Registration Dossier

Toxicological information

Repeated dose toxicity: oral

Currently viewing:

Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
28 February 2012 to 04 April 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: study performed under GLP compliance

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2012

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Deviations:
no
Principles of method if other than guideline:
Commission Regulation (EC) No. 440/2008, L 142, Annex Part B, May 30, 2008
Commission Directive 2001/59/EC of 6 August 2001
GLP compliance:
yes (incl. certificate)
Remarks:
(Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Germany)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
Test System:

Species/strain: healthy Wistar rats, Crl: WI(Han) (Full Barrier) Source:Charles River, 97633 Sulzfeld, Germany
Sex: male and female; the female animals were non-pregnant and nulliparous.
Age at the first administration: males and females: 7-8 weeks old.
Body weightat the beginning of the study: males:162 - 181 g; females:126-151 g
The animals were derived from a controlled full-barrier maintained breeding system (SPF). According to Art. 9.2, No. 7 of the German Act on Animal Welfare [7] the animals were bred for experimental purposes.

Housing and Feeding Conditions:

- Full barrier in an air-conditioned room
- Temperature: 22 3°C
- Relative humidity: 55 10%
- Artificial light, sequence being 12 hours light, 12 hours dark
- Air change: 10 x / hour
- Free access to Altromin 1324 maintenance diet for rats and mice (lot no. 1114)
- Free access to tap water, sulphur acidified to a pH of approximately 2.8 (drinking water, municipal residue control, microbiological controls at regular intervals)
- The animals were kept individually in IVC cages, type III H, polysulphone cages on Altromin saw fibre bedding (lot no. 110811)
- Certificates of food, water and bedding are filed at BSL BIOSERVICE
- Adequate acclimatisation period (at least 5 days)

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on oral exposure:
The animals were treated with the test item or vehicle on 7 days per week for a period of 28 days. The test item formulation or vehicle was administered at a single dose to the animals by oral gavage at an application volume of 5 mL/kg bw.
For each animal the individual dosing volume was calculated on the basis of the body weight most recently measured.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples for the nominal concentration verification were taken in study week 1, week 2, study week 3 and study week 4 of freshly prepared control, low, medium, and high dose groups (16 samples in total).
Samples for homogeneity were taken from the top, middle and bottom of the high dose, mid dose and low dose preparation in study week 1 (9 samples in total).
Samples for stability analysis were taken from high dose, mid dose and low dose group in study week 1 at 0 hr and 6 hrs (6 samples in total).
All formulation samples were stored frozen (approximately -20°C) until the analysis was performed.
Analysis of FAT 40858/ A TE in aqueous samples was performed using a HPLC-UV method.

The determination of test item concentration in the dosing formulations was performed by the analytics department of BSL Bioservice in accordance with GLP.
Duration of treatment / exposure:
28 days
Frequency of treatment:
7 days per week for a period of 28 days
No. of animals per sex per dose:
40 animals (20 males and 20 females) were included in this study (5 male and 5 female animals per group). The study included one control (C) and three dose groups (LD, MD, HD).
Details on study design:
Preparation of the Animals:
Prior to the start of the treatment period a detailed clinical observation outside the home cage was made. Animals showing pathological signs before the first administration were excluded from the study. Supplementary animals from the same delivery were provided in exchange. Before the first administration all animals used for the study were weighed and assigned to the experimental groups with achieving a most homogenous variation in body weight throughout the groups of males and females.

According to the results of the dose range finding study (BSL project no. 115997) and in consultation with the sponsor the following doses were selected for the 3 dose groups (LD, MD, HD) and 1 control group (C): 100, 300, 1000 mg/kg bw.

The animals in the control group were handled in an identical manner to the test group subjects and received the vehicle using the same volume as used for the high dose group.

Administration of Doses:
The animals were treated with the test item or vehicle on 7 days per week for a period of 28 days. The test item formulation or vehicle was administered at a single dose to the animals by oral gavage at an application volume of 5 mL/kg bw.
For each animal the individual dosing volume was calculated on the basis of the body weight most recently measured.

Body Weight, Food Consumption:
The body weight was recorded once before the assignment to the experimental groups, on the first day of administration and weekly during the treatment period. Food consumption was measured weekly during the treatment period.

