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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Remarks:
based on test type (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
no data
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2001

Materials and methods

Test guideline
Qualifier:
according to guideline
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

Constituent 1
Chemical structure
Reference substance name:
Methacrylonitrile
EC Number:
204-817-5
EC Name:
Methacrylonitrile
Cas Number:
126-98-7
Molecular formula:
C4H5N
IUPAC Name:
methacrylonitrile
Details on test material:
- Name of test material (as cited in study report): 2-methyl-2-propenenitrile (MAN)
- Substance type: Colourless liquid at room temperature
- Physical state: Liquid
- Analytical purity: 99 %
- Stability under test conditions: Tested by the provider of the test material
- Storage condition of test material: In a hermetic container, shield from the light at 4 - 10 degrees Centigrade

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Crj:CD(SD)IGS rat, Charles River (Japan) Ltd, Atsugi, Japan
- Age at study initiation: 10 weeks
- Weight at study initiation: Mean of 387.6 g (348 to 422 g) for males, mean of 234.3 g (212 to 260 g) for females
- Housing: Initially, all animals were housed individually in metal wire netting cages (260W x 380D x 180H, mm). During the mating phase, animals were transferred to cages on a one male: one female basis within each dose group. Mated females were housed individually during gestation and lactation., with white flakes (Charles River (Japan) Ltd).
- Diet: CRF-1 (Lot No. of 990109, 990203, Oriental Yeast Co., Ltd.), ad libitum. The diet was analysed by Japan Food Research laboratories and Oriental Yeast Co., Ltd and thye were considered not to contain any contaminants that would exceed the standard levels of contaminants.
- Water: Mains water (Sapporo-shi, Hokkaido, Japan), ad libitum. The mains water was analysed by Nihoneisei Co., Ltd and were considered not to contain any contaminants that would exceed the standard levels of contaminants.
- Acclimation period: 14 days.

ENVIRONMENTAL CONDITIONS
- Temperature: 23 ± 3degrees Centigrade (21 -26 degrees Centigrade)
- Humidity: 55 ± 10 % (37 - 62 %).
- Air changes: 10 to 15 changes per hour
- Photoperiod (hrs dark / hrs light): Twelve hours continuous light (08:00 to 20:00) and 12 hours darkness.


IN-LIFE DATES: From Day 0 to Day 47 (males); Day 0 to Day 5 post partum (females) or to Day 53 (females with unsuccessful mating).

Administration / exposure

Route of administration:
oral: gavage
Type of inhalation exposure (if applicable):
other: not applicable
Vehicle:
olive oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: The test material was prepared at 7.5, 15 and 30 mg/kg/day as a suspension in olive oil. The stability and homogeneity of the test material formulations were determined at the test laboratory. The formulations to be stable for 3 hours at room temperature and 8 days at 14 degrees Centigrade in the dark. Formulations were therefore prepared at least once every 8 days and kept in a hermetic container, shield fand stored at cool temperature in the dark.

VEHICLE
- Concentration in vehicle: 1.5, 3 and 6 mg/mL.
- Amount of vehicle (if gavage): 5 mL/kg.
- Lot/batch no.: 811152, 812143, 810152 and 808128 (Yakuhan Pharmaceutical Co., Ltd., Japan).
Details on mating procedure:
- M/F ratio per cage: 1:1.
- Length of cohabitation: Up to 14 days.
- Proof of pregnancy: The presence of sperm within the vaginal smear and/or vaginal plug in situ was taken as positive evidence of mating (Day 0 of gestation).


Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The dosing solution was tested for the initial and final administration at each concentration. It was identified to be 97.7 - 103 % of nominal concentrations and considered to be stable under test conditions.
Duration of treatment / exposure:
From Day 0 To: Day 46 (males); Day 0 To: Day 4 post partum (females) or To: Day 52 (females with unsuccessful mating).

Frequency of treatment:
Once daily.
Details on study schedule:
MATING
Animals were 12 weeks old at the time of mating and were paired on a 1 male: 1 female basis within each dose group on Day 14, for a period of up to 14 days. The presence of sperm within the vaginal smear and/or vaginal plug in situ was taken as positive evidence of mating (Day 0 of gestation).

