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Description of key information

There is no valid repeat dose study on the registered substance.  An apparently well conducted GLP 28-day study oral gavage study in rats, on the analog substance CAS# 27138-01-8 is considered the key study.  

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:
16 July 2012 - 03 December 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Compliant to GLP and testing guidelines; adequate consistence between data, comments and conclusions.
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: breeder: Charles River Laboratories France, l’Arbresle, France
- Age at study initiation: approximately 5 weeks old on the first day of treatment
- Mean body weight at study initiation: the males had a mean body weight of 150 g (range: 136 g to 163 g) and the females had a mean body weight of 118 g (range: 109 g to 129 g)
- Fasting period before study: no
- Housing: the animals were housed by five, in polycarbonate cages with stainless steel lids containing autoclaved sawdust
- Diet: SSNIFF R/M-H pelleted diet (free access)
- Water: tap water filtered with a 0.22 µm filter (free access)
- Acclimation period: 7 days before the beginning of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2°C
- Humidity (%): 50 ± 20%
- Air changes (per hr): approximately 12 cycles/hour of filtered, non-recycled air
- Photoperiod (hrs dark / hrs light): 12 h/12 h.

IN-LIFE DATES: 14 August 2012 to 11 September 2012.
Route of administration:
oral: gavage
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The test item was administered as a suspension in the vehicle. The test item was ground to a fine powder, using a mortar and pestle, and then mixed with the required quantity of vehicle.
The frequency of dose formulation preparation was based on available stability data. The dose formulations were stored and delivered to the study room at room temperature and protected from light.

VEHICLE
- Concentration in vehicle: 20, 60 and 200 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg/day.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Type of method: HPLC-UV
Test item concentrations: within an acceptable range of variation compared to nominal values.
Homogeneity: homogenous
Stability: stable for 10 days at room temperature and protected from light
Duration of treatment / exposure:
4 weeks
Frequency of treatment:
Daily
Remarks:
Doses / Concentrations:
100, 300 and 1000 mg/kg/day
Basis:
actual ingested
No. of animals per sex per dose:
5 animals per sex per dose.
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
The dose-levels were selected in agreement with the Sponsor based on the results of a previous 7-day toxicity study performed in the same species and strain. In this study, four groups of four males and females received the test item at 0, 100, 300 or 1000 mg/kg/day as a suspension in corn oil. There were no premature deaths and no test item-related clinical signs. There were no obvious test item-related effects on mean body weight or mean food consumption. At necropsy, there were no test item-related macroscopic findings but dose-related increases in mean liver weights in males from 100 mg/kg/day (up to +23% from controls) and in females at 1000 mg/kg/day (up to +18%).
Therefore, the dose 1000 mg/kg/day was selected as the high dose-level for this 4-week study. Mid and low dose-levels were selected in order to cover approximately 3-fold intervals.

- Rationale for animal assignment: computerized stratification procedure.
Positive control:
no (not required)
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS / MORTALITY:
- Time schedule: once a day during the acclimation period and at least twice a day during the treatment period.

CLINICAL SIGNS:
- Time schedule: once a day.

DETAILED CLINICAL OBSERVATIONS:
- Time schedule: at the beginning of the treatment period and then once a week until the end of the study.

BODY WEIGHT:
- Time schedule: the body weight of each animal was recorded once before the beginning of the treatment period, on the first day of treatment, then at least once a week until the end of the study.

FOOD CONSUMPTION:
- Time schedule: the quantity of food consumed by the animals in each cage was recorded once a week, until the end of the study.
Food consumption was calculated per animal and per day.

NEUROBEHAVIOURAL EXAMINATION:
- Time schedule: each animal was evaluated once in week 4.

HAEMATOLOGY, CLINICAL CHEMISTRY:
- Time schedule: at the end of the treatment period.
Sacrifice and pathology:
ORGAN WEIGHTS: see table below.

GROSS PATHOLOGY:
Complete macroscopic post-mortem examination of all study animals.

