Registration Dossier

Toxicological information

Repeated dose toxicity: oral

Currently viewing:

Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Date of study initiation: September 13, 1988; Date of Final report development: January 31, 1989
Reliability:
1 (reliable without restriction)
Justification for data waiving:
other:
Cross-referenceopen allclose all
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1989
Report Date:
1989

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
other: Japanese 28 repeated dose guidelines
Principles of method if other than guideline:
"The Amendment to the Testing Guidelines for New Chemical Substances (Notification No. 700 of the Planning and Coordination Bureau, Agency of Environment, No. 1039 of the PAB, Ministry of Health and Welfare, and No. 1014 of 61 the Basic Industries Bureau, Ministry of International Trade and Industry dated December 5, 1986)", (establishment of screening toxicity study procedures), and “'Testing institution specified by Article 3 of the “Ministerial Ordinance Specifying items concerning the Testing of New Chemical Substances" (No. 39 of the Planning and Coordination Bureau, Agency of Environment, No. 229 of the PAB, Ministry of Health and Welfare, and No. 85 of 59 the Basic Industries Bureau, Ministry of International Trade and Industry dated March 31, 1984)" (Good Laboratory Practice: GLP).
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
- Name of test material (as cited in study report): 1, 2-diphenoxyethane
- Lot/batch No.: 80402
- Test substance supplied from Kanzaki Paper Co., Ltd. (3-1, Jokoji 4 chome, Amagasaki, Hyogo, Japan)

Test animals

Species:
rat
Strain:
other: Crj:CD(SD) SPF
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories Japan Inc. (Shimofurusawa 795, Atsugi, Kanagawa, Japan)
- Age at study initiation: aged 4 weeks when purchased and habituated in the test conditions for 13 or 14 days. Subsequently, 36 males and 36 females with good growth and general conditions were chosen at the age of 5 to 6 weeks to use in this study.
- Weight at study initiation: The mean body weight (the range of body weight) of males and females on administration was 189 g (172-200 g) and 161 g (146-172 g), respectively.
- Housing: 3 animals were housed in a stainless metal cage (W 276 x D 426 x H 200 mm) separately by sex.
- Diet (e.g. ad libitum): Animals were fed ad libitum with pellet (Lab M R Stock, lot No.: 63.7.61 and 63.9.59, Nosan Corporation, Japan) and water (tapping water sterilized by filtration with 1-μ cartridge filter and UV irradiation) from feeder and automatic water supplying system.
- Water (e.g. ad libitum): Animals were fed ad libitum with pellet (Lab M R Stock, lot No.: 63.7.61 and 63.9.59, Nosan Corporation, Japan) and water (tapping water sterilized by filtration with 1-μ cartridge filter and UV irradiation) from feeder and automatic water supplying system.
- Acclimation period: habituated in the test conditions for 13 or 14 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 24±2
- Humidity (%): 55±10
- Air changes (per hr): more than 10 times/hr (all fresh air) of ventilation
- Photoperiod (hrs dark / hrs light): 12-hr light and 12-hr dark cycle (light on: 6 AM, light off: 6 PM)

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: methyl cellulose
Details on oral exposure:
Preparation and administration procedures of test substance: The test substance 1, 2-diphenoxyethane was suspended in the 1% methylcellulose solution using a centrifugal ball mill (Model: 06-101, Fritsch) to prepare a testing solution at a designated concentration. The test substance was orally administered to animals once a day (9:30 AM - 11:00 AM) by an injection syringe with gastric gavage for 28 days. The volume of a testing solution was 10 mL/g bw and the volume for each animal was calculated from the body weight that was measured once a week.

