Registration Dossier

Toxicological information

Endpoint summary

Currently viewing:

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames test (study similar to OECD TG 471): negative

In vitro micronucleus test (OECD TG 487): not clastogenic or aneugenic

In vitro gene mutation using read across from Eucalyptus oil (OECD TG 476): negative

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
23 October - 06 December 2013
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:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay
Target gene:
None
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Details on mammalian cell type (if applicable):
Not applicable
Additional strain / cell type characteristics:
other: see table 7.6.1/1
Metabolic activation:
with and without
Metabolic activation system:
10 % (v/v) S9 mix; S9 fraction prepared from liver homogenates of rats induced with Phenobarbitone/β-Naphthoflavone at 80/100 mg/kg bw/day by oral route
Test concentrations with justification for top dose:
Preliminary Toxicity Test: 50, 150, 500, 1500 and 5000 μg/plate in TA100 or WP2uvrA strains, with and without S9- mix using preincubation method.

Mutation Test:
Experiment 1 (preincubation method):
- All strains (absence of S9-mix): 0.05, 0.15, 0.5, 1.5, 5, 15, 50, 150 and 500 µg/plate.
- All strains (presence of S9-mix): 0.5, 1.5, 5, 15, 50, 150, 500 and 1500 µg/plate.

Experiment 2 (preincubation method):
- All strains (absence of S9-mix): 0.05, 0.15, 0.5, 1.5, 5, 15, 50 and 150 µg/plate.
- All Salmonella strains (presence of S9-mix) and E.coli strain WP2uvrA (absence and presence of S9-mix): 0.15, 0.5, 1.5, 5, 15, 50, 150 and 500 µg/plate.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Dimethyl sulphoxide (DMSO)
- Justification for choice of solvent/vehicle: Test item was immiscible in sterile distilled water at 50 mg/mL but was fully miscible in DMSO at the same concentration. DMSO was selected as the vehicle.
- Formulation preparation: Test item was accurately weighed and approximate half-log dilutions prepared in DMSO by mixing on a vortex mixer and 5 minutes sonication at 40 °C on the day of each experiment. All formulations were used within four hours of preparation and were assumed to be stable for this period.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Remarks:
See Table 7.6.1/2
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
N-ethyl-N-nitro-N-nitrosoguanidine
Remarks:
without metabolic activation
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Remarks:
See Table 7.6.1/2
Positive control substance:
benzo(a)pyrene
other: 2-Aminoanthracene
Remarks:
with metabolic activation
Details on test system and experimental conditions:
SOURCE OF TEST SYSTEM: All strains of bacteria used in the test were obtained from the University of California, Berkeley, on culture discs, on 04 August 1995 or from the British Industrial Biological Research Association, on nutrient agar plates, on 17 August 1987.

METHOD OF APPLICATION: Preincubation (test item, vehicle and positive controls) and plate incorporation (negative control) methods

DURATION
- Preincubation period: 20 minutes at 37 ± 3 °C
- Incubation period: Plates were placed in anaerobic jars or bags (one jar/bag for each concentration of test item/vehicle) and incubated at 37 ± 3 °C for approximately 48 h.

NUMBER OF REPLICATIONS:
-1 plate/dose for preliminary toxicity test and 3 plates/dose for treatment, vehicle and positive controls in mutation test (Experiment 1 & 2).

DETERMINATION OF CYTOTOXICITY
- Method: Toxicity was determined on the basis of growth of the bacterial background lawn.

OTHER: After approximately 48 h incubation at 37 °C the plates were assessed for numbers of revertant colonies using a Domino colony counter.
Evaluation criteria:
There are several criteria for determining a positive result. Any one, or all of the following can be used to determine the overall result of the study:
1. A dose-related increase in mutant frequency over the dose range tested (De Serres and Shelby (1979)).
2. A reproducible increase at one or more concentrations.
3. Biological relevance against in-house historical control ranges.
4. Statistical analysis of data as determined by UKEMS (Mahon et al (1989)).
5. Fold increase greater than two times the concurrent solvent control for any tester strain (especially if accompanied by an out-of-historical range response, Cariello and Piegorsch, 1996).
- A test item will be considered non-mutagenic (negative) in the test system if the above criteria are not met.
- Although most experiments will give clear positive or negative results, in some instances the data generated will prohibit making a definite judgement about test item activity. Results of this type will be reported as equivocal.
Statistics:
- Statistical analysis of data as determined by UKEMS (Mahon et al (1989)).
Key result
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
initially from 50 µg/plate (Salmonella strains) and 150 µg/plate (Escherichia coli strain WP2uvrA)
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No test item precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix.

CYTOTOXICITY:
Preliminary Toxicity Test: Test item was initially toxic to TA100 from 50 µg/plate (absence of S9-mix), to WP2uvrA from 150 µg/plate (absence of S9-mix), and to TA100 and WP2uvrA from 500 µg/plate (presence of S9-mix).
Mutation Test: Test item caused a visible reduction in the growth of the bacterial background lawns of all of the tester strains, initially from 50 µg/plate (Salmonella strains) and 150 µg/plate (Escherichia coli strain WP2uvrA). The sensitivity of the tester strains to the toxicity of the test item varied slightly between strain type and exposures with or without S9 mix. The test item was tested up to to the toxic limit.

COMPARISON WITH HISTORICAL CONTROL DATA:
- Results were compared with historical negative, solvent and positive control data (2011 and 2012).

OTHERS:
- Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory). The amino acid supplemented top agar and S9-mix used in both experiments was shown to be sterile.
- The culture density for each bacterial strain was also checked and considered acceptable.
- Test item formulation and S9-mix used in this experiment were both shown to be sterile.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

See attached Document for Tables of Results

Conclusions:
Under the test conditions, Rosemary oil is not considered as mutagenic in S. typhimurium (TA 1535, TA 1537, TA 98 and TA 100) and E. coli (WP2uvrA) strains with and without metabolic activation.
Executive summary:

In a reverse gene mutation assay performed according to the OECD test guideline No. 471 and in compliance with GLP, S. typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and E.coli strain WP2 uvrA- were exposed the test material diluted in dimethyl sulfoxide both in the presence and absence of metabolic activation system (10% liver S9 in standard co-factors) using the pre-incubation method (under anaerobic conditions). The dose range for the first experiment was determined in a preliminary toxicity assay and ranged between 0.05 and 1500 µg/plate depending on bacterial strain type and presence or absence of metabolic activation. The experiment was repeated on a separate day using fresh cultures of the bacterial strains and fresh test material formulations. The dose range was based on the results of Experiment 1 and ranged between 0.05 and 500 µg/plate, depending on bacterial strain type and presence or absence of S9-mix. Additional dose levels and an expanded dose range were selected in both experiments in order to achieve both four non-toxic dose levels and the toxic limit of the test item.

