Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In reverse gene mutation assays in bacteria, performed according to OECD Guideline 471 and in compliance with GLP, dipentene multiconstituent and d-limonene were found as non mutagenic in the presence or absence of metabolic activation.

In a chromosome aberration test in human lymphocytes and in a study on sister chromatid exchange in CHO cells, no genotoxic effects were observed with d-limonene.

In two gene mutation tests in mouse lymphoma L5178Y cells, d-limonene did not demonstrate any mutagenic potential.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1990
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
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
Qualifier:
equivalent or similar to
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:
mammalian cell gene mutation assay
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:
- Trail 1: 0, 10, 20, 30, 40, 50 and 60 nL/mL
- Trail 2: 0, 30, 40, 50, 60, 80 and 100 nL/mL
- Trail 3: 0, 5, 10, 20, 30, 40 and 50 nL/mL
- Trail 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 Migrated to IUCLID6: 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 Migrated to IUCLID6: 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:
- Trail 1: Inconclusive (i)
- Trail 2: Questionable (?)
- Trail 3 and 4: Negative (-)
- Overall response: Negative (-)

Response in trials with S9:
- Trail 1 and 2: Negative (-)
- Trail 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
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

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:
d-Limonene was not considered as mutagenic in mouse lymphoma L5178Y cells and does not need to be classified according to Directive 67/548/EEC and CLP Regulation (EC) No 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 concentrations below

Without S9:

- 0, 10, 20, 30, 40, 50 and 60 nL/mL (trial 1)

- 0, 30, 40, 50, 60, 80 and 100 nL/mL (trial 2)

- 0, 5, 10, 20, 30, 40 and 50 nL/mL (trial 3)

- 0, 5, 10, 20, 30, 40, 50 and 60 nL/mL (trial 4)

With S9:

- 0, 10, 20, 30, 40, 50, 60 and 80 nL/mL (trial 1)

- 0, 10, 20, 30, 40, 50, 60 and 80 nL/mL (trial 2)

- 0, 30, 40, 50, 60, 80 and 100 nL/mL (trial 3)

 

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.

 

Therefore, d-limonene was not considered as mutagenic in mouse lymphoma L5178Y cells and does not need to be classified according to Directive 67/548/EEC and CLP Regulation (EC) No 1272/2008.

Endpoint:
in vitro DNA damage and/or repair study
Remarks:
Type of genotoxicity: DNA damage and/or repair
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1990
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
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
Qualifier:
equivalent or similar to
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
Principles of method if other than guideline:
Not applicable
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 Migrated to IUCLID6: 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 Migrated to IUCLID6: 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
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

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:
d-Limonene is not considered as cytogenetic in CHO cells according toDirective 67/548/EEC and CLP Regulation (EC) No 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 concentrations below.

Without S9:

- 0, 16.2, 54 and 162 µg/mL (trial 1)

- 0, 30, 50 and 100 µg/mL (trial 2)

- 0, 15, 30 and 50 µg/mL (trial 3)

With S9:

- 0, 16.2, 54 and 162 µg/mL (trial 1)

 

Clear increases in mean SCE/cell were induced by the positive control chemicals mitomycin C (without S9) and cyclophosphamide (with S9). In trail 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).

 

Therefore, d-limonene is not considered as cytogenetic in CHO cells according to Directive 67/548/EEC and CLP Regulation (EC) No 1272 /2008.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1990
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
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
Qualifier:
equivalent or similar to
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
Principles of method if other than guideline:
Not applicable
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 Migrated to IUCLID6: 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 Migrated to IUCLID6: 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.
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
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

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:
d-Limonene is not considered as cytogenetic in CHO cells according to Directive 67/548/EEC and CLP Regulation (EC) No 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 concentrations below.

- without S9: 0, 10, 30 and 100 µg/mL ;

- 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.

 

Therefore, d-limonene is not considered as cytogenetic in CHO cells according to Directive 67/548/EEC and CLP Regulation (EC) No 1272 /2008.

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
supporting study
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
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
Qualifier:
equivalent or similar to
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
Principles of method if other than guideline:
Not applicable
GLP compliance:
not specified
Type of assay:
mammalian cell gene mutation assay
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:
100 µg or nL/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
Remarks:
Migrated from field 'Test system'.

