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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2000
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2002

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Lot/batch No.of test material: 4879

Test animals

Species:
rat
Strain:
Sprague-Dawley
Remarks:
Crj; CD(SD)
Details on species / strain selection:
This strain was selected because this species is usually used in this kind of test and the laboratory has a high experience with this rat strain.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories Japan, Inc. Atsugi 6/27 breeding center
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 10 wks
- Weight at study initiation: Males: 335 - 386 g; Females: 208 - 249 g
- Housing: individually (except for acclimatisation and mating period)
- Diet: ad libitum, γ-ray irradiation fixed feed CRF-1 (Oriental Yeast Co., Ltd)
- Water: ad libitum, tap water
- Acclimation period: 14 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23 ± 3 (actual measured range of 20 - 24 °C)
- Humidity (%): 55 ± 10 (actual measured range of 45 - 66 %)
- Air changes (per hr): 10 to 15
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: 1% carmellose sodium aqueous solution
Remarks:
hereinafter referred to as 1% CMC-Na aqueous solution
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
After grinding the test substance in a mortar, it was weighed accurately per dose, suspended in 1% CMC-Na aqueous solution to receive a predetermined concentration, and was dispersed using an ultrasonic device.

VEHICLE
- Concentration in vehicle: 10, 100, 200 mg/mL
- Amount of vehicle (if gavage): 10 mL/kg bw
- Lot/batch no. : 9318
Details on mating procedure:
- M/F ratio per cage: 1/1
- Length of cohabitation: 1 d
- Proof of pregnancy: vaginal plug or sperm in vaginal smear
- M/F ratio per cage: 1/1
- Length of cohabitation: 14 d
- Proof of pregnancy: vaginal plug or sperm in vaginal smear referred to as day 0 of pregnancy
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged (how): individually
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
No details on the analytical measurements were given.
The analysis of the stability performed prior to administration for the prepared solutions of concentrations of 10 and 200 mg/mL, confirmed that the test substance is stable for 24 hours at room temperature and for 7 days at refrigeration. The prepared solutions were used within 7 days after preparation. Further, the uniformity of the prepared solutions of 10, 100 and 200 mg/mL was confirmed.
The concentration of the test substance in the prepared solutions of each concentration of the initial and final preparation was analysed. The content was 98.3 to 101.7 % of the prescribed concentration, and therefore was regarded as appropriate.
Duration of treatment / exposure:
Males: 46 days
Females: 42-46 days
Frequency of treatment:
Once daily
Doses / concentrationsopen allclose all
Dose / conc.:
250 mg/kg bw/day (nominal)
Dose / conc.:
500 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
12
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The doses of the test substance were set on the basis of the results of the preliminary test for the Combined Repeated Dose and Reproductive/Developmental Toxicity Screening Test of C.I. Pigment Yellow 53 in Rats (Test No. SR-9984P). In the preliminary test, the test substance was administered once a day for 14 days at doses of 0, 500, 1000, and 2000 mg/kg to five male and five female SD rats per group. No abnormalities beside yellow feces were observed. Therefore the high dose in the main test was set to 1000 mg/kg bw/d and the lower doses were set at 500 and 250 mg/kg by dividing by a common ratio of 2, for both males and females.

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily
- Cage side observations checked: mortality, behaviour, external appearance

BODY WEIGHT: Yes
- Time schedule for examinations:
Males: administration days 1, 2, 3, 5, 7, 10 and 14; then weekly until necropsy
Females: administrations days 1, 2, 3, 5, 7, 10 and 14; pregnancy days 0, 1, 3, 5, 7, 10, 14, 17 and 20 and lactation days 0, 1 and 4

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

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at necropsy
- Anaesthetic used for blood collection: Yes (ether)
- Animals fasted: Yes
- How many animals: all male animals; 6 lactating females per dosing group
- Parameters examined: Number of red blood cells (RBC), Hematocrit (Ht), Hemoglobin (Hb), Mean corpuscular volume (MCV), Mean corpuscular hemoglobin (MCH), Mean corpuscular hemoglobin concentration (MCHC), Reticulocyte count (Ret), Platelet count (Plat), White blood cell count (WBC), White blood cell percentage (Hemogram of WBC), Prothrombin time (PT), Activated partial thromboplastin time (APTT)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at necropsy
- Animals fasted: Yes
- How many animals: all male animals; 6 lactating females per dosing group
- Parameters examined: GOT, GPT, Alkaline phosphatase (ALP), γ-GTP, Glucose (Glu), Choline esterase (ChE), Total cholesterol (T-Cho), Phospholipid (PL), Triglyceride (TG), Total bilirubin (T-Bil), Urea nitrogen (UN), Creatinine (Crea), Sodium (Na), Potassium (K), Chloride (Cl), Calcium (Ca), Inorganic phosphorus (IP), Total protein (TP), A/G ratio (A/G)

