were scanned to determine the length of the whole comet, diameter of the head and mean migration of 50 nuclei per organ per animal.

Evaluation criteria:

Migration was calculated as the difference between length and diameter for each of the 50 nuclei. Mean migration of 50 nuclei from each organ was calculated for each individual animal. The differences between the averages of four treated animals and the untreated control animals were compared with the Dunnett test after one-way ANOVA. A P-value less than 0.05 was considered statistically significant.

Statistics:

Dunnett test after one-way ANOVA.

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

not applicable

Table 1. Migration of nuclear DNA from organs of mice treated with vehicles

Species	Treatment	Sampling time (h)		Migration (µm, mean of 12 animals)
				Stomach	Colon	Liver	Kidney	Bladder	Lung	Brain	Bone marrow
Mouse	–	–	Mean	6.19	5.15	2.55	2.32	4.66	2.70	1.48	0.94
			S.E.M.	0.44	0.44	0.24	0.34	0.42	0.24	0.20	1.01
	Olive oil	3	Mean	6.46	4.56	2.06	1.90	4.33	2.33	1.56	0.90
			S.E.M.	0.57	0.45	0.29	0.32	0.33	0.37	0.38	0.30
		8	Mean	7.22	5.09	2.86	2.64	5.63	2.49	1.83	1.08
			S.E.M.	0.49	0.40	0.43	0.43	0.49	0.60	0.41	0.41
		24	Mean	6.23	4.70	2.49	1.80	4.20	2.27	1.70	1.23
			S.E.M.	0.40	0.35	0.20	0.24	0.39	0.27	0.41	0.27

Table 2. Migration of nuclear DNA from organs of mice treated with d-limonene

Chemical	Species	Dose (mg/kg)	Sampling time (h)		Migration (µm, mean of 4 animals)
					Stomach	Colon	Liver	Kidney	Bladder	Lung	Brain	Bone marrow
d-limonene	Mouse	2000 (limit dose)	0	Mean	6.87	4.71	2.76	2.60	4.98	2.58	1.70	1.19
				S.E.M.	0.52	0.33	0.65	0.59	1.19	0.37	0.22	0.57
			3	Mean	5.74	6.03	2.18	2.88	3.10	2.99	1.50	1.94
				S.E.M.	1.15	0.45	0.65	0.75	0.44	1.01	1.50	0.55
			8	Mean	6.85	5.34	2.35	1.48	4.88	3.38	1.03	2.05
				S.E.M.	0.89	0.36	0.65	0.53	0.47	1.14	1.03	0.80
			24	Mean	6.02	5.99	2.15	2.02	4.34	2.15	1.01	1.14
				S.E.M.	1.03	1.44	0.56	0.47	0.47	0.37	0.59	1.14

Conclusions:

Under the test conditions, d-limonene is not considered as mutagenic in the comet assay and does not need to be classified according to the criteria of the CLP Regulation (EC) N° (1272-2008).

Executive summary:

In an in vivo comet assay, 4 male ddY mice were administered a single oral dose of d-limonene in olive oil by gavage at dose levels of 2000 mg/kg bw. Animals were then observed for pharmacotoxic signs and were macroscopically necropsied 3, 8 and 24 hours after treatment. Stomach, colon, liver, kidney, urinary bladder, lung, brain and bone marrow were isolated and the prepared slides were scanned to determine the length of the whole comet, diameter of the head and mean migration of 50 nuclei per organ per animal. A preliminary range-finding test was also conducted using 4-5 male mice/dose to determine the LD50 value. No death, morbidity or distinctive clinical and microscopic signs were observed. D-limonene did not induced DNA damage in the studied organs. Under the test conditions, d-limonene is not considered as mutagenic in the comet assay and does not need to be classified according to the criteria of the CLP Regulation (EC) N° (1272-2008).

Reference 2

Endpoint:

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

Remarks:

Comet assay

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Principles of method if other than guideline:

Method: Comet assay (Tice et al., 2000).

