Registration Dossier

Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
haematology and clinical chemistry parameters only assessed in male rats
Cross-reference
Reason / purpose:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1999

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
1996
Deviations:
yes
Remarks:
haematology and clinical chemistry parameters only assessed in male rats
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
-Appearance: colorless to pale yellow
-Molecular weight: 136.15
-Melting point: 11.7 °C
-Boiling point: 263 °C
- soluble in organic solvents, almost insoluble in vegetable oils, sparingly water soluble
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: sealed at a cool (4 °C) and dark place
- Stability under test conditions: stable during use period
- Solubility and stability of the test substance in the solvent/vehicle: stable for at least 7 days


TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: dissolved in 1 % methylcellulose in water

Test animals

Species:
rat
Strain:
Crj: CD(SD)
Remarks:
SPF
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Co., Ltd., Japan
- Females nulliparous and non-pregnant: yes
- Age at study initiation:
males: 9 weeks
females: 8 weeks
- Weight at study initiation:
males: mean weight 357 g (range: 342 - 372 g)
females: mean weight 222 g (range: 209 - 237 g)
- Fasting period before study:
- Housing:
-housed individually in stainless steel wire mesh cages
-after confirmation of mating, females were housed individually in polycarbonate cages, nest-making material (obtained from Charles River Co., Ltd., Lot No.: 9623) was provided
- Diet (ad libitum): diet was obtained from Japan Agricultural Association Company, national feed laboratory MR stock, Lot. No.: 71064 (assumed)
- Water (ad libitum): UV irradiated tap water
- Acclimation period: 5 days

DETAILS OF FOOD AND WATER QUALITY: quality of feed, water and nest-making materials was confirmed by analysis

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 - 23 °C
- Humidity (%): 53 - 60 %
- Air changes (per hr): 10 times or more per hour
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: 1 % methylcellulose i(aqueous solution)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
- Rate of preparation of dosing solution (frequency): once or twice a week
- Storage temperature and stability of dosing solution: stored sealed in the dark at 4 °C, used within 7 days of preparation

VEHICLE
- Concentration in vehicle:
- 30 mg/kg bw/ day group: 0.6 % (w/v) m-toluic acid
- 100 mg/kg bw/day group: 2 % (w/v) m-toluic acid
- 300 mg/kg bw/day group: 6 % (w/v) m-toluic acid
- 1000 mg/kg bw/day group: 20 % (w/v) m-toluic acid

- Amount of vehicle (if gavage): 5 mL/ kg bw
- Lot/batch no.: methylcellulose was obtained from Wako Pure Chemical Industries, Ltd. (Lot No.: DLH 5715) (assumed)
Details on mating procedure:
- M/F ratio per cage: one male/one female
- Length of cohabitation: 14 days
- 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 individually in polycarbonate cages, nest-making material (obtained from Charles River Co., Ltd., Lot No.: 9623) was provided
Analytical verification of doses or concentrations:
yes
Duration of treatment / exposure:
Exposure period:
Males: 14 days before mating
Females: from 14 days before mating to day 3 of lactation
Premating exposure period (males): 14 days
Premating exposure period (females): 14 days
Duration of test: Males: 44 days from 14 days before mating
Females: 41 - 45 days from 14 days before mating to day 3 of lactation
Details on study schedule:
once a day
Doses / concentrationsopen allclose all
Dose / conc.:
30 mg/kg bw/day (actual dose received)
Dose / conc.:
100 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 males/ 10 females
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
- a 14-day study was conducted at doses of 0, 80, 150, 300, 600 and 1000 mg/kg bw/day in groups of 4 male and 4 female rats. One male and one female rat were cohoused from day 7. In the 1000 mg/kg bw/day group effects were observed (males: decreased platelet coun; females: decreased platelet counts, tendency of decreased body weight gain, tendency of decreased food consumption), consequently this dose was selected as high dose for the present study. Acidification of urine was observed in males and females in the 600 and 1000 mg/kg bw/day groups. 30 mg/kg bw/day were selected as low dose, which is expected to display no treatment-related effects.
- Rationale for animal assignment: based on body weights

