Registration Dossier

Administrative data

Description of key information

Repeated dose toxicity using read-across from Eucalyptus oil (OECD TG 422): NOAEL = 300 mg/kg bw

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Referenceopen allclose all

Endpoint:
short-term repeated dose toxicity: oral
Remarks:
combined repeated dose and reproduction / developmental screening
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
Dose descriptor:
NOAEL
Remarks:
systemic toxicity
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: Based on effects on bodyweight, food consumption.
Dose descriptor:
NOAEL
Remarks:
systemic toxicity
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: Hyaline droplet nephropathy at all dose levels; however this response is considered to be rat specific and to have no counterpart in man.
Critical effects observed:
not specified
Conclusions:
The oral repeated dose toxicity endpoint of Rosemary oil is assessed by using read across from Eucalyptus oil resulting in a No Observed Adverse Effect Level (NOAEL) for systemic toxicity of 300 and 1000 mg/kg bw/day in female and males rats, respectively.
Endpoint:
short-term repeated dose toxicity: oral
Remarks:
combined repeated dose and reproduction / developmental screening
Type of information:
experimental study
Adequacy of study:
key study
Study period:
4 October 2012 - 22 March 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Well conducted and well described study in accordance with GLP and OECD Guideline 422 without any deviation.
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to same study
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. QA statement)
Remarks:
12 feb 2013
Limit test:
no
Species:
rat
Strain:
other: Crl:CD(SD) rat
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River (UK) Limited
- Age of F0 animals at study initiation: Approximately 70 days
- Weight of F0 animals at study initiation: Males: 342-390 g; Females: 232-276 g
- Housing: No. of animals per cage - Males: acclimatisation, pre pairing and post pairing - up to 5 animals; Females: acclimatisation and pre pairing - up to 5 animals; During pairing - one male and one female; Gestation - one female; Lactation: one female + litter. Cages comprised of a polycarbonate body with a stainless steel mesh lid; changed at appropriate intervals. Solid (polycarbonate) bottom cages were used during the acclimatisation, pre-pairing, gestation, littering, lactation and maturation periods. Grid bottomed cages were used during pairing. These were suspended above absorbent paper which was changed daily during pairing.
- Diet (e.g. ad libitum): SDS VRF1 Certified pelleted diet, ad libitum
- Water (e.g. ad libitum): Potable water from the public supply via polycarbonate bottles with sipper tubes, ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 19-23 °C
- Humidity: 40-70 %
- Air changes: Filtered fresh air which was passed to atmosphere and not recirculated.
- Photoperiod: 12 h dark / 12 h fluorescent light
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
- The required amount of test material, for each formulation was pre-weighed into suitable containers. Approximately 50 % of the final volume of vehicle was added to the test material and magnetically stirred. This formulation was then made up to the required volume using further quantities of the vehicle and mixed until visually homogenous. Each concentration was formulated in ascending order using the same method for preparation. The test substance was used as supplied. All formulations were prepared freshly each week and were stored refrigerated (nominally 4 °C).

