Registration Dossier

Administrative data

Description of key information

Subacute:
SAS-40: OECD 407: NOAEL 50 mg/kg
SAS-305: OECD 407: NOAEL: 15 mg/kg
Phenyl-tolyl-ethane: OECD 422: NOAEL: 30 mg/kg
SAS-296: OECD 422: LOAEL males: 12.5 mg/kg, NOAEL females: 50 mg/kg
Chronic:
SAS-296: similar to OECD 453: NOAEL: 4.83~5.72 mg/kg/day

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
chronic toxicity: oral
Remarks:
combined repeated dose and carcinogenicity
Type of information:
migrated information: read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Study period:
1981-06-04 - 1983-06-17
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: This study has been performed similar to OECD and/or EC guidelines. Nevertheless, according to the ECHA's practical guide 6: "How to report read-across and categories" the maximum for read-cross is 2.
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies)
Deviations:
no
GLP compliance:
no
Limit test:
no
Species:
rat
Strain:
Fischer 344/DuCrj
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories Japan, Inc.
- Age at study initiation: 4 weeks old
- Weight at study initiation: 100 to 118 g (male) and 90 to 106 g (female).
- Fasting period before study: no
- Housing: wire mesh cage (3 per cage up to the first 6 months and 2 per cage thereafter)
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 1 week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22±1°C
- Humidity (%):55±5% of relative humidity
- Air changes (per hr): 13 times/hour of ventilation
- Photoperiod (hrs dark / hrs light): 12 hours light period (7:00 am to 7:00 pm)
Route of administration:
oral: feed
Vehicle:
other: CRF-1(feed): 95%, Olive oil: 3% and Cellulose: 2%
Details on oral exposure:
The test article was mixed with basic feed (CRF-1: 95%, Olive oil: 3% and Cellulose: 2%) to obtain designated concentrations and was fed ad libitum to the animals for 104 weeks. Control animals were fed ad libitum with the basic feed only. The test article mixture was prepared every 2 months by Oriental Yeast Co., Ltd. At the time of mixing, feed was randomly sampled to confirm the mixture rate of the test article at Research Laboratory, Japan Oil Chemicals, Inc.
Analytical verification of doses or concentrations:
yes
Duration of treatment / exposure:
24 month
Frequency of treatment:
ad libitum in the diet
Remarks:
Doses / Concentrations:
30, 100, 300 ppm
Basis:
nominal in diet
No. of animals per sex per dose:
Number of animals in 1 group was to be 80 each for male and female rats, and 10 male and female animals each were allocated to a 26-week (6-months) dose group, 52-week (12-month) dose group, and 78-week (18-month) dose group, and 50 male and female animals each were allocated to a 104-week (24-month) dose group. The allocation was conducted with a random sampling method.
Control animals:
yes
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: once per day

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once per day

BODY WEIGHT: Yes
- Time schedule for examinations: 2 times per week from the initiation of administration to Week 26, and once per week thereafter until test completion.

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: No data

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: Food efficiency and test article intake were calculated based on the body weight and food consumption.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: Daily water consumption was measured once every 4 weeks (mL/rat/day) for male and female rats (12 each) of each group from the initiation of the study to Week 26, and for male and female rats (10 each) of each group thereafter until the test completion.

OPHTHALMOSCOPIC EXAMINATION: No


HAEMATOLOGY: Yes
- Time schedule for collection of blood: Urinalysis, and hematology and clinical chemistry examinations were conducted to 10 male and 10 female rats of each group at Week 26 (6 months), Week 52 (12 months), Week 78 (18 months), and Week 104 (24 months).
- Anaesthetic used for blood collection: Yes: under ether anesthesia
- Animals fasted: Yes
- How many animals: see above
- Parameters checked in table [1] were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Urinalysis, and hematology and clinical chemistry examinations were conducted to 10 male and 10 female rats of each group at Week 26 (6 months), Week 52 (12 months), Week 78 (18 months), and Week 104 (24 months).
- Animals fasted: Yes
- How many animals: see above
- Parameters checked in table [2] were examined.

URINALYSIS: Yes
- Time schedule for collection of urine: Urinalysis, and hematology and clinical chemistry examinations were conducted to 10 male and 10 female rats of each group at Week 26 (6 months), Week 52 (12 months), Week 78 (18 months), and Week 104 (24 months).
- Metabolism cages used for collection of urine: No
- Animals fasted: No data
- Parameters checked in table [3] were examined.

NEUROBEHAVIOURAL EXAMINATION: No


Sacrifice and pathology:
GROSS PATHOLOGY: Yes
Necropsy:
Necropsy was conducted immediately after finding dead animