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Description of key information

In a Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test (OECD Guideline 422), the NOAEL for systemic toxicity was7000 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.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 401 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:
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
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
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
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
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".
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.
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

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).
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.

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:
Recent GLP study conducted according to OECD Guideline No 422 without any deviation (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 3000, 6000 or 12000 ppm.

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. Uterus and cervix weights were higher for females when compared to Controls.

The effects on bodyweight and food consumption at the start of treatment, from which the animals quickly recovered, were considered likely to be related to the palatability of the test item.

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).

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 1750, 3500 or 7000 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.

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.

Based on the results of this study it is concluded that a No-Observed-Effect-Level (NOEL) for systemic effects could not be achieved as hyaline droplets were observed in the kidneys of males given 1750, 3500 or 7000 ppm, but this is of no relevance to humans and this finding was not observed in females at the same levels, and was not observed following a 14-day recovery period.

The No Observed Adverse Effect Level (NOAEL) for repeated dose toxicity is therefore 7000 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:

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 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) which is above the classification threshold of 300 mg/kg bw/day for a sub-acute study.

Therefore the registered substance is not classified for repeated dose toxicity according to CLP Regulation (EC) No 1272 /2008 and UN GHS criteria.

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