VEHICLE
- Concentration in vehicle: 25, 75 and 250 mg/mL
- Amount of vehicle (if gavage): 4 mL/kg bw/day
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
- Before treatment commenced, the suitability of the proposed mixing procedure was determined and specimen formulations at 1 and 250 mg/mL were analysed to assess the homogeneity and stability of the test material in the liquid matrix. Samples of each formulation prepared for administration in Weeks 1 and 5 of treatment were analysed for achieved concentration of the test substance. The homogeneity and stability was confirmed for Eucalyptus Oil in Corn oil formulations at nominal concentrations of 1 mg/mL and 250 mg/mL for up to1 day at ambient temperature storage and refrigerated storage for up to 15 days.
Results:
- The homogeneity and stability was confirmed for Eucalyptus Oil in Corn oil formulations at nominal concentrations of 1 mg/mL and 250 mg/mL during distribution between the bottles, during magnetic stirring for 2 hours, ambient temperature storage for 1 day and refrigerated storage for up to 15 days. In addition, the stability of discrete 1 mL samples was confirmed following refrigerated storage for 15 days.
- The mean concentrations of Eucalyptus Oil in test formulations analysed for the study was within +10 %/-15 % of nominal concentrations, confirming accurate formulation.
Duration of treatment / exposure:
- F0 males were treated for two weeks before pairing up to necropsy after a minimum of five weeks.
- F0 females were treated daily for two weeks before pairing, throughout pairing and gestation until Day 6 of lactation.
Frequency of treatment:
Once daily, at approximately the same time each day.
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dose levels were selected based on the results of preliminary study (Study No.: RAJ0012). In that study doses up to 1000 mg/kg bw/day, the limit dose for OECD 422 studies, were well tolerated.
- Rationale for animal assignment: Randomly allocated on arrival. Using the sequence of cages in the battery, one animal at a time was placed in each cage with the procedure being repeated until each cage held the appropriate number of animals.
Positive control:
Not applicable
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Animals were inspected visually at least twice daily for evidence of ill-health or reaction to treatment. Cages were inspected daily for evidence of animal ill-health amongst the occupant(s). During the acclimatisation period, observations of the animals and their cages were recorded at least once per day.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily during the first week of treatment, weekly from week 2 for all F0 animals and on Days 0, 6, 13 and 20 of gestation and days 1 and 6 of lactation for F0 females. Viability check was performed near the start and end of each working day. Before treatment commenced and during each week of treatment and for females during the Gestation phase on Days 0, 6, 13 and 20 and during the Lactation phase on Days 1 and 6, detailed physical examination and arena observations were performed on each animal.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule: Sensory reactivity and grip strength assessments were performed (before dosing) on the five lowest numbered surviving males in each group during week 5 of treatment and on the five lowest numbered lactating females in each group at Days 4-6 of lactation. During Week 5 of treatment for males and at Days 4-6 of lactation for females, the motor activity of the five lowest numbered surviving males and the five lowest numbered lactating females in each group was measured (before dosing).

BODY WEIGHT: Yes
- Time schedule for examinations:
F0 males: Before dosing on the day that treatment commenced, weekly thereafter and on the day of necropsy.
F0 females: Before dosing on the day that treatment commenced, weekly thereafter prior to positive mating evidence. Days 0, 6, 13 and 20 after mating and Days 1, 4 and 7 of lactation.

FOOD CONSUMPTION: Yes
- Time schedule for examinations:
F0 males: Weekly until paired for mating. Food consumption was not recorded during pairing (Week 3) and then recommenced in Week 4.
F0 females: Weekly until paired for mating. Days 0-5, 6-12, 13-19 after mating and Days 1-3 and 4-6 of lactation.

HAEMATOLOGY AND CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Week 2 prior to pairing. The five lowest numbered surviving F0 males and females per group.
- Animals fasted: Yes, overnight fasting
- Animals were held under light general anaesthesia induced by isoflurane. Blood samples were withdrawn from the sublingual vein.
- Haematology parameters: Haematocrit, Haemoglobin concentration, Erythrocyte count (RBC), Absolute reticulocyte count, Percentage reticulocyte count, Mean cell haemoglobin, Mean cell haemoglobin concentration, Mean cell volume, Red cell distribution width, Total leucocyte count, Differential leucocyte count: Neutrophils, Lymphocytes, Eosinophils, Basophils, Monocytes, Large unstained cells, Platelet count, Morphology: Anisocytosis, Macrocytosis, Microcytosis, Hypochromasia, Hyperchromasia, Prothrombin time and Activated partial thromboplastin time.
- Blood Chemistry parameters: Alkaline phosphatase (ALP), Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Total bilirubin, Bile acids, Urea, Creatinine, Glucose, Total cholesterol, Triglycerides, Sodium (Na), Potassium (K), Chloride (Cl), Calcium (Ca), Inorganic phosphorus, Total protein, Albumin and Albumin/globulin ratio (A/G Ratio).