HISTOPATHOLOGY:
- all tissues listed in the Tissue Procedure Table for the control and high-dose animals (groups 1 and 4) sacrificed at the end of the treatment period,
- kidneys, liver and adrenals from the low- and intermediate-dose animals (groups 2 and 3) sacrificed on completion of the treatment period,
- all macroscopic lesions from all low- and intermediate-dose animals (groups 2 and 3) sacrificed on completion of the treatment period.

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
for clinical signs but no mortality
Mortality:
mortality observed, treatment-related
Description (incidence):
for clinical signs but no mortality
Body weight and weight changes:
no effects observed
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:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
not examined
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:
not examined
Details on results:
MORTALITY:
There were no unscheduled deaths during the study.

CLINICAL SIGNS:
Piloerection and staggering gait were mostly observed from days 1 to 4. Ptyalism generally appeared between weeks 2 and 4 of treatment and lasted until the end of the study.
All these clinical signs were considered to be test item-related but non adverse as not critical and/or because they lasted only a few days out of 4 weeks of treatment in less than half of the animals.
Although the observation was not required in the study, towards the middle of the treatment period water consumption was noticed to be higher in a dose-related manner at 300 and 1000 mg/kg/day when compared with controls.
Incidental findings including wound, scabs, abnormal growth of teeth, abnormal reddish color of urine, dyspnea, soft feces, thinning of hair, increase in size of right hindlimb and locomotory difficulties were generally observed in isolated animals.

BODY WEIGHT (GAIN):
There were no test item-related effects on mean body weight and mean body weight changes.

FOOD CONSUMPTION:
There were no toxicologically relevant effects on mean food consumption.

NEUROBEHAVIOURAL EXAMINATION:
There were no test item-related effects at the Functional Observation Battery and motor activity.

HAEMATOLOGY:
There were no toxicologically relevant effects in hematology parameters.
In the female control group, only data from two females were available because of coagulated blood or insufficient amount of blood for analysis. However, data from test item-treated females were including in or close to the historical control data.

CLINICAL CHEMISTRY:
The mean cholesterol level was statistically higher than in controls in both sexes at 1000 mg/kg/day. However, this was considered to be of minor toxicological relevance in view of the slight increase.
At 1000 mg/kg/day, males had slightly higher mean protein and albumin blood concentrations and a lower bile acid level than controls. In view of the slight variations from controls and/or in the absence of any adverse correlating effects, these findings were considered to be non adverse.
The statistical significances obtained in females for sodium, chloride and bile acid levels and for aspartate aminotransferase activity were considered to be fortuitous as there was no dose relationship. The statistical significances obtained in males for the alkaline phosphatase activity was considered not to be toxicologically important as the reduction was slight and not associated with correlating findings.

ORGAN WEIGHTS:
When compared with controls, the following changes in the mean organ weights were noted:
- the mean absolute and relative liver weights were statistically significantly higher in animals given 1000 mg/kg/day (p<0.01),
- the mean absolute and relative kidney weights were statistically higher in males given 1000 mg/kg/day (p<0.01). In females treated at 1000 mg/kg/day, they were also higher, reaching statistically significant value for the relative weight only (p<0.05),
- the mean absolute and relative adrenal weights were slightly higher in males given 1000 mg/kg/day, although not statistically significant.
These changes were considered to be test item-related and correlated with microscopic findings in these organs.

The mean absolute and relative weights of uterus were lower in all test item-treated groups, reaching significant value at 100 and 300 mg/kg/day (p<0.01 and p<0.05 for the absolute and relative values, respectively) but not at 1000 mg/kg/day. This was due to the estrus cycle variations (2/5 control females with dilated uterus macroscopically vs. none in the groups given 100 and 300 mg/kg/day).
The mean absolute and relative testes weights were higher in males given 1000 mg/kg/day, reaching significant value for the absolute weight. In this group, absolute values of all but one males were within the control range. As a consequence and in the absence of histopathological test item-related changes, any relationship with the treatment was considered to be excluded.
Other organ weight changes were considered not to be related to the test item as they were small in amplitude, had no gross or microscopic correlates and/or were not consistent for the sexes.

