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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Remarks:
combined repeated dose and reproduction / developmental screening (OECD 422)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
30 March 2017 to 20 February 2018.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Well conducted and well described study in accordance with GLP and OECD Guideline 422 without any deviation.
Cross-referenceopen allclose all
Reason / purpose for cross-reference:
reference to other study
Reference
Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
From 06 to 26 June, 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail
Remarks:
Preliminary study, used as range-finder experiment for OECD 422 screening test performed in GLP laboratory.
Reason / purpose for cross-reference:
reference to other study
Reason / purpose for cross-reference:
reference to other study
Qualifier:
no guideline required
Principles of method if other than guideline:
The purpose of this study was to assess the systemic toxic potential of Elemi Oil, in a 14-day oral dietary study in Sprague-Dawley rats, to select a suitable high dose for a subsequent OECD 422 combined repeated dose toxicity study with the reproductive/developmental toxicity screening study.

GLP compliance:
no
Limit test:
no
Species:
rat
Strain:
other: Crl:CD(SD)
Details on species / strain selection:
The rat was chosen as the test species because it is accepted as a predictor of toxic change in man and the requirement for a rodent species by regulatory agencies. The Sprague-Dawley [Crl:CD(SD)] strain was used because of the historical control data available at this laboratory.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River (UK) Ltd.
- Age at study initiation: Males: 68 to 75 days; Females: 82 to 89 days
- Weight at study initiation: Males: 327-383 g; Females: 229-254 g
- Housing: Animals were housed in groups of 4 (same sex) in polycarbonate cages. Cages comprised of a polycarbonate body with a stainless steel mesh lid, changed at
appropriate intervals.
- Diet: SDS VRF1 Certified powdered diet, ad libitum
- Water: Potable water from public supply via polycarbonate bottles with sipper tubes, ad libitum
- Acclimation period: 5 days

DETAILS OF FOOD AND WATER QUALITY:
Certificates of analysis for the diet were scrutinized and approved before any batch of diet was released for use. Certificates of analysis are routinely provided by the water supplier. No specific contaminants were known that may have interfered with or prejudiced the outcome of the study and therefore no special assays were performed.

ENVIRONMENTAL CONDITIONS
- Temperature: 20-24 °C
- Humidity: 40-70%
- Air changes: Filtered fresh air which was passed to atmosphere and not recirculated.
- Photoperiod: 12 h dark / 12 h light
- Environmental Enrichment
Aspen chew block: Provided to each cage throughout the study and replaced when necessary.
Plastic shelter: Provided to each cage throughout the study and replaced when necessary.

IN-LIFE DATES: 07 June to 26 June 2017
Route of administration:
oral: feed
Details on route of administration:
The oral (dietary), route of administration was chosen as it is a possible route of human exposure.
Vehicle:
other: feed
Details on oral exposure:
DIET PREPARATION
- Diet: SDS VRF1 Certified powdered diet.
- Stabiliser: Corn oil (test material to corn oil ratio 5:1).
- Correction factor: None.
- Method of preparation: The test substance was incorporated into the diet to provide the required concentrations by initial preparation of a premix at a concentration of 30000 ppm test item and 6000 ppm corn oil. The amount of test substance and corn oil required for the premix were added to an equal amount of plain diet and stirred. An amount of plain diet equal to the weight of the mixture was added and the mixture was stirred again until visibly homogenous. The doubling up process was repeated until approximately half the premix diet was added. At this stage the mixture was ground with a mechanical grinder. The mixture was made up to the weight of the premix with plain diet. The premix was then mixed using a turbula mixer for 100 cycles.
This premix was diluted with further quantities of plain diet using the doubling up process to prepare the required concentration test mixes. Each formulation was mixed using a Turbula mixer for 100 cycles. The Control diet contained the same level of corn oil as the 12000 ppm diet.
- Frequency of preparation: Weekly
- Storage of formulation: Frozen (-10 to -30°C). Four days ambient (15 to 25°C) and 15 days frozen (-10 to -30°C) stability confirmed at 500 ppm to 20000 ppm.

STABILITY AND HOMOGENEITY
Before commencement of treatment, the suitability of the proposed mixing procedures was determined and specimen formulations were analyzed to assess the stability and homogeneity of the test item in the diet matrix as part of the main OECD 422 study (Envigo Study Number: KH93MY). The specimen formulations were analyzed to assess the stability and homogeneity of the test item in the diet matrix at concentrations of 500 ppm and 20000 ppm.
Stability and homogeneity of the formulations was confirmed for 15 days when stored frozen (-10 to -30°C) and for four days at ambient temperature (15°C to 25°C).
Analytical verification of doses or concentrations:
no
Details on analytical verification of doses or concentrations:
No formulation analysis was performed in this study. However, 200 g samples of first preparations per group were stored at (-10 to 30°C).
Duration of treatment / exposure:
14 days
Frequency of treatment:
Continuously
Dose / conc.:
0 ppm
Remarks:
Group 1 (control)
Dose / conc.:
3 000 ppm
Remarks:
Group 2 (low dose)
Dose / conc.:
6 000 ppm
Remarks:
Group 3 (mid dose)
Dose / conc.:
12 000 ppm
Remarks:
Group 4 (high dose)
No. of animals per sex per dose:
4
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale: In an acute oral toxicity study in rats for a similar test item, the LD50 exceeded 2000 mg/kg bw, therefore, in conjunction with the Sponsor, the dietary concentrations used in this study were 0, 3000, 6000 or 12000 ppm.
- 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. Each sex was allocated separately.
- Animal Replacement: On Day 1 (before dosing) variations in body weight of the animals were checked to ensure that they did not exceed ±20% of the mean for the appropriate sex. Any individuals rejected during the acclimatization period were replaced with spare animals of suitable weight from the same batch.
Replacement before treatment commenced: Body weight range extremes - one males and one females.
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 occupants. During the acclimatization period, observations of the animals and their cages were recorded at least once per day.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: A detailed physical examination was performed on each animal to monitor general health on Days 1, 4, 8, 11 and 15.

BODY WEIGHT: Yes
- Time schedule for examinations: The weight of each animal was recorded for three days before treatment commenced, on the day that treatment commenced (Day 1), daily throughout the study and before necropsy.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- The weight of food supplied to each cage, that remaining and an estimate of any spilled was recorded daily from Day -3 up to termination.

WATER CONSUMPTION: Yes
- Time schedule for examinations: Fluid intake was assessed by daily visual observation. No effect was observed and consequently quantitative measurements were not performed.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: No

CLINICAL CHEMISTRY: No

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No

OTHER: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes; All animals were killed following 14 days of treatment by carbon dioxide asphyxiation with subsequent exsanguination and subjected to detailed necropsy. Only the thoracic and abdominal cavities were opened. The cranial cavity was not opened as there were no observations during the study to indicate a possible neurotoxic action. 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. The retained tissues were checked before disposal of the carcass.

ORGAN WEIGHTS: For bilateral organs, left and right organs were weighed individually and presentated in the table 7.5.1/1. Requisite organs were weighed for all animals.

HISTOPATHOLOGY: No; but tissues were routinely preserved for microscopic examinations in 10% neutral buffered formalin (except testes: In modified Davidson’s fluid).
Other examinations:
None
Statistics:
Summary statistics (e.g. means and standard deviations) presented in this report were calculated from computer-stored individual raw data. Group mean values and standard deviations were frequently calculated using a greater number of decimal places than presented in the appendices. It is, therefore, not always possible to derive exact group values from the data presented in the appendices.
Clinical signs:
no effects observed
Description (incidence and severity):
There were no clinical signs observed that were considered to be related to treatment with the test item.
Mortality:
no mortality observed
Description (incidence):
No mortality occured during the study.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
- Group mean bodyweight loss was evident for males and females receiving 12000 ppm during Days 1-4 of treatment and group mean bodyweight gain was lower for animals receiving 3000 or 6000 ppm when compared with controls. Good bodyweight gain was evident for all treatment groups from Day 4 of treatment.

- Group mean bodyweight gain was similar or greater than Controls during Days 8-14 of treatment (except for males at 3000 ppm), resulting in overall bodyweight being similar to Controls in all treated groups with the exception of males receiving 3000 or 12000 ppm which were 70 or 82% of Controls.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
Overall food consumption was considered unaffected by treatment with the test item.

- Food consumption for males receiving 6000 or 12000 ppm was significantly lower on Day 1 when compared with Controls and remained slightly lower on Day 2 for males receiving 12000 ppm when compared with Controls.

- Food intake was significantly lower until Day 3 of treatment for all groups of treated females and remained slightly low until Day 6 of treatment for females receiving 3000 or 12000 ppm when compared with Controls.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
There were no visual water consumption effects.
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
- Higher absolute and bodyweight relative liver weights were observed in males at 6000 ppm (mean increase of 19% from Controls) and at 12000 ppm (mean increase of 25 and 28% from Controls, respectively).
- Higher absolute and bodyweight relative liver weights were observed at 12000 ppm in females (mean increase of 21% and 25% compared to Controls, respectively).
- Uterus and cervix weights were higher for females that received 12000 ppm when compared to Controls.
Gross pathological findings:
no effects observed
Description (incidence and severity):
Macroscopic examination of animals on Day 15 of study did not reveal any findings that were considered related to treatment with the test item.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Key result
Dose descriptor:
other: 7000 ppm is considered as the high dose level in the OECD 422 screening study
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
food consumption and compound intake
organ weights and organ / body weight ratios
Key result
Critical effects observed:
no

Achieved dose:

The overall mean achieved doses in animals receiving 3000, 6000 and 12000 ppm were 201, 403 and 763 mg/kg bw/day in males and 212, 425 and 800 mg/kg bw/day in females, respectively.