The vehicle (dimethyl sulfoxide) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

The test item caused a visible reduction in the growth of the bacterial background lawns of all of the tester strains, initially from 50 µg/plate (Salmonella strains) and 150 µg/plate (Escherichia coli strain WP2uvrA). The sensitivity of the tester strains to the toxicity of the test item varied slightly between strain type and exposures with or without S9 mix. The test item was tested up to the toxic limit. No test item precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix.

No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation.

Under the test condition, Rosemary oil was not mutagenic to S. thyphimurium strains TA1535, TA1537 TA98, TA100, and E.coli WP2 uvrA-, in the presence and absence of metabolic activation.

This study is considered as acceptable and satisfies the requirement for reverse gene mutation endpoint.

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:
15 January - 25 February 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose for cross-reference:
reference to same study
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
14 Apr 2012
Type of assay:
mammalian cell gene mutation assay
Target gene:
TK gene
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
- Source: American Type Culture Collection (ATCC), Virginia.
- Type and identity of media: RPMI 1640 medium
- Properly maintained: Yes
- Periodically checked for Mycoplasma contamination: Yes
- Spontaneous thymidine kinase deficient mutants, TK -/-, were eliminated from the cultures by a 24 h incubation in the presence of methotrexate, thymidine, hypoxanthine and glycine two days prior to storage at -196 °C, in heat-inactivated donor horse serum (HiDHS) containing 10 % DMSO.
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9 fraction (5% v/v); S9 fraction was prepared from liver homogenates of male Sprague Dawley rats treated with phenobarbital and 5,6 benzoflavone.
Test concentrations with justification for top dose:
Preliminary toxicity test:
- 9.77, 19.53, 39.06, 78.13, 156.25, 312.5, 625, 1250, 2500 and 5000 μg/mL, with S9 mix (3 h exposure) and without S9 mix (3 and 24 h exposure)

Mutation tests:
- Without S9 mix (3 h exposure): 10, 100, 150, 200, 225, 250, 275 and 300 μg/mL
- Without S9 mix (3 h exposure, additional test): 10, 100, 115, 130, 145, 160, 175, 190, 210, 225, 250 and 300 μg/mL
- With S9 mix (3 h exposure): 10, 100, 115, 130, 145, 160, 175, 190, 210, 225, 250 and 300 μg/mL
- Without S9 mix (24 h exposure): 10, 50, 100, 150, 175, 200, 225, 250, 275 and 300 μg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Acetone
- Formulation preparation: Eucalyptus oil was dissolved and diluted in acetone (analytical grade), shortly before dosing. The final concentration of acetone added to the cultures was 1 % v/v. Eucalyptus oil was found to be miscible at 500 mg/mL in acetone.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
Acetone
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
10 μg/mL (3 h exposure); 5 μg/mL (24 h exposure) - without metabolic activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
Acetone
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
1 μg/mL (3 h exposure); with metabolic activation
Details on test system and experimental conditions:
METHOD OF APPLICATION: RPMI 1640 medium
- R0: RPMI 1640, buffered with 2 mg/mL sodium bicarbonate, supplemented with 2.0 mM L-glutamine and 50 μg/mL gentamicin.
- R10p: R0, supplemented with 0.1 % v/v Synperonic F68, 1.0 mM sodium pyruvate and HiDHS at 10 % v/v.
- R30p: R0, supplemented with 0.02 % v/v Synperonic F68, 1.0 mM sodium pyruvate and HiDHS at 30 % v/v.
- R10p medium was used for cell culture unless otherwise specified.
- R20p medium was used for the cloning efficiency plating. This was prepared by mixing equal volumes of R10p and R30p.

DURATION
- Exposure duration: Preliminary toxicity test: 3 and 24 h exposure (without S9 mix) and 3 h exposure (with S9 mix); Mutation tests: 3 and 24 h exposure (without S9 mix); 3 h exposure (with S9 mix)
- Expression time (cells in growth medium): 48 h
- Selection time (if incubation with a selection agent): 7 days for viability plates and approximately 10 to 14 days for mutant plates
- All incubations were performed at 37 °C in a humidified atmosphere of 5 % CO2 in air.

SELECTION AGENT (mutation assays): Selective medium consisted of R10p containing 4 μg/mL trifluorothymidine (TFT).

NUMBER OF REPLICATIONS:
- Preliminary toxicity test: Single culture/dose for test item and 2 cultures for vehicle control
- Main test: 4 cultures for vehicle control, 2 cultures/dose for test item and positive controls

NUMBER OF CELLS EVALUATED: 1.6 and 2000 cells per well plated for assessing cloning efficiency (CE) and mutant frequency (MF), respectively.

DETERMINATION OF CYTOTOXICITY
- Method: Relative suspension growth (RSG), Cloning efficiency (CE), Relative cloning efficiency (RCE) and Relative total growth (RTG)
RSG = (Individual SG x 100) / Mean vehicle control SG
CE = - InP(0) / No. of cells per well
RCE = (Individual CE x 100) / Mean vehicle control CE
RTG = (RSG x Day2 RCE) / 100