GLP compliance:

no

Type of assay:

mammalian comet assay

Species:

rat

Strain:

other: OFA Sprague-Dawley

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River (Saint-Germain-sur-l’Arbresle, France)
- Age at study initiation: 5-6 weeks
- Assigned to test groups randomly: Yes
- Housing: Housed in groups of 2-3 in polypropylene cages
- Diet (e.g. ad libitum): Commercial pellets (SAFE, Augy, France), ad libitum
- Water (e.g. ad libitum): Tap water, ad libitum
- Acclimation period: 1 week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 55 ± 15%
- Air changes (per hour): 20/hour
- Photoperiod (hours dark / hours light): 20 hours dark / 20 hours light

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: 0.1% CMC (carboxymethyl cellulose)
- Amount of vehicle (if gavage): 10 mL/kg bw

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: Test solutions were prepared with 0.1% CMC.

Duration of treatment / exposure:

3-6 or 22-26 hours

Frequency of treatment:

Once

Post exposure period:

No

Dose / conc.:

0 mg/kg bw (total dose)

Remarks:

In 0.5% CMC.

Dose / conc.:

1 000 mg/kg bw (total dose)

Remarks:

In 0.5% CMC.

Dose / conc.:

2 000 mg/kg bw (total dose)

Remarks:

In 0.5% CMC.

No. of animals per sex per dose:

- Vehicle control and treatment groups: Four males
- Positive control group: Three males

Control animals:

yes, concurrent vehicle

Positive control(s):

Streptozotocin
- Justification for choice of positive control(s): Known renal epigenetic carcinogen
- Route of administration: Intravenous
- Doses / concentrations: 20 mg/kg bw

Tissues and cell types examined:

Kidney cells

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: A preliminary range-finding test was conducted using 4 male rats/dose and animals were observed at least 2 days for mortality and clinical signs of toxicity. Maximum tolerated dose (MTD) determined in the preliminary test was selected as the highest dose for the main study.

TREATMENT AND SAMPLING TIMES: After an exposure period of 3-6 or 22-26 hours, treated animals were sacrificed and kidney cells were isolated by specific enzymatic method (Bruggeman et al., 1989). Cytotoxicity was determined on a small sample of each isolated cell suspension following the trypan blue vital dye-exclusion technique.

DETAILS OF SLIDE PREPARATION: Slides (16/dose/expression period) with the cell suspensions (3 × 10^4 cells), embedded in a layer of 0.5% of low melting-point agarose, were immersed in a lysing solution for at least 1 hour at +4 °C in the dark and then run in a horizontal gel electrophoresis unit for 20 min at 0-4 °C by applying an electric current of 0.7 V/cm (25 V/300 mA). After electrophoresis, the slides were neutralized with 0.4 M Tris (pH 7.5) and the DNA was exposed for 5 min to absolute ethanol in order to preserve all the Comet assay slides.

METHOD OF ANALYSIS: Prepared slides were stained with propidium iodide (20 µg/mL distilled water; 25 µL/slide) and scanned using a fluorescent microscope (Leica Microscopy and Scientific Instruments Group, Switzerland), connected through a gated CCD camera to Comet Image Analysis System version 4.0 software (Kinetic Imaging Ltd., UK), to determine mean Olive Tail Moment (OTM) median value in 150 cells per animal (Tice et al., 2000).

Evaluation criteria:

- Olive Tail Moment (OTM) preconised by Olive (1990) was used to evaluate DNA damage.
- OTM, expressed in arbitrary units, is calculated by multiplying the percent of DNA (fluorescence) in the tail by the length of the tail in µm. The tail length is measured between the edge of Comet head and the end of the Comet tail.

Statistics:

- Kruskall-Wallis test was used to display a possible dose–effect relationship.
- Statistical significance of differences in the median values between each group versus the control was determined with the non-parametric Mann-Whitney U-test.

