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Repeated dose toxicity: oral

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Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Study conducted to GLP and in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not effect the quality of the relevant results. This study has been performed according to OECD 407 guidelines and GLP principles. Reliability given as 2 due to results being obtained on a read-across substance.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1992

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Deviations:
not specified
Qualifier:
according to
Guideline:
other: Notification on Partial Revision of Testing Methods Relating to New Chemical Substances (Notification No 700 of Kanpogyo, No 1039 of Yakuhatsu and No 1014 of 61 Kikyoku, December 5 1986
Deviations:
not specified
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Test material form:
other: Solid

Test animals

Species:
rat
Strain:
Crj: CD(SD)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Male and female Crj:CD(SD) strain rats were supplied by Charles River Japan Inc
- Age at study initiation: five weeks of age
- Weight at study initiation: males 149 - 167g and females 121 - 142g..
- Housing: The animals were housed individually in suspended wire mesh cages.
- Diet : Food MF pelleted diet (Oriental Yeast Co, Ltd) was allowed throughout the study.
- Water : Free access to mains chlorinated drinking water was allowed throughout the study.
- Acclimation period: At least five days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): Set to achieve limits of 23 ± 2°C
- Humidity (%): Set to achieve limits of 55 ± 10%
- Air changes (per hr): 10 - 15 air changes per hour
- Photoperiod (hrs dark / hrs light): artificial light for twelve hours light (07:00 to 19:00) and twelve hours darkness.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Remarks:
purified
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The test item was prepared at the appropriate concentrations dissolved in purified water.
The stability of the test item formulations was confirmed by Chemicals Inspection and Testing Institute, Japan .
Results showed the formulations to be stable for at least 7 days and therefore solutions were prepared once a week.

The test item was administered daily by gavage using a Nelaton catheter and syringe. Control animals were treated in an identical manner with purified water.
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
The test item was administered to rats for 28 days. A recovery group for the control and high dose group (1000mg/kg/day) included a 14 day recovery period after treatment ceased.
Frequency of treatment:
Daily during the 28 day treatment period
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
8 mg/kg bw day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
40 mg/kg bw day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
200 mg/kg bw day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
1000 mg/kg bw day
Basis:
actual ingested
No. of animals per sex per dose:
Six males and six females per dose group. An additional control and 1000mg/kg bw day recovery group also included six males and six females.
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels were chosen based on the results of a 14-day range finding study.
- Rationale for animal assignment: Healthy animals were allocated to groups using bodyweight-stratified random sampling.
- Rationale for selecting satellite groups: The high dose (1000 mg/kg bw day) and control recovery groups were conducted to comply with the Japanese MHW method for use in a Japanese MITI/MHW notification
- Post-exposure recovery period in satellite groups: 14 days

Examinations

Observations and examinations performed and frequency:
CLINICAL OBSERVATIONS:
The general condition of all animals was examined daily.

BODY WEIGHT:
Individual body weights of all animals were recorded on Day 1 (at the start of dosing) and then days 3, 5, 8, 10, 12, 15, 17, 19, 22, 24, 26 and 28.
Individual body weights of all animals from the recovery groups were recorded on Days 1 (at the start of the recovery period), 3, 5, 8, 10, 12 and 14 of the recovery period.
Bodyweights were also recorded immediately before necropsy.

FOOD CONSUMPTION:
Food consumption was measured twice a week during the dosing and recovery periods.

LABORATORY INVESTIGATIONS:
Haematological and blood chemical investigations were performed on all males and females at the end of the dosing and recovery periods. Animals were fasted overnight and blood samples taken via the abdominal aorta from rats under anesthesia.