OTHER:
Mutant frequency per 10^6 survivors (MF) was calculated as:: CE selective medium / CE non-selective medium
Evaluation criteria:
The following criteria were applied for assessment of individual assay results using data for MF where the RTG normally exceeded 10 %:
Definitions:
GEF = Global Evaluation Factor. For microwell assays this is 126 x 10^-6 (Moore et al., 2006).
The assay was considered valid in accordance with the assay acceptance criteria.
The test agent was regarded as negative if:
- The mean mutant frequency of all test concentrations was less than the sum of the mean concurrent vehicle control mutant frequency and the GEF.
If the mutant frequency of any test concentrations exceeded the sum of the mean concurrent solvent control mutant frequency and the GEF, a linear trend test was applied:
- If the linear trend test was negative, the result was regarded as negative.
- If the linear trend test was positive, this indicated a positive, biologically relevant response.
- Where appropriate, other factors were considered in the interpretation of the results, for example, the reproducibility within and between tests, the overall number of mutant colonies (as opposed to mutation frequency) and the nature of any concentration-related effect(s).
- Results that only partially satisfied the assessment criteria described above were considered on a case-by-case basis. In cases where the results were inconclusive, further testing and/or a test modification may have been required to better define the assay response.
Statistics:
The data were analysed using Fluctuation application SAFEStat (SAS statistical applications for end users) version 1.1, which follows the methods described by Robinson et al. (1989) using a one-sided F-test, where p<0.001. Statistics were only reported if the sum of the mean concurrent vehicle control mutant frequency and the Global Evaluation Factor was exceeded, and this was accompanied by a significant positive linear trend.
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No change in the pH of the medium of more than 1.0 unit compared with the vehicle control was observed at 5000 μg/mL.
- Effects of osmolality: The osmolality of the test substance in medium was tested at a concentration of 5000 μg/mL; no change in osmolality of the medium of more than 50 mOsm/kg was observed when compared with the vehicle control.
- Precipitation: A solution of 500 mg/mL, dosed at 1 % in medium, showed no precipitate in the culture medium.

PRELIMINARY TOXICITY TEST:
- In preliminary toxicity test, precipitate (observed by eye at the end of treatment) was observed at 312.5 μg/mL and above and 625 μg/mL and above in the absence and presence of S9 mix respectively, following a 3 h exposure. Exposure to Eucalyptus oil at concentrations from 9.77 to 5000 μg/mL in the absence and presence of S9 mix (3 h exposure) resulted in relative suspension growth (RSG) values between 99 and 0 % and between 91 and 0 % respectively. Following a 24 h exposure in the absence of S9 mix (from 9.77 to 5000 μg/mL), no precipitation (assessed by eye at the end of treatment) was observed at any concentration and RSG was reduced from 98 to 0 %.

COMPARISON WITH HISTORICAL CONTROL DATA:
- Results were compared with historical control data (14 March 2011 - 18 February 2013)
Remarks on result:
other: strain/cell type: TK^+/-
Remarks:
Migrated from field 'Test system'.

Table 7.6.1/1: Results summary

Test Article

Dose Level

µg/mL

3 h Treatment ‑S9‑mix

3 h Treatment +S9‑mix

24 h Treatment ‑S9‑mix

Mean Relative Total Growth
(%)

Mean Mutant Freq. (x10‑6)

Mean Relative Total Growth
(%)

Mean Mutant
Freq. (x10‑6)

Mean Relative Total Growth
(%)

Mean Mutant
Freq. (x10‑6)

Acetone

0

100

86

100

75

100

61

Eucalyptus Oil

10

110

67

96

73

106

83

50

 

 

 

 

77

80

100

 

 

 

 

51

66

115

95

74

 

 

 

 

145

89

66

84

81

 

 

150

 

 

 

 

13

68

160

51

89

 

 

 

 

175

18

84

76

93

 

 

225

 

 

53

84

 

 

250

 

 

22

117

 

 

Methyl methanesulphonate

10

128

699

 

 

 

 

5

 

 

 

 

78

746

Benzo[a]pyrene

1

 

 

96

380

 

 

 

Conclusions:
Under the test conditions, Eucalyptus oil is not considered as mutagenic at the tk locus of L5178Y mouse lymphoma cells in the presence and absence of metabolic activation.
Executive summary:

In an in vitro mammalian cell gene mutation test performed according to OECD Guideline 476 and in compliance with GLP, L5178Y tk+/-(3.7.2C) mouse lymphoma cells were exposed to Eucalyptus oil at the following concentrations:

 

Preliminary toxicity test:
- 9.77, 19.53, 39.06, 78.13, 156.25, 312.5, 625, 1250, 2500 and 5000 μg/mL, with S9 mix (3 h exposure) and without S9 mix (3 and 24 h exposure)

Mutation tests:
- Without S9 mix (3 h exposure): 10, 100, 150, 200, 225, 250, 275 and 300 μg/mL
- Without S9 mix (3 h exposure, additional test): 10, 100, 115, 130, 145, 160, 175, 190, 210, 225, 250 and 300 μg/mL
- With S9 mix (3 h exposure): 10, 100, 115, 130, 145, 160, 175, 190, 210, 225, 250 and 300 μg/mL
- Without S9 mix (24 h exposure): 10, 50, 100, 150, 175, 200, 225, 250, 275 and 300 μg/mL

 

Vehicle (acetone) and positive control groups were also included in each mutation test. Metabolic activation system used in this test was 5 % (v/v) S9 mix; S9 fraction was prepared from liver homogenates of male Sprague Dawley rats treated with phenobarbital and 5,6 benzoflavone.

 

In preliminary toxicity test, precipitate (observed by eye at the end of treatment) was observed at 312.5 μg/mL and above and 625 μg/mL and above in the absence and presence of S9 mix respectively, following a 3 h exposure. Exposure to Eucalyptus oil at concentrations from 9.77 to 5000 μg/mL in the absence and presence of S9 mix (3 h exposure) resulted in relative suspension growth (RSG) values between 99 and 0 % and between 91 and 0 % respectively. Following a 24 h exposure in the absence of S9 mix (from 9.77 to 5000 μg/mL), no precipitation (assessed by eye at the end of treatment) was observed at any concentration and RSG was reduced from 98 to 0 %. Following 3 h treatment in the absence and presence of S9 mix, there were no increases in the mean mutant frequencies of any of the test concentrations assessed that exceeded the sum of the mean concurrent vehicle control mutant frequency and the Global Evaluation Factor (GEF) (212x 10-6 and 201 x 10-6, respectively), within acceptable levels of toxicity. The maximum concentrations assessed for mutant frequency in the 3 h treatment in the absence and presence of S9 mix were 175 and 250 µg/mL respectively. In the absence and presence of S9 mix RTG was reduced to 18 and 22 % respectively. In the 24 h treatment, the maximum concentration assessed for mutant frequency was 150 µg/mL. No increase in mutant frequency exceeded the sum of the mean concurrent vehicle control mutant frequency and the GEF (187 x 10-6), within acceptable levels of toxicity. The RTG was reduced to 13 %. Mutant frequencies in vehicle control cultures fell within acceptable ranges and clear increases in mutation were induced by the positive control chemicals [Methyl methanesulphonate (without S9 mix) and Benzo[a]pyrene (with S9 mix)] indicating the validity of the study.