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

- No deaths, morbidity, or distinctive clinical signs were observed after any of the treatments.
- Viability, using the trypan-blue exclusion method, was >70% for each cell suspension in all control and treated groups up to the MTD.
- See table 1

Table 1: DNA damage measured by the Comet assay in isolated rat kidney cells 3–6 or 22–26 hours after a single administration of d-limonene at dose levels of 1000 and 2000 mg/kg bw

 

Sampling time (h)	Group	Dose (mg/kg)	OTM
3-6	d-limonene	0	1.76
		1000	1.81
		2000	1.35
	Streptozotocin	20	41.1***
22-26	d-limonene	0	1.87
		1000	1.91
		2000	2.21
	Streptozotocin	20	40.8***

Significant difference (Mann–Whitney U-test) as compared with the vehicle control; ***p < 0.001.

OTM: mean Olive Tail Moment median value

Conclusions:

Under the test conditions, d-limonene is not considered as mutagenic in the comet assay on isolated kidney cells and does not need to be classified according to the criteria of the CLP Regulation (EC) N° (1272-2008).

Executive summary:

In an in vivo comet assay, groups of 4 OFA Sprague-Dawley male rats were administered a single oral dose of d-limonene in 0.5% CMC by gavage at dose levels of 0, 1000 and 2000 mg/kg bw. After an exposure period of 3-6 or 22-26 hours, treated animals were sacrificed and the kidney cells were isolated and the prepared slides were scanned to determine mean Olive Tail Moment (OTM) median value in 150 cells per animal using the method described by Tice et al (2000). A preliminary range-finding test has also been conducted using 4 males rats/dose and animals were observed at least 2 days for any clinical signs of toxicity and any mortalities in order to determine the maximum tolerated dose (MTD). Positive control (streptozotocin, 20 mg/kg bw) caused a clear increase in the mean OTM median value. D-limonene showed no substantial increase in the mean OTM median value. Under the test conditions, d-limonene is not considered as mutagenic in the comet assay on isolated kidney cells and does not need to be classified according to the criteria of the CLP Regulation (EC) N° (1272-2008).

Reference 3

Endpoint:

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

Remarks:

Comet assay.

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)

Version / remarks:

Methodology described in several publications (Sasaki et al., 1997; Sasaki et al., 1999; Sasaki et al., 2000; Tsuda et al., 2000)

GLP compliance:

no

Type of assay:

mammalian comet assay

Species:

rat

Strain:

Wistar

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Japan SLC Co. (Shizuoka, Japan)
- Age at study initiation: 8 weeks
- Weight at study initiation: no available
- Fasting period before study: not reported
- Diet (e.g. ad libitum): commercial pellets MF (Oriental Yeast Industries Co., Tokyo, Japan) ad libitum
- Water (e.g. ad libitum): tap water ad libitum
- Acclimation period: 1 week.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24ºC
- Humidity (%): 55-65%
- Photoperiod (hrs dark / hrs light): 12 h light–dark cycle

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: Olive oil
- Amount of vehicle (if gavage or dermal): 10 mL/kg

Duration of treatment / exposure:

3, 8 and 24 hours (sampling times after single dose)

Frequency of treatment:

Once

Post exposure period:

No data

Dose / conc.:

2 000 mg/kg bw (total dose)

Remarks:

(for sampling times of 3, 8 and 24 hours)

No. of animals per sex per dose:

- Treatment groups: 4 males per sampling time
- Vehicle control: 12 males per sampling time
- Untreated control group: 12 males

Control animals:

yes, concurrent no treatment

yes, concurrent vehicle

Positive control(s):

None.

Tissues and cell types examined:

Stomach, colon, liver, kidney, urinary, bladder, lung, brain and bone marrow.

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
A single dose was set at 0.5 X LD50 (with a limit of 2000 mg/kg) with the purpose of using a dose at which gross necrosis was not observed in order to avoid false-positive results of the comet assay due to cytotoxicity.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
A preliminary range-finding test was conducted using 4-5 male rats/dose to determine the LD50 value.
Animals were observed for pharmacotoxic signs and were macroscopically necropsied 3, 8 and 24 hours after treatment.

METHOD OF ANALYSIS:
Stomach, colon, liver, kidney, urinary bladder, lung, brain and bone marrow were isolated and the prepared slides were scanned to determine the length of the whole comet, diameter of the head and mean migration of 50 nuclei per organ per animal.