Conclusions:
Based on the results of this study, it is considered that < 1200 ppm and > 600 ppm should be a suitable high dose to be tested in the subsequent OECD 422 study (Envigo Study No. KH93MY).
Executive summary:

In a repeated dose toxicity range-finding study, groups of Crl:CD(SD) rats (4/sex/dose) received test item orally, via the diet at concentrations of 3000, 6000 or 12000 ppm. A similarly constituted control group received the vehicle, basal diet with added corn oil. During the study, clinical condition, body weight, food consumption, visual water consumption, organ weight and macropathology investigations were undertaken.

The overall mean achieved doses in animals receiving 3000, 6000 and 12000 ppm were 201, 403 and 763 mg/kg bw/day in males and 212, 425 and 800 mg/kg bw/day in females, respectively.

All animals survived the dosing period, with no adverse clinical signs or macroscopic changes in organ appearance at any dose level. There were no visual water consumption effects.

Following the administration of test diet at 3000 ppm, mean bodyweight gain was lower when compared with Controls. Bodyweight gain was evident from Day 4 of treatment. Group mean bodyweight gain in females was greater than Controls during Days 8-14 of treatment for females, resulting in overall bodyweight being similar to Controls. For males, body weight gain during Days 8-14 was lower than in Controls. Food intake was significantly lower until Day 3 of treatment and remained slightly low until Day 6 of treatment for females when compared with Controls. Male food intake was similar to that of the Controls. There were no effects on organ weights at this dose level.

Following the administration of test diet at 6000 ppm, mean bodyweight gain was significantly lower when compared with Controls. Bodyweight gain was evident from Day 4 of treatment. Group mean bodyweight gain was similar or greater than Controls during Days 8-14 of treatment, resulting in overall bodyweight being similar to Controls. Food intake was reduced in both sexes on Day 1 when compared with Controls. However, food intake remained low until Day 3 for females when compared with Controls. Absolute and bodyweight relative liver weights were higher when compared to Controls.

Following the administration of test diet at 12000 ppm, bodyweight loss was recorded during Days 1-4 of treatment, but bodyweight gain was recorded from Day 4 of treatment. Group mean bodyweight gain was similar or greater than Controls during Days 8-14 of treatment, resulting in overall bodyweight being similar to Controls with the exception of males which were 82% of Controls. Food consumption was significantly lower on Day 1 for males and until Day 3 for females, and remained slightly lower on Day 2 for males and until Day 6 of treatment for females when compared with Controls. Absolute and bodyweight relative liver weights were higher for males and females when compared to Controls (above 20% increases when compared to Controls). 

The effects on body weight and food consumption were seen at 12000 ppm in males and females at the start of treatment, from which the animals recovered from Day 4 (with an overall bodyweight in males being 82% of Controls at the end of the treatment period). These effects were considered likely to be related to the palatability of the Elemi oil. At 12000 ppm, absolute and bodyweight relative liver weights were markedly higher in both males and females when compared to Controls (above 20% increases), especially in males (up to 28% increase after 14 days). These effects prevented from using 12000 ppm for the longer treatment period of the main study (Envigo Study No. KH93MY).

Reason / purpose for cross-reference:
reference to same study
Reference
Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
30 March 2017 To 20 February 2018.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Well conducted and well described study in accordance with GLP and OECD Guideline 422 without any deviation.
Reason / purpose for cross-reference:
reference to other 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)
Version / remarks:
Adopted 29 July 2016
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. QA statement)
Remarks:
Inspected on 2018-01-16 / Signed on 2018-06-05
Limit test:
no
Justification for study design:
- Basis for dose level selection:
Dietary levels were selected following the completion of the preliminary toxicity study (Envigo Study number: FP11DD) and following consultation with the Sponsor.
All dose levels were well tolerated and all animals survived the dosing period, with no adverse clinical signs or macroscopic changes in organ appearance were detected at any dose level. There were no visual water consumption effects. Effects of treatment at 12000 ppm included initial body weight loss from Days 1-4 of treatment in males and females, associated with lower food consumption, which persisted until Day 2 in males and until Day 6 of treatment in females, probably due to low palatability of the treated diet. Among males and females at 3000 or 6000 ppm, body weight gain was significantly lower during Days 1-4, but good weight gain was recorded from Day 4 of treatment, and food intake was low on the first day of treatment for males and females at these dose levels but remained low until Day 3 for females receiving 6000 ppm, and Day 6 of treatment for females receiving 3000 ppm. There was an increase in absolute (up to 25% and 21% in males and females at 12000 ppm, respectively and 19% in males at 6000 ppm) and body weight relative (up to 28% and 25% in males and females at 12000 ppm, respectively and 19% in males at 6000 ppm) liver weights for animals that received 6000 or 12000 ppm when compared to Controls.
The high dietary concentration of 7000 ppm was expected to elicit initial mean body weight loss or stasis and initial low food consumption as well as a significant increase in liver weight at the end of treatment.
The lowest dietary concentration of 1750 ppm was expected to be a no effect level for target organ toxicity. The intermediate dietary concentration of 3500 ppm allowed evaluation of any concentration related trends and provided a geometric progression of dietary concentrations.

- Route of administration: The dietary route of administration was chosen to simulate the conditions of potential human exposure.
- Animal model: The rat was chosen as the test species because of the requirement for a rodent species by regulatory agencies. The Crl:CD(SD) was used because of the historical control data available at this laboratory.
Species:
rat
Strain:
other: Crl:CD(SD)
Details on species / strain selection:
The rat was chosen as the test species because of the requirement for a rodent species by regulatory agencies. The Crl:CD(SD) strain was used because of the historical control data available at this laboratory.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River (UK) Ltd.
- Females (if applicable) nulliparous and non-pregnant: Yes
- Age at study initiation: Males: 71 to 78 days old; Females: 85 to 92 days old.
- Weight at study initiation: Males: 327-392 g; Females: 237-333 g
- Housing: Solid (polycarbonate) bottom cages were used during the acclimatization, pre-pairing, treatment, gestation, littering and lactation periods. Grid bottomed polypropylene cages were used during pairing; Cages comprised of a polycarbonate body with a stainless steel mesh lid.
- Number of animals per cage: Pre-pairing: up to five animals of one sex; Pairing: one male and one female; Males after mating: up to five animals; Gestation: one female; Lactation: one female + litter
- Diet: SDS VRF1 Certified powdered diet, ad libitum (removed overnight before blood sampling for hematology and blood chemistry investigations and during urine collection)
- Water: Potable water from the public supply via polycarbonate bottles with sipper tubes, ad libitum (removed overnight during urine collection)
- Acclimation period: Females: 22 days before commencement of treatment; Males: eight days prior to the commencement of treatment.

DETAILS OF FOOD AND WATER QUALITY:
Certificates of analysis for the diet were scrutinized and approved before any batch of diet was released for use. Certificates of analysis are routinely provided by the water supplier. No specific contaminants were known that may have interfered with or prejudiced the outcome of the study and therefore no special assays were performed.

ENVIRONMENTAL CONDITIONS
- Temperature: 20-24 °C
- Humidity: 40-70 %
- Air changes: Filtered fresh air which was passed to atmosphere and not recirculated.
- Photoperiod: Artificial lighting, 12 h light : 12 h dark
- Environmental Enrichment:
Aspen chew block: A soft white untreated wood block; provided to each cage throughout the study [(except during pairing and lactation (returned on Day 13 of lactation, after dispatch of the litter)] and replaced when necessary.
Plastic shelter: Provided to each cage throughout the study [(except during pairing and lactation (returned on Day 13 of lactation, after dispatch of the litter)] and replaced at the same time as the cages.

