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Description of key information

An oral diet repeated dose toxicity study (combined with a reproduction/developmental toxicity screening test) was conducted with the registered substance according to guideline OECD 422 and in compliance with GLP. The NOAEL for systemic  toxicity was 8000 ppm, the highest dose tested, corresponding to 490 mg/kg bw/day for males and 501 mg/kg bw/day for toxicity phase females.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
repeated dose toxicity: oral, other
Remarks:
OECD 422
Type of information:
experimental study
Adequacy of study:
key study
Study period:
12 January 2017 - 26 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)
Limit test:
no
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Sponsor - 1002888716
- Appearance: Paste, pale yellow
- Expiration date of the lot/batch: 15 December 2017

STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Dry area, closed containers, room temperature 15 to 25°C
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: 69 to 76 days old; Females: 83 to 90 days old.
- Weight at study initiation: Males: 339-391 g; Females: 237-305 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 thyroid hormone, hematology and blood chemistry investigations)
- Water: Potable water from the public supply via polycarbonate bottles with sipper tubes, ad libitum (removed overnight during urine collection)
- Acclimation period: Females: 20 days before the commencement of treatment; Males: six days before 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: 19 July 2017 to 10 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: The required amount of test item was weighed out into a suitable container. With magnetic stirring, the required amount of solvent was added until the test item was fully dispersed. The dispersion was added to the appropriate amount of plain diet, followed by the addition of a further appropriate amount of plain diet.
The bulk was stirred well and then the solvent evaporated off in a fume hood, until constant weight was achieved.
The required amount of corn oil was then added to the mixture and the bulk mixed firstly using a mechanical grinder, and then with a Turbula mixer after making to the required weight with plain diet.
Appropriate amounts of plain diet was then added to aliquots of this pre-mix, with mixing, until the requisite test item concentration was achieved.
- Frequency of preparation: Weekly.
- Storage of formulation: Frozen (-10 to -30 °C). Stability and homogeneity of the formulations was confirmed for 15 days when stored frozen (-10 to -30°C) and for eight 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.:
2 000 ppm
Remarks:
Group 2 (low dose)
Dose / conc.:
4 000 ppm
Remarks:
Group 3 (mid dose)
Dose / conc.:
8 000 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: SN57HH) following consultation with the Sponsor.
Effects on
body weight and food consumption were seen at 7500 ppm in males at the start of treatment, from which they did not completely recover. The same trend was observed at 10000 ppm in males and females, with slight recovery at the end of treatment. These effects were considered likely to be related to the palatability of test item. Also, high kidneys and liver weights (mean increase of 9 and 28% from controls, respectively) were observed in males at 10000 ppm and even in liver weights at 5000 ppm (mean increase of 15% from controls).
The high dietary concentration of 8000 ppm was expected to elicit initial mean body weight loss or stasis and initial low food consumption and potential adverse effects in target organs, especially in males. The lowest dietary concentration of 2000 ppm was expected to be a no effect level for effects on body weight and food consumption. The intermediate dietary concentration of 4000 ppm allowed evaluation of any concentration related trends and provides 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.

- 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: Body weight range extreme Four males and two females
Irregular estrous cycles Five females
Acyclic One female
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.

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 (Week 3), but recommenced for males on Week 4.
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 (Females only in Recovery phase), Erythrocyte count (RBC), Absolute reticulocyte count, Reticulocyte %, Mean cell haemoglobin, Mean cell haemoglobin concentration, Mean cell volume, Red cell distribution width (Males and Females in recovery phase), Total leucocyte count (Females only in recovery phase), Differential leucocyte count (Males and Females in recovery phase): Neutrophils, Lymphocytes, Eosinophils, Basophils, Monocytes, Large unstained cells, Platelet count, Prothrombin time (Males only in recovery time) and Activated partial thromboplastin time.
- Blood Chemistry parameters: Alkaline phosphatase (ALP), Alanine aminotransferase (ALT), Aspartate aminotransferase (AST) (Males only in recovery phase), Gamma-glutamyl transpeptidase (gGT), Total bilirubin, Bile acids, Urea (Males only in recovery phase), Creatinine (Females only in recovery phase), Glucose (Males and Females in recovery phase), Total cholesterol (Males and Females in recovery phase), Triglycerides, Sodium (Na), Potassium (K), Chloride (Cl), Calcium (Ca) (Males only in recovery phase), Inorganic phosphorus, Total protein (Males only in recovery phase), Albumin (Males only in recovery phase) and Albumin/globulin ratio (A/G Ratio) (Females only in recovery phase).

