Essential oil of Boswellia carterii (Burseraceae) obtained from gum by distillation

Endpoint summary

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Effects on fertility

Description of key information

Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening test: NOAEL Fertility = 15000 ppm (equivalent to 905 mg/kg bw/day during gestation and 1962 mg/kg bw/day during lactation), (OECD 422, GLP, K, Rel. 1).

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Remarks:

OECD 422

Type of information:

experimental study

Adequacy of study:

key study

Study period:

22 February to 19 October, 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 same study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

Inspected on / Signed on 2019-04-02 / Signed on 2019-08-01

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: VB48HY). In that study doses of 5000, 10000 and 15000 ppm were investigated and animals treated for 14 days. Overall body weight gains were slightly low at all dietary concentrations in both males and females. The food intake at 10000 or 15000 ppm was markedly lower after 1 or 2 days but improved thereafter, indicating that
palatability was influential but not a considerable factor on body weights. Liver weights were high in all females or in males at 15000 ppm. Therefore, dietary concentrations of 3000, 7500 and 15000 ppm were selected for this main OECD 422 study.
- 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) 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: 69 to 76 days old. Females: 83 to 90 days old.
- Weight at study initiation: Males: 322 to 382 g; Females: 244 to 306 g
- Housing: Solid (polycarbonate) bottom cages were used during the acclimatization, pre-pairing, 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: Males: six days before commencement of treatment. Females: 20 days before commencement treatment.
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) and replaced when necessary.
Plastic shelter: Provided to each cage throughout the study (except during pairing and lactation) and replaced at the same time as the cages.

IN-LIFE DATES: From 05 March To 14 August, 2018

Route of administration:

oral: feed

Vehicle:

other: Feed

Details on exposure:

DIET PREPARATION
- Diet: SDS VRF1 Certified powdered diet
- Correction factor: A correction factor was not required.
- Stabilizer: Corn oil (test material to corn oil ratio 5:1).
- Method of preparation: The test item was incorporated into the diet to provide the required concentrations by initial preparation of a premix. The amount of test item and corn oil required for the premix were added to an equal amount of plain (basal) 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.
- Frequency of preparation: Weekly.
- Storage of formulation: Frozen (-10 to -30°C). Diet was allowed to thaw before feeding commenced. 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).

Details on mating procedure:

- Animals: Toxicity phase and Recovery phase males 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: Presence of sperm within the vaginal smear and/or ejected copulation plugs referred to as Day 0 of gestation.
- 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.

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 at 500 and 20000 ppm were analyzed to assess the stability and homogeneity of the test item in the diet matrix. Formulations were found to be homogenous and stable for 15 days when stored frozen (10 to -30°C) and for four days when stored at ambient temperature (15 to 25°C).
- Achieved concentration: Samples of each formulation prepared for administration in Week 1 and in 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

Dose / conc.:

0 ppm

Remarks:

Group 1 (Control: Basal diet + corn oil)

Dose / conc.:

3 000 ppm

Remarks:

Group 2 (Low dose)

Dose / conc.:

7 500 ppm

Remarks:

Group 3 (Mid dose)

Dose / conc.:

15 000 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: VB48HY). In that study doses of 5000, 10000 and 15000 ppm were investigated and animals treated for 14 days. Overall body weight gains were slightly low at all dietary concentrations in both males and females. The food intake at 10000 or 15000 ppm was markedly lower after 1 or 2 days but improved thereafter, indicating that
palatability was influential but not a considerable factor on body weights. Liver weights were high in all females or in males at 15000 ppm. Therefore, dietary concentrations of 3000, 7500 and 15000 ppm were selected for this main OECD 422 study.
- Rationale for animal assignment: On arrival and non-selective allocation to cages.
Estrous cycles were evaluated prior to treatment. After 14 days evaluation, animals that failed to exhibit typical 4-5 day cycles were not allocated to the reproductive phase of 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.
- Post-exposure recovery period in satellite groups: 14 days
- Other: Each adult animal was assigned a number and identified uniquely within the study using a microchip 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: five females.
Body weight range extremes: two 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.
A viability check was performed near the start and end of each working day. Animals were killed for reasons of animal welfare where necessary.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Before treatment commenced, during each week of treatment and recovery, on Days 0, 6, 13 and 20 after mating and on 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 (before feeding of the treated diets on Day 1).

