Registration Dossier

Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
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 guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Version / remarks:
Version 27 Jul 1995
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Version / remarks:
Version 30 May 2008
Deviations:
no
Qualifier:
according to
Guideline:
other: Japan/MHW: Guidelines for Toxicity Testing of Chemicals, twenty-eight-day repeated dose toxicity in mammalian species; MITI/MHW, 1987 (Translation), pp. 183-188
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
- Identifier: CAS 80675-49-6
- Lot/batch No.: L-7015-20
- Yellow powder
- Storage conditions: Ambient temperature, no protection from light necessary
Specific details on test material used for the study:
- Identifier: CAS 80675-49-6
- Lot/batch No.: L-7015-20
- Analytical purity: 95%
- Yellow powder
- Storage conditions: Ambient temperature, no protection from light necessary

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Wistar (Crl:WI[Han]) rats from Charles River Laboratories, Research Models and Services, Germany GmbH, 97633 Sulzfeld, Germany
- Age at study initiation: 36 ± 1 days at arrival, 42 ± 1 days at the start of the administration period (day 0).
- Housing:
Housed together (5 animals per cage) in H-Temp polysulfonate cages (TECNIPLAST, Hohenpeißenberg, Germany). Floor area about 2065 cm².
Motor activity measurements were conducted in polycarbonate cages with wire covers (Ehret, Emmendingen, Germany). Floor area about 800 cm².
- Diet: ad libitum, ground Kliba maintenance diet mouse/rat “GLP”, meal (Provimi Kliba SA, Kaiseraugst, Switzerland)
- Water: ad libitum, drinking water (from water bottles)
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24
- Humidity (%): 30 - 70
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12 / 12

Administration / exposure

Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
DIET PREPARATION
- Rate of preparation of diet: the test substance preparations were usually mixed weekly.
- Mixing appropriate amounts with: for each concentration, the test substance was weighed out and mixed with a small amount of food. Then corresponding amounts of food, depending on test group, were added to this premix in order to obtain the desired concentrations. Mixing was carried out for about 10 minutes in a laboratory mixer.
- Storage temperature of food: room temperature
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The stability of the test substance in the diet for a period of 10 days was proven before the start of the administration period (HPLC). The concentration control analyses of all concentrations revealed that the values were in the range of the target concentrations, i.e. were always in a range of about 91.8 - 110.6 % of the nominal concentrations.
Duration of treatment / exposure:
4 weeks
Frequency of treatment:
daily in the diet
Doses / concentrationsopen allclose all
Dose / conc.:
1 500 ppm
Dose / conc.:
5 000 ppm
Dose / conc.:
15 000 ppm
Dose / conc.:
108 mg/kg bw/day (actual dose received)
Remarks:
♂ other: calculated mean daily intake (analytical)
Dose / conc.:
373 mg/kg bw/day (actual dose received)
Remarks:
♂ other: calculated mean daily intake (analytical)
Dose / conc.:
1 160 mg/kg bw/day (actual dose received)
Remarks:
♂ other: calculated mean daily intake (analytical)
Dose / conc.:
102 mg/kg bw/day (actual dose received)
Remarks:
♀ other: calculated mean daily intake (analytical)
Dose / conc.:
342 mg/kg bw/day (actual dose received)
Remarks:
♀ other: calculated mean daily intake (analytical)
Dose / conc.:
1 126 mg/kg bw/day (actual dose received)
Remarks:
♀ other: calculated mean daily intake (analytical)
No. of animals per sex per dose:
5 (low and mid dose groups) or 10 (control and high dose groups)
Control animals:
yes, plain diet
Details on study design:
- Rationale for selecting satellite groups: recovery + detection of possible late effect
- Post-exposure recovery period in satellite groups: maintained for another two weeks with ground diet (control and high dose groups, 5 animals per dose per sex)
Positive control:
None

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: performed in all animals prior to the administration period and weekly thereafter. The findings were ranked according to the degree of severity, if applicable. The animals were transferred to a standard arena (50 x 37.5 cm with sides of 25 cm high).
The following parameters were examined: abnormal behaviour during “handling”, fur, skin, posture, salivation, respiration, activity/arousal level, tremors, convulsions, abnormal movements, impairment of gait, lacrimation, palpebral closure, exophthalmus, faeces (appearance/consistency), urine and pupil size.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: a check for moribund and dead animals was made twice daily on working days and once daily on saturdays, sundays and public holidays. If animals were in a moribund state, they were sacrificed and necropsied. All animals were checked daily for any clinically abnormal signs. Abnormalities and changes were documented for each animal.