PARTURITION AND LITTER RESPONSES
The following was recorded for each mated female at 9:00 on Day 21 of pregnancy to the end of parturition: stage of parturition, condition of nursing, number of live/dead offspring, sex of live offspring and external observation. Day 0 of the post partum period was assumed when parturition had completed by 9:00am.
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
7.5 mg/kg/day
Basis:
other: nominal in vehicle
Remarks:
Doses / Concentrations:
15 mg/kg/day
Basis:
other: nominal in vehicle
Remarks:
Doses / Concentrations:
30 mg/kg/day
Basis:
other: nominal in vehicle
No. of animals per sex per dose:
12 animals per sex per dose and the control group.
Control animals:
yes, concurrent vehicle
Details on study design:
The dose levels were chosen based on the results of a preliminary study performed as part of this investigation plus the preliminary study for the acute oral study (Study number SR-9874, Safety Research Institute for Chemical Compounds Co., Ltd, 1999).
Positive control:
No data

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS / ETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once a day for all animals.
- Cage side observations checked: External observation and palpation for behavioural change and general conditions.


BODY WEIGHT: Yes
- Time schedule for examinations: Days 1, 2, 5, 7, 10 and 14 of treatment (before dosing) for males and females, thereafter once a week and at autopsy for males, or at days 0, 1, 3, 5, 7, 10, 14, 17 and 20 of gestation period and at days 0, 1 and 4 of lactation period and at autopsy for females. Females with unsuccessful mating were examined on days 28, 35, 42 and 49 (before dosing) and at autopsy.


FOOD CONSUMPTION: Yes
- Time schedule for examinations: At days 1, 2, 5, 7, 10 and 14 of treatment (before dosing) for males and females, thereafter once a week for males except mating period, or at days 1, 3, 5, 7, 10, 14, 17 and 20 of gestation period and at days 1 and 4 of lactation period for females.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: On Day 46 for males and on Day 5 post partum for females.
- Anaesthetic used for blood collection: Yes, with ether. Blood samples were obtained from the abdominal aorta. Plasma and serum were discarded after the assay.
- Animals fasted: Yes, 16 - 22 hours before autopsy.
- How many animals: All males and 6 females per group.
- Parameters checked: RBC, Ht, Hb, MCV, MCH, MCHC, ret, Plat, WBC and hemogram of WBC, blood was treated with EDTA.2K. PT and APTT, blood was treated with 3.8 % sodium citrate.


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: On Day 46 for males and on Day 5 post partum for females.
- Anaesthetic used for blood collection: Yes, with ether. Blood samples were obtained from the abdominal aorta. Plasma and serum were stored at -20ºC after the assay.
- Animals fasted: Yes, 16 - 22 hours before autopsy.
- How many animals: All males and 6 females per group.
- Parameters checked: GOT, GPT, ALP, gamma-GTP and Glu, blood was treated with heparin natrium (Mochida Pharmaceutical Co., Ltd), Ch-E, T-Cho, PL, TG, T-Bil, UN, Crea, Na, K, CI, Ca, IP, TP, Protein fraction and A/G ratio, blood was treated with separating agent (Sepa clean, Eiken Kizai Co., Ltd).


URINALYSIS: Yes
- Time schedule for collection of urine: Day 43 - 44.
- Metabolism cages used for collection of urine: Yes (KN-646 B-1 type, Natsume Seisakujo Co Ltd.)
- Animals fasted: No
- How many animals: 6 selected males at each group.
- Parameters checked: pH, Pro, Glu, Ket, Uro, Bil, Occult blood, urinary sediments, colour, U-Vol, specific gravity and water consumption of 3- and 21-hour urine collections.


