Rectified Hydrocarbons by-products from synthetic process of Turpentine and acid

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In vitro bacteria reverse mutation: Weight of evidence: Experimental results from studies performed with the components or analogue substances camphene, d-limonene, l-limonene, alpha terpinene, alpha pinene, l-alpha pinene and cineole are available. All studies were negative. Based on these results, the substance is considered to be non-mutagenic to bacteria.

In vitro mammalian chromosome aberration/sister chromatid exchange: Weight of evidence: Experimental results from studies performed with components or analogue substances d-limonene, camphene, 5-Ethylidene-2-norbornene and cineole are available. Based on these results, the test substance is considered to be non-cytogenic to mammalian cells.

In vitro gene mutation in mammalian cells: Weight of evidence: Experimental results from studies performed with d-limonene and 5-Ethylidene-2-norbornene are available. All studies were negative. Based on these results, the test substance is considered to be non-mutagenic to mammalian cells.

Other studies: alpha pinene and gamma terpinene were determined non-mutagenic in an unscheduled DNA synthesis assay in rat hepatocytes.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

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 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

(Only two of the recommended strains were tested)

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine-requiring gene in Salmonella typhimurium

Species / strain / cell type:

S. typhimurium TA 100

Additional strain / cell type characteristics:

other: uvrB and rfa mutated

Species / strain / cell type:

S. typhimurium TA 98

Additional strain / cell type characteristics:

other: uvrB and rfa mutated

Species / strain / cell type:

S. typhimurium, other: UTH 8414

Species / strain / cell type:

S. typhimurium, other: UTH 8413

Metabolic activation:

with and without

Metabolic activation system:

liver homogenate (S9) from Aroclor-induced male Sprague-Dawley rats (100 µL/plate)

Test concentrations with justification for top dose:

Five concentrations, from 10 µg/plate to 500 µg/plate.
Toxicity and/or solubility determined the upper limit of the dose tested.

Vehicle / solvent:

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

Untreated negative controls:

yes

Remarks:

(With and without metabolic activation)

Positive controls:

yes

Positive control substance:

sodium azide

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene

Positive controls:

yes

Positive control substance:

other: Cisplatin

Details on test system and experimental conditions:

DURATION
- Exposure duration: The plates were incubated at 37 ºC for 48 h, at which time the number of colonies per plate was determined.

SELECTION AGENT (mutation assays): the lack of amino-acid in the medium. Only the mutants can grow due to their capability to synthesize the essential amino acid.

NUMBER OF REPLICATIONS: 2

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not examined

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not examined

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

other: UTH 8414

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not examined

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

other: UTH 8413

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not examined

Untreated negative controls validity:

valid

Positive controls validity:

valid

Alpha pinene was not mutagenic in all strains tested with and without metabolic activation.

Conclusions:

Alpha pinene was not mutagenic in all strains tested with and without metabolic activation.

Executive summary:

Alpha pinene was tested for mutagenecity (doses: 10 -500µg/plate). The mutagenicity assay employed was that described by Maron and Ames using Salmonella typhimurium strains TAl00 and TA98, which are DNA-repair deficient, and two additional strains, UTH 8414 and UTH 8413, developed by Matney, which have full DNA repair capacity. The mutagenicity assays were carried out both with and without metabolic activation (S9). Alpha pinene was not mutagenic in all strains tested with and without metabolic activation.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

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 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

(Only two of the recommended strains were tested)

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine-requiring gene in Salmonella typhimurium

Species / strain / cell type:

S. typhimurium TA 100

Additional strain / cell type characteristics:

other: uvrB and rfa mutated

Species / strain / cell type:

S. typhimurium TA 98

Additional strain / cell type characteristics:

other: uvrB and rfa mutated

Species / strain / cell type:

S. typhimurium, other: UTH 8414

Species / strain / cell type:

S. typhimurium, other: UTH 8413

Metabolic activation:

with and without

Metabolic activation system:

liver homogenate (S9) from Aroclor-induced male Sprague-Dawley rats (100 µL/plate)

Test concentrations with justification for top dose:

Five concentrations, from 10 µg/plate to 1000 µg/plate.
Toxicity and/or solubility determined the upper limit of the dose tested.

Vehicle / solvent:

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

Untreated negative controls:

yes

Remarks:

(With and without metabolic activation)

Positive controls:

yes

Positive control substance:

sodium azide

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene

Positive controls:

yes

Positive control substance:

other: Cisplatin

Details on test system and experimental conditions:

DURATION
- Exposure duration: The plates were incubated at 37 ºC for 48 h, at which time the number of colonies per plate was determined.

SELECTION AGENT (mutation assays): the lack of amino-acid in the medium. Only the mutants can grow due to their capability to synthesize the essential amino acid.

NUMBER OF REPLICATIONS: 2

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not examined

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not examined

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

other: UTH 8414

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not examined

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

other: UTH 8413

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not examined

Untreated negative controls validity:

valid

Positive controls validity:

valid

Camphene was not mutagenic in all strains tested with and without metabolic activation.

Conclusions:

Camphene was not mutagenic in all strains tested with and without metabolic activation.

Executive summary:

Camphene was tested for mutagenecity (doses: 10 -1000µg/plate). The mutagenicity assay employed was that described by Maron and Ames using Salmonella typhimurium strains TAl00 and TA98, which are DNA-repair deficient, and two additional strains, UTH 8414 and UTH 8413, developed by Matney, which have full DNA repair capacity. The mutagenicity assays were carried out both with and without metabolic activation (S9). Camphene was not mutagenic in all strains tested with and without metabolic activation.

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

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 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

(Only two of the recommended strains were tested)

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine-requiring gene in Salmonella typhimurium

Species / strain / cell type:

S. typhimurium TA 100

Additional strain / cell type characteristics:

other: uvrB and rfa mutated

Species / strain / cell type:

S. typhimurium TA 98

Additional strain / cell type characteristics:

other: uvrB and rfa mutated

Species / strain / cell type:

S. typhimurium, other: UTH 8414

Species / strain / cell type:

S. typhimurium, other: UTH 8413

Metabolic activation:

with and without

Metabolic activation system:

liver homogenate (S9) from Aroclor-induced male Sprague-Dawley rats (100 µL/plate)

Test concentrations with justification for top dose:

three concentrations, from 10 µg/plate to 500 µg/plate.
Toxicity and/or solubility determined the upper limit of the dose tested.

Vehicle / solvent:

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

Untreated negative controls:

yes

Remarks:

(With and without metabolic activation)

Positive controls:

yes

Positive control substance:

sodium azide

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene

Positive controls:

yes

Positive control substance:

other: Cisplatin

Details on test system and experimental conditions:

DURATION
- Exposure duration: The plates were incubated at 37 ºC for 48 h, at which time the number of colonies per plate was determined.

SELECTION AGENT (mutation assays): the lack of amino-acid in the medium. Only the mutants can grow due to their capability to synthesize the essential amino acid.

NUMBER OF REPLICATIONS: 2

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not examined

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not examined

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

other: UTH 8414

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not examined

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

other: UTH 8413

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not examined

Untreated negative controls validity:

valid

Positive controls validity:

valid

d-limonene was not mutagenic in all strains tested with and without metabolic activation.

Conclusions:

d-limonene was not mutagenic in all strains tested with and without metabolic activation.

Executive summary:

d-limonene was tested for mutagenecity (doses: 10 - 500µg/plate). The mutagenicity assay employed was that described by Maron and Ames using Salmonella typhimurium strains TAl00 and TA98, which are DNA-repair deficient, and two additional strains, UTH 8414 and UTH 8413, developed by Matney, which have full DNA repair capacity. The mutagenicity assays were carried out both with and without metabolic activation (S9). D-limonene was not mutagenic in all strains tested with and without metabolic activation.

Reference 4

Endpoint:

in vitro gene mutation study in bacteria

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 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

(only one replicate was conducted)

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine-requiring gene in Salmonella typhimurium

Species / strain / cell type:

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

Remarks:

"Spot test"

Additional strain / cell type characteristics:

other: uvrB and rfa mutated

Species / strain / cell type:

S. typhimurium TA 98

Remarks:

"Quantitative test"

Additional strain / cell type characteristics:

other: uvrB and rfa mutated

Species / strain / cell type:

S. typhimurium TA 100

Remarks:

"Quantitative test"

Additional strain / cell type characteristics:

other: uvrB and rfa mutated

Metabolic activation:

with and without

Metabolic activation system:

rat liver S9 fraction induced by Aroclor 1254 (S-9A)

Test concentrations with justification for top dose:

Spot test: 3 μmol/plate (408 μg/plate)
Quantitative test: 0.03, 0.3, 3 and 30 μmol /plate (4.08, 40.8, 408, and 4080 μg /plate)

Vehicle / solvent:

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

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene (+S9); N-methyl-N'-nitro-N-nitrosoguanidin (-S9)

Details on test system and experimental conditions:

METHOD OF APPLICATION:
Cultures were grown in Oxoid nutrient broth No. 2. Revertants were scored on glucosenminimal salts medium supplemented with 0.05 μmol histidine and 0.05 μmol biotin. Plates used for viable counts contained 10 μmol histidine (and 0.05 μmol biotin). The experiments were carried out essentially as described by Ames.

SELECTION AGENT (mutation assays): the lack of amino-acid in the medium. Only the mutants can grow due to their capability to synthesize the essential amino acid.

NUMBER OF REPLICATIONS: 1

DETERMINATION OF CYTOTOXICITY
Toxicity was determined based on the absence of a background lawn of bacteria on the plates. If absence of a background lawn was found the test was repeated with a lower concentration of the substance.

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

at 3 μmol/plate (spot test)

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

at 3 μmol/plate (spot test)

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

at 3 μmol/plate (spot test)

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

at 3 μmol/plate (spot test)

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

(precipitates at 30 μmol/plate)

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at doses equal and higher than 3 μmol/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

(precipitates at 30 μmol/plate)

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at doses equal and higher than 3 μmol/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: test item precipitates at top dose of 30 μmol/plate in the quantitative test

Remarks on result:

other: spot test (rat liver S9 fraction induced by Aroclor 1254 (S-9A))

Alpha pinene was not mutagenic in all strains tested with and without metabolic activation.

Conclusions:

Alpha pinene was not mutagenic in all strains tested with and without metabolic activation.

Executive summary:

Alpha pinene was tested using the Ames assay for mutagenecity on Salmonella typhimurium strains TA 98, TA 100, TA 1535 and TA 1537 with and without metabolic activation (S9). The test item was initially tested (spot test) at 3 μmol/plate on strains TA 98, TA 100, TA 1535 and TA 1537 both with and without metabolic activation using a liver fraction (S-9) from Aroclor 1254. As negative results were obtained, the test item was tested quantitatively at doses of 0.03, 0.3, 3 and 30 μmol /plate using TA 98 and TA 100 with and without metabolic activation using a liver fraction (S-9) from 3-methylcholanthrene. Alpha pinene was found toxic to the bacteria at doses equal and higher than 3 μmol/plate and precipitated at top dose of 30 μmol/plate. Under these test conditions, alpha pinene was found not mutagenic in all strains tested with and without metabolic activation.

Reference 5

Endpoint:

in vitro gene mutation study in bacteria

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 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

(only one replicate was conducted)

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine-requiring gene in Salmonella typhimurium

Species / strain / cell type:

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

Remarks:

"Spot test"

Additional strain / cell type characteristics:

other: uvrB and rfa mutated

Species / strain / cell type:

S. typhimurium TA 98

Remarks:

"Quantitative test"

Additional strain / cell type characteristics:

other: uvrB and rfa mutated

Species / strain / cell type:

S. typhimurium TA 100

Remarks:

"Quantitative test"

Additional strain / cell type characteristics:

other: uvrB and rfa mutated

Metabolic activation:

with and without

Metabolic activation system:

rat liver S9 fraction induced by Aroclor 1254 (S-9A)

Test concentrations with justification for top dose:

Spot test: 3 μmol/plate
Quantitative test: 0.03, 0.3, 3 and 30 μmol /plate

Vehicle / solvent:

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

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene (+S9); N-methyl-N'-nitro-N-nitrosoguanidin (-S9)

Details on test system and experimental conditions:

METHOD OF APPLICATION:
Cultures were grown in Oxoid nutrient broth No. 2. Revertants were scored on glucosenminimal salts medium supplemented with 0.05 μmol histidine and 0.05 μmol biotin. Plates used for viable counts contained 10 μmol histidine (and 0.05 μmol biotin). The experiments were carried out essentially as described by Ames.