Evaluation criteria:

Migration was calculated as the difference between length and diameter for each of the 50 nuclei. Mean migration of 50 nuclei from each organ was calculated for each individual animal. The differences between the averages of four treated animals and the untreated control animals were compared with the Dunnett test after one-way ANOVA. A P-value less than 0.05 was considered statistically significant.

Statistics:

Dunnett test after one-way ANOVA.

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

not applicable

Table 1. Migration of nuclear DNA from organs of rats treated with vehicles

Species	Treatment	Sampling time (h)		Migration (µm, mean of 12 animals)
				Stomach	Colon	Liver	Kidney	Bladder	Lung	Brain	Bone marrow
Rat	–	–	Mean	12.5	11.4	2.09	2.29	8.54	3.43	1.72	0.94
			S.E.M.	0.84	0.78	0.43	0.41	0.95	0.33	0.39	0.30
	Olive oil	3	Mean	12.2	10.9	2.00	2.58	9.45	2.74	2.06	0.94
			S.E.M.	1.09	0.69	0.34	0.45	0.48	0.34	0.36	0.22
		8	Mean	11.9	11.0	1.75	2.34	9.14	2.89	1.74	0.86
			S.E.M.	0.73	0.75	0.23	0.44	0.84	0.46	0.38	0.24
		24	Mean	11.6	11.4	1.87	2.31	9.37	2.67	2.16	1.02
			S.E.M.	0.96	0.95	0.41	0.40	0.70	0.38	0.31	0.28

Table 2. Migration of nuclear DNA from organs of rats treated with d-limonene

Chemical	Species	Dose (mg/kg)	Sampling time (h)		Migration (µm, mean of 4 animals)
					Stomach	Colon	Liver	Kidney	Bladder	Lung	Brain	Bone marrow
d-limonene	Rat	2000 (limit dose)	0	Mean	12.6	11.5	1.24	2.37	9.66	2.01	1.70	1.06
				S.E.M.	0.69	1.64	0.19	0.55	2.04	0.95	0.72	0.39
			3	Mean	12.5	13.1	1.50	2.99	10.9	3.95	2.25	0.93
				S.E.M.	2.20	1.58	0.66	0.66	2.02	1.56	0.59	0.93
			8	Mean	10.1	10.6	3.55	2.06	7.23	2.92	1.43	0.98
				S.E.M.	0.38	2.91	0.87	1.31	1.68	1.62	0.50	0.68
			24	Mean	13.5	13.3	1.40	3.62	9.86	3.09	2.14	2.45
				S.E.M.	1.38	1.91	0.39	0.87	2.31	2.27	0.78	0.58

Conclusions:

Under the test conditions, d-limonene is not considered as mutagenic in the comet assay and does not need to be classified according to the criteria of the CLP Regulation (EC) N° (1272-2008).

Executive summary:

In an in vivo comet assay, 4 male Wistar rats were administered a single oral dose of d-limonene in olive oil by gavage at dose levels of 2000 mg/kg bw. Animals were then observed for pharmacotoxic signs and were macroscopically necropsied 3, 8 and 24 hours after treatment. Stomach, colon, liver, kidney, urinary bladder, lung, brain and bone marrow were isolated and the prepared slides were scanned to determine the length of the whole comet, diameter of the head and mean migration of 50 nuclei per organ per animal. A preliminary range-finding test was also conducted using 4-5 male rats/dose to determine the LD50 value. No death, morbidity or distinctive clinical and microscopic signs were observed. D-limonene did not induced DNA damage in the studied organs. Under the test conditions, d-limonene is not considered as mutagenic in the comet assay and does not need to be classified according to the criteria of the CLP Regulation (EC) N° (1272-2008).

