[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 - 14 January 2005; Experiment end date - 19 May 2005; Study completion date - 24 May 2005.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2005

Report date:

2005

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: Japanese Guidelines for Screening Toxicity Testing of Chemicals: Testing Methods for new Substances, enacted July 13, 1974, amended December 5, 1986

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

FAT40819/A TE

IUPAC Name:

FAT40819/A TE

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

- Name of test material (as cited in study report): FAT 40819/A
- Purity: Approx. 77%
- Lot/batch No.: Red ROE 420 BOP 01/04
- Expiration date: 2 November 2009
- Stability: Stable under storage conditions.
- Storage conditions: At room temperature (range of 20 ± 5 °C), away from direct sunlight. Stored in an exsiccator.

Specific details on test material used for the study:

Identity: FAT 40819/A
Description: Red brown powder
Batch number: Red ROE 420 BOP 01/04
Purity: approx. 77 %
Stability of test item: Stable under storage condition
Expiry date: 02 November 2009
Stability of test item dilution: Stable in PEG 300 for at least 7 days at room temperature
Storage conditions: At room temperature.

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: RCC Ltd, Laboratory Animal Services, CH-4414 Füllinsdorf / Switzerland
- Age at delivery: 6 weeks
- Weight at acclimatization: Males: 132.8-156.3 grams (mean 145.0 grams), Females: 113.9- 129.5 grams (mean 122.4 grams)
- Housing: In groups of five in Makrolon type-4 cages with wire mesh tops and standardized softwood bedding ('Lignocel' Schill AG, CH-4132 Muttenz/Switzerland).
- Diet: Pelleted standard Provimi Kliba 3433 (batch nos. 69/04 from 14 January 2005, 92/04 from 11 February 2005, and 94/04 from 25 February 2005) rat maintenance diet (Provimi Kliba AG, CH-4303 Kaiseraugst/Switzerland) was available ad libitum.
- Water: Community tap-water from Itingen was available ad libitum in water bottles.
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 30-70
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

bidistilled water

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The dose formulations were prepared weekly. The test item was weighed into a tared glass beaker on a suitable precision balance and the vehicle added (weight: volume). The mixtures were prepared using a magnetic stirrer or homogenizer, as appropriate. Homogeneity of the test item in the vehicle (bidistilled water) was maintained during the daily administration period using a magnetic stirrer.

Dose volume: 10 mL/kg body weight.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Concentration, homogeneity and stability (after 2 hours and 7 days) of the dose formulations were determined in samples taken after experimental start. Concentration and homogeneity of the dose formulations were determined in samples taken during week 3 of the treatment.

Duration of treatment / exposure:

28 days

Frequency of treatment:

daily

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.:

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

Remarks:

Group 2 - Low dose group

Doses / concentrations 3

Dose / conc.:

200 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:

Groups 0 mg/kg/day and 1000 mg/kg/day: 10 males; 10 females
Groups 50 mg/kg/day and 200 mg/kg/day: 5 males; 5 females

Control animals:

yes, concurrent vehicle

Details on study design:

- In this subacute toxicity study, the test substance was administered daily by oral gavage to SPF-bred Wistar rats of both sexes at dose levels of 50, 200 and 1000 mg/kg body weight/day for a period of 28 days. A control group was treated similarly with the vehicle, bidistilled water, only. The groups comprised 5 animals per sex that were sacrificed after 28 days of treatment. Additional 5 rats per sex and group were used at 0 and 1000 mg/kg. These animals were treated for 28 days and then allowed a 14 day treatment-free recovery period after which they were sacrificed.

- Rationale for dose level selection: Based upon the results of a non-GLP 5-day dose range-finding study (RCC Study Number 858104) in which FAT 40819/A was administered by gavage to 2 rats per group and sex.

Examinations

Observations and examinations performed and frequency:

- Mortality/viability: Observations for mortality/viability were recorded twice daily.

- Cage side observations: The animals were observed for clinical signs once before commencement of administration; twice daily on days 1-3; as well as once daily on days 4-28 and once daily during days 29-42 (recovery).

- Clinical 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 in random sequence once before the first test item administration and once weekly (during weeks 1-3) thereafter.

- Food consumption: The food consumption was recorded once during the acclimatization period and weekly thereafter.

- Body weight: Body weights were recorded weekly during the acclimatization, treatment and recovery and before necropsy.

