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Description of key information

A sub-acute 28 days repeated dose study was performed according to OECD guideline and GLP principles. Based on effects seen at 300 mg/kg bw (enlarged liver with dose-related changes in biochemical parameters, but in absence of histopathological changes), the NOAEL was found to be 100 mg/kg bw. 

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:
November 14th, 2012 - January 18th, 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Qualifier:
according to
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Qualifier:
according to
Guideline:
other: United States Environmental Protection Agency (EPA). Health Effects Test Guidelines, OPPTS 870.3050, Repeated dose 28-day oral toxicity study in rodents. Office of Prevention, Pesticides and Toxic Substances (7101), EPA 712-C-00-366, July 2000
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Age at study initiation: 8 weeks
- Weight at study initiation: males 223 - 282g; females 147 - 192g
- Fasting period before study: no
- Housing: Group houses (5 animals per sex) in Macrolon cages
- Diet: Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany); During motor activity measurements, animals had no access to food; Animals were deprived of food overnight (with a maximum of 24 hours) prior to scheduled necropsy
- Water: ad libitum, tap water
- Acclimation period: at least 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18-24
- Humidity (%): 40-70
- Air changes (per hr): approximately 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 14NOV 2012 To: 18JAN2013
Route of administration:
oral: gavage
Vehicle:
polyethylene glycol
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:

VEHICLE
- Justification for use and choice of vehicle: Based on trial formulations performed at WIL Research Europe and on information from the sponsor and on the results of the dose range finding study.
- Concentration in vehicle: 20, 60, 120, 200 mg/ml
- Amount of vehicle: 5 mL/kg
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The stability of the test substance in formulations was determined. The accuracy and homogeneity of the the formulations were determined on a single occasion after the treatment phase, according to a validated method (WIL Research project 501365).
The accuracy of preparation was considered acceptable if the mean measured concentrations were 90-110% of the target concentration for solutions. Homogeneity was demonstrated if the coefficient of variation is ≤ 10%. Formulations were considered stable if the relative difference before and after storage was maximally 10%.
Duration of treatment / exposure:
At least 28 days
Frequency of treatment:
Once daily
Remarks:
Doses / Concentrations:
100, 300, 600, 1000 mg/kg bw
Basis:

No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Based on the results from the the dose range finding study
Positive control:
No.
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: At least twice daily.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Clinical observations were conducted after dosing at no specific time point, but within a similar time period after dosing for the respective animals, based on the range finding study.

BODY WEIGHT: Yes
- Time schedule for examinations: Weekly

FOOD CONSUMPTION: Yes
- Time schedule for examinations: Weekly

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Yes

WATER CONSUMPTION AND COMPOUND INTAKE: Yes
- Time schedule for examinations: Subjective appraisal was maintained during the study, but no quantitative investigation introduced as no effect was suspected.

OPHTHALMOSCOPIC EXAMINATION: No
- Time schedule for examinations:
- Dose groups that were examined:

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at the end of the treatment
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals: all
- Parameters following OECD guideline were checked

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at the end of the treatment
- Animals fasted: Yes
- How many animals: all
- Parameters following OECD guideline were checked

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Motor activity testing was performed
Sacrifice and pathology:
GROSS PATHOLOGY: Yes, all animals
HISTOPATHOLOGY: Yes
The following slides were examined by a pathologist:
- all tissues collected at the scheduled sacrifice from all control animals and animals exposed to 600 mg/kg bw.
- all tissues from all animals of control group and animals exposed to 100, 300 and 600 mg/kg bw which died spontaneously or were terminated in extremis,
- all gross lesions.
No histopathology was performed on animals exposed to 1000 mg/kg bw that died spontaneously or that were sacrificed.

On detection of treatment-related morphological changes in the urinary bladder of the females in the high dose group (600 mg/kg) the histological examination was extended to that particular organ of all females exposed to 100 and 300 mg/kg bw. All abnormalities were described and included in the report. An attempt was made to correlate gross observations with microscopic findings.
Statistics:
The following statistical methods were used to analyze the data:
- If the variables could be assumed to follow a normal distribution, the Dunnett-test (many-to-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
- The Steel-test (many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution.
- The Fisher Exact-test was applied to frequency data.
- The Kruskal-Wallis nonparametric ANOVA test (Ref. 4) was applied to motor activity data to determine intergroup differences. In case intergroup differences were seen, the Wilcoxon test was applied to compare the treated groups to the control group.

All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance. Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables. Test statistics were calculated on the basis of exact values for means and pooled variances. Individual values, means and standard deviations may have been rounded off before printing. Therefore, two groups may display the same printed means for a given parameter, yet display different test statistics values.
Clinical signs:
effects observed, treatment-related
Mortality:
mortality observed, treatment-related
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
no effects observed
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:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Details on results:
CLINICAL SIGNS AND MORTALITY
At the highest concentration (1000 mg/kg bw) two males and three females died on day 1. The highest dose was lowered to 600 mg/kg bw and animals were replaced. At 600 mg/kg bw one male and one female died died after first exposure. No other mortality occurred in the animals that survived the treatment with 1000 mg/kg bw, or in the animals that replaced the animals that died on the first day.
No mortality occurred in control animals and animals at 100 and 300 mg/kg.