IN-LIFE DATES: 02 August 2017 to 27 October 2017
Route of administration:
oral: feed
Vehicle:
other: feed
Details on exposure:
DIET PREPARATION
- Diet: SDS VRF1 Certified powdered diet
- Stabilizer: Corn oil (test material to corn oil ratio 5:1).
- Correction factor: A correction factor was not required.
- Method of preparation: Starting with the lowest diet concentration, the required amount of test item and corn oil was weighed out into a suitable container. An approximately equal amount of plain diet was added and the bulk then stirred together with a spoon.
Further aliquots of plain diet was then added, approximately doubling the bulk at each addition. After each addition the mixture was stirred until homogenous.
When the mixture appeared dry, the mixture was ground with a mechanical grinder.
Further plain diet was added followed by grinding until the requisite weight was obtained. The mixture was then further mixed in a Tubula mixer until completely homogenous. The Control diet contained untreated diet of the same batch with corn oil.
- Frequency of preparation: Weekly.
- Storage of formulation: Frozen (-10 to -30 °C). Stability and homogeneity of the formulations was confirmed for15 days when stored frozen (-10 to -30°C) and for four days at ambient temperature (15°C to 25°C).
Details on mating procedure:
- Animals: Toxicity phase and Recovery phase males paired with Reproductive phase females (Toxicity and Recovery phase females were not paired for mating).
- M/F ratio per cage: 1:1 from within the same treatment groups
- Pairing commenced: After a minimum of three weeks of treatment.
- Length of cohabitation: Up to 2 weeks
- Proof of pregnancy: Ejected copulation plugs in cage tray and sperm in the vaginal smear.
- 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.
- Day 0 of gestation: When positive evidence of mating was detected.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
- Stability and homogeneity Before commencement of treatment, the suitability of the proposed mixing procedures was determined and specimen formulations were analyzed to assess the stability and homogeneity of the test item in the diet matrix at concentrations of 500 ppm and 20000 ppm.
- Achieved concentration Samples of each formulation prepared for administration in Week 1 and the final week of treatment were analyzed for achieved concentration of the test item.
Duration of treatment / exposure:
- Reproductive phase (females): Three weeks before pairing, then throughout pairing and gestation until Day 12 of lactation (necropsy on Day 13 of lactation (the treated diet was available to the animals until the morning of necropsy)).
- Toxicity phase (males): Three weeks before pairing up to necropsy after a minimum of six weeks.
- Toxicity phase (females): A minimum of six weeks.
- Recovery phase (males): Three weeks before pairing up to necropsy after minimum of six weeks followed by a minimum 14-day recovery.
- Recovery phase (females): At least six weeks followed by a minimum 14-day recovery.
Frequency of treatment:
Daily
Dose / conc.:
0 ppm
Remarks:
Group 1 (control)
Dose / conc.:
1 750 ppm
Remarks:
Group 2 (low dose)
Dose / conc.:
3 500 ppm
Remarks:
Group 3 (mid dose)
Dose / conc.:
7 000 ppm
Remarks:
Group 4 (high dose)
No. of animals per sex per dose:
Reproductive phase females: 10 animals/dose
Toxicity phase females: 5 females/dose in all groups; 5 males/dose in control and high dose groups; 10 males/dose in low and mid dose groups
Recovery phase animals: 5 animals/sex/dose in control and high dose groups
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale: Dietary levels were selected following the completion of the preliminary toxicity study (Envigo Study number: FP11DD) and following consultation with the Sponsor.
All dose levels were well tolerated and all animals survived the dosing period, with no adverse clinical signs or macroscopic changes in organ appearance were detected at any dose level. There were no visual water consumption effects. Effects of treatment at 12000 ppm included initial body weight loss from Days 1-4 of treatment in males and females, associated with lower food consumption, which persisted until Day 2 in males and until Day 6 of treatment in females, probably due to low palatability of the treated diet. Among males and females at 3000 or 6000 ppm, body weight gain was significantly lower during Days 1-4, but good weight gain was recorded from Day 4 of treatment, and food intake was low on the first day of treatment for males and females at these dose levels but remained low until Day 3 for females receiving 6000 ppm, and Day 6 of treatment for females receiving 3000 ppm. There was an increase in absolute (up to 25% and 21% in males and females at 12000 ppm, respectively and 19% in males at 6000 ppm) and body weight relative (up to 28% and 25% in males and females at 12000 ppm, respectively and 19% in males at 6000 ppm) liver weights for animals that received 6000 or 12000 ppm when compared to Controls.
The high dietary concentration of 7000 ppm was expected to elicit initial mean body weight loss or stasis and initial low food consumption as well as a significant increase in liver weight at the end of treatment.
The lowest dietary concentration of 1750 ppm was expected to be a no effect level for target organ toxicity. The intermediate dietary concentration of 3500 ppm allowed evaluation of any concentration related trends and provided a geometric progression of dietary concentrations.

- Rationale for animal assignment: On arrival and non-selective allocation to cages. Estrous cycles were evaluated pre-treatment. After 14 days evaluation, animals that failed to exhibit typical 4-5 days cycles were not allocated to the study. On Day 1 of study all animals were weighed and body weights were reviewed before feeding of the treated diets by Study Management to ensure variations in body weight of animals did not exceed +/-20% of the mean for each sex. Groups were adjusted to reduce inter-/intra-group variation.

- Rationale for selecting satellite groups: Females within the toxicity and recovery groups were not paired.

- Post-exposure recovery period in satellite groups: 14 days

- Identification of animals: Each adult animal was assigned a number and identified uniquely within the study by a tail tattoo before Day 1 of treatment. The offspring were numbered individually within each litter on Day 1 of age, using a toe tattoo.

- Animal Replacement: Before the commencement of treatment, study allocation was revised to reduce inter/intra group body weight variation by replacement of animals with spares and moving animals within groups. Any individuals rejected during the acclimatization period were replaced with spare animals of suitable weight from the same batch.
Replacement before allocation: Irregular estrous cycles Four females
Ophthalmic lesion One male and one female
Acyclic Three females
Positive control:
Not applicable
Parental animals: Observations and examinations:
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 acclimatization period, observations of the animals and their cages were recorded at least once per day.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Before treatment commenced and during each week of treatment and on Days 0, 6, 13 and 20 after mating and Days 1, 6 and 12 of lactation, detailed physical examination and arena observations were performed on each animal. On each occasion, the examinations were performed at approximately the same time of day, by an observer unaware of the experimental group identities.
After removal from the home cage, animals were assessed for physical condition and behavior during handling and after being placed in a standard arena. Any deviation from normal was recorded with respect to the nature and, where appropriate, degree of severity. Particular attention was paid to possible signs of neurotoxicity, such as convulsions, tremor and abnormalities of gait or behavior.
Findings were either reported as "present" or assigned a severity grade - slight, moderate or marked.

BODY WEIGHT: Yes
- Time schedule for examinations:
F0 Toxicity and Recovery phase males and females: Weekly during acclimatization; Before feeding of the treated diets on the day that treatment commenced (Day 1) and twice weekly thereafter (including the recovery phase), and on the day of necropsy.
F0 Reproductive phase females: Weekly during acclimatization; Before feeding of the treated diets on the day that treatment commenced (Day 1) and twice weekly before pairing; Days 0, 7, 14 and 20 after mating; Day 1, 4, 7 and 13 of lactation.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- The weight of food supplied to each cage, that remaining and an estimate of any spilled was recorded as follows:
F0 animals: Daily, including the recovery phase. Food consumption was not recorded for males and females during the period when paired for mating (Days 22 to 28), but recommenced for males on Day 29. For Reproductive females after mating food consumption was recorded daily until Day 12 of lactation.
From these records the mean daily consumption per animal (g/animal/day) was calculated for each relevant phase.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations and dose groups: Pre-treatment: All Toxicity and Recovery phase animals and spare animals; Week 6: All Toxicity phase females and the first five Toxicity phase males of Groups 1 and 4.
The eyes of the animals were examined by means of a binocular indirect ophthalmoscope. Prior to each examination, the pupils of each animal were dilated using tropicamide ophthalmic solution (Mydriacyl). The adnexae, conjunctiva, cornea, sclera, anterior chamber, iris (pupil dilated), lens, vitreous and fundus were examined.

HAEMATOLOGY AND CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Week 6: Five lowest numbered surviving Toxicity phase males and females in each group; Recovery: All Recovery phase animals
- Anaesthetic used for blood collection: Yes, Animals were held under light general anaesthesia induced by isoflurane.
- Animals fasted: Yes, blood samples were collected after overnight withdrawal of food; animals were also deprived of water overnight but had access to water for a minimum period of one hour prior to the commencement of blood sampling procedures.
- Blood samples were withdrawn from the sublingual vein, collected into tubes containing EDTA anticoagulant.
- Haematology parameters: Haematocrit, Haemoglobin concentration, Erythrocyte count (RBC), Absolute 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, Prothrombin time and Activated partial thromboplastin time.
- Blood Chemistry parameters: Alkaline phosphatase (ALP), Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Gamma-glutamyl transpeptidase (gGT), 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).

URINALYSIS: Yes
- Time schedule for collection of urine: Week 6: Five lowest numbered surviving Toxicity phase males and females in each group
- Metabolism cages used for collection of urine: Yes; animals were placed in an individual metabolism cage, without access to food or water. Urine samples were collected overnight.
- Parameters:
Using manual methods: Clarity and Color/Appearance (App) - by visual assessment; Volume (Vol) - using a measuring cylinder; pH - using a pH meter; Specific gravity (SG) - by direct refractometry using a SG meter
Using Multistix reagent strips interpreted using the Clinitek®500 instrument: Ketones, Bile pigments, Urobilinogen, Blood pigments
Using a Roche P Modular Analyzer: Protein (T-Prot and U-Prot), Creatinine (T-Creat and U-Creat), Glucose (T-Gluc and U-Gluc), Sodium, Potassium, Chloride
A microscopic examination of the urine sediment was performed: Epithelial cells, Leucocytes (WBC), Erythrocytes (RBC), Casts and Other abnormal components (A)
The slide was also examined for abnormalities in spermatozoa and crystals.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations and dose groups:
Sensory reactivity and grip strength: Sensory reactivity and grip strength assessments were performed on all recovery animals in Group 1 and 4, five lowest numbered surviving toxicity phase males and females in Group 2 and 3 during Week 6 of treatment.
Motor activity: During Week 6 of treatment, the motor activity of the all recovery animals in Group 1 and 4, five lowest numbered surviving toxicity phase males and females in Group 2 and 3 was measured.
- Battery of functions tested: sensory activity / grip strength / motor activity

IMMUNOLOGY: No

OTHER: Mating Procedure
- Animals: Toxicity phase and Recovery phase males paired with Reproductive phase females (Toxicity and Recovery phase females were not paired for mating).
- Pairing commenced: After a minimum of three weeks of treatment.
- Male/female ratio: 1:1 from within the same treatment groups.
- Duration of pairing: Up to two weeks.
- Daily checks for evidence of mating: Ejected copulation plugs in cage tray and sperm in the vaginal smear.
- Day 0 of gestation: When positive evidence of mating was detected.
- 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.