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 weekly thereafter, including the recovery phase. On Day 5 of recovery the animals were fed diets prepared for the females in the lactation phase in error (recovery control animals received control diet with the corn oil stabiliser, and recovery animals in Group 4 received treated diet (8000 ppm); 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 weekly before pairing; Days 0, 7, 14 and 20 after mating; Day 1, 4, 7 and 13 of lactation; On the day of 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 as follows:
F0 animals: Daily, including the recovery phase. On Day 5 of recovery the animals were fed diets prepared for the females in the lactation phase in error (recovery control animals received control diet with the corn oil stabiliser, and recovery animals in Group 4 received treated diet (8000 ppm). Food consumption was not recorded for Toxicity phase males and Reproductive phase females during the period when paired for mating (Week 3), but recommenced for males in Week 4. Food consumption was recorded continuously for Toxicity and Recovery phase females. For Reproductive phase females after mating food consumption was performed daily throughout gestation and lactation (until Day 12).
From these records the mean daily consumption per animal (g/animal/day) was calculated for each phase.

OTHER:
NEUROBEHAVIOURAL EXAMINATION:
- Time schedule:
Sensory reactivity and grip strength: Sensory reactivity and grip strength assessments were performed on all recovery animals in Groups 1 and 4 and on the lowest numbered toxicity phase males and females in Groups 2 and 3 during Week 6 of treatment.
Motor activity: During Week 6 of treatment, the motor activity of all recovery animals in Groups 1 and 4 and on the lowest numbered toxicity phase males and females in Groups 2 and 3 was measured.

OPHTHALMOLOGY
- Time schedule:
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:
- Time schedule for collection of blood:
Week 6: Five lowest numbered surviving Toxicity phase males and females in each group; Recovery: All male Recovery animals
- 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.
- Animals were held under light general anaesthesia induced by isoflurane. Blood samples were withdrawn from the sublingual vein. Sampling was performed on the morning after overnight collection of urine.
- 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
- Time schedule for collection of urine:
Week 6: Five lowest numbered surviving Toxicity phase males and females in each group; Recovery: All Recovery animals
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, Creatinine, Glucose, 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.

Thyroid Hormone Analysis
- Time schedule for examination
At termination: F0 males, All F0 Reproductive phase females
Day 4 of age - F1 offspring, two females per litter (where possible) - no pups were eliminated when litter size dropped below ten/litter
- one for T4 (serum)#
- one for TSH (plasma - optional analysis)
# priority given to serum sample
Day 13 of age: F1 offspring, two males and two females per litter (where possible)
- two for T4 (serum): where possible one male and one female#
- two for TSH (plasma): where possible one male and one female
# priority given to serum sample

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):

Estrous Cycle
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 the Reproductive phase of the study.
- After pairing until mating.
- For four days before scheduled termination (all Reproductive phase, Toxicity phase and Recovery phase females).

Dry smears:
Reproductive phase females: from beginning of treatment until animals were paired for mating, using cotton swabs (approximately three weeks).

Litter observations:

Clinical observations: Examined at approximately 24 h after birth (Day 1 of age) and then daily thereafter for evidence of ill health or reaction to 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 failing to produce a viable litter: Day 25 after mating.
F0 females: 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.
Main phase females
The following were recorded:
Each uterine horn: Number of implantation sites was counted and confirmed.

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: All F0 animals killed or dying prematurely; Toxicity phase males and females in Groups 1 and 4 at scheduled termination.
Abnormalities: All remaining adult animals.
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 (external and internal) with an assessment of stomach for milk content was performed. Abnormal tissues retained.
- F1 offspring on Day 4 of age:
Blood sampling required
Externally normal offspring discarded without examination.
Externally abnormal offspring identified on despatch to necropsy, examined externally, and retained pending possible future examination.
- F1 offspring on Day 13 of age
Blood sampling required
All animals (but not including those selected for thyroid hormone analysis) 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.
Animals selected for thyroid hormone analysis: externally normal offspring discarded without examination; externally abnormal offspring examined.

