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Toxicological information

Repeated dose toxicity: oral

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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:
1986
Report Date:
1986

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Version / remarks:
May 12, 1981
Qualifier:
according to
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Version / remarks:
84/449/EC
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Animal Production, Ciba-Geigy, Stein Switzerland
- Strain: Tif: RAIf
- Females (if applicable) nulliparous and non-pregnant: no data
- Housing: individually in Macrolon type 3 cages with granulated soft wood bedding
- Age at study initiation: 7-8 weeks
- Weight at study initiation: males 171-195 g; females 160-182 g
- Diet : pelleted standard diet (Nafag. 890 Tox) ad libitum (quality checks on regular basis)
- Water: drinking water ad libitum (quality check under routine investigations)
- Acclimation period: 8 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2 °C
- Humidity (%): 55 ± 10 %
- Air changes (per hr): 16-20
- Photoperiod (hrs dark / hrs light):12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: water with 0.5% CMC and 0.1% Tween 80
Details on oral exposure:
VEHICLE
- Justification for use and choice of vehicle: necessary to make suspension
- Concentration in vehicle: 1 mg/mL, 4 mg/mL, 10 mg/mL
- Amount of vehicle (if gavage): 10 mL/kg bw
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
LC with UV detection:
pump: Perkin Elmer Series 4
sampler: Perkin Elmer ISS 100
detector: Kratos Spectroflow 773 (λ = 250 nm)
integrator: Spectra-Physics (SP 4270) 5 mm/min
Column: Hypochrome (250 mm Ø 4.6 mm) packed with Nucleosil C18 (5 um)
Mobile phase: acetonitrile/acetate buffer (22/78%)
column temperature: 40 °C
flow rate: 2.0 mL/min
substance retention time: 3.6 min

Accuracy
method suitable between 1 and 15 mg/L (no recovery samples reported)

Stability
stability over a 4 hr period is measured at 1 mg/mL and 50 mg/mL in samples prepared at nominal concentrations in the vehicle and diluted after 4 hours with methanol (final nominal concentration 0.4 and 40 mg/L) . Thereafter the samples were measured (standard 4 mg/L).
stability : 91-92% at 1 mg/ml and 100.2-100.8% at 50 mg/mL

In the tests for accuracy of preparation (on day 1 and 19) the results after storage at room temperature for (17 days and 1 day) were 1.5 and 68% at 1 mg/mL; 12.0 and 87.5% at 4 mg/mL; 54.4 and 93.6% at 15 mg/mL. When stored on dry ice for 1 day all samples (taken on day 23 and 28) were within 84.5 to 98.5% of nominal
Duration of treatment / exposure:
28 days
Frequency of treatment:
daily
Doses / concentrationsopen allclose all
Dose / conc.:
10 mg/kg bw/day (nominal)
Remarks:
measured concentration when stored on ice 84.5-92.5% of nominal
Dose / conc.:
40 mg/kg bw/day (nominal)
Remarks:
measured concentration when stored on ice 89.8-91.8% of nominal
Dose / conc.:
150 mg/kg bw/day (nominal)
Remarks:
measured concentration when stored on ice 95.3-98.5% of nominal
No. of animals per sex per dose:
5 males and 5 females
Control animals:
yes, concurrent vehicle
Details on study design:
standard 28 day study
Positive control:
NA

Examinations

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

DETAILED CLINICAL OBSERVATIONS: No outside cage examinations

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION: Yes
- Time schedule: weekly

WATER CONSUMPTION: Yes
- Time schedule: weekly

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: pre-test and on day 27
- Dose groups that were examined: controls and high dose group

HAEMATOLOGY: Yes
- Time schedule for collection of blood: on day 28
- Anaesthetic used for blood collection: Yes (ether)
- Animals fasted: Yes for 18 hours
- How many animals: all
- Parameters checked: White blood cells, Differential leucocyte count (neutrophils, lymphocytes, monocytes, eosinophils, basophils, plasma cells), Red blood cells (including morphology), Reticulocytes, Red blood cell distribution width Haemoglobin, Haematocrit, Mean corpuscular volume, Mean corpuscular haemoglobin, Mean corpuscular haemoglobin concentration, Heinz bodies, Platelets and PT


CLINICAL CHEMISTRY: Yes / No / Not specified
- Time schedule for collection of blood: on day 28
- Anaesthetic used for blood collection: Yes (ether)
- Animals fasted: Yes for 18 hours
- How many animals: all
- Parameters checked: Alanine aminotransferase, Aspartate aminotransferase, Alkaline phosphatase, Total Protein, Albumin, Globulin, A/G ratio, Urea, Glucose, Cholesterol, Sodium, Potassium, Calcium, Inorganic Phosphate

