[No public or meaningful name is available]

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experiment start date - 27 July 2010; Experiment completion date - 22 November 2010; Study completion date - 30 November 2010.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Specific details on test material used for the study:

Identification: FAT 40851/A TE
Batch Number: TZ 5891 / BOP 02-09
Purity: 69.9 % all coloured components
Appearance: Orange powder
Expiry Date: July 31, 2014
Storage Conditions: At room temperature at about 20 °C

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS: Rat, RccHanTM: WIST(SPF)
- Source: Harlan Laboratories B.V. Kreuzelweg 53 5961 NM Horst / Netherlands
- Age at study initiation: ca. 7 weeks
- Weight at study initiation: Males: 188 to 215 g (mean 198 g), Females: 147 to 161 g (mean 153 g)
- Fasting period before study: no data
- Housing: In groups of five in Makrolon type-4 cages with wire mesh tops and standard softwood bedding including paper enrichment
- Diet (e.g. ad libitum): Pelleted standard Harlan Teklad 2914C rat / mouse maintenance diet was available ad libitum.
- Water (e.g. ad libitum): Community tap-water from Itingen was available ad libitum in water bottles.
- Acclimation period: 7 days
- Acclimatization: Under test conditions after health examination. Only animals without any visible signs of illness were used for the study
- Pre-Randomization Phase: 1 day
- Randomization: Randomly allocated to groups by body weight.
- Group 10: reserve, not used in the study

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 30 - 70 %). The relative humidity briefly exceeded this value (once), which is commonly seen following room cleaning, and was considered not to have any influence on the study
- Air changes (per hr): Air-conditioned with 10 - 15 air changes per hour
- Photoperiod (hrs dark / hrs light): There was 12-hour fluorescent light/12-hour dark cycle with music during the light period.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

bidistilled

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:

Dosing solution
- Rate of preparation of dosing solution (frequency):The dose formulations were prepared daily.
- Mixing appropriate amounts with (Type of food):
- Stability: Based upon the results of dose formulation analyses performed during a non-GLP dose range finding study (Harlan Laboratories study C93593), the stability of the test item formulations was considered to require daily preparation: Stability of Test Item in Vehicle: Up to 4 hours at room temperature (15 - 25 °C)
- Preparation: FAT 40851/A TE was weighed into a glass beaker on a tared Mettler balance. The vehicle was added and the mixtures were stirred using a magnetic stirrer and used at room temperature (20 ± 5 °C). Homogeneity of the test item in the vehicle was maintained during the daily administration period using a magnetic stirrer.

VEHICLE
- Justification for use and choice of vehicle (if other than water): none (water)
- Concentration in vehicle:0 mg/mL, 10 mg/mL, 30 mg/mL and 100 mg/mL
- Amount of vehicle (if gavage): 10 mL/kg body weight
- Purity: bidistilled water

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

After experimental start and during week 3, duplicate samples of the control group as well as three samples (top, middle and bottom) of about 2 g of each concentration were taken prior to dosing for analysis of homogeneity and concentration. Duplicate samples of about 2 g of each concentration were taken to confirm stability (4 hours). The samples will be delivered to the analytical laboratory (Harlan Laboratories Ltd., Analytics, Itingen / Switzerland) at room temperature (20 ± 5 °C) and stored there at -20 ± 5 °C until analysis.

The following acceptance criteria will be applied to analytical results:
sample contents should be within a range of ± 20 % of nominal content. Formulations will be considered homogeneous if the maximum deviation from mean calculated from top, middle and bottom samples is not more than 15 %. The results obtained from storage stability samples should not deviate more than 10 % from time-zero reference (content or mean of homogeneity samples). The application formulations investigated during the study were found to comprise FAT 40851/A in the range of 94.4 % to 110.5 %, meeting the required content limit of ±20 % with reference to the nominal concentration. Because single results found did not deviate more than 2.4 % (<15 %) from the corresponding mean, FAT 40851/A was considered to be homogeneously distributed in the vehicle. In addition, the test item was found to be stable in application formulations when kept four hours at room temperature due to recoveries which met the variation limit of 10 % from the time-zero (homogeneity) mean.