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

DETAILED CLINICAL OBSERVATIONS: Not specified

BODY WEIGHT: Yes
- Time schedule for examinations:
males: once before administration period, and weekly thereafter and at termination
females: once before administration, and weekly thereafter (covering pregnancy day 0, 7, 14 and 20), and on day 0 and day 4 postpartum

FOOD CONSUMPTION: Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: 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: No


OPHTHALMOSCOPIC EXAMINATION: Not specified

HAEMATOLOGY: Yes, males only
- Time schedule for collection of blood: before termination (45 days after administration), from abdominal aorta
- Anaesthetic used for blood collection: Yes (ether)
- Animals fasted: Yes
- How many animals: all male rats
- The following parameters were examined: red blood cell count (RBC), hemoglobin (Hb), Hematocrit (Ht), mean red blood cell volume (MCV), mean, red blood cell hemoglobin content (MCH), mean red blood cell hemoglobin concentration (MCHC), white blood cell count (WBC), platelet count (Plat), reticulocyte count (Ret), differential keukocyte counts (basophiles, eosinophiles, neutrophiles, lymphocytes, monocytes, prothrombin time (PT), activated partial thromboplastin time (APTT)

CLINICAL CHEMISTRY: Yes, males only
- Time schedule for collection of blood: before termination (45 days after administration), from abdominal aorta
- Animals fasted: Yes
- How many animals: all male rats
- The following parameters were examined: total protein, albumin, A/G ratio, glucose, triglycerides, total cholesterol level, total bilirubin, urea nitrogen, creatinine, aspartate aminotransferase (AST or GOT), alanine aminotransferase (ALT or GPT), gamma-glutamyltransferase (gamma-GTP), lactate dehydrogenase (LDH), alkaline phosphatase (ALP), cholinesterase, calcium, inorganic phosphorus, sodium, potassium, chloride

URINALYSIS: Yes, males only
- Time schedule for collection of urine: on day 39 or day 42 of administration
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Not specified
- The following parameters were examined: colour, cloudiness, specific gravity, pH, protein, glucose, ketones, blood, urobilinogen, bilirubin, erythrocytes, leukocytes, crystals (ammonium magnesium phosphate, calcium phosphate, amorphous), epithelial cells (squamous, round, spindle), casts (granule, hyaline, waxy), fat globules

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations:daily (cage side observations)
- Dose groups that were examined: all rats
- Battery of functions tested: locomotor activity

IMMUNOLOGY: Not specified
Oestrous cyclicity (parental animals):
Estrous cyclicity was examined daily during mating period of 14 days by analysis of vaginal smear
Sperm parameters (parental animals):
Parameters examined in male rats:
testis weight, epididymis weight
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- All pups/litter were examined

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
number and sex of pups (including sex ratio), stillbirths, live births, postnatal mortality, presence of gross anomalies (external and visceral findings), weight gain; anogenital distance (AGD)

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities; possible cause of death was not determined for pups born or found dead.

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: not specified

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: not specified
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals were sacrificed 45 days after first administration
- Maternal animals: All surviving animals were sacrificed at day 4 postpartum

GROSS PATHOLOGY: Yes
- Necropsy was conducted on all male and female rats.
- The whole body including all organs was examined for abnormalities. For females, corpora lutea and implantation sites were examined
- The following organs were weighed: brain, liver, kidney, spleen, heart, thymus, pituitary gland, adrenal gland, testes, epididymes