SELECTION AGENT (mutation assays): the lack of amino-acid in the medium. Only the mutants can grow due to their capability to synthesize the essential amino acid.

NUMBER OF REPLICATIONS: 1

DETERMINATION OF CYTOTOXICITY
Toxicity was determined based on the absence of a background lawn of bacteria on the plates. If absence of a background lawn was found the test was repeated with a lower concentration of the substance.

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

at 3 μmol/plate (spot test)

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

at 3 μmol/plate (spot test)

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

at 3 μmol/plate (spot test)

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

at 3 μmol/plate (spot test)

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

(precipitates at 30 μmol/plate)

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at doses equal and higher than 3 μmol/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

(precipitates at 30 μmol/plate)

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at doses equal and higher than 3 μmol/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: test item precipitates at top dose of 30 μmol/plate in the quantitative test

Remarks on result:

other: spot test (rat liver S9 fraction induced by Aroclor 1254 (S-9A))

Limonene was not mutagenic in all strains tested with and without metabolic activation.

Conclusions:

Limonene was not mutagenic in all strains tested with and without metabolic activation.

Executive summary:

Limonene was tested using the Ames assay for mutagenecity on Salmonella typhimurium strains TA 98, TA 100, TA 1535 and TA 1537 with and without metabolic activation (S9). The test item was initially tested (spot test) at 3 μmol/plate on strains TA 98, TA 100, TA 1535 and TA 1537 both with and without metabolic activation using a liver fraction (S-9) from Aroclor 1254. As negative results were obtained, the test item was tested quantitatively at doses of 0.03, 0.3, 3 and 30 μmol /plate using TA 98 and TA 100 with and without metabolic activation using a liver fraction (S-9) from 3-methylcholanthrene. Limonene was found toxic to the bacteria at doses equal and higher than 3 μmol/plate and precipitated at top dose of 30 μmol/plate. Under these test conditions, limonene was found not mutagenic in all strains tested with and without metabolic activation.

Reference 6

Endpoint:

in vitro gene mutation study in bacteria

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 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine-requiring gene in Salmonella typhimurium

Species / strain / cell type:

S. typhimurium TA 98

Additional strain / cell type characteristics:

other: uvrB and rfa mutated

Species / strain / cell type:

S. typhimurium TA 100

Additional strain / cell type characteristics:

other: uvrB and rfa mutated

Species / strain / cell type:

S. typhimurium TA 1535

Additional strain / cell type characteristics:

other: uvrB and rfa mutated

Species / strain / cell type:

S. typhimurium, other: TA97a

Additional strain / cell type characteristics:

other: uvrB and rfa mutated

Metabolic activation:

with and without

Metabolic activation system:

rat liver S9 fraction induced by Aroclor 1254

Test concentrations with justification for top dose:

First set of experiments: 0, 10, 20, 35, 50, 75, 100, 200, 250, 300, 400, 500, 750, 1000, 1250, 1500, 2000, 2500 and 5000 μg/plate
Second set of experiments (complementary number of doses within the non-toxic dose interval determined in first set of experiments): 0, 5, 10, 20, 25, 35, 50, 75, 100, 200, 250, 500, 750 and 1500 μg/plate

Vehicle / solvent:

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

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

(100 µL ethanol)

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

2-nitrofluorene

sodium azide

other: 2-aminoanthracene (TA100/+S9 and TA1535/+S9 (1 μg/plate), TA98/+S9 (0.5 μg/plate)); 2-aminofluorene (TA97a/+S9 (10 μg/plate))

Details on test system and experimental conditions:

METHOD OF APPLICATION:
Direct plate incorporation method: 100 μl of an overnight grown 100 μl of the test substance (diluted in analytical grade ethanol, Vetec™, Rio de Janeiro, Brazil), the negative (solvent) control, or the positive control (PC) and 500 μl of the sodium-phosphate buffer or the S9 mix were mixed with 2ml of top agar which was poured onto a minimal glucose plate. Plates were incubated at 37ºC for 72h in the dark and then scored for revertant his+ bacteria colonies. Each determination was made in triplicate and two independent experiments were carried out.
- Cell density at seeding: For all assays, an inoculum (200 μl) of a thawed permanent culture was added to 20ml of Nutrient Broth No. 2 and incubated at 37ºC with shaking until a concentration of approximately 1–2 x10^9 bacteria per millilitre was obtained.

DURATION
- Exposure duration: 72h

SELECTION AGENT (mutation assays): the lack of amino-acid in the medium. Only the mutants can grow due to their capability to synthesize the essential amino acid.

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
Toxicity of (-) alpha pinene to S. typhimurium strains TA97a, TA98, TA100 and TA1535 was investigated in a first set of experiments. Toxicity was apparent either as a reduction in the number of his+ revertant bacteria colonies and or as a change of auxotrophic background growth (i.e. the background lawn).
Doses at which toxicity appeared as an alteration of the background lawn are marked with an asterisk in table 1 (Any other information on results incl. Tables).
Then, a second set of experiments was conducted which included a complementary number of doses within the non-toxic dose interval determined in first set of experiments.

OTHER:
-Lyophilized rat liver S9 fraction induced by Aroclor 1254 was purchased from Moltox™ (Molecular Toxicology Inc., Boone, NC, USA). The S9 mixture was prepared as follows: 7.0ml of ultrapure water; 10.5ml of 200mM sodium phosphate buffer 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 purification system.

Evaluation criteria:

The criteria for a positive mutagenic response, was a clear dose-dependent increase in the number of revertants within the non-toxic range (Maron and Ames, 1983).

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

without

Genotoxicity:

negative

Remarks:

(up to highest dose tested of 100 μg/plate)

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

(at 100 μg/plate)

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with

Genotoxicity:

negative

Remarks:

(up to highest dose tested of 2000 μg/plate)

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

(at 100 μg/plate and above)

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

without

Genotoxicity:

negative

Remarks:

(up to highest dose tested of 2000 μg/plate)

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

(at 500 μg/plate and above)

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with

Genotoxicity:

negative

Remarks:

(up to highest dose tested of 5000 μg/plate)

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

(at 1500 μg/plate and above)

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium, other: TA97a

Metabolic activation:

without

Genotoxicity:

negative

Remarks:

(up to highest dose tested of 500 μg/plate)

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

(at 200 μg/plate and above)

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium, other: TA97a

Metabolic activation:

with

Genotoxicity:

negative

Remarks:

(up to highest dose tested of 5000 μg/plate)

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

(at 1000 μg/plate and above)

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

without

Genotoxicity:

negative

Remarks:

(up to highest dose tested of 5000 μg/plate)

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

(at 250 μg/plate and above)

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with

Genotoxicity:

negative

Remarks:

(up to highest dose tested of 5000 μg/plate)

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

(at 750 μg/plate)

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Table 1. Testing of alpha-terpinene (1-isopropyl-4-methyl-1,3-cyclohexadiene) in the Salmonella/microsome assay

Dose (µg/plate)		Number of revertants (Mean ± SD)
		TA 100		TA 98		TA 97a		TA 1535
		- S9	+ S9	- S9	+ S9	- S9	+ S9	- S9	+ S9
Set 1 experiments	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	-	-	-	-	-	-	-
	0	167 ± 10	206	43 ± 12	58 ± 8	150 ± 2	202 ± 28	18 ± 4	27 ± 5
	PC	804 ± 59	614 ± 97	173 ± 35	430 ± 15	845 ± 17	928 ± 24	506 ± 8	154 ± 4
Set 2 experiments	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	-
	0	158 ± 26	148 ± 19	37 ± 7	47 ± 1	152 ± 13	190 ± 4	30 ± 4	23 ± 5
	PC	449 ± 32	774 ± 54	88 ± 8	183 ± 22	752 ± 87	841 ± 24	696 ± 81	182 ± 10

Negative control (dose 0) = 100μl ethanol; Positive control (PC): TA100/-S9 and TA1535/-S9, SA (1μg/plate); TA100/+S9 and TA1535/+S9, 2AA (1μg/plate); TA98/-S9, 2-NF (1.5μg/plate); TA98/+S9; 2AA (0.5μg/plate); TA97a/-S9, 4-NQNO (1μg/plate); TA97a/+S9, 2AF (10μg/plate).

(-) not tested. (*) Toxicity apparent as an alteration of the background lawn. Values are means ± SD of three plates.

Conclusions:

Alpha terpinene was not mutagenic in all strains tested with and without metabolic activation.

Executive summary:

Alpha terpinene was tested for mutagenecity on Salmonella typhimurium strains TA100, TA98, TA97a and TA1535 with and without metabolic activation (S9). The experiment was performed using the Ames Salmonella assay for mutagenicity. The test substance was not mutagenic in all strains tested with and without metabolic activation.

Reference 7

Endpoint:

in vitro gene mutation study in bacteria

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 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine-requiring gene in Salmonella typhimurium

Species / strain / cell type:

S. typhimurium TA 98

Additional strain / cell type characteristics:

other: uvrB and rfa mutated

Species / strain / cell type:

S. typhimurium TA 100

Additional strain / cell type characteristics:

other: uvrB and rfa mutated

Species / strain / cell type:

S. typhimurium TA 1535

Additional strain / cell type characteristics:

other: uvrB and rfa mutated

Species / strain / cell type:

S. typhimurium, other: TA97a

Additional strain / cell type characteristics:

other: uvrB and rfa mutated

Metabolic activation:

with and without

Metabolic activation system:

rat liver S9 fraction induced by Aroclor 1254

Test concentrations with justification for top dose:

First set of experiments: 0, 100, 250, 500, 750, 1000, 1250, 1500, 2000, 2500 and 5000 μg/plate
Second set of experiments (complementary number of doses within the non-toxic dose interval determined in first set of experiments): 0, 1, 5, 10, 25, 50, 100, 200, 250, 400, 500,600, 700, 750, 800, 900, 1000, 1250, 1500, 2000, 4000 μg/plate

Vehicle / solvent:

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

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

2-nitrofluorene

sodium azide

other: 2-aminoanthracene (TA100/+S9 and TA1535/+S9 (1 μg/plate), TA98/+S9 (0.5 μg/plate)); 2-aminofluorene (TA97a/+S9 (10 μg/plate))

Details on test system and experimental conditions:

METHOD OF APPLICATION:
Direct plate incorporation method: 100 μl of an overnight grown culture, 100 μl of the test substance (diluted in analytical grade ethanol, Vetec™, Rio de Janeiro, Brazil), the negative (solvent) control, or the positive control (PC) and 500 μl of the sodium-phosphate buffer or the S9 mix were mixed with 2ml of top agar which was poured onto a minimal glucose plate. Plates were incubated at 37ºC for 72h in the dark and then scored for revertant his+ bacteria colonies. Each determination was made in triplicate and two independent experiments were carried out.
- Cell density at seeding: For all assays, an inoculum (200 μl) of a thawed permanent culture was added to 20ml of Nutrient Broth No. 2 and incubated at 37ºC with shaking until a concentration of approximately 1–2 x10^9 bacteria per millilitre was obtained.

DURATION
- Exposure duration: 72h

SELECTION AGENT (mutation assays): the lack of amino-acid in the medium. Only the mutants can grow due to their capability to synthesize the essential amino acid.

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
Toxicity of (-) alpha pinene to S. typhimurium strains TA97a, TA98, TA100 and TA1535 was investigated in a first set of experiments. Toxicity was apparent either as a reduction in the number of his+ revertant bacteria colonies and or as a change of auxotrophic background growth (i.e. the background lawn).
Doses at which toxicity appeared as an alteration of the background lawn are marked with an asterisk in table 1 (Any other information on results incl. Tables).
Then, a second set of experiments was conducted which included a complementary number of doses within the non-toxic dose interval determined in first set of experiments.

OTHER:
-Lyophilized rat liver S9 fraction induced by Aroclor 1254 was purchased from Moltox™ (Molecular Toxicology Inc., Boone, NC, USA). The S9 mixture was prepared as follows: 7.0ml of ultrapure water; 10.5ml of 200mM sodium phosphate buffer 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 purification system.

Evaluation criteria:

The criteria for a positive mutagenic response, was a clear dose-dependent increase in the number of revertants within the non-toxic range (Maron and Ames, 1983).