- Functional observational battery: During week 4, relevant parameters from a modified Irwin screen test were evaluated in all animals. Grip strength: Forelimb and hind limb grip strength measurements were performed using a push-pull strain gauge (Mecmesin, AFG 25N). The animals were placed with the forepaws inside a triangular grasping ring and with the hind paws outside a triangular grasping ring. Using one hand, the animals were held towards the base of the tail and steadily pulled away or towards the ring until the grip was broken. Each measurement was repeated three times, the means were calculated and recorded. Locomotor activity: Locomotor (decreased or increased) activity was measured quantitatively with AMS Föhr Medical Instruments GmbH (FMI) and DeMeTec GmbH Activity monitor System. Animals were monitored during the fourth treatment week for a 60-minute period and the total activity of this time period was recorded. Low beams count was reported in 10-minute intervals as well as the total activity of the measuring period.

- Clinical laboratory investigations: Blood samples for haematology and clinical chemistry were collected from all animals under light isoflurane anesthesia. The animals were fasted for approximately 18 hours before blood sampling but allowed access to water ad libitum. Blood samples were collected early in the working day to reduce biological variation caused by circadian rhythms. Blood samples were drawn from the retro-orbital plexus using a micro-haematocrit glass capillary tube. The following haematology parameters were determined: Erythrocyte count, Haemoglobin, Haematocrit, Mean corpuscular volume, Red cell volume distribution width, Mean corpuscular hemoglobin, Mean corpuscular haemoglobin concentration, Haemoglobin concentration distribution width, Platelet (thrombocyte) count, Reticulocyte count, Reticulocyte maturity index, Total leukocyte count, Differential leukocyte count, Methaemoglobin, Thromboplastin time, Activated partial thromboplastin time. The following clinical biochemistry parameters were determined: Glucose, Urea, Creatinine, Bilirubin, Cholesterol, Triglycerides, Phospholipids, Aspartate aminotransferase, Alanine aminotransferase, Lactate dehydrogenase, Glutamate dehydrogenase, Creatine kinase, Alkaline phosphatase, Gamma-glutamyl-transferase, Sodium, Potassium, Chloride, Calcium, Phosphorus inorganic, Protein, Albumin, Globulin, Albumin/Globulin ratio. The following urinalysis parameters were determined: Volume (18 hours), Specific gravity (relative density), Color, Appearance, pH, Nitrite, Protein, Glucose, Ketones, Urobilinogen, Bilirubin, Erythrocytes, Leukocytes.

Sacrifice and pathology:

- All animals were weighed and necropsied. Descriptions of all macroscopic abnormalities were recorded.
- All animals surviving to scheduled necropsy were anesthetized by intraperitoneal injection of sodium pentobarbitone and killed by exsanguination.
Samples of the following tissues and organs were collected from all animals at necropsy and fixed in neutral phosphate buffered 4 % formaldehyde solution (unless otherwise indicated): Adrenal glands, Aorta, Bone (sternum, femur including joint), Bone marrow (femur), Brain (multiple levels), Cecum, Colon, Duodenum, Epididymides (fixed in Bouin's solution), Esophagus, Eyes with optic nerve (fixed in Davidson's solution), Harderian gland (fixed in Davidson's solution), Heart, Ileum with Peyer's patches, Jejunum with Peyer's patches, Kidneys, Larynx, Lacrimal gland (exorbital), Liver, Lungs (infused with formalin at necropsy), Lymph nodes (mesenteric, mandibular), Mammary gland area, Nasal cavity, Ovaries, Pancreas, Pituitary gland, Prostate gland (incl. coagulating gland), Rectum, Salivary glands (mandibular, sublingual), Sciatic nerve, Seminal vesicles, Skeletal muscle, Skin, Spinal cord (cervical, mid-thoracic, lumbar), Spleen, Stomach, Testes (fixed in Bouin's solution), Thymus, Thyroid (incl. parathyroid gland), Tongue, Trachea, Urinary bladder (infused with formalin at necropsy), Uterus, Vagina, Gross lesions.
-The following organ weights were recorded on the scheduled dates of necropsy: Brain, Heart, Liver, Thymus, Kidneys, Adrenals, Spleen, Testes, Epididymides, Ovaries. The organ to terminal body weight ratios as well as organ to brain weight ratios were determined. The determination of the terminal body weight was performed immediately prior to necropsy.
- Slides of organs and tissues from the animals of control and high-dose groups were examined by a pathologist. Since test item-related morphologic changes were detected in some organs of the high-dose animals, those same organs from the mid- and low-dose group were examined to establish a no-effect level, if possible.

