Registration Dossier

Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Remarks:
combined repeated dose and reproduction / developmental screening
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study Study in Japanese with English translation
Cross-reference
Reason / purpose:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1997

Materials and methods

Test guideline
Qualifier:
according 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
Details on test material:
Lot/batch No.: 33041
Purity: 99.6 %
Physical state: colorless and aromatic solution which can be dissolved in aromatic hydrocarbon, ethanol and acetone, but not in water
Stability under test conditions: The test substance was stored in the dark at room temperature and confirmed to be stable during the study period by the manufacturer.

Test animals

Species:
rat
Strain:
Crj: CD(SD)
Sex:
male/female
Details on test animals and environmental conditions:
Male and female SD rats (Crj:CD) were obtained from Charles River Japan, Inc. After quarantine and acclimatization for 6 days, the animals were used for the study. Ten rats from each sex were assigned to each group using a stratified-by-weight randomization method on a day before administration. The animals were at 8 weeks of age for both males and females at the beginning of the administration and the body weights ranged from 309 to 337 g for males and from 181 to 225 g for females.
During the entire husbandry period including quarantine and acclimatization periods, the animals were housed in an animal room, which was automatically maintained at a temperature of 20 to 25 degrees C and relative humidity of 40 to 70 %, with ventilation of about 12 times/hr, and 12 hr lighting (7:00-19:00). The animals were housed and kept in polycarbonate cages with experimental animal beddings (Beta Chip, Charles River Japan, Inc.) individually after administration, in a pair of male and female during mating period, or with a litter during lactation period in one cage.
The animals were given autoclave-sterilized solid diet for experimental animals (CRF-1, Oriental Yeast Co. ,) and tap water passed through a 5 um filter and irradiated with UV light ad libitum.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
olive oil
Details on oral exposure:
When a 10-day repeated dose study (doses: 0, 100, 300, 1000 mg/kg) was conducted using SD rats, trends of suppression of body weight gain and increase of liver size were observed in males in the 1000 mg/kg group. Based on the results, the dose of 1000 mg/kg was set as the highest dose, with 200mg/kg and 40mg/kg for the middle and low doses, respectively, with a common ratio of 5. In addition, the control group was designed as the group receiving vehicle only.
Administration period included 14 days prior to mating and mating period in both sexes, more specifically a total of 43 days until the day before scheduled sacrifice in males and from the day of successful mating, followed by delivery until post-partum day 3 in females. The test substance, which was dissolved in an olive oil, was gavaged once a day in the morning using a gastric tube. Administration volume was set to 5 ml/kg and determined based on the latest measured body weight.
Analytical verification of doses or concentrations:
not specified
Details on analytical verification of doses or concentrations:
The administrative solution was prepared under yellow lamp illumination and refrigerated in dark until use. In addition, stability and concentrations of the administrative solution were confirmed prior to use.
Duration of treatment / exposure:
Oral administration for 43 days from 14 days prior to mating to days after mating in males and for the periods including 14 days prior to mating and from gestation period, followed by delivery until post-partum day 3 in females.

Terminal kill:
m: day 44
f: day 4 of lactation
Frequency of treatment:
once daily
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 40, 200 or 1000 mg/kg bw
Basis:
other: Concentrations of the administrative solution were confirmed prior to use
No. of animals per sex per dose:
10 m / 10 f per group
Control animals:
yes, concurrent vehicle
Details on study design:
Administration period included 14 days prior to mating and mating period in both sexes, more specifically a total of 43 days until the day before scheduled sacrifice in males and from the day of successful mating, followed by delivery until post-partum day 3 in females. The test substance, which was dissolved in an olive oil, was gavaged once a day in the morning using a gastric tube. Administration volume was set to 5 ml/kg and determined based on the latest measured body weight.
Positive control:
No

Examinations

Observations and examinations performed and frequency:
General Conditions:
All animals were observed every day for survival, external anomalies and behaviours. Animals that died were necropsied promptly upon discovery.

Body weight and food consumption:
Body weight was measured at the beginning of the administration and once a week thereafter for males and at the beginning of the administration, once weekly before mating and on days 0 (day of copulation), 7, 14 and 20 of gestation and days 0 (day of parturition) and 4 post partum for mated females. Food consumption was measured on the same days that body weights were measured except during mating period.