 

Under the test conditions, Eucalyptus oil is not considered as mutagenic at the tk locus of L5178Y mouse lymphoma cells in the presence and absence of metabolic activation.

Endpoint:
in vitro cytogenicity / micronucleus study
Type of information:
experimental study
Adequacy of study:
key study
Study period:
23 October 2014 - 20 November 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)
GLP compliance:
yes
Type of assay:
in vitro mammalian cell micronucleus test
Target gene:
n.a.
Species / strain / cell type:
mammalian cell line, other:
Details on mammalian cell type (if applicable):
human peripheral blood lymphocytes
Additional strain / cell type characteristics:
not applicable
Cytokinesis block (if used):
Cytochalasin B
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254-induced rat liver S9
Test concentrations with justification for top dose:
Non-activated:
4 hr: 10, 25, 50, 100, 125, 150, 200, 250, 300, 350, 400, 450, 500, 700 µg/mL
24 hr: 5, 10, 25, 35, 50, 60, 75, 100, 125 µg/mL
S9-activated:
4 hr: 25, 50, 100, 150, 175, 200, 225, 250, 275, 300, 350, 400, 450 µg/mL
Vehicle / solvent:
- Vehicle/solvent used: Ethanol
- Justification for choice of solvent/vehicle: Ethanol was used as the vehicle based on the solubility of the test substance and compatibility with the target cells. In a solubility test, the test substance was soluble in ethanol at a concentration of approximately 500 mg/mL, the maximum concentration tested for solubility. Since the vehicle has limited historical control data, an untreated control was included in the study in order to compare the toxicity and micronucleus data with that of the vehicle control.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
other: Vinblastine
Remarks:
MCC: Positive control substance in the non-activated test system for clastogenicity. VB: Non-activated test system for aneugenicity. CP: Positive control substance in the S9-activated test system.
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 44-48 hours.
- Exposure duration: Non-activated 4h or 24h. S9-activated: 4h
- Fixation time: overnight or longer, Methanol: glacial acetic acid, 25:1 v/v

STAIN: acridine orange

NUMBER OF REPLICATIONS:
2 Replicate Cultures

NUMBER OF CELLS EVALUATED:
Cell Cycle Kinetics Scoring: For the preliminary toxicity test, at least 500 cells were evaluated to determine the CBPI at each dose level and the control. For the micronucleus assay, at least 1,000 cells (500 cells per culture) were evaluated to determine the CBPI at each dose level and the control.
Micronucleus Scoring: A minimum of 2000 binucleated cells from each concentration (1000 binucleated cells from each culture) were examined and scored for the presence of micronuclei.

CRITERIA FOR MICRONUCLEUS IDENTIFICATION:
Micronuclei in a binucleated cell (MN-BN) were recorded if they meet the following criteria:
• the micronucleus should have the same staining characteristics as the main nucleus.
• the micronuclei should be separate from the main nuclei or just touching (no cytoplasmic bridges).
• the micronuclei should be of regular shape and approximately 1/3 or less than the diameter of the main nucleus.

DETERMINATION OF CYTOTOXICITY
Preliminary Toxicity Test for Selection of Dose Levels: Dose levels for the micronucleus assay were based upon post-treatment toxicity (CBPI relative to the vehicle control).

OTHER EXAMINATIONS:
Preliminary Toxicity Test for Selection of Dose Levels: The precipitation in the treatment medium was determined using unaided eye at the beginning and conclusion of treatment. The osmolality of the solvent, the highest dose level, the highest precipitating dose level, and the highest soluble dose level in treatment medium was measured.
Rationale for test conditions:
The in vitro mammalian cell micronucleus assay was conducted using standard procedures (Kirsch-Volders et al. 2000; Parry and Sors 1993; Fenech and Morley, 1986; Fenech 1993) by exposing HPBL to appropriate concentrations of the test substance as well as the concurrent positive and vehicle controls, in the presence and absence of an exogenous metabolic activation system.
Evaluation criteria:
The test substance would be considered positive if it induced a statistically significant and dose-dependent increase the frequency of MN-BN cells (p ≤ 0.05). If only one criterion was met (statistically significant OR dose-dependent increase), the result was considered equivocal. If neither criterion was met, the results were considered to be negative.
Other criteria were also used in reaching a conclusion about the study results (e.g., comparison to historical control values, biological significance, etc.). In such cases, the Study Director used sound scientific judgment and clearly reported and described any such considerations.
Statistics:
Statistical analysis of the percentage of micronucleated cells was performed using the Fisher's exact test. The Fisher's test was used to compare pairwise the percent micronucleated cells of each treatment group with that of the vehicle control. The Cochran-Armitage test was used to measure dose-responsiveness.
Key result
Species / strain:
mammalian cell line, other: HPBL
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING/SCREENING STUDIES:
Preliminary Toxicity Test
A preliminary toxicity test was conducted to observe the cytotoxicity profile of the test substance and to select suitable dose levels for the definitive micronucleus assay. HPBL cells were first exposed to nine concentrations of Rosemary Oil ranging from 0.5 to 5000 μg/mL, as well as vehicle and untreated controls, in both the absence and presence of an Aroclor-induced S9 activation system for 4 hours, or continuously for 24 hours in the absence of S9 activation. The test substance was soluble in ethanol at all concentrations tested. Visible precipitate was observed in treatment medium at dose levels ≥ 500 μg/mL, while dose levels ≤ 150 μg/mL were soluble in treatment medium at the beginning and conclusion of the treatment period. At the conclusion of the treatment period, hemolysis was observed at dose levels ≥ 500 μg/mL in all three treatment conditions.

The osmolality in treatment medium of the highest dose level tested, 5000 μg/mL, was 275 mmol/kg. The osmolality in treatment medium of the lowest precipitating dose level, 500 μg/mL, was 278 mmol/kg. The osmolality in treatment medium of the highest soluble dose level, 150 μg/mL, was 278 mmol/kg. The osmolality of the vehicle (ethanol) in the treatment medium was 287 mmol/kg. The osmolality of the test substance dose levels in treatment medium is acceptable because it did not exceed the osmolality of the vehicle by more than 20%. The pH of the highest concentration of test substance in treatment medium was 7.5.

Substantial cytotoxicity [≥ 50% cytokinesis-blocked proliferation index (CBPI) relative to the vehicle control] was observed at dose levels 150, 1500, and 5000 μg/mL μg/mL in the non-activated 4-hour exposure group, at dose levels ≥ 500 μg/mL in the S9-activated 4-hour exposure group, and at dose levels ≥ 150 μg/mL in the non-activated 24-hour exposure group.