HAEMATOLOGY:
The following parameters were measured on blood collected into tubes containing potassium EDTA anti-coagulant:
Haemoglobin (Hb)
Red blood cell count (RBC)
Haematocrit (Ht)
Erythrocyte indices
- mean corpuscular haemoglobin (MCH)
- mean corpuscular volume (MCV)
- mean corpuscular haemoglobin concentration (MCHC)
White blood cell count (WBC)
Differentiation of leukocyte count
- Stab neutrophil (Stab)
- Segmental neutrophil (Seg)
- lymphocytes (Lymph)
- monocytes (Mono)
- eosinophils (Eos)
- basophils (Bas)
Platelet count
Reticulocyte count (%)
Prothrombin time (PT)
Activated partial thromboplastin time (APTT)

BLOOD CHEMISTRY:
Serum samples were separated from the blood samples for hematology and examined as follows:
Glutamic Oxaloacetic Transaminase (GOT) (also known as Aspartate aminotransferase (ASAT))
Glutamic Pyruvic Transaminase (GPT) (also known as Alanine aminotransferase (ALAT))
γ Glutamic Pyruvic Transaminase (γ GTP)
Cholinesterase (Che)
Blood Urea Nitrogen (BUN)
Calcium (Ca)
Glucose
Inorganic phosphorus (IP)
Total protein (T Protein)
Albumin
Albumin/Globulin ratio (A/G ratio)
Alkaline phosphatase (AP)
Sodium (Na)
Creatinine
Potassium (K)
Total cholesterol ( T-Cho)
Triglyceride (TG)
Chloride (Cl)
Total bilirubin (T Bil)

URINALYSIS
BLOOD CHEMISTRY:
Sixteen hour urine samples were collected once at the end of the treatment and recovery period from each animal in individual metabolic cages and examined as follows:
Volume
Colour
pH
Protein
Ketone bodies
Bilirubin
Occult blood
glucose
Urobilinogen
















Sacrifice and pathology:
PATHOLOGY:
Animals were necropsied at the end of either the treatment or recovery period.

ORGAN WEIGHTS:
The following organs were weighed wet for all animals.
Brain
Liver
Spleen
Kidneys
Adrenals
Testis or Ovary


HISTOPATHOLOGY:
The following organs and tissues of all animals were preserved in 10% formalin fixative:
Brain
Pituitary
Eyeball
Thyroid (with parathyroid)
Heart
Lung
Liver
Kidneys
Spleen
Adrenals
Stomach
Intestine (duodednum - rectum)
Testis or Ovary
Urinary bladder
bone marrow (femur)
Gross lesions

Light microscopic examinations were performed on the following organs and tissues after paraffin embedding and sectioning follwed by hematoxylin and eosin staining
Treatment Period
Control and 1000 mg/kg bw day: Liver, Spleen, Kidneys, Heart, Stomach, Intestine ( duodenum, jejenum, ileum, cecum, colon, rectum) and adrenal.
200 mg/kg bw day : Liver, Spleen, Kidneys (males only)
40 mg/kg bw day : Spleen (females only)

Recovery animals
Control and 1000 mg/kg bw day: Liver, Spleen, Kidneys (males only).

In addition the spleens in the following groups were also stained with Berlins blue
1000 mg/kg bw day group
1000 mg/kg bw day recovery group
200 mg/kg bw day group
40 mg/kg bw day group
8 mg/kg bw day group (females only)
Control group
Control recovery group

Statistics:
Bodyweight, food consumption, haematological examinations, blood chemistry, urine volume and organ weights, Bartletts test for homogeneity of variance was used. If the variances were homogenous at the 5% significance level, one way analysis of varince was performed. When there were significant differences between the control and the dose groups in the analysis of variance, Dunnetts test between groups with equal number of data or Scheffes test between groups with equal number of data was carried out.
If the variances were not homogenous, Kruskal - Walliss test was used. When there were significant differences between the control group and each group in Kuskal-Walliss test, non parametric Dunnetts test between groups with equal number of data or non parametric Scheffes test between groups with unequal number of data was performed.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
See details on results
Mortality:
mortality observed, treatment-related
Description (incidence):
See details on results
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 examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
See details on results
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
See details on results
Urinalysis findings:
no effects observed
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
See details on results
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
See details on results
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
See details on results
Histopathological findings: neoplastic:
not examined
Details on results:
ADULT RESPONSES:
MORTALITY:
There were no deaths during the study in any dose group.

