Registration Dossier

Administrative data

Description of key information

In a Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test) (OECD 422) (Covance, 2018b), the NOAEL for systemic toxicity for Cyrene™ was established at the dose level of 1000 mg/kg bw/day (Covance, 2018b).

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
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Study Initiation Date: 02 October 2017 - Experimental Completion Date: May 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
Adopted 29 July 2016
Deviations:
yes
Remarks:
But of minor nature. None of the minor deviations affected the integrity or interpretation of the results of the study.
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
other: Crl:WI(Han)
Details on species / strain selection:
The rat was selected because it is a readily available rodent species acceptable to the regulatory authorities and is recommended for repeated dose toxicity studies. Crl:WI(Han) was selected as the Lab has experience with this type of stain and has been using in such studies.
Sex:
male/female
Details on test animals and environmental conditions:
Animal Specifications and Acclimation:
Forty-three male and 46 female Crl:WI(Han) rats were obtained from Charles River Laboratories, Margate, United Kingdom, in order to provide sufficient animals for study selection. Upon arrival, males were approximately 9 to 10 weeks of age, while females were approximately 7 to 9 weeks of age.
Males weighed between 290.0 and 436.8 g and females weighed between 170.5 and 217.1 g at the start of dosing. Animals were 10 to 12 weeks of age and considered sexually mature. Animals were acclimated for at least 14 days prior to initiation of dosing (males) or 7 days prior to initiation of sme
aring (females).
Housing:
Animals were housed in cages. During the pre-pairing phase, animals were housed in groups of up to four by sex and dose group. During the pairing phase, one female was housed with one male from the same dose group until mating was confirmed. Following mating, females were housed individually during gestation and with their litter during the lactation phase. Males were returned to group-housing after the pairing phase.
During neurobehavioral assessments, animals remained in their home cage, except when placed in the testing apparatus.
Bedding was provided on a weekly basis to each cage by use of clean Aspen wood chips or European Softwood bedding during the gestation and lactation phases (Datesand Ltd, Manchester, United Kingdom).
Water: Water from the main tap supply was provided ad libitum via water bottles.
Diet: Animals had ad libitum access to VRFI diet (Special Diets Services Ltd, Witham, United K
ingdom). A 50 g sample of each batch of diet used on study was collected and stored at ambient temperature, and discarded after finalization of the study report.
Environment:
Animals were housed in a single, exclusive room. The room was air conditioned to provide a minimum of 15 to 20 air changes/hour. The temperature and relative humidity ranges were maintained in the specified ranges of 22°C +/- 3°C and 30 to 70%, respectively.
Fluorescent lighting was controlled automatically to give a cycle of 12 hours of light and 12 hours of dark, with the exception of when experimental procedures dictated.
Environmental Enrichment:
Animals were provided with wooden Aspen chew blocks and rodent retreats. During gestation, nesting materials were provided as forms of environmental enrichment.
Animal Identification and Assignment to Study:
Upon arrival, animals were assigned to dose groups using a total randomization procedure. Animals were individually identified by electronic implant.
Cages were placed in dose group order across the batteries.
Route of administration:
oral: gavage
Details on route of administration:
Cyrene™ was administered once daily by oral gavage.
Vehicle:
corn oil
Details on oral exposure:
Rats were administered Cyrene™ once daily by oral gavage. The control (vehicle) article was corn oil, and formulations were administered at a dose volume of 4 mL/Kg.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Stability and Homogeneity:
Formulations of 4 and 250 mg/mL concentration were found to be homogenous and stable for 4 days at room temperature (15 to 25°C) in validation study 8367720 (non Covance). Formulations of 7.5 and 250 mg/mL were prepared after the initial sampling for homogeneity; formulations were split into two aliquots. The second aliquot was stored refrigerated (2 to 8°C) until report finalization. Samples for stability were collected from each formulation as soon as possible after preparation and after 4 and 10 days. Homogeneity assessments were performed on samples used on Day 1 of dosing; samples were taken in triplicate from the top, middle, and bottom.
Achieved Concentration:
Samples (2 x 5 mL [random] aliquots from all formulations) prepared for use during Weeks 1 and 6 of dosing were taken for achieved concentration analysis.
Samples were dispatched at room temperature (15 to 25°C) for analysis.
Duration of treatment / exposure:
Male rats were dosed for 42 consecutive days (2 weeks prior to pairing, during pairing, and approximately 3 weeks post-pairing) and were sent to necropsy on Day 43. Female rats were dosed for up to 55 days (2 weeks prior to pairing, during pairing, throughout gestation, and upto Lactation Day [LD] 13) and were sent to necropsy on LD 14.
Frequency of treatment:
Once daily
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Control (corn oil) Group
Dose / conc.:
30 mg/kg bw/day (nominal)
Remarks:
Low Dose Group
Dose / conc.:
300 mg/kg bw/day (nominal)
Remarks:
Intermediate Dose Group
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Remarks:
High Dose Group
No. of animals per sex per dose:
Four groups of 10 male and 10 female rats per dose.
Control animals:
yes, concurrent vehicle
Details on study design:
A dose range-finding (DRF) study was previously performed with once daily oral gavage administration of 0, 250, 500, or 1000 mg/kg bw/day Cyrene™ for 14 consecutive days. The test article was well tolerated at the highest dose level. The oral route of administration was chosen because it is an acceptable and commonly used route exposure for regulatory studies of this type.
Before study initiation, estrous cycle data were reviewed to ensure all females allocated to the study showed regular estrous cycles. Any female that did not show a regular estrous cycle was replaced by a spare female showing a regular estrous cycle. The high-dose level of 1000 mg/kg bw/day was selected as the limit dose as this dose level was well tolerated in the previous dose range-finding study and was expected to be well tolerated over the longer dosing duration in this study. The intermediate-dose level of 300 mg/kg bw/day allowed a 3-fold increase to the high dose. The lowdose level of 30 mg/kg bw/day was anticipated to be the no observed adverse effect level (NOAEL). The prior to dose initiation body weights were calculated and inspected to ensure no unacceptable differences occurred between groups. Cages were appropriately identified with study information, including study number and animal number(s).
Observations and examinations performed and frequency:
Clinical Observations: Animals were observed at the beginning and end of the working day for signs of ill health or overt toxicity.
Clinical Examinations: Each animal was given a detailed physical examination once during acclimation then once daily from the start of dosing, including the day of necropsy.
Postdose Observations: Animals were observed daily for the first 3 days of dosing; upon return to the home cage; and approximately 0.5, 1, 2, and 4 hours postdose.
In the absence of any postdose observations recorded during the first 3 days of dosing, no further postdose observations were scheduled.
Body Weights: Male body weights were recorded once during acclimation, before dosing on the first day of dosing, at weekly intervals, and before necropsy. Female body weights were recorded once during acclimation; before dosing on the first day of dosing, at weekly intervals prior to pairing, and until confirmation of mating; on GD 0, 7, 14, and 20; and on LD 0 (where applicable), 1, 4, 7, 13, and 14 (prior to necropsy).