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.
- 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, unless specified below. 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 only: All remaining adult animals.
°Processing - Adrenals: Toxicity phase males and females in Group 2 and 3 and all Recovery phase males and females.
°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 (no samples were obtained from animals which failed to litter) 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 into Toxicity animals.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Males receiving 8000 ppm had statistically significantly reduced mean body weight gain between Days 1-4 of treatment and slight body weight loss between Days 36-39; however, subsequent body weight gains were similar to Controls.
Animals receiving 2000 or 4000 ppm had slightly higher group mean bodyweight gain between Days 1-46, (which was statistically significant for females) when compared with Controls.
Statistically significant group mean bodyweight loss was observed during Days 1-4 for toxicity and recovery phase females receiving 4000 or 8000 ppm, when compared with Controls. Subsequent body weight loss was also observed during Days 15-22 for females receiving 8000 ppm .
Statistically significant body weight loss was observed in reproductive phase females receiving 4000 or 8000 ppm between Days 1-4, and reduced bodyweight gain was observed in all groups of treated females during Days 4-8, when compared to Controls. Overall body weight gains for females treated at all dose levels remained slightly lower than Controls between Days 1-22 prior to pairing.
During Days 0-20 of gestation body weight gain was similar to the Control group. Body weight gain was higher during Days 1-7, and overall (Days 1-13) during the lactation phase, in all groups treated with the test item, when compared with the Controls.
During the recovery phase (Days R1-R15) the body weight gains of both the Group 4 males and females remained similar to the gains of the Controls.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Group mean food consumption during Days 1-45 was similar to Controls for males receiving 2000 and 4000 ppm of the test item administered via the diet. A slight reduction in food intake was observed in male animals receiving 8000 ppm on Day 1 of treatment, however subsequent food consumption values were similar to Controls.
Toxicity phase and recovery phase females receiving 8000 ppm had slightly reduced food consumption on Day 1 to 5 of treatment when compared with Controls; however food intake was generally unaffected thereafter. Food consumption for females receiving 2000 or 4000 ppm was generally similar to that of Controls.
During Days 1 to 4 of treatment, a reduction in food consumption was observed in reproductive phase female animals receiving 8000 and to a lesser extent 4000 ppm, and on Day 2 and 4 for females receiving 2000 ppm. Following Day 4, food intake was generally similar to that of the Controls up until Day 21 prior to pairing. Females receiving 8000 ppm also showed a statistically significant reduction in food intake on Day 2 of gestation. Food intake of all groups receiving test item were statistically significantly lower on Days 16 and 17 of gestation, when compared to Controls.
Group mean food consumption during Days 1-12 of lactation for females receiving test item was generally similar to that of the Controls. However, on Day 10 of lactation all groups of treated females had statistically significant reduced food intake, when compared with Controls.
Food consumption was similar to the Controls through Days R1-R14 in the recovery phase males and females.
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 animals receiving the test item, when comprae to Controls.
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 both males and females treated at 4000 or 8000 ppm (statistical significance being attained at 8000 ppm). In males, this was predominantly due to increased neutrophils and lymphocytes observed at both the 4000 and 8000 ppm levels. In females, lymphocyte counts were also increased at the 4000 and 8000 ppm dose levels.
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 hematological examinations during the recovery phase revealed a slight decrease in prothrombin time in males previously treated at 8000 ppm, when compared with the Controls. Females previously receiving 8000 ppm of test item had an increase in large unstained cells following two weeks of recovery when compared to the Control group.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
The biochemical examination of the blood plasma indicated an increase in creatinine and plasma cholesterol levels in animals treated at 4000 or 8000 ppm. Glucose levels were statistically significantly lower in animals receiving 2000, 4000 or 8000 ppm when compared to Control values.
For males receiving 8000 ppm, urea levels were found to be statistically significantly higher than Controls, in addition to an increase in calcium, albumin and total protein levels. Statistically significantly lower albumin or globulin ratios were observed for females receiving the test item, when compared with Controls.
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.
Females previously receiving 8000 ppm of test item had an increase in total protein following two weeks of recovery when compared to the Control group.
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):
effects observed, non-treatment-related
Description (incidence and severity):
Sensory reactivity observations: In females receiving 8000 ppm one female showed a normal response to the tail pinch, one female showed an exagerated response and three females showed a weak response. This finding is not considered normal, but as a finding on its own, does not indicate a treatment related effect.