Haematological Examination in Male Rats:
All surviving male rats were fasted approximately 21 hours before sacrifice and blood was collected from posterior aorta under anaesthesia with the intraperitoneal administration of thiopental sodium (Ravonal: Tanabe Pharmaceutical Inc.). After treating some of the blood with an anti-coagulator, EDTA-2K, we calculated RBC (sheath flow DC impedance detection method), WBC (RF/DC impedance detection method), platelet count (sheath flow DC impedance detection method), haemoglobin concentration (SLS haemoglobin method) and haematocrit value (WBC pulse wave peak detection method) using a multi-channel auto-analyzer (NE-4500: Toa Medical Electronics Inc), WBC percentage (Wright's stained blood smears) using a blood cell auto-analyzer (MICROX HEG-70A: Tateichi Electronics) and reticulocyte count (Argon Laser Flow cytometry method) using a reticulocyte auto-analyzer (R-2000: Toa Medical electronics Inc.). In addition, mean corpuscular volume (MCV) and mean corpuscular haemoglobin (MCV) and mean corpuscular haemoglobin concentration (MCHC) were obtained based on the test values.

Blood chemical examination in male rats:
Blood was collected from all surviving male rats on the day of sacrifice. After incubating at room temperature for approximately 30 minutes, the blood was centrifuged at 3000 rpm (2050G) for 10 minutes to obtain serum. The serum was analyzed for GOT (SSCC modified method), GPT (SSCC modified method), ALP (GSCC modified method), gamma-GTP (SSCC modified method), urea nitrogen (Urease-GLDH method), glucose (GK-G6PDH method), total cholesterol (CES-CO-POD method), triglyceride (LPL-GK-G3PO-POD method), creatinine (Jaffe), total bilirubin (Jendrassik modified method), total protein (Biuret method), albumin (BCG method), A/G ratio (calculate from the amounts of total protein and albumin), calcium (O-CPC method), inorganic phosphate (UV method), sodium, potassium, chloride (ion selective electrode method) using an autoanalyzer (Hitachi 736-10 model: Hitachi Inc.).
Sacrifice and pathology:
Both surviving female and male rats were sacrificed by exsanguination from the abdominal aorta under anaesthesia with the intraperitoneal administration of thiopental sodium on the next day of the last day of administration and the weights of the thymus, liver, kidneys, testes and epididymides were measured. Furthermore, in addition to these organs, the brain, heart, spleen, adrenals, ovaries, and urinary bladder of the males with abnormal necropsy findings were collected and stored after fixing with 10 % PBS neutral buffered formalin solution (testes and epididymides were fixed with Bouin's solution). Haematoxylin and eosin stained specimens were conventionally prepared for the brain, heart, liver, spleen, kidneys, adrenals, testes and epididymides of the control group and the 1000 mg/kg group in both sexes, and for the urinary bladders of both sexes and for the thymus of female for the rats with suspected influence of the test substances based on the necropsy findings and the results of organ weight measurement, and they were analyzed microscopically. As a result, since there were changes attributable to the test substance observed in the liver, kidney and adrenals of both sexes, the urinary bladder of males and the thymus of females, the same organs of the rats in the 200 and 40 mg/kg groups were examined. For the male rat that died in the 1000 mg/kg group before the end of the study, seminal vesicles, prostate grand, lung, stomach, duodenum and urinary duct were examined in addition to the above organs. Furthermore, the ovaries of the non-pregnant female and the skin of the rat in the 1000 mg/kg group in which crust was observed in necropsy findings were examined as well. In addition, some of the liver, kidneys and adrenals were stained with oil red O.
Statistics:
Metrical data were tested for homogeneity of variance by Bartlett’s test. When the variance was homogeneous, the one-way analysis of variance was performed, and when heterogeneous, the Kruskal-Wallis test was used. When a significant inter-group difference was found and if the number of samples in each group are the same, Dunnett’s test or the Dunnett-type multiple comparison test was employed, and if not, Scheffe test or the Scheffe type multiple comparison test was employed. However, Kruskal-Wallis test was used for the following items with *. The numerical data were analyzed with Fisher’s exact probability test. The level of significance was set at 5 % for all analyses. For the data pertaining neonates, a mean value calculated for each dam was used as a statistical unit. The following items were statistically analyzed.

Multiple comparison tests
Bodyweight, food consumption, haematological examination ,blood chemical examination, organ weight, pairing days until mating *, number of oestrous stages without mating *, gestation period *, number of corpora lutea, number of implantation sites, implantation index*, delivery index*, number of offspring delivered, live birth index* and viability index on day 4*.