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
- 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 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 considered to be no signs seen that were related to treatment during detailed physical examination and arena observations for animals in the treatment, gestation or lactation period.

Mortality:

no mortality observed

Description (incidence):

No deaths were observed during the study.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

- Overall body weight gain in males was unaffected by treatment. However, there was a statistically significant lower body weight gain following initial treatment (Days 1 to 4 at 15000 ppm and Days 4 to 8 at 3000 or 7500 ppm). Subsequent body weight gains of males at any dietary concentration were similar to Controls - with the exception of males treated at 15000 ppm in Week 6 (Days 36-43).
Overall body weight gain in females of toxicity and recovery phase during treatment was unaffected by treatment at 3000 ppm. Group mean body weight loss was evident from Days 1-4 for toxicity and recovery phase females receiving 7500 or 15000 pm and body weight stasis was observed for females at
3000 ppm. Thereafter, the body weight gains in females at all dietary concentrations improved and were generally similar to Controls, however the extent of the body weight loss experienced by females receiving 15000 ppm resulted in a significant lower overall body weight gain, leading to statistically significant lower mean bodyweight (-7% of Controls).
- Reproductive phase females receiving 7500 or 15000 ppm started gestation with slightly low body weights (0.93X of Control, both); and, the overall body weight gains in females at 7500 and 15000 ppm were low (-10% and -21% of Controls, respectively) with statistical significance attained at 7500 ppm due to statistically significant lower values during the first 2 weeks of gestation. Females at 3000 or 7500 ppm were unaffected during the gestation period.
Overall body weight gain for reproductive phase females in lactation was unaffected by treatment. The body weight loss observed in females treated at 7500 ppm and the slightly high body weight gain in females receiving 3000 ppm, during lactation Days 1 to 4 were considered incidental and not related to treatment. There was a higher body weight gain in females at the end of the recovery period (+50% of Controls) due to higher bodyweight gain during the first week of recovery period. The body weight gain in males that previously received 15000 ppm was higher than Controls throughout recovery period, leading to statistical significance on the overall mean values (+59% of Controls).

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Food consumption in males was generally unaffected by treatment with Olibanum oil. Following initial treatment, there was a slight reduction in food intake in males receiving 3000 or 7500 ppm, and a marked reduction of food intake in males receiving 15000 ppm - with slightly low food intake persisting until Day 2 or 3 of treatment. There was also a period of marginally reduced food intake in males receiving 15000 ppm between Days 27 and 34. For females, the pattern of food consumption was similar, although slightly more pronounced, at 7500 or 15000 ppm and persisted for a longer (Day 4 or 7, respectively) period, than the males. During the recovery period food intake for males, that previously received 15000 ppm, was higher than Control (+10%), especially during the first 5 days of recovery period while food intake was globally similar to Control in females, although higher intake was also observed during the first 2 days of recovery period. In the gestation period, food consumption for reproductive phase females receiving treated diet at 7500 or 15000 ppm was marginally lower than Controls during the first 2 weeks of gestation, leading to an overall reduction of -9% and -17% of Control, respectively. Food intake at 3000 ppm was slightly low on Days 0 to 1 of gestation. Throughout the lactation period, food consumption was low in females receiving 7500 or 15000 ppm with an overall reduction of -20% and -28% of Control, respectively.

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 5 of treatment for animals receiving Olibanum oil, when compared to Controls.

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

The hematological examination of the blood plasma in Week 5 did not reveal any toxicological differences from Controls. All inter-group differences from Controls, including those that attained statistical significance, were generally small, confined to one sex, or the magnitudes were not doserelated and, consequently, were considered to represent normal biological variation. Such differences included the statistically significant differences, at 15000 ppm, for large unstained cells in males and in neutrophil or eosinophil counts in females, although there was clearly no relationship to treatment, differences were minor, exclusive to one sex, and all values were within the HCD range

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

The biochemical examination of the blood plasma in Week 5 did not reveal any toxicological differences from Controls. All inter-group 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.

Urinalysis findings:

effects observed, non-treatment-related

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 Olibanum oil, 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 not considered to be associated with treatment.