URINALYSIS: Yes
- Time schedule for collection of urine: on administration days 44 - 45
- Metabolism cages used for collection of urine: Yes
- Animals fasted: No
- Parameters examined: pH, protein, glucose, ketone bodies, urobilinogen, bilirubin, occult blood, sediment, color, volume, specific gravity
Oestrous cyclicity (parental animals):
For all females, every day from 10 days prior to dosing until successful copulation, vaginal smear samples with Giemsa staining were prepared and their estrous cycle phases (proestrus, estrus, metestrus, diestrus) were determined under an optical microscope and a cycle of repeating each phase of estrous cycle two or more times at an interval of 4 to 5 days was regarded as normal. Continuation of diestrus for 7 days or more was judged to be an abnormal estrous cycle. Estrus interval was calculated from estrous cycle of each female.
Litter observations:
PARAMETERS EXAMINED
The following parameters were examined in F1:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, viability index, body weight and necroscopy

Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals (on the day after administration Day 46)
- Maternal animals: All surviving animals (until Day 5 post partum)

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated below were prepared for microscopic examination:
Brain (cerebrum, cerebellum), pituitary, thymus, thyroid (left and right), parathyroid (left and right), adrenal gland (left and right), spleen, heart, thoracic aorta, tongue, esophagus, stomach (forestomach and glandular stomach), liver, pancreas, duodenum, jejunum, ileum (including Peyer's patches), cecum, colon, rectum, larynx, trachea, lung (including bronchial), kidney (left and right), bladder, testes (left and right), epididymis (left and right), prostate, seminal vesicles (including coagulation glands, left and right), eye (left and right), harderian gland (left and right), skin (right abdomen), sternum (including bone marrow), femur (including bone marrow, right), spinal cord (neck), skeletal muscle (right thigh), mesenteric lymph node, mandibular lymph nodes (left and right), submandibular gland (left and right), sublingual gland (left and right), parotid gland (left and right) and the sciatic nerve (right).

The following organs were weighed:
Brain, heart, liver, kidney (left and right), spleen, adrenal glands (left and right), thymus, testis (left and right) and epididymis (left and right), ovary (left and right)
Postmortem examinations (offspring):
SACRIFICE
- The offspring was sacrificed at 4 days of age.
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows:

GROSS NECROPSY
- Gross necropsy consisted of external examination (including the inside of the mouth) and internal examinations including the cervical, thoracic, and abdominal viscera
Statistics:
Please refer to "Any other information on materials and methods incl. tables"
Reproductive indices:
Estrous cycle, copulation index (number of pairs with successful copulation/number of pairs mated X 100), fertility index (number of pregnant animals/number of pairs with successful copulation X 100), gestation index (number of females with live pups/number of living pregnant females X 100), gestation length, nursing index, number of pregnant females, corpora lutea and implantation sites, implantation index (number of implantation sites/number of corpora lutea X 100), delivery index (number of pups born/number of implantation sites X 100),
Offspring viability indices:
Number of live pups on day 4/number of live pups on day 0 X 100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Males: After administration day 2 until the necropsy day, yellowish brown feces in the 500 mg/kg group, and yellow or yellowish brown feces in the 1000 mg/kg group were observed in all of 12 rats of each group. In addition to this, fracture of incisor teeth was observed in one animal of the 250 mg/kg group on administration day 42, but this was considered accidental.