CONTROL DATA
The results of the CBPI and micronucleus data from the untreated control were comparable to that of the vehicle control. Therefore use of ethanol as vehicle control in this assay was justified. The results for the positive and vehicle controls indicate that all criteria for a valid assay were met. Based on these criteria, the results are justified and do not require a repeat of any portions of the study.
Conclusions:
Under the conditions of the assay described in this report, Rosemary Oil was concluded to be negative for the induction of micronuclei in the non-activated and S9-activated test systems in the in vitro mammalian micronucleus test using human peripheral blood lymphocytes.
Executive summary:

The test substance, Rosemary Oil (CAS# 8000-25-7), was tested in the in vitro mammalian cell micronucleus test according to OECD TG 478, using human peripheral blood lymphocytes (HPBL) in both the absence and presence of an Aroclor-induced S9 activation system. A preliminary toxicity test was performed to establish the dose range for testing in the micronucleus test. The micronucleus assay was used to evaluate the aneugenic and clastogenic potential of the test substance. In the preliminary toxicity and the micronucleus assays, HPBL cells were treated for 4 and 24 hours in the non-activated test system and for 4 hours in the S9-activated test system. All cells were harvested 24 hours after treatment initiation.

Based on the result of a preliminary toxicity assay, the doses chosen for the micronucleus assay ranged from 10 to 700 μg/mL for the non-activated 4-hour exposure group, from 25 to 450 μg/mL for the S9-activated 4-hour exposure group, and from 5 to 125 μg/mL for the non-activated 24-hour exposure group.

In the micronucleus assay, substantial cytotoxicity was observed at dose levels ≥ 150 μg/mL in the non-activated 4-hour exposure group, at dose levels ≥ 250 μg/mL in the S9-activated 4-hour exposure group, and at dose levels ≥ 75 μg/mL in the non-activated 24-hour exposure group. The highest dose analyzed under each treatment condition produced 50 to 60% reduction in CBPI, which met the dose limit as recommended by testing guidelines for this assay. A minimum of 1000 binucleated cells from each culture were examined and scored for the presence of micronuclei.

The percentage of cells with micronucleated binucleated cells in the non-activated 4 and 24-hour exposure groups was not statistically significantly increased relative to vehicle control at any dose level (p > 0.05, Fisher’s Exact test).

The percentage of cells with micronucleated binucleated cells in the S9-activated 4-hour exposure group was statistically significantly increased relative to vehicle control at 50 μg/mL (p ≤ 0.05, Fisher’s Exact test). However, the Cochran-Armitage test was negative for a dose response (p > 0.05). In addition, the percentage of cells with micronucleated binucleated cells test substance-treated group (0.6%) was within the historical solvent control range of 0.0% to 1.5%. Therefore, the statistically significant increase was considered to be biologically irrelevant.

The results for the positive and negative controls indicate that all criteria for a valid assay were met.

Based on the findings of this study, Rosemary Oil was concluded to be negative for the induction of micronuclei in both non-activated and S9-activated test systems in the in vitro mammalian cell micronucleus test using human peripheral blood lymphocytes.

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: read across
Justification for type of information:
The read across justification is presented in the endpoint summary and the acompanying files are also attached there.
Reason / purpose for cross-reference:
read-across source
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
other: strain/cell type: TK^+/-
Remarks:
Migrated from field 'Test system'.
Conclusions:
The mutagenicity in bacterial cells of Rosemary oil is assessed by using read across from Eucalyptus oil, and is considered not mutagenic at the tk locus of L5178Y mouse lymphoma cells in the presence and absence of metabolic activation.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Mode of Action Analysis / Human Relevance Framework

All in vitro genotoxicity tests are negative and therefore the substance does not have a genotoxic mode of action.

Additional information

Ames test

A Bacterial Reverse mutation Assay (Ames test) was performed according to OECD test guideline No 471 with Rosemary oil (Test No. 1). No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose, either in the presence or absence of metabolic activation. Rosemary oil does not induce gene mutations in bacteria under the test conditions whereas the positive control chemical (with and without metabolic activation) induced significant increase of colonies. Rosemary oil is therefore considered as non-mutagenic according to the Ames test.

In vitro mammalian micronucleus test

The test substance, Rosemary Oil (CAS# 8000-25-7), was tested in the in vitro mammalian cell micronucleus test according to OECD TG 478, using human peripheral blood lymphocytes (HPBL) in both the absence and presence of an Aroclor-induced S9 activation system. A preliminary toxicity test was performed to establish the dose range for testing in the micronucleus test. The micronucleus assay was used to evaluate the aneugenic and clastogenic potential of the test substance. In the preliminary toxicity and the micronucleus assays, HPBL cells were treated for 4 and 24 hours in the non-activated test system and for 4 hours in the S9-activated test system. All cells were harvested 24 hours after treatment initiation.

Based on the result of a preliminary toxicity assay, the doses chosen for the micronucleus assay ranged from 10 to 700 μg/mL for the non-activated 4-hour exposure group, from 25 to 450 μg/mL for the S9-activated 4-hour exposure group, and from 5 to 125 μg/mL for the non-activated 24-hour exposure group.

In the micronucleus assay, substantial cytotoxicity was observed at dose levels ≥ 150 μg/mL in the non-activated 4-hour exposure group, at dose levels ≥ 250 μg/mL in the S9-activated 4-hour exposure group, and at dose levels ≥ 75 μg/mL in the non-activated 24-hour exposure group. The highest dose analyzed under each treatment condition produced 50 to 60% reduction in CBPI, which met the dose limit as recommended by testing guidelines for this assay. A minimum of 1000 binucleated cells from each culture were examined and scored for the presence of micronuclei.

The percentage of cells with micronucleated binucleated cells in the non-activated 4 and 24-hour exposure groups was not statistically significantly increased relative to vehicle control at any dose level (p > 0.05, Fisher’s Exact test).

The percentage of cells with micronucleated binucleated cells in the S9-activated 4-hour exposure group was statistically significantly increased relative to vehicle control at 50 μg/mL (p ≤ 0.05, Fisher’s Exact test). However, the Cochran-Armitage test was negative for a dose response (p > 0.05). In addition, the percentage of cells with micronucleated binucleated cells test substance-treated group (0.6%) was within the historical solvent control range of 0.0% to 1.5%. Therefore, the statistically significant increase was considered to be biologically irrelevant.