OTHER: 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.
Oestrous cyclicity (parental animals):
Dry and wet smears were taken as follows:
- Dry smears: Reproductive phase females: from beginning of treatment until animals were paired for mating, using cotton swabs.
- Wet smears: Using pipette lavage during the following phases: For 14 days before treatment (all females including spares); animals that failed to exhibit 4-5 day cycles
were not allocated to study / After pairing until mating / For four days before scheduled termination (all Reproductive phase, Toxicity phase and Recovery phase females).
Litter observations:
Clinical observations: Examined at approximately 24 hours after birth (Day 1 of age) and then daily thereafter for evidence of ill health or reaction to maternal 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 1-13 of age.
Sex ratio of each litter Recorded on Days 1, 4, 7 and 13 of age.
Individual offspring body weights: Days 1, 4, 7 and 13 of age.
Ano-genital distance: Day 1 - all F1 offspring.
Nipple/areolae count: Day 13 of age - male offspring.
Postmortem examinations (parental animals):
SACRIFICE
Time of necropsy
- Toxicity phase: F0 males and females - after Week 6 investigations were completed.
- Reproductive phase females:
°F0 females killed at termination - Day 13 of lactation.
- Recovery phase: F0 Males and females - after at least 14 days without treatment.
- Method of sacrifice: All adult animals were killed by Carbon dioxide asphyxiation with subsequent exsanguination. (No animal was exposed to carbon dioxide until after completion of thyroid hormone assays).

GROSS NECROPSY
- Necropsy: All adult animals were subject to a detailed necropsy. After a review of the history of each animal, a full macroscopic examination of the tissues was performed. 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.
Reproductive Phase Females: Each uterine horn Number of implantation sites was counted and recorded.

ORGAN WEIGHTS
- For bilateral organs, left and right organs were weighed together. Requisite organs were weighed for animals killed at scheduled intervals.

HISTOPATHOLOGY
- Fixation: Tissues were routinely preserved in 10% Neutral Buffered Formalin with the exception of those detailed below: Testes - initially in modified Davidson’s fluid; Eyes - in Davidson’s fluid.
- Histology:
°Processing: Tissue samples were dehydrated, embedded in paraffin wax and sectioned at a nominal four to five micron thickness. For bilateral organs, sections of both organs were prepared. A single section was prepared from each of the remaining tissues required.
°Full List: Toxicity phase males and females in Groups 1 and 4 at scheduled termination.
°Abnormalities: All remaining adult animals.
°Processing - Kidneys: toxicity phase males in Group 2 and 3 and all Recovery phase males.
°Routine staining: Sections were stained with hematoxylin and eosin; in addition samples of the testes were stained using a standard periodic acid/Schiff (PAS) method.
Postmortem examinations (offspring):
SACRIFICE
Time of necropsy:
Selected offspring for Day 4 thyroid hormone analysis - Day 4 of age.
Scheduled kill - Day 13 of age.
Method of sacrifice:
- Offspring- selected for thyroid hormone sampling on Day 4 or Day 13 of age: Decapitation
- Offspring - not selected for thyroid hormone sampling: Intraperitoneal injection of sodium pentobarbitone.

GROSS NECROPSY
Where possible, a fresh macroscopic examination with an assessment of stomach for milk content was performed. Abnormal tissues retained.
- F1 offspring on Day 4 of age:
Blood sampling required
All animals were subject to an external macroscopic examination; particular attention was paid to the external genitalia.
- F1 offspring on Day 13 of age
Blood sampling required
All animals were subject to an external macroscopic examination; particular attention was paid to the external genitalia. Thyroid glands were preserved from one male and one female in each litter.
Statistics:
See "Any other information on materials and methods incl. tables".
Reproductive indices:
Mating Performance and Fertility:
- Percentage mating = (Number animals mating / Animals paired) x 100
- Conception rate (%) = (Number animals achieving pregnancy / Animals mated) x 100
- Fertility index (%) = (Number animals achieving pregnancy / Animals pairing) x 100

Gestation Length and Index:
Gestation length was calculated as the number of gestation days up to and including the day on which offspring were first observed, with Day 1 = day of mating for calculation purposes. Where parturition had started overnight, this value was adjusted by subtracting half of one day. Gestation index was calculated for each group as:
- Gestation index (%) = (Number of live litters born / Number pregnant ) x 100
Offspring viability indices:
Survival indices:
- Post-implantation survival index (%) = (Total number of offspring born / Total number of uterine implantation sites) x 100
Post-implantation survival index was expressed as 100% where the number of offspring exceeded the number of implantation sites recorded.
- Live birth index (%) = (Number of live offspring on Day 1 after littering / Total number of offspring born) x 100
- Viability index (%) = (Number of live offspring on Day 4 (before blood sampling) / Number live offspring on Day 1 after littering) x 100
- Lactation index (%) = (Number of live offspring on Day 13 of lactation / Number live offspring on Day 4 (after blood sampling)) x 100

Sex ratio: The percentage of male offspring in each litter was calculated at Day 1, and for live offspring on Days 1, 4 (before and after blood sampling) and 13 of age.
- Percentage males = (Number of males in litter / Total number of offspring in litter) x 100
Clinical signs:
no effects observed
Description (incidence and severity):
There were no clinical signs observed that were considered to be related to dietary administration of the test item.
Mortality:
no mortality observed
Description (incidence):
There were no mortalities at any level.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
The body weight performance of males and females receiving Elemi Oil at 1750, 3500 and 7000 ppm were generally similar to that of the Controls throughout the treatment period.
Reduced group mean body weight gain was observed from Days 1-5, 15-19 and 36-40 (with statistical significance being obtained during Day 36-40) for toxicity and recovery phase males receiving 3500 or 7000 ppm when compared with Controls. Bodyweight loss was observed during Days 1-5, 12-15, 19-22, 47-50 and reduced bodyweight gain was observed during Days 26-29 for toxicity and recovery phase females receiving 7000 ppm when compared with Controls. Bodyweight loss was observed during Days 5-8 and 47-50 for females receiving 3500 ppm, when compared with Controls.
Overall group mean body weight gain between Days 1-47/50 of treatment for animals receiving 7000 ppm was lower than Controls, with the magnitude being statistically significantly greater in females.
Body weight and body weight gain for females receiving test item prior to pairing and during gestation and lactation were generally similar to that of the Controls. However, bodyweight gain during Days 0-7 of gestation and Day 7-13 and 1-13 of lactation were lower (statistical significance being attained during gestation) for females receiving 7000 ppm when compared with Controls.
During the recovery period, the group mean overall body weight performance of animals previously treated at 7000 ppm was higher than that of the Control group.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Group mean food consumption for animals receiving all dietary concentrations of the test item was generally similar to Controls during Days 1-49 of treatment, Days 1-21 prior to pairing and Days 0-19 of gestation and 1-12 lactation.
Toxicity and recovery phase males and females treated with the test item at 7000 ppm had slight decreases in food intake during Days 1-2 of treatment in males, and Days 1-5 and 32, 33, 36 and 45 in females when compared with Controls. Food intake was slightly reduced for females receiving 7000 ppm during Days 1-4 of the pre pairing period, and statistically significantly lower on Day 10 gestation and Day 11 of lactation when compared with Controls.
Group mean food consumption of animals that previously received Elemi Oil was generally similar to Controls during Days 1-14 of recovery.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Description (incidence and severity):
No effects of treatment were observed during ophthalmic examinations in Week 6 of treatment for male and female animals treated at all dose levels.
Further ophthalmic investigations during the recovery period are therefore not considered to be necessary.
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
The haematological examinations in Week 6 of treatment revealed, when compared with Controls, increased white blood cell counts in females treated at 7000 ppm. This was predominantly due to increased neutrophils and lymphocytes observed at this level.
All other differences from Controls, including those that attained statistical significance, were generally small, confined to one sex, or the magnitudes were not dose-related and, consequently, were considered to represent normal biological variation.
Further haematological examinations during the recovery phase revealed a slight decrease in white blood cell counts in males previously treated at 7000 ppm. This was due to decreased neutrophils and lymphocytes observed at this level when compared with the Controls.
In contrast, females previously treated with 7000 ppm of the test item had increased white blood cell counts during recovery when compared to the Control group.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
The biochemical examination in Week 6 of treatment did not reveal any findings that were considered to be related to treatment with the test item, when compared with Controls. All differences from Control values, were generally small, confined to one sex, or the extent of the difference from Controls was not dose-related and, consequently, was considered to not be associated with treatment.
Urinalysis findings:
no effects observed
Description (incidence and severity):
Urinalysis investigations conducted in Week 6 of treatment did not reveal any findings that were considered to be related to treatment with the test item, when compared with Controls. All differences from Control values, were generally small, confined to one sex, or the extent of the difference from Controls was not dose-related and, consequently, was considered to not be associated with treatment.
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
Sensory reactivity observations conducted in Week 6 of treatment did not reveal any findings that were considered to be related to treatment with the test item.
Grip strength was considered unaffected by treatment with the test item, when compared with Controls.
The motor activity assessment conducted during Week 6 of treatment revealed no treatment related effects in animals at all dose levels.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Changes related to treatment with the test item were seen in the kidneys.