Grip strength observations: The group mean forelimb grip strength value for males receiving 8000 ppm was statistically significantly low when compared with Controls. However, this value was within the Historical Control Data range. The Control values were considered atypically high, and were above the Historical Control Data range. Hindlimb grip strength values were similar to Controls and all within the Historical Control Data range.

The motor activity assessment conducted during Week 6 of treatment revealed no treatment related effects in male animals at all dose levels and females receiving 2000 or 4000 ppm.
All group mean low beam and to a lesser extent high beam activity scores for females treated at 8000 ppm were slightly high when compared with Controls, with statistical significance being attained at the 12-minute interval for high beams and the 6 and 42-minute interval for low beams. However, with the exception of the 42-minute interval (for both high and low beams) all scores were within the Historical Control Data range. The total scores were within the Historical Control Data range, therefore no effect of treatment was inferred.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
The analysis of absolute and adjusted organ weights in the toxicity phase males and females that received the test item had an increase in adrenal and liver weights, when compared to the Controls, with statistical significance being attained for adjusted liver weights for animals that received 8000 ppm.
For the reproductive phase females that received 8000 ppm, the combined weight of the uterus, cervix and oviducts was low, when compared to the Controls.
In the recovery phase males, there were no treatment-related organ weight changes. The adrenal and liver weights remained slightly but not statistically significantly higher in the females previously treated at 8000 ppm, following 14 days of recovery.
All other differences from Controls were minor and were therefore attributed to normal biological variation.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
The macroscopic examination performed after 6 weeks of treatment revealed no test item-related lesions.
The macroscopic examination performed after 14 days of recovery revealed no test item-related lesions.
The incidence and distribution of all other findings were considered to be unrelated to treatment.
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Changes related to treatment with the test item were seen in the adrenal glands of males and females and the kidneys of males.

Adrenal glands:
There was an increase in zona glomerulosa hypertrophy in males and females treated with the test item at 8000 ppm and in males treated with the test item at 4000 ppm. There was an increase in cortical vacuolation in males treated with the test item at 2000 ppm and above, occurring in a dose related pattern.

Kidneys:
There was an increase in cortical tubular hyaline droplets in males treated with the test item at 8000 ppm.

All other findings are considered to be incidental.

After 14 days of recovery, a complete recovery was apparent in the kidneys of males and the adrenal glands of females. However, zona glomerulosa hypertrophy was still apparent in males previously treated with Elemi Resinoid at 8000 ppm. It occurred in one female previously treated with Elemi Resinoid at 8000 ppm but this was similar to main study Control females. There was an increase in cortical vacuolation in males previously treated with Elemi Resinoid at 8000 ppm. All other histological changes were considered to be unrelated to treatment.
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:
8 000 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
histopathology: non-neoplastic
Key result
Critical effects observed:
no

Formulation Analysis:

The homogeneity and stability 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 500ppm and 20000 ppm 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% of nominal concentrations, confirming accurate formulation.

