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Description of key information

Subchronic (90-d) oral repeated dose toxicity: NOAEL 10 mg/kg bw/d (OECD408)

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
30 March 2009 - 23 December 2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study was conducted according to OECD guideline 408 and under GLP conditions.
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories Inc., Raleigh, NC, USA
- Age at study initiation: approx. 37 days
- Weight at study initiation:
Male: 89-138 g
Female: 72-108 g
- Housing: Individually
- Diet (e.g. ad libitum): Ad libitum, certified rodent diet
- Water (e.g. ad libitum): Ad libitum, local water (with chlorine as bacteriostat)
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18-26
- Humidity (%): 30-70
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES:
From: 02 March 2009
To: 13 - 17 July 2009 or 10 - 11 August 2009 (recovery animals)
Route of administration:
oral: gavage
Vehicle:
other: Canola oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
Suspensions of test substance were prepared at least weekly and stored refrigerated (2-8°C) and protected from light. Prepared formulations were equilibrated to ambient temperature for at least 30 minutes prior to use, and stirred continuously while equilibrating as well as during dosage administration.

VEHICLE
- Justification for use and choice of vehicle (if other than water): No data
- Amount of vehicle (if gavage): 10 ml/kg (including test substance)
- Lot/batch no.: XR0054 / J-073
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Concentrations were within acceptable limits of ±15% error. Homogeneity was within the acceptable range of ≤5%. Stability of formulations from 1-100 mg/mL and storage conditions was confirmed for 24 hours when stored at room temperature and protected from light, and for 10 days at 5±3°C and protected from light.
Duration of treatment / exposure:
90 days
Frequency of treatment:
Once daily
Remarks:
Doses / Concentrations:
10, 50, 125, 250 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
10 (+5 for the control and high dose group, for the recovery study)
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: 14-day toxicity study
- Rationale for animal assignment: At random
- Rationale for selecting satellite groups: No data
- Post-exposure recovery period in satellite groups: 4 weeks
- Section schedule rationale (if not random): No data
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS:
- Time schedule: Twice daily
- Cage side observations: Viability

DETAILED CLINICAL OBSERVATIONS:
- Time schedule: Weekly

BODY WEIGHT:
- Time schedule for examinations: Weekly

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes / No / No data
- Time schedule for examinations:

OPHTHALMOSCOPIC EXAMINATION:
- Time schedule for examinations: Prior to dosage, at the completion of the dosage period and at completion of the recovery period
- Dose groups that were examined: Control and high dose group

HAEMATOLOGY:
- Time schedule for collection of blood: During week 7 (day 43) and on the day of scheduled sacrifice (day 91 or 118 for the recovery group)
- Anaesthetic used for blood collection: No data
- Animals fasted: Yes
- How many animals: All animal
- Parameters checked: erythrocyte count, mean corpuscular volume, hematocrit, total leukocyte count, hemoglobin, differential leukocyte count, mean corpuscular hemoglobin, platelet count, mean corpuscular hemoglobin concentration, mean platelet volume and cell morphology.

CLINICAL CHEMISTRY:
- Time schedule for collection of blood: During week 7 (day 43) and on the day of scheduled sacrifice (day 91 or 118 for the recovery group)
- Animals fasted: Yes
- How many animals: All animals
- Parameters checked: total protein, creatinine, triglycerides, alanine aminotransferase, albumin, aspartate aminotransferase, globulin, alkaline phosphatase, albumin/globulin ratio, calcium, glucose, phosphorus, cholesterol, sodium, total bilirubin, potassium, urea nitrogen and chloride.

URINALYSIS:
- Time schedule for collection of urine: During week 7 (day 43) and on the night before scheduled sacrifice (day 91 or 118 for the recovery group)
- Metabolism cages used for collection of urine: No data
- Animals fasted: Yes
- Parameters checked: color, clarity, quantitative analysis of specific gravity and volume, semiquantitative analysis of glucose, bilirubin, ketone, blood, pH, protein, urobilinogen, nitrite and leukocytes and microscopic examination of sediment.