Since 1, 2-diphenoxyethane was confirmed to be stable in a testing solution at least 7 days by Sponsor, the use of testing solutions prepared was for 7 days and testing solutions were dispensed into small portions by concentration and stored in a dark and cool room.
Analytical verification of doses or concentrations:
not specified
Details on analytical verification of doses or concentrations:
no data
Duration of treatment / exposure:
See attached report
Frequency of treatment:
see attached report
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
10mg/kg/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
100mg/kg/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
1000mg/kg/day
Basis:
actual ingested
No. of animals per sex per dose:
0 mg/kg/day (solvent control): 12 males and 12 females
10 mg/kg/day: 6 males and 6 females
100 mg/kg/day: 6 males and 6 females
1,000 mg/kg/day: 12 males and 12 females
Control animals:
yes, concurrent vehicle
Details on study design:
- Duration of observation period following administration: 14 days
- Frequency of observations and weighing:
- General conditions observation: Viability and moribundity, appearance and behaviour were observed once a day.
- Body weight measurement: Measured on the first day of administration and once a week thereafter
- Feed consumption measurement: Feed consumption per day (24 hr) per cage was determined once a week and the mean feed consumption per animal per day was calculated.
- Urinary test: In general conditions observation, the appearance of urine that was spontaneously excreted showed no abnormal finding. To confirm this finding, animals of the control and high-dose (1,000 mg/kg) groups were forced to excrete the urine by pressing the lower back the 7 days before the completion of administration and the appearance of urine was observed again. Consequently, no change suggesting toxic effect was not found and the full urinary test was not conducted.
- Hematology: The sample blood was collected from all surviving animals at autopsy on the next day of the completion of administration and recovery period. Animals were fasted from 5 PM of the day before autopsy and given only water. The blood was drawn from the abdominal aorta after laparotomy in animals anesthetized with ether and the following items in blood were determined. Anticoagulant for measurement of prothrombin time and activated partial thromboplastin time was 3.8% sodium citrate solution and that for other determination was EDTA-2K.
- Biochemistry: The serum was isolated from the remaining blood of the blood sample for the hematology detailed in the attached report.
- Pathology:
(1) Autopsy: All animals were sacrificed by bleeding after blood sampling and autopsied.
(2) Organ weight measurement: The weight (absolute weight) of the following organs of all surviving animals were measured by an electronic balance (AK160, Mettler) and the ratio to body weight on the day of sacrifice (relative weight) was calculated: brain, liver, kidneys, adrenal glands and testes/ovaries, however, right and left kidneys, spleens, testes/ovaries were weighed together.
(3) Histopathology: The following organs of all animals were collected and fixed with 10% neutral phosphate buffered formalin: brain, pituitary glands, eyeball, Harderian gland, thyroid gland (including parathyroid gland), salivary gland, thymus, trachea, lungs, heart, tongue, esophagus, stomach, intestine, liver, spleen, pancreas, adrenal glands, kidneys, bladder, testes, epididymus, prostate, gland, ovaries, uterine, vagina, aorta (thoracic), spinal cord (cervical and lumbar ampulla), sciatic nerve, bone and bone marrow (sternum and femur), lymph nodes (cervical and mesenteric lymph nodes), skeletal muscle (triceps surae muscle), skin (low back) and mammary gland (abdomen).
- With regard to histopathology, paraffin sections of the following organs were prepared in accordance with common procedures and stained with hematoxylin or eosin and observed by microscope.
- The preparation of histopathological specimens was commissioned to the Hist Science Laboratory Co., Ltd. (984-1, Kurosawa 2-chome, Ome, Tokyo, Japan).

- Necropsy of survivors performed: yes
Positive control:
No

Examinations

Observations and examinations performed and frequency:
See attached report
Sacrifice and pathology:
see attached reprot
Statistics:
1) Case of 3 groups or more: Quantitative data were analyzed by Bartlett's test of variance. As a result, when equal variance was confirmed, one-way analysis of variance was performed. When the difference was significant, Dunnett multiple comparison was performed between the control. When equal variance was not confirmed, Kruskal-Wallis rank sum test was performed. When a significant difference was shown between groups, Dunnett’s paired comparison with the control was performed. Autopsy and histopathological findings were analyzed by chi-square test.