None

Conclusions:
d-Limonene is not considered as mutagenic in L5178Y cells according to Directive 67/548/EEC and CLP Regulation (EC) No 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 the presence or absence of S9 mix.

 

Therefore, d-limonene is not considered as mutagenic in L5178Y cells according to Directive 67/548/EEC and CLP Regulation (EC) No 1272/2008.

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
supporting study
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
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
Qualifier:
equivalent or similar to
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
Principles of method if other than guideline:
Not applicable
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay
Target gene:
Not applicable
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
Key result
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

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:
d-Limonene is not considered as mutagenic in S. typhimurium TA 1535, TA 1537, TA 98 and TA 100 according to Directive 67/548/EEC and CLP Regulation (EC) No 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 concentrations below.

- 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 the presence or the absence of S9 mix.  

Therefore, d-limonene is not considered as mutagenic in this bacterial system according to Directive 67/548/EEC and CLP Regulation (EC) No 1272/2008. 

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

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

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

Table 7.6.1/4:Results for the strainTA1535

Dipentene concentration plate (µg/plate)

Without metabolic activation

With metabolic activation

First experiment (direct plate incorporation)

Second experiment (direct plate incorporation)

First experiment (direct plate incorporation)

Second experiment (pre-incubation)

Ratio mean revertant treated/revertant solvent

Cytotoxicity*

Ratio mean revertant treated/revertant solvent

Cytotoxicity*

Ratio mean revertant treated/revertant solvent

Cytotoxicity*

Ratio mean revertant treated/revertant solvent

Cytotoxicity*

0

-

No

-

No

-

No

-

No

0.8

0.9

No

0.9

No

-

-

-

-

2.3

1.1

No

1.0

No

1.2

No

1.6

No

6.9

0.9

No

0.9

No

0.7

No

1.2

No

20.6

0.9

No

0.9

No

0.8

No

1.5

No

61.7

0.7

No

0.9

St

0.7

No

1.1

No

185.2

0.4

St

0.2

St

0.6

No

1.2

Mt

555.6

-

-

-

-

0.9

No

0.5

St

Postive controls

 

 

 

 

 

 

 

 

NaN3 (1µg)

28.6

No

17.1

No

-

-

-

 

2AM (2µg)

-

-

-

-

13.6

No

16.5

 

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

Table 7.6.1/5:Results for the strainTA1537

Dipentene concentration plate (µg/plate)

Without metabolic activation

With metabolic activation

First experiment (direct plate incorporation)

Second experiment (direct plate incorporation)

First experiment (direct plate incorporation)

Second experiment (pre-incubation)

Ratio mean revertant treated/revertant solvent

Cytotoxicity*

Ratio mean revertant treated/revertant solvent

Cytotoxicity*

Ratio mean revertant treated/revertant solvent

Cytotoxicity*

Ratio mean revertant treated/revertant solvent

Cytotoxicity*

0

-

No

-

No

-

No

-

No

0.3

-

-

0.3

No

-

-

-

 

0.8

1.0

No

0.7

No

-

-

1.2

-

2.3

1.1

No

1.1

No

-

-

0.9

No

6.9

0.7

No

0.8

No

1.3

No

1.5

No

20.6

0.7

Mt

0.5

Mt

1.3

No

1.4

No

61.7

0.0

St

1.7

St

1.5

No

1.1

No

185.2

0.0

St

-

-

1.7

No

0.7

Mt

555.6

-

-

-

-

0.9

No

-

-

1666.7

 

 

 

 

1.0

Mt

-

-

Postive controls

 

 

 

 

 

 

 

 

9AA (50µg)

18.1

No

32.9

No

-

-

-

-

2AM (2µg)

-

-

-

-

8.7

No

17.1

No

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

Table 7.6.1/6:Results for the strainTA98

Dipentene concentration plate (µg/plate)

Without metabolic activation

With metabolic activation

First experiment (direct plate incorporation)

Second experiment (direct plate incorporation)

First experiment (direct plate incorporation)

Second experiment (pre-incubation)