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

Sensory reactivity and grip strength were considered unaffected by treatment.
Motor activity scores for animals given the Test Item were considered to be unaffected by treatment. A slight increase total high beam breaks in females at 7500 ppm or 15000 ppm was observed; however, the difference did not attain statistical significance, was not present in the males and there was no consistent difference in the pattern of breaks over time.

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):

- Animals Killed After 6 Weeks of Treatment:
Treatment Related Findings Changes related to treatment with Olibanum Oil were seen in the kidneys.
* Kidney: in the kidneys, accumulation of hyaline droplets in the renal tubular epithelium was observed at similar incidence across the groups of males given Olibanum Oil at 3000, 7500 or 15000 ppm. The severity of these changes showed a dose response pattern. Basophilic tubules in the kidneys were also observed at increased incidence and severity in males given the test item at 3000, 7500 or 15000 ppm, when compared with control.
- Animals Killed After 14 days of Recovery:
Treatment Related Findings:
Test item related changes were observed in the kidneys of males previously treated with Olibanum Oil at 15000 ppm.
* Kidney:
Tubular changes such as basophilia and dilatation (with intraluminal tubular casts), associated with minimal mononuclear cells inflammatory infiltrate in the interstitium were observed in the kidneys of recovery males previously treated with Olibanum Oil at 15000 ppm.

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 Mid samples.

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

Estrous cycles was considered unaffected by treatment with Olibanum oil, when compared with Controls. Prior to treatment all females had a regular estrus cycle of 4 to 5 days, with the exception of one toxicity phase female in the 7500 ppm group and one recovery phase Control female.
During the treatment period all females had a regular estrus cycle of 4 to 5 days, with the exception of one female (4F 143) receiving 15000 ppm which had an irregular estrous cycle. As this was an isolated incidence, the exception was considered not to be treatment related.

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 Olibanum oil, when compared with Controls Gestation lengthand gestation index were considered unaffected by treatment at any dose level.

Key result

Dose descriptor:

NOAEL

Effect level:

7 500 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

food consumption and compound intake

Remarks on result:

other: 7500 ppm, equivalent to 428 mg/kg bw/day for males and 442 mg/kg bw/day for females.

Key result

Critical effects observed:

no

Clinical signs:

no effects observed

Description (incidence and severity):

There were no clinical signs observed among the F1 litters that were clearly attributable to parental treatment of the Test Item.

Mortality / viability:

no mortality observed

Description (incidence and severity):

The total litter size at 7500 or 15000 ppm was slightly low as a result of the low implantation counts, although statistical significance was not achieved. The number of live young on Day
1 was slightly low at 15000 ppm which resulted in a slightly low live birth index for this group, but not at 7500 ppm.
There was considered to be no effect of parental treatment on offspring survival or

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Mean offspring body weight on Day 1 of age was unaffected by parental treatment, however overall body weight gain (Days 1 to 13 of age) was low in offspring of parents treated with Olibanum oil with relationship to dietary concentration. Group mean male or female offspring bodyweights on Day 13 of age were statistically significantly low in the 7500 (0.90X or 0.89X Control for males or females, respectively) or 15000 ppm (0.82X Control for both sexes) groups.

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

Anogenital distance (AGD):

no effects observed

Description (incidence and severity):

There was considered to be no effects of parental treatment on the ano-genital distances of offspring.

Nipple retention in male pups:

no effects observed

Description (incidence and severity):

No nipples were observed in male offspring of parents treated with Olibanum oil.

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no macroscopic findings observed in the offspring that died prior to scheduled termination or among those offspring killed on Day 13 of age that were attributable to parental treatment with the test item.

Histopathological findings:

not examined

Other effects:

no effects observed

Description (incidence and severity):

Sex ratios at first litter check and on day 4 post partum were not considered to have been affected by the test item.

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Key result

Dose descriptor:

NOAEL

Remarks:

Screening for development (foetal and pup growth survival until day 4)

Generation:

F1

Effect level:

15 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects observed

Remarks on result:

other: 15000 ppm: equivalent to 905 mg/kg bw/day during gestation and 1962 mg/kg bw/day during lactation..