2) Case of 2 groups: Quantitative data were analyzed by F test. As a result, when equal variance was confirmed, Student t test was performed. When equal variance was not confirmed, Aspin-Welch t test was performed. Autopsy and histopathological findings were analyzed by Fisher's exact probability test.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not specified
Ophthalmological findings:
not specified
Haematological findings:
no effects observed
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
effects observed, treatment-related
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
not specified
Histopathological findings: neoplastic:
not specified
Details on results:
Mortality:
- General conditions and death: No change in general conditions or death was found during the administration and recovery periods in all males and females of all groups.

Body weight:
- Body weight was steadily gained throughout the administration period in males and females of all groups, showing no significant difference between the control and the test substance groups. Furthermore, body weight during the recovery period in males and females of the 1,000 mg/kg group had no influence on gain, showing no significant difference between the control and the test substance groups.

Gross Pathology:
- Autopsy findings: In the animals that were sacrificed after the completion of administration period, no change that was caused by the test substance was observed in males and females of the 10 and 100 mg/kg groups. In the 1,000 mg/kg group, mild enlarged liver was observed in 5 males and 1 female of 6 each.
- Urethral protein plug associated with urinary retention in the bladder and hyperplasia in the bladder mucosa was observed in one male of the control group and diaphragmatic hernia in the liver was found in one male and one female of the 100 mg/kg group, however, these findings were not considered to be related to administration of the test substance.
- No gross abnormal finding was observed in organs of animals that were sacrificed after the completion of recovery period..

Organ weight:
- In the animals that were sacrificed after the completion of administration period, no change was observed in males and females of the 10 and 100 mg/kg groups. In animals of the 1,000 mg/kg group, slight increase in absolute and relative liver weight was found in males and increase in relative liver weight alone was observed in females.
- In animals of the 1,000 mg/kg group that were sacrificed after the completion of recovery period, increased liver weight that was found in animals that were sacrificed after the completion of administration period was not observed. However, increased relative kidney weight alone was observed in males and decreased absolute brain weight alone was found in females.

Histopathological findings:
- (1) Animals sacrificed after the administration period: The heart, liver, kidney, adrenal gland and spleen were examined. Consequently, no effect of the test substance was found in animals of the 10 and 100 mg/kg groups, however, the effect on the liver was found in animals of the 1,000 mg/kg group. The details are shown as follows.
a. Liver (males and females of all groups): Mild hepatocellular swelling was found in 5 males and one female of 6 each of the 1,000 mg/kg group. Hepatocellular swelling was diffusely observed over the hepatic lobule. However, hepatocellular degenerative change, cell infiltration or changes related to the bile duct were not marked. These changes were not observed in animals of the control and 10 and 100 mg/kg groups. Diaphragmatic hernia that was found in one male and one female of the 100 mg/kg group was not associated with marked hepatocellualr changes, however, mild hyperplasia was observed in the coat.
b. Heart and kidney (males and females of the control and 1,000 mg/kg groups): changes that were assumed to be caused by the test substance were not found. Changes that spontaneously occurred in rats of this strain and were considered to be incidental findings included myocardial fibrosis in the heart in males, focal basophilic lesions in the tubular epithelium of the kidney in males and females, Eosinophilic bodies in the proximal renal tubular epithelium, renal tubular dilation, stromal cell infiltration and hyperplasia in the renal pelvis epithelium in males.
c. Adrenal gland and spleen (males and females of the control and 1,000 mg/kg groups): no changes were found.
- (2) Animals sacrificed after the recovery period: The liver in which the effect of the test substance was found of animals that were sacrificed after the completion of administration period was examined by histopathology. Furthermore, the kidney of males and the brain of females were examined by histopathology due to increased relative kidney weight in males and decreased absolute brain weight in females that were sacrificed after the completion of recovery period. Consequently, changes in the liver recovered and no change that was caused by the test substance was observed in the kidney and brain. The results are summarized as follows.
a. Brain (females of the control and 1,000 mg/kg groups): no abnormal findings were found.
b. Liver (males and females of the control and 1,000 mg/kg groups): no abnormal findings were found.
c. Kidney (males of the control and 1,000 mg/kg groups): changes that were assumed to be caused by the test substance were not found.
- Changes that are considered to be spontaneous lesions were basophilic lesions in the tubular epithelium and focal renal tubular dilation in males, which were similar to those observed in animals that were sacrificed after the completion of administration period.