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

without

Genotoxicity:

negative

Remarks:

(up to highest dose tested of 2500 μg/plate)

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

(at 1250 μg/plate and above)

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with

Genotoxicity:

negative

Remarks:

(up to highest dose tested of 2500 μg/plate)

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

(at 2000 μg/plate and above)

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

(up to highest dose tested of 5000 μg/plate)

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium, other: TA97a

Metabolic activation:

without

Genotoxicity:

negative

Remarks:

(up to highest dose tested of 2000 μg/plate)

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

(at 1250 μg/plate and above)

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium, other: TA97a

Metabolic activation:

with

Genotoxicity:

negative

Remarks:

(up to highest dose tested of 5000 μg/plate)

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

without

Genotoxicity:

negative

Remarks:

(up to highest dose tested of 5000 μg/plate)

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

(at 100 μg/plate and above)

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with

Genotoxicity:

negative

Remarks:

(up to highest dose tested of 5000 μg/plate)

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

(at 5000 μg/plate)

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Table 1 Testing of (-)-alpha-pinene in the Salmonella/microsome assay

Dose (µg/plate)		Number of revertants (Mean ± SD)
		TA 100		TA 98		TA 97a		TA 1535
		- S9	+ S9	- S9	+ S9	- S9	+ S9	- S9	+ S9
Set 1 experiments	5000	-	-	33±8	53±6	-	124±22	10±4*	13±4*
	2500	140±8*	147±52*	35±9	46±5	-	159±11	8±2*	19±8
	2000	138±32*	162±11*	-	-	134±21*	-	-	-
	1500	144*	164±1	-	-	103±17*	-	-	-
	1250	125±23*	-	-	-	122±25*	-	-	-
	1000	-	175±34	43±9	49±8	106±6	133±20	11±5*	19±5
	750	-	-	-	-	99±3	-	-	-
	500	-	143±1	34±3	47±3	124±15	153±16	12±2*	19±4
	250	-	-	-	-	-	173±22	-	21±3
	100	-	-	33±6	51±13	-	-	16±5*	-
	0	214±6	212±25	37±6	59±4	194±8	201±11	19±1	20±1
	PC	1112±64	716±40	618±28	278±19	799±47	879±33	362±27	165±24
Set 2 experiments	4000	-	-	-	-	-	-	-	18±3
	2000	-	-	37±7	-	-	-	-	19±6
	1500	-	-	33±3	-	-	-	-	-
	1250	-	-	35±5	-	-	-	-	-
	1000	174±17	-	42±1	45±4	92±11	153±14	-	21±3
	900	190±21	165±32	-	51±6	101±5	124±2	-	-
	750	-	-	33±8	-	-	-	-	-
	800	171±8	167±19	-	39±8	106±15	111±7	-	-
	700	166±30	167±13	-	49±4	105±16	113±28	-	-
	600	165±17	156±12	-	45±5	109±3	100±4	-	-
	500	170±26	174±6	27±4	48±13	89±6	109±18	-	18±2
	400	187±3	165±16	-	39±6	110±8	-	-	-
	250	-	-	-	-	-	-	-	18±3
	100	-	-	-	-	-	-	13±4	23±4
	50	-	-	-	-	-	-	10±1	-
	25	-	-	-	-	-	-	19±2	-
	10	-	-	-	-	-	-	19±7	-
	5	-	-	-	-	-	-	19±7	-
	1	-	-	-	-	-	-	20±13	-
	0	171±17	142±21	43±2	54±3	121±26	199±37	25±2	24±3
	PC	775±69	716±73	173±35	423±25	583±27	866±114	899±44	193±13

Negative control (dose 0) = 100μl ethanol; Positive control (PC): TA100/-S9 and TA1535/-S9, SA (1μg/plate); TA100/+S9 and TA1535/+S9, 2AA (1μg/plate); TA98/-S9, 2-NF (1.5μg/plate); TA98/+S9; 2AA (0.5μg/plate); TA97a/-S9, 4-NQNO (1μg/plate); TA97a/+S9, 2AF (10μg/plate).

(-) not tested. (*) Toxicity. Values are means ± SD of three plates.

Conclusions:

(-) alpha pinene was not mutagenic in all strains tested with and without metabolic activation.

Executive summary:

(-) alpha pinene was tested for mutagenecity on Salmonella typhimurium strains TA100, TA98, TA97a and TA1535 with and without metabolic activation (S9). The experiment was performed using the Ames Salmonella assay for mutagenicity. (-) alpha pinene was not mutagenic in all strains tested with and without metabolic activation.

Reference 8

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

1983

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

Study performed similarly to OECD Guideline 471 with deviations: one strain missing; no data on number of bacterial cells per culture; individual plate counts not reported

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

one strain missing; no data on number of bacterial cells per culture; individual plate counts not reported

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine-requiring gene in Salmonella typhimurium

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

not applicable

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9 fraction of Aroclor 1254-induced adult male Sprague Dawley rats (RLI) and Syrian hamsters (HLI) liver

Test concentrations with justification for top dose:

- Without S9: 0, 0.3, 1, 3, 10 and 33 µg/plate
- With S9 (RLI): 0, 33, 100, 333, 1000 and 3333 µg/plate
- With S9 (HLI): 0, 10, 33, 100, 333 and 1000 µg/plate

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

ethanol (95%)

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene (1 µg/plate with TA 98 and TA 100; 2.5 µg/plate with TA 1535 and TA 1537)

Remarks:

with metabolic activation

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

ethanol (95%)

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: 4-Nitro-o-phenylenediamine (5 µg/plate with TA 98), sodium azide (1 µg/plate with TA 100 and TA 1535), 9-aminoacridine (50 µg/plate with TA 1537)

Remarks:

without metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: preincubation
- Cell density at seeding (if applicable): no data

DURATION
- Preincubation period: 20 min at 37 °C
- Exposure duration: 48 hours at 37 °C

SELECTION AGENT (mutation assays): the lack of amino-acid in the medium. Only the mutants can grow due to their capability to synthesize the essential amino acid.

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other:
- Any supplementary information relevant to cytotoxicity:

- OTHER:
Dose-range finding experiment: Test substance was checked for toxicity to TA 100 up to a concentration of 10 mg/plate or the Iimit of solubility, both in the presence and absence of S-9 mix.

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

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

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

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

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

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

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

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

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Table 1: Mean plate count

 

Dose

TA100

TA 1535

TA 1537

TA 98

NA

RLI

HLI

NA

RLI

HLI

NA

RLI

HLI

NA

RLI

HLI

0

121 ± 15.4

151 ± 11.6

150 ± 6.6

24 ± 4.2

24 ± 4.3

24 ± 5

4 ± 1.5

5 ± 1.2

5 ± 0.9

18 ± 1.2

32 ± 0.6

32 ± 2.3

0.3

132 ± 3.8

14 ± 4.3

5 ± 0.3

18 ± 1.7

1

117 ± 9.4

15 ± 0.6

3 ± 1.2

21 ± 4.7

3

131 ± 4.2

13 ± 2.1

28 ± 1.5

3 ± 0.6

17 ± 4.6

10

122 ± 7.5

136 ± 10.7

17 ± 2.3

21 ± 2.2

6 ± 1.7

10 ± 2.7

23 ± 2

31 ± 3.5

33

129 ± 4.6

153 ± 21

125 ± 4.5

0 ± 0s

31 ± 1.9

24 ± 3.3

4 ± 0.7s

5 ± 0.9

6 ± 0.7

13 ± 4.3s

39 ± 1.2

26 ± 3.0

100

143 ± 1.8

138 ± 12.5

20 ± 2.6

19 ± 4.5

7 ± 1.5

6 ± 0.9

34 ± 1.8

27 ± 5.8

333

129 ± 13.6

110 ± 9.9

24 ± 3.5

t

7 ± 3.2

6 ± 1.5

26 ± 3.1

28 ± 3.9

1000

112 ± 21.1s

105 ± 9.6s

25 ± 0.5

4 ± 1.2

6 ± 2.8s

16 ± 8.4

20 ± 2.1s

3333

133 ± 2.5

25 ± 4.4

10 ± 2.0

14 ± 8.1s

POS

410 ± 27.1

601 ± 37.7

1401 ± 53.4

406 ± 4.0

163 ± 12.2

309 ± 8.7

172 ± 18.5

193 ± 11.6

506 ± 3.4

728 ± 67.6

380 ± 16.6

1276 ± 33.1

 

Abbreviations: POS: Positive control; NA: not activated; RLI: rat liver S-9, Aroclor 1254 induced; HLI: hamster liver S-9, Aroclor 1254 induced; s: slight clearing of background lawn; t: complete clearing of background lawn

Conclusions:

Under the test conditions, d-limonene is not considered as mutagenic in S. typhimurium TA 1535, TA 1537, TA 98 and TA 100 according to the criteria of the CLP Regulation (EC) N° (1272-2008).

Executive summary:

In a reverse gene mutation assay in bacteria, performed similarly to OECD guideline 471, strains of S. typhimurium (TA 1535, TA 1537, TA 100 and TA 98) were exposed to d-limonene in 95% ethanol with and without S9 metabolic activation [S9 fraction of Aroclor 1254-induced adult male Sprague Dawley rats (RLI) and Syrian hamsters (HLI) liver] according to the preincubation method (20 min) at the following concentrations: - Without S9: 0, 0.3, 1, 3, 10 and 33 µg/plate - With S9 (RLI): 0, 33, 100, 333, 1000 and 3333 µg/plate - With S9 (HLI): 0, 10, 33, 100, 333 and 1000 µg/plate   The positive controls induced the appropriate responses in the corresponding strains. D-limonene showed no substantial increases in revertant colony numbers over control count obtained with any of the tester strains at any concentrations in either presence or absence of S9 mix.   Under the test conditions, d-limonene is not considered as mutagenic in this bacterial system according to the criteria of the CLP Regulation (EC) N° (1272-2008). 

Reference 9

Endpoint:

in vitro gene mutation study in bacteria

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:

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

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

not specified

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine-requiring gene in Salmonella typhimurium

Species / strain / cell type:

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

Additional strain / cell type characteristics:

other: uvrB and rfa mutated

Species / strain / cell type:

S. typhimurium TA 1538

Additional strain / cell type characteristics:

other: uvrB and rfa mutated

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced rat liver S9

Test concentrations with justification for top dose:

25000 μg/plate

Vehicle / solvent:

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

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

not specified

True negative controls:

not specified

Positive controls:

not specified

Details on test system and experimental conditions:

DURATION
- Exposure duration: 48h

SELECTION AGENT (mutation assays): the lack of amino-acid in the medium. Only the mutants can grow due to their capability to synthesize the essential amino acid.

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Key result

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Alpha pinene was not mutagenic in all strains tested with and without metabolic activation.

Conclusions:

Alpha pinene was not mutagenic in all strains tested with and without metabolic activation.

Executive summary:

Alpha pinene was tested for mutagenecity on Salmonella typhimurium strains TA 1535, TA 1537, TA 1538, TA 98 and TA 100 with and without metabolic activation (S9). The experiment was performed using the Ames Salmonella assay for mutagenicity. Alpha pinene was not mutagenic in all strains tested with and without metabolic activation.

Reference 10

Endpoint:

in vitro gene mutation study in bacteria

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 471 (Bacterial Reverse Mutation Assay)

Deviations:

not specified

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine-requiring gene in Salmonella typhimurium

Species / strain / cell type:

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

Additional strain / cell type characteristics:

other: uvrB and rfa mutated

Species / strain / cell type:

S. typhimurium TA 1538

Additional strain / cell type characteristics:

other: uvrB and rfa mutated

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced rat liver S9

Test concentrations with justification for top dose:

150000 μg/plate

Vehicle / solvent:

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

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

not specified

True negative controls:

not specified

Positive controls:

not specified

Details on test system and experimental conditions:

DURATION
- Exposure duration: 48h

SELECTION AGENT (mutation assays): the lack of amino-acid in the medium. Only the mutants can grow due to their capability to synthesize the essential amino acid.

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Key result

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

d-limonene was not mutagenic in all strains tested with and without metabolic activation.

Conclusions:

d-limonene was not mutagenic in all strains tested with and without metabolic activation.

Executive summary:

D-limonene was tested for mutagenecity on Salmonella typhimurium strains TA 1535, TA 1537, TA 1538, TA 98 and TA 100 with and without metabolic activation (S9). The experiment was performed using the Ames Salmonella assay for mutagenicity. D-limonene was not mutagenic in all strains tested with and without metabolic activation.

