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Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Remarks:
28-Day Repeated Dose Toxicity Study
Type of information:
experimental study
Adequacy of study:
key study
Study period:
31 May 1999 - 1 November 1999
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study

Data source

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

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
other: The Guidelines for screening toxicity testing of chemicals that describe the partially revised test procedures required for testing new chemical compounds (establishment of the method for screening toxicity testing)
Version / remarks:
(Kanpogyo No. 700, Yakuhatsu No. 1039, 61 Kikyoku No. 1014: December 5, 1986)
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
-
EC Number:
439-270-3
EC Name:
-
Cas Number:
260408-02-4
Molecular formula:
CAS formula: (C12 H10 O4 S . C6 H6 O . Cl5 P . Cl H4 N)x Molecular formula of the reaction products: (C12 H10 N O2 P)n (n=3-15)
IUPAC Name:
439-270-3
Test material form:
solid
Details on test material:
- Appearance: Yellowish-white, semisolid
- Storage condition: At room temperature away from light

Test animals

Species:
rat
Strain:
Sprague-Dawley
Remarks:
Crj:CD(SD)IGS
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Japan, Co., Ltd.,
- Age at study initiation: 6 weeks
- Weight at study initiation: 225.0-258.5g (males); 141.6-185.3g (females)
- Housing: Animals were housed individually in stainless steel bracket cages
- Diet (ad libitum)
: Powdered diet for experimental animals (CE-2, Clea Japan, Inc, Lot No. E2029-F4)
- Water (ad libitum): Tap water
- Acclimation period: 12 days

DETAILS OF FOOD AND WATER QUALITY:
Analysis for impurities and contaminants was performed in diet and water. All results were within the permitted levels specified in this facility and fulfilled the water quality standards specified in the ministerial ordinance, and there were no abnormal values considered to have affected the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24
- Humidity (%): 41 -70
- Air changes (per hr): 10 or more times per hour
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: feed
Vehicle:
acetone
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The test substance was dissolved in acetone in 5% amount of the base diet and mixed with powdered diet (Clea Japan Inc.: 2-20-14, Aobadai, Meguro-ku, Tokyo) for experimental animals. The mixture was mixed in a rotating drum for 30 min (60 min for a dose of 20000 ppm), and left for more than 12 hours to evaporate acetone. Preparation was made per dose. The prepared diet was collected in a plastic bag, and stored away from the light (in containers).

DIET PREPARATION
- Rate of preparation of diet (frequency): once every 10 days
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The stability, concentration, and homogeneity of the test substance in diet were analyzed.
The results confirmed that the test substance in the diet at 2000 or 20000ppm was stable at room temperature for at least 10 days.
Each diet batch concentration of SPS-100 was 80-85.7% of the target range.
The coefficient of variation of the test substance concentration, which was determined at for the upper, middle and lower layer) of the preparation diet of each dose, was 2.4-4.9%. Both the concentration and homogeneity were within the acceptable limits
Duration of treatment / exposure:
Test duration: 28 days
Frequency of treatment:
Daily
Doses / concentrationsopen allclose all
Dose / conc.:
2 000 ppm
Remarks:
nominal in diet
Dose / conc.:
7 000 ppm
Remarks:
nominal in diet
Dose / conc.:
20 000 ppm
Remarks:
nominal in diet
No. of animals per sex per dose:
6/sex/dose (low and mid dose)
6/sex/dose (control and high dose)
6/sex/dose (control (14 day recovery group) and high dose (14 day recovery group))
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale: A dose range finder (not reported) indicated that no overt toxic signs were manifested up to the highest dose 30000 ppm over a 1 week period. Therefore the highest dose selected in the present study was 20000 ppm, the maximum limit dose specified in the KaShin Law guideline.
- Fasting period before blood sampling for clinical biochemistry: 20 hours.
- Following the administration period, a 14-day recovery period was set for 6 animals of each sex in the 0 and 20000 ppm groups.

Examinations

Observations and examinations performed and frequency:
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Animals were observed for clinical signs including mortality and moribundity, twice (a.m. and p.m.) daily in the administration period, and once (a.m.) daily in the recovery period.