Ratio mean revertant treated/revertant solvent

Cytotoxicity*

Ratio mean revertant treated/revertant solvent

Cytotoxicity*

Ratio mean revertant treated/revertant solvent

Cytotoxicity*

Ratio mean revertant treated/revertant solvent

Cytotoxicity*

0

-

No

-

No

-

No

-

No

0.8

2.1

No

-

-

-

-

-

-

2.3

1.8

No

0.9

No

-

-

0.9

No

6.9

0.9

No

0.7

No

0.9

No

0.6

No

20.6

0.7

No

0.6

No

1.0

No

0.7

No

61.7

0.9

Mt

0.9

No

1.1

No

0.7

Mt

185.2

0.9

Mt

0.2

Mt

1.7

No

0.0

St

555.6

-

-

0.4

St

0.8

Mt

0.0

St

1666.7

-

-

-

-

0.8

Mt

-

-

Postive controls

 

 

 

 

 

 

 

 

2NF (0.5µg)

7.8

No

4.1

No

-

-

-

 

2AM (2µg)

-

-

-

-

26.4

No

45.6

N o

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

Table 7.6.1/7:Results for the strainTA100

Dipentene concentration plate (µg/plate)

Without metabolic activation

With metabolic activation

First experiment (direct plate incorporation)

Second experiment (direct plate incorporation)

First experiment (direct plate incorporation)

Second experiment (pre-incubation)

Ratio mean revertant treated/revertant solvent

Cytotoxicity*

Ratio mean revertant treated/revertant solvent

Cytotoxicity*

Ratio mean revertant treated/revertant solvent

Cytotoxicity*

Ratio mean revertant treated/revertant solvent

Cytotoxicity*

0

-

No

-

No

-

No

-

No

0.8

1.1

No

-

-

-

-

-

-

2.3

1.4

No

0.9

No

1.4

-

1.1

No

6.9

1.0

No

1.1

No

1.5

No

1.1

No

20.6

1.1

No

0.9

No

1.1

No

1.3

No

61.7

0.8

No

0.7

Mt

1.4

No

1.1

No

185.2

0.3

Mt

0.4

St

1.1

No

1.0

Mt

555.6

-

-

0.6

St

1.1

No

0.3

St

Postive controls

 

 

 

 

 

 

 

 

NaN3 (1µg)

4.7

No

7.7

No

-

-

-

 

BAP (5µg)

-

-

-

-

8.6

No

5.6

N o

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

Table 7.6.1/8:Results for the strainTA102

Dipentene concentration plate (µg/plate)

Without metabolic activation

With metabolic activation

First experiment (direct plate incorporation)

Second experiment (direct plate incorporation)

First experiment (direct plate incorporation)

Second experiment (pre-incubation)

Ratio mean revertant treated/revertant solvent

Cytotoxicity*

Ratio mean revertant treated/revertant solvent

Cytotoxicity*

Ratio mean revertant treated/revertant solvent

Cytotoxicity*

Ratio mean revertant treated/revertant solvent

Cytotoxicity*

0

-

No

-

No

-

No

-

No

0.8

1.4

No

-

-

-

-

-

-

2.3

1.8

No

-

-

-

-

0.8

No

6.9

1.9

No

1.0

No

-

-

1.1

No

20.6

1.3

No

1.0

No

1.0

No

1.1

No

61.7

1.2

No

0.9

No

1.5

No

1.0

No

185.2

1.6

No

0.7

Mt

1.3

No

1.0

No

555.6

-

-

0.6

Mt

1.6

No

0.6

Mt

1666.7

-

-

0.4

Mt

1.0

No

-

-

5000

-

-

-

-

1.4

No

-

-

Postive controls

 

 

 

 

 

 

 

 

MMC (0.5µg)

7.6

No

5.2

No

-

-

-

 

2AM (10µg)

-

-

-

-

3.8

No

5.5

N o

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

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

In a reverse gene mutation assay in bacteria (No. 36799 MMO), performed according to the OECD No. 471 and EC No. B13/14 guidelines, the test item 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.

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

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

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

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

 

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

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

Genetic toxicity in vivo

Description of key information

d-Limonene was also found negative in in vivo testing in comet assays.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vivo mammalian cell study: DNA damage and/or repair
Remarks:
Type of genotoxicity: DNA damage and/or repair
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2007
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
Study well documented, meets generally accepted scientific principles, acceptable for assessment
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
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 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
Remarks:
Doses / Concentrations:
0, 1000 and 2000 mg/kg bw in 0.5% CMC
Basis:
actual ingested
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:
d-Limonene is not considered as mutagenic in Comet assay on isolated kidney cells and does not need to be classified according to Directive 67/548/EEC and CLP Regulation (EC) No 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.