ESTROUS CYCLICITY:
- Dry smears: Daily smears were taken for 15 days before pairing, using cotton swabs moistened with saline. Smears were subsequently examined to establish the duration and regularity of the oestrous cycle.
- Wet smears: After pairing with the male, daily smearing was continued using pipette lavage, until evidence of mating was observed.

PARTURITION OBSERVATIONS AND GESTATION LENGTH:
- Duration of gestation: Time elapsing between the detection of mating and commencement of parturition.
- Parturition observations: From Day 20 after mating, females were inspected three times daily for evidence of parturition. The progress and completion of parturition was monitored, numbers of live and dead offspring were recorded and any difficulties observed were recorded.
Sacrifice and pathology:
SACRIFICE
- F0 males: After week 5 investigations completed.
- F0 females: Scheduled kill - Day 7 of lactation; Failing to produce viable litter - Day 25 after mating
- Method of sacrifice: F0 animals were killed by carbon dioxide asphyxiation with subsequent exsanguination.

GROSS NECROPSY
- All F0 animals were subject to a detailed necropsy. All external features and orifices were examined visually. Any abnormality in the appearance or size of any organ and tissue (external and cut surface) was recorded and the required tissue samples preserved in appropriate fixative. In addition all F0 females had the number of uterine implantation sites recorded.

HISTOPATHOLOGY / ORGAN WEIGHTS
- The organs weighed, tissue samples fixed and sections examined microscopically are detailed in table 7.8.1/1 and 7.8.1/2.
- Routine staining: Sections were stained with haematoxylin and eosin; in addition samples of the testes were stained using a standard periodic acid Schiff (PAS) method.
- Tissues were routinely preserved in 10 % Neutral Buffered Formalin with the exception of Testes in modified Davidson’s fluid; Eyes In Davidson’s fluid.
Other examinations:
DETAILS ON MATING PROCEDURE
- M/F ratio per cage: 1: 1
- Pairing commenced: After 2 weeks of treatment for males and females
- Length of cohabitation: Up to 2 weeks
- Proof of pregnancy: Presence of sperm within the vaginal smear and/or ejected copulation plugs referred to as Day 0 of pregnancy.
- Male/female separation: Day when mating evidence was detected.
- Pre-coital interval: Calculated for each female as the time between first pairing and evidence of mating.

LITTER OBSERVATIONS
- Clinical observations: Examined at approximately 24 h after birth (Day 1 of age) and then daily thereafter for evidence of ill health or reaction to treatment; these were on an individual offspring basis or for the litter as a whole, as appropriate.
- Litter size: Daily records were maintained of mortality and consequent changes in litter size from Days 1-7 of age.
- Sex ratio: The sex ratio of each litter was recorded on Days 1, 4 and on Day 7 of age.
- Individual offspring bodyweights: F1 offspring - Days 1, 4 and 7 of age.

Postmortem examinations (Offspring)
SACRIFICE:
- F1 offspring scheduled kill: Day 7 of age
- Method of sacrifice: Intraperitoneal injection of sodium pentobarbitone
- Gross necropsy: All F1 offspring were examined externally; any offspring found to be externally abnormal were also examined internally. Where possible, sporadic deaths in early neonates were also examined internally, including an assessment of stomach for milk content.

Statistics:
See section "Any other information on materials and methods incl. tables”.
Clinical signs:
effects observed, treatment-related
Mortality:
mortality observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Details on results:
CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
- After dosing during the first week of study animals receiving Eucalyptus oil at 1000 mg/kg bw/day displayed signs of under activity and unsteady muscle reactions. Males and females receiving 1000 mg/kg bw/day also displayed chin rubbing and salivation; salivation was also recorded in females receiving 300 mg/kg bw/day.
- One pregnant female receiving 1000 mg/kg bw/day was found dead on Day 15 after mating. This death was not attributed to treatment.