Fisher’s exact probability test
Mating index, fertility index, gestation index and sex ratio (male /female)

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Mortality:
mortality observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
effects observed, treatment-related
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
no effects observed
Details on results:
Mortality:
One of the male rats in the 1000 mg/kg group died on day 23 of administration. There was no other case of death.

Clinical signs:
Persistent slight or moderate level of salivation was observed in females at 1000mg/kg immediately after administration and in almost all males at >= 200 mg/kg at day 3 of administration or after. In addition, intermittent slight level of salivation was observed in a small number of the females at 200 mg/kg and males at 40 mg/kg. Some of these rats were found to start salivate immediately before administration. The dead male in the 1000mg/kg group showed anaemia, haematuria, decreased locomotor activity, and bradypnea on day 22 of administration and died on the next day.
Furthermore, possible incidental findings of hair loss, wound or crusts were observed in one male in each of the control and the 1000 mg/kg groups as well as in females in the 200mg/kg.

Bodyweight:
In the 1000 mg/kg group, male rats showed suppression of body weight gain from day 7 of administration until the end of the study. Although there were no significant differences, female rats showed a slight suppression of body weight gain on day 20 of gestation and a trend of suppression of body weight gain in the late gestation period. In addition, despite the significantly high value, observed in body weight gain on day 4 of lactation in the 200 mg/kg group, it was judged to be incidental due to the lack of correlation with the dose.

Food consumption:
Although in the 1000 mg/kg group, male rats showed a decrease in food consumption on day 7 of administration, the changes thereafter was almost similar to the control group. In females, there were no significant differences between the control group and the test substance treatment group in terms of the pre-mating period, gestation period and lactation period.

Haematological Examination in Male Rats:
There were no significant differences between the control group and the test substance treatment groups.

Blood Chemical Examination in Male Rats:
Increase in GPT at >= 200 mg/kg and increases in urea nitrogen and potassium, and a decrease in triglyceride in the 1000 mg/kg group were observed. In addition, despite the high value in total cholesterol in the 200 mg/kg group and the low values in glucose and chloride in the 1000 mg/kg group observed, they were judged to be incidental because the dose response relationship was not clear or the changes were within the physiological range.

Organ weights:
Increases of absolute organ weight and organ weight relative to body weight of the liver were observed in males of the 1000 mg/kg groups and females at >= 200 mg/kg. Furthermore, increases of absolute organ weight and organ weight relative to body weight of the kidneys were observed in both sexes of the 1000 mg/kg group and the increase of organ weight relative to body weight was observed in females of the 200 mg/kg group. In addition, decrease of absolute organ weight and organ weight relative to body weight of the thymus was observed in female of the 1000 mg/kg group. In addition, despite the high value of organ weight relative to body weight of the testes in the 1000 mg/kg group, it was judged to be a superficial change reflecting the suppression of body weight gain because there were no significant differences in absolute organ weight compared to the control group and there were no changes attributable to the test substance in histological and reproductive performance studies.

Necropsy findings:
In the animals had scheduled to be sacrificed, changes attributable to the test drug were observed in the liver, kidneys, urinary bladder, adrenals and thymus.
Liver enlargement was observed in both sexes of the 1000 mg/kg group and dark reddish change was observed only in males at >= 200 mg/kg.
Kidney enlargement was observed in both sexes of the 1000 mg/kg group and discoloration of the cortico-medullary junction was observed only in females at >= 200 mg/kg. In addition, although there was one female case of enlargement in the 40 mg/kg group, it was judged to be an incidental lesion since the occurrence was unilateral.
In the urinary bladder, yellow micro-granular calculi was observed only in male of the 1000 mg/kg group.
Enlargement and grayish change in the adrenals and atrophy in the thymus were individually observed in females of the 1000 mg/kg group. The female rat, that showed thymus atrophy, also showed spleen atrophy.
In addition, despite a case of unilateral atrophy in the testes in a male of the control group and another case of skin crust in a male of the 1000 mg/kg group, they were judged to be incidental lesions due to the circumstances of occurrence.
For the male rat died in the 1000 mg/kg group, a large amount of blood-like discoloration of urine was found in the distended urinary bladder and 7-8 urinary calculi with 1-2 mm in diameter were observed. Enlarged kidneys, distinct bleeding in renal pelvis, urinary ducts, urinary bladder and epididymides and prostate grand adjacent to the urinary bladder were found, indicating ischuria. In addition, pulmonary oedema and spleen atrophy were observed.