HISTOPATHOLOGY: Yes
- Histopathological examination was conducted on male and female rats of the control groups and of the high dose groups (1000 mg/kg bw/day). Male and female rats with unsuccessful mating and pregnancy were additionally examined.
-The following organs were prepared: all gross lesions, heart, tongue, lung, liver, pancreas, kidney, urinary bladder, testes, epididymes, prostate, thymus, spleen, uterus, adrenal gland, skin, eye, harder's gland, thyroid (including parathyroid) gland, salivary gland, trachea, esophagus, stomach, intestine, seminal vesicles, ovaries, aorta, spinal cord, sciatic nerve, bone/bone marrow, lymph nodes (cervical, mesenteric), skeletal muscle, mammary gland
-The following organs were histopathologically examined: all gross lesions, brain, pituitary gland, thymus, heart, liver, lung, kidney, spleen, urinary bladder, adrenal gland, bone marrow, testes epididymes, prostate, seminal vesicles, ovaries, uterus, skin
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring was sacrificed at 4 days of age.
- These animals were subjected to postmortem examinations (macroscopic examination) as follows:

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the main organs in the chest and abdomen
Statistics:
- Bartlett's variance test was performed on parametric data
- Dunnett method or Scheffe method was performed for one-way analysis of variance
- Kruskal-Wallis test was performed on non-parametric data
Reproductive indices:
Copulation index = (No. of pairs with successful copulation/No. of pairs mated) x 100
Fertility index = (No. of pregnant females/No. of pairs with successful copulation) x 100
Implantation index =(No. of implantation sites/ No. of corpora lutea) x 100
Gestation index = (No. of females with live pups/No. of pregnant females) x 100
Offspring viability indices:
Delivery index = (No. of pups born/No. of implantation sites) x 100
Live birth index = (No. of live pups on day 0/No. of pups born) x 100
Viability index = (No. of live pups on day 4/No. of live pups on day 0) x 100

Results and discussion

Results: P0 (first parental animals)

General toxicity (P0)

Clinical signs:
no effects observed
Dermal irritation (if dermal study):
not examined
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 specified
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Behaviour (functional findings):
effects observed, treatment-related
Description (incidence and severity):
Males: decreased locomotor activity was observed in 4 male rats after 16 days of administration in the high dose group (1000 mg/kg bw/day)
Immunological findings:
not specified
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Males:
- significant increase of relative weight of the pituitary gland at 1000 mg/kg bw/day
Females:
- significant increase of absolute and relative liver weights at 1000 mg/kg bw/day
Gross pathological findings:
no effects observed
Neuropathological findings:
not specified
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Females:
- periportal hepatocellular vacuolar degeneration in liver was observed in 7 females at 1000 mg/kg bw/day
- periportal hepatocellular vacuolar degeneration in liver was observed in 3 females at 300 mg/kg bw/day
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed

Reproductive function / performance (P0)

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

Details on results (P0)

Please refer to attached background material in section 'Overall remarks, attachements'

Effect levels (P0)

Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects

Target system / organ toxicity (P0)

Critical effects observed:
not specified

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Dermal irritation (if dermal study):
not examined
Mortality / viability:
mortality observed, non-treatment-related
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
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:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings:
not examined
Other effects:
no effects observed

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not specified

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not specified

Details on results (F1)

Please refer to attached background material in section 'Overall remarks, attachements'

Effect levels (F1)

Dose descriptor:
NOAEL
Generation:
F1
Effect level:
1 000 mg/kg bw/day (actual dose received)
Remarks on result:
not determinable due to absence of adverse toxic effects

Target system / organ toxicity (F1)

Critical effects observed:
not specified

Overall reproductive toxicity

Reproductive effects observed:
no

Applicant's summary and conclusion

Conclusions:
In the combined repeated dose/ reproductive and developmental toxicity test, rats were exposed to 30, 100, 300 and 1000 mg/kg bw/day m-toluic acid for 41 to 45 days. Reproductive parameters in parental animals were examined and no adverse effects were observed. Offspring parameters wer examined and no adverse effects were observed. Thus, the reproductive NOAEL was considered at 1000 mg/kg bw/day.