BODY WEIGHT (PARENTAL ANIMALS)
- Bodyweight gain of males receiving 1000 mg/kg bw/day was low for the Week 0-1 period. Weight gain in males after Week 1 was similar or superior to controls.
- Bodyweight gain of females at all dose levels during the first two weeks of dosing was variable, no dose trends were apparent. During gestation, bodyweight gain was lower than Control in females receiving 1000 mg/kg bw/day, statistical significant differences to the Control were apparent in absolute and bodyweight gain values from Day 6 of gestation. On Day 1 of lactation absolute bodyweight was statistically lower than Control reflecting the low gains during gestation however following superior bodyweight gain statistical significance was no longer present on Day 7 of lactation.

FOOD CONSUMPTION (PARENTAL ANIMALS)
- Food consumption appeared slightly low for females receiving 1000 mg/kg bw/day during Week 1 of study, and significantly low during Days 6-19 of gestation and Days 4-6 of lactation.
- No effects on food consumption were detected in males.

NEUROBEHAVIOURAL EXAMINATION (PARENTAL ANIMALS)
- There were no changes associated with the test material during sensory reactivity, grip strength and motor activity assessments.

HAEMATOLOGY (PARENTAL ANIMALS)
- The haematological investigations conducted during Week 2 of dosing showed high lymphocyte, basophil, monocyte and large unstained cell counts (resulting in an associated increase in total white blood cell counts) in females receiving 300 or 1000 mg/kg bw/day, compared with Controls, however these findings lacked any dose-relationship trend, were not apparent in males and are considered not to be adverse at the degree observed. Activated partial thromboplastin time was lower in males receiving 1000 mg/kg bw/day, no clear dose trend was apparent, this difference was not seen in females and was considered not to be adverse at the degree observed.

BLOOD CHEMISTRY (PARENTAL ANIMALS)
- Biochemical analysis of blood plasma during Week 2 of dosing showed high alanine amino transferase activity and bile acid concentration in females at 1000 mg/kg bw/day, high urea and low triglyceride concentrations in males at 1000 mg/kg bw/day.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
- Reproductive assessments on oestrous cycles, mating performance and fertility, gestation length and parturition observations and reproductive performance did not detect any adverse effects of treatment.

ORGAN WEIGHTS (PARENTAL ANIMALS)
- Dose related higher kidney (males) and liver weights (males and females) were observed. Adrenal weights were high in females at 1000 mg/kg bw/day.
- Low adjusted spleen weight attained statistical significance for females in all dose groups; however no dose trend was apparent. The uterus (including uterine cervix and oviducts) weight in females receiving 1000 mg/kg bw/day was statistically higher than control though this was considered not adverse at the degree observed. There was considered to be no effect on male reproductive organ weights, the apparent slightly high testes and epididymal weights in treated animals are considered to partially be an effect of the single control male which had atypically small reproductive organs.

GROSS PATHOLOGY (PARENTAL ANIMALS)
- Depressed areas were present on the kidneys of four males dosed with 1000 mg/kg bw/day. These correlated with foci of tubular degeneration/regeneration that were related to test article administration. The incidence and distribution of all the other findings were consistent with the common background findings seen at these laboratories.

HISTOPATHOLOGY (PARENTAL ANIMALS)
- Changes related to treatment with Eucalyptus oil were seen in the kidneys of males and in the liver of males and females.
- Kidneys: All treated male groups had hyaline droplets in the proximal tubules of the cortex, with a dose-related incidence and severity. Multifocal tubular degeneration/regeneration was also noted in all treated male groups, but without a dose-relationship. Tubular casts of cell debris, seen at 100 and 1000 mg/kg bw/day in males, were considered to originate from the degenerating tubules, causing dilation of the lumens of occasional tubules at the corticomedullary junction. The changes in the kidney were probably responsible for the increased weight of the kidneys compared with controls. There were no similar changes in the kidneys of females dosed with Eucalyptus oil.
- Liver: Centrilobular hepatocyte hypertrophy was present in all treated male groups with a dose relationship. Glycogenic vacuolation was recorded in all treated female groups but not in control females. These findings are probably partially responsible for the increased weight of the liver in the Eucalyptus oil treated animals.
- All other microscopic findings were considered incidental and unrelated to the test article.