Key result

Critical effects observed:

no

Key result

Reproductive effects observed:

no

Formulation Analysis:

The mean concentrations of Olibanum oil for Week 1 and the final week of treatment were within the applied limits of +10/-15%, confirming the accuracy of formulation. The difference from mean remained within 2%, confirming precise analysis. The procedural recoveries remained within the validated range, confirming the continued accuracy of the analytical procedure.

Conclusions:

Under the test condition, the No Observed Adverse Effect Level (NOAEL) for systemic toxicity was considered to be 7500 ppm (equivalent to 428 mg/kg/day for males and 422 mg/kg/day for females) and 15000 ppm for the reproductive/developmental toxicity (equivalent to 905 mg/kg bw/day during gestation and 1962 mg/kg bw/day during lactation).

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 3000, 7500 and 15000 ppm. An additional subgroup was used to assess reversibility, persistence or delayed occurrence of systemic effects for 14 days post treatment. A similarly constituted control group was assigned to each phase, and received the vehicle, powdered SDS VRF1 Certified diet with corn oil, throughout the same relative treatment period.

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.

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

Mean achieved doses for males at 3000, 7500 or 15000 ppm were 178, 428, 837 mg/kg bw/day, respectively. For Toxicity and Recovery phase females during treatment and Reproductive phase females before pairing, mean achieved doses were 184, 442, 863 mg/kg bw/day, respectively. Mean achieved doses for females during gestation were 189, 476 and 905 mg/kg bw/day, respectively, and for females during lactation were 459, 1015 or 1962 mg/kg bw/day, respectively.

Analyses of samples for thyroxine (T4) obtained from Main study male animals and F1 offspring on Day 13 of age did not reveal any differences that could be attributed to treatment. Administration of Olibanum oil at dietary levels of 3000, 7500 or 15000 ppm had no effect on clinical condition, sensory reactivity and grip strength, motor activity, ophthalmology, hematology, blood chemistry, urinalysis, estrous cycles, pre-coital interval, mating performance, fertility and gestation length or macropathology of the adult animals.

Food intake was low after initial treatment on Day 1 to 4 and resulted in group mean body weight stasis/loss in treated females, and a lower initial body weight gain in males at 15000 ppm. Overall, there was no effect on body weight gains for males. The reproductive females at 7500 or 15000 ppm commenced gestation with body weights that were slightly low as a result of the initial body weight losses; and, for females at 15000 ppm, the performance at the start of treatment contributed to the difference in overall body weight gains across the study.

Liver weights in all treated groups, except for females at 3000 ppm were high, and kidney weights in males at 7500 or 15000 ppm were slightly high.

Microscopic changes related to treatment with the test item were seen in the kidneys of males at all treated groups, although for males with 14 days of recovery hylaline droplets were no longer present. Implantation counts in females at 7500 or 15000 ppm were slightly low but was considered not to be due to the intrinsic property of the test item. This lower level of implantations resulted in a slightly low total litter size for both groups. The live birth index was slightly low at 15000 ppm. There was no effect of parental treatment on offspring clinical signs, sex ratio, offspring body weight on Day 1, survival, ano-genital distance, nipple counts or macropathology. However, offspring body weight by Day 13 from treated parents was lower than Controls, with relationship to dose.

In the adults, test article-related histopathological changes seen in the kidneys of males were considered to be a typical species- and sex-specific response to general xenobiotic exposure, however the changes were deemed reversible and not relevant to humans. The effect on overall F0 body weights was adverse since this was had an influence on the implantation counts at 15000 ppm and, therefore, the fecundity of females. The offspring body weights of litters from parents treated at 15000 ppm was considered adverse however this was attributed to the effect of treatment on the maternal system.

Based on these considerations, the No Observed Adverse Effect Level (NOAEL) was concluded to be 7500 ppm for the systemic toxicity (equivalent to 428 mg/kg bw/day for males and 442 mg/kg bw/day for females) and 15000 ppm for the reproductive/developmental toxicity (equivalent to 905 mg/kg bw/day during gestation and 1962 mg/kg bw/day during lactation).

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

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

905 mg/kg bw/day

Study duration:

subacute

Species:

rat

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

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 3000, 7500 and 15000 ppm. An additional subgroup was used to assess reversibility, persistence or delayed occurrence of systemic effects for 14 days post treatment. A similarly constituted control group was assigned to each phase, and received the vehicle, powdered SDS VRF1 Certified diet with corn oil, 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 examinations, hematology (peripheral blood), blood chemistry, urinalysis, thyroid hormone analysis, estrous cycles, pre-coital interval, mating performance, fertility, gestation length, organ weight, macropathology 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.

