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Repeated dose toxicity: oral

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short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
November 14th, 2012 - January 18th, 2013
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference Type:
study report
Report Date:

Materials and methods

Test guidelineopen allclose all
according to
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
according to
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
according to
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:
Limit test:

Test material

Test material form:
liquid: viscous
Details on test material:
- Appearance: clear, slightly yellowish to brown, viscous liquid
- Storage condition of test material: at room temperature in the dark

Test animals

Details on test animals and environmental conditions:
- 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

- 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

Administration / exposure

Route of administration:
oral: gavage
polyethylene glycol
Details on oral exposure:

- 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:
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
Doses / concentrations
Doses / Concentrations:
100, 300, 600, 1000 mg/kg bw

No. of animals per sex per dose:
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:


Observations and examinations performed and frequency:
- Time schedule: At least twice daily.

- 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.

- Time schedule for examinations: Weekly

- Time schedule for examinations: Weekly

- 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

- Time schedule for examinations: Subjective appraisal was maintained during the study, but no quantitative investigation introduced as no effect was suspected.

- Time schedule for examinations:
- Dose groups that were examined:

- 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

- 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


- Motor activity testing was performed
Sacrifice and pathology:
GROSS PATHOLOGY: Yes, all animals
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.
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.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
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:
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.

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.

No toxicologically significant changes in food consumption before or after correction for body weight were noted.

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.

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,

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.

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.

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.

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.

Effect levels

Key result
Dose descriptor:
Effect level:
100 mg/kg bw/day (actual dose received)
Based on:
test mat.
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).

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

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%).

Applicant's summary and conclusion

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.