LITTER SIZE, OFFSPRING SURVIVAL AND SEX RATIO (OFFSPRING)
- There were no significant effects of the test material on litter size, offspring survival indices or sex ratio.

BODYWEIGHT (OFFSPRING)
- Bodyweight of offspring on Day 1 of age was similar to Control but bodyweight gain of male and female offspring derived from Dams receiving 1000 mg/kg bw/day was low and by Day 4 of age absolute bodyweight of this group was also significantly lower than Control.

MACROPATHOLOGY (OFFSPRING)
- There were no findings attributed to treatment for offspring examined before or at scheduled termination.
Key result
Dose descriptor:
NOAEL
Remarks:
systemic toxicity
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: Based on effects on bodyweight, food consumption.
Dose descriptor:
NOAEL
Remarks:
systemic toxicity
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: Hyaline droplet nephropathy at all dose levels; however this response is considered to be rat specific and to have no counterpart in man.
Critical effects observed:
not specified

None

Conclusions:
Under the test condition, the No Observed Adverse Effect Level (NOAEL) for systemic toxicity was considered to be 300 and 1000 mg/kg bw/day in female and males rats, respectively.
Executive summary:

In a Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test conducted according to OECD Guideline 422 and in compliance with GLP, Eucalyptus oil was administered to groups of Crl:CD(SD) rats at 0, 100, 300 and 1000 mg/kg bw/day by oral (gavage). The F0 males were treated for two weeks before pairing up to necropsy after a minimum of five weeks. The F0 females were treated daily for two weeks before pairing, throughout pairing, gestation and lactation until the day prior to termination on Day 6 of lactation. During the study, data was recorded on mortality, clinical signs, behavioural assessments, body weight change, food consumption, haematology, blood chemistry. All animals were subjected to a gross necropsy examination, selected organs were weighed and histopathological evaluation of selected tissues was performed.

One female receiving 1000 mg/kg bw/day was found dead on Day 15 after mating, this death was not attributed to treatment. During the first week of dosing, animals receiving 1000 mg/kg bw/day displayed transient post dosing signs of under activity and unsteady muscle reactions. Males and females receiving 1000 mg/kg bw/day also displayed chin rubbing and salivation; salivation was also recorded in females receiving 300 mg/kg bw/day. Detailed physical and arena observations, sensory reactivity, grip strength or motor activity assessments of the animals did not detect any changes attributed to the test material. Bodyweight gain of males receiving 1000 mg/kg bw/day was low for the Week 0-1 period. During gestation bodyweight gain and food consumption was low in females receiving 1000 mg/kg bw/day. Food consumption remained low for females receiving 1000 mg/kg bw/day during lactation.

Changes in haematology parameters were considered not to be adverse at the degree observed.

Biochemical analysis of blood plasma during Week 2 of dosing showed high alanine amino transferase activity and bile acid concentration in females receiving Eucalyptus oil at 1000 mg/kg bw/day. Urea concentration was high and triglyceride concentration was low in males receiving 1000 mg/kg bw/day. These changes may be associated with the microscopic changes to the liver and kidneys.

Eucalyptus oil orally administered to male rats at all doses resulted in hyaline droplet nephropathy in the kidneys, accompanied by tubular casts and/or tubular degeneration/regeneration. Hyaline droplet nephropathy in the kidneys of male rats is caused by accumulation of alpha 2 microglobulin (produced by the male rat liver) in the proximal tubules, which leads to subsequent damage and regeneration of the tubular epithelium. It has been reported with a number of organic chemicals but it appears to be a male, rat-specific toxicological response that has no counterpart in man (for reviews see Hard et al 1993, Swenberg 1993). The absence of any tubular injury in the test article treated females supports the conclusion that the tubular degeneration is secondary to the male specific hyaline droplet accumulation.

