Registration Dossier

Administrative data

Endpoint:
short-term repeated dose toxicity: oral
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
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 males
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:
methylcellulose
Remarks:
1 % in aqueous solution
Details on oral 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)
Analytical verification of doses or concentrations:
yes
Duration of treatment / exposure:
males: 44 days
females: 41-45 days (day 3 postpartum)
Frequency of treatment:
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

Observations and examinations performed and frequency:
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
Sacrifice and pathology:
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
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

Results and discussion

Results of examinations

Clinical signs:
no effects observed
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:
effects observed, treatment-related
Description (incidence and severity):
Males:
- significant increase in prothrombin time and decrease in platelet count in the high dose group (1000 mg/kg bw/day)
- increased leucocyte counts were observed in the 30 and 1000 mg/kg bw/day groups
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Males:
- significant increase of aspartate aminotransferase value (GOT or AST) in the high dose group (1000 mg/kg bw/day)
- significant increase of sodium in the high dose group (1000 mg/kg bw/day)
- significant increase of alkaline phosphatase (ALP) at 300 mg/kg bw/day
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). The severity was graded as being slight.
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 swelling in livers was observed in 7 females at 1000 mg/kg bw/day. The severity was graded in 5 females as slight and in two females as moderate.
- periportal hepatocellular vacuolar swelling in livers was observed in 3 females at 300 mg/kg bw/day. The severity was graded in 2 females as slight and in 1 female as moderate.
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed

Effect levels

open allclose all
Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
organ weights and organ / body weight ratios
histopathology: non-neoplastic
Based on:
test mat.
Sex:
male
Remarks on result:
not determinable due to absence of adverse toxic effects

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
In the combined repeated dose/ reproductive and developmental toxicity test, 10 male and 10 female SD rats were exposed orally via gavage to 0, 30, 100, 300 and 1000 mg/kg bw/day m-toluic acid for 41 to 45 days.
No deaths were observed in male and female animals in each group.
In males of the 1000 mg/kg bw/day dose group, the following effects were observed: slightly decreased locomotor activity, increased prothrombin time and increased aspartate aminotransferase values. The statistical significantly increased prothrombin time (14.3 sec) is, although being statistical significant, still within the historical control range for that rat strain (13.3 - 16.5 sec, Lee et al. 2012), thus should be regarded as incidental finding with no biological relevance. The slightly decreased locomotor activity is regarded as transient finding, in the absence of further interrelated clinical or pathological findings should be regarded as not biological relevant. The increased aspartate aminotransferase (AST, 77 ±13 IU/L) might indicate a correlation with the histopathological findings in the liver in the female animals, however the value is still well within the historical control range of that rat strain (48.9-122.7 IU/L). Thus, an NOAEL of 1000 mg/kg bw/day was identified for male animals.
In females of the 1000 mg/kg bw/day dose group, the following effects were observed: significant increase of absolute and relative liver weights and periportal hepatocellular vacuolar degeneration. In females of the 300 mg/kg bw/day dose group, periportal hepatocellular vacuolar degeneration was observed with a grading of minimal to moderate severity. Vacuolar degeneration, also termed cell swelling, characterises a reversible cell injury, occurs in most types of acute injury (Ed.: Haschek, WM et al. 2007 Fundamentals in toxicologic pathology, AP; p. 13). In combination with increased liver weight, this is a common finding in animals being exposed to high doses of xenobiotics and commonly reverses if the injurious stimulus is removed (Ed.: Haschek, WM et al. 2007 Fundamentals in toxicologic pathology, AP; p. 201f). In the absence of degenerative cell lesions, such as necrosis or apoptosis, and based on the minimal to mild manifestation of that injury, this finding is considered a very mild adverse effect. Based on the increased relative and absolute liver weight and the periportal hepatocyte vacuolar swelling, the NOAEL for females was considered at 100 mg/kg bw/day.