Statistics:

The following statistical methods were used to analyze the grip strength, locomotor activity, body weight, organ weights and ratios:
- The Dunnett-test (many to one t-test) based on a pooled variance estimate were 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.
- Fisher's exact-test were applied to the macroscopic findings.
The following statistical methods were used for statistical analysis of clinical laboratory data:
- Quantitative data were analyzed by a one-way analysis of variance (ANOVA) when the variances are considered homogeneous according to Bartlett. Alternatively, if the variances are considered to be heterogenous (p≤0.05), a non-parametric Kruskal-Wallis test was used. Treated groups were compared to the control groups using Dunnett's test if the ANOVA was significant at the 5 % level and by Dunn's test in the case of a significant Kruskal-Wallis test (p ≤0.05).

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

No clinical signs of toxicological relevance were noted. Clinical signs were restricted to red feces in all males and females of the mid (with a mean severity between 1.00 and 1.67) and high dose groups (with a mean severity between 1.00 and 1.67 from day 1 through 3, and between 1.68 and 2.33 from day 4 through 28). The occurrence of red feces had disappeared in males and females of the high dose groups on the fourth day of the recovery period. The mean severity on the first recovery day was between 1.68 and 2.33, and on recovery days 2 and 3 between 1.00 and 1.67.

Mortality:

no mortality observed

Description (incidence):

All animals survived until their respective scheduled necropsy.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

The overall mean body weights in all treated male and female groups were slightly decreased by ≤5 % besides of the low dosed female group which revealed a slight increase of +4 %. Because there was no dose-response relationship visible and in the absence of any statistically significant effects on body weight development the effects encountered were considered to be not caused by treatment with the test item. Similarly, body weight development in the high dosed males and females during the recovery period revealed no significant difference when compared with the controls. No toxicological relevance was attributed to the significantly (at the 5 % level) lowered body weight gain in the low dosed males which was decreased by -12.2 %, -15.6 % and -15.8 % after 2, 3 and 4 weeks of treatment, respectively, when compared to controls. Body weight gain in the high dosed females was also significantly (at the 1 % level) reduced by -21.4 %, - 22.1 % and -22.4 % after 2, 3 and 4 weeks of treatment, respectively. At the end of the recovery period body weight gain was still slightly reduced in the high dosed males by -5.6 % but increased in the high dosed females by +22.2 %, when compared to the controls but without achieving any statistical significance. Thus, it can be stated that body weight development was not affected by the treatment with the test item but revealed only some slight biological variation during the course of the study.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

The mean food intake in all treated male and female groups was not affected by the administration of the test item. The mean food intake in the high dosed male and female groups was slightly increased by +4 % and +7 %, respectively, during the recovery period. However, this slight increase in food consumption was considered incidental and considered to be within the normal biological variation. Equally, relative food consumption in the high dosed male and female groups was slightly increased by +3 % and +7 %, respectively.