BODY WEIGHT: Yes
- Time schedule for examinations: before the start of the administration period in order to randomize the animals, then on day 0 (start of the administration period) and weekly thereafter. The difference between the body weight on the respective day of weighing and the body weight on day 0 was calculated as body weight change.

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Yes

WATER CONSUMPTION: Yes

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at the end of the administration period. At the end of the recovery period only platelet counts were measured in the surviving males.
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals: all
- Parameters were examined: leukocyte count (WBC), erythrocyte count (RBC), haemoglobin (HGB), haematocrit (HCT), mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC), platelet count (PLT), differential blood count, reticulocytes, prothrombin time. Furthermore differential blood smears were prepared and stained according to Wright without being evaluated.

CLINICAL CHAEMISTRY: Yes
- Time schedule for collection of blood: at the end of the administration period.
- Animals fasted: Yes
- How many animals: all
- Parameters examined: alanine aminotransferase (ALT; L-alanine: 2-oxoglutarate aminotransferase), aspartate aminotransferase (AST; L-aspartate: 2-oxoglutarate aminotransferase), alkaline phosphatase (ALP; orthophosphoric acid monoester phosphohydrolase), γ-Glutamyltransferase (GGT; (γ -glutamyl) peptide: aminoacid-γ-glutamyl-transferase), sodium (NA), potassium (K), chloride (CL), inorganic phosphate (INP), calcium (CA), urea (UREA), creatinine (CREA), glucose (GLUC), total bilirubin (TBIL), total protein (TPROT), albumin (ALB), globulins (GLOB), triglycerides (TRIG), cholesterol (CHOL), magnesium (MG) and bile acids (BILE).

URINALYSIS: Yes
- Time schedule for collection of urine: at the end of the administration period.
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters examined: pH, proteins, ketones, glucose, urobilinogen, bilirubin, blood, specific gravity, sediment, color, turbidity, volume

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: at the end of the administration period.
- Dose groups that were examined: all
- Battery of functions tested: Functional observational battery (FOB; home cage observations, open field observations, sensorimotor tests/reflexes) and motor activity assessment.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
The animals were sacrificed by decapitation under isoflurane anaesthesia. The exsanguinated animals were necropsied and assessed by gross pathology. Weight assessment was carried out on all animals sacrificed and the following weights were determined: anesthetized animals, adrenal glands, brain, epididymides, heart, kidneys, liver, ovaries, prostate, and seminal vesicles with coagulation glands, spleen, testes, thymus, thyroid glands, and uterus with cervix.

HISTOPATHOLOGY: Yes
The following organs/tissues were preserved in neutral buffered 4 % formaldehyde or modified Davidson’s solution: all gross lesions, adrenal glands, aorta, bone marrow (femur), brain, caecum, cervix, colon, duodenum, epididymides, oesophagus, extraorbital lacrimal glands, eyes with optic nerve, femur with knee joint, heart, ileum, jejunum, kidneys, larynx, liver, lungs, lymph nodes (mesenteric and axillary lymph nodes), mammary gland (males and females), nose (nasal cavity), ovaries, oviducts, pancreas, parathyroid glands, pharynx, pituitary gland, prostate, rectum, salivary glands (mandibular and sublingual glands), seminal vesicle with coagulation glands, sciatic nerve, skeletal muscle, skin, spinal cord (cervical, thoracic and lumbar cords), spleen, sternum with marrow, stomach (fore-stomach and glandular stomach), testes (modified Davidson’s solution), thymus, thyroid glands, trachea, urinary bladder, uterus and vagina. From the liver, each one slices of the Lobus dexter medialis and the Lobus sinster lateralis was fixed in Carnoy’s solution and embedded in paraplast. Fixation was followed by histo-technical processing, examination by light microscopy and assessment of findings.
Statistics:
Two-sided DUNNETT's test (body weight, body weight change, food consumption, food efficiency); t-test (two-sided) for the hypothesis of equal means (body weight and body weight change); Non-parametric one-way analysis using KRUSKAL-WALLIS test (faeces, rearing, grip strength length forelimbs, grip strength length hind limbs, foots play test, motor activity, clinical pathology parameters, urine volume, urine specific gravity, weight parameters at pathology); pair wise comparison of each dose group with the control group using FISHER's exact test for the hypothesis of equal proportions (Urinalysis, except colour, turbidity, volume and specific gravity).

