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Diss Factsheets

Administrative data

short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: well performed GLP and OECD guideline study

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guideline
according to guideline
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)
GLP compliance:
Limit test:

Test material

Test animals

Details on test animals or test system and environmental conditions:
- Source: IFFA CREDO, BP 0109, 69592 L'Arbresle Cedex, FRANCE.
- Age at study initiation: 6 weeks
- Weight at study initiation: males: 174 to 200 g, females: 158 to 180 g
- Fasting periods: Animals were fasted (at least 16 hours) before blood sampling and before necropsy
- Housing: in groups of 5 of the same sex and dose group in stainless steel mesh cages
- Diet (e.g. ad libitum): Rat & Mouse pelleted complete diet, ad libitum
- Water (e.g. ad libitum): filtered (0.2 µm) mains water, ad libitum
- Acclimation period:

- Temperature (°C): 19 to 25°C
- Humidity (%): 30 to 70 % (target range)
- Air changes (per hr): minimum 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
corn oil
Details on oral exposure:


- Concentration in vehicle: the test article was prepared as a suspension in the vehicle at concentrations of 5, 15 and 100 mg/ml.
- Amount of vehicle (if gavage): The dose volume was 10 ml/kg/day
Analytical verification of doses or concentrations:
Details on analytical verification of doses or concentrations:
Storage: at room temperature or at refrigerator temperature when the preparations were stored for any length of time in the dispensary

Homogeneity of the test article in the vehicle: homogeneity of the suspensions was determined at the testing facility before initiation of treatment for the low and high concentrations.

Stability of the test article in the vehicle : stability over 24 hours was determined at the testing facility before initiation of treatment for the lowest and highest concentrations at + 4°C and at room temperature.

Frequency of preparations: daily; the suspensions were administered to the animals within 4 hours of preparation.

Analysis of preparations: a sample (2 x 2 g) from each preparation (including the control group) was taken:
- at week 1 (day 1),
- at week 4 (day 22),
and was stored at approximately -20°C. These samples were analysed at the testing facility for achieved concentration.
Duration of treatment / exposure:
Frequency: once daily
Duration: 28 days
Frequency of treatment:
Frequency: once daily
Duration: 28 days
Doses / concentrations
Doses / Concentrations:
0, 50, 150 or 1000 mg/kg/day
actual ingested
No. of animals per sex per dose:
Male: 5 animals at 0 mg/kg bw/day
Male: 5 animals at 50 mg/kg bw/day
Male: 5 animals at 150 mg/kg bw/day
Male: 5 animals at 1000 mg/kg bw/day
Female: 5 animals at 0 mg/kg bw/day
Female: 5 animals at 50 mg/kg bw/day
Female: 5 animals at 150 mg/kg bw/day
Female: 5 animals at 1000 mg/kg bw/day
Control animals:
yes, concurrent no treatment
Details on study design:
- Dose selection rationale:
- - The high dose level of 1000 mg/kg/day is the maximum required by the guidelines and is expected to produce signs of toxicity.
- - The low dose level of 50 mg/kg/day is expected to be a no obserable adverse effect level and
- - The intermediate dose of 150 mg/kg/day is a dose which is considered useful in order to determine the safety classification of the test article.
Positive control:


Observations and examinations performed and frequency:
All animals were observed twice daily, at the beginning and at the end of each working day to detect any which were dead or moribund.

All animals were observed daily, before and at least once after dosing to detect any clinical signs or reaction to treatment. A full clinical examination was performed weekly.

Individual body weights were recorded weekly during the treatment period.

Food consumption was measured weekly for each cage of animals during the treatment period.

Animals examined and frequency:
- all animals at termination (day 29)
Samples taken:
- blood. blood was withdrawn from the retro-orbital sinus following light ether anaesthesia of fasted animals (at least 16 hours).
Samples were collected:
- on EDTA for haematology
- in tubes without anticoagulant for clinical chemistry.
Parameters examined:
- Haematology:
- - Haemoglobin
- - Mean corpuscular haemoglobin (MCH)
- - Mean corpuscular haemoglobin concentration (MCHC)
- - Mean corpuscular volume (MCV)
- - Packed cell volume
- - Red blood cell count
- - Platelet count
- - Total white blood cell count
- - Differenflal white blood cell count

Blood smears for reticulocyte count were prepared at week 4.
They were not examined, after consideration of the results obtained for other parameters.

- Blood clinical chemistry:
- - Sodium
- - Potassium
- - Chloride
- - Calcium
- - Inorganic phosphorus
- - Glucose
- - Blood urea nitrogen
- - Total cholesterol
- - Total bilirubin
- - Total protein
- - Albumin
- - Globulin (calculated)
- - Albumin/globulin ratio
- - Creatinine
- - Alkaline phosphatase (AP)
- - Aspartate aminotransferase (ASA T)
- - Alanine aminotransferase (ALAT)
Sacrifice and pathology:
- After 4 weeks of treatment. all animals were necropsied in random order, after fasting
- The animals were weighed before necropsy
- The animals were killed by carbon dioxide inhalation and exsanguination