Clinical Observation:
All animals were observed for clinical signs during the entire treatment period of 28 days.
General clinical observations were made once a day. Twice daily all animals were observed for morbidity and mortality except for weekends and public holidays when observations were made once daily.

Ophthalmological examination, using an ophthalmoscope was made on all animals before the first administration and in the last week of the treatment period.

Functional Observation:
Once before the first exposure, as well as once in the fourth week of exposure multiple detailed behavioural observations were made outside the home cage using a functional observational battery of tests . These tests were conducted in all animals.

The haematological parameters examined were : haematocrit value (Hct), haemoglobin content (Hb), red blood cell count (RBC), mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC), reticulocytes (Re), platelet count (PLT), white blood cells (WBC), neutrophils (Neu), lymphocytes (Lym), monocytes (Mono), eosinophils (Eos), basophils (Baso).

The coagulation parameters examined were: prothrombin time (PT), activated partial thromboplastin time (aPTT).

The clinical biochemistry parameters examined were: alanine aminotransferase (ALAT), aspartate-aminotransferase (ASAT), alkaline phosphatase (AP), creatinine (Crea), total protein (TP), albumin (Alb), urea, total bilirubin (TBIL), total bile acids (TBA), total cholesterol (Chol), glucose (Gluc), sodium (Na), potassium (K).

Urinalysis:
A urinalysis was performed with samples collected from all animals prior to or as part of the sacrifice of the animals.

Examinations

Sacrifice and pathology:
Pathology
Gross necropsy
All animals were subjected to a detailed gross necropsy on day 29 which included careful examination of the external surface of the body, all orifices and the cranial, thoracic and abdominal cavities and their contents.
The wet weight of the organs of all sacrificed animals was recorded as soon as possible. Paired organs were weighed separately.
The following tissues from all animals were preserved in 10% neutral buffered formalin except eyes, testes and epididymides that were fixed in Modified Davidson’s Fixative for 24 hours and then transferred to 10% neutral buffered formalin.

Preserved and Examined Tissues:

lesions, lung, brain (cerebrum, cerebellum and pons), trachea, spinal cord, uterus with cervix (females), thyroid/parathyroid glands, ovaries (females), thymus, vagina (females), oesophagus, testes (males), stomach, epididymides (males), small and large intestines (including Peyer´s patches), prostate with seminal vesicles with coagulating glands as a whole (males), liver, heart, kidneys, urinary bladder, adrenal glands, lymph nodes (mesenteric and axillary), spleen, peripheral nerve (e.g. sciatic nerve) with skeletal muscle, eyes, bone with bone marrow (sternum)

Histopathology:

The afore-listed organs of all animals were examined histologically after preparation of paraffin sections and haematoxylin-eosin staining.
A full histopathology was carried out on the preserved organs and tissues of all animals of the control and high dose groups which were sacrificed at the end of the treatment period. The stomach (nonglandular and glandular) evaluation was extended to animals of all other dosage groups, because treatment-related changes were observed in the high dose group.
All gross lesions identified in any animal were examined.
Histological processing of tissues to microscope slides was performed at the GLP-certified contract laboratory Propath UK Ltd. (test site for tissue processing), Willow Court, Netherwood Road, Hereford HR2 6JU, England. Histopathological evaluation was performed at the GLP-certified contract laboratory KALEIDIS – Consultancy in Histopathology (test site for histopathology), 6 rue du Gers, 68300 Saint-Louis, France. Blocking, embedding, cutting, H&E staining and scientific slide evaluation were performed according to the corresponding SOP’s of the test sites.
Statistics:
A statistical assessment of the results of the body weight, food consumption, parameters of haematology, blood coagulation and clinical biochemistry and absolute and relative organ weights were performed for each gender by comparing values of dosed with control animals of the main groups using a one-way ANOVA and a post-hoc Dunnett Test. These statistics were performed with GraphPad Prism 5.01 software (p<0.05 was considered as statistically significant).

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Details on results:
Animal Survival:

There was no mortality observed in male- female treatment and control groups.

Clinical Observations.