Kidneys:
In males receiving 1750, 3500 or 7000 ppm, there were hyaline droplets (minimal to slight severity) in the cytoplasm of the tubular epithelium lining the cortical tubules and cortical tubular basophilia (minimal to slight severity).

Incidental Findings:
Vacuolation was present in the adrenal cortex (zona fasciculata) bilaterally in one Control male and in three males given 7000 ppm. The severity, distribution and appearance of the vacuolation was similar in all cases and is considered to reflect an incidental finding that is not related to administration of the test item.
The incidence and distribution of all other findings were considered to be unrelated to treatment.

Animals Killed After 2 Weeks of Recovery:
There were no findings considered to be related to treatment in the tissues examined. All other histological changes were considered to be unrelated to treatment.
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Description (incidence and severity):
Thyroid Hormone Analysis:
All samples taken from adult males at termination and Day 13 of age male and female offspring in Groups 1 to 4 had concentrations that were comparable with the endogenous levels observed in the control matrix used to prepare the QC samples.
As the T4 investigations conducted indicated no treatment related effects, no further analysis of T4 or TSH was required.
Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
Prior to treatment all females had a regular oestrus cycle of 4 to 5 days, with the exception of one Control female that had an irregular cycle and one female receiving 3500 ppm which was acyclic. During the treatment period all females had a regular oestrus cycle of 4 to 5 days, with the exception of one female receiving either 1750 ppm or 3500 ppm and three females receiving 7000 ppm that had irregular cycles and one Control female two females receiving 3500 ppm and two females receiving 7000 ppm which were acyclic.
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed
Description (incidence and severity):
Pre-coital interval and mating performance and fertility was considered unaffected by treatment with the test item, when compared with Controls.
All females had a pre-coital interval of 1-4 days, with the exception of one female receiving 3500 ppm and one female receiving 7000 ppm which had a pre-coital interval of 5-8 days. At termination, toxicity and recovery phase females showed estrous and all reproductive females were in diestrus.
Gestation length and gestation index were considered unaffected by treatment at any dose level.
Key result
Dose descriptor:
NOAEL
Effect level:
7 000 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
histopathology: non-neoplastic
Key result
Critical effects observed:
no
Clinical signs:
no effects observed
Description (incidence and severity):
The distribution of signs at physical examination showed no relationship to parental treatment.
Mortality / viability:
no mortality observed
Description (incidence and severity):
Litter size was not affected by treatment with the test item. The number of females raising their litters to Day 13 of age was 10, 10, 10 and 10 for Control, 1750, 3500 and 7000 ppm, respectively.
Group mean post implantation survival index, mean live birth index, viability index and lactation index were unaffected by treatment with the test item.
There were no effects on sex ratio that were associated to treatment with the test item.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Mean offspring bodyweight and mean bodyweight gain for male and female offspring of parents treated with the test item was considered unaffected by treatment when compared with Controls.
Bodyweight gain during Days 7-13 of age of F1 female offspring from parents treated at 7000 ppm were slightly but statistically significantly lower when compared to concurrent Controls. However, the difference from Control was minor and only evident in one sex with no impact on overall gain, and therefore attributed to normal biological variation.
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
no effects observed
Anogenital distance (AGD):
no effects observed
Description (incidence and severity):
There was no conclusive effect on ano-genital distance in the males and females offspring of parents treated with the test item.
Nipple retention in male pups:
no effects observed
Description (incidence and severity):
No nipples were observed in male offspring of parents treated with the test item.
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Description (incidence and severity):
Macroscopic examination of offspring that either died prematurely or at scheduled termination on Day 13 of age did not reveal any findings that could be attributed to treatment.
Histopathological findings:
not examined
Other effects:
not examined
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
Key result
Generation:
F1
Sex:
male/female
Remarks on result:
not determinable due to adverse toxic effects at highest dose / concentration tested
Key result
Critical effects observed:
no
Key result
Reproductive effects observed:
no

Formulation Analysis:

The analytical procedure was successfully validated with respect to specificity of chromatographic analysis, limits of detection and quantification, linearity of detector response, repeatability, method accuracy and precision.

The homogeneity was confirmed for the test item in SDS VRF1 Certified with a corn oil stabilizer at a ratio of test item to corn oil of 5 to 1 formulations at nominal concentrations of 500 ppm and 20000 ppm. Stability was confirmed during ambient temperature storage for 8 days and frozen storage for up to 15 days.

The mean concentrations of the test item in test formulations analyzed for the study were within +10/-15% of nominal concentrations, confirming accurate formulation.

 

Achieved Dose:

Mean achieved doses for toxicity and recovery phase males were 100, 203 or 403 mg/kg bw/day respectively at 1750, 3500 or 7000 ppm.

Mean achieved doses for toxicity and recovery phase females were 104, 203 or 392 mg/kg bw/day respectively at 1750, 3500 or 7000 ppm.

Mean achieved doses for reproductive phase females at 1750, 3500 or 7000 ppm were 106, 203 or 407 mg/kg bw/day before pairing, 114, 230 or 462 mg/kg bw/day during gestation and 252, 496 or 1001 mg/kg bw/day during Days 1-13 of lactation.

Achieved doses generally maintained the interval between dietary concentrations. As expected, the achieved doses declined slightly as the animals mature - this is more obvious in males.

Conclusions:
Based on the results of this study it is concluded that the NOAEL of test item for systemic toxicity was 7000 ppm (mean achieved doses of 403 mg/kg bw/day for males, 392 mg/kg bw/day for toxicity phase females, 407 mg/kg bw/day for females during pre-paring phase, 462 mg/kg bw/day for females during gestation and 1001 mg/kg bw/day for females during lactation). The NOAEL of test item for reproductive/developmental effects was concluded to be 7000 ppm.
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, the test item was administered to groups of Crl:CD(SD) rats at dietary concentrations of 1750, 3500 and 7000 ppm.

 

Toxicity phase males were treated for three weeks before pairing up to necropsy after a minimum of six weeks. Toxicity phase females were treated for at least six weeks. Recovery phase males were treated for three weeks before pairing up to necropsy after a minimum of six weeks followed by a minimum 14-day recovery. Recovery phase females were treated for six weeks followed by a minimum 14-day recovery.

Reproductive phase females were treated for three weeks before pairing, then throughout pairing and gestation until Day 12 of lactation. Females were allowed to litter and rear their offspring to weaning and were killed on Day 13 of lactation (the treated diet was made available until the morning of necropsy). Selected F1 offspring were killed on Day 4 and Day 13 of age for blood sampling collection for thyroid hormone analysis. The remaining F1 offspring were killed on Day 13 of age. The offspring received no direct administration to the test item; any exposure was in utero or via the milk.

A similarly constituted Control group was assigned to each phase, and received the vehicle, untreated diet, throughout the same relative treatment period.

During the study, clinical condition, detailed physical examination and arena observations, sensory reactivity observations, grip strength, motor activity, body weight, food consumption, ophthalmic examination, hematology (peripheral blood), blood chemistry, urinalysis, thyroid hormone analysis, estrous cycles, pre-coital interval, mating performance, fertility, gestation length, organ weight and macroscopic pathology and histopathology investigations were undertaken.

The clinical condition, litter size and survival, sex ratio, body weight, ano-genital distance and macropathology for all offspring were also assessed. Nipple counts were performed on male offspring on Day 13 of age.

The mean concentrations of test item in test formulations analyzed for the study were within +10/-15% of nominal concentrations, confirming accurate formulation.

There were no mortalities at any level. No effects that could be clearly related to treatment were observed on sensory reaction, grip strength, motor activity, ophthalmic examination and blood chemistry and urinalysis parameters. In addition there were no treatment related clinical signs observed, no effects on pre-coital interval, mating performance, fertility, gestation length, litter size, offspring survival or external morphology. There were no adverse effects on estrous cycles.

The body weight performance of males and females receiving the test item at 1750, 3500 and 7000 ppm were generally similar to that of the Controls throughout the treatment period. Group mean food consumption for animals receiving all dietary concentrations of test item was generally similar to Controls during Days 1-49 of treatment, Days 1-21 prior to pairing and Days 0-19 of gestation and 1-12 lactation.

The absolute and adjusted organ weights in the toxicity phase males that received 3500 or 7000 ppm or females that received 7000 ppm had an increase in liver weight when compared to the Controls, with the adjusted weights attaining statistical significance. Following a 14-day recovery phase males that previously received 7000 ppm had a statistically significantly higher adjusted epididymis weight, when compared to Controls.

Changes considered to be related to administration of the test item were present in the kidneys. Males given 1750, 3500 or 7000 ppm had hyaline droplets (minimal to slight severity) in the cortical tubules of the kidneys. Cortical tubular basophilia (minimal to slight severity) was also present in some male rats given 1750, 3500 or 7000 ppm. In this study, tubular basophilia was of minimal to slight severity, but was not present in Control. However, following a 14-day recovery period there were no findings considered to be related to treatment in the kidneys of males examined.

Dietary administration of the test item in males and toxicity phase females for 6 weeks and reproductive phase females for two weeks before pairing, throughout pairing, gestation and until Day 13 of lactation at levels up to 7000 ppm was generally well tolerated and no mortalities occurred.

There were initial effects on bodyweight and food consumption at the start of treatment, but this had no impact on terminal bodyweight and showed full recovery. Administration of the test item was associated with test item-related changes in the kidneys of males given 1750, 3500 or 7000 ppm.