Reference 11

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

significant methodological deficiencies

Remarks:

Method similar to OECD guideline 471, but only two strains were tested with metabolic activation, no data on test item doses tested, no data on results with controls.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

(Only two strains were tested with metabolic activation, no data on test item doses tested, no data on results with controls)

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine-requiring gene in Salmonella typhimurium

Species / strain / cell type:

S. typhimurium TA 98

Additional strain / cell type characteristics:

other: uvrB and rfa mutated

Species / strain / cell type:

S. typhimurium TA 100

Additional strain / cell type characteristics:

other: uvrB and rfa mutated

Metabolic activation:

with

Metabolic activation system:

rat liver microsome fraction, S9, prepared from Aroclor 1254-treated animals according to the procedure of Ames et al. (800 μg/plate)

Test concentrations with justification for top dose:

No data

Vehicle / solvent:

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

Untreated negative controls:

yes

Remarks:

untreated

Negative solvent / vehicle controls:

not specified

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

other: picrolonic acid

Details on test system and experimental conditions:

METHOD OF APPLICATION:
Direct plate incorporation method: a test sample of 10^8 bacterial cells, and S9 at a concentration of 800 μg protein per plate were incorporated into a tube containing top agar prepared with minimal medium (Minimal Broth Davis, Difco) and 0.05 mM histidine and 0.05 mM biotin. The top agar was then poured on a petri dish containing minimal medium supplemented with 20% glucose. After a 48-hour incubation at 37°C, each assay plate was counted and the number of spontaneous mutants for either TA98 (40) or TA100 (180) were subtracted from the total number of revertants.

DURATION
- Exposure duration: 48h

SELECTION AGENT (mutation assays): the lack of amino-acid in the medium. Only the mutants can grow due to their capability to synthesize the essential amino acid.

NUMBER OF REPLICATIONS: at least 2

DETERMINATION OF CYTOTOXICITY
An additional tube of top agar was prepared as explained above and plated on nutrient agar (Difco) to examine the background lawn of bacterial growth for the presence of toxic effects.

OTHER:
-Plates containing aflatoxin B1 were also included in each experiment to confirm enzyme activation by the S9 fraction.

Evaluation criteria:

The positive response to mutagenicity with TA100 is defined as any deviation above the upper 99.9% confidence limits of the mean control value. This value (180) is the average number of spontaneous TA100 revertants observed on the control plates.

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Alpha pinene was not mutagenic in all strains tested with metabolic activation.

Conclusions:

Alpha pinene was not mutagenic in all strains tested with metabolic activation.

Executive summary:

Alpha pinene was tested for mutagenecity on Salmonella typhimurium strains TA100 and TA98 with metabolic activation (S9). The experiment was performed using the Ames Salmonella assay for mutagenicity. Alpha pinene was not mutagenic in all strains tested with metabolic activation.

Reference 12

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

significant methodological deficiencies

Remarks:

Method similar to OECD guideline 471, but only two strains were tested with metabolic activation, no data on test item doses tested, no data on results with controls.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

(Only two strains were tested with metabolic activation, no data on test item doses tested, no data on results with controls)

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine-requiring gene in Salmonella typhimurium

Species / strain / cell type:

S. typhimurium TA 98

Additional strain / cell type characteristics:

other: uvrB and rfa mutated

Species / strain / cell type:

S. typhimurium TA 100

Additional strain / cell type characteristics:

other: uvrB and rfa mutated

Metabolic activation:

with

Metabolic activation system:

rat liver microsome fraction, S9, prepared from Aroclor 1254-treated animals according to the procedure of Ames et al. (800 μg/plate)

Test concentrations with justification for top dose:

No data

Vehicle / solvent:

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

Untreated negative controls:

yes

Remarks:

untreated

Negative solvent / vehicle controls:

not specified

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

other: picrolonic acid

Details on test system and experimental conditions:

METHOD OF APPLICATION:
Direct plate incorporation method: a test sample of 10^8 bacterial cells, and S9 at a concentration of 800 μg protein per plate were incorporated into a tube containing top agar prepared with minimal medium (Minimal Broth Davis, Difco) and 0.05 mM histidine and 0.05 mM biotin. The top agar was then poured on a petri dish containing minimal medium supplemented with 20% glucose. After a 48-hour incubation at 37°C, each assay plate was counted and the number of spontaneous mutants for either TA98 (40) or TA100 (180) were subtracted from the total number of revertants.

DURATION
- Exposure duration: 48h

SELECTION AGENT (mutation assays): the lack of amino-acid in the medium. Only the mutants can grow due to their capability to synthesize the essential amino acid.

NUMBER OF REPLICATIONS: at least 2

DETERMINATION OF CYTOTOXICITY
An additional tube of top agar was prepared as explained above and plated on nutrient agar (Difco) to examine the background lawn of bacterial growth for the presence of toxic effects.

OTHER:
-Plates containing aflatoxin B1 were also included in each experiment to confirm enzyme activation by the S9 fraction.

Evaluation criteria:

The positive response to mutagenicity with TA100 is defined as any deviation above the upper 99.9% confidence limits of the mean control value. This value (180) is the average number of spontaneous TA100 revertants observed on the control plates.

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Camphene was not mutagenic in all strains tested with metabolic activation.

Conclusions:

Camphene was not mutagenic in all strains tested with metabolic activation.

Executive summary:

Camphene was tested for mutagenecity on Salmonella typhimurium strains TA100 and TA98 with metabolic activation (S9). The experiment was performed using the Ames Salmonella assay for mutagenicity. Camphene was not mutagenic in all strains tested with metabolic activation.

Reference 13

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

14 July 2010 - 20 September 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

Two test item flasks opened for two different uses instead of for only one use, but no major differences between the results obtained with a flask open for only one use and those obtained with a re-used flasks (study validity or integrity not compromised)

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

Two test item flasks opened for two different uses instead of for only one use, but no major differences between the results obtained with a flask open for only one use and those obtained with a re-used flasks (study validity or integrity not compromised)

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

Each strain derived from S. typhimurium LT2 contains one mutation in the histine operon, resulting in a requirement for histidine.

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

other: additional mutations in rfa and uvrB genes. For the strains TA98 and TA100 presence of an additional plasmid pKM101 in order to enhance their sensitivity of detection of some mutagens. See Table 1.

Species / strain / cell type:

S. typhimurium TA 102

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

other: mutated in rfa gene and presence of an additional plasmid pKM101 in order to enhance the sensitivity of detection of some mutagens. See Table 1.

Metabolic activation:

with and without

Metabolic activation system:

S9 fraction was purchased from Moltox (Molecular Toxicology, INC, Boone, NC 28607, USA) and obtained from the liver of rats treated with Aroclor 1254 (500 mg/kg) by the intraperitoneal route.

Test concentrations with justification for top dose:

Preliminary test: First preliminary test: 10, 100, 500, 1000, 2500, 5000 µg/plate with and without S9 mix.
Second preliminary test: 1 to 2500 µg/plate with and without S9 mix.
Mutagenicity experiments: First experiment = 0.3 to 555.6 µg/plate without S9 mix (see details in Table 2); 6.9 to 5000 µg/plate with S9 mix (see details in Table 2). Second experiment = 0.3 to 185.2 µg/plate with and without S9 mix (see details in Table 2).

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Dimethylsulfoxide (DMSO), batch Nos. K39250750906 and K41063050021 (VWR, Fontenay Sous Bois, France).
- Justification for choice of solvent/vehicle: During the solubility assay, the test item was found soluble in DMSO at 50 mg/mL. Consequently, using a treatment volume of 100 µL/plate, it was possible to reach the dose-level of 5000 µg/plate which is the highest recommended dose-level in the international guidelines. DMSO was therefore selected as the vehicle to be used in this study.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: sodium azide, 9-Aminoacridine, 2-Nitrofluorene, Mitomycin C for without S9 mix efficacy control. 2-Anthramine for with S9 mix efficacy control. See details in Table 3.

Remarks:

no remarks

Details on test system and experimental conditions:

METHOD OF APPLICATION:
direct plate incorporation: test item solution (0.1 mL), S9 mix when required or phosphate buffer pH 7.4 (0.5 mL) and bacterial suspension (0.1 mL) were mixed with 2 mL of overlay agar (containing traces of the relevant aminoacid and biotin and maintained at 50°C). After rapid homogenization, the mixture was overlaid onto a Petri plate containing minimum medium.
or preincubation method: test item solution (0.1 mL), S9 mix (0.5 mL) and the bacterial suspension (0.1 mL) were incubated for 60 minutes at 37°C, under shaking, before adding the overlay agar and pouring onto the surface of a minimum agar plate.
DURATION
- Preincubation period: 60 min at 37°C
- Exposure duration: 48 or 72 hours
SELECTION AGENT (mutation assays): not applicable
SPINDLE INHIBITOR (cytogenetic assays): not applicable
STAIN (for cytogenetic assays): not applicable
NUMBER OF REPLICATIONS: Preliminary assays: two assays were performed: one with one petri dish per dose per strain and the other one with 2 petri dishes/dose/strain. Main tests: 2 independent tests with 3 plates/dose/strain.
NUMBER OF CELLS EVALUATED: not applicable
DETERMINATION OF CYTOTOXICITY
- Method: observation of the decrease in the number of revertant colonies and/or thinning of the bacterial lawn
OTHER EXAMINATIONS:
- Determination of polyploidy: not required
- Determination of endoreplication: not required
OTHER: All the Petri dishes obtained were placed in a sealed jar using one jar for each dose-level tested, one jar for the vehicle control and another jar for the positive controls. The jars were then incubated at 37°C. The precaution of using jars in this study was due to the volatile characteristic of the test item and to limit the oxidation of the test item.
The revertants were scored with an automatic counter (Cardinal counter, Perceptive Instruments, Suffolk CB9 7 BN, UK). Manual counting was used as needed.

Evaluation criteria:

A reproducible 2-fold increase (for the TA 98, TA 100 and TA 102 strains) or 3-fold increase (for the TA 1535 and TA 1537 strains) in the number of revertants compared with the vehicle controls, in any strain at any dose-level and/or evidence of a dose-relationship was considered as a positive result. Reference to historical data, or other considerations of biological relevance may also be taken into account in the evaluation of the data obtained.

Statistics:

no data

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

See tables 4 to 8

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

See tables 4 to 8

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

See tables 4 to 8

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

See tables 4 to 8

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

See tables 4 to 7

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

See tables 4 to 7

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

See tables 4 to 8

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

See tables 4 to 8

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

See tables 4 to 7

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

See tables 4 to 7

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES: For the first preliminary test (Table 9), no precipitate was observed in the Petri plates when scoring the revertants at any of the tested dose-levels. Without S9 mix, a moderate to strong cytotoxicity was observed at dose-levels ≥ 100 µg/plate with the TA 98, TA 100 and TA 102 strains. With S9 mix, a moderate to strong cytotoxicity was observed at dose-levels ≥ 10 µg/plate with the TA 102 strain, at dose-levels ≥ 100 µg/plate with the TA 100 strain and at dose-levels ≥ 500 µg/plate with the TA 98 strain.
Based on the cytotoxicity results obtained with one plate/dose in the first preliminary test, a second test was performed using two plates/dose and a lower range of dose-levels (Table 10). No precipitate was observed in the Petri plates when scoring the revertants at any of the tested dose-levels. Without S9 mix, a moderate to strong cytoxicity was observed at dose-levels ≥ 10 µg/plate with the TA 100 strain, at dose-levels ≥ 50 µg/plate with the TA 98 strain and at dose-levels ≥ 100 µg/plate with the TA 102 strain. With S9 mix, a moderate cytotoxicity (thinning of the bacterial lawn) was observed at dose-levels ≥ 750 µg/plate with the TA 98 strain, and a moderate to strong cytotoxicity was observed at dose-levels ≥ 250 µg/plate with the TA 100 strain and at 500 µg/plate with the TA 102 strain.

COMPARISON WITH HISTORICAL CONTROL DATA: The numbers of revertants for the vehicle and positive controls were as specified in the acceptance criteria. The study was therefore considered valid.