BODY WEIGHT: Yes
- Time schedule for examinations:
Animals were weighed prior to treatment on day 0, and thereafter once weekly during treatment and recovery periods.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: 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: Yes


OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Eye exemination was performed once prior to the start of administration, in week 4 of administration and week 2 of recovery. The anterior portion of the eye (cornea, conjunctiva, sclera, iris, lens) was observed using a ophthalmoscope, and the fundus were observed under mydriasis using a midriatic agent.
- Dose groups that were examined: All

HAEMATOLOGY: Yes
- Time schedule for collection of blood: At the end of the treatment or recover period.
- Anaesthetic used for blood collection: Yes (ether)
- Animals fasted: Yes (20 hours)
- How many animals: All
- Parameters: Blood was collected into EDTA-2K-treated tubes or 3.8% sodium citrate-treated tubes.The following parameters were examined.
EDTA-2K-treated blood: erythrocyte count (RBC), leukocyte count (WBC) and platelet count, hematocrit (Ht), hemoglobin (Hb), mean corpuscular volume (MCV). mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), differential leukocyte percentage (Lympho, Eosino, Mono, Baso, Stab, Seg) (Wright stain), reticulocyte ratio.
3.8% sodium citrate-treated blood: prothrombin time (P T) and activated partial thromboplastine time (APTT)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: At the end of the treatment or recover period.
- Animals fasted: Yes (20 hours)
- How many animals:All
- Parameters: Blood was either treated with heparin sodium or left untreated, samples were centrifuged (3000rpm, 150, approximately 10 min), and the serum or plasma was obtained.
Serum: glutamic-oxaloacetic transaminase (GOT) and glutamic-pyruvic transaminase (GPT), alkaline phosphatase (ALP), 7 -glutamyl transpeptidase (7-GTP) , cholinesterase (ChE) , total protein, albumin, glucose, total cholesterol, triglyceride, phospholipid , total bilirubin, blood urea nitrogen (BUN), creatinine, inorganic phosphorus, calcium (Ca), sodium (Na), potassium (K), chloride (Cl), protein fraction (albumin, ß 7 -globulin), A/G ratio
Plasma: lactate dehydrogenase (LDH), creatine phosphokinase (CPK)


URINALYSIS: Yes
- Time schedule for collection of urine: After the treatment or recovery period.
- Animals fasted: Yes (3 hours for fresh urine, 17 hours for accumulated urine)
- Metabolism cages used for collection of urine: Yes
- Parameters: Fresh urine; occult blood, ketone body. glucose, protein, pH, urobilinogen, bilirubin, sediment (microscopy, optical microscope), and color.
Accumulated urine: urine volume,specific gravity,sodium, potassium, and chloride
Sacrifice and pathology:
GROSS PATHOLOGY: Yes

The body surface, and the internal organs/ tissues of the cranium, thorax and abdomen were macroscopically examined.
Organ weights: Brain, pituitary, thyroid gland (including parathyroid), thymus, salivary gland (submaxillary and sublingual gland), heart, liver, spleen, kidney* adrenal* testis* prostate, epididymis* seminal vesicle, ovary* and uterus. *Bilateral organs were weighed separately

HISTOPATHOLOGY: Yes
Examined: Lung, trachea, bronchus, tongue, esophagus, stomach, duodenum, jejunum, ileum, cecum, colon, rectum, spleen, urinary bladder, bone and bone marrow (sternum and femur), skeletal muscle (femoral muscle), thoracic aorta, spinal cord, sciatic nerve, vas deferens, vagina, mammary gland, skin, submaxillary and mesenteric lymph node, eye ball (both, including optic nerve) and Harderian gland (both).

Histopathological examination was performed on all organs/ tissues of all males and females in the control and 20000 ppm groups, and on the liver of males and females in the 2000 ppm, 7000 ppm and recovery groups as treatment-related effects were observed in the liver of the animals in the 20000 ppm group. The organs/ tissues were embedded in paraffin, sectioned, and stained with Hematoxylin and Eosin. Further, the uterus, in which macroscopical abnormality was noted, was also examined histopathologically.
Statistics:
Quantitative data obtained in this study were compared to those of the controls using the following statistical tests. The level of significance was set to < 5% and < 1%.

In 28-day treatment phase, data distribution was tested by Bartlett's test, and then one way layout analysis of variance was applied in case of homogenous variance. If significant difference was observed between groups, means were compared pair-wise between each treatment group and control group by Dunett's method.
In case of heterogeneous variance, Kruskal-Wallis H-test was applied; and if significant difference was observed between groups, means were compared pair-wise between each treatment group and control group by Dunnet's test of rank order.
In 14-day recovery phase, data distribution was tested by F-test, and then the treatment group was compared to the control group by Student t-test in case of homogenous variance or Aspin-Welch's t-test in case of heterogeneous variance.