Oestrous cyclicity (parental animals):
A vaginal smear was prepared for each female using Giemsa staining from10 days before dosing until successful mating and the stage of the oestrous cycle was examined microscopically. Oestrous stages with 4 – 6 days interval at least 2 times consecutively were considered to be normal, and oestrous or anoestrous stages of 7 days or more were considered to be abnormal.
Sperm parameters (parental animals):
Not examined
Litter observations:
PARAMETERS EXAMINED
LITTER OBSERVATION
The number of live and dead offspring, clinical sign and external observation was recorded on Days 0 – 4 post partum. The day of parturition was considered to be Day 0 of post partum.

PHYSICAL DEVELOPMENT
All live offspring were weighed on Day 0, 1 and 4 post partum and the mean weight of all live pups and the mean weight of male and female pups were calculated for each female rat.

GROSS EXAMINATION OF DEAD PUPS: Yes (as for live pups)


Postmortem examinations (parental animals):
SACRIFICE
All animals were sacrificed, anaesthetised with ether.

GROSS PATHOLOGY: Yes

External examination was performed. Organs and tissue were observed macroscopically. Brain, pituitary gland, thymus, thyroid glands, parathyroid glands, adrenal glands, spleen, heart, thoracic aorta, tongue, esophagus, stomach, liver, pancreas, duodenum, jejunum, ileum, cecum, colon, rectum, larynx, trachea, lung, kidneys, urinary bladder, testes, epididymides, prostate, seminal vesicle, ovaries, uterus, vagina, eyes, harderian gland, mammarian gland, skin, sternum, femur, spinal cord, skeletal muscle, mesentery lymph node, mandibular lymph nodes, submandibular glands, sublingual glands, parotid glands and right ischiadic nerve were fixed and stored in 10 % formalin neutral buffer solution. Bulb of eyes and harder gland were fixed and stored in Davidson solution.

Organ weight examined for brain, heart, liver, kidneys, spleen, adrenals, thymus, testes and epididymides.

HISTOPATHOLOGY: Yes
Males: All animals at control and the highest dose groups. All the stored organs and tissues, glandular stomach, pituitary gland. Spleen and the left testis of the male rat (No. 308 at 15 mg/kg/day) which showed cyst in the spleen and atrophy in testes. Haematoxylin-eosin staining. The spleen from control and 30 mg/kg/day groups were also stained with berlin blue.

Females: All animals at control and the highest dose groups. All the stored organs and tissues, glandular stomach, pituitary gland. Right back skin of the female rat (No. 361 at 15 mg/kg/day) which showed white subcutaneous mass and right thoracic skin of the female rat (No. 452 at 30 mg/kg/day) which showed yellow subcutaneous mass. Haematoxylin-eosin staining. The spleen from dose groups were also stained with berlin blue if any treatment-related effects were observed in any animals at doses.
Postmortem examinations (offspring):
Dead pups were autopsied immediately. The whole body was fixed in 10 % formalin neutral buffer solution. The rest of offspring were observed externally on Day 4 of post partum, followed by anaesthesis with carbon dioxide for macroscopic observation of whole organs and tissues in the bodies.
Statistics:
- Bodyweight (gain), food consumption, urinalysis, haematology, clinical chemistry, organ weight (absolute and relative), numbers of corpus luteum/implantation sites/rate of successful implantation, birth rate, number of live pups on Day 0 of pos purtum, delivery index, parturition index, viability index (Day 0 and 4 of post partum), sex ratio, number of dead pups born, duration of pregnancy: Bartlett test (one-factor ANOVA analysis of variance for homoscedasticity, Kruskal-Wallis for heteroscedasticity). In case of significancy, Dunnett test for ANOVA, and U-test of Mann-Whitney for Kruskal-Wallis.
- Urinalysis and histopathology: Kruskal-Wallis follwed by U-test of Mann-Whitney in case of significance.
- Osterous cycle, mating index, pregnancy index, delivery index, viability index on Day 4 of post partum and histopathology, in case 1-st class of positive response have been observed: chi-test or Fisher's exact test.
- A probability value of 0.05 or below was considered to be statistically significant. The statistical analysis of reproductive parameters were based on the mean value in each maternal body.
Reproductive indices:
Mating Index (%) = (Number of females mated ÷ Number of femailes paired) x 100
Pregnancy Index (%) = (Number of pregnant females ÷ Number of females mated) x 100
Gestation Length: Calculated as the number of days of gestation including the day for observation of mating and the end of parturition.
Parturition Index (%) = (Number of females delivering live offspring ÷ Number of pregnant females) x 100
Delivery index (%) = (number of offspring born ÷ number of implantation site) x 100
Birth Index (%) = (Number of offspring alive on observation of parturition ÷ Number of offspring born) x 100
Nuring rate on Day 4 of post partum (%) = (Number of females with live offspring on Day 4 of post partum ÷ Number of females delivering live offspring) x 100
Sex ratio (%) = (Number of male offspring ÷ Number of female offspring) x 100
Offspring viability indices:
Viability Index (%) = (Number of live offspring on Day 4 of post partum ÷ Number of offspring alive on Day 0) x 100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Other effects:
not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed

Details on results (P0)

CLINICAL SIGNS AND MORTALITY
(Males/females) No treatment-related effects were observed.

BODY WEIGHT AND WEIGHT GAIN
(Males/females) No treatment-related effects were observed (see Figure 1, attached).

FOOD CONSUMPTION
(Males/females) No treatment-related effects were observed (see Figure 2, attached).

HAEMATOLOGY
(Males) Decreases in RBC, hematocrit and haemoglobin concentration in 30mg/kg group (see Table 1, attached).
(Females) Non treatment-related effects: decrease in MCHC at 30 mg/kg and decrease in Stab at ≤ 15 mg/kg (see Table 2, attached).

CLINICAL CHEMISTRY
(Males) Treatment-related effects: decreases in potassium in 15 and 30mg/kg groups, increase in creatinine in 30mg/kg group (see Table 3, attached)
(Females) Increase in T-Bil and Glu at 30 mg/kg (see Table 4, attached).

URINALYSIS
(Males) No treatment-related effects observed.

ORGAN WEIGHTS
(Males) Increase in the relative liver weight at 30 mg/kg (see Table 5, attached).
(Females) Increase in relative liver weight in 7.5, 15 and 30mg/kg groups and in absolute liver weight in 30mg/kg group, in absolute heart weight in 7.5, 15 and 30mg/kg groups and in relative heart weight in 15 and 30mg/kg groups and in absolute and relative spleen weight in 30mg/kg group (see Table 6, attached).

GROSS PATHOLOGY
(Males) Dark red patches on the mucosa of the glandular stomach in one out of twelve animals given 30mg/kg.
(Females) Dark red patches on the mucosa of the glandular stomach in one out of twelve animals given 7.5 or 15mg/kg and in two out of twelve animals given 30mg/kg.

HISTOPATHOLOGY: NON-NEOPLASTIC
(Males) Slight erosion (not statistically significant) was observed in the glandular stomach in animals showed dark red patches on the mucosa at necropsy (see Table 7, attached).
(Females) Extramedullary hematopoiesis in the spleen was observed in 3 females given 15mg/kg, and 7 females given 30mg/kg (see Table 7, attached).

HISTOPATHOLOGY: NEOPLASTIC
(Females) Spontaneous neoplasms at ≥ 15 mg/kg.

REPRODUCTIVE PARAMETERS
No treatment-related effects in any of reproductive and developmental parameters tested (see Tables 8 and 9, attached)

Effect levels (P0)

Dose descriptor:
NOEL
Remarks:
reproductive and development toxicity
Effect level:
>= 30 mg/kg bw/day
Sex:
male/female
Basis for effect level:
other: No treatment-related effects in any of reproductive and developmental parameters tested.
Remarks on result:
other: Generation: parents and offspring (migrated information)

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
not examined

Details on results (F1)

No treatment-related effects in any of reproductive and developmental parameters tested. The dead offspring were born from a female rat however no abnormalities were observed in the general condition or autopsy of the maternal rat or the live offspring from this female, therefore the relationship between the effects and the administration of the test substance were not established.

Overall reproductive toxicity

Reproductive effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
The NOEL for reproductive and developmental toxicity of the test substance (P & F1) was considered to be 30 mg/kg or above.