NEUROBEHAVIOURAL EXAMINATION:
- Time schedule for examinations: Week 11 and 17
- Dose groups that were examined:
- Battery of functions tested: autonomic functions (lacrimation, salivation, palpebral closure, prominence of eye, pupillary reaction to light, piloerection, respiration, urination and defecation, body temperature) / reactivity and sensitivity (sensorimotor responses to visual, acoustic, tactile and painful stimuli) / excitability (reactions to handling and behavior in the open field) / gait and sensorimotor coordination (gait pattern in the open field, severy / forelimb and hindlimb grip tests / motor activity (test)
Sacrifice and pathology:
GROSS PATHOLOGY: Physical examination of external surfaces and all orifices, as well as an internal examination of tissues and organs in situ. Additionally, the cranial, thoracic and abdominal cavities were examined.

SPERM ANALYSIS: Sperm concentration, motility and morphology and testicular spermatid concentration were analyzed for all male rats.

ORGAN WEIGHTS: The weight of the following organs was determined: Adrenals, brain, right epididymis, left epididymis (whole and cauda), heart, kidneys, liver, ovaries, spleen, right tesits, thymus and uterus (with cervix).

HISTOPATHOLOGY: Histological examinations were performed on tissues of all control and high-dose group rats, for the following tissues: adrenals, aorta (thoracic), femur, sternum, brain, cecum, colon, duodenum, right epididymis, left epididymis (corpus and caput), esophagus, eyes (with optic nerves), Harderian glands, heart, ileum, jejunum, kidneys, liver, lungs, lymph nodes (mandibular and mesenteric), mammary gland (female rats, with skin), nasal cavity and sinuses, ovaries, pancreas, pituitary, prostate, rectum, salivary glands, sciatic nerve, seminal vesicles, skeletal muscle, skin, spinal cord (cervical, lumbar and thoracic), spleen, stomach, right testis, thymus, thyroid lobes and parathyroids, tongue, trachea, urinary bladder, uterus (with cervix) and vagina.

Rats that were found dead or died before termination were directly examined and necropsied to the extent possible.
Statistics:
- Bartlett's test
- ANOVA
- Krukal-Wallis test
- Fisher's Exact test
Clinical signs:
effects observed, treatment-related
Mortality:
mortality observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
effects observed, treatment-related
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY
No treatment-related mortality occurred in the male and female animals. Four males died due to intubation errors (2) and a broken palate (2), while 2 female animals died after blood collection.

Slight but statistically significant excess in salivation was noted for both males and females of the 50 mg/kg bw/day group, while this excess was moderate and significant in the 125 and 250 mg/kg bw/day groups. A statistically significant increase in urine-stained abdominal fur was observed in the 125 and 250 mg/kg bw/day groups, while the latter group showed also rales (males) and chromorhinorrhea (females). Females of the 50, 125 and 250 mg/kg bw/day dose group showed an ungroomed coat.

No test substance-related clinical signs were observed during the recovery period in both males and females.

BODY WEIGHT AND WEIGHT GAIN
Absolute body weights in males of the 125 and 250 mg/kg bw/day group were significantly reduced from day 28, as well as body weight gain during several periods throughout the study. The latter also accounted for the females of the same groups, but their absolute body weights did not differ from the control group (as well as the other female groups).

No effects that were considered treatment-related were observed on the body weight (gain) of males and females of the other two test groups (10 and 50 mg/kg bw/day).

During recovery the absolute body weights remained significantly reduced in the 250 mg/kg bw/day group, although body weight gains were significantly increased.

FOOD CONSUMPTION
Absolute feed consumption was comparable between the control and 250 mg/kg bw/day group. Relative feed consumption was significantly increased for the 125 and 250 mg/kg bw/day group at several intervals and the entire dose period. No effects were observed for the other dose groups.

No difference in absolute and relative feed consumption was observed for females of the control and 250 mg/kg bw/day group.

During recovery the relative feed consumption was significantly higher for both males and females in the 250 mg/kg bw/day group.

OPHTHALMOSCOPIC EXAMINATION
No effects found in males and females of any dose group, at the end of study (including recovery).