ADDITIONAL INFORMATION ON CYTOTOXICITY: Without metabolic activation, in the first experiment (Table 4), a moderate to strong toxicity was noted at dose-levels  61.7 µg/plate in the TA 102 strain and a strong toxicity was noted at dose-levels  61.7 µg/plate in all the other strains.
In the second experiment (Table 6), a moderate toxicity was noted at 185.2 µg/plate in the TA 102 strain and a strong toxicity was noted at 61.7 µg/plate in all the other strains.
With metabolic activation, in the first experiment (Table 5), using the direct plate incorporation method, a moderate to strong toxicity was noted at dose-levels  555.6 µg/plate in the TA 1535, TA 1537, TA 98 and TA 100 strains and a moderate toxicity was noted at 1666.7 µg/plate in the TA 102 strain.
In the second experiment performed using the preincubation method, a moderate to strong toxicity was observed at dose-levels  20.6 µg/plate in all the tested strains (Table 8). Since this toxicity was noted at five out of six dose-levels, less than five analysable dose-levels were obtained in this experiment. This requirement of the international guidelines being not met, the results obtained were not retained and the treatment was repeated using lower ranges of dose levels (Table 7). In this second assay using the preincubation method with lower doses, a moderate toxicity was noted at 61.7 µg/plate in the TA 1535 and TA 100 strains, and a strong toxicity was observed at 61.7 µg/plate in the TA 98 strain. No noteworthy toxicity was induced in the TA 1537 and TA 102 strains up to the highest tested dose-levels of 61.7 and 185.2 µg/plate, respectively.

Table 4: Number of revertants per plate in the absence of metabolic activation in the first test (direct plate incorporation method)

 

Laevo Limonene Concentration (µg/plate)	TA 1535		TA1537		TA 98		TA 100		TA 102
	Mean$	Standard deviation	Mean$	Standard deviation	Mean$	Standard deviation	Mean$	Standard deviation	Mean$	Standard deviation
0*	15	6	11	6	31	8	124	39	380	25
0.3	18	2	-	-	-	-	144	18	-	-
0.8	22	3	10	5	26	4	159	59	-	-
2.3	21	4	7	2	22	4	114	8	349	25
6.9	24	7	16	3	23	7	114	13	430	56
20.6	14	3	11	2	25	4	101	22	426	19
61.7	7St	3	4St	2	12St	5	43St	21	199Mt	9
185.2	-	-	0St	1	7St	0	-	-	232St	6
555.6	-	-	-	-	-	-	-	-	209St	3
Positive control**	953	69	400	154	128	10	922	4	2009	329

$: Mean of triplicate

*Solvent control = negative control: DMSO

**Mutagens positive controls:

- NaN₃(1 µg/plate) in TA1535 and TA100 strains

- 9AA (50 µg/plate) in TA1537 strain

- 2NF (0.5 µg/plate) in TA 98 strain

- MMC ( 0.5 µg/plate) in TA 102 strain

Mt: Moderate cytoxicity

St: Strong cytotoxicity 

 

Table 5:Number of revertants per plate in the presence of metabolic activation (S9 mix) in the first test (direct plate incorporation method) 

 

Laevo Limonene Concentration (µg/plate)	TA 1535		TA1537		TA 98		TA 100		TA 102
	Mean$	Standard deviation	Mean$	Standard deviation	Mean$	Standard deviation	Mean$	Standard deviation	Mean$	Standard deviation
0*	16	6	8	3	35	12	121	27	427	46
6.9	22	9	-	-	-	-	116	9	423	60
20.6	18	3	14	8	32	8	139	11	479	140
61.7	19	6	13	5	29	5	127	10	504	116
185.2	12	6	15	3	34	13	118	23	487	94
555.6	11Mt	2	9Mt	3	23Mt	6	111Mt	5	376	69
1666.7	12St	5	13Mt	4	22St	8	106St	15	511Mt	12
5000	-	-	3St	5	14St	16	-	-	-	-
Positive control**	410	50	126	10	855	196	242	13	2202	20

$: Mean of triplicate

*Solvent control = negative control: DMSO

**Mutagens positive controls:

- 2AM (2µg/plate) in TA1535, TA1537, TA98

- 2AM (10µg/plate) in TA102 strain

- BAP (5µg/plate) in TA100 strain

Mt: Moderate cytotoxicity

St: Strong cytotoxicity

  

Table 6:Number of revertants per plate in the absence of metabolic activation in the second test (direct plate incorporation method)

Laevo Limonene Concentration (µg/plate)	TA 1535		TA1537		TA 98		TA 100		TA 102
	Mean$	Standard deviation	Mean$	Standard deviation	Mean$	Standard deviation	Mean$	Standard deviation	Mean$	Standard deviation
0*	26	11	10	3	24	7	106	8	417	22
0.3	25	4	12	5	39	8	114	7	-	-
0.8	33	3	11	3	28	4	108	13	424	48
2.3	24	1	21	6	27	8	101	13	439	31
6.9	23	12	14	2	25	6	91	10	416	100
20.6	32	14	24	7	22	6	103	15	413	19
61.7	27St	9	6St	2	14St	7	103St	10	390	40
185.2	-	-	-	-	-	-	-	-	344Mt	30
Positive control**	706	99	193	87	141	20	778	61	1524	220

$: Mean of triplicate

*Solvent control = negative control: DMSO

- NaN₃(1 µg/plate) in TA1535 and TA100 strains

- 9AA (50 µg/plate) in TA1537 strain

- 2NF (0.5 µg/plate) in TA 98 strain

- MMC ( 0.5 µg/plate) in TA 102 strain

Mt: Moderate cytotoxicity

St: Strong cytotoxicity 

 

Table 7: Number of revertants per plate in the presence of metabolic activation (S9 mix) in the second test (pre-incubation method)

 

Laevo Limonene Concentration (µg/plate)	TA 1535		TA1537		TA 98		TA 100		TA 102
	Mean$	Standard deviation	Mean$	Standard deviation	Mean$	Standard deviation	Mean$	Standard deviation	Mean$	Standard deviation
0*	21	4	11	5	34	8	108	12	428	71
0.3	19	5	14	2	32	5	121	8	-	-
0.8	18	2	13	6	38	10	142	9	527	115
2.3	20	5	8	6	30	7	129	6	571	34
6.9	16	5	14	4	36	5	138	21	511	54
20.6	22	2	13	3	34	5	130	14	480	93
61.7	18Mt	5	10	3	29St	7	127Mt	15	452	131
185.2	-	-	-	-	-	-	-	-	496	122
Positive control**	230	30	164	45	832	56	325	8	1577	175

$: Mean of triplicate

*Solvent control = negative control: DMSO

**Mutagens positive controls:

- 2AM (2µg/plate) in TA1535, TA1537, TA98 strains

- 2AM (10µg/plate) in TA102 strain

- BAP (5 µg/plate) in TA100 strain

Mt: Moderate cytotoxicity

St: Strong cytotoxicity

Table 8: Number of revertants per plate in the presence of metabolic activation (S9 mix) in the second test (pre-incubation method): this assay was considered non-valid as less than five analyzable dose-levels were obtained in this experiment (Remark: TA102 was not tested in this experiment).

 

Laevo Limonene Concentration (µg/plate)	TA 1535		TA1537		TA 98		TA 100
	Mean$	Standard deviation	Mean$	Standard deviation	Mean$	Standard deviation	Mean$	Standard deviation
0*	21	4	13	4	39	8	131	4
6.9	25	7	16	6	48	3	133	22
20.6	17 Mt	3	17 Mt	3	41 St	13	103 St	8
61.7	4 St	3	9 St	6	17 St	5	89 St	9
185.2	10 St	1	13 St	4	31 St	7	95 St	6
555.6	10 St	5	0 X+St	0	0 St	0	6 St	10
1666.7	0 St	0	1 St	1	5 St	2	1 St	1
Positive control**	189	9	170	24	1014	28	451	32

$: Mean of triplicate

*Solvent control = negative control: DMSO

**Mutagens positive controls:

- 2AM (2µg/plate) in TA1535, TA1537, TA98 strains

- BAP (5 µg/plate) in TA100 strain

Mt: Moderate cytotoxicity

St: Strong cytotoxicity

X: too many microcolonies

Conclusions:

Under the test conditions, the test item Laevo Limonene did not show mutagenic activity in the bacterial reverse mutation test with Salmonella typhimurium strains.Laevo Limonene is not considered as mutagenic in this bacterial system according to the criteria of the Annex VI of the CLP Regulation (EC) N° (1272-2008).

Executive summary:

In a reverse gene mutation assay in bacteria (No. 36956 MMO), performed according to the OECD No.471 and EC No.B13/14 guidelines, Laevo Limonene (purity of 81.1%) diluted in Dimethylsulfoxide (DMSO) was tested in S. typhimurium TA1535, TA1537, TA100, TA98 and TA102 in the presence and the absence of mammalian metabolic activation (S9) using the direct plate incorporation or the preincubation method. All the concentrations and dose-levels were expressed as active item, taking into account the purity of the test item (81.1%).

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

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

Two independent preliminary tests were performed. Since the test item was cytotoxic (observation of a decrease in the number of revertant colonies and of thinning of the bacterial lawn) in these preliminary tests for all strains with or without metabolic activation, the choice of the highest dose-level to be used in the main test was based on the level of toxicity, according to the criteria specified in the international guidelines.

Two independent main experiments with and without metabolic activation were performed. In the second main experiment, two assays in presence of S9 mix according to the preincubation method were necessary. Indeed, in the first assay with S9 mix using this specific method, a moderate to strong toxicity was observed at dose-levels higher than 20.6 µg/plate in TA1535, TA1537, TA98 and TA100. Since this cytotoxicity was noted at five out of six dose-levels, less than five analyzable dose-levels were obtained in this assay. The results were therefore not retained and the treatment was repeated using lower ranges of dose-levels. In the second treatment with S9 mix using the preincubation method, there was sufficient analysable doses and the assay was considered as acceptable.

During the two main tests, no induced revertant over background was observed in any strains of S. typhimurium with or without metabolic activation whereas the cytotoxic dose-level was reached.

In conclusion, under the test conditions, Laevo Limonene did not show any mutagenic activity in the bacterial reverse mutation test using Salmonella typhimurium. Laevo Limonene is not considered as mutagenic in this bacterial system according to the criteria of the Annex VI of CLP Regulation (EC) N° (1272-2008) .

 

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

Reference 14

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 15

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

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

GLP compliance:

not specified

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

Not applicable

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: CHO cells were obtained from the Oak Ridge National Laboratory with a designation CHO-K1-BH4 (subclone D1)

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: Ham`s modified F12 medium supplemented with 10% (v/v) heated inactivated fetal bovine serum, lacking in hipoxanthine. (+S9: F12 medium with 50 units/ml penicillin, 50 µg/ml streptomycin and 5% (v/v) dyalized bovine serum; -S9: without serum)
- Properly maintained: yes

Metabolic activation:

with and without

Metabolic activation system:

S-9 liver homogenate prepared from Arochlor 1254-induced Sprague-Dawley male rats (Microbi ological Associates, Bethesda, MD)

Test concentrations with justification for top dose:

With and without metabolic activation: 0.01, 0.02, 0.04, 0.06 and 0.08 mg/mL.
The selection of a suitable range of concentrations for testing was based upon a preliminary cytotoxicity study.

Vehicle / solvent:

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

Untreated negative controls:

yes

Remarks:

cell culture medium

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

triethylenemelamine

cyclophosphamide

Details on test system and experimental conditions:

METHOD OF APPLICATION: in culture medium.
Duplicate cultures of CHO were incubated 36-48 h before treatment into 75-cm2 culture flasks. A range of test compound concentration was added and cultured for 6 h and 10 h before sampling with and without metabolic activation.

- Cell density at seeding (if applicable): 3-5 x 10^5 cells/75-cm2 culture flask.

DURATION
- Exposure duration: 6 h and 10 h

SPINDLE INHIBITOR (cytogenetic assays): Colchicine was added to culture flasks 2 h prior to cell harvesting.

STAIN (for cytogenetic assays): Dilute Geimsa solution (1:25)

NUMBER OF REPLICATIONS: 2 cultures

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: After treatment with colchicine, cells were removed from flasks by brief incubation with 0.01% trypsin and gentle agitation. They were centrifuged and suspended in 0.075 M KCl and incubated for 20-25 min at 37ºC. Cells were then centrifuged, fixed in 3 or 4 changes of Carnoy’s fixative and chromosome spreads prepared. Chromosomes were stained for 10 min in dilute Geimsa solution (1:25) and rinsed with water.

NUMBER OF CELLS EVALUATED: minimum of 50 cells/culture for each sampling time and each dosage.

DETERMINATION OF CYTOTOXICITY
Cytotoxicity was determined by inoculating 3-5 x 10^5 cells into 75-cm2 culture flasks and treating for 6-8 h with test compound in dosage ranges of 0.006-0.06 mg/mL (test 1) and 0.07-0.10 mg/mL (test 2) in the absence and presence of metabolic activation. Both growth inhibition and inhibition of mitosis (mitotic index) were performed for cytotoxicity.
- Growth inhibition: It was expressed as the relative number of surviving cells in untreated (DMSO control) compared to test item-treated cells.
- Inhibition of mitosis (mitotic index): after harvesting of cells for chromosome preparations, the mitotic inhibition was determined as the proportion of cells in metaphase.

