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Repeated dose toxicity: oral

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Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Sept 2008 - Jan 2009
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
Qualifier:
according to guideline
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Reference substance name:
Hostavin N 20
IUPAC Name:
Hostavin N 20
Specific details on test material used for the study:
Batch No.: DEFD097971
Purity: > 99.9%
Expiry date: 26. July 2012

Test animals

Species:
rat
Strain:
Sprague-Dawley
Remarks:
Hsd: SD
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Harlan Italy S.r.l, San Pietro al Natisone, Italy
- Age at study initiation: 27-29 days
- Weight at study initiation: 75-99 g
- Housing: up to 5 of one sex in clear polycarbonate cages with stainless steel mesh lid and floor
- Diet (e.g. ad libitum): laboratory rodent diet (4 RF 21, Mucedola S.r.l, Settimo Milanese, Italy), ad libitum
- Water (e.g. ad libitum): drinking water, ad libitum
- Acclimation period: 18 days

DETAILS OF FOOD AND WATER QUALITY:
Records of analyses of water and diet are kept on file at RTC

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22+/-2°C
- Humidity (%): 55+/-15%
- Air changes (per hr): 15-25
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES:
From: 23. Sept. 2008 To: 26. Jan. 2009

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: sesame oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:

VEHICLE
- Justification for use and choice of vehicle (if other than water): solubility
- Concentration in vehicle:
males: 2.5, 7.5 and 22.5 mg/mL
females: 1.0, 2.5 and 7.5 mg/mL
- Amount of vehicle (if gavage): 10 mL/kg
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Prior to commencement of treatment the proposed formulation procedure for the test item was checked in the range 1 - 22.5 mg/mL (concentration and homogeneity) and stability by chemical analysis.
Samples of the formulations prepared in weeks 1 and 13 of the study were analysed to check the homogeneity and concentration.
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
daily
Doses / concentrationsopen allclose all
Dose / conc.:
25 mg/kg bw/day (actual dose received)
Remarks:
males
Dose / conc.:
75 mg/kg bw/day (actual dose received)
Remarks:
males
Dose / conc.:
225 mg/kg bw/day (actual dose received)
Remarks:
males
Dose / conc.:
10 mg/kg bw/day (actual dose received)
Remarks:
females
Dose / conc.:
25 mg/kg bw/day (actual dose received)
Remarks:
females
Dose / conc.:
75 mg/kg bw/day (actual dose received)
Remarks:
females
No. of animals per sex per dose:
10 + 5 animals/sex for 4 weeks recovery (control and high dose group)
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
Based on the results of a 28 day dose range finder
- Post-exposure recovery period in satellite groups:
4 weeks
Positive control:
none

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: once daily for clinical signs, twice daily for mortality

DETAILED CLINCAL EXAMINATION:
weekly

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

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

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes / No / Not specified
- Time schedule for examinations:

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations:prior to treatment and suring week 13
- Dose groups that were examined: all groups

HAEMATOLOGY: Yes
- Time schedule for collection of blood: during week 13 and week 4 of recovery
- Anaesthetic used for blood collection: Yes (isofluorane)
anminals under food and water deprivation
- How many animals: all animals
- Parameters examined:
Haematocrit, haemoglobin, red blood cell count, reticulocyte count, reticulocyte maturation index, mean red blood cell volume, red cell volume distribution width, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration, Haemoglobin concentration width, Heinz bodies, white blood cell count, differential leucocyte count, platelets, prothrombin time, activated partial thromboplastin time

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: during week 13 and week 4 of recovery
- How many animals: all animals
animals under food and water deprivation
- Parameters examined:
Alkaline phosphatase, alanine aminotransferase, aspartate aminotransferase, gamma-glutamyltransferase, lactate dehydrogenase, glutamat dehydrogenase, creatine kinase, urea, creatinine, glucose, triglycerides, phosphorus, total bilirubin, total cholesterol, total protein, albumin, globulin, A/G ratio, sodium, potassium, calcium, chloride

URINALYSIS: Yes
- Time schedule for collection of urine: during week 13 and week 4 of recovery (overnight)
- Metabolism cages used for collection of urine: Not specified
- Animals fasted: Yes
- Parameters examined:
Appearance, color, volume, specific gravity, pH, protein, total reducing substances, nitrite, glucose, ketones, bilirubin, urobilinogen, blood
Sediment examination:
epithelial cells, erythrocytes, leucocytes, crystals, spermatozoa and precursors, other abnormal components