Other findings:
- Feed consumption: No effect of the test substance on feed consumption was found during the administration and recovery periods in all males and females of the test substance-treated groups.

Hematological findings: No change that was caused by the test substace was observed in males and females that were sacrificed after the completion of administration and recovery periods. The white blood cell and lymphocyte counts in males and females of the 10 and 100 mg/kg groups were lower and red blood blood cell count in males of the 100 mg/kg group was higher than those of the control group, however, no dose-dependency was found. In comparison with the control group, the prothrombin time was lower in males of the 10 and 1,000 mg/kg groups but that in males of the 100 mg/kg group was no change, in contrast, higher in females of the 100 mg/kg group, showing no dose-dependency or consistent tendency.

Biochemical findings: In animals that were sacrificed after the completion of administration period, no change that was caused by the test substance was observed in males and females of the 10 and 100 mg/kg groups. GOT activity slightly decreased only in females of the 1,000 mg/kg group. Potassium level in males of the 10 and 100 mg/kg groups was lower than that in the control group, however, no dose-dependency was found. In animals that were sacrificed after the completion of recovery period, decreased GOT activity, which was observed in animals that were sacrificed after the completion of administration period, was not found in animals of the 1,000 mg/kg group. However, mild increase in sodium and decrease in creatinine were observed in males.

Effect levels

Dose descriptor:
NOEL
Effect level:
100 other: mg/kg
Based on:
test mat.
Sex:
male/female

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
Based on the above results, no toxic change was found in the 28-day repeated oral dose study of 1, 2-diphenoxyethane in rats, however, considering mild effects on the liver in animals of the 1,000 mg/kg group, the no-observed-adverse-effect level (NOEL) was concluded to be 100 mg/kg.
Executive summary:

1, 2-diphenoxyethane was orally administered to Crj:CD (SD) rats at doses of 0 (solvent control: 12 males and 12 females) , 10 (6 each), 100 (6 each) and 1,000 mg/kg/day (12 each) by gavage for 28 days in order to evaluate the toxicity. To evaluate the reversibility of changes, 6 males and 6 females of the control and the 1,000 mg/kg/day groups were assigned again to the recovery group for a 14-day recovery study after the completion of administration. The results are summarized as follows.

 

1) In males and females of the 10 and 100 mg/kg groups, changes that were assumed to be caused by the test substance were not found.

 

2) In males of the 1,000 mg/kg group, the effect on the liver was found, i.e., gross hypertrophy associated with increased weight and diffuse hepatocellualr swelling by histopathology were observed. Similar tendency was found also in females, however, it was slightly mild.

 

3) However, no biochemical change indicating hepatic dysfunction was found in males and females. Mild decrease in GOT observed only in females had little importance in pathology.

 

4) No changes in growth and general conditions of males and females were found, therefore, these effects were considered to be mild.

 

5) The white blood cell and lymphocyte counts in males and females of the 10 and 100 mg/kg groups were lower than those of the control group, however, such changes were not found in animals of the 1,000 mg/kg group. Consequently, these findings were considered not to be caused by the test substance.

 

6) Changes observed in animals that were sacrificed after the completion of administration period were not found those sacrificed after the completion of recovery period, therefore, the changes were reversible.

 

7) Changes with statistically significant difference were found in animals that were sacrificed after the completion of administration and recovery periods except the above findings, however, these changes were not dose-dependent and had no related changes, therefore, the changes were considered not to be problem.

 

8) In conclusion, the no-observed-adverse-effect level (NOEL) for rats in a 28-day repeated oral administration of 1, 2-diphenoxyethane was concluded to be 100 mg/kg.