There were no effects on parental reproductive parameters or fertility. All offspring were macroscopically normal. Elemi oil showed no evidence of being an endocrine disruptor and did not affect thyroid hormone levels.

The major toxicological effect observed in the OECD guideline 422 study is hyaline droplets observed in the kidneys of males given 1750, 3500 or 7000 ppm, but this finding was not observed in females at the same levels, and was not observed following a 14-day recovery period. Also, it is a well-known sex and species specific effect, of no relevance to humans.

Therefore, the No-Observed-Adverse-Effect-Level (NOAEL) for systemic toxicity is 7000 ppm (mean achieved doses of 403 mg/kg bw/day for males, 392 mg/kg bw/day for toxicity phase females). The NOAEL for reproductive and developmental toxicity is 7000 ppm, corresponding to mean achieved doses of 403 mg/kg bw/day for males, 392 mg/kg bw/day for toxicity phase females, 407 mg/kg bw/day for females during pre-paring phase, 462 mg/kg bw/day for females during gestation and 1001 mg/kg bw/day for females during lactation.

Under the test conditions, Elemi Oil is not classified according to the annex I of the Regulation (EC) No. 1272/2008 (CLP).

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report date:
2019

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
Adopted 29 July 2016
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. QA statement)
Remarks:
Inspected on 2018-01-16 / Signed on 2018-06-05
Limit test:
no

Test material

Constituent 1
Reference substance name:
Essential oil of Canarium commune (Burseraceae) obtained from gum by steam distillation
EC Number:
945-898-3
Cas Number:
97675-63-3
Molecular formula:
not relevant for a UVCB substance
IUPAC Name:
Essential oil of Canarium commune (Burseraceae) obtained from gum by steam distillation
Test material form:
liquid
Details on test material:
- Appearance: slightly pale yellow liquid
- Homogeneity: Homogeneous
- Storage condition of test material: at ambient temperature (15 to 25°C), in the dark.

Test animals

Species:
rat
Strain:
other: Crl:CD(SD)
Details on species / strain selection:
The rat was chosen as the test species because of the requirement for a rodent species by regulatory agencies. The Crl:CD(SD) strain was used because of the historical control data available at this laboratory.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River (UK) Ltd.
- Females (if applicable) nulliparous and non-pregnant: Yes
- Age at study initiation: Males: 71 to 78 days old; Females: 85 to 92 days old.
- Weight at study initiation: Males: 327-392 g; Females: 237-333 g
- Housing: Solid (polycarbonate) bottom cages were used during the acclimatization, pre-pairing, treatment, gestation, littering and lactation periods. Grid bottomed polypropylene cages were used during pairing; Cages comprised of a polycarbonate body with a stainless steel mesh lid.
- Number of animals per cage: Pre-pairing: up to five animals of one sex; Pairing: one male and one female; Males after mating: up to five animals; Gestation: one female; Lactation: one female + litter
- Diet: SDS VRF1 Certified powdered diet, ad libitum (removed overnight before blood sampling for hematology and blood chemistry investigations and during urine collection)
- Water: Potable water from the public supply via polycarbonate bottles with sipper tubes, ad libitum (removed overnight during urine collection)
- Acclimation period: Females: 22 days before commencement of treatment; Males: eight days prior to the commencement of treatment.

DETAILS OF FOOD AND WATER QUALITY:
Certificates of analysis for the diet were scrutinized and approved before any batch of diet was released for use. Certificates of analysis are routinely provided by the water supplier. No specific contaminants were known that may have interfered with or prejudiced the outcome of the study and therefore no special assays were performed.

ENVIRONMENTAL CONDITIONS
- Temperature: 20-24 °C
- Humidity: 40-70 %
- Air changes: Filtered fresh air which was passed to atmosphere and not recirculated.
- Photoperiod: Artificial lighting, 12 h light : 12 h dark
- Environmental Enrichment:
Aspen chew block: A soft white untreated wood block; provided to each cage throughout the study [(except during pairing and lactation (returned on Day 13 of lactation, after dispatch of the litter)] and replaced when necessary.
Plastic shelter: Provided to each cage throughout the study [(except during pairing and lactation (returned on Day 13 of lactation, after dispatch of the litter)] and replaced at the same time as the cages.

IN-LIFE DATES: 02 August 2017 to 27 October 2017

Administration / exposure

Route of administration:
oral: feed
Details on route of administration:
The dietary route of administration was chosen to simulate the conditions of potential human exposure.
Vehicle:
other: feed
Details on oral exposure:
DIET PREPARATION
- Diet: SDS VRF1 Certified powdered diet
- Stabilizer: Corn oil (test material to corn oil ratio 5:1).
- Correction factor: A correction factor was not required.
- Method of preparation: Starting with the lowest diet concentration, the required amount of test item and corn oil was weighed out into a suitable container. An approximately equal amount of plain diet was added and the bulk then stirred together with a spoon.
Further aliquots of plain diet was then added, approximately doubling the bulk at each addition. After each addition the mixture was stirred until homogenous.
When the mixture appeared dry, the mixture was ground with a mechanical grinder.
Further plain diet was added followed by grinding until the requisite weight was obtained. The mixture was then further mixed in a Tubula mixer until completely homogenous. The Control diet contained untreated diet of the same batch with corn oil.
- Frequency of preparation: Weekly.
- Storage of formulation: Frozen (-10 to -30 °C). Stability and homogeneity of the formulations was confirmed for15 days when stored frozen (-10 to -30°C) and for four days at ambient temperature (15°C to 25°C).
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
- Stability and homogeneity: Before commencement of treatment, the suitability of the proposed mixing procedures was determined and specimen formulations were analyzed to assess the stability and homogeneity of the test item in the diet matrix at concentrations of 500 ppm and 20000 ppm.
- Achieved concentration: Samples of each formulation prepared for administration in Week 1 and the final week of treatment were analyzed for achieved concentration of the test item.
Duration of treatment / exposure:
- Reproductive phase (females): Three weeks before pairing, then throughout pairing and gestation until Day 12 of lactation (necropsy on Day 13 of lactation (the treated diet was available to the animals until the morning of necropsy)).
- Toxicity phase (males): Three weeks before pairing up to necropsy after a minimum of six weeks.
- Toxicity phase (females): A minimum of six weeks.
- Recovery phase (males): Three weeks before pairing up to necropsy after minimum of six weeks followed by a minimum 14-day recovery.
- Recovery phase (females): At least six weeks followed by a minimum 14-day recovery.
Frequency of treatment:
Continuously
Doses / concentrationsopen allclose all
Dose / conc.:
0 ppm
Remarks:
Group 1 (control)
Dose / conc.:
1 750 ppm
Remarks:
Group 2 (low dose)
Dose / conc.:
3 500 ppm
Remarks:
Group 3 (mid dose)
Dose / conc.:
7 000 ppm
Remarks:
Group 4 (high dose)
No. of animals per sex per dose:
Reproductive phase females: 10 animals/dose
Toxicity phase females: 5 females/dose in all groups; 5 males/dose in control and high dose groups; 10 males/dose in low and mid dose groups
Recovery phase animals: 5 animals/sex/dose in control and high dose groups
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale: Dietary levels were selected following the completion of the preliminary toxicity study (Envigo Study number: FP11DD) and following consultation with the Sponsor.
All dose levels were well tolerated and all animals survived the dosing period, with no adverse clinical signs or macroscopic changes in organ appearance were detected at any dose level. There were no visual water consumption effects. Effects of treatment at 12000 ppm included initial body weight loss from Days 1-4 of treatment in males and females, associated with lower food consumption, which persisted until Day 2 in males and until Day 6 of treatment in females, probably due to low palatability of the treated diet. Among males and females at 3000 or 6000 ppm, body weight gain was significantly lower during Days 1-4, but good weight gain was recorded from Day 4 of treatment, and food intake was low on the first day of treatment for males and females at these dose levels but remained low until Day 3 for females receiving 6000 ppm, and Day 6 of treatment for females receiving 3000 ppm. There was an increase in absolute (up to 25% and 21% in males and females at 12000 ppm, respectively and 19% in males at 6000 ppm) and body weight relative (up to 28% and 25% in males and females at 12000 ppm, respectively and 19% in males at 6000 ppm) liver weights for animals that received 6000 or 12000 ppm when compared to Controls.
The high dietary concentration of 7000 ppm was expected to elicit initial mean body weight loss or stasis and initial low food consumption as well as a significant increase in liver weight at the end of treatment.
The lowest dietary concentration of 1750 ppm was expected to be a no effect level for target organ toxicity. The intermediate dietary concentration of 3500 ppm allowed evaluation of any concentration related trends and provided a geometric progression of dietary concentrations.

- Rationale for animal assignment: On arrival and non-selective allocation to cages. Estrous cycles were evaluated pre-treatment. After 14 days evaluation, animals that failed to exhibit typical 4-5 days cycles were not allocated to the study. On Day 1 of study all animals were weighed and body weights were reviewed before feeding of the treated diets by Study Management to ensure variations in body weight of animals did not exceed +/-20% of the mean for each sex. Groups were adjusted to reduce inter-/intra-group variation.

- Rationale for selecting satellite groups: Females within the toxicity and recovery groups were not paired.

- Post-exposure recovery period in satellite groups: 14 days

- Identification of animals: Each adult animal was assigned a number and identified uniquely within the study by a tail tattoo before Day 1 of treatment. The offspring were numbered individually within each litter on Day 1 of age, using a toe tattoo.