Qualitative data in urinalysis, general conditions, ophthalmoscopy, necropsy, and histopathology were not statistically analyzed.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Description (incidence and severity):
No abnormalities were observed in males or females in the control group or each treatment group throughout the treatment and recovery periods.
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Description (incidence and severity):
In males and females, the body weights of treatment groups did not significntly differ from those of the controls throughout the treatment and recovery periods
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
In males and females, the food consumption of treatment groups did not significantly differ from those of the controls throughout the treatment and recovery periods.
Food efficiency:
no effects observed
Description (incidence and severity):
In males and females, the food efficiency of treatment groups did not significantly differ from those of the controls throughout the treatment and recovery periods.
Ophthalmological findings:
no effects observed
Description (incidence and severity):
No abnormalities were observed in males or females in the control group or each treatment group at the examination in week 4 of administration or week 2 of recovery.
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
A significantly lower MCH level was observed in females at 20000ppm at the end of treatment. Significantly lower MCH and MCHC levels in males at 20000ppm, and significantly shortened prothrombin time in females at at 20000 ppm were observed only in the recovery group.
The decreased MCH level at treatment end recorded for females at 20000ppm was not considered treatment-related either because RBC and Hb values were not affected and because of the absence of histopathologic effects on the bone marrow or other hematopoietic system.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Significantly higher Y -GTP and ChE levels were recorded for females at 20000ppm.
A significant decrease in glucose level was noted for males at 7000ppm, but this change was considered incidental because of the absence of a similar change in the 20000ppm group. A significant increase in y -GTP level was noted in males at 20000 ppm, which was considered incidental because of the absence of a similar change in the treament groups and because the value was lower than those found in the 20000 ppm group and the control group recorded.
Urinalysis findings:
no effects observed
Description (incidence and severity):
In all of the parameters examined at both treatment end and recovery end, no changes were found in males or females of the treatment groups compared to controls.
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
In all the organs weighed, no changes were observed in males or females of the treatment groups compared to controls.
A statistically significant reduction in the absolute right kidney weight was observed and increases in relative liver weight and in the absolute left and right kidney weight in females were recorded at 20000 ppm in the recovery group.

The increased liver weight in females was not considered treatment-related either because the slight increase was noted only for the relative weight or because of the absence of histopathological alteration in the liver. the absolute kidney weight was decreased in males and increased in females at 20000ppm. The changes in kidney weight were considered incidental either because of the absence of alteration in the other kidney-related parameters and because similar changes were not observed at the end of treatment period.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
At treatment end, softness and atrophy of the testes and epididymides was observed in 1 male at 20000 ppm and swelling of the uterus in 2, 1 and 1 females at 2000, 7000 and 20000ppm, respectively.
The incidence of these changes did not correlate to the doses, and therefore, they were considered to be spontaneous in nature.
In the control group, swelling of the uterus was observed in one female after recovery, which however, was a change that commonly occurs spontaneously.
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
At treatment end, centrilobular hepatocyte hypertrophy (minimal) was observed in the liver of 6/6 males at 20000 ppm. Other findings noted in the treatment groups were considered incidental lesions because they were commonly noted in the controls and did not correlate to doses. No treatment-related changes were observed in the recovery group

Effect levels

open allclose all
Dose descriptor:
NOAEL
Effect level:
7 000 ppm
Based on:
test mat.
Sex:
male
Basis for effect level:
organ weights and organ / body weight ratios
Key result
Dose descriptor:
NOAEL
Effect level:
20 000 ppm
Based on:
test mat.
Sex:
female
Basis for effect level:
other: Absence of adverse effects up to and including the highest dose level tested. (equivalent to 2090 mg/kw bw/day)

Target system / organ toxicity

Critical effects observed:
no

Applicant's summary and conclusion

Conclusions:
Based on the results of a 28-Day Repeated Dose Toxicity Study, the NOAEL of SPS-100 is considered to be 627 mg/kg bw/day in male rats and 2090 mg/kg bw/day in female rats
Executive summary:

A 28-Day Repeated Dose Toxicity Study with a 14 day recovery group was performed in accordance with GLP principles. Male and female rats (6/sex/dose) were exposed via diet to 0 (control), 2000 , 7000 and 20000 ppm of the test substance. For the control and 20000 ppm group an additional 6/sex/dose rats were added for the 14 day recovery group. The stability, concentration, and homogeneity of the test substance in diet were analyzed and were within acceptable limits. There were no unscheduled deaths. No treatment-related changes were noted the following parameters investigated in this study (i.e. mortality/moribundity, body weight, food consumption, food efficiency, hematology, ophthaloscopy, urinalysis, clinical biochemistry, macroscopic examinations, organ weights). Centrilobular hepatocyte hypertrophy was observed in males at 20000 ppm equivalent to 1910 mg/kg bw/day (high dose). This was reversible within 14 days as no treatment-related changes were observed in the recovery groups.


Based on the results of this 28-day repeated dose toxicity study the NOAEL of SPS-100 is considered to be 627 mg/kg bw/day in male rats and 2090 mg/kg bw/day in female rats.