GROSS PATHOLOGY:
There were no test item-related macroscopic findings.
The macroscopic findings had no histological correlates or correlated with common histological findings in control rats, and were considered to be incidental.
Specifically the scabs and the ovarian cyst seen only in test item-treated groups are not uncommon in untreated rats of this age and strain. Consequently they were considered to be of no toxicological importance.

HISTOPATHOLOGY: NON-NEOPLASTIC:
Microscopic findings were seen in liver, kidney and adrenals.

. liver
Minimal to moderate hepatocellular hypertrophy was seen in males and females treated at 1000 mg/kg/day.
There were no associated degenerative changes at any of the dose-levels.

. kidney
Minimal to moderate hyaline droplets were found in tubular epithelium (renal cortex) from males treated at 300 or 1000 mg/kg/day.
Hyaline droplets were variably sized, round, dense eosinophilic and intracytoplasmic. Hyaline droplets were not observed in any of the females.
In addition, there was a trend toward an increase in regenerative/inflammatory lesions in males treated at 1000 mg/kg/day, i.e. in tubule dilation, tubular basophilia (suggesting an evidence of regeneration) and infiltrate of mononuclear cell infiltrate.
In females, the differences between controls and test item-treated animals were considered to be of low magnitude and consequently not to be toxicologically relevant.

. adrenals
Minimal to slight increase in vacuolation (macro- or microvacuoles) was seen in the adrenal cortex from occasional males and females treated at 1000 mg/kg/day.

Other microscopic findings noted in test item-treated animals were considered incidental changes, as they also occurred in controls, were of low incidence, and/or are common background findings for the Sprague-Dawley rat. Among these was the slight to moderate sero-cellular crusts noted in test item-treated males and females while it was not seen in controls. This lesion is frequent in untreated rats of this age and strain and was considered not as drug-related.
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
body weight and weight gain
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
clinical signs
Critical effects observed:
not specified
Conclusions:
The test item was administered daily for 4 weeks by oral route to male and female Sprague-Dawley rats at dose-levels of 100, 300 or 1000 mg/kg/day in corn oil.
Under the experimental conditions of this study, the No Observed Adverse Effect Level (NOAEL) was considered to be at 1000 mg/kg/day in females and at 300 mg/kg/day in male rats as renal hyaline droplets were associated with increased tubular basophilia at 1000 mg/kg/day in males, suggesting previous chronic cell damage and increased cell turnover. There were no other effects that could be considered as adverse at any doses. Renal hyaline droplets were considered to be specific to male rats and therefore not relevant for human.
Executive summary:

The objective of this study was to evaluate the potential toxicity of the test item following daily oral administration to rats for 4 weeks.

 

Methods

Three groups of five male and five female Sprague-Dawley rats received the test item by daily oral administration for 28 days at dose-levels of 100, 300 or 1000 mg/kg/day. The test item was administered as a suspension in the vehicle (corn oil) at a constant dosage-volume of 5 mL/kg/day. A control group of five males and five females received the vehicle alone under the same experimental conditions.

 

Test item concentrations were checked on formulations used in weeks 1 and 4.

The animals were checked at least twice daily during the dosing period for mortality and morbidity and once daily for clinical signs. In addition, detailed clinical examinations were performed at least once weekly. Body weight was recorded once before the beginning of the treatment period, and then at least once a week during the study as food consumption. Towards the end of the dosing period, a Functional Observation Battery, motor activity measurement, hematology and blood biochemistry were performed on all animals.

On completion of the treatment period, the animals were euthanized and submitted to a full macroscopic post-mortem examination. Designated organs were weighed and selected tissues were preserved. A microscopic examination was performed on selected tissues from control- and high‑dose animals and on kidneys, liver and adrenals from the low- and intermediate-dose animals sacrificed at the end of the treatment period, as well as on all macroscopic lesions.