There were few clinical signs observed in treatment groups when compared to control. The predominant clinical sign observed in HD group was discoloured skin (reddish in colour). The clinical signs observed in treatment groups are listed as, LD group: slight piloerection (1/5 males); MD groups: slightly increased spontaneous activity (2/5 females), alopecia on abdomen (1/5 females), eschar on right side of abdomen (1/5 females); HD group: exophthalmos (1/5 males), discoloured skin (5/5 males and 5/5 females), moderately increased spontaneous activity (2/5 females), red urine (1/5 females).
Slight piloerection observed in 1/5 males of LD group and slightly increased spontaneous activity in 2/5 females of MD group were considered incidental. There were also unspecific findings like alopecia and eschar in 1/5 females. These findings were spontaneous in nature and were considred not to be treatment related.
The exophthalmos (1/5 males, animal no. 19) in HD group revealed no histopathological changes and was considered to be spontaneous in nature.
At HD group, all male and female animals’ skin turned red colour. This was considered to be due to absorbed test item in the body.
During the weekly detailed clinical observation, no significant changes or differences between the groups were found.
There were no ophthalmoscopic findings in any of the animals of this study.

Functional Observation Battery:

No relevant effects were observed in any of the parameters of the functional observation battery before and at the end of the treatment period. There were no biologically relevant differences in body temperature between the groups.

Body Weight Development

In males, there was marginal decrease in mean body weight observed in HD group on day 22 and 28, but statistically there was no significant difference when compared to the corresponding control. Statistically no significant difference was observed for the body weight gain during the study period.

In females, all mean values of body weight and body weight gain in treatment and control groups were comparable and statistically there was no significant difference.

Food Consumption:

There was statistically no significant difference observed for food consumption for any of the groups of male and female animals when compared to the control. However, there was marginal decrease in food consumption observed in MD group females between the study days 8-15 and 15-22 when compared to the control group. But this finding was not assumed to be of toxicological relevance.

Haematology and Blood Coagulation:


In males and females, all haematological parameters were within the normal range of variation. However there were statistically significant differences observed for mean MCHC and eosinophil count in male HD group and mean MCHC in female MD group. These values were within the historical control range and not related to treatment.
Blood coagulation was not affected by test item in both males and females. However, there was severe increase in mean values of male LD group. This finding was due to high variation of values among the individuals of the group. Hence, is not related to treatment.

Clinical Biochemistry:

In males, there was statistically significant decrease in mean ASAT value in LD and HD groups; statistically significant decrease in mean ALAT and AP values in HD group and statistically significant increase in mean TP, TBIL and Chol values in HD group. The decrease in ASAT and ALAT value is considered to have no biological relevance. The mean values of AP, TP and Chol were within historical control range and the difference in HD group from corresponding control was very likely to be related to treatment.
In females, there was statistically significant increase in mean AP value in MD group; statistically significant increase in mean TP value in HD group and statistical significant increase in mean TBIL values in MD and HD groups. There was no toxicological relevance considered for AP due to the absence of dose response pattern and the TP value in HD group was within historical control range.
There was considerable decrease in mean TBA value in male and female HD group. Due to the absence of most of the individual values of male HD group and heterogeneity in female HD group, the finding was likely to be related to treatment.
There was consistent increase in TBIL values noted in both male and female LD, MD and HD groups showing clear dose response pattern and attaining statistical significance at higher dose levels. In the absence of histopathological liver changes, this finding was considered to be less adverse.

Besides, all parameters of clinical chemistry were within the normal range of variation for this strain and statistically no significant differences between dose and control groups were observed.

Urinalysis:

In males and females, the urine colour changed from yellowis to orange and rose/ reddish in LD, MD and HD group animals. This change in urine colour was due to excreted test item. There was high erythrocyte level noted in one isolated male of MD group, which was considered to be incidental. There was high UBG and BIL level noted in one individual female (animal 40) of HD group. There was also high leukocyte level in two females (animal 36 and 40) of HD group. As there were no corresponding effects noted during microscopic examination of kidneys or in hemathology and clinical biochemistry, the toxicological relevance of these findings was not clear.