Duration of treatment / exposure:

28 d

Frequency of treatment:

daily, 7d/week

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (actual dose received)

Remarks:

Group - 1 : Control group

Doses / concentrations 2

Dose / conc.:

100 mg/kg bw/day (actual dose received)

Remarks:

Group - 2 : Low dose group

Doses / concentrations 3

Dose / conc.:

300 mg/kg bw/day (actual dose received)

Remarks:

Group - 3 : Mid dose group

Doses / concentrations 4

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

Remarks:

Group - 4 : High dose group

No. of animals per sex per dose:

Group 1: 5 males and 5 females
Group 2: 5 males and 5 females
Group 3: 5 males and 5 females
Group 4: 5 males and 5 females

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels were selected based on a previous non-GLP dose range finding toxicity study in Wistar rats, Harlan Laboratories study C93593
- Rationale for animal assignment: Randomly allocated to groups by body weight.
- Rationale for selecting satellite groups: not applicable
- Post-exposure recovery period in satellite groups: not applicable
- Section schedule rationale: Sacrifice: After 4 Weeks. All animals were weighed and necropsied. Descriptions of all macroscopical abnormalities were recorded. All animals were anesthetized by intraperitoneal injection of pentobarbitone and killed by exsanguination. Slides of all organs and tissues collected at scheduled sacrifices from all animals of the control and high-dose groups and all gross lesions from all animals were examined by the study pathologist. From the animals of the low and mid-dose groups, the stomach, kidneys and vagina were evaluated to establish a no-effect level.

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily
- Cage side observations checked in table No 1 were included.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: The animals were observed for clinical signs once before commencement of administration as well as daily on days 1 - 28 (twice daily during days 1 - 3) during the treatment period.
Weekly Behavioral Observations
The animals were observed in their home cages, outside their home cages in a standard arena and in the hand. These observations were performed once before commencement of administration and once weekly (weeks 1 to 3) thereafter.

BODY WEIGHT: Yes
- Time schedule for examinations: Body weights were recorded weekly during the acclimatization and treatment periods and before necropsy.

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, The food consumption was recorded once during the acclimatization period and weekly thereafter.
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No data

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; Body weight gain in %

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): not applicable
- Time schedule for examinations:

OPHTHALMOSCOPIC EXAMINATION: Yes, see table 1

HAEMATOLOGY: Yes
- Time schedule for collection of blood: after 4 weeks
- Anaesthetic used for blood collection: Yes (identity) Blood samples were drawn from the retro-orbital plexus from all animals under light isoflurane anesthesia
- Animals fasted: Yes, The animals were fasted in metabolism cages for approximately 18 hours before blood sampling but allowed access to water ad libitum
- How many animals: all
- Parameters checked in table No.3 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: after 4 weeks
- Animals fasted: Yes, in metabolism cages for approximately 18 hours before blood sampling but allowed access to water ad libitum.
- How many animals: all
- Parameters checked in table No.4 were examined.

URINALYSIS: Yes
- Time schedule for collection of urine: Urine was collected during the 18 hours fasting period into a specimen vial, using a metabolism cage
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters checked in table No.5 were examined.

NEUROBEHAVIOURAL EXAMINATION: Yes, see table 1
- Time schedule for examinations: During week 4, relevant parameters from a modified Irwin screen test were evaluated in all animals.
- Dose groups that were examined: see table 1
- Battery of functions tested: reflexes / grip strength / locomotor activity

OTHER: Vaginal Smear for Estrus Stage
Vaginal smears were taken during week 4 over four days from all females, and the stage of estrus was evaluated.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes see table 2
HISTOPATHOLOGY: Yes see table 2

Statistics:

The following statistical methods were used to analyze body weight, grip strength, locomotor activity, clinical laboratory data, organ weights and ratios as well as macroscopic findings:
• The Dunnett-test (many to one t-test) based on a pooled variance estimate was applied if the variables could be assumed to follow a normal distribution for the comparison of the treated groups and the control groups for each sex.
• The Steel-test (many-one rank test) was applied instead of the Dunnett-test when the data could not be assumed to follow a normal distribution.
• Fisher's exact-test

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Description (incidence and severity):

No test item-related findings of toxicological relevance were noted at any dose level during daily observations or during weekly behavioral observations (weeks 1 - 3).