The animals treated at 1000 mg/kg for one day showed slight lethargy, severe clonic spasms, tremor, flat posture, and/or hypersensitivity to touch on Day 1 prior to death. Hunched posture or piloerection was noted in the surviving animals at 1000 mg/kg on Days 2 and/or 3.

The animals at 600 mg/kg showed during the study period: lethargy, clonic spasms, tremors, flat and/or hunched posture, quick breathing, rales, laboured and/or shallow respiration, piloerection, salivation and/or chromodacryorrhoea.

The males at 300 mg/kg showed incidentally over de study period flat posture, quick breathing, rales and/or chromodacryorrhoea. The females showed lethargy, tremor, hunched posture, uncoordinated movements, laboured or shallow respiration, rales, piloerection, salivation and/or ptosis during the study period predominantly in the first three days.

No toxicologically relevant clinical signs were noted in control animals and animals treated at 100 mg/kg. Scabs were noted in one male at 100 mg/kg in a short period. This finding occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study. At the incidence observed, this was considered a sign of no toxicological significance.

BODY WEIGHT AND WEIGHT GAIN
No toxicologically significant changes in body weights and body weight gain were noted.
The body weight gain in the first week appeared to be lower at 600 mg/kg compared to controls but recovered thereafter.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
No toxicologically significant changes in food consumption before or after correction for body weight were noted.

HAEMATOLOGY
No toxicologically relevant changes occurred in haematological parameters of treated rats.

In animals at 600 mg/kg increased red blood cell counts and hematocrit level was noted. These changes were considered to be of no toxicological significance as they occurred in the absence of a treatment-related distribution and remained within the range considered normal for rats of this age and strain.

Minor statistically significant differences (platelet count and red blood cell distribution width) arising between controls and animals receiving 100 and 300 mg/kg were considered not to represent a change of biological significance.

CLINICAL CHEMISTRY
The following (statistically significant) changes in clinical biochemistry parameters distinguished treated animals at 600 mg/kg from control animals and were outside the range considered normal for rats of this age and strain:
- higher total bilirubin in males and females
- higher cholesterol in females (not statistically significant in males)

Statistically significant changes (at 600 mg/kg) remaining within the range considered normal for rats of this age and strain included:
- higher alanine aminotransferase level in females (ALAT); increased in all exposed groups, dose-related increase
- higher bile acid, calcium and inorganic phosphate level in females (higher inorganic phosphate also noted at 300 mg/kg)

Any statistically significant changes at 100, 300 or 600 mg/kg were considered to be of no toxicological significance as they occurred in the absence of a treatment-related distribution and remained within the range considered normal for rats of this age and strain. These findings included lower glucose level, higher sodium and chloride level,

ORGAN WEIGHTS
Higher liver weight (absolute and relative) were noted at 300 (females; >10% larger than control) and 600 mg/kg (male and female). Higher thyroid weight (absolute and relative) were found at 600 mg/kg bw (males and females)
Other organ weights and organ to body weight ratios among the dose groups were similar to control levels.

GROSS PATHOLOGY
The animals treated at 1000 mg/kg, which were found death on Day 1, showed discoloration of the stomach and/or foci in the thymus. The animals at 600 mg/kg, which were sacrificed on Day 1 showed foci in the thymus and/or lungs and thickened thymus.

In the other animals necropsy revealed the following findings in single animals: nodules in stomach (control animal), discoloration of clitoral gland (100 mg/kg), fluid in the uterus (100 and 300 mg/kg), foci in stomach (300 mg/kg), and enlarged liver, pelvic dilation in the kidney, reduced size of testes and epididymides, foci in clitorial gland and/or adrenal glands and enlarged clitorial gland (600 mg/kg). These necropsy findings were considered to be of no toxicological relevance the incidence was very low and within the expected range of findings that are encountered among rats of this age and strain.

HISTOPATHOLOGY: NON-NEOPLASTIC
In 2/5 female rats at 600 mg/kg a minimal degree of diffuse hyperplasia of the urothelium of the urinary bladder was recorded. Based on the low grade and the absence of additional histopathologic findings, this was considered to be a non-adverse treatment-related effect.
All remaining microscopic findings recorded were considered to be within the normal range of background pathology encountered in rats of this age and strain.

NEUROBEHAVIOUR
Hearing ability, pupillary reflex, static righting reflex and grip strength were normal in all animals.
Motor activity in males was similar between treated and control groups. The females showed lower total movements and lower ambulation counts at 600 mg/kg
All groups showed a similar motor activity habituation profile with high activity in the first interval that decreased over the duration of the test period.
Key result
Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Increased liver weights (female) at 300 mg/kg bw coinciding with elevated levels of ALAT, total bilirubin, and cholesterol (levels increased in exposed groups in a dose-dependent way).
Critical effects observed:
not specified

Analytical verification of dose formulations:

Formulations at the entire range were found to be stable when stored at room temperature under normal laboratory light conditions for at least 6 hours (maxmum relative difference -2.3%).