 

Therefore, d-limonene is not considered as mutagenic in Comet assay on isolated kidney cells and does not need to be classified according to Directive 67/548/EEC and CLP Regulation (EC) No 1272/2008.

Endpoint:
in vivo mammalian cell study: DNA damage and/or repair
Remarks:
Type of genotoxicity: DNA damage and/or repair
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
2002
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Remarks:
Study well documented, meets generally accepted scientific principles, acceptable for assessment
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
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
Duration of treatment / exposure:
24 hours
Frequency of treatment:
Once
Post exposure period:
No data
Remarks:
Doses / Concentrations:
2000 mg/kg bw
Basis:
actual ingested
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
Key result
Sex:
male
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
not applicable

None

Conclusions:
d-Limonene is not considered as mutagenic in Comet assay and does not need to be classified according to Directive 67/548/EEC and CLP Regulation (EC) No 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.

 

Therefore, d-limonene is not considered as mutagenic in Comet assay and does not need to be classified according to Directive 67/548/EEC and CLP Regulation (EC) No 1272/2008.

Endpoint:
in vivo mammalian cell study: DNA damage and/or repair
Remarks:
Type of genotoxicity: DNA damage and/or repair
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
2002
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Remarks:
Study well documented, meets generally accepted scientific principles, acceptable for assessment
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
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
Duration of treatment / exposure:
24 hours
Frequency of treatment:
Once
Post exposure period:
No data
Remarks:
Doses / Concentrations:
2000 mg/kg bw
Basis:
actual ingested
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
Sex:
male
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
not applicable

None

Conclusions:
d-Limonene is not considered as mutagenic in Comet assay and does not need to be classified according to Directive 67/548/EEC and CLP Regulation (EC) No 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.

 

Therefore, d-limonene is not considered as mutagenic in Comet assay and does not need to be classified according to Directive 67/548/EEC and CLP Regulation (EC) No 1272/2008.

Endpoint:
in vivo mammalian germ cell study: gene mutation
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
2001
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Remarks:
Test procedure in accordance with generally accepted scientific standards with minor deviations: no data on housing conditions
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
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 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
Remarks:
Doses / Concentrations:
1% w/w (corresponding to about 525 mg/kg bw/day)
Basis:
nominal in diet
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.
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 BlueTM 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 BlueTM 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 BlueTM 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 BlueTM rats 14 days after administration of the final dose of compound. Data were analysed for statistical significance as described herein; **P < 0.001.

Conclusions:
d-Limonene is not considered as mutagenic in the transgenic animal mutagenicity assay and does not need to be classified according to Directive 67/548/EEC and CLP Regulation (EC) No 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% of limonene, or 4-aminobiphenyl (20 mg/kg bw/day) was 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 d-limonene. Positive control induced the appropriate response.

Therefore, d-limonene is not considered as mutagenic in the transgenic animal mutagenicity assay and does not need to be classified according to Directive 67/548/EEC and CLP Regulation (EC) No 1272/2008.

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

Additional information

Negative results were obtained with dipentene multiconstituent in reverse mutation tests in bacterial strains TA1535, TA1537, TA100, TA98 and TA102. These results are confirmed with a supporting study showing no genotoxic effects of d-limonene in TA 1535, TA 1537, TA 100 and TA 98. These in vitro tests showed no effects in absence and in presence of metabolic activation.

Other in vitro genotoxicity tests conducted with d-limonene did not show any genotoxic effects in the presence and absence of metabolic activation:

- chromosome aberration test in human lymphocytes and sister chromatid exchange in CHO cells

- two gene mutation tests in mouse lymphoma L5178Y cells.

d-Limonene was also found negative in in vivo testing in one well conducted comet assay. This result was confirmed by supporting studies: negative results were found for d-limonene in 2 other comet assays and one test for mutation frequency of liver and kidney DNA samples of treated animals.

For further information on read-across justification, see section 13: point "read-across approach"

Justification for classification or non-classification

Negative results were obtained with d-limonene in Ames tests. d-Limonene was also negative in the other in vitro tests (cytogenicity and gene mutation tests in mammalian cells) and in vivo tests (comet assays). Therefore, d-limonene is not classified for mutagenicity according to CLP Regulation (EC) No 1272/2008 and Directive 67/548/EEC.