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Haematology data revealed no test item-related effects after 4 weeks of treatment or at termination of the treatment-free recovery period. The following minor changes observed in hematology parameters were considered to be of no toxicological significance as these findings were considered to be incidental and to lie within the range of normal background data and/or within the normal range of biological variation encountered in animals of this strain and age: Hemoglobin concentration distribution width (HDW) was slightly increased by 7 % in males of the high dose group. This difference in males was statistically significant at the 5 % level but within the range of historical control data. L, M or H reticulocyte counts (L, M or H reti, relative) in low dosed or mid dosed males L reti were significantly increased by 9 % (at the 5% level) and M and H reti were decreased by -30 % or -53 % (at the 5 % level), respectively. However, because the control value was already higher than the historical range as was the case for the L reticulocytes or the values were below the historical control values as for the M and H reticulocytes these values were considered of not being relevant since there was no dose response-relationship present. Similarly, relative counts of L and M reticulocytes in the mid dosed female group were significantly (at the 5 % level) higher or lower, respectively, than the range of historical control data but in the absence of any dose response-relationship these findings were considered to be without any toxicological relevance. Relative lymphocyte counts (Lympho) in the low dosed males were significantly (at the 5 % level)-decreased by -6 % but the value was within the range of historical control data and thus considered to be of no toxicological relevance. Partial thromboplastin times (PTT) were prolongated in males of the low, mid and high dose groups by +22 %, +28 % and +27 %, respectively. This finding was not considered as a treatment-related effect since there was no clear dose response-relationship present and there were no treatment-related changes seen in other hematological parameters but the PTT value in males of the control group was incidentally lower. PTT values in females of the control group were more prolongated when compared with the range of historical control data. Differential leukocyte counts (absolute): Basophiles (Baso) in the high dosed females were slightly higher (at 5 % level) after 4 weeks of treatment but still within the range of historical control data and, therefore, not considered to be of any toxicological relevance. Monocytes (Mono) and Large Unstained Cells (Luc) were significantly (at 5 % level) increased at the end of the recovery period or after the 4 week treatment period, respectively, but still within the range of historical control data and of no relevance.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Slightly higher levels for cholesterol (+26 % for males, +63 % for females) and phospholipids (+13 % for males +37 % for females) were noted after 4 weeks of treatment in both sexes of the high dose groups as well as for phospholipids (+29 %) in females of the mid dose group. An increase (+71 %) in the triglyceride concentration was also observed in the high dose females. At termination of the treatment-free recovery period the effects observed in males and for triglyceride in females had returned to control levels, but in the high dosed females significantly increased levels for cholesterol (+41 %) and phospholipids (+24 %) were still present. The following minor differences were within the 95 % tolerance limit of the historical control data and considered to be of no toxicological relevance: increased (+24 %) plasma glucose concentration (p <0.01) in high dosed males at the end of the recovery period; increased (+23 %) plasma urea concentration (p <0.01) in high dosed males at the end of the recovery period, but decreased (-18 %) plasmas urea concentration in the high dosed females at the end of the recovery period. This latter value was slightly below the range of historical control data but still considered of no relevance; decreased (-22 %) Alanine aminotransferase (ALAT) concentration (p <0.01) in high dosed females at the end of the treatment period; decreased (by -26 %) glutamate dehydrogenase concentration (p <0.05) in high dosed females at the end of the treatment period; the changes encountered in electrolytes (Na+ and CI- in males, and Na+, CI- and Ca++ in females) in various treatment groups were rather small and all within the range of historical control data. These findings were considered incidental and/or revealed no clear dose response-relationship.

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

Urinalysis indicated urine discoloration (ranging from yellow-brown to red-brown), a slight increase in pH, a moderate increase in bilirubin and a marked increase in erythrocytes in males and females of the high dose groups. In addition, a slight increase in urinary protein, ketones and leukocytes was noted in males of group 4 which was for all these parameters within the reference range and therefore not considered to be of any toxicological relevance. At termination of the treatment-free recovery period yellow-brown urine discoloration and the increase in erythrocytes were still evident in males and females of the high dose groups. All other findings had returned to normal after discontinuation of treatment. It should be noted, that due to the test item related urine discoloration some of the changes observed in the urine may reflect artifactual changes.

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

There was no test item-related effect on absolute organ weights noted besides of a slight decrease of -22 % (at the 5 % level) in absolute thymus weight in the high dose females. In males and females of the high dose group, relative kidney weights were increased by +17 % (at 1 % level) and +15 % (at 5 % level), respectively. In addition, in males of the mid dose group relative kidney weight was increased by +10 % (at 5 % level). Relative thymus weight was slightly decreased by -19 % (at 5 % level) in females of the mid dose group.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

At necropsy, a bilateral dark red discoloration of the kidneys was present in all males and females of the mid and high dose groups. A reddish discoloration of the mesenteric lymph node was noted in 4 males and 5 females of the high dose groups. A reddish discoloration at different parts of the gastro-intestinal tract was recorded in one male and 2 or 4 females of the high dose groups, whereas a reddish discoloration of the whole subcutis were recorded at necropsy in 3 high dosed females performed at the end of the treatment period. The dark red discoloration of the kidneys was still persistent in all males and females of the high dose groups at the end of the treatment-free recovery period. And the discloration of the rectum was still noted in 3 males and 2 females of the high dose groups after the recovery period.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Microscopic examination revealed kidneys and mesenteric lymph nodes as target organs. In the kidneys, a minimal to moderate vacuolar tubulus cell degeneration was recorded in all main study and recovery animals of the high dose group. In addition, an increased mean grade of tubular basophilia was noted in main study females of the high dose group, as well as in the recovery males and females of the high dose group. Both changes were characterized by cells with fine-granular, red-brown pigment in cytoplasmic vacuoles. The mesenteric lymph node in main study and recovery animals of the high dose groups revealed minimal to slight amounts of foamy macrophages with fine-granular red-brown cytoplasmic pigment. All other microscopic findings recorded in various organs of animals treated with 50, 200 and 1000 mg/kg/day did not distinguish significantly treated rats from control rats and were considered to be spontaneous in nature and within the normal background pathology commonly seen in rats of this strain and age.