The results for the positive and negative controls indicate that all criteria for a valid assay were met.

Based on the findings of this study, Rosemary Oil was concluded to be negative for the induction of micronuclei in both non-activated and S9-activated test systems in the in vitro mammalian cell micronucleus test using human peripheral blood lymphocytes.

In vitro gene mutation

The in vitro gene mutation of Rosemary oil was assessed by using read across from Eucalyptus oil (CAS no. 84625-32-1). First the experimental toxicity information of Eucalyptus oil will be summarised. Thereafter the read across justification is presented. The accompanying files are attached in the present endpoint summary.

Read across with Eucalyptus oil

In an in vitro mammalian cell gene mutation test performed according to OECD Guideline 476 and in compliance with GLP, L5178Y tk+/-(3.7.2C) mouse lymphoma cells were exposed to Eucalyptus oil at the following concentrations:

Preliminary toxicity test:
- 9.77, 19.53, 39.06, 78.13, 156.25, 312.5, 625, 1250, 2500 and 5000 μg/mL, with S9 mix (3 h exposure) and without S9 mix (3 and 24 h exposure)
Mutation tests:
- Without S9 mix (3 h exposure): 10, 100, 150, 200, 225, 250, 275 and 300 μg/mL
- Without S9 mix (3 h exposure, additional test): 10, 100, 115, 130, 145, 160, 175, 190, 210, 225, 250 and 300 μg/mL
- With S9 mix (3 h exposure): 10, 100, 115, 130, 145, 160, 175, 190, 210, 225, 250 and 300 μg/mL
- Without S9 mix (24 h exposure): 10, 50, 100, 150, 175, 200, 225, 250, 275 and 300 μg/mL

Vehicle (acetone) and positive control groups were also included in each mutation test. Metabolic activation system used in this test was 5 % (v/v) S9 mix; S9 fraction was prepared from liver homogenates of male Sprague Dawley rats treated with phenobarbital and 5,6 benzoflavone.

In preliminary toxicity test, precipitate (observed by eye at the end of treatment) was observed at 312.5 μg/mL and above and 625 μg/mL and above in the absence and presence of S9 mix respectively, following a 3 h exposure. Exposure to Eucalyptus oil at concentrations from 9.77 to 5000 μg/mL in the absence and presence of S9 mix (3 h exposure) resulted in relative suspension growth (RSG) values between 99 and 0 % and between 91 and 0 % respectively. Following a 24 h exposure in the absence of S9 mix (from 9.77 to 5000 μg/mL), no precipitation (assessed by eye at the end of treatment) was observed at any concentration and RSG was reduced from 98 to 0 %. Following 3 h treatment in the absence and presence of S9 mix, there were no increases in the mean mutant frequencies of any of the test concentrations assessed that exceeded the sum of the mean concurrent vehicle control mutant frequency and the Global Evaluation Factor (GEF) (212x 10-6and 201 x 10-6, respectively), within acceptable levels of toxicity. The maximum concentrations assessed for mutant frequency in the 3 h treatment in the absence and presence of S9 mix were 175 and 250 µg/mL respectively. In the absence and presence of S9 mix RTG was reduced to 18 and 22 % respectively. In the 24 h treatment, the maximum concentration assessed for mutant frequency was 150 µg/mL. No increase in mutant frequency exceeded the sum of the mean concurrent vehicle control mutant frequency and the GEF (187 x 10-6), within acceptable levels of toxicity. The RTG was reduced to 13 %. Mutant frequencies in vehicle control cultures fell within acceptable ranges and clear increases in mutation were induced by the positive control chemicals [Methyl methanesulphonate (without S9 mix) and Benzo[a]pyrene (with S9 mix)] indicating the validity of the study.

Under the test conditions, Eucalyptus oil is not considered as mutagenic at the tk locus of L5178Y mouse lymphoma cells in the presence and absence of metabolic activation.

Read across justification

The genotoxicity of Rosemary oil (CAS 84604-14-8; target) using read across from Eucalyptus oil (CAS 84265-32-1; source)

 

Introduction

Rosemary oil is a UVCB containing hydrocarbon type of substances. The main substances contain an ether group (cineol), solely hydrocarbon (alpha-pinene and camphene) or a ketone (camphor). For this substance no genotoxicity data are available. In accordance with Article 13 of REACH, lacking information should be generated whenever possible by means other than vertebrate animal tests, i.e. applying alternative methods such as in vitro tests, QSARs, grouping and read-across. For assessing the genotoxicity of Rosemary oil the analogue substance-based read-across approach is selected because for one closely related UVCB substance, Eucalyptus oil, genotoxicity information is available which can be used for read across.

 

Hypothesis: Rosemary oil is expected to have a similar genotoxicity profile as Eucalyptus oil because both have common constituents in comparable concentrations in the oil.

Available information: The source substance Eucalyptus oil has been tested in a well conducted in vitro gene mutation study in mammalian cells (OECD TG 490 under GLP). The study consisted of a preliminary toxicity test and three independent mutagenicity assays. Mouse lymphoma cells (L5178Y tk+/-(3.7.2C) were exposed to Eucalyptus oil, with S9 mix (3 h exposure) and without S9 mix (3 and 24 h exposure). Vehicle (acetone) and positive control groups were also included in each mutation test. From the preliminary toxicity test, concentrations of were derived using toxicity as the primary determinant. The observed mutation frequencies after exposure to Eucalyptus oil were:

- 3 hours: 10 to 175 µg/mL in absence of S9 mix: no increases in the mean mutant frequencies, 10 to 250 µg/mL in the presence of S9 mix: no increases in the mean mutant frequencies.

- 24 hours: 10 to 150 µg/mL in absence of S9 mix: no increases in the mean mutant frequencies.

Positive controls induced an acceptable increase in mutation frequency and an acceptable increase in the number of small colony mutants. From the results it was concluded that Eucalyptus oil did not demonstrate mutagenic potential in this in vitro cell mutation assay, under the experimental conditions described.

 

Target chemical and source chemical(s)

Introduction: The composition of the target chemical (Rosemary oil) and the source chemical (Eucalyptus oil) are shown in Table 1. Rosemary oil and Eucalyptus oil are both UVCBs, obtained by steam distillation from the leaves, flowers and twigs of Rosmarinus officinalis (Lamiaceae) and the leaves and stems of Eucalyptus globulus (Myrtaceae), respectively.