 

Achieved Dose:

Mean achieved doses for toxicity and recovery phase males were 118, 242 or 490 mg/kg bw/day respectively at 2000, 4000 or 8000 ppm.

Mean achieved doses for toxicity and recovery phase females were 123, 239 or 501 mg/kg bw/day respectively at 2000, 4000 or 8000 ppm.

Mean achieved doses for reproductive phase females at 2000, 4000 or 8000 ppm were 126, 247 or 478 mg/kg bw/day before pairing, 139, 282 or 536 mg/kg bw/day during gestation and 309, 601 or 1186 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 8000 ppm (mean achieved doses of 490 mg/kg bw/day for males, 501 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 2000, 4000 and 8000 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.

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% of nominal concentrations, confirming accurate formulation.

There were no treatment-related 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 urinalysis parameters. In addition there were no treatment-related clinical signs observed.

No adverse effects of treatment were evident on the circulating levels of thyroxine. No significant changes were identified at the microscopic examination of thyroid and pituitary glands and the testes and ovaries.

There was an initial reduction of body weight gain/ body weight loss in toxicity phase females receiving 4000 ppm and in both sexes receiving at 8000 ppm, associated with an initial reduction in food consumption during this period, when compared to the Controls.

Haematological findings at termination included increased white blood cell counts in animals treated at 4000 or 8000 ppm, which was predominantly due to increased neutrophils and lymphocytes in males and increased lymphocytes observed at these levels and following a 14 day recovery period lymphocytes levels were still slightly elevated.

Biochemical effects of the blood plasma indicated an increase in creatinine and plasma cholesterol levels in animals treated at 4000 or 8000 ppm. Glucose levels were statistically significantly lower in animals receiving 2000, 4000 or 8000 ppm. Urea levels were found to be statistically significantly higher than Controls for males receiving 8000 ppm, in addition to an increase in calcium, albumin and total protein levels. Statistically significantly lower albumin or globulin ratios were observed for females receiving test item, when compared with Controls. Following a 14 day recovery period females previously receiving 8000 ppm maintained an increase in total protein, when compared to the Control group, but all other parameters were similar to Controls.

The analysis of absolute and adjusted organ weights in the toxicity phase males and females that received the test item had an increase in adrenal and liver weights, when compared to the Controls, with statistical significance being attained for adjusted liver weights for animals that received 8000 ppm. In the recovery phase the adrenal and liver weights remained slightly but not statistically significantly higher in the females previously treated at 8000 ppm, following 14 days of recovery.

Changes considered to be related to administration of the test item were present in the adrenal glands of males and females and kidneys of males. There was an increase in zona glomerulosa hypertrophy seen in the adrenals of males and females treated at 8000 ppm and in males treated at 4000 ppm, and was persistent in males after 14 days of recovery. Vacuolation of the adrenal gland cortex was increased in males given the test item at 2000 ppm and above in a dose-related incidence. This persisted after 14 days of recovery.

There was an increase in renal cortical tubular hyaline droplets in males treated with the test item at 8000 ppm.

Dietary administration of 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 8000 ppm was generally well tolerated and there were no treatment-related mortalities at any level.

There were initial effects on bodyweight and food consumption at the start of treatment, but this had no impact on overall bodyweight and showed full recovery. Test item showed no evidence of being an endocrine disruptor and did not affect thyroid hormone levels.

Administration of test item was associated with minimal to slight changes in the adrenal glands of males and females and kidneys of males receiving 8000 ppm. Slight changes were noted in the adrenal glands of males treated at 2000 ppm and above that persisted in the adrenal glands of males after a 14-day recovery period. However, these changes are considered likely to be non-adverse.