Evaluation criteria:

The test compound was considered positive if:
a) at least one of the test concentrations exhibits a statistically significant increase in chromosome aberrations compared with the concurrent negative control,
b) the increase is dose-related,
c) any of the results are outside the range of the historical negative control

Statistics:

One-tailed Fisher Exact probability test.

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Other results: When present, chromatid breaks and chromosome fragment were the predominant aberration.

RANGE-FINDING/SCREENING STUDIES: A concentration of 0.08 mg/ml produced a 22.8% reduction of culture growth of CHO cells tested without S9 activation relative to control values and a 30.6% reduction of growth with metabolic activation. Excessive mitotic inhibition was produced without S9 activation above 0.08 mg/mL and with S9 activation 0.06 mg/mL produced a 70% reduction in metaphase mitoses. A concentration range of 0.01-0.07 mg/ml test compound was used without metabolic activation and 0.01-0.08 mg/ml with metabolic activation.

HISTORICAL DATA:
The concurrent negative and positive controls were within the range for historical controls.

Table 1: Summary of incidence of aberrant CHO cells in the in vitro clastogenicity study with ENB

		+S9		-S9
Material	Dosage	Cells scored	Percent of aberrant cells (mean - SD)	Cells scored	Percent of aberrant cells (mean - SD)
6 h sampling time
DMSO		100	4.0 – 5.66	100	4.0 – 2.83
ENB	0.01 mg/mL	100	1.0 – 1.-41	NE	-
ENB	0.02 mg/mL	100	5.0 – 1.41	NE	-
ENB	0.04 mg/mL	100	3.0 – 1.41	100	6.0 – 0.0
ENB	0.06 mg/mL	NE	-	100	5.0 – 4.24
ENB	0.08 mg/mL	NE	-	100	1.0 – 1.41
10 h sampling time
DMSO		100	2.0 – 0.0	100	4.0 – 2.83
ENB	0.01 mg/mL	100	2.0 – 0.0	100	5.0 – 1.41
ENB	0.02 mg/mL	100	4.0 – 0.0	100	4.0 – 0.0
ENB	0.04 mg/mL	100	3.0 – 4.24	100	4.0 – 0.0
Medium	-	50	4.0 – 0.0	50	2.0 – 0.0
TEM	1.5 µg/mL	50	36.0 – 0.3c	NE	-
CP	12.0 µg/mL	NE	-	50	26.0 – 0.0c

TEM, triethylenemelamine; CP, cyclophosphamide; NE, not evaluated

c Significant at p < 0.001 (compared to solvent control)

Conclusions:

In an in vitro chromosome aberration study, the tested material ENB was not clastogenic when tested in CHO cells in vitro with or without metabolic activation.

Executive summary:

5-ethylidene-2-norbornene (ENB) was tested in an in vitro chromosome aberration study with cultured Chinese hamster ovary CHO cells in presence and absence of metabolic activation using a method comparable to OECD guideline 473. A preliminary cytotoxicity study consisting on growth inhibition and inhibition of mitosis (mitotic index) was conducted. Based on these results, the tested concentrations were 0.01, 0.02, 0.04, 0.06 and 0.08 mg/mL both with and without metabolic activation. Duplicate cultures were incubated 36-48 h before treatment into 75-cm2 culture flasks. The test compound was added and cultured for 6 h and 10 h before sampling. There were no statistically significant or dosage-related increases in chromosome aberrations compared with the concurrent control (DMSO), with and without metabolic activation at both sampling times. When present, chromatid breaks and chromosome fragment were the predominant aberration. Both positive controls, triethylenemelamine without metabolic activation and cyclophosphamide with metabolic activation, produced highly significant increases in the numbers of aberrant cells compared to control. The concurrent negative and positive controls were within the range for historical controls. Based on these results, it can be concluded that ENB is not a clastogen.

Reference 16

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

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 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

GLP compliance:

not specified

Type of assay:

in vitro mammalian cell gene mutation test using the Hprt and xprt genes

Target gene:

HGPRT (hypoxanthine-guanine-phosphoribosyl transferase) gene

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: CHO cells were obtained from the Oak Ridge National Laboratory with a designation CHO-K1-BH4 (subclone D1)

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: Ham`s modified F12 medium supplemented with 10% (v/v) heated inactivated fetal bovine serum, lacking in hipoxanthine. (+S9: F12 medium with 50 units/ml penicillin, 50 μg/ml streptomycin and 5% (v/v) dyalized bovine serum; -S9: without serum)
For determination of mutant frecuencies, F-12-D5 medium containing 2.0 mg/ml 6-TG was used as the selective medium.
- Properly maintained: yes

Metabolic activation:

with and without

Metabolic activation system:

S-9 liver homogenate prepared from Arochlor 1254-induced Sprague-Dawley male rats (Microbiological Associates, Bethesda, MD)

Test concentrations with justification for top dose:

Without metabolic activation: 0.02, 0.04, 0.05, 0.06, 0.07 and 0.08 µL/mL.
With metabolic activation: Test 1: 0.02, 0.04, 0.05, 0.06, 0.07 and 0.10 µL/mL; test 2: 0.06, 0.07, 0.08 and 0.09 µL/mL
The selection of a suitable range of concentrations for testing was based upon a preliminary cytotoxicity study.

Vehicle / solvent:

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

Untreated negative controls:

yes

Remarks:

(Cell culture medium)

Negative solvent / vehicle controls:

yes

Remarks:

DMSO (20 µL/mL)

Positive controls:

yes

Positive control substance:

N-dimethylnitrosamine

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in culture medium.
For the test without metabolic activation, 20-24 h before treatment 5x10^5 CHO cells were inoculated into 25-cm2 culture flasks containing F12-D5 medium and incubated at 37 ºC in a 5-6% CO2 atmosphere. Appropriate amounts of ENB or control materials (DMSO solvent, cell culture medium, or positive controls) were added, and the cultures were exposed for 5 h. For testing in the presence of metabolic activation, the procedure used F12 medium without serum, but containing 1.0 mL S9 activation mix per 4 mL of medium.

- Cell density at seeding (if applicable): 5 x 10^5 cells/25-cm2 culture flask.

DURATION
- Exposure duration: 5 h
- Selection time (if incubation with a selection agent): The mutant fraction was determined in duplicate cultures for each treatment group after a 9- and 12-day subculturing period. At 2- and 3-day intervals post-treatment ca. 3-5 x 10^5 cells were subcultured and incubated for 7 days before dissociation and seeding to plates in medium containing 6-thioguanine (6-TG) or without 6-TG to assess plating efficiency of the treated cell population.All cultures were incubated for an additional 6-8 days to allow cell growth.

SELECTION AGENT (mutation assays): 6-thioguanine (6-TG)


NUMBER OF REPLICATIONS: 2 cultures

NUMBER OF CELLS EVALUATED: The number of mutants per 10^6 total cells and per 10^6 viable cells were calculated

DETERMINATION OF CYTOTOXICITY
- Groth inhibition test: a preliminary test was conducted in order to select the highest dosage that produced a maximum of 80-90% cell death. It was expressed as the relative number of surviving cells in untreated (DMSO control) compared to test item-treated cells.
- Cytotoxicity, as relative survival of ENB-treated cells compared with DMSO controls, was determined 1 d after exposure ("surviving fraction"). This colony-forming potential of treated cells was used as a measure of treatment-induced cytotoxicity, using 4 plates/culture and 100 cells/plate.

Statistics:

Analysis of mutation frequencies followed the procedure for Irr and Snee (1979), employing Box-Cox transformation (Box & Cox, 1964) before parametric analysis 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:

(From 0.06 µL/mL)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

(at 0.1 µL/mL)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

(From 0.08 µL/mL)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

In the mutagenicity test, ENB produced a dosage-related cytotoxicity to CHO cells with and without metabolic activation. The findings indicated a steep slope in the dose-response relationship between 0.06 and 0.07 µl/mL without metabolic activation, and 0.07-0.10 µl/mL with metabolic activation. In the study without metabolic activation, ENB did not produce any statistically significant or dosage-related increase in the number of mutants/10^5 clonable cells. In the test with metabolic activation, increases in the incidence of mutants were seen with only one of the duplicate cultures at each concentration. However, these increases were not statistically significant. A repeat test was therefore conducted to confirm the absence of a mutagenic effect with 0.06-0.09 µl/mL dosage range with metabolic activation. The 0.08 and 0.09 µl/mL doses were completely cytotoxic but no mutagenic effects were observed in duplicate cultures with ENB concentrations of 0.06 and 0.07 µl/mL.

Remarks on result:

other: Test 1

Conclusions:

Under test conditions, ENB did not produce any statistically significant or dosage-related increase in the number of mutants cells with and without metabolic activation.

Executive summary:

To determine the potential for 5-Ethylidene-2-norbornene (ENB) to cause forward gene mutations, a CHO cell line was used for the detection of mutations at the HGPRT (hypoxanthine-guanine-phosphoribosyl transferase) gene locus in a medium containing the purine analog 6-thioguanine (6-TG). For the test without metabolic activation, 20-24 h before treatment 5E5 CHO cells were inoculated into 25-cm2 culture flasks containing F12-D5 medium and incubated at 37 ºC in a 5-6% CO2 atmosphere. Appropriate amounts of ENB or control materials (DMSO solvent, cell culture medium, or positive controls) were added, and the cultures were exposed for 5 h. For testing in the presence of metabolic activation, the procedure used F12 medium without serum, but containing 1.0 mL S9 activation mix per 4 mL of medium. The tested concentrations were: without metabolic activation: 0.02, 0.04, 0.05, 0.06, 0.07 and 0.08 µL/mL; and with metabolic activation: 0.02, 0.04, 0.05, 0.06, 0.07 and 0.10 µL/mL (test 1) and 0.06, 0.07, 0.08 and 0.09 µL/mL (test 2). Positive controls were dimethylnitrosamine (DMN) with metabolic activation, and ethylmethanesulfonate (EMS) without metabolic activation. The mutant fraction was determined in duplicate cultures for each treatment group. A dosage-related cytotoxicity to CHO cells with and without metabolic activation was found. In the study without metabolic activation, ENB did not produce any statistically significant or dosage-related increase in the number of mutants/1E5 clonable cells. In the test 1 with metabolic activation, increases in the incidence of mutants were seen with only one of the duplicate cultures at each concentration. However, these increases were not statistically significant. Test 2 was therefore conducted to confirm the absence of a mutagenic effect. The 0.08 and 0.09 µl/mL doses were completely cytotoxic but no mutagenic effects were observed in duplicate cultures with ENB concentrations of 0.06 and 0.07 µl/mL.

Reference 17

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

1989

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 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 18

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 19

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 20

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
DL-camphene	+	81.7	80.9	80.5	81.9	82.0	T
	-	9.0	9.0	8.8	9.2	9.3	T

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

Each value represents the mean of 3 independent experiments.

T, toxic; -, not tested.

Conclusions:

Camphene did not induce sister chromatid exchanges in CHO cells tested without metabolic activation.

Executive summary:

Camphene 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 high dose of 1000 μM. Under these test conditions, camphene did not induce sister chromatid exchanges in CHO cells at any of the doses tested.

Reference 21

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 22

Endpoint:

in vitro DNA damage and/or repair study

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

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:

0.001, 0.003, 0.01, 0.03, 0.1, 10 μl/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.

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

Conclusions:

Alpha pinene was not mutagenic based on the results of the rat hepatocyte unscheduled DNA synthesis assay.

Executive summary:

Alpha pinene 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 0.001, 0.003, 0.01, 0.03, 0.1, 10 μl/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, alpha pinene is considered to be negative in the unscheduled DNA synthesis assay.

Reference 23

Endpoint:

in vitro DNA damage and/or repair study

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

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 24

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

1983

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

Study performed similarly to OECD Guideline 471 with deviations: one strain missing; no data on number of bacterial cells per culture; individual plate counts not reported

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

one strain missing; no data on number of bacterial cells per culture; individual plate counts not reported

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine-requiring gene in Salmonella typhimurium

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254 induced liver S-9 fractions obtained from male Sprague-Dawley rats and male Syrian hamsters, injected, i.p.

