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Toxicological information

Repeated dose toxicity: oral

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Administrative data

sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference Type:
study report

Materials and methods

Test guideline
according to guideline
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
GLP compliance:
yes (incl. QA statement)
Limit test:

Test material

Constituent 1
Chemical structure
Reference substance name:
EC Number:
EC Name:
Cas Number:
Molecular formula:

Test animals

Details on test animals or test system and environmental conditions:
- Wistar Han rats with appropriate bodyweight at study start.
- Source: Charles River, Wilmington MA, USA
- Age at study initiation (treatment start): 7-9 weeks.
- Assignment to dose groups: Randomization by a computer-generated random algorithm
- Weight at study initiation (treatment start): Males: minimum 217 g, maximum 247 g,
Females: minimum 158 g, maximum 180 g.
- Housing: In a single experimental room, in groups of 2/cage by sex in polysulfone cages with stainless steel top grill.
During urine collection individual housing in special cages (overnight, water allowed, prior to sacrifice)
- Bedding material: steam sterilized clean corn cob
- Diet (ad libitum)*: Pelleted Teklad Certified Global 14% Protein rodent maintenance diet, Harlan, 5800 AN Venray, The Netherlands
- Water (ad libitum): Deep bore-well water passed through activated charcoal and exposed to UV rays
- Acclimation period: 5 days before treatment start, after clinical examination for health and suitability of all animals.
- Animal wellfare: AAALAC-approved laboratory in accordance with the USA Animal Protection Law.

Routine analysis of the batch of diet used and of water produced results within acceptable ranges.

Animals were fasted overnight (water was available ad libitum) prior to blood sampling for haematology and clinical biochemistry at the end of the treatment period).

ENVIRONMENTAL CONDITIONS (continuously monitored)
The animal room was maintained at (target ranges for temperature and relative humidity):
- Temperature (°C): 19 - 24°C
- Relative Humidity (%): 57 - 67 %
- Photoperiod (artificial lighting): 12 hrs day / 12 hrs night
- Rate of air exchange: 13 to 14 changes/h

Administration / exposure

Route of administration:
oral: gavage
polyethylene glycol
PEG 300
Details on oral exposure:
- Concentration in vehicle: The concentration of the test material in vehicle varied between dose groups thus allowing constant dosage volume in terms of mL/kg bw/day. Concentrations amounted to 15, 30, and 100 mg/ml vehicle, accordingly.

- Amount (dose volume by gavage): 10 mL/kg bw/day.

- Dosing formulations were prepared fresh weekly.
Analytical verification of doses or concentrations:
Details on analytical verification of doses or concentrations:
For analysis of the test substance in the vehicle GC / FID was used with a DB-35 column (30 m long, 0.53 mm i.d., 0.5 μm film thickness). The analytical method was validated. The mean contents of the test substance in dose formulations were found to be well within the acceptance limit (within +/- 15%) of dose theoretical concentration; the relative standard deviation was equal to or less than 10%.
Duration of treatment / exposure:
Animals were dosed for 90 consecutive days.
Frequency of treatment:
Once daily
Doses / concentrationsopen allclose all
Dose / conc.:
150 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
10 animals
Control animals:
yes, concurrent vehicle
Details on study design:
- dose selection was based on the result of the 28 day study
- study design was according to OECD 408 guideline
Positive control:
not applicable


Observations and examinations performed and frequency:
The following observations and examinations were performed on all animals:
- Mortality/morbidity: twice daily
- General clinical observations: twice daily (pre and post dose) during the first 26 days, then three times daily on Treatment Days 27-90.
- Detailed clinical observations (in their home cages, in standard arena outside the home cage and in the hand, in random sequence):
once before treatment start and once per week until Week 13; in Week 13 observation was carried out twice.
- Functional observation battery (FOB)*: During treatment week 12.
- Body weights: Weekly and before necropsy
- Food consumption: Weekly
- Haematology/Clinical chemistry
(after overnight fasting with water available ad libitum)** , all surviving animals: At completion of the treatment period (Day 91).

* FOB including sensory reactivity tests (reflexes), appearance, behavioural, respiratory and a number of other relevant parameters from a modified
Irvin screen, landing hindlimbs footsplay, fore and hindlimb grip strength and locomotor activity (over 60 minutes).

**Hematology parameters examined:
Red blood cell count, hemoglobin concentration, hematocrit value, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, platelet count, reticulocyte count, white blood cell count, differential leukocyte counts, prothrombin time, activated partial thromboplastin time.