Food Consumption: The amount of food consumed was determined twice weekly prior to pairing (both sexes) and during the post-pairing phase for males. Daily food consumptions were recorded for females from GD 0 to 20 and from LD 1 to 13. Consumption was calculated as g/animal/day.
Functional Observational Battery (FOB): FOB assessments were performed to allow blind testing (in a manner so the observer did not know the dose group of animals during testing). Observations were performed at the same time on each occasion (approximately 2 hours postdose), where possible.
Detailed Clinical Observational Measurements: All males were assessed for detailed clinical observational measurements once prior to dose initiation and once weekly thereafter. All females were assessed once prior to dose initiation; once weekly during the pre-pairing and pairing phases; on GD 0, 7, 14, and 20; and on LD 1, 7, and 13.
Locomotor Activity: Locomotor activity was assessed in an automated photocell activity recorder for
30 minutes and was undertaken for five selected animals/sex/group (five males with the highest identification numbers and the first five littered females/group). Assessments were performed during Week 6 of dosing (Post Pairing Day 16) for males and on LD 7 for females. Activity counts were recorded at 5-minute intervals. The following parameters were determined:
- Total activity counts
- Total rears
- Total mobile counts
Quantitative Assessments: Animals were observed in the hand and in an arena. Assessments were performed during Week 6 of dosing (Post Pairing Day 20) for males and on LD 7 for females.
Quantitative assessment parameters were as follows:
- Hind limb foot splay
- Fore and hind limb grip strength
Sacrifice and pathology:
Males were sacrificed on Study Day 43 (Post-Pairing Day 22). Females were sacrificed on LD 14 (those that achieved pregnancy) or Day 26 post coitum (those that did not litter). Animals were fasted overnight prior to sacrifice. Animals were sacrificed in a controlled randomization sequence, where possible, by isoflurane anaesthesia. Once a suitable deep plane of anaesthesia was established, major blood vessels were severed to exsanguinate the animal. After sacrifice, macroscopic examinations were conducted, and all lesions were recorded.
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
A transient instance of hunched posture, increased activity, or raised hair was recorded between GD 8 and 9 for three dams administered 1000 mg/kg bw/day, with regression observed thereafter. One male administered 300 mg/kg bw/day also showed hunched posture for 5 days during the post-pairing phase, with regression to normal observed thereafter. These findings were isolated to a few animals and occurred without a dose-response; as such, they were considered incidental and of no toxicological importance. Thin fur, skin/fur staining, and vocalization were noted throughout the dose groups, including controls, and were of no toxicological importance.
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Statistically significant lower body weight gain was noted on Post-Pairing Days 8 through 15 for males administered 300 or 1000 mg/kg bw/day, compared with controls. Furthermore, statistically significant changes in body weight were noted during the lactation phase for females administered 300 mg/kg bw/day. In the absence of a convincing dose-related response, these changes were considered to have arisen incidentally.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
No adverse effect on food consumption was noted.
Food consumption for dams administered 1000 mg/kg bw/day was statistically significantly lower than controls between GD 4 and 5; however, due to the transient nature of this observation it was considered not toxicologically significant.
Food efficiency:
effects observed, non-treatment-related
Description (incidence and severity):
Food consumption for dams administered 1000 mg/kg bw/day was statistically significantly lower than controls between GD 4 and 5; however, due to the transient nature of this observation it was consid ered not toxicologically significant.
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Males administered 1000 mg/kg bw/day showed a statistically significant reduction in percentage reticulocytes and red cell distribution width, compared with controls (P < 0.01). Platelets, platelet crit, and fibrinogen were also elevated for males administered 1000 mg/kg bw/day, compared with controls (P < 0.001), with the effect also evident for males administered 300 mg/kg bw/day (P < 0.01 to P < 0.001). A decrease in activated partial thromboplastin time was also observed for males administered 1000 mg/kg bw/day, compared with controls (P < 0.05).
No test article-related effects were noted for females from any dose group. Remaining statistically significant changes observed (increased haematocrit distribution width for males administered 300 mg/kg bw/day; increased white blood cell counts, specifically in the lymphocyte fraction, for males administered 30 mg/kg bw/day; increased mean cell volume for females administered 30 mg/kg bw/day; and reduced neutrophil counts for females administered 1000 mg/kg bw/day), did not show a convincing dose response or any supporting correlations with the parameters investigated and, as such, were considered to have arisen incidentally, with no toxicological relevance.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
Cholesterol was increased for males and females administered 1000 mg/kg bw/day, compared with controls (P < 0.001 and P < 0.01, respectively). Males administered 300 mg/kg bw/day were similarly affected (P < 0.001). Other changes in the clinical chemistry parameters were evident for males administered 1000 mg/kg bw/day, compared with controls. These included increased alanine aminotransferase (P < 0.05) and increased alkaline phosphatase activity (P < 0.01). Total protein, globulin, and calcium levels were also increased (P < 0.001), with a corresponding reduction in albumin:globulin ratio (P < 0.05).
Furthermore, chloride levels were significantly reduced (P < 0.001), whereas urea and bile acids were elevated for males administered 1000 mg/kg bw/day, compared with controls, although statistical significance was only achieved for the elevation in urea (P < 0.01). For males administered 300 mg/kg bw/day, total protein (P < 0.05) and globulin (P < 0.001) were increased, compared with controls, with a corresponding reduction in albumin:globulin ratio (P < 0.01). No test article related change in clinical chemistry was evident for females administered 300 mg/kg bw/day or both sexes administered 30 mg/kg bw/day.
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
No toxicologically significant findings were noted during the weekly open field arena assessments.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Neuropathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Description (incidence and severity):
Hormone Assessments: No effect in thyroid hormones was noted for either sex administered Cyrene™, compared with controls. Furthermore, no effect on estradiol or testosterone was noted for males following DOBCO administration, compared with controls.
Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No effects observed.
Remarks on result:
other: The liver and enzyme changes noted were considered to represent an adaptive response to xenobiotic administration, and in the absence of any microscopic correlates the changes were considered not to represent an adverse effect
Key result
Critical effects observed:
no
Conclusions:
In conclusion, once daily oral gavage administration of 0, 30, 300, or 1000 mg/kg bw/day Cyrene™ to male rats for 42 consecutive days and to female rats for up to 55 days (pre pairing, throughout gestation, and during the first 2 weeks of lactation) did not result in any systemic toxicity to adults. Cyrene™ related effects consisted of haematology, clinical chemistry, and organ weight changes following administration of 300 or 1000 mg/kg bw/day. The liver and enzyme changes were considered to represent an adaptive response to xenobiotic administration, and in the absence of any microscopic correlates the changes were considered not to represent an adverse effect. Therefore, based on the results of this study, the NOAEL for systemic toxicity was established at the dose level of 1000 mg/kg bw/day.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subacute
Species:
rat