Reference 4

Endpoint:

in vivo mammalian somatic cell study: gene mutation

Remarks:

Transgenic animal mutagenicity assay

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2001

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Remarks:

Test procedure in accordance with generally accepted scientific standards with minor deviations: no data on housing conditions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 488 (Transgenic Rodent Somatic and Germ Cell Gene Mutation Assays)

GLP compliance:

no

Type of assay:

transgenic rodent mutagenicity assay

Species:

rat

Strain:

other: Big Blue transgenic rats

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Stratagene Taconic Farms, Germantown, USA
- Age at study initiation: 12 weeks old
- Weight at study initiation: 300 g
- Diet: Standard diet (CT1)

Route of administration:

oral: feed

Vehicle:

No

Details on exposure:

DIET PREPARATION
- Mixing appropriate amounts with (Type of food): Test material was ground into the standard diet (CT1) using an automatic pestle and mortar to give final dose level of 1% in diet.
- Storage temperature of food: Room temperature

Duration of treatment / exposure:

10 days

Frequency of treatment:

Diet containing limonene administered daily

Post exposure period:

14 days

Dose / conc.:

1 other: % w/w

Remarks:

Corresponding to about 525 mg/kg bw/day.

No. of animals per sex per dose:

10 males

Control animals:

yes, plain diet

Positive control(s):

4-aminobiphenyl (4AB)
- Justification for choice of positive control(s): 4AB previously shown to induce a positive response in Muta-Mouse transgenic mice liver and kidney (Fletcher et al., 1998).
- Route of administration: Oral (gavage)
- Dose: 20 mg/kg bw/day
- Source: Lancaster Synthesis (Morecambe, UK)

Tissues and cell types examined:

Liver and kidney tissues

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: Based on data in literature (NTP, 1990)

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
- Animals were killed 14 days after the final dose and DNA was isolated from liver and kidney tissue using the Recoverease kit (Stratagene). Mutation assays were carried out as described by Tinwell et al (1994).
- Mutant frequency (MF) was determined for the liver and kidney.
- Approximately 200000 plaque forming units (PFU) were analysed for the presence of mutations for liver and kidney DNA samples.

Evaluation criteria:

No data

Statistics:

Statistical analyses were performed as per the methods described by Piegorsch et al (1997) with modifications.

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

not examined

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

- See table 1 and 2

 Table 1. Induction of lacI mutations in the liver of Big Blue^TM rats

 

Compound	Dose	Animal no.	Total PFU	Mutant PFU	MF X 10–6	Mean ± SD
CT1 diet	–	1	223 665	6	26.8	14.4 ± 8.9
		2	195 500	4	20.5
		3	218 650	4	18.3
		4	194 425	2	10.3
		5	162 325	1	6.2
		6	239 175	1	4.2
Limonene	1% in diet	11	165 075	4	24.2	16.2 ± 10.4
		14	190 050	2	10.5
		15	285 500	6	21
		16	199 400	4	20
		17	225 000	0	0
		19	201 925	6	30
		20	124 900	1	8
4-aminobiphenyl	20 mg/kg bw/day	31	212 300	8	37.7	44.9 ± 12.7**
		32	193 900	7	36.1
		33	183 250	8	43.7
		34	241 975	8	33
		37	119 575	8	67
		40	231 425	12	51.9

Induction of lacI mutations in the liver of Big Blue^TM rats 14 days after the last of 10 daily exposures to the appropriate compound. Data were analysed for statistical significance as described herein, **P < 0.01.

 

Table 2. Induction of lacI mutations in the kidney of Big Blue^TM rats

 

Compound		Dose Animal no.	Total PFU	Mutant PFU	MF X 10–6	Mean ± SD
CT1 diet	_	1	224 475	4	17.8	17.75 ± 8.0
		2	221 550	2	9.03
		3	243 450	5	20.5
		4	230 075	4	17.4
		5	299 075	7	20.1
		6	185 300	4	21.6
		7	211 275	7	33.1
		8	198 850	1	5.02
		9	197 150	2	15.2
Limonene	1% in diet	11	211 625	3	14.2	21.7 ± 12.8
		12	294 100	6	15.2
		13	238 775	8	33.5
		14	190 225	8	42
		15	199 525	6	30.1
		16	227 225	1	4.4
		17	313 525	10	31.9
		18	254 400	1	3.93
		19	236 150	6	25.4
		20	250 225	4	16
4-aminobiphenyl	20 mg/kg bw/day	31	227 975	15	65.8	123 ± 106**
		32	227 450	22	96.7
		33	266 350	19	71.3
		34	230 375	16	69.5
		36	36 950	4	108.3
		37	12 675	2	157.8
		38	12 050	1	83
		39	2540	1	394
		40	15 400	1	64.9

The induction of lacI mutations in the kidney of Big Blue^TM rats 14 days after administration of the final dose of compound. Data were analysed for statistical significance as described herein; **P < 0.001.