HAEMATOLOGY
Hematology and coagulation parameters for male and female rats were generally comparable between the vehicle control and four treatment groups and no toxicologically important differences were observed on day 43 and/or day 91.

CLINICAL CHEMISTRY
Male rats showed significantly increased total bilirubin in the 125 and 250 mg/kg/day treatment groups. Cholesterol was also increased in the 250 mg/kg/day treatment group male rats. Blood urea nitrogen was significantly increased in the 250 mg/kg/day treatment group. Female rats showed significantly increased cholesterol and total bilirubin in the 250 mg/kg/day treatment group. After the recovery period, clinical chemistry values were comparable between the vehicle control and the 250 mg/kg bw/d treatment groups and no toxicologically important differences were observed.

URINALYSIS
A significant increase in the number of male rats in the 250 mg/kg bw/day group with cloudy orange urine was observed in week 7. There was also a tendency for reduced pH and an increase of ketones in urine in the 125 and 250 mg/kg bw/day groups (males). In females the pH was significantly reduced in all dose groups. The number of female rats with absent ketones was significantly reduced in the 50 and 250 mg/kg bw/day groups, while in the latter group there was a slight increase in females with 40 mg/dl ketones.

During week 13 the pH of urine was significantly decreased for the males of the 125 mg/kg bw/day group, but not for the 250 mg/kg bw/day group. For females this effect was observed in all dose groups. Number of rats with absent ketones were significantly reduced in the 50, 125 and 250 mg/kg bw/day dose groups for both males and females. Number of male rats with clear light orange urine was significantly increased in the highest dose group.

The number of male rats with ketones in urine (5 mg/dl) was significantly increased in the 10 and 50 mg/kg bw/day groups, but the effect was not dosage dependent and therefore not considered toxicologically relevant. Number of male rats in the 250 mg/kg bw/day group with 40 mg/dl ketones was (not significantly) increased.

No effects on urinalysis were observed during the recovery period for males, while for females the specific gravity of urine was significantly reduced in the 250 mg/kg bw/day group.

NEUROBEHAVIOUR
During the functional observational battery (FOB) conducted in week 11 of the dosage period, the mean scores for appearance were increased for male rats in the 125 and 250 mg/kg/day treatment groups, as were the numbers of unkempt rats or rats with urine and/or fecal staining. No other treatment-related differences were observed among the vehicle control and four treatment groups in any of the functional observational battery (FOB) parameters during week 11 of the dosage period. The total number of movements and the time spent in movement were increased for male and female rats in the 250 mg/kg/day treatment group during week 11 of the dosage period.

No test substance-related effects were observed in the performance of male and female rats in the functional observational battery conducted during week 17 (recovery period). During week 17 of the study, the total number of movements and the total time spent in movement continued to be increased (17% above control) for female rats in the 250 mg/kg/day treatment group, while the motor activity of male rats was not affected in the same treatment group during this same period.

ORGAN WEIGHTS
In males, no effects on absolute kidney weight were observed, while the relative kidney weight to BW ratio increased in 50, 125 and 250 mg/kg bw/d and relative kidney weight to brain ratio increased in 50, and 125 mg/kg bw/d. In females, absolute kidney weight was increased at all doses, while relative kidney weight to BW ratio was significantly increased in 125 and 250 mg/kg/d and relative kidney weight to brain weight ratio wassignificantly increased at 250 mg/kg/d
In males no effects were observed on absolute liver weight, while the relative liver weight to BW ratio was increased in 50, 125, and 250 mg/kg bw/d. When the relative liver weight to brain ratio was calculated, no effects were observed. In females, absolute and relative liver weight to BW ratio were increased at 50, 125 and 250 mg/kg bw/d. Relative liver weight to brain ratio was increased at 50 and 250 mg/kg bw/d.
Furthermore, an increased relative adrenal weight to BW ratio was observed in males, while in females no effects were observed on adrenal weight, absolute or relative.

No toxicologically important differences were observed in the organ weights and the ratios of the organ weights to terminal body weight and brain weight for male rats after the recovery portion of the study. For female rats, the liver weight was significantly reduced and the ratio of the thymus weight to the terminal body weight was significantly increased in the 250 mg/kg/day treatment group on DS 118.