Details on results:

- Functional Observational Battery: Grip Strength: No test item-related changes were noted in mean fore- and hind limb grip strength of treated males and females when compared to the controls. Locomotor Activity: There were no test-item related effects on locomotor activity. A slight decrease (at 1% level) in the mean locomotor activity was observed in males treated with 200 mg/kg/day at the 20 minutes measurement interval and in high dosed males (1000 mg/kg) at the 60 minutes interval. In high dosed females (1000 mg/kg) there was a slight increase (at 1 % level) at the 60 minutes measurement interval which was considered as an incidental effect as there was no dose response-relationship present and the effect was in the wrong direction. A moderate increase (+24 %) in total motor activity of the females of the high dose group was present when compared to the controls. These changes were considered to be incidental events as there was no dose response relationship present or the values were within the control data.

Effect levels

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Toxic effects observed in the kidneys and mesenteric lymph nodes.

Target system / organ toxicity

Critical effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

The NOAEL was determined to be 200 mg/kg bw/day.

Executive summary:

In a GLP-compliant repeated toxicity study, performed according to OECD guideline 407, Wistar rats were treated with the test substance (50, 200, 1000 mg/kg bw) by repeated oral gavage for a period of 28 days. The study was comprised of 4 groups, the groups comprised 5 animals per sex that were sacrificed after 28 days of treatment. Additional 5 rats per sex and group were used at 0 and 1000 mg/kg bw. These animals were treated for 28 days and then allowed a 14-day treatment-free recovery period after which they were sacrificed. Oral administration resulted in no unscheduled mortality, and apart from the secondary effect of a dose-dependently reddish discoloration of the feces in animals of the mid and high dose groups no clinical signs of any adverse nature were recorded during daily or weekly observations and during functional observational battery. In addition, there were no test item related effects on fore- and hind limb grip strength, nor on locomotor activity. Food consumption and body weight were not affected by treatment. There was no test item-related effect on any haematological parameters. Clinical chemistry parameters revealed slightly higher levels for cholesterol and phospholipids in animals of the high dose groups and a slightly increased triglyceride concentration in females of the high dose group. At termination of the treatment-free recovery period the cholesterol and phospholipid concentrations in the high dosed males and the triglyceride concentration in the high dosed females had returned to control levels. In contrast, increased levels for cholesterol and phospholipids were still present in the high dosed females after the recovery period. These findings are suggestive of metabolic adaptive changes and in absence of any corroborative histopathological findings not interpreted as being toxicologically significant. The findings noted in urinalysis were restricted to the secondary effect of urine discoloration, a slight increase in pH, a moderate increase in bilirubin and a marked increase in erythrocytes in both sexes of the high dose group. At termination of the recovery period, a yellow-brown urine discoloration and an increase in erythrocytes were still evident in both sexes of the high dose group. The slight increase in urinary protein may reflect a tubular effect, whereas the observed increase in the level of ketones could refer to a slight starvation. The increased leukocyte counts is considered an incidental finding as there was no corroborative histopathological finding present. Due to the test item-related presence of urine discoloration it cannot be excluded that some of the changes observed in the urine may represent artifactual changes. A primary toxicological relevance is attributed to the relative organ weight changes seen for kidneys as corroborative histopathological findings were reported. In absence of any histopathological correlates no toxicological relevance is attributed to the decreased absolute (in female high dose group) and relative (in female mid dose group) thymus weights nor to the slightly increased relative kidney weight (in male mid dose group). Microscopic examination revealed kidneys and mesenteric lymph nodes as target organs at 1000 mg/kg/day.

Under the conditions the test substance did produce evidence of a toxic effect of the test item mainly in the kidneys and mesenteric lymph nodes of almost all animals of the high dose group. Based on the results of this study, 200 mg/kg body weight/day established as the no-observed-adverse-effect-level (NOAEL), and equally, 200 mg/kg body weight/day could be established as the no-observed-effect-level (NOEL).