- Animal Replacement: Before the commencement of treatment, study allocation was revised to reduce inter/intra group body weight variation by replacement of animals with spares and moving animals within groups. Any individuals rejected during the acclimatization period were replaced with spare animals of suitable weight from the same batch.
Replacement before allocation: Irregular estrous cycles Four females
Ophthalmic lesion One male and one female
Acyclic Three females
Positive control:
Not applicable

Examinations

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 acclimatization period, observations of the animals and their cages were recorded at least once per day.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Before treatment commenced and during each week of treatment and on Days 0, 6, 13 and 20 after mating and Days 1, 6 and 12 of lactation, detailed physical examination and arena observations were performed on each animal. On each occasion, the examinations were performed at approximately the same time of day, by an observer unaware of the experimental group identities.
After removal from the home cage, animals were assessed for physical condition and behavior during handling and after being placed in a standard arena. Any deviation from normal was recorded with respect to the nature and, where appropriate, degree of severity. Particular attention was paid to possible signs of neurotoxicity, such as convulsions, tremor and abnormalities of gait or behavior.
Findings were either reported as "present" or assigned a severity grade - slight, moderate or marked.

BODY WEIGHT: Yes
- Time schedule for examinations:
F0 Toxicity and Recovery phase males and females: Weekly during acclimatization; Before feeding of the treated diets on the day that treatment commenced (Day 1) and twice weekly thereafter (including the recovery phase), and on the day of necropsy.
F0 Reproductive phase females: Weekly during acclimatization; Before feeding of the treated diets on the day that treatment commenced (Day 1) and twice weekly before pairing; Days 0, 7, 14 and 20 after mating; Day 1, 4, 7 and 13 of lactation.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
-- The weight of food supplied to each cage, that remaining and an estimate of any spilled was recorded as follows:
F0 animals: Daily, including the recovery phase. Food consumption was not recorded for males and females during the period when paired for mating (Days 22 to 28), but recommenced for males on Day 29. For Reproductive females after mating food consumption was recorded daily until Day 12 of lactation.
From these records the mean daily consumption per animal (g/animal/day) was calculated for each relevant phase.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations and dose groups: Pre-treatment: All Toxicity and Recovery phase animals and spare animals; Week 6: All Toxicity phase females and the first five Toxicity phase males of Groups 1 and 4.
The eyes of the animals were examined by means of a binocular indirect ophthalmoscope. Prior to each examination, the pupils of each animal were dilated using tropicamide ophthalmic solution (Mydriacyl). The adnexae, conjunctiva, cornea, sclera, anterior chamber, iris (pupil dilated), lens, vitreous and fundus were examined.

HAEMATOLOGY AND CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Week 6: Five lowest numbered surviving Toxicity phase males and females in each group; Recovery: All Recovery phase animals
- Anaesthetic used for blood collection: Yes, Animals were held under light general anaesthesia induced by isoflurane.
- Animals fasted: Yes, blood samples were collected after overnight withdrawal of food; animals were also deprived of water overnight but had access to water for a minimum period of one hour prior to the commencement of blood sampling procedures.
- Blood samples were withdrawn from the sublingual vein, collected into tubes containing EDTA anticoagulant.
- Haematology parameters: Haematocrit, Haemoglobin concentration, Erythrocyte count (RBC), Absolute 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, Prothrombin time and Activated partial thromboplastin time.
- Blood Chemistry parameters: Alkaline phosphatase (ALP), Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Gamma-glutamyl transpeptidase (gGT), 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).

URINALYSIS: Yes
- Time schedule for collection of urine: Week 6: Five lowest numbered surviving Toxicity phase males and females in each group
- Metabolism cages used for collection of urine: Yes; animals were placed in an individual metabolism cage, without access to food or water. Urine samples were collected overnight.
- Parameters:
Using manual methods: Clarity and Color/Appearance (App) - by visual assessment; Volume (Vol) - using a measuring cylinder; pH - using a pH meter; Specific gravity (SG) - by direct refractometry using a SG meter
Using Multistix reagent strips interpreted using the Clinitek®500 instrument: Ketones, Bile pigments, Urobilinogen, Blood pigments
Using a Roche P Modular Analyzer: Protein (T-Prot and U-Prot), Creatinine (T-Creat and U-Creat), Glucose (T-Gluc and U-Gluc), Sodium, Potassium, Chloride
A microscopic examination of the urine sediment was performed: Epithelial cells, Leucocytes (WBC), Erythrocytes (RBC), Casts and Other abnormal components (A)
The slide was also examined for abnormalities in spermatozoa and crystals.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations and dose groups:
Sensory reactivity and grip strength: Sensory reactivity and grip strength assessments were performed on all recovery animals in Group 1 and 4, five lowest numbered surviving toxicity phase males and females in Group 2 and 3 during Week 6 of treatment.
Motor activity: During Week 6 of treatment, the motor activity of the all recovery animals in Group 1 and 4, five lowest numbered surviving toxicity phase males and females in Group 2 and 3 was measured.
- Battery of functions tested: sensory activity / grip strength / motor activity

IMMUNOLOGY: No
Sacrifice and pathology:
SACRIFICE
Time of necropsy
- Toxicity phase: F0 males and females - after Week 6 investigations were completed.
- Reproductive phase females:
°F0 females killed at termination - Day 13 of lactation.
°F1 offspring scheduled kill - Selected offspring for thyroid hormone analysis - Day 4 of age; Scheduled kill - Day 13 of age.
- Recovery phase: F0 Males and females - after at least 14 days without treatment.
- Method of sacrifice: All adult animals were killed by Carbon dioxide asphyxiation with subsequent exsanguination. (No animal was exposed to carbon dioxide until after completion of thyroid hormone assays).

GROSS NECROPSY
- Necropsy: All adult animals were subject to a detailed necropsy. After a review of the history of each animal, a full macroscopic examination of the tissues was performed. 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.

ORGAN WEIGHTS
- For bilateral organs, left and right organs were weighed together. Requisite organs were weighed for animals killed at scheduled intervals.

HISTOPATHOLOGY
- Fixation: Tissues were routinely preserved in 10% Neutral Buffered Formalin with the exception of those detailed below: Testes - initially in modified Davidson’s fluid; Eyes - in Davidson’s fluid.
- Histology:
°Processing: Tissue samples were dehydrated, embedded in paraffin wax and sectioned at a nominal four to five micron thickness. For bilateral organs, sections of both organs were prepared. A single section was prepared from each of the remaining tissues required.
°Full List: Toxicity phase males and females in Groups 1 and 4 at scheduled termination.
°Abnormalities: All remaining adult animals.
°Processing - Kidneys: toxicity phase males in Group 2 and 3 and all Recovery phase males.
°Routine staining: Sections were stained with hematoxylin and eosin; in addition samples of the testes were stained using a standard periodic acid/Schiff (PAS) method.
Other examinations:
Thyroid Hormone Analysis:
Blood samples were collected as follows:
- At termination: All surviving F0 males, all surviving F0 Reproductive phase females and all Recovery phase animals
Statistics:
See "Any other information on materials and methods incl. tables".