CLINICAL OBSERVATIONS:
During the treatment period
Male: Salivation was observed in the 200 mg/kg bw/day (1/6 after dosing on day 26 and 27) and 1000 mg/kg bw/day (12/12 after dosing on day 4 or 6 and occasionally before dosing from day 16)
Female : Salivation was observed in the 1000 mg/kg bw/day (12/12 after dosing on day 3 or 6 and occasionally from day 17). One female from the 8 mg/kg bw/day showed decreased spontaneous locomotion, increased respiratory rate, abnormal gait and sub normal temperature.

During the recovery period
No abnormalities were detected in rats of either sex.

BODY WEIGHT:
During the treatment period
No abnormalities were detected in rats of either sex.

During the recovery period
No abnormalities were detected in rats of either sex.

FOOD CONSUMPTION:
During the treatment period
No abnormalities were detected in rats of either sex.

During the recovery period
No abnormalities were detected in rats of either sex.

LABORATORY INVESTIGATIONS:
HEAMATOLOGY:
At the end of the dosing period
Male: There were decreases in red blood cell count, haemoglobin concentration and haematocrit value, an increase in reticulocyte and prolonged prothrombin time in the 1000 mg/kg bw /day group.
Female: There were decreases in red blood cell count, haemoglobin concentration and haematocrit value and mean corpuscualr haemoglobin and an increase in reticulocyte in the 1000 mg/kg bw /day group. In addition, a decrease in lymphocytes and an increase in segmental neutrophils were found in the 1000 mg/kg bw/day. Another change included a prolongation of activated thromboplastin time in the 40 mg/kg bw /day group.

At the end of the recovery period
Male: There were decreases in red blood cell count and haemoglobin concentration, and an increase in platelet count in the 1000 mg/kg bw /day group.


BLOOD CHEMISTRY:
At the end of the dosing period
Male: .There was a decrease in alkaline phosphatase in the 200 mg/kg bw /day group.
Female: There was a decrease in GOT in the 8 mg/kg bw /day and 200 mg/kg bw /day groups. A decrease in GOT and an increase in glucose in the 40 mg/kg bw /day group.

At the end of the recovery period
Male: No abnormalities were detected.
Female : There was an increases in alkaline phosphatase and Na in the 1000 mg/kg bw /day group.

URINANALYSIS
At the end of the dosing period
No abnormalities were detected in rats of either sex.

At the end of the recovery period
No abnormalities were detected in rats of either sex.


PATHOLOGY:
NECROPSY:
At the end of the dosing period
Male: Blackish change (5/6) of the spleen and apparent spotty pattern of surface (2/6) in the kidneys were noted in the 1000 mg/kg bw /day group.
Female : Blackish change of the spleen in the 200 mg/kg bw /day group (1/6) and 1000 mg/kg bw /day group(6/6) were observed.

At the end of the recovery period
Male : Whitish nodule in the kidneys in the 1000 mg/kg bw /day group (1/6).
Female : No abnormalities were detected.

ORGAN WEIGHTS:
At the end of the dosing period
Male: There were increases in absolute and relative weights of the liver and spleen, and an increase in absolute weight of the kidneys in the 1000 mg/kg bw /day group. An increase in absolute weight of the kidneys was observed in the 200 mg/kg bw /day group.
Female : There was an increase in absolute spleen weight in the 200 mg/kg bw /day group, and an increases in absolute and relative weights of the liver and spleen in the 1000 mg/kg bw /day group.

At the end of the recovery period
Male : No abnormalities were detected.
Female : There was an increase in absolute and relative spleen weights and an increase in absolute adrenal weight in the 1000 mg/kg bw /day group.

HISTOPATHOLOGY:
At the end of the dosing period
Male: Congestion (6/6) and increased haemosiderin- laden cells (6/6) in the spleen, increased eosinophilic bodies (6/6) in the kidneys and swelling of hepatocytes (4/6) were observed in the 1000 mg/kg bw /day group. Microgranuloma was obserevd (1/6) in the liver in the 200 mg/kg bw /day group.
Female : Congestion (3/6) and increased haemosiderin- laden cells (2/6) in the spleen, swelling of hepatocytes (2/6) were observed in the 1000 mg/kg bw /day group. Other changes included erilobular lipid droplets (1/6) in the liver in the control group and perilobular lipid droplets (3/6) in the liver in the 200 mg/kg bw /day group.