All animals were submitted to full necropsy procedures including examination of:
- the external surface
- all orifices
- the cranial cavity
- the carcass
- the thoracic and abdominal cavities and organs

Organ weights:
The following organs were weighed at scheduled necropsy for all animals:
- adrenals
- kidneys
- liver
- testes

Organ/tissue preservation and histopathology:
The following organs/tissues were sampled for all animals:
- adrenals
- heart
- idneys
- liver
- lungs
- spleen
- testes
- all gross lesions

Histopathological examinations were performed on all selected organs/tissues listed above (with the exception of testes in the first instance):
- for all animals in groups 1 (control) and 4 (high dose) killed after 4 weeks of treatment

In each group, histopathological examination was performed for all gross lesions, except those for which the diagnosis was judged unnecessary for the outcome of the study by the pathologist.
For body we"ght, body weight gains, food consumption, haematology, blood clinical chemistry parameters and organ weights arithmetic mean and standard deviation was calculated for each group and sex.

The following parameters were analysed statistically:
- mean body weight on days 1 and 28 and mean body weight gains over days 1 to 28
- haematology (where appropriate) and blood clinical chemistry at week 4
- absolute and relative organ weights at the terminal kill

For each parameter Levene's test was used to test the equal',ty of va(,ance across groups.

As Levene's test showed no significant difference in the group variances, data were analysed using parametric procedures. Such analysis consisted of a one way analysis of variance (ANOVA) allowing for a group effect, followed by pairwise comparisons using a protected t-test (pre-treatment body weight data) or Dunnett's t-test to assess the significance of any intergroup differences. A dose-response test was also performed.

Any intergroup differences which attained statistical significance are identivied, using the following criteria: P (probability) < 0.05

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Description (incidence and severity):
except orange coloured fur and tails as well as orange-coloured faeces
no mortality observed
Description (incidence):
except orange coloured fur and tails as well as orange-coloured faeces
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
on food consumption
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
but not examined in detail
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:
not examined
Details on results:
There were no unscheduled deaths.

From soon after treatment on day 1, group 4 (high dose) animals had stained (orangecoloured) fur. The extent and intensity of the coloration tended to increase over the first week of the study until end of the week, the entire animal was generally coloured orange. At the end of the treatment period the tails of high dose animals were coloured deep orange. Animals in other treated groups began to have similarly stained fur from about day 5. Orange-coloured faeces were present under the cages of treated animals from day 2. There were no other treatment-related clinical signs.

High dose females gained slightly less body weight than controls such that there was a 7 % difference between the means at the end of the treatment period. These differences were not statistically significant and therefore are considered of doubtful toxicological significance. There were no other differences in body weight between the groups.

There were no consistent differences in the amount of food eaten between the groups.

Treated and control animals had similar haematology profiles.

The minor differences in some clinical chemistry parameters between treated and control groups. some of which reached the level of statistical significance. were inconsistent or of small magnitude and in the absence of any consistent pattern ot change were considered unrelated to treatment.

There were no treatment-related differences in the weight of the organs.

Macroscopic findings were infrequent and were considered unrelated to treatment with the test article.

There were no treatment-related microscopic findings.

Effect levels

Dose descriptor:
Effect level:
>= 1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: no toxic effects up to the highest dose tested

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

In conclusion, oral (gavage) administration of the test item to the rat at dose levels up to 1000 mg/kg/day was not associated with any toxic change. The only change associated with treatment was on orange coloration of the fur and tail. This sign was considered to be related to the animals grooming behaviour after the administration of a deeply-coloured test compound. Under the defined experimental conditions. 1000 mg/kg/day can be considered the no-effect level for the test item in the rat.

Based on these findings the test item is not subject to classification and labelling.
Executive summary:

The objective of the study was to determine the toxicity and, if possible, a no-effect level of the test article following daily oral (gavage) administration to the Sprague-Dawley rat for 4 consecutive weeks.

For that purpose 5 males and 5 female rats per group received doses of 0, 50, 150, and 1000 mg/kg bw. at a dose volume of 10 ml/kg bw.

The control group animals received the neat vehicle (i.e. corn oil).

Mortality !morbidity checks were performed twice daily. Clinical examinations were performed daily. A full clinical examination was performed weekly. Individual body weights were recorded weekly. Food consumption was measured weekly for each cage of animals. Clinical pathology investigations were performed at termination. All animals were killed and necropsied after 4 weeks of treatment; selected organs were weighed and tissue samples were fixed and preserved at necropsy. Selected tissues from group and 4 animals killed after 4 weeks of treatment were examined histopathologically.

There were no unscheduled deaths.

All treated animals had orange fur staining. The time of onset of this sign and the intensity of the coloration were generally dose-related. The tails of high dose animals were coloured deep-orange at the end of the treatment period. Orange coloured faeces were present under the cages of treated animals from day 2. There were no other treatment- related clinical signs.

There were no differences in body weight gain between the groups which were of sufficient magnitude to indicate on effect of treatment.

Food consumption tended to be similar in all groups.

There were no differences in the haematology or clinical chemistry parameters to indicate on effect of treatment.

The weight of the organs was similar in control and treated groups.

There was no macroscopic or microscopic evidence of change associated with treatment.