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No
Sacrifice and pathology:
ORGAN WEIGHTS: from all animals:
Adrenal glands, Spleen, Brain, Testes, Thymus, Heart, Kidneys, Liver, Ovaries

GROSS PATHOLOGY: in all animals:
Skin, mammary gland area, spleen, mesenteric lymphnode, axillary lymphnode, popliteal lymphnode, sternum with bone marrow, femur with joint, skeletal muscle, trachea, lung, heart, aorta, submandibular salivary gland, liver, pancreas, oesophagus, stomach, small intestine, large intestine, kidney, urinaty bladder, prostate, seminal vesicle, testis, epididymus, vagina, uterus, ovary, pituitary gland, adrenal gland, thyroid with parathyroid gland, thymus, peripheral nerve, brain, spinal cord, eye with optic nerve, orbital gland, extra orbital lacrimal gland, zymbal gland, muzzle with tongue

HISTOPATHOLOGY: in control and high dose animals:
Skin, mammary gland area, spleen, mesenteric lymphnode, axillary lymphnode, sternum with bone marrow, femur with joint, skeletal muscle, trachea, lung, heart, aorta, submandibular salivary gland, liver, pancreas, oesophagus, stomach, small intestine, large intestine, kidney, urinaty bladder, prostate, seminal vesicle, testis, epididymus, uterus, ovary, pituitary gland, adrenal gland, thyroid with parathyroid gland, thymus, peripheral nerve, brain, spinal cord, eye with optic neree, orbital gland, extra orbital lacrimal gland

in animals dosed at 15 and 40 mg/kg bw: liver, spleen, kidneys, testes and epididymus

For kidneys in addition a PAS screen was included.
Statistics:
included uni-variate statistical analytics (Jonckhere 1954, Lepage, 1971)

Results and discussion

Results of examinations

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
small wounds due to scratching
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
no effects observed
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
Females at 150 mg/kg bw: sign. increased numbers of Heinz bodies, sign decreased number of erythrocytes and haemoglobin, sign increased MCV and MCH, sign increase of number of reticulocytes
Females at 40 and 150 mg/kg bw: non-treatment related changes in white blood cells
Males at 150 mg/kg bw: sign increase of number of reticulocytes
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Females at 150 mg/kg bw: increased ALAT and increased glucose
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Males at 40 and 150 mg/kg bw: increased spleen weights (abs and rel to BW), increased kidney weights (abs)
Females at 150 mg/kg bw: increased spleen weights (abs and rel to BW)
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Males at 150 mg/kg bw:
spleen: congestion and (extramedulary) hemosiderosis increased (incidence and severity) -> dose related
kidneys: eosinic bodies in the proximal tubulus
epididymus: cellular debris and spermatic granuloma
testis: Leydig cell hyperplasia
Males at 40 mg/kg bw:
spleen: congestion and (extramedulary) hemosiderosis increased (incidence and severity) -> dose related
kidneys: eosinic bodies in the proximal tubulus
epididymus: cellular debris in 1/5 males

Females at 150 mg/kg bw
spleen: congestion and (extramedulary) hemosiderosis increased (incidence and severity) -> dose related
Liver: hemosiderosis
Females at 40 mg/kg bw:
spleen: congestion and (extramedulary) hemosiderosis increased (incidence and severity) -> dose related
Histopathological findings: neoplastic:
not examined

Effect levels

Key result
Dose descriptor:
NOAEL
Effect level:
10 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
haematology
histopathology: non-neoplastic
Remarks on result:
other: the effects on the testes were observed at the same dose levels, thus the derived NOAEL also protects for these male specific effects

Target system / organ toxicity

open allclose all
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
40 mg/kg bw/day (nominal)
System:
haematopoietic
Organ:
spleen
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
yes
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
150 mg/kg bw/day (nominal)
System:
male reproductive system
Organ:
testes
Leydig cells
cauda epididymis
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
yes

Applicant's summary and conclusion

Conclusions:
The NOAEL as derived from this study is 10 mg/kg bw
Executive summary:

Rats (5/sex/dose) received the substance by gavage at 0, 10, 40 and 150 mg/kg bw during 28 days. No treatment related effects were reported related to mortality, clinical signs, body weight, food and water consumption, clinical chemistry and macroscopy. Effects seen were related to the the maturation of red blood cells (reduction of number of red blood cells, reduction of hemoglobin, increased number of reticulocytes, increased spleen weights and spleen congestion and (extramedulary) hemosiderosis) at 40 and 150 mg/kg bw in both males and females. In addition eosinophylic bodies were found in the kidneys of males at 40 and 150 mg/kg bw. The male reporductive systenm (testes (hyperplasia of Leydig cells) and epididymus (debris and granuloma)) was also affected at 150 mg/kg bw (and very slightly at 40 mg/kg bw).

Based on these findings the NOAEL for males and females is considered to be 10 mg/kg bw.