Similarities in composition: The source and target UVCBs share four key common constituents:

1,8 Cineole (Eucalyptol), Alpha-Pinene, Camphene and Beta-Pinene.All other common constituents are present in comparable concentrations in Rosemary oil and Eucalyptus oil. Both Rosemary oil and Eucalyptus oil have comparable amount of unknowns < 10% w/w.

Differences in composition: 1,8-cineole is present in a lower amount in Rosemary oil than in Eucalyptus oil. Camphene and Beta-pinene are present in a higher amounts in Rosemary oil than in Eucalyptus oil.The other differences in composition will be addressed in the analogue justification section.

- The following constituents > 1% are present in Rosemary oil but not in Eucalyptus oil:Camphor (5.00-23.00%)Caryophyllene-β (0.00-9.50%), Verbenone (0.00-3.00%), borneol-levo (1.00-5.00%), Linalool (0.00-4.00%), terpinol-4 (0.00-2.50%) and bornyl acetate levo / (isobornyl acetate) (min. 0.00-3.00%).

- The following constituents are not present in Rosemary oil but are in Eucalyptus oil: Isovaleric acid (min 0.01-1.00%), alloaromadendrene (0.01-2.00%), (±)-2(10)-pinen-3-one (0.01-0.50%), and 3,7-dimethylocta-1,3,6-triene (min 0.01-0.50%), because these are all <=2% these are not considered further

The focus will be on the five key constituents 1,8 Cineole (Eucalyptol), Alpha-Pinene, Camphor, Camphene and Beta-Pinene, as well as theconstituents present in Rosemary oil but not in Eucalyptus oil. The details for theseconstituents are given in Table 2.

 

Purity / Impurities

Constituents are applicable for UVCB. For Rosemary oil the constituents are presented. The similarity and differences in constituents between Target and source will be discussed below in the analogue justification.

 

Analogue justification

Substance-based approach justification

According to Annex XI 1.5, read across can be used to replace testing when the similarity between the substances based on composition can be established. When using read across the result derived should be applicable for C&L and/or risk assessment and it should be presented with adequate and reliable documentation.

Analogue justification: Rosemary oil and Eucalyptus oil have many constituents in common in comparable amounts which justifies the analogue approach. The constituents that are present in significant concentrations in Rosemary oil but not Eucalyptus oil will be addressed in the Toxico-dynamic section.

Toxicokinetics, Oral absorption: In view of the molecular weight of the constituents of both Rosemary and Eucalyptus oil, their liquid appearances, the water solubility and log Kow of all constituents have a high oral absorption of these UVCBs is expected despite variation in properties of some constituents.

Metabolism: In view of the similarity in constituents, similar metabolic pathways are applicable: During Phase I metabolism constituents with methyl groups outside the skeleton of the backbone can become oxidised into primary alcohols and subsequently acids (all 4 key constituents of Rosemary oil). Cineol is one of the common constituents that will be reduced into a tertiary alcohol and is likely a metabolite that will be distributed by alpha-2u globulins.

Toxico-dynamics:Both the source and target substances have common constituents in similar amounts. The source substance, as well as its main constituents were reported negative in the available MLA genotoxicity tests (table 2). Furthermore, no genotoxicity was reported for any of the constituents that are present in Rosemary oil but not in Eucalyptus oil. This indicates that the expected potential for genotoxicity of the source and target substance are similar.

In addition to the toxico-dynamic features presented above, the profiling of constituents using OECD QSAR toolbox 4.1 predicts no genotoxic or DNA binding alerts for most constituents. Isobornyl acetate esters of Bornylis were assigned for a DNA alert but this alert is for certain aromatic acetates and Isobornyl acetate levo is is not in this applicability domain.  

Uncertainty of the prediction: In Table 2 a summary of the other relevant toxicological data are presented to show that there are no large differences in the toxicological effects of the common constituents. Therefore, despite the variability in the composition profile of UVCB substances, there are no rmaining uncertainties regarding the read across for this endpoint.

 

Data matrix

The relevant information on constituents, physico-chemical properties and toxicological characteristics are presented in the data matrix in Table 1 and 2.

 

Conclusions for genotoxicity

For Rosemary oil no in vitro gene mutation study in mammalian cells is available and read across from Eucalyptus oil is used. When using read across the result derived should be applicable for C&L and/or risk assessment and be presented with adequate and reliable documentation. The current document presents such documentation. The source substance Eucalyptus oil has been tested in a well conductedin vitro gene mutation study inmammalian cells (OECD TG 490 under GLP). Under the test conditions it was concluded that Eucalyptus oil did not demonstrate mutagenic potential in this in vitro cell mutation assay. Therefore, the substance does not need to be classified or labelled for this endpoint. Based on the similarity between the source and target substances, this data can be used to fulfil the requirement for the genotoxicity endpoint for Rosemary oil, and therefore classification of the target substance is not needed.

Final conclusion on hazard and application in the risk characterization: Based on this read-acrossRosemary oilis considered not mutagenic in mammalian cells.


Data matrix 1 for the comparison of the constituents in Rosemary oil (target) and Eucalyptus oil (source)

NAME

CAS

Rosemary oil

Min-max range of both qualities (Spanish and North African) together

 

Eucalyptus oil

Min-max range of both qualities (Spanish and North African) together

 

Similar constituents

 

 

 

 

 

1,8-cineole

470-82-6

16.00%

56.00%

55.00%

95.00%

alpha pinene

80-56-8

7.00%

26.00%

0.01%

18.00%

Limonene

138-86-3

0.50%

7.00%

4.00%

12.00%

γ-terpinene

99-85-4

0.00%

3.00%

0.01%

6.00%

terpineol α/β/γ

8000-41-7

0.50%

5.00%

1.00%

3.00%

β-pinene

127-91-3

1.00%

12.00%

0.01%

2.00%

p-cymene

99-87-6

0.00%

3.50%

1.00%

8.00%

α-phellandrene

 99-83-2

0.00%

2.00%

0.10%

3.00%

β-myrcene

123-35-3

0.00%

6.00%

0.50%

2.00%

Camphene

79-92-5

2.00%

13.00%

0.01%

0.50%

Different constituents

 

 

 

 

 