Conclusions:

Under the test conditions, limonene is not considered as mutagenic in the transgenic animal mutagenicity assay and does not need to be classified according to the criteria of the CLP Regulation (EC) N° (1272-2008).

Executive summary:

In a transgenic animal mutagenicity assay, groups of male Big BlueTM rats (10/dose) received CT1 diet (negative control), diet containing 1% limonene, or 4-aminobiphenyl (20 mg/kg bw/day) administered by oral gavage (positive control agent) daily for 10 consecutive days. Animals were killed 14 days after the final dose and DNA was isolated from liver and kidney tissue using the Recoverease kit (Stratagene). Mutation assays were carried out as described by Tinwell et al (1994). Mutant frequency (MF) was determined for the liver and kidney. Approximately 200000 plaque forming units (PFU) were analysed for the presence of mutations for liver and kidney DNA samples. There was no evidence of a significant increase in the MF in either the liver or kidney of rats exposed to limonene. Positive control induced the appropriate response.

Under the test conditions, limonene is not considered as mutagenic in the transgenic animal mutagenicity assay and does not need to be classified according to the criteria of the CLP Regulation (EC) N° (1272-2008).

Reference 5

Endpoint:

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

Remarks:

Comet assay.

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH
The analogue substance (R)-p-mentha-1,8-diene (d-limonene) which shares the same functional groups with the substance p-mentha-1,3-diene (alpha terpinene) also has comparable values for the relevant molecular properties.
See attached the reporting format.

Reason / purpose for cross-reference:

read-across source

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Remarks:

(Read across from an analogue)

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

not applicable

Remarks on result:

other: read across from an analogue for which a negative result was obtained in the comet assay.

Conclusions:

Based on the read-across approach from the analogue d-limonene, alpha terpinene is not considered as mutagenic in the comet assay and does not need to be classified according to the criteria of the CLP Regulation (EC) N° (1272-2008).

Executive summary:

In an in vivo comet assay, 4 male ddY mice were administered a single oral dose of d-limonene in olive oil by gavage at dose levels of 2000 mg/kg bw. Animals were then observed for pharmacotoxic signs and were macroscopically necropsied 3, 8 and 24 hours after treatment. Stomach, colon, liver, kidney, urinary bladder, lung, brain and bone marrow were isolated and the prepared slides were scanned to determine the length of the whole comet, diameter of the head and mean migration of 50 nuclei per organ per animal. A preliminary range-finding test was also conducted using 4-5 male mice/dose to determine the LD50 value. No death, morbidity or distinctive clinical and microscopic signs were observed. D-limonene did not induced DNA damage in the studied organs. Based on these results, the read-across was applied and alpha terpinene is not considered as mutagenic in the comet assay and does not need to be classified according to the criteria of the CLP Regulation (EC) N° (1272-2008).

Reference 6

Endpoint:

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

Remarks:

Comet assay

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH
The analogue substance (R)-p-mentha-1,8-diene (d-limonene) which shares the same functional groups with the substance p-mentha-1,3-diene (alpha terpinene) also has comparable values for the relevant molecular properties.
See attached the reporting format.

Reason / purpose for cross-reference:

read-across source

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Remarks:

(read across from an analogue)

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other: read across from an analogue for which a negative result was obtained in the comet assay.

Conclusions:

Based on the read-across approach from the analogue d-limonene, alpha terpinene is not considered as mutagenic in the comet assay on isolated kidney cells and does not need to be classified according to the criteria of the CLP Regulation (EC) N° (1272-2008).