Females: After administration day 2 until the necropsy day, yellowish brown feces in the 500 mg/kg group, and yellow or yellowish brown feces in the 1000 mg/kg group were observed in all 12 rats of each group. In addition, alopecia was observed in one female in the control group after day 12 of pregnancy (5 days after delivery) until the necropsy day. The scab-formation on the back in the 250 mg/kg group and the trauma and scab-formation in the lower eyelid in the 500 mg/kg group were observed each in one female. These effects were not considered as toxicologically relevant.
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):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Males: In the 250 and 1000 mg/kg groups, a significant shortening of the activated partial thromboplastin time (APTT) was observed.
Females: In the 250 mg/kg group, significant high values of mean corpuscular hemoglobin (MCH) and platelet were observed, but these changes were not observed in the higher dosing groups.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
Males: A significant low value of potassium (K) was observed in the 1000 mg/kg group, but it was considered to have no toxicological significance.
Females: Compared with the control group, a significant low value of cholinesterase (ChE) in the 250 mg/kg group, a significant low value of sodium (Na) in the 500 mg/kg group, and a significant low value of GPT in the 1000 mg/kg group were observed, but, since there were no changes found in the related organs, this was considered as non treatment related.
Urinalysis findings:
effects observed, non-treatment-related
Description (incidence and severity):
In the 250 mg/kg group, a significant increase was observed in urine specific gravity, but this was not observed in the higher dosing groups. No other urine parameter was altered compared to the control group.
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
Males: In the control group, myocardial degeneration of the heart, hyaline cast of the kidney, eosinophilic bodies of the proximal tubule epithelium, atrophy of the seminiferous tubules of testes and sperm granuloma of the epididymis were observed each in one male. Lymphocyte infiltration of the prostate was found in six other males. In the 1000 mg/kg group, myocardial degeneration of heart in three males, eosinophilic bodies of the proximal tubule epithelium in one male, atrophy of the seminiferous tubules of testes in three males, intraluminal cell debris of the epididymis in one male, and lymphocyte infiltration of the prostate in four males were observed.
No other significant differences were observed compared to the control group.
Females: In the control group, erosion of the tongue was observed in three animals, and focal necrosis of the liver, kidney cysts, dilatation of the renal pelvis, dilatation of the renal tubules, focal fibrosis of the kidney, mineralization in the cortico-medullary junction of the kidney, granuloma of the cervix, atrophy of the thymus, atrophy of the skin hair follicles and inflammation of the mammary gland were observed each in one case. In the 1000 mg/kg group, erosion of the tongue was observed in one animal, and mineralization in the cortico-medullary junction of the kidney was observed in three females. In addition, the atresia of vagina and the inflammation of the uterine horn were observed in one animal in which the atresia of vagina was seen at necropsy (infertility example). The atresia of vagina was a congenital anomaly and it was considered as the cause of infertility.
As for all the above changes, no significant difference was observed in the frequency of appearance, as compared to the control group.
Histopathological findings: neoplastic:
not examined
Other effects:
not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
not examined
Reproductive performance:
effects observed, non-treatment-related
Description (incidence and severity):
As for the incidence of females with normal estrous cycle, estrus interval, copulation index, fertility index, gestation index, length of gestation, and nursing index on lactation day 4, there was no significant difference observed in each dose group, compared to the control group. Unsuccessful copulation was observed in the 250 and 500 mg/kg groups, each in one pair. In the estrous cycle inspection of females of these pairs, continuous diestrus was observed. But from the fact that it was not observed in the 1000 mg/kg group, it was not considered as substance related. One non-pregnant female was observed in the 1000 mg/kg group. For this female, the atresia of vagina was observed in the pathological examination. This was assumed to cause infertility and it was considered to be the cause of infertility.
As for the number of corpora lutea, number of implantation sites, implantation index, total number of pups born, the sex ratio of pups born, number of live pups on lactation day 0, live birth index, number of live pups on lactation day 4, newborn viability index, there was no significant difference observed in each administration group, compared with the control group. Delivery index was significantly decreased in the 250 mg/kg group, but this was considered as an incidental variation since there was no significant difference observed in the dose groups of 500 mg/kg or more.

Effect levels (P0)

Key result
Dose descriptor:
NOAEL
Effect level:
>= 1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects observed in the parental generation.

Target system / organ toxicity (P0)

Key result
Critical effects observed:
no

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Except for the scab observed in one male of the 1000 mg/kg group, there was no abnormality observed in any of the dead pups.
Yellowish white portion of the liver was observed in one male of 250 mg/kg group and in one male of the 1000 mg/kg group, for the pups necropsied on day 4 of lactation. Trauma was noted for one female of the 500 mg/kg group. But no other abnormalities were observed.
Histopathological findings:
not examined

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Details on results (F1)

No effects observed in the offspring.

Effect levels (F1)

Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
>= 1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No effects observed in the offspring.

Target system / organ toxicity (F1)

Key result
Critical effects observed:
no

Overall reproductive toxicity

Key result
Reproductive effects observed:
no

Applicant's summary and conclusion