Pathology:

Few specific gross pathological changes were recorded for the male and female animals during necropsy.
Male: MD- discoloured skin salmon colour (Animal 13), nodule on stomach wall (animal 15); HD group- discoloured skin reddish colour (animal 16), discoloured red- all organs (animal 17, 18, 19, 20).
Female: LD: fluid distended uterus with oviduct and cervix (animal 27, 28), discoloured axillary lymph node (dark) and discoloured red thymus (28); MD- fluid distended uterus with oviduct and cervix (animal 34, 36), discoloured red all organ (animal 36, 38), discoloured red skin (animals 37).
The discolouration of organs was considered to be due to absorbed test item. Other necropsy findings revealed no hitopathological changes and were not related to treatment.

Organ Weight:

In males and females, statistically there were no significant changes observed for absolute and relative (to terminal body weight and brain weight) organ weights in the treatment groups when compared to the control.
There was mild decrease observed for absolute organ weight for thymus in male MD and HD groups. This finding was supported by relative brain and body weight of thymus.
In females, there was marginal increase in absolute adrenal gland in LD group, marginal increase in absolute spleen weight in LD group, marginal increase in absolute ovary weight in MD group, there was marked increase in uterus weight in HD group and mild increase in MD and HD group. This finding was supported by relative brain and body weights of mentioned organs.

Histopathologically no treatment related changes were noted in any of the above organs that could be associated with the changes in organ weight. Hence, the organ weight change was not related to treatment.

Histopathology:

Histopathological findings considered to be of toxicological relevance were restricted to the stomach at HD group. In the nonglandular part of the stomach, minimal or mild epithelial hyperplasia of the limiting ridge was observed in a proportion of both sexes. In the females concerned, there was also submucosal edema and mixed cell inflammation in the glandular part of the stomach. In view of their nature and limited degree of severity, both findings were considered to be indicative of a minor local irritation by the test item formulation and not to be sufficient evidence of a toxic effect.
Other findings related to test item administration were restricted to the presence of minor amounts of red-brown pigment in a number of organs in animals at HD group. Pigment deposition was seen in the corticotubular epithelium of the kidney, axillary and mandibular lymph node, cecum, interstitium of the testis and epididymis, and endometrium of the uterus. It was considered to be due to absorbed test item. Under the conditions of this study, it was considered to be without toxicological significance.

Dose Formulation Analytics:

The concentration analysis of formulation samples was determined in study week 1, 2, 3 and 4 for all dose groups. The mean recoveries observed in LD, MD and HD groups were 93.5%, 99.4% and 98.3% of the nominal concentration, respectively.
Stability of the formulation samples was determined in study week 1 for LD, MD and HD groups. The mean recoveries observed after 6 hours storage at room temperature for LD group was 98.5% for MD group was 99.7% and for HD group was 99.5% of the concentration without storage.
Homogeneity of the formulation samples was determined in study week 1 for LD, MD and HD groups. The mean recoveries observed for LD group was 99.2% for MD group was 100.9%, and for HD group was 96.4% of the nominal value. The coefficients of variation of the different sampling locations (top, middle, and bottom) in LD group were 0.8% in MD group was 1.7% and in HD group was 1.4%.

Effect levels

Dose descriptor:
NOAEL
Remarks:
1000 mg/kg body weight
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see 'Remark'
Remarks on result:
not measured/tested
Remarks:
Effect level not specified (migrated information)

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
On the basis of the present study, the 28-Day Repeated Dose Oral Toxicity study with FAT 40858/A TE in male and female rats, with dose levels of 100, 300, and 1000 mg/kg body weight the following conclusions can be made:
There were slight clinical signs (mostly reddish discolouration of skin) noted at 1000 mg/kg body weight; changed urine colour (yellowish or orange or rose/reddish) in males and females at and above 100 mg/kg bw and macroscopically discoloured organs at and above 300 mg/kg bw. These findings were due to absorbed test item and under the conditions of this study, it was considered to be without toxicological significance.
The serum TBIL in males and females showed consistent increase in treatment groups at and above 100 mg/kg body weight. However, there were no associated histopathological changes supporting this finding and hence, was considered to be less adverse.
There was minimal or mild epithelial hyperplasia of the limiting ridge observed in nonglandular part of the stomach in majority of males and females at 1000 mg/kg body weight. This effect might be related to a local irritation by the test item formulation.
Based on the data generated, the dose of 1000 mg/kg body weight is considered to be the NOAEL (No Observed Adverse Effect Level) for the 28-Day repeated dose oral toxicity study with FAT 40858/A TE in male and female rats.
Executive summary:

No mortality occurred in the control or any of the dose groups during the treatment period of this study. There were few clinical signs observed in treatment groups when compared to control. The predominant clinical sign observed in HD group was discoloured skin (reddish in colour). Slight piloerection observed in 1/5 males of LD group and slightly increased spontaneous activity in 2/5 females of MD group were considered incidental. There were also few unspecific findings like alopecia, eschar (1/5 females) in MD group and exophthalmos (1/5 males) in HD group. These findings were spontaneous in nature and were considred not to be related treatment. During the weekly detailed clinical observation, no significant changes or differences between the groups were found. No relevant effects were observed in any of the parameters of the functional observation battery before and at the end of the treatment period. There were no ophthalmoscopic findings in any of the animals of this study. In males and females, there were no treatment related changes noted for body weight, body weight gain and food intake in treatment groups when compared to the corresponding control group.

No toxicological relevance was considered for the hematology and blood coagulation parameters in both male and female animals in treatment group when compared to the control. However, there was statistical significance observed for mean MCHC and eosinophil count in male HD group and mean MCHC in female MD group. No toxicological relevance was considered for the clinical biochemistry parameters. However, there was statistically significant decrease in mean ASAT value in male LD and HD groups; statistically significant decrease in mean ALAT and AP values in male HD group; statistical significant increase in mean TP, TBIL and Chol values in HD group. In females, statistically significant increase were observed for mean AP and TP values in MD and HD groups, respectively. The mean AP, TP and Chol values in males or females were within historical control range and the difference was very likely to be related to treatment. The decrease in ASAT and ALAT value is considered to have no biological relevance. There was considerable decrease in mean TBA value in male and female HD group. In the absence of most individual values of male HD group and heterogeneity in female HD group, the finding was likely to be related to treatment. There was consistent increase in TBIL values noted in both male and female LD, MD and HD groups showing clear dose response pattern and attaining statistical significance at higher dose levels. In the absence of histopathological liver changes, this finding was considered to be less adverse. Changed urine colouration from yellowish to orange and rose or reddish was observed in males or females of LD, MD and HD groups. This change in urine colour was considered to be due to excreted test item. There was high UBG and BIL level noted in one individual female (animal 40) of HD group and high leukocyte level in two females (animal 36 and 40) of HD group. In the absence of corresponding effects noted during microscopic examination of kidneys or in hemathology and clinical biochemistry, the toxicological relevance of these findings was not clear. Few specific gross pathological changes were recorded for the male and female animals during necropsy. Male: MD- discoloured skin salmon colour (Animal 13), nodule on stomach wall (animal 15); HD group- discoloured skin reddish colour (animal 16), discoloured red- all organs (animal 17, 18, 19, 20). Female: LD: fluid distended uterus with oviduct and cervix (animal 27, 28), discoloured axillary lymph node (dark) and discoloured red thymus (28); MD- fluid distended uterus with oviduct and cervix (animal 34, 36), discoloured red all organs (animal 36, 38), discoloured red skin (animals 37). The discolouration of organ was due to absorbed test item and the other necropsy findings were not related to treatment. No treatment related changes were observed for absolute and relative (to terminal body weight and brain weight) organ weights of males and females. Histopathological findings considered to be of toxicological relevance were restricted to the stomach in HD group. In the nonglandular part of the stomach, minimal or mild epithelial hyperplasia of the limiting ridge was observed in a proportion of both sexes. In the females concerned, there was also submucosal edema and mixed cell inflammation in the glandular part of the stomach. These findings were considered to be indicative of a minor local irritation by the test item formulation and not to be sufficient evidence of a toxic effect.

There was presence of minor amounts of red-brown pigment in a number of organs in animals of HD group. Pigment deposition was seen in the corticotubular epithelium of the kidney, axillary and mandibular lymph node, cecum, interstitium of the testis and epididymis, and endometrium of the uterus. It was considered to be due to absorbed test item. Under the conditions of this study, it was considered to be without toxicological significance.