Histopathological Findings:
In the animals had scheduled to be sacrificed, changes attributable to the test substance were observed in the liver, kidneys and adrenals of males and females, in the testes of males and in the thymus of females.
In the liver, acidophilic change of the hepatocytes was observed in both sexes at >= 200 mg/kg. In males, micro-granular acidophilic cells were diffusely spread in the entire small lobules, which was coincided with the loss of fatty droplets that otherwise usually exist. On the other hand, in females, micro-granular acidophilic cells were distinctively observed around the centre of the lobules and enlarged hepatocytes were found.
In the kidneys, increase of hyaline droplets in the renal tubular epithelium was found in males of the 200 mg/kg group and most of the hyaline droplets were accompanied with the basophilic changes of the renal tubular epithelium. In addition, vacuolation in the renal tubular epithelium was found in females at >= 200 mg/kg, and infiltrated lymphocytes were found in an adjacent area in some cases. Purple colour in oil red O stain indicated that the vacuolation is fatty droplets. In addition, there were no such changes observed in one case of female in the 40 mg/kg group, in which necropsy findings indicated enlarged kidneys.
In the adrenals, increase of small and large fatty droplets in the fascicular zone was observed in both sexes of the 1000 mg/kg group.
In the urinary bladder, hyperplasia of the mucosal epithelium was observed in males of the 1000 mg/kg group, including the thickened mucosal epithelium layer by 2 to 3 folds, erosion and infiltration of inflammatory cells to submucosal tissues.
In the thymus, atrophy was observed in females at >= 200 mg/kg and the boundary between the cortex and medulla were unclear.
Other observed changes were all minor case and they were judged to be non-specific incidental cases because of the circumstances of occurrences. In addition, there were no pathological changes observed in the ovaries of non-pregnant female rats.
In dead animals, bleeding was observed in the kidneys, urinary duct, urinary bladder, epididymides and prostate gland, and furthermore, in kidneys, expanded urinary duct and renal papillary necrosis were observed in the kidneys. In addition, pulmonary oedema and spleen atrophy were observed.

Effect levels

open allclose all
Dose descriptor:
NOEL
Effect level:
40 mg/kg bw/day (actual dose received)
Sex:
male/female
Dose descriptor:
LOAEL
Effect level:
200 mg/kg bw/day (actual dose received)
Sex:
male/female
Basis for effect level:
other: Histological changes in liver and kidneys of both sexes and in thymus of female rats. Increase in GPT in male rats.

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
The LOAEL for repeated dose toxicity was given with 200 mg/kg bw/d for both sexes. The NOEL was 40 mg/kg bw/d. Therefore, the NOAEL is expected to be >100 mg/kg bw/d.
Executive summary:

In this study performed according to OECD Guideline 422, 1-methylethenylbenzene was given to SD (Crj:CD) rats of both sexes at dose levels of 0, 40, 200 or 1000 mg/kg/day by oral administration for 43 days from 14 days prior to mating to days after mating in males and for the periods including 14 days prior to mating and from gestation period, followed by delivery until post-partum day 3 in females.

At 1000 mg/kg, male rats showed a suppression of body weight gain, and a decrease in food consumption, and one animal died due to ischuria with urinary calculi. Female rats showed slight suppression of body weight gain in the late gestation period. Histopathological examination demonstrated increases of liver and kidney sizes, acidophilic change of hepatocytes and increase of fatty droplets in the fascicular zone of the adrenals in both sexes, increase of hyaline droplets and basophilic change in the renal tubular epithelium, formation of urinary calculi and hyperplasia of the mucosal epithelium in the urinary bladder in male rats, and vacuolation and infiltration of lymphocytes in the renal tubular epithelium and atrophy of the thymus in female rats. Blood chemical examination in male rats showed increases in GPT, urea nitrogen and potassium, and a decrease in triglyceride.

At 200 mg/kg, similar histological changes were found in the liver and kidneys of both sexes, and the thymus of female rats, and an increase in GPT was observed in male rats. Haematological examination showed that the test substance had no effects in males.

The LOAEL for repeated dose toxicity was given with 200 mg/kg/day for both sexes. The NOEL was 40 mg/kg bw/d. The NOAEL is expected to be >100 mg/kg bw/d.