 

Results

The test item concentrations in the administered dose formulations analyzed in weeks 1 and 4 were within the acceptance criteria.

 

There were no unscheduled deaths during the study.

Piloerection and staggering gait were observed at the very beginning of the treatment period in 4/10 animals treated at 1000 mg/kg/day and ptyalism was noted in all animals from this group. These clinical signs were considered to be test item-related but non adverse.

Although the observation was not required in the study, water consumption was noticed to be higher towards the middle of the treatment period in a dose-related manner at 300 and 1000 mg/kg/day.

There were no test item-related effects at Functional Observation Battery and motor activity or onmean body weight and mean body weight changes.There were no toxicologically relevant effects on mean food consumption and mean hematology parameters.

At 1000 mg/kg/day, males had slightly higher mean protein (66 g/L vs. 60, p<0.05) and albumin (40 g/L vs. 36, p<0.01) blood concentrations and a lower bile acid level (23.6 µmol/L vs. 55.1, p<0.01) than controls. The cholesterol level was slightly higher than in controls in both sexes (males: 2.0 mmol/L vs. 1.5, p<0.05; females: 2.1 mmol/L vs. 1.7, p<0.01) and was considered to be of minor toxicological relevance. These blood biochemical findings were considered to be non adverse.


Mean liver (from +28 to +34% from controls, p<0.01), kidney (up to +23% in males with p<0.01, +10% in females with p<0.05 for the mean relative weight) and adrenal (up to +16%) weights were higher in males and/or females given 1000 mg/kg/day. There were no test item-related macroscopic findings. Microscopic findings were seen in liver (non adverse hepatocellular hypertrophy in 5/5 males and 2/5 females at 1000 mg/kg/day), kidney (hyaline droplets in 4/5 and 5/5 males at 300 and 1000 mg/kg/day, respectively, together with an increase in tubular basophilia in 3/5 males at 1000 mg/kg/day considered as adverse, tubular dilation and infiltrate of mononuclear cells), and adrenals (non adverse increased vacuolation in 2/5 males at 1000 mg/kg/day).

 

Conclusion

The test item was administered daily for 4 weeks by oral route to male and female Sprague-Dawley rats at dose-levels of 100, 300 or 1000 mg/kg/day in corn oil.

Under the experimental conditions of this study, the No Observed Adverse Effect Level (NOAEL) was considered to be at 1000 mg/kg/day in females and at 300 mg/kg/day in male rats as renal hyaline droplets were associated with increased tubular basophilia at 1000 mg/kg/day in males, suggesting previous chronic cell damage and increased cell turnover. There were no other effects that could be considered as adverse at any doses. Renal hyaline droplets were considered to be specific to male rats and therefore not relevant for human.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Quality of whole database:
There is no valid repeat dose study on the registered substance. An apparently well conducted GLP 28-day study, on the analog substance CAS# 27138-01-8 is considered the key study.

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Quality of whole database:
No study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Quality of whole database:
No study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Quality of whole database:
No study avaiable.

Additional information

The analog was administered daily for 4 weeks by oral route to male and female Sprague-Dawley rats at dose-levels of 100, 300 or 1000 mg/kg/day in corn oil.

Under the experimental conditions of this study, the No Observed Adverse Effect Level (NOAEL) was considered to be at 1000 mg/kg/day in females and at 300 mg/kg/day in male rats as renal hyaline droplets were associated with increased tubular basophilia at 1000 mg/kg/day in males, suggesting previous chronic cell damage and increased cell turnover. There were no other effects that could be considered as adverse at any doses. Renal hyaline droplets were considered to be specific to male rats and therefore not relevant for human.

Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:

Apparently well conducted GLP study.  

Justification for classification or non-classification

There were no effects, in the repeat dose study on the analog substance, that could be considered as adverse at any doses. Renal hyaline droplets were considered to be specific to male rats and therefore not relevant for human.