Mortality:

no mortality observed

Description (incidence):

All animals survived the scheduled treatment or recovery periods.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

The mean absolute body weights of the test item-treated males and females were similar to those of the respective controls. The mean body weight gain values were also unaffected.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

The mean daily food consumption and relative food consumption values were unaffected by the treatment with the test item.

Ophthalmological findings:

effects observed, treatment-related

Description (incidence and severity):

red fecal dicoloration

Haematological findings:

no effects observed

Description (incidence and severity):

There were no effects upon the hematology parameters of the test item-treated males and females when compared with the respective control values.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

No test item-related changes were noted in the clinical biochemistry parameters. The mean potassium level was significantly higher in males at 1000 mg/kg/day (p <0.05) when compared with the controls. In females treated with 1000 mg/kg/day, the mean calcium, protein and albumin levels were significantly reduced (all p <0.01) when compared with controls. The globulin level was also significantly lower (p <0.05) in females at 1000 mg/kg/day. The mean activity of aspartate aminotransferase was significantly elevated in males at 300 mg/kg/day (p <0.05). Females treated with 300 mg/kg/day had significantly decreased cholesterol and phospholipid levels (both p <0.05). At 100 mg/kg/day, females showed significant reductions in mean cholesterol (p <0.01), mean triglyceride (p <0.05) and mean phospholipid (p<0.05) levels when compared with the controls. The mean calcium level was also significantly reduced (p <0.05) in females at this dose level.

Urinalysis findings:

no effects observed

Description (incidence and severity):

At 1000 mg/kg/day, the urine volume of males was significantly reduced (p <0.05) when compared with the controls but remained within the ranges of the historical control data. This was considered to be of no toxicological relevance. No differences were noted in the females at any dose level.

Behaviour (functional findings):

effects observed, treatment-related

Description (incidence and severity):

Functional Observational Battery (Screen): There were no test item-related changes in the functional observational battery performed at week 4.

Grip Strength: The mean fore and hind limb grip strength of the test item treated males and females compared favorably with those of the respective controls.

Locomotor Activity: In test item-treated males, elevated locomotor activity was noted briefly in males treated with 100 mg/kg/day (30-40 minutes, p <0.05) and in males treated with 300 mg/kg/day (20 - 30 minutes, p <0.05). This transient difference was considered to be unrelated to the test item. The mean locomotor activity values of the test item-treated females were similar to the controls.

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

There were no test item-related differences in the mean absolute organ weights, in the mean organ-to-body weight ratios or in the organ-to-brain weight ratios at any dose level.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

There were no test item-related macroscopic findings.
Discoloration of the thymus was noted in one male at 100 mg/kg/day. Lung foci were noted in one control male and one male treated with 300 mg/kg/day. One control male, one male treated at 100 mg/kg/day and one female treated with 1000 mg/kg/day had dilation of the renal pelvis.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Stomach: Substantially increased acanthosis of the limiting ridge, increased submucosal inflammatory infiltrate and increased epithelial hyaline inclusions (glandular mucosa) were observed in both genders at 300 and 1000 mg/kg/day. Increased vacuolation of the limiting ridge was recorded in females at 300 mg/kg/day and both genders at 1000 mg/kg/day.

Kidneys: Brownish pigment was recorded in the tubular epithelia of males and females at 1000 mg/kg/day.

Vagina: Anestrus and mucification of vaginal epithelium was found at 300 mg/kg/day and 1000 mg/kg/day.

Description (incidence and severity):

Vaginal Smear for Estrus Stage: The incidence and pattern of the estrus cycles were comparable in the test item-treated and control females. Persistent diestrus noted in two females treated with 1000 mg/kg/day was considered to be a possible artifact of sampling.