Treatment at all dose levels also resulted in centrilobular hepatocytic hypertrophy in the liver of males and an increase in glycogenic vacuolation in the liver of females. Minimal centrilobular hepatocytic hypertrophy of the male livers associated with liver weight increase is considered an adaptive change likely associated with microsomal enzyme induction. A slight increase in the incidence and severity of glycogenic vacuolation in the test article treated female livers compared with controls may be partially responsible for the liver weight increase. Although centrilobular hepatocytic hypertrophy was not recorded in the females, a minimal diffuse hypertrophy could account for the liver weight increase in this sex, but would be difficult to detect histologically. The liver changes are considered not adverse.

There were no microscopic correlates for the decrease in spleen weight and the increase in adrenal weight of the 1000 mg/kg/day females.

Under the test condition, the No Observed Adverse Effect Level (NOAEL) were considered to be:

- 300 mg/kg bw/day for systemic toxicity (female), based on lower body weight gain and food consumption during gestation. Both findings appeared to be associated with pregnancy status. It was not possible to link this effect to the taste of the substance since females had shown a significant duration of normal bodyweight and food performance prior to Day 6 of gestation and after birth of the pups. These latter observations appeared to indicate recovery in females.

- 1000 mg/kg bw/day for systemic toxicity (males) since hyaline droplet nephropathy observed at all dose levels is considered to be rat specific and to have no counterpart in man.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
300 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
Read across from Eucalyptus oil, OECD 422 guideline study in compliance with GLP, no restrictions, fully adequate for assessment.

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

The repeated dose toxicity toxicity 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.

 

Repeated dose toxicity (oral)

In a Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test conducted according to OECD Guideline 422 and in compliance with GLP, Eucalyptus oil was administered to groups of Crl:CD(SD) rats at 0, 100, 300 and 1000 mg/kg bw/day by oral (gavage). The F0 males were treated for two weeks before pairing up to necropsy after a minimum of five weeks. The F0 females were treated daily for two weeks before pairing, throughout pairing, gestation and lactation until the day prior to termination on Day 6 of lactation. During the study, data was recorded on mortality, clinical signs, behavioural assessments, body weight change, food consumption, haematology, blood chemistry. All animals were subjected to a gross necropsy examination, selected organs were weighed and histopathological evaluation of selected tissues was performed.

 

One female receiving 1000 mg/kg bw/day was found dead on Day 15 after mating, this death was not attributed to treatment. During the first week of dosing, animals receiving 1000 mg/kg bw/day displayed transient post dosing signs of under activity and unsteady muscle reactions. Males and females receiving 1000 mg/kg bw/day also displayed chin rubbing and salivation; salivation was also recorded in females receiving 300 mg/kg bw/day. Detailed physical and arena observations, sensory reactivity, grip strength or motor activity assessments of the animals did not detect any changes attributed to the test material. Bodyweight gain of males receiving 1000 mg/kg bw/day was low for the Week 0-1 period. During gestation bodyweight gain and food consumption was low in females receiving 1000 mg/kg bw/day. Food consumption remained low for females receiving 1000 mg/kg bw/day during lactation. Changes in haematology parameters were considered not to be adverse at the degree observed.

Biochemical analysis of blood plasma during Week 2 of dosing showed high alanine amino transferase activity and bile acid concentration in females receiving Eucalyptus oil at 1000 mg/kg bw/day. Urea concentration was high and triglyceride concentration was low in males receiving 1000 mg/kg bw/day. These changes may be associated with the microscopic changes to the liver and kidneys.

Eucalyptus oil orally administered to male rats at all doses resulted in hyaline droplet nephropathy in the kidneys, accompanied by tubular casts and/or tubular degeneration/regeneration. Hyaline droplet nephropathy in the kidneys of male rats is caused by accumulation of alpha 2 microglobulin (produced by the male rat liver) in the proximal tubules, which leads to subsequent damage and regeneration of the tubular epithelium. It has been reported with a number of organic chemicals but it appears to be a male, rat-specific toxicological response that has no counterpart in man (for reviews see Hard et al 1993, Swenberg 1993). The absence of any tubular injury in the test article treated females supports the conclusion that the tubular degeneration is secondary to the male specific hyaline droplet accumulation.