The concentrations analysed in the test item formulations were in agreement with target concentrations (i.e. mean accuracies between 94% and 105%).

A small response at the retention time of the test substance was observed in the chromatograms of the control group formulation. Maximum contribution to the other samples was 0.055% based on peak area.

The formulations of the mid and the high dose group were analyse to be homogeneous (i.e. maximum coefficient of variation 1.2%).

Conclusions:
A sub-acute 28 days repeated dose study was performed according to OECD guideline and GLP principles. Based on effects seen at 300 mg/kg bw (enlarged liver with dose-related changes in biochemical parameters), the NOAEL was found to be 100 mg/kg bw.
Executive summary:

In a sub-acute repeated dose study performed according to OECD guideline and GLP principles, rats were treated with 100, 300 or 1000 mg/kg bw Accelerator (PT 25E or PT 25E/2). Due to mortality in the highest dose group on day 1 (2/5 males and 3/5 females), the highest dose was lowered to 600 mg/kg bw and the animals were replaced. One male and one female died shortly after the first dose of 600 mg/kg bw. At macroscopic examination these animals showed foci in the thymus and/or lungs and thickened thymus. No further mortality occurred. Clinical signs were noted during the study period at 600 mg/kg (lethargy, clonic spasms, tremors, flat and/or hunched posture, quick breathing, rales, laboured and/or shallow respiration, piloerection, salivation and/or chromodacryorrhoea). During the first week the body weight gain was slightly lower but recovered from week 2 onwards. No changes were noted in food consumption, or functional behaviour were noted compared to control animals. At the highest dose level of 600 mg/kg higher liver weights and thyroid gland weights were noted in males and females, coinciding with changes in total bilirubin and cholesterol levels. Liver weights were also increased in females at 300 mg/kg. In females ALAT-values, total bilirubin, and cholesterol were elevated in all groups and increased in a dose-dependent way (significant at 600 mg/kg bw).

Microscopic examination revealed in some females diffuse hyperplasia of the urothelium of the urinary bladder (2/5 females at 600 mg/kg). Based on the low grade and the absence of additional histopathologic findings, this was considered to be a non-adverse treatment-related effect. Based on the effects on liver at 300 mg/kg with dose-related changes in biochemical parameters (ALAT, cholesterol, total bilirubin and bile acids), a No Observed Adverse Effect Level (NOAEL) for Accelerator (PT 25E or PT 25E/2) of 100 mg/kg was established.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
100 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
The study is conducted in accordance with the OECD guideline and GLP principles (reliability 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 sub-acute repeated dose study performed according to OECD guideline and GLP principles, rats were treated with 100, 300 or 1000 mg/kg bw Accelerator (PT 25E or PT 25E/2). Due to mortality in the highest dose group on day 1 (2/5 males and 3/5 females), the highest dose was lowered to 600 mg/kg bw and the animals were replaced. One male and one female died shortly after the first dose of 600 mg/kg bw. At macroscopic examination these animals showed foci in the thymus and/or lungs and thickened thymus. No further mortality occurred. Clinical signs were noted during the study period at 600 mg/kg (lethargy, clonic spasms, tremors, flat and/or hunched posture, quick breathing, rales, laboured and/or shallow respiration, piloerection, salivation and/or chromodacryorrhoea). During the first week the body weight gain was slightly lower but recovered from week 2 onwards. No changes were noted in food consumption, or functional behaviour were noted compared to control animals. At the highest dose level of 600 mg/kg higher liver weights and thyroid gland weights were noted in males and females, coinciding with changes in total bilirubin and cholesterol levels. Liver weights were also increased in females at 300 mg/kg. In females ALAT-values, total bilirubin, and cholesterol were elevated in all groups and increased in a dose-dependent way (significant at 600 mg/kg bw).

Microscopic examination revealed in some females diffuse hyperplasia of the urothelium of the urinary bladder (2/5 females 600 mg/kg). Based on the low grade and the absence of additional histopathologic findings, this was considered to be a non-adverse treatment-related effect. Based on the effects on liver at 300 mg/kg with dose-related changes in biochemical parameters (ALAT, cholesterol, total bilirubin and bile acids), a No Observed Adverse Effect Level (NOAEL) for Accelerator (PT 25E or PT 25E/2) of 100 mg/kg was established. Since no histopathological changes were observed in the affected livers and taking into account the severity of the changes in biochemical parameters, the obserevd effects were not found to clearly indicate functional disturbance.

Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
One study available, study was performed according to OECD guideline and GLP principles (Klimisch score = 1).

Repeated dose toxicity: via oral route - systemic effects (target organ) digestive: liver

Justification for classification or non-classification

Based on the available data, the substance is not classified for STOT-RE according to Regulation (EC) No 1272/2008.