Based on the results of this study it is concluded that the No-Observed-Adverse-Effect- Level (NOAEL) for systemic toxicity is 8000 ppm (mean achieved doses of 490 mg/kg bw/day for males, 501 mg/kg bw/day for toxicity phase females).

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
490 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 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 2000, 4000 and 8000 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.

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% of nominal concentrations, confirming accurate formulation.

There were no treatment related 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 urinalysis parameters. In addition there were no treatment related clinical signs observed.

No adverse effects of treatment were evident on the circulating levels of thyroxine. No significant changes were identified at the microscopic examination of thyroid and pituitary glands and the testes and ovaries.

There was an initial reduction of body weight gain/ body weight loss in toxicity phase females receiving 4000 ppm and in both sexes receiving at 8000 ppm, and an initial reduction in food consumption during this period, when compared to the Controls.

Haematological findings at termination included increased white blood cell counts in animals treated at 4000 or 8000 ppm, which was predominantly due to increased neutrophils and lymphocytes in males and increased lymphocytes observed at these levels and following a 14 day recovery period lymphocytes levels were still slightly elevated.

Biochemical effects of the blood plasma indicated an increase in creatinine and plasma cholesterol levels in animals treated at 4000 or 8000 ppm. Glucose levels were statistically significantly lower in animals receiving 2000, 4000 or 8000 ppm. Urea levels were found to be statistically significantly higher than Controls for males receiving 8000 ppm, in addition to an increase in calcium, albumin and total protein levels. Statistically significantly lower albumin or globulin ratios were observed for females receiving test item, when compared with Controls. Following a 14 day recovery period females previously receiving 8000 ppm maintained an increase in total protein, when compared to the Control group, but all other parameters were similar to Controls.

The analysis of absolute and adjusted organ weights in the toxicity phase males and females that received the test item had an increase in adrenal and liver weights, when compared to the Controls, with statistical significance being attained for adjusted liver weights for animals that received 8000 ppm. In the recovery phase the adrenal and liver weights remained slightly but not statistically significantly higher in the females previously treated at 8000 ppm, following 14 days of recovery.

Changes considered to be related to administration of the test item were present in the adrenal glands of males and females and kidneys of males. There was an increase in zona glomerulosa hypertrophy seen in the adrenals of males and females treated at 8000 ppm and in males treated at 4000 ppm, and was persisted in males after 14 days of recovery. Vacuolation of the adrenal gland cortex was increased in males given the test item at 2000 ppm and above in a dose related incidence. This persisted after 14 days of recovery.

There was an increase in renal cortical tubular hyaline droplets in males treated with the test item at 8000 ppm.

Dietary administration of 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 8000 ppm was generally well tolerated and there were no treatment related mortalities at any level.

There were initial effects on bodyweight and food consumption at the start of treatment, but this had no impact on overall bodyweight and showed full recovery. Test item showed no evidence of being an endocrine disruptor and did not affect thyroid hormone levels.

Administration of test item was associated with changes in the adrenal glands of males and females and kidneys of males receiving 8000 ppm. Changes were noted in the adrenal glands of males treated at 2000 ppm and above. Changes persisted in the adrenal glands of males after a 14 day recovery period. All changes are considered likely to be non-adverse.

Based on the results of this study it is concluded that a No-Observed-Effect-Level (NOEL) for systemic effects could not be achieved. Therefore, the No-Observed-Adverse-Effect- Level (NOAEL) for systemic toxicity is 8000 ppm (mean achieved doses of 490 mg/kg bw/day for males, 501 mg/kg bw/day for toxicity phase females).

Justification for classification or non-classification

The NOAEL obtained in the OECD guideline 422 study is mainly based on changes observed in the adrenal glands of males and females and kidneys of males receiving 8000 ppm, as this changes persisted in the adrenal glands of males after a 14 day recovery period.

Therefore, the NOAEL for systemic toxicity is 8000 ppm (mean achieved doses of 490 mg/kg bw/day for males, 501 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.

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