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: during week 12 and once during week 4 of recovery
- Dose groups that were examined: all
- Battery of functions tested: sensory activity / grip strength / motor activity
additionally (weekly):
handling reactivity, lachrymation, palperal closure, salivation, piloerection, rearing, spasms, myclonia, mobility impairment, arousal, vocalisation, stereotypies, unusual repsiratory pattern, bizarre behavior, urination, defecation, tremors, gait

Sacrifice and pathology:
GROSS PATHOLOGY:
Yes, all animals

ORGAN WEIGHT:
From all animals completing the scheduled test period the following organs were weighed:
adrenal glands, brain, epididymides, heart, kidneys, liver, ovaries, parathyroid glands,spleen, testes, thymus, thyroid, uterus

HISTOPATHOLOGY: Yes
from all main phase animals in the control and high dose groups, from all animals in low and medium dose groups died during the treatment and all abnormalities in all main phase animals.
Histopathologically examined organs/tissues:
adrenal glands, aorta, bone, boe marrow, brain, caecum, colon, duodenum, epididymides, heart, ileum, jejunum, kidneys, lungs, lymph nodes (mandibular and mesenteric), mammary gland, oesophagus, ovaries, oviducts, pancreas, prathyroid gland, pituitatry gland, prostate glandm rectum, salivary glands, sciatic nerve, skin, spinal cord, spleen, stomach, testes, thymus, thyroid, trachea, urinary bladder, uterus/cervix, vagina
Statistics:
For continuous variables the significance of the differences amongst groups was assessed by analysis of variance. Differences between each treated group and the control group were assessed by Dunnett’s test. The homogeneity of the data was verified by Bartlett’s test before Dunnett’s test. If data were found to be inhomogeneous a Modified t test (Cochran and Cox) was applied. The mean values, standard deviations and statistical analysis were calculated from the actual values in the computer without rounding off. Statistical analysis of histopathology findings was carried out by means of the non-parametric Kolmogorov-Smirnov
test.

Results and discussion

Results of examinations

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Hairloss was observed in some animals of both sexes of the high dose group. Other minor clinical signs, such as damaged eye, corneal opacity, broken/missing teeth, swollen head and skin/fur staining were observed in individual animals during the treatment period. These signs were not considered to be treatment-related.
No significant clinical signs were noted in recovery animals during the treatment-free period.
Mortality:
no mortality observed
Description (incidence):
No mortality occurred during the study.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Very slight but statistically significant body weight reductions were observed in the high dose males from Day 15 (-4%) to Day 92 (-10%) of the treatment period. Very slight reductions were also observed in the high dose females, treated at 75 mg/kg/day, from Day 85 (-6% to -4%). Terminal body weight showed a statistically significant reduction (- 9%) in the high dose males, treated at 225 mg/kg/day.

Although all the above mentioned body weight reductions were treatment-related, they were considered to be of no toxicological importance as they were very slight (< 10%) and comparable to the historical control data.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
Very slight decreases in food consumption (from -9% to -17%) were observed in the high dose males (225 mg/kg/day) from Day 8 to Day 43. Differences in food consumption disappeared by Day 50 and were no longer detected up to the end of the recovery period.
These changes were not considered of toxicological importance as they were slight and comparable to the historical control data.
No significant food consumption differences were observed in female animals at any stage of the study.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
No changes of toxicological significance were observed.
Slight leucopenia was observed in all treated females and comprised almost all leukocyte cell types (approximately 30%-35% when compared with controls), whereas the decrement of white blood cells was not dose-related and the results obtained were within the range of historical data.
In addition, a slight increase in Heinz Bodies was observed in treated groups when compared to controls. However, due the lack of relation with the dose and of further related changes (anaemia, reticulocytosis) this change was considered to be of no toxicological significance.
The other statistically significant differences between control and treated animals (increase in haemoglobin in males treated with 25 mg/kg/day, haematocrit and mean corpuscular haemoglobin concentration in males receiving 25 and 225 mg/kg/day, 37% decrease in monocytes in males treated with 25 mg/kg/day and in mean corpuscular haemoglobin concentration) were of low severity (1-5%) and/or with no dose-relation, therefore they were considered to be incidental.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
In general, minor fluctuations of some biochemical parameters were recorded in treated animals as showed below.
Males treated with 225 mg/kg/day showed a slight increase in alkaline phosphatase (+30%), alanine aminotransferase (+45%), urea (+23%), globulin (+10%) and chloride (+2%) and a decrease in bilirubin (-26%), cholesterol (-17%) and sodium (-2%). Cholesterol was also decreased in the other treated male groups and changes in globulin and sodium were observed even in those receiving 75 mg/kg/day. Females treated with 75 mg/kg/day showed an increase in alkaline phosphatase (+29%), cholesterol (+16%), chloride (+2%) and sodium (+2%) and a decrease in lactate dehydrogenase (-48%) and bilirubin (-23%).

