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

Endpoint summary

Description of key information

In a Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test (OECD Guideline 422, GLP, Rel.1, K), the NOAEL for systemic toxicity was 7500 ppm (mean achieved doses of 428 mg/kg bw/day for males, 442 mg/kg bw/day for toxicity and recovery phase females during treatment and reproductive phase females before pairing , 476 mg/kg bw/day for females during gestation and 1015 mg/kg bw/day for females during lactation). The NOAEL of test item for reproductive/developmental effects was concluded to be 7500 ppm.The mean of the mean achieved doses for males, toxicity phase females and reproductive phase females before mating, obtained during the OECD 422 study corresponds to mg/kg bw/day.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

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:

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

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

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
- 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).

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

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.
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.

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 male Recovery 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. 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: Yes
- 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.

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 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.
- 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 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.

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.

Other examinations:

Thyroid Hormone Analysis:
At termination: F0 males, All F0 Reproductive phase females

Statistics:

See "Any other information on materials and methods incl. tables".

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 this 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, non-treatment-related

Description (incidence and severity):

Analysis of organ weights at scheduled termination after 6 weeks of treatment revealed high adjusted liver weights in females receiving 7500 or 15000 ppm (1.1X or 1.2X Controls, respectively) or in males at any dietary concentration (between 1.2 to 1.3X Controls).
Absolute and adjusted adrenal weights were slightly low in all groups of treated females (between 0.80 to 0.81X of Control for adjusted weights) but without dose relationship and adjusted kidney weights were slightly high in males receiving 7500 or 15000 ppm (1.1X or 1.2X Controls, respectively) with statistical significance attained at 15000 ppm only. These differences in adrenal or kidney weights were not apparent in the opposite sex.
Adjusted heart weights were marginally low in males at 7500 or 15000 ppm but not doserelated (91 or 94% of Control, respectively).
At the end of the recovery period, mean adjusted liver weights remained slightly high in males previously treated at 15000 ppm.

Gross pathological findings:

no effects observed

Description (incidence and severity):

- Animals Killed After 6 Weeks of Treatment:
The macroscopic examination performed after 6 weeks of treatment revealed no intergroup differences.
- Animals Killed After 14 Days of Recovery:
The macroscopic examination performed after 14 days of recovery revealed no intergroup differences.

Neuropathological findings:

not examined

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.

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

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 (mean achieved doses of 428 mg/kg bw/day for males, 442 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 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.

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, organ weight, macropathology and histopathology investigations were undertaken.

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, 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/day, respectively, and for females during lactation were 459, 1015 or 1962 mg/kg/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 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.

The 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.

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).

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 UN GHS.

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

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

401 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

The key study is GLP-compliant and of high quality (Klimisch score = 1)

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

In the dose range-finding study for the Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test (Envigo, 2018, rel.1), groups of Crl:CD(SD) rats (4/sex/dose) received test item orally, via the diet at 5000, 10000 or 15000 ppm.

All dietary concentrations were well tolerated and all animals survived the treatment period, with no adverse clinical sign and no visual water consumption effects. 

Group mean body weight loss was evident in males and females receiving 10000 or 15000 ppm following the start of treatment which resulted in low weight gains during the first week except in females receiving 10000 ppm; low weight gains persisted in week 2 for males receiving 10000 ppm. Body weight gains of males receiving 5000 ppm were low for the first day of treatment. Food intake on Day 1 of treatment was markedly low for males and females receiving 15000 ppm and for females receiving 10000 ppm; intake was also low for males receiving 10000 ppm. By Day 3 of study, food intake had improved and had generally returned to pre-treatment levels.

There was a slight increase in absolute and body weight adjusted liver weights in males at 15000 ppm or females at all dietary concentrations which may reflect a general adaptive response to a xenobiotic. There were some other slight differences from Controls in terms of organ weights including kidneys, thymus and spleen with macroscopic findings seen in the kidneys of two treated animals. It is however acknowledged that there was no clear relationship to treatment, and the findings seen are common background findings.

The high-dose level to be used in the main study was 15000 ppm.

In the main study, considered as the key study (Envigo, 2018, rel. 1), in this study the systemic toxic potential of Olibanum oil was assessed when administered

orally, in the diet, to Sprague Dawley (Crl: CD (SD)) rats at dietary concentrations of 3000, 7500 or 15000 ppm for a minimum of five weeks. Reversibility, persistence or delayed occurrence of systemic effects was also assessed during a 2-week off-dose period.

Treatment with Olibanum oil was generally well tolerated with no premature deaths in adults, no test article-related signs observed during the detailed physical examination and arena observations, and no effects on sensory reactivity and grip strength, motor activity, ophthalmology, hematology, blood chemistry, urinalysis or macropathology of the adult animals.

Considering the body weight gains of females after Day 4 were similar to Control, the reason for the lower body weights for females commencing treatment was due to the loss of body weight from Days 1 to 4 for females at 7500 or 15000 ppm. Females receiving 15000 ppm then gained slightly less weight during days 0 to 14 of gestation, probably due to their slightly smaller size. There was considered to be no adverse effect on body weights in males at any dietary concentration since overall body weight was unaffected. Food consumption reflected the patterns seen in body weights. Liver weights were high, when compared to Controls, in males at any dietary concentration or in females receiving 7500 or 15000 ppm; although since there were no differences in clinical pathology parameters and the microscopic examination of the liver did not reveal any findings this was not considered to be adverse. In the kidney, accumulation of hyaline droplets in the tubular epithelium and tubular basophilia was seen in all treated males, and this correlated with the increased kidney weights seen at 7500 or 15000 ppm. For males at 15000 ppm given 14 days of recovery, the hyaline droplets in the kidneys were no longer observed and the difference in kidney weights did not achieve statistical significance; tubular changes (basophilia and dilatation) were observed but, overall, this was indicative of, at least, partial recovery. The lower adrenal weights in all groups of treated females and the marginally lower adjusted heart weights in males at 7500/15000 ppm were not supported by any microscopic pathology changes and are therefore of uncertain relationship to treatment and not adverse findings.

In the adults, test article-related histopathological changes seen in the kidneys of males were considered to be a typical xenobiotic response to general insult, however the changes were deemed reversible. 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. Therefore, based on the body weights effects observed in females at 15000 ppm, the No Observed Adverse Effect Level (NOAEL) was

concluded to be 7500 ppm for the systemic toxicity (equivalent to 428 mg/kg/day for males and 442 mg/kg/day for females)

The No Observed Adverse Effect Level (NOAEL) for repeated dose toxicity is therefore 7500 ppm.

Justification for classification or non-classification

Harmonized classification:

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

Self-classification:

Based on the available data, no additional classification is proposed regarding the specific target organ toxicity after oral dose-repeated exposure acording to the Annex I of the regulation (EC) No. 1272/2008 (CLP) and UN GHS.