Treatment at all dose levels also resulted in centrilobular hepatocytic hypertrophy in the liver of males and an increase in glycogenic vacuolation in the liver of females. Minimal centrilobular hepatocytic hypertrophy of the male livers associated with liver weight increase is considered an adaptive change likely associated with microsomal enzyme induction. A slight increase in the incidence and severity of glycogenic vacuolation in the test article treated female livers compared with controls may be partially responsible for the liver weight increase. Although centrilobular hepatocytic hypertrophy was not recorded in the females, a minimal diffuse hypertrophy could account for the liver weight increase in this sex, but would be difficult to detect histologically. The liver changes are considered not adverse.

There were no microscopic correlates for the decrease in spleen weight and the increase in adrenal weight of the 1000 mg/kg/day females.

 

Under the test condition, the No Observed Adverse Effect Level (NOAEL) were considered to be:

- 300 mg/kg bw/day for systemic toxicity (female), based on lower body weight gain and food consumption during gestation. Both findings appeared to be associated with pregnancy status. It was not possible to link this effect to the taste of the substance since females had shown a significant duration of normal bodyweight and food performance prior to Day 6 of gestation and after birth of the pups. These latter observations appeared to indicate recovery in females.

- 1000 mg/kg bw/day for systemic toxicity (males) since hyaline droplet nephropathy observed at all dose levels is considered to be rat specific and to have no counterpart in man.

Read across justification - The repeated dose toxicity 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 repeated dose toxicity 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 repeated dose toxicity of Rosemary oil the analogue substance-based read-across approach is selected because for one closely related UVCB substance, Eucalyptus oil, repeated dose toxicity information is available which can be used for read across.

Hypothesis:Rosemary oil is expected to have a similar repeated dose toxicity profile and similar NOAEL 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 conductedCombined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity ScreeningTest (OECD TG 422 under GLP).Eucalyptus oil was administered to groups of Crl:CD(SD) rats at 0, 100, 300 and 1000 mg/kg bw/day by oral (gavage)and the test result receives a reliability of 1.At 1000 mg/kg bwlower body weight gain and food consumption during gestationis seen on which the NOAEL of 300 mg/kg bw is derived.

 

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 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) (0.00-3.00%).

- The following constituents are not present in Rosemary oil but are in Eucalyptus oil: Isovaleric acid ( 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 ( 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:Increased liver weight is anticipated because all constituents will be metabolised here at doses up to 4 or 5 mMols. The alpha-hydrocarbon nephropathy seen for Eucalyptus oil is indicative for metabolism in the liver lysosomes and transport of the substances via alpha2u globulins into the kidneys as can be derived from the alphahydrocarbon nephropathy seen in the kidneys. These features have also been observed in other constituents of both UVCBs such as Camphene, Camphor, Linalool and Bornyl acetate levo (isobornyl acetate). This indicates that the expected toxicodynamics 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 show a similar profile for Hazard Evaluation Support System (HESS), predicting alpha 2u-globulin nepropathy for alpha and beta pinene and camphor as hazard for the repeated dose toxicity.

Other data:

In Table 2 a summary of the other toxicological data are presented to show that there are no large differences in the toxicological effects of the common constituents between Rosemary and Eucalyptus oil. Both Rosemary and Eucalyptus oil have oral LD50 > 2000 mg/kg bw; Eucalyptus oil has a somewhat lower LD50:3320 versus 5000 mg/kg bw, respectively. There are some constituents that have LD50s < 2000 mg/kg bw, however a tested LD50 = 5000 mg/kg was obtained for Rosemary oil which indicated no acute toxicity for this UVCB as a whole. Both are not mutagenic in Ames. They are both skin sensitizers based on the presence of the sensitizing constituent 1,8-Cineole. The nucleophilic addition predicted for Verbenone can be relevant for skin sensitisation and is reflected in the classification of Rosemary oil as a skin sensitiser. Isobornyl acetate is assigned for a DNA alert but this alert is for certain aromatic acetates and Isobornyl acetate is not in this applicability domain. 