Effect levels

Dose descriptor:

NOAEL

Effect level:

100 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

Target system / organ toxicity

Critical effects observed:

no

Any other information on results incl. tables

Table 1 Incidence and Mean Severity of Main Findings in Stomach

Finding / Groups	1 0 mg/kg/day		2 100 mg/kg/day		3 300 mg/kg/day		4 1000 mg/kg/day
Total Affected / Mean Severity	(5) M	(5) F	(5 M	(5) F	(5) M	(5) F	(5) M	(5) F
Vacuolation limiting ridge	1 1.0	3 1.0	2 1.0	2 1.5	3 1.0	5 1.6	5 2.2	5 2.4
Acanthosis limiting ridge	-	1 1.0	-	-	3 1.0	3 1.0	5 1.0	5 1.0
Inflammatory infiltrate, submucosal	3 1.0	3 1.0	2 1.0	3 1.3	4 1.5	5 1.2	5 2.0	5 2.2
Hyaline inclusions	4 1.0	4 1.0	5 1.0	3 1.0	5 1.6	5 1.8	5 1.2	5 2.0

Table 2: Incidence and Mean Severity of Main Findings in Kidneys

Finding / Groups	1 0 mg/kg/day		2 100 mg/kg/day		3 300 mg/kg/day		4 1000 mg/kg/day
Total Affected / Mean Severity	(5) M	(5) F	(5) M	(5) F	(5) M	(5) F	(5) M	(5) F
Extramedullary hemopoiesis	-	-	-	-	-	-	5 1.2	5 1.2

Table 3: Incidence and Mean Severity of Main Findings in Vagina

Finding / Groups	1 0 mg/kg/day		2 100 mg/kg/day		3 300 mg/kg/day		4 1000 mg/kg/day
Total Affected / Mean Severity	(5) M	(5) F	(5) M	(5) F	(5) M	(5) F	(5) M	(5) F
Mucification/ Anestrus	-	-	-	-	-	5 1.2	-	5 1.2

Applicant's summary and conclusion

Conclusions:

The NOAEL (No Observed Adverse Effect Level) for 28-day repeated dose toxicity was established at 100 mg/kg.

Executive summary:

An oral repeated dose toxicity study in accordance with EU Method B.7 and under GLP conditions was performed in male and female Wistar rats. Oral administration of FAT 40851/A to Wistar rats at doses of 100, 300 and 1000 mg/kg/day, for 28 days resulted in passive, non-adverse red discoloration of the feces at all dose levels, and microscopical findings in the stomach and kidneys of males and females at 300 and 1000 mg/kg/day and in the vagina of females at 300 and mg/kg/day. In the stomach, acanthosis of the limiting ridge, increased submucosal inflammatory infiltrate and increased epithelial hyaline inclusions (glandular mucosa) were observed in males and females at 300 and 1000 mg/kg/day. Increased vacuolation of the limiting ridge was recorded in females at 300 mg/kg/day and 1000 mg/kg/day and in males at 1000 mg/kg/day. While the relevance of the findings in the forestomach / limiting ridge are disputed (Greaves P, 2000) and may be not significant for man, the inflammatory changes of the glandular submucosal region indicate a potential for irritation of the stomach by the test item with a certain relevance for man. In the kidneys: brownish pigment was recorded in the tubular epithelia of males and females at 1000 mg/kg/day. This finding was not accompanied by signs of de- or regeneration, and there was no increased inflammatory reaction. Although it was considered likely to represent some form of physiological clearance of the test item, the exact nature of pigment and its significance was unclear. Anestrus and mucification of vaginal epithelium was found in two animals at 300 mg/kg/day and two females at 1000 mg/kg/day and may point at a progestational effect of the test item, but the exact cause of this finding was unclear. In this study, a NOEL (No Observed Effect Level) could not be established, but the NOAEL (No Observed Adverse Effect Level) could be established at 100 mg/kg.