GROSS PATHOLOGY
There were no gross test substance-related pathology findings in the animals evaluated.

HISTOPATHOLOGY: NON-NEOPLASTIC
In males, dose dependent increase in chronic progressive nephropathy was observed from 10mg/kg/d (4 minimal), 50 mg/kg/d (8 minimal, 1 mild), 125 mg/kg/d (5 minimal, 5 mild) and 250 mg/kg/d (5 minimal/5 mild). Minimal hyaline droplet change was noted in the cortical tubules of male rats in the 125 mg/kg/d and 250 mg/kg/d dose groups. Furthermore, dose dependent increase in dilatation of tubules was observed from 10mg/kg/d (1 minimal), 50 mg/kg/d (3 minimal, 1 mild), 125mg/kg/d (5 minimal) and 250 mg/kg/d (5 minimal, 4 mild).
In females, dose dependent dilatation of kidney tubules was observed in 50 (4 minimal), 125(9 minimal) and 250 mg/kg/d (7minimal, 2 mild, 1 moderate).
No histopathological effects were observed on liver and adrenals in both males and females.

OTHER FINDINGS
Not relevant
Dose descriptor:
NOAEL
Effect level:
10 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Adverse clinical signs, increase in organ weight, changes in urine, histological changes.
Critical effects observed:
not specified
Conclusions:
In a 90-d oral repeated dose toxicity study, Cashmeran was administered by gavage to male and female Sprague-Dawley rats. Based on increased adverse clinical observations, decreased pH and increased ketones in the urine of female rats, increased kidney weights, increased liver weights for female rats only and increased incidence and/or severity of histological changes in the kidney at the three highest doses, a NOAEL of 10 mg/kg bw/day was established. After a four week recovery period, none of these effects were observed and they were therefore considered to be reversible.
Executive summary:

In a 90-d oral repeated dose toxicity study, Cashmeran was administered to male and female Sprague-Dawley rats by gavage at 0, 10, 50, 125, and 250 mg/kg bw. Viability, clinical observations (including detailed clinical observations), body weights, body weight changes, feed consumption, and necropsy observations were recorded, and rats were evaluated for ophthalmological changes, estrous cycling, functional observational battery, motor activity, urinalysis and sperm analysis. Blood samples were collected for hematology and clinical chemistry analysis. A four-week recovery study was included to study reversibility of effects.

In the three highest dose levels of 50, 125 and 250 mg/kg/day increased adverse clinical observations (principally excess salivation, urine-stained abdominal fur, rales and ungroomed coat), decreased pH and increased ketones in the urine of female rats, increased kidney weights, increased liver weights for female rats only and increased incidence and/or severity of histological changes in the kidney (i.e., changes consistent with chronic progressive nephropathy and increased tubular dilatation in the renal outer medulla) were observed. Furthermore, in the 125 and 250 mg/kg/day treatment group male rats reduced body weight gain, increased relative feed consumption, decreased pH and increased urinary ketones, and reduced heart weights were observed. A minimal hyaline droplet change was also observed in the cortical tubules of individual males in the 125 and 250 mg/kg/day treatment groups. In the 250 mg/kg/day treatment group, male and female rats displayed increased motor activity and changes in clinical chemistry parameters.

During the four-week recovery period, body weight gains and relative feed consumption values were increased for male and female rats in the 250 mg/kg/day treatment group. Increases in motor activity continued for female rats during the recovery period. Test substance-related changes in clinical signs, urine pH and ketones, kidney weight/kidney histopathology and clinical pathology were not observed in the recovery animals and were therefore considered to be reversible.

Although decreased urine pH for female rats and increased kidney weight for male and female rats occurred at the lowest dose level of 10 mg/kg bw/d, the changes were considered to be small and were not associated with histopathological changes in the kidneys. Therefore, the 10 mg/kg/day treatment level was considered to be the NOAEL. 