Test concentrations with justification for top dose:

The maximum concentration dosed was 10mg/plate unless toxicity was observed at this dose level.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: [acetone; dimethyl sulphoxide; ethanol(95%); water (distilled)]
- Justification for choice of solvent/vehicle: The solvent of choice was distilled water, DMSO was used if the chemical was insoluble. Ethanol or acetone was used if the substance was not soluble or stable in DMSO. The final choice of solvent for this substance was not reported.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene (1 µg/plate with TA 98 and TA 100; 2.5 µg/plate with TA 1535 and TA 1537)

Remarks:

with metabolic activation

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 4-Nitro-o-phenylenediamine (5 µg/plate with TA 98), sodium azide (1 µg/plate with TA 100 and TA 1535), 9-aminoacridine (50 µg/plate with TA 1537)

Remarks:

without metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: preincubation
- Cell density at seeding (if applicable): no data

DURATION
- Preincubation period: 20 min at 37 °C
- Exposure duration: 48 hours at 37 °C

SELECTION AGENT (mutation assays): the lack of amino-acid in the medium. Only the mutants can grow due to their capability to synthesize the essential amino acid.

NUMBER OF REPLICATIONS: 3

- OTHER:
Dose-range finding experiment: Test substance was checked for toxicity to TA 100 up to a concentration of 10 mg/plate or the Iimit of solubility, both in the presence and absence of S-9 mix.

Evaluation criteria:

A positive response was indicated by a reproducible, dose-related increase, whether it be twofold over background or not.

Statistics:

The data were evaluated using analysis based on the models presented by Margolin et al (1981).

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Conclusions:

Under the test conditions, cineole is not considered as mutagenic in S. typhimurium strains TA 1535, TA 1537, TA 98 and TA 100.

Executive summary:

In a reverse gene mutation assay in bacteria, performed similarly to OECD guideline 471, strains of S. typhimurium (TA 1535, TA 1537, TA 100 and TA 98) were exposed to cineole with and without S9 metabolic activation [S9 fraction of Aroclor 1254-induced adult male Sprague Dawley rats and Syrian hamsters liver] according to the preincubation method (20 min). The positive controls induced the appropriate responses in the corresponding strains. Cineole showed no substantial increases in revertant colony numbers over control count obtained with any of the tester strains in either presence or absence of S9 mix. Under the test conditions, cineole is not considered as mutagenic in this bacterial system according to the criteria of the CLP Regulation (EC) N° (1272-2008). 

Reference 25

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

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

yes

Remarks:

only 2-h exposure with test substance with S9 was performed and only 100 cells per concentration were scored.

GLP compliance:

not specified

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):

CELLS USED
- Cell line: CHO-W-B1

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: McCoy's 5a medium with 10 % foetal calf serum, L-glutamine and antibiotics.

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9 mix consisting of 15 µL/mL liver homogenate (from male Sprague-Dawley rats, induced with Aroclor 1254), 2.4 mg/mL NADP, and 4.5 mg/mL isocitric acid in serum-free medium.

Test concentrations with justification for top dose:

- Without S9: 479 µg/mL - 663 µg/mL.
- With S9: 630 µg/mL - 810 µg/mL.

- Doses were chosen for the aberration test based on a preliminary test of cell survival 24 hrs after treatment. For most tests, doses were based on observations of cell confluence and mitotic cell availability in the SCE test (reported elsewhere).

Vehicle / solvent:

- Solvents used: either water, dimethyl sulfoxide (DMSO), ethanol or acetone, in that order of preference. It is unclear which solvent was used for the test material.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Remarks:

Solvents used: either water, dimethyl sulfoxide (DMSO), ethanol or acetone, in that order of preference. It is unclear which solvent was used for the test material.

Untreated negative controls:

no

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

without metabolic activation

Untreated negative controls:

no

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

with metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: In medium

DURATION
- Exposure duration: with S9 mix: 2 hr; without S mix: throughout incubation period
- Fixation time (start of exposure up to fixation or harvest of cells): 8 to 12 hr standard (cells in first mitosis). In cases where experience suggested that mitosis was delayed by the presence of the test material, harvesting was delayed to "e.g. 18-26 hr".

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

NUMBER OF REPLICATIONS: 1

NUMBER OF CELLS EVALUATED: 100 cells were scored from each of the three highest dose groups having sufficient metaphases for analysis and from positive and solvent controls.

OTHER EXAMINATIONS:
- Determination of polyploidy: Aberrations from polyploid cells not scored, but metaphases with 19-23 chromosomes were used.

- OTHER:
- All types of aberrations were recorded separately, but for data analysis they were grouped into categories of "simple" (breaks and terminal deletions), "complex" (exchanges and rearrangements), “other” (including pulverised chromosomes), and "total".

Evaluation criteria:

The analyses examined the evidence for a dose relation and absolute increase over the solvent control at each dose.

Statistics:

Linear regression analysis of the percentage of cells with aberrations vs the log-dose was used as the test for trend. To examine absolute increases over control levels at each dose, a binomial sampling assumption (as opposed to Poisson) was used, Margolin et al. (1983). The P values were adjusted by Dunnett's method to take into account the multiple dose comparisons. For data analysis, the "total" aberration category was used and the criterion for a positive response was that the adjusted P value be ≤ 0.05.

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:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: precipitation observed at concentrations ≥ 630 µg/mL.

Metabolic activation	Result	Range (µg/mL)	Least effect concentration (µg/mL)
Without S9 mix	-	479 - 663	-
With S9 mix	-	630 - 810	-

The least effective concentration tested (LEC) is the lowest dose to give a statistically significant increase (P ≤ 0.05) in aberrations. For chemicals with which the lowest dose tested gave a positive response, the LEC is preceded by "<". “-”, no LEC observed.

Conclusions:

The test substance was negative for chromosome aberrations with and without metabolic activation.

Executive summary:

In an in vitro mammalian chromosome aberration test performed similarly to OECD guideline 473, Chinese hamster Ovary (CHO) cells were exposed to 1,8 -cineole in McCoys 5a medium with and without metabolic activation [S9 fraction of livers from Aroclor 1254-induced male Sprague-Dawley rats (15 µL/mL)] at the following range concentrations: without S9: 479 - 663 µg/mL, and with S9: 630 - 810 µg/mL. Positive controls (mitomycin C without S9 and cyclophosphamide 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.

Reference 26

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)

GLP compliance:

not specified

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):

CELLS USED
- Cell line: CHO-W-B1

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: McCoy's 5a medium with 10% foetal calf serum, L-glutamine and antibiotics.

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9 mix consisting of 15 μL/mL liver homogenate (from male Sprague-Dawley rats, induced with Aroclor 1254), 2.4 mg/mL NADP, and 4.5 mg/mL isocitric acid in serum-free medium.

Test concentrations with justification for top dose:

- Without S9: 50 μg/mL - 500 μg/mL.
- With S9: 600 μg/mL - 800 μg/mL.

Doses selected on one of two bases (unclear on which basis the concentrations of test material were determined):
-- Preliminary growth inhibition test, counting cells excluding trypan blue 24 hr after treatment. Top dose was that estimated to reduce growth by 50 %.
-- Observation of cell monolayer and confluence activity in the cultures used for analysis of SCEs. Aim to obtain results at the highest dose at which sufficient metaphase cells would be available for analysis.

Vehicle / solvent:

- Solvents used: either water, dimethyl sulfoxide (DMSO), ethanol or acetone, in that order of preference. It is unclear which solvent was used for the test material.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Remarks:

Solvents used: either water, dimethyl sulfoxide (DMSO), ethanol or acetone, in that order of pre ference. It is unclear which solvent was used for the test material.

Untreated negative controls:

no

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

without metabolic activation

Untreated negative controls:

no

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

with metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: No data
- Exposure duration: without metabolic activation: approx. 25 hr; with metabolic activation: approx. 2 hr. 5-Bromodeoxyuridine (BrdUrd; 10 pM) was added 2 hr after addition of the test chemical (without S9) or immediately after the S9 mix plus chemical had been removed.
- Fixation time (start of exposure up to fixation or harvest of cells): 28 hrs. Immediately before harvesting, the cell monolayers were examined and the degree of confluence and availability of mitotic cells were noted.

SPINDLE INHIBITOR (cytogenetic assays): colcemid (0.1 µg/ml) present during the final 2-3 hr of total incubation time with 5-Bromodeoxyuridine (BrdUrd).

STAIN (for cytogenetic assays): 10 min in concentrated Hoechst 33258 (5 µg/mL in pH 6.8 buffer) and exposure to “black light” at 55 to 60 °C for approx. 5 min, then slides were stained with Giemsa.

NUMBER OF REPLICATIONS: 1

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: For a preliminary assessment of cell cycle delay, test slides were prepared from cells treated at the highest dose levels to see if later harvests were necessary. These test slides were stained with “dilute” Hoeschst 33258 (0.5 pg/ml in Sorensen’s buffer, pH 6.8) and examined by fluorescence microscopy to assess cell cycle kinetics. In control cultures, almost all cells completed two cycles in BrdUrd (M2 cells) in 25-26 hr, whereas, in treated cultures, cell cycle delay was common. In cases of severe delay, additional harvests were made from the same cultures at a later time to obtain sufficient second metaphase (M2) cells for SCE analysis. Later harvests were not necessary for this test substance.

NUMBER OF CELLS EVALUATED: 50 cells per dose scored from the 3 highest doses at which sufficient M2 cells available.

Statistics:

A linear regression test (trend test) of SCEs per chromosome vs the log of the dose was used. For individual doses, absolute increases in SCEs per chromosome of 20% or more over the solvent control were considered significant.

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation observed at concentrations ≥ 500 µg/mL.

The SCE test was positive only without activation at doses that induced cell cycle delay. No aberration induction was detected even after extending the incubation time without S9 to 20 hours.

Metabolic activation	Result	Range (µg/mL)	Least effective concentration (LEC) (µg/mL)
Without S9 mix	+	50 - 500	500 / 200
With S9 mix	-	600 - 800	-

The least effective concentration tested (LEC) is the lowest dose to give a 20% increase in SCEs.

Conclusions:

The test material was positive without metabolic activation but negative with metabolic activation in a sister chromatid exchange test. The SCE test was positive only without activation at doses that induced cell cycle delay. No aberration induction was detected even after extending the incubation time without S9 to 20 hours.

Executive summary:

Cloned Chinese hamster ovary cells were cultured in McCoy's 5a medium with 0 % foetal calf serum, L-glutamine and antibiotics. 5-Bromodeoxyuridine was added 2 hours after addition of the test chemical (without S9) or immediately after the S9 mix plus chemical had been removed. The chemical treatment period was approximately 25 hours without S9 ad 2 hours with S9. The total incubation time with 5-Bromodeoxyuridine was 25 - 26 hours, with colcemid present during the final 2-3 hours. Immediately before the cells were harvested, the cell monolayers were examined, and the degree of confluence and availability of mitotic cells were noted. Cells were collected by mitotic shake-off at doses up to the maximum considered likely to yield enough metaphase cells for analysis. Slides were stained, coded and scored.

The test material was positive without metabolic activation but negative with metabolic activation in a sister chromatid exchange test for genotoxicity. The SCE test was positive only without activation at doses that induced cell cycle delay. No aberration induction was detected even after extending the incubation time without S9 to 20 hours.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

Genetic toxicity in vivo: Weight of evidence: Several experimental results from studies performed with the main component d-limonene and the analogue substance alpha pinene are available. All study results were negative. Based on these results, no evidence of genotoxicity is predicted for the test substance.

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:

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

Principles of method if other than guideline:

Method: Comet assay (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

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: olive oil

Duration of treatment / exposure:

24 hours

Frequency of treatment:

Once

Post exposure period:

No data

Dose / conc.:

2 000 mg/kg bw (total dose)

No. of animals per sex per dose:

- Treatment groups: 4 males
- Vehicle control and untreated control groups: 12 males

Control animals:

yes, concurrent no treatment

yes, concurrent vehicle

Tissues and cell types examined:

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

Details of tissue and slide preparation:

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.

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

not applicable

Conclusions:

Under the test conditions, d-limonene is not considered as mutagenic in 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 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 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 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:

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

Principles of method if other than guideline:

Method: Comet assay (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

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: Olive oil

Duration of treatment / exposure:

24 hours

Frequency of treatment:

Once

Post exposure period:

No data

Dose / conc.:

2 000 mg/kg bw (total dose)

No. of animals per sex per dose:

- Treatment groups: 4 males
- Vehicle control and untreated control groups: 12 males

Control animals:

yes, concurrent no treatment

yes, concurrent vehicle

Tissues and cell types examined:

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

Details of tissue and slide preparation:

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.