Executive summary:

In an in vivo comet assay, groups of 4 OFA Sprague-Dawley male rats were administered a single oral dose of d-limonene in 0.5% CMC by gavage at dose levels of 0, 1000 and 2000 mg/kg bw. After an exposure period of 3-6 or 22-26 hours, treated animals were sacrificed and the kidney cells were isolated and the prepared slides were scanned to determine mean Olive Tail Moment (OTM) median value in 150 cells per animal using the method described by Tice et al (2000). A preliminary range-finding test has also been conducted using 4 males rats/dose and animals were observed at least 2 days for any clinical signs of toxicity and any mortalities in order to determine the maximum tolerated dose (MTD). Positive control (streptozotocin, 20 mg/kg bw) caused a clear increase in the mean OTM median value. D-limonene showed no substantial increase in the mean OTM median value. Based on these results, the read-across was applied and alpha terpinene is not considered as mutagenic in the comet assay on isolated kidney cells and does not need to be classified according to the criteria of the CLP Regulation (EC) N° (1272-2008).

Reference 7

Endpoint:

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

Remarks:

Comet assay.

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH
The analogue substance (R)-p-mentha-1,8-diene (d-limonene) which shares the same functional groups with the substance p-mentha-1,3-diene (alpha terpinene) also has comparable values for the relevant molecular properties.
See attached the reporting format.

Reason / purpose for cross-reference:

read-across source

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Remarks:

(read across from an analogue)

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

not applicable

Remarks on result:

other: read across from an analogue for which a negative result was obtained in the comet assay.

Conclusions:

Based on the read-across approach from the analogue d-limonene, alpha terpinene is not considered as mutagenic in the comet assay and does not need to be classified according to the criteria of the CLP Regulation (EC) N° (1272-2008).

Executive summary:

In an in vivo comet assay, 4 male Wistar rats were administered a single oral dose of d-limonene in olive oil by gavage at dose levels of 2000 mg/kg bw. Animals were then observed for pharmacotoxic signs and were macroscopically necropsied 3, 8 and 24 hours after treatment. Stomach, colon, liver, kidney, urinary bladder, lung, brain and bone marrow were isolated and the prepared slides were scanned to determine the length of the whole comet, diameter of the head and mean migration of 50 nuclei per organ per animal. A preliminary range-finding test was also conducted using 4-5 male rats/dose to determine the LD50 value. No death, morbidity or distinctive clinical and microscopic signs were observed. D-limonene did not induced DNA damage in the studied organs. Based on these results, the read-across was applied and alpha terpinene is not considered as mutagenic in the comet assay and does not need to be classified according to the criteria of the CLP Regulation (EC) N° (1272-2008).

Reference 8

Endpoint:

in vivo mammalian somatic cell study: gene mutation

Remarks:

Transgenic animal mutagenicity assay

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH
The analogue substance (R)-p-mentha-1,8-diene (d-limonene) which shares the same functional groups with the substance p-mentha-1,3-diene (alpha terpinene) also has comparable values for the relevant molecular properties.
See attached the reporting format.

Reason / purpose for cross-reference:

read-across source

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

not examined

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: read across from an analogue for which a negative result was obtained in the transgenic animal mutagenicity assay.

Conclusions:

Based on the read-across approach from the analogue d-limonene, alpha terpinene is not considered as mutagenic in the transgenic animal mutagenicity assay and does not need to be classified according to the criteria of the CLP Regulation (EC) N° (1272-2008).

Executive summary:

In a transgenic animal mutagenicity assay, groups of male Big BlueTM rats (10/dose) received CT1 diet (negative control), diet containing 1% limonene, or 4-aminobiphenyl (20 mg/kg bw/day) administered by oral gavage (positive control agent) daily for 10 consecutive days. Animals were killed 14 days after the final dose and DNA was isolated from liver and kidney tissue using the Recoverease kit (Stratagene). Mutation assays were carried out as described by Tinwell et al (1994). Mutant frequency (MF) was determined for the liver and kidney. Approximately 200000 plaque forming units (PFU) were analysed for the presence of mutations for liver and kidney DNA samples. There was no evidence of a significant increase in the MF in either the liver or kidney of rats exposed to limonene. Positive control induced the appropriate response.