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
10 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
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 90-d oral repeated dose toxicity study, Cashmeran was administered to male and female Sprague-Dawley rats by gavage at 0, 10, 50, 125, and 250 mg/kg bw. Viability, clinical observations (including detailed clinical observations), body weights, body weight changes, feed consumption, and necropsy observations were recorded, and rats were evaluated for ophthalmological changes, estrous cycling, functional observational battery, motor activity, urinalysis and sperm analysis.Blood samples were collected for hematology and clinical chemistry analysis. A four-week recovery study was included to study reversibility of effects.

In the three highest dose levels of 50, 125 and 250 mg/kg/day increased adverse clinical observations (principally excess salivation, urine-stained abdominal fur, rales and ungroomed coat), decreased pH and increased ketones in the urine of female rats, increased kidney weights, increased liver weights for female rats only and increased incidence and/or severity of histological changes in the kidney (i.e., changes consistent with chronic progressive nephropathy and increased tubular dilatation in the renal outer medulla) were observed.Furthermore, in the 125 and 250 mg/kg/day treatment group male rats reduced body weight gain, increased relative feed consumption, decreased pH and increased urinary ketones, and reduced heart weights were observed.A minimal hyaline droplet change was also observed in the cortical tubules of individual males in the 125 and 250 mg/kg/day treatment groups.In the 250 mg/kg/day treatment group, male and female rats displayed increased motor activity and changes in clinical chemistry parameters.

During the four-week recovery period, body weight gains and relative feed consumption values were increased for male and female rats in the 250 mg/kg/day treatment group.Test substance-related changes in clinical signs, urine pH and ketones, kidney weight/kidney histopathology and clinical pathology were not observed in the recovery animals and were therefore considered to be reversible.

Although decreased urine pH for female rats and increased kidney weight for male and female rats occurred at the lowest dose level of 10 mg/kg bw/d, the changes were considered to be small and were not associated with histopathological changes in the kidneys. Therefore, the 10 mg/kg/day treatment level was considered to be the NOAEL. 


Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
The 90-day oral repeated dose toxicity study available is adequate to cover the repeated dose toxicity

Repeated dose toxicity: via oral route - systemic effects (target organ) urogenital: kidneys

Justification for classification or non-classification

An extensive review of the observed effects in the 90 day oral repeated dose toxicity study is available in the attached document. Please find below the summary and conclusion with regard to classification in accordance with the criteria outlined in Annex VI of 67/548/EEC and Annex I of 1272/2008/EC.

A NOAEL of 10 mg/kg bw and a LOAEL of 50 mg/kg bw is derived in the 90 day oral repeated dose toxicity study and the Reproscreen study. This LOAEL is within guidance values ranges mentioned in the CLP for which classification and labelling may be warranted. In both studies the target organ is the kidney. The effects seen in the kidney at 50 mg/kg bw (in a 90-day oral study) are not considered to be significant health effects that impair the function of the kidney and therefore STOT 2 classification is not warranted. The kidney effects of Cashmeran have been evaluated according to the CLP criteria in section 3.9.1.1 and in 3.9.2.7.3 for the presence of classification and 3.9.2.8.1 for its absence (CLP, 2008). The effects seen at this dose in urinalysis (decrease of pH and increase of ketones) are minor and of doubtful toxicological relevance and are therefore not considered to be significant adverse changes. At necropsy there are no macroscopically effects seen indicating the absence of severe organ dysfunction at any dose. The microscopically CPN seen (in male rats only) is minimal in nature at 50 mg/kg bw dose and is only increased to mild at the higher doses. The CPN associated regeneration processes are also minimal in nature (minimal tubular dilatation and basophilic tubili) at the 50 and 40 mg/kg bw in the 90-day and Reproscreen study, respectively. Though the NOAEL is 10 mg/kg bw, the effects seen at 50 mg/kg bw and higher doses are not indicating significant health effects impairing organ function.

In evaluating the kidney effects following 3.9.2.7.3 it can be seen that the criteria set in 3.9.1.1 “All significant health effects that can impair function, both reversible and irreversible, should be included to conclude on classification” are not met. Therefore, Cashmeran does not have to be classified for repeated dose toxicity in accordance with the criteria outlined in Annex VI of 67/548/EEC and Annex I of 1272/2008/EC.