At the end of the recovery period
Male : Increased haemosiderin- laden cells (6/6) and congestion (5/6) in the spleen were observed in the 1000 mg/kg bw /day group. Basophilic change of the tubular epithelium (1/6) and fibrosis (1/6) in the kidneys in the control group. Nephroblastoma (1/6) in the kidneys in the 1000 mg/kg bw /day group.
Female : Perilobular lipid droplets (3/6) and microgranuloma (1/6) in the liver in the control group and perilobular lipid droplets (1/6) in the liver in the 1000 mg/kg bw /day group.




Effect levels

Dose descriptor:
NOEL
Effect level:
40 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Treatment related findings in animals of either sex at 200 and 1000 mg/kg bw day

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
The oral administration of the test substance to rats by gavage resulted in treatment-related findings in animals of either sex in the 200 mg/kg bw/day and 1000 mg/kg bw/day groups. A ‘No Observed Effect Level’ (NOEL) for systemic toxicity was considered to be 40 mg/kg bw/day.
Executive summary:

Introduction.

The purpose of the study was to assess the toxicity of the test substance study by observing the functional and morphological changes in animals receiving repeated doses for 28 days. The study was conducted in accordance with the "28 day Repeated Dose Toxicity Study in Mammalian Species" prescribed in "The Notification on Partial Revision of Testing Methods Relating to New Chemical Substances" and is compatible with the requirements of the OECD Guidelines for Testing of Chemicals No. 407 Repeated Dose Oral Toxicity Rodent : 28 day or 14 day study (May 12 1981).

Methods.

A 28 day repeated dose oral toxicity study of the test substance followed by a 14 day recovery test was conducted in male and female Crj : CD(SD) rats. The test item was administered by gavage to four groups, each of six male and six female at either 0, 8, 40, 200 or 1000 mg/kg bw/day. Separate recovery groups for the control and 1000 mg/kg bw /day each of six male and six females were provided.

The control group received the vehicle purified water.

Clinical signs, body weight change and food consumption were monitored during the study.

Haematology and blood chemistry and urinanalysis were evaluated prior to termination on all animals from each group.

Animals were terminated on Day 28, all animals were subjected to a necropsy examination and histopathological evaluation of selected tissues was performed.

Results.

There were no unscheduled deaths.

Salivation was observed in males of the 200 and 1000 mg/kg bw/day groups, and in females of the 1000 mg/kg bw/day group.

No treatment related effects were observed in body weight and food consumption during the dosing period.

Laboratory Investigations:

Haematological examination revealed the following observations in the 1000 mg/kg bw day group at the end of the dosing period: decrease in red blood cell count, haemoglobin concentration and haematocrit value, and an increase in reticulocyte in both sexes : prolonged prothrombin times in males : and decrease in mean corpuscular haemoglobin concentration and lymphocyte ratio, and an increase in segmental neutrophil ratio in females.

At the end of the dosing period there was an increase in the spleen weight of females at 200 mg/kg bw/day. Spleen and liver weight in males and females of the 1000 mg/kg bw/day was increased.

Necropsy at the end of the dosing period revealed blackish change of the spleen in females of the 200 mg/kg bw/day and in both males and females of the 1000 mg/kg bw day.

Histopathologcal examination at the end of the dosing period revealed congestion and increased hemosiderin laden cells of the spleen in females of the 200 mg/ kg bw/day and 1000 mg/kg bw/day. Swelling of hepatocytes and congestion and increased

hemosiderin laden cells of the spleen in both males and females of the 1000 mg/kg bw/day.

In the 1000 mg/kg bw/day group at the end of the recovery period the following findings persisted : decrease in red blood cell count and haemoglobin concentration, congestion and increased hemosiderin laden cells of the spleen in males and an increased spleen weight in females.

Conclusion. The No Observed Effect Level’ (NOEL) for systemic toxicity was considered to be 40 mg/kg bw/day.