Mean achieved doses for males at 3000, 7500 or 15000 ppm were 178, 428, 837 mg/kg bw/day, respectively. For Toxicity and Recovery phase females during treatment and Reproductive phase females before pairing, mean achieved doses were 184, 442, 863 mg/kg bw/day, respectively. Mean achieved doses for females during gestation were 189, 476 and 905 mg/kg bw/day, respectively, and for females during lactation were 459, 1015 or 1962 mg/kg bw/day,

respectively. Analyses of samples for thyroxine (T4) obtained from Main study male animals and F1 offspring on Day 13 of age did not reveal any differences that could be attributed to treatment. Administration of Olibanum oil at dietary levels of 3000, 7500 or 15000 ppm had no effect on clinical condition, sensory reactivity and grip strength, motor activity, ophthalmology, hematology, blood chemistry, urinalysis, estrous cycles, pre-coital interval, mating performance, fertility and gestation length or macropathology of the adult animals. Food intake was low after initial treatment on Day 1 to 4 and resulted in group mean body weight stasis/loss in treated females, and a lower initial body weight gain in males at 15000 ppm. Overall, there was no effect on body weight gains for males. The reproductive females at 7500 or 15000 ppm commenced gestation with body weights that were slightly low as a result of the initial body weight losses; and, for females at 15000 ppm, the performance at the start of treatment contributed to the difference in overall body weight gains across the study.

Liver weights in all treated groups, except for females at 3000 ppm were high, and kidney weights in males at 7500 or 15000 ppm were slightly high.

Microscopic changes related to treatment with the test item were seen in the kidneys of males at all treated groups, although for males with 14 days of recovery hylaline droplets were no longer present. Implantation counts in females at 7500 or 15000 ppm were slightly low but was considered not to be due to the intrinsic property of the test item. This lower level of implantations resulted in a slightly low total litter size for both groups. The live birth index was slightly

low at 15000 ppm. There was no effect of parental treatment on offspring clinical signs, sex ratio, offspring body weight on Day 1, survival, ano-genital distance, nipple counts or macropathology. However, offspring body weight by Day 13 from treated parents was lower than Controls, with relationship to dose.

In the adults, test article-related histopathological changes seen in the kidneys of males were considered to be a typical species- and sex-specific response to general xenobiotic exposure, however the changes were deemed reversible and not relevant to humans. The effect on overall F0 body weights was adverse since this was had an influence on the implantation counts at 15000 ppm and, therefore, the fecundity of females. The offspring body weights of litters from parents treated at 15000 ppm was considered adverse however this was attributed to the effect of treatment on the maternal system.

Based on these considerations, the No Observed Adverse Effect Level (NOAEL) was concluded to be 7500 ppm for the systemic toxicity (equivalent to 428 mg/kg bw/day for males and 442 mg/kg bw/day for females) and 15000 ppm for the reproductive/developmental toxicity (equivalent to 905 mg/kg bw/day during gestation and 1962 mg/kg bw/day during lactation).

Effects on developmental toxicity

Description of key information

One screening study for reproductive toxicity was available, showing no adverse effect on development (Iuclid §7.8.1). Therefore a waiver is submitted for this endpoint.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

905 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

The study used for endpoint conclusion is GLP-compliant and of high quality. Additional developmental toxicity studies are not deemed necessary.

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Justification for classification or non-classification

Harmonized classification:

The substance has no harmonized classification according to the Regulation (EC) No 1272/2008 (CLP).

Self-classification:

In a recent GLP repeated dose toxicity study (OECD guideline 422), no adverse systemic toxicity changes relevant to humans were observed in males and females rats exposed to the registered substance. Furthermore, no effects on fertility and post-natal effects were observed. In this OECD guideline 422 study, the NOAEL was set at 7500 ppm.

Therefore, the registered substance does not need to be classified for reproductive toxicity according to CLP Regulation (EC) n° 1272/2008 and UN GHS criteria.