Camphor

200-945-0

5.00%

23.00%

-

-

caryophyllene-β

87-44-5

0.00%

9.50%

-

-

borneol-levo

464-45-9

1.00%

5.00%

-

-

Linalool

78-70-6

0.00%

4.00%

-

-

Verbenone

80-57-9

0.00%

3.00%

-

-

bornyl acetate levo / (isobornyl acetate)

5655-61-8

0.00%

3.00%

-

-

terpinol-4

562-74-3

0.00%

2.50%

-

-

Constituents in Eucalyptus but not in Rosemary

 

 

 

 

 

Isovaleric acid

503-74-2

-

-

0.01%

1.00%

alloaromadendrene

489-39-4

-

-

0.01%

2.00%

(±)-2(10)-pinen-3-one

30460-92-5

 

 

0.01%

0.50%

3,7-dimethylocta-1,3,6-triene

3338-55-4

 

 

0.01%

0.50%

Unknowns

 

0.00%

< 10.00%

0.50%

8.00%


Data matrix 2 for the read across to Rosemary oil (target) from Eucalyptus oil (source)*

 

Target

Source

Common main constituent

Common main constituent

Common main constituent

Common main constituent

Rosemary only constituents

Rosemary only constituents

Rosemary only constituents

Rosemary only constituents

Rosemary only constituents

Rosemary only constituents

Rosemary only constituents

Common names

Rosemary oil

Eucalyptus oil

1,8 Cineole (Eucalyptol)

Alpha-Pinene

Camphene

Beta-Pinene

Caryophyllene-β

Verbenone

Borneol-levo

Linalool

Terpinol-4

Bornyl acetate levo / (isobornyl acetate)

Camphor

Chemical structures

UVCB

UCVB

 

CAS no

8000-25-7,84604-14-8

8000-48-4,84625-32-1

470-82-6

80-56-8

79-92-5

127-91-3

87-44-5

80-57-9

464-45-9

78-70-6

562-74-3

5655-61-8

76-22-2

Molecular formula

N/A

N/A

C10H18O

C10H16

C10H16

C10H16

C15H24

C10H14O

C10H18O

C10H18O

C10H18O

C12H20O2

C10H16O

Molecular weight (g/mol)

N/A

N/A

154.25

136.24

136.24

136.24

204.36

150.22

154.25

154.25

154.25

196.29

152.24

Physical state

liquid

liquid

liquid

liquid

solid

liquid

liquid

Not available, no REACH dossier

solid

liquid

liquid

liquid

solid

Water solubility, mg/l

 

 

0.54 – 1767.3 mg/L (QSAR)

 

3500 mg/L (cineole)
2.66 mg/L (limonene, QSAR)

2.4E+03 ppm (M)

3.5E+03 mg/L (M)

÷0.04 mg/L (M)

2.49 mg/L (M)

4.2 mg/L (M)

4.6 mg/L (M)

-

0.088mg/L (M)

 

Not available

585.7 mg/L (M)

1560 ± 90 mg/Lat25 °C (M)

1767 mg/Lat20 °C(M, WOE)

386.6 mg/Lat25 °C(M, WOE)

 

62.6 ± 4.7 mg/Lat20 °C(M)

1537 mg/Lat25 °C(M)

 

Log Kow

 

2.85 – 6.30 (QSAR)

2.84 forCineoleREACH dossier

3.4 (M)

4.487 + 0.004 (M)

4.22(M)

4.16(M)

6.23 (M)

Not available

2.75 (M)

2.9 (M)

3.26 (M, WOE)

2.749 (M,WOE)

 

3.74 ± 0.11at20 °C(M)

2.42 at25 °C(M)

Genotoxicity – Ames test

RA from Eucalyptus oil

Ames / CA / MLA: negative

Ames / MLA / Micronucleus (in vivo): negative (RA to Clarycet for micronucleus)

Ames / HPRT / Micronucleus (in vitro): negative

Ames / CA / MLA: negative

Ames / CA / MLA / Micronucleus (in vivo): negative (all RA)

Ames: negative (RA)

Not available

Not available

Ames / MLA / Micronucleus (in vivo): negative

Not available

RA Ames / MLA / Micronucleus (in vivo): negative

Ames / MLA / Micronucleus (in vivo): negative

Repeated dose toxicity mg/kg bw

RA from Eucalyptus oil

NOAEL 300 mg/kg bw/day (OECD 422)

NOAEL 600 mg/kg bw/day (OECD 407)

NOAEC 50 ppm (inhalation, mouse, NTP 90-day)

NOAEL 250 mg/kg bw/day (OECD 407)

NOAEC 50 ppm (inhalation, mouse, RA to alpha-pinene, NTP 90-day)

Not available

Not available

Not available

NOAEL 117 mg/kg bw/day (Coriander oil OECD 407),

without male nephropathy 400 mg/kg bw/day)

Not available

NOAEL 15 mg/kg bw/day (isobornyl acetate OECD 408), without male nephropathy 270 mg/kg bw/day.

NOAEL 250 mg/kg bw/day (dermal, rat, NTP 90-day)

Inhalation (NOAEC 330 mg/m3) / oral study K3 (NOEL 25 mg/kg bw/day)

Protein binding by OASIS**

 

 

No alert found

No alert found

No alert found

No alert found

No alert found

Nucleophilic addition

No alert found

No alert found

No alert found

No alert found

No alert found

Protein binding by OECD**

 

 

No alert found

No alert found

No alert found

No alert found

No alert found

No alert found

No alert found

No alert found

No alert found

No alert found

No alert found

DNA alert for AMES by OASIS**

 

 

No alert found

No alert found

No alert found

No alert found

No alert found

No alert found

No alert found

No alert found

No alert found

No alert found

No alert found

DNA binding by

OASIS**

 

 

No alert found

No alert found

No alert found

No alert found

No alert found

No alert found

No alert found

No alert found

No alert found

Structural alert: Specific Acetate Esters (not relevant for Bornyl acetate levo)

No alert found

DNA binding by OECD**

 

 

No alert found

No alert found

No alert found

No alert found

No alert found

No alert found

No alert found

No alert found

No alert found

No alert found

No alert found

*The data used to fill this table has been derived from the available disseminated dossiers on the ECHA website (March 2018), as well as OECD QSAR Toolbox V4.1

**OECD QSAR TOOLBOX V4.1

M=Measured; WoE = Weight of Evidence

Justification for classification or non-classification

Based on the available data, the substance does not need to be classified for genotoxicity in accordance with the criteria outlined in EU CLP (EC. no 1272/2008 and its amendments).