Repeated dose toxicity: inhalation - systemic 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 an OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test) study (Covance, 2018b), once daily oral gavage administration of 0, 30, 300, or 1000 mg/kg bw/day Cyrene™ to male rats for 42 consecutive days and to female rats for up to 55 days (pre pairing, throughout gestation, and during the first 2 weeks of lactation) did not result in any systemic toxicity to adults. Cyrene™ related effects consisted of haematology, clinical chemistry, and organ weight changes following administration of 300 or 1000 mg/kg bw/day. The liver and enzyme changes were considered to represent an adaptive response to xenobiotic administration, and in the absence of any microscopic correlates the changes were considered not to represent an adverse effect. Therefore, based on the results of this study, the NOAEL for systemic toxicity was established at the dose level of 1000 mg/kg bw/day.

In a dose range finding study (Covance, 2018a), once daily oral gavage administration of 250, 500, or 1000 mg/kg bw/day Cyrene™ to rats over 14 consecutive days was well tolerated. Based on this study, dose levels of 0, 30, 300, and 1000 mg/kg bw/day were recommended as suitable dose levels for a subsequent combined repeated-dose toxicity study with a reproduction /developmental toxicity screening test (OECD Test Guideline 422) (Covance, 2018b).

Justification for classification or non-classification

Based on the available data, no classification is required for specific target organ toxicity following repeated exposures for Cyrene™ according to Regulation (EC) No 1272/2008.