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Description (incidence and severity):
There were no clinical signs observed that were considered to be related to dietary administration of the test item.
Mortality:
no mortality observed
Description (incidence):
There were no mortalities at any level.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
The body weight performance of males and females receiving the test item at 1750, 3500 and 7000 ppm were generally similar to that of the Controls throughout the treatment period.
Reduced group mean body weight gain was observed from Days 1-5, 15-19 and 36-40 (with statistical significance being obtained during Day 36-40) for toxicity and recovery phase males receiving 3500 or 7000 ppm when compared with Controls. Bodyweight loss was observed during Days 1-5, 12-15, 19-22, 47-50 and reduced bodyweight gain was observed during Days 26-29 for toxicity and recovery phase females receiving 7000 ppm when compared with Controls. Bodyweight loss was observed during Days 5-8 and 47-50 for females receiving 3500 ppm, when compared with Controls.
Overall group mean body weight gain between Days 1-47/50 of treatment for animals receiving 7000 ppm was lower than Controls, with the magnitude being statistically significantly greater in females.
Body weight and body weight gain for females receiving test item prior to pairing and during gestation and lactation were generally similar to that of the Controls. However, bodyweight gain during Days 0-7 of gestation and Day 7-13 and 1-13 of lactation were lower (statistical significance being attained during gestation) for females receiving 7000 ppm when compared with Controls.
During the recovery period, the group mean overall body weight performance of animals previously treated at 7000 ppm was higher than that of the Control group.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Group mean food consumption for animals receiving all dietary concentrations of the test item was generally similar to Controls during Days 1-49 of treatment, Days 1-21 prior to pairing and Days 0-19 of gestation and 1-12 lactation.
Toxicity and recovery phase males and females treated with the test item at 7000 ppm had slight decreases in food intake during Days 1-2 of treatment in males, and Days 1-5 and 32, 33, 36 and 45 in females when compared with Controls. Food intake was slightly reduced for females receiving 7000 ppm during Days 1-4 of the pre pairing period, and statistically significantly lower on Day 10 gestation and Day 11 of lactation when compared with Controls.
Group mean food consumption of animals that previously received the test item was generally similar to Controls during Days 1-14 of recovery.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Description (incidence and severity):
No effects of treatment were observed during ophthalmic examinations in Week 6 of treatment for male and female animals treated at all dose levels.
Further ophthalmic investigations during the recovery period are therefore not considered to be necessary.
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
The haematological examinations in Week 6 of treatment revealed, when compared with Controls, increased white blood cell counts in females treated at 7000 ppm. This was predominantly due to increased neutrophils and lymphocytes observed at this level.
All other differences from Controls, including those that attained statistical significance, were generally small, confined to one sex, or the magnitudes were not dose-related and, consequently, were considered to represent normal biological variation.
Further haematological examinations during the recovery phase revealed a slight decrease in white blood cell counts in males previously treated at 7000 ppm. This was due to decreased neutrophils and lymphocytes observed at this level when compared with the Controls.
In contrast, females previously treated with 7000 ppm of the test item had increased white blood cell counts during recovery when compared to the Control group.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
The biochemical examination in Week 6 of treatment did not reveal any findings that were considered to be related to treatment with the test item, when compared with Controls. All differences from Control values, were generally small, confined to one sex, or the extent of the difference from Controls was not dose-related and, consequently, was considered to not be associated with treatment.
Urinalysis findings:
no effects observed
Description (incidence and severity):
Urinalysis investigations conducted in Week 6 of treatment did not reveal any findings that were considered to be related to treatment with the test item, when compared with Controls. All differences from Control values, were generally small, confined to one sex, or the extent of the difference from Controls was not dose-related and, consequently, was considered to not be associated with treatment.
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
Sensory reactivity observations conducted in Week 6 of treatment did not reveal any findings that were considered to be related to treatment with the test item.
Grip strength was considered unaffected by treatment with the test item, when compared with Controls.
The motor activity assessment conducted during Week 6 of treatment revealed no treatment related effects in animals at all dose levels.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
The absolute and adjusted organ weights in the toxicity phase males that received 3500 or 7000 ppm or females that received 7000 ppm had an increase in liver weight when compared to the Controls, with the adjusted weights attaining statistical significance.
The organ weights of reproductive phase females treated with Elemi Oil were similar to the Control group.
Following a 14 day recovery phase males that previously received 7000 ppm had a statistically significantly higher adjusted epididymis weight, when compared to Controls. For females previously treated at 7000 ppm there were no differences in organ weights when compared with Controls.
All other difference from Controls were minor and were therefore attributed to normal biological variation.
Gross pathological findings:
no effects observed
Description (incidence and severity):
The macroscopic examination performed after 6 weeks of treatment revealed no test item related lesions.
The macroscopic examination performed after 2 weeks of recovery revealed no test item related lesions.
The incidence and distribution of all findings were considered to be unrelated to treatment.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Changes related to treatment with the test item were seen in the kidneys.

Kidneys:
In males receiving 1750, 3500 or 7000 ppm, there were hyaline droplets (minimal to slight severity) in the cytoplasm of the tubular epithelium lining the cortical tubules and cortical tubular basophilia (minimal to slight severity).

Incidental Findings:
Vacuolation was present in the adrenal cortex (zona fasciculata) bilaterally in one Control male and in three males given 7000 ppm. The severity, distribution and appearance of the vacuolation was similar in all cases and is considered to reflect an incidental finding that is not related to administration of the test item.
The incidence and distribution of all other findings were considered to be unrelated to treatment.

Animals Killed After 2 Weeks of Recovery:
There were no findings considered to be related to treatment in the tissues examined. All other histological changes were considered to be unrelated to treatment.
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Description (incidence and severity):
Thyroid Hormone Analysis:
All samples taken from adult males at termination and Day 13 of age male and female offspring in Groups 1 to 4 had concentrations that were comparable with the endogenous levels observed in the control matrix used to prepare the QC samples.
As the T4 investigations conducted indicated no treatment related effects, no further analysis of T4 or TSH was required.

Effect levels

Key result
Dose descriptor:
NOAEL
Effect level:
7 000 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
histopathology: non-neoplastic

Target system / organ toxicity

Key result
Critical effects observed:
no

Any other information on results incl. tables

Formulation Analysis:

The analytical procedure was successfully validated with respect to specificity of chromatographic analysis, limits of detection and quantification, linearity of detector response, repeatability, method accuracy and precision.

The homogeneity was confirmed for the test item in SDS VRF1 Certified with a corn oil stabilizer at a ratio of test item to corn oil of 5 to 1 formulations at nominal concentrations of 500 ppm and 20000 ppm. Stability was confirmed during ambient temperature storage for 8 days and frozen storage for up to 15 days.

The mean concentrations of the test item in test formulations analyzed for the study were within +10/-15% of nominal concentrations, confirming accurate formulation.

 

Achieved Dose:

Mean achieved doses for toxicity and recovery phase males were 100, 203 or 403 mg/kg bw/day respectively at 1750, 3500 or 7000 ppm.

Mean achieved doses for toxicity and recovery phase females were 104, 203 or 392 mg/kg bw/day respectively at 1750, 3500 or 7000 ppm.

Mean achieved doses for reproductive phase females at 1750, 3500 or 7000 ppm were 106, 203 or 407 mg/kg bw/day before pairing, 114, 230 or 462 mg/kg bw/day during gestation and 252, 496 or 1001 mg/kg bw/day during Days 1-13 of lactation.

Achieved doses generally maintained the interval between dietary concentrations. As expected, the achieved doses declined slightly as the animals mature - this is more obvious in males.

Applicant's summary and conclusion

Conclusions:
Based on the results of this study it is concluded that the NOAEL of test item for systemic toxicity was 7000 ppm (mean achieved doses of 403 mg/kg bw/day for males, 392 mg/kg bw/day for toxicity phase females).
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, the test item was administered to groups of Crl:CD(SD) rats at dietary concentrations of 1750, 3500 and 7000 ppm.

 

Toxicity phase males were treated for three weeks before pairing up to necropsy after a minimum of six weeks. Toxicity phase females were treated for at least six weeks. Recovery phase males were treated for three weeks before pairing up to necropsy after a minimum of six weeks followed by a minimum 14-day recovery. Recovery phase females were treated for six weeks followed by a minimum 14-day recovery.

Reproductive phase females were treated for three weeks before pairing, then throughout pairing and gestation until Day 12 of lactation. Females were allowed to litter and rear their offspring to weaning and were killed on Day 13 of lactation (the treated diet was made available until the morning of necropsy).

A similarly constituted Control group was assigned to each phase, and received the vehicle, untreated diet, throughout the same relative treatment period.

During the study, clinical condition, detailed physical examination and arena observations, sensory reactivity observations, grip strength, motor activity, body weight, food consumption, ophthalmic examination, hematology (peripheral blood), blood chemistry, urinalysis, thyroid hormone analysis, organ weight and macroscopic pathology and histopathology investigations were undertaken.

The mean concentrations of test item in test formulations analyzed for the study were within +10/-15% of nominal concentrations, confirming accurate formulation.

There were no mortalities at any level. No effects that could be clearly related to treatment were observed on sensory reaction, grip strength, motor activity, ophthalmic examination and blood chemistry and urinalysis parameters.

The body weight performance of males and females receiving the test item at 1750, 3500 and 7000 ppm were generally similar to that of the Controls throughout the treatment period. Group mean food consumption for animals receiving all dietary concentrations of test item was generally similar to Controls during Days 1-49 of treatment, Days 1-21 prior to pairing and Days 0-19 of gestation and 1-12 lactation.

The absolute and adjusted organ weights in the toxicity phase males that received 3500 or 7000 ppm or females that received 7000 ppm had an increase in liver weight when compared to the Controls, with the adjusted weights attaining statistical significance. Following a 14-day recovery phase males that previously received 7000 ppm had a statistically significantly higher adjusted epididymis weight, when compared to Controls.

Changes considered to be related to administration of the test item were present in the kidneys. Males given 1750, 3500 or 7000 ppm had hyaline droplets (minimal to slight severity) in the cortical tubules of the kidneys. Cortical tubular basophilia (minimal to slight severity) was also present in some male rats given 1750, 3500 or 7000 ppm. In this study, tubular basophilia was of minimal to slight severity, but was not present in Control. However, following a 14-day recovery period there were no findings considered to be related to treatment in the kidneys of males examined.

Dietary administration of the test item in males and toxicity phase females for 6 weeks and reproductive phase females for two weeks before pairing, throughout pairing, gestation and until Day 13 of lactation at levels up to 7000 ppm was generally well tolerated and no mortalities occurred.

There were initial effects on bodyweight and food consumption at the start of treatment, but this had no impact on terminal bodyweight and showed full recovery. Administration of the test item was associated with test item-related changes in the kidneys of males given 1750, 3500 or 7000 ppm.

The major toxicological effect observed in the OECD guideline 422 study ishyaline droplets observed in the kidneys of males given 1750, 3500 or 7000 ppm, but this finding was not observed in females at the same levels, and was not observed following a 14-day recovery period. Also, it is a well-known sex and species specific effect, of no relevance to humans. Therefore, the No-Observed-Adverse-Effect-Level (NOAEL) for systemic toxicity is 7000 ppm (mean achieved doses of 403 mg/kg bw/day for males, 392 mg/kg bw/day for toxicity phase females).

Based on the results of this study, the test substance is not classified for damage to organs through prolonged oral repeated exposure according to the criteria of the Regulation (EC) No. 1272/2008 (CLP) and to the GHS.

This study is considered as acceptable and satisfies the requirement for sub-acute oral toxicity endpoint.