Based on these results, the read-across was applied and alpha terpinene is not considered as mutagenic in the transgenic animal mutagenicity assay and does not need to be classified according to the criteria of the CLP Regulation (EC) N° (1272-2008).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Summary of results of the experimental studies used to assess the genotoxicity of alpha terpinene:

In vitro gene mutation in bacteria:

Alpha terpinene was tested for mutagenecity on Salmonella typhimurium strains TA100, TA98, TA97a and TA1535 with and without metabolic activation (S9) (Gomes-Carneiro, 2005). The test substance was not mutagenic in all strains tested with and without metabolic activation.

In vitro mammalian chromosomal aberration/sister chromatid exchange:

In an in vitro mammalian chromosome aberration test performed similarly to OECD guideline 473, Chinese hamster Ovary (CHO) cells were exposed to d-limonene with and without metabolic activation (Anderson, 1990). D-limonene is not considered as cytogenetic in CHO cells according to the criteria of the CLP Regulation (EC) N° (1272-2008).

The test substance tea tree oil (TTO) composed of terpinen-4-ol (42.8%), γ-terpinene (20.4%), p-cymene (9.6%), α-terpinene (7.9%), 1,8-cineole (3%), α-terpineol (2,8%) and α-pinene (2.4%) as major compounds, was tested in an in vitro mammalian chromosomal aberration test performed accordingly with OECD guideline 473 (Pereira, 2014). None of the tested TTO concentrations caused significant differences in the frequencies of both structural and numerical (polyploidy and endorreduplication) chromosomal aberrations when compared to those of the negative control. Thus, it was concluded that TTO is not genotoxic in in vitro mammalian cells.

D-limonene was tested in a study on sister chromatid exchange assay in cultured Chinese hamster ovary (CHO) cells with and without metabolic activation (Anderson, 1990). D-limonene did not induce sister chromatid exchanges in CHO cells at any of the doses tested.

D-limonene was tested in another studiy on sister chromatid exchange assay in cultured Chinese hamster ovary (CHO) cells without metabolic activation (Sasaki, 1989). D-limonene did not induce sister chromatid exchanges in CHO cells at any of the doses tested.

In vitro gene mutation in mammalian cells:

Two different studies on d-limonene were conducted for in vitro mammalian cell gene mutation test with mouse lymphoma L5178Y TK+/- cells with and without metabolic activation (Myhr, 1990 and Heck, 1989). D-limonene was not considered as mutagenic in mouse lymphoma L5178Y cells.

Gamma terpinene was tested on the rat hepatocyte unscheduled DNA synthesis assay following the OECD Guideline 482. Gamma terpinene was considered to be negative in this assay.

In vivo genetic toxicity:

d-limonene was tested in a transgenic rodent mutagenicity assay with male Big BlueTM rats (10/dose) (Turner, 2001). There was no evidence of a significant increase in the mutant frequency in either the liver or kidney of rats exposed to test item. Thus, d-limonene is not considered as mutagenic in the transgenic animal mutagenicity assay.

 

In two different in vivo comet assays (Sekihashi, 2002), 4 male mouse or 4 male rats were administered a single oral dose of d-limonene in olive oil by gavage at dose levels of 2000 mg/kg bw. D-limonene was not considered as mutagenic in Comet assay.

 

In another in vivo comet assay (Nesslany, 2007), groups of 4 OFA Sprague-Dawley male rats were administered a single oral dose of d-limonene in 0.5% CMC by gavage at dose levels of 0, 1000 and 2000 mg/kg bw. D-limonene was not considered as mutagenic in Comet assay on isolated kidney cells.

Based on all the above information, weight of evidence and read across approach was applied, and it is concluded that no genotoxicity is predicted for alpha terpinene.

Justification for classification or non-classification

Based on the available information, the substance is considered to be negative for genetic toxicity, and therefore the substance is not classified in accordance with CLP Regulation (EC) no 1272/2008.