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Description (incidence and severity):
Yellow discoloration was observed in all animals of both sexes of test group 3 (15000 ppm), i.e. faeces and urine during the entire study period and fur from day 22 onwards, of test group 2 (5000 ppm), i.e. faeces and urine during the entire study period, and of test group 1 (1000 ppm), i.e. urine from day 7 onwards.
During the recovery period, the discoloration of faeces, urine and fur disappeared in all animals of test group 3 (15000 ppm; study day 31). This finding was clearly attributable to the ingestion and excretion of the test substance as the test substance is a dye. Its physical (tinctorial) properties do not represent a toxicologically relevant finding or adverse effect.
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Water consumption and compound intake (if drinking water study):
no effects observed
Haematological findings:
no effects observed
Description (incidence and severity):
In males of test groups 2 (5000 ppm) and 3 (15000 ppm) the platelet counts were increased dose-dependently. However, the increase was within the historical control range (741 - 1025 G/l). Therefore, it was regarded as not adverse. After a two weeks recovery period the platelet counts median was lower compared to the platelet counts median directly after the administration period.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
- In males of test group 2 (5000 ppm) total bilirubin values were higher compared to controls. This increase was not dose-dependent and within the historical control range (1.57 - 2.78 μmol/L) and was therefore regarded as not treatment-related.
- In males of test group 1 (1500 ppm) serum total bile acid levels were lower compared to controls, but not dose-dependently decreased. Therefore, these lower bile acid values were regarded as not treatment-related.
- In females of test group 1 (1500 ppm) the potassium concentration as well as the triglyceride levels were higher compared to controls. The potassium levels (4.43 mmol/L) were marginally above the historical control range (3.83 - 4.39 mmol/L), whereas the triglyceride median was within the historical control range (0.29 - 0.52 mmol/L). Both parameters were not dose-dependently altered. Therefore, the potassium as well as the triglyceride values were regarded as not treatment-related
Urinalysis findings:
no effects observed
Description (incidence and severity):
- In males of test group 2 (5000 ppm) the urine volumes was significantly decreased and the specific gravity of the urine was increased. Both parameters were not altered dose-dependently. No other urinalysis parameters were changed. Therefore, the mentioned changes reflect the capacity of the kidneys to concentrate the primary urine, when a lower volume had been produced. The alterations of these parameters were regarded as non-adverse.
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
In test group 3 (15000 ppm) rearing of female animals was significantly decreased by -37.8 %, but, with regard to non-impaired motor activity in female animals of test group 3 (15000 ppm) it was concluded that this deviation was incidental in nature and not related to the test substance.
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
The ovaries’ weight in females of test group 3 (15000 ppm) revealed a significant weight decrease (84 %) compared to controls (set to 100 %). Since ovaries were not found as target organs in the main groups (neither weight nor histo-morphological changes were detected) the weak weight decrease in the ovaries was considered to be incidental and not related to treatment, as this parameter varies during the female sexual cycle.
Gross pathological findings:
no effects observed
Description (incidence and severity):
At gross pathology, male and female animals of test group 3 (15000 ppm) showed “light green” discoloration of the contents in jejunum, ileum, caecum and colon. This finding was related to the physical characteristics of the test substance (yellow) and was therefore regarded as not adverse. No other macroscopic changes were noted in test animals.
Histopathological findings: non-neoplastic:
no effects observed
Details on results:
HISTORICAL CONTROL DATA
All data were generally within the available historical control range from the testing laboratory.

Effect levels

open allclose all
Key result
Dose descriptor:
NOAEL
Effect level:
>= 15 000 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
mortality
Dose descriptor:
NOAEL
Effect level:
1 160 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
mortality
Dose descriptor:
NOAEL
Effect level:
1 026 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
mortality

Target system / organ toxicity

Key result
Critical effects observed:
no

Applicant's summary and conclusion