Changes were considered to be of no toxicological importance, since they were of low magnitude and the direction of some of the alterations has no pathological importance. In addition, there were no histopathological findings which could be related with the liver and kidney marker changes.
Urinalysis findings:
no effects observed
Behaviour (functional findings):
no effects observed
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
A slight but statistically significant decrease in absolute and relative weight of the spleen (-23% and -16% respectively) was observed in the males dosed at 225 mg/kg/day. In addition, an increase in testes relative weight (+10%) was noted in the high dose males.
No other changes of toxicological significance were observed.
Partial or total recovery was observed at the end of the 4-week treatment-free period. As no correlated lesion was noted at histology, this change was not considered to be of toxicological importance.
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Description (incidence and severity):
The histopathological examination of the testes and epididymides did not reveal any evident differences between the findings observed in treated and control animals that could be considered treatment-related.
Seminiferous tubules were evaluated with respect to their stage in the spermatogenic cycle and to the integrity of the various cell types within the different stages and no abnormalities were noted.

Effect levels

open allclose all
Key result
Dose descriptor:
NOAEL
Effect level:
225 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: no effects of toxicological relevance observed
Key result
Dose descriptor:
NOAEL
Effect level:
75 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: no effects of toxicological relevance observed

Target system / organ toxicity

Key result
Critical effects observed:
no

Any other information on results incl. tables

For clarification on the substance`s mode of action please refer to attached document "Assessment of toxicological mode of action_Hostavin N 20"

Applicant's summary and conclusion

Conclusions:
Based on the changes observed in the present 13 week oral toxicity study performed in Sprague Dawley rats the NOAEL was considered to be 225 mg/kg bw/d in males and 75 mg/kg bw/d in females (highest doses tested).
Executive summary:

The oral toxicity of the test substancewhen given by daily administration to rats, has been investigated over a period of 13 consecutive weeks and recovery from any potential treatment-related effects over a period of 4 consecutive weeks.

No mortality occurred and no significant clinical signs were observed during the in vivo phase of the study. The slight reductions in body weight and food consumption observed in the high dose animals were not considered to be of toxicological importance due to the low magnitude (<20%).

The statistically significant changes observed in haematological parameters during dosing and/or recovery phase were considered to be of no toxicological significance, as they were of low severity and not dose-related. Minor fluctuations of some biochemical parameters were recorded in treated animals during the dosing and/or recovery phase. However, changes were of low magnitude and not in a direction to have pathological importance. In addition, there were no histopathological findings which could be related with the liver and kidney markers changes. Therefore they were considered to be of no toxicological importance. No changes of toxicological significance were observed in urine parameters. A slight but statistically significant decrease of the absolute and relative weight of the spleen was observed in the males dosed at 225 mg/kg/day. The toxicologically significance of this change was not supported by the microscopic examination of this organ. No changes of toxicological significance were found at post mortem macroscopic observation and microscopic examination performed at the end treatment and recovery periods. No abnormalities were noted at the evaluation in the seminiferous tubules of the stage in the spermatogenic cycle and in the integrity of the various cell types within the different stages.

On the basis of these results, some slight changes were seen at the high doses of 225 mg/kg/day for males and 75 mg/kg/day for females. However, these changes were of low magnitude and/or inconsistent between sexes. In addition, no lesions were observed at histopathological examination of tissues/organs.