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

not applicable

Conclusions:

Under the test conditions, d-limonene is not considered as mutagenic in 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 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: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

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 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

yes

Remarks:

no positive controls were tested

GLP compliance:

yes

Remarks:

in compliance with Food and Drug Administration Good Laboratory Practice Regulations (21 CFR, Part 58)

Type of assay:

other: Mammalian Erythrocyte Micronucleus Test

Species:

mouse

Strain:

B6C3F1

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: NTP colony maintained at Taconic Farms, Inc. (Germantown, NY)
- Age at study initiation: 5-6 weeks
- Weight at study initiation: 22.5-23 g (male) and 19.3-19-7 g (female)
- Housing: individually. Cages: Stainless steel, wire bottom (Lab Products, Inc., Seaford, DE); rotated weekly; cageboard: Untreated paper cage pan liner (Shepherd Specialty Papers, Kalamazoo, MI), changed daily
- Diet (e.g. ad libitum): NTP-2000 irradiated wafers (Zeigler Brothers, Inc., Gardners, PA), available ad libitum (except during exposure periods)
- Water (e.g. ad libitum): Tap water (Richland, WA, municipal supply) via automatic watering system (Edstrom Industries, Waterford, WI); available ad libitum
- aclimatation period: Animals were quarantined for 12 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 72 ± 3ºF
- Humidity (%): 50% ± 15%
- Room fluorescent light: 12 hours/day
- Chamber air changes: 15 ± 2/hour

Route of administration:

inhalation: vapour

Vehicle:

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

Details on exposure:

TYPE OF INHALATION EXPOSURE: whole body

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Test item was held in an 8-gallon stainless-steel chemical reservoir. Test item was pumped into a heated glass column filled with glass beads that increased the surface area for vaporization. Heated nitrogen entered the column from below and assisted in vaporizing the chemical while conveying it into a short distribution manifold. Concentration in the manifold was determined by the chemical pump rate, nitrogen flow rate, and dilution air flow rate. The pressure in the distribution manifold was kept fixed to ensure constant flow through the manifold and into all chambers as the flow of vapor to each chamber was adjusted.
Metering valves at the manifold controlled flow to each chamber through individual Teflon® delivery lines that carried the vapor from the manifold to three-way exposure valves at the chamber inlets. The exposure valves diverted vapor delivery to exposure chamber exhaust until the generation system was stable and exposures were ready to proceed. To initiate exposure, the chamber exposure valves were rotated to allow the test item vapor to flow to each exposure chamber inlet duct where it was further diluted with filtered, conditioned air to achieve the desired exposure concentration.
- Temperature, humidity, pressure in air chamber: 72 ± 3ºF; 50% ± 15%.
- Air change rate: 15 air changes per hour
- Method of particle size determination: A condensation particle detector (Model 3022A, TSI, Inc., St. Paul, MN) was used with and without animals in the exposure chambers. No particle counts above the minimum resolvable level (approximately 200 particles/cm3) were detected.

TEST ATMOSPHERE
- Brief description of analytical method used: on-line gas chromatograph. Samples were analyzed using GC/FID to measure the stability and purity of test item in the generation and delivery system. To assess whether impurities or degradation products coeluted with test item or the solvent, a second GC/FID analysis of the samples was performed using a polar column capable of resolving compounds with similar boiling points and polarities.
- Samples taken from breathing zone: yes.

Duration of treatment / exposure:

14 weeks; 6 hours plus T90 (10 minutes) per day

Frequency of treatment:

five times per week, weekdays only

Dose / conc.:

0 ppm

Dose / conc.:

25 ppm

Remarks:

(0.14 mg/L)

Dose / conc.:

50 ppm

Remarks:

(0.28 mg/L)

Dose / conc.:

100 ppm

Remarks:

(0.56 mg/L)

Dose / conc.:

200 ppm

Remarks:

(1.13 mg/L)

Dose / conc.:

400 ppm

Remarks:

(2.26 mg/L)

No. of animals per sex per dose:

5

Control animals:

yes, concurrent no treatment

Positive control(s):

none

Tissues and cell types examined:

Peripheral blood samples

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
2-week preliminary study were conducted to determine the highest administrable non lethal dose level. 5 mice per sex and per dose were exposed to 0, 100, 200, 400, 800, and 1,600 ppm test item.

TREATMENT AND SAMPLING TIMES:
At the end of the 3-month toxicity study, peripheral blood samples were obtained from male and female mice. Smears were immediately prepared and fixed in absolute methanol.

DETAILS OF SLIDE PREPARATION:
Slides were air-dried, fixed and stained with a fluorescent DNA-specific stain (acridine orange).

METHOD OF ANALYSIS:
Slides were scanned to determine the frequency of micronuclei in 2000 normochromatic erythrocytes (NCEs) in each of five animals per exposure group. In addition, the percentage of polychromatic erythrocytes (PCEs) among a population of 1000 erythrocytes was scored for each exposure group as a measure of bone marrow toxicity.

Evaluation criteria:

In the micronucleus test, an individual trial is considered positive if the trend test P value is less than or equal to 0.025 or if the P value for any single exposed group is less than or equal to 0.025 divided by the number of exposed groups. A final call of positive for micronucleus induction was preferably based on reproducibly positive trials. Ultimately, the final call was determined by the scientific staff after considering the results of statistical analyses, the reproducibility of any effects observed, and the magnitudes of those effects.

Statistics:

The results were tabulated as the mean of the pooled results from all animals within a treatment group plus or minus the standard error of the mean. The frequency of micronucleated cells among NCEs was analyzed by a statistical software package that tested for increasing trend over exposure groups with a one-tail Cochran-Armitage trend test, followed by pairwise comparisons between each exposed group and the control group. In the presence of excess binomial variation, as detected by a binomial dispersion test, the binomial variance of the Cochran-Armitage test was adjusted upward in proportion to the excess variation

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

not specified

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

not applicable

Additional information on results:

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): See table 1
- Ratio of PCE/NCE (for Micronucleus assay): See table 1

Table 1: Frequency of Micronuclei in Peripheral Blood Erythrocytes of Mice Following Treatment with alpha Pinene by Inhalation for 3 Months a

	Concentration (ppm)	Number of Mice with Erythrocytes Scored	Micronucleated NCEs/1,000 NCEs b	P Value c	PCEs b (%)
Male
Aird	0	5	1.6 ± 0.33		2.50 ± 0.39
Alpha pinene	25	5	1.8 ± 0.30	0.3657	2.34 ± 0.19
	50	5	1.9 ± 0.53	0.3059	2.20 ± 0.26
	100	5	2.1 ± 0.43	0.2053	2.88 ± 0.31
	200	5	1.9 ± 0.29	0.3059	2.74 ± 0.19
	400	5	1.4 ± 0.40	0.6426	3.10 ± 0.20
			P=0.742e
Female
Air	0	5	1.4 ± 0.19		2.40 ± 0.19
Alpha pinene	25	5	2.1 ± 0.43	0.1182	2.16 ± 0.26
	50	5	1.8 ± 0.25	0.2396	2.16 ± 0.20
	100	5	1.7 ± 0.44	0.2949	2.74 ± 0.36
	200	5	1.7 ± 0.30	0.2949	2.06 ± 0.29
	400	5	1.1 ± 0.19	0.7259	2.16 ± 0.06
			P=0.899

a Study was performed at ILS, Inc. The detailed protocol is presented by MacGregor et al. (1990). NCE=normochromatic erythrocyte; PCE=polychromatic erythrocyte

b Mean ± standard error

c Pairwise comparison with the chamber control group, significant at P≤0.005

d Chamber control

e Significance of micronucleated NCEs/1,000 NCEs tested by the one-tailed trend test; significant at P≤0.025

Conclusions:

Alpha-Pinene was not mutagenic in the mouse peripheral blood micronucleus test

Executive summary:

In a peripheral blood micronucleus test conducted similarly to OECD Guideline 474, alpha pinene was administered through inhalation to groups of B6C3F1 mice (5/sex/dose) at dose levels of 0, 25, 50, 100, 200 or 400 ppm; 5 days/week for 14 weeks. At the end of the study, peripheral blood samples were obtained from mice. Smear slides were air-dried, fixed, stained with fluorescent DNA-specific stain (acridine orange) and scanned to determine the frequency of micronuclei in 2000 normochromatic erythrocytes (NCEs) per animal. In addition, the percentage of polychromatic erythrocytes (PCEs) in a population of 1000 erythrocytes was determined.

No increase in the frequency of micronucleated erythrocytes and no significant changes in the percentages of polychromatic erythrocytes were observed in peripheral blood samples in male or female B6C3F1 mice administered alpha-pinene.

Therefore, alpha-pinene was not mutagenic in the mouse peripheral blood micronucleus test.

Reference 5

Endpoint:

in vivo mammalian germ 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

Principles of method if other than guideline:

Transgenic animal mutagenicity assay

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).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Weight of evidence from experimental results with individual components/analogue substances:

In vitro gene mutation in bacteria:

Camphene was tested in two different studies for mutagenecity on Salmonella typhimurium strains TA100 and TA98 and also two additional strains, UTH 8414 and UTH 8413, with and without metabolic activation (S9). Camphene was not mutagenic in all strains tested with or without metabolic activation.

L-limonene was tested in Salmonella typhimurium TA1535, TA1537, TA100, TA98 and TA102 in the presence and the absence of mammalian metabolic activation (S9). L-limonene was not mutagenic in all strains tested with or without metabolic activation.

D-limonene was tested in several studies for mutagenecity on Salmonella typhimurium strains TA 1535, TA 1537, TA 1538, TA 98 and TA 100 and also two additional strains, UTH 8414 and UTH 8413, with and without metabolic activation (S9). D-limonene was not mutagenic in all strains tested with and without metabolic activation.

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

L-alpha pinene was tested for mutagenecity on Salmonella typhimurium strains TA100, TA98, TA97a and TA1535 with and without metabolic activation (S9). L-alpha pinene was not mutagenic in all strains tested with and without metabolic activation.

Alpha pinene was tested in several studies for mutagenecity on Salmonella typhimurium strains TA 1535, TA 1537, TA1538, TA 98 and TA 100, and also two additional strains, UTH 8414 and UTH 8413,

with and without metabolic activation (S9). Alpha pinene was not mutagenic in all strains tested with and without metabolic activation.

Cineole was tested for mutagenecity on Salmonella typhimurium strains TA100, TA98, TA1535 and TA1537 with and without metabolic activation (S9). Cineole was not mutagenic in all strains tested with and without metabolic activation.

Based on a weight of evidence approach, the substance is determined to be non-mutagenic to bacteria.

In vitro mammalian chromosome 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. D-limonene is not considered as cytogenetic in CHO cells according to the criteria of the CLP Regulation (EC) N° (1272-2008).

The analogue 5-ethylidene-2-norbornene (ENB) was tested in an in vitro chromosome aberration study with cultured Chinese hamster ovary CHO cells in presence and absence of metabolic activation. It was concluded that ENB is not a clastogen.

Camphene was tested on sister chromatid exchange assay in cultured Chinese hamster ovary CHO-K1 cells without metabolic activation. Camphene did not induce sister chromatid exchanges in CHO cells at any of the doses tested.

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

Cineole was found positive without metabolic activation but negative with metabolic activation in a sister chromatid exchange assay. The SCE test was positive only without activation at doses that induced cell cycle delay. However, no aberration induction was detected in an in vitro mammalian chromosome aberration test performed similarly to OECD guideline 473, even after extending the incubation time without S9 to 20 hours.

Based on a weight of evidence approach, the substance is determined to be non-clastogenic to mammalian cells.

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. D-limonene was not considered as mutagenic in mouse lymphoma L5178Y cells.

 

To determine the potential for 5-Ethylidene-2-norbornene (ENB) to cause forward gene mutations, a CHO cell line was used for the detection of mutations at the HGPRT gene locus in a medium containing the purine analog 6-thioguanine (6-TG) with and without metabolic activation. ENB did not produce any statistically significant or dosage-related increase in the number of mutants cells with and without metabolic activation.

 

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

 

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.

Based on a weight of evidence approach, the substance is determined to be non-mutagenic to mammalian cells.

In vivo genetic toxicity:

In a peripheral blood micronucleus test conducted similarly to OECD Guideline 474, alpha pinene was administered through inhalation to groups of B6C3F1 mice (5/sex/dose). Alpha-pinene was found not mutagenic in this test.

 

d-limonene was tested in a transgenic rodent mutagenicity assay with male Big BlueTM rats (10/dose). 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, 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, 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 a weight of evidence approach, no evidence of genotoxicity is predicted for the substance.

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.