**Blood chemistry parameters examined:
Glucose, urea, creatinine, total bilirubin, total cholesterol, triglycerides, aspartate aminotransferase, alanine aminotransferase, lactate dehydrogenase, glutamate dehydrogenase, creatine kinase, alkaline phosphatase, sodium, potassium, chloride, calcium, inorganic phosphorus, total protein, albumin, globulin.
Sacrifice and pathology:
All surviving animals were killed on Day 91 after 90 consecutive days of oral (gavage) treatment.
- Sacrifice and gross pathology, i.e. full macroscopic external and internal examination with tissue collection, on all animals.
- Organs Weights: Brain, heart, liver, spleen, thymus, adrenals, kidneys, testes, epididymides, ovaries, lungs, pituitary, prostate, seminal vesicles and coagulating glands, thyroid and parathyroid, uterus with cervix.

- Histopathology on the vehicle control and 1000 mg/kg bw/day groups (organs microscopically examined):
Adrenal glands; Bone marrow; Brain [cerebrum, cerebellum, pons, medulla oblongata]; Caecum; Colon; Diaphragm; Duodenum; Epididymides; Esophagus; Eyes with optic nerve; Femoral muscles; Femur bone with joint; Heart; Ileum with Peyer's patches; Jejunum; Kidneys; Liver; Lungs; Lymph nodes – mandibular, mesenteric; Ovaries; Pancreas; Prostate gland; Pituitary; Preputial gland; Rectum; Salivary gland; Sciatic nerve; Seminal vesicles; Skin; Spinal cord -cervical, midthoracic, lumbar; Spleen; Sternum with marrow; Stomach; Testes; Thymus; Thyroid including parathyroid; Trachea; Urinary bladder; Uterus with cervix; Vagina; gross lesions.
Other examinations:
The Data captured using Provantis™ for the parameters namely body weights, food consumption and organ weights were analyzed using built-in statistical tests. Derived data like net body weight change and organ weight ratios were also analyzed using above mentioned methods.
The statistical analysis of the experimental data captured other than Provantis™, was carried out using the developed and validated package in Excel and/or using licensed copies of SYSTAT Statistical package Ver.12.0. All quantitative variables like neurological observations (neuromuscular observation and body temperature) and clinical pathology (haematology, coagulation and clinical chemistry) data was tested for normality and homogeneity of variances (Levene’s test) within the group before performing a one-factor ANOVA modeling by treatment groups. Comparison of means between treatment groups and control group was done using Dunnett’s test when the overall treatment, ‘F’ test is found to be significant.
All analyses and comparisons were evaluated at the 5% (P<0.05) level.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
semi-solid feces in 8/10 males at 1000 mg/kg/day on few occasions in the first week considered as adverse effect.
mortality observed, non-treatment-related
Description (incidence):
One female of high dose group found dead on Day 68; death was due to misdosing / intubation error.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Significantly lower mean body weights and body weight gains were observed during several weeks in males and females at 300 and 1000 mg/kg bodyweight/day. See attached Tables and Figures in "Attached background material"
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Statistically significantly lower food consumption was observed in males during weeks 1, 8 and 12 and in females during weeks 1 and 8 at 1000 mg/kg Bwt/day.See attached Tables in "Attached background material"
Food efficiency:
effects observed, treatment-related
Description (incidence and severity):
Food efficiency was not determined in the study. However, calculation of food efficiency based on available data indicates a clear reduction of efficiency with increasing dose.
Water consumption and compound intake (if drinking water study):
not examined
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:
Weights of male and female reproductive organs did not show significant differences between control and dosed animals up to and including the highest dose level (see attached Tables on organ weights in "Attached background material"

Effect levels

Key result
Dose descriptor:
Effect level:
> 150 - < 300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
body weight and weight gain

Target system / organ toxicity

Key result
Critical effects observed:
Lowest effective dose / conc.:
300 mg/kg bw/day (actual dose received)
other: body weight gain
other: body weight gain
Treatment related:
Dose response relationship:

Applicant's summary and conclusion

In this well performed sub-chronic oral toxicity study in rats no systemic toxic effects to organs or blood parameters were observed. Also no effects on neurobehaviour were observed. The only significant effects observed were related to body weight and body weight gain in males and females of the mid and high dose groups. Based on these observations the NOAEL was derived at the low dose group, i.e. 150 mg/kg bodyweight/day.