 

Uncertainty of the predictions;

Rosemary and Eucalyptus oil contain many constituents in common, in similar concentrations. The constituents of Rosemary oil that are not present in Eucalyptus oil can be categorised in similar groups as the key constituents, and are not expected to have an effect on the NOAEL derivation.

The constituents Camphor and Camphene are present in high concentrations in the target substance (resp. 5.00-23.00% and 2.00-13.00%). The long term systemic NOAELs reported for these constituents are of a similar order of magnitude as for the source substance Eucalyptus oil. For both substances the NOAELs are based on similar long term toxico-dynamics (body weight effects, male rat specific nephropathy and liver effects) and no effects such as CNS depression were reported in this repeated dose study (despite the known STOT-SE classification for Camphor). Based on the previous, these constituents are not expected to impair the reliability of this read-across prediction.

 

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 repeated dose toxicity

For Rosemary oil no repeated dose toxicity information 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 conducted 28-day repeated dose toxicity tests (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test OECD TG 422, Reliability 1). The overall NOAEL for systemic toxicity is 300 mg/kg bw/day Based on these data and the read across justification, the target substance Rosemary oil also has a NOAELs for of 300 mg/kg bw/day 

Final conclusion on hazard and application in the risk characterization:Rosemary oil has a NOAEL of 300 mg/kg bw.


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)

Acute oral tox

LD50 = 5000 mg/kg bw

LD50: 3320 & 4400 mg/kg bw

LD50 = 4500 mg/kg bw (RA to Tetrahydro-4-methyl-2-propyl-2H-pyran-4-yl acetate (Clarycet))

LD50 = 300-1000 mg/kg bw (OECD 423)

LD50 > 2000 mg/kg bw (RA to 5-ethylidenebicyclo[2.2.1]hept-2-ene)

LD50 > 2000 mg/kg bw (5 studies K4)

LD50 ≥ 5,000 mg/kg bw

Notified Acute tox 4. But no REACH dossier/ study available

Not available

LD50 = 2790 mg/kg bw

LD50 = 1300 mg/kg bw

LD50 > 5 000 mg/kg bw

LD50: 1310 mg/kg bw (key)
LD50: 3687.62 mg/kg bw (supporting)

 

Skin sensitisation

Sensitising (based on C&L of constituents)

Sensitising (based on C&L of constituents)?

Sensitising (EC3 = 65.9%)

RA to beta-pinene

Not sensitising (QSAR + 2 studies K4)

Sensitising (EC3 = 29%)

Sensitising (EC3 = 26.2%)

Notified, but no REACH dossier/ study available

Notified, but no REACH dossier/ study available

Sensitising (EC3 = 35.5 %)

Not available

RA not sensitising (isobornyl acetate)

Not sensitising (QSAR)

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)

Reproductive toxicity

RA from Eucalyptus oil

NOAEL dev 300 mg/kg bw/day (OECD 422) NOAEL repr 1000 mg/kg bw/day

NOAEL dev + repr 600 mg/kg bw/day (highest dose tested; OECD 421)

Being performed (OECD 421) / planned (OECD 414)

NOAEL dev 250 mg/kg bw/day (OECD 414)

RA to Camphene (OECD 414)

Not available

Not available

Not available

NOAEL 365 mg linalool/kg bw/day (OECD 421)

Developm: 1000 mg/kg/day (ICH 1994)

 

 

Not available

NOAEL 1000 mg/kg bw/day (isobornyl acetate, OECD 414)

NOAEL dev >400 mg/kg bw/day rabbit (OECD 414)

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 repeated dose toxicity in accordance with the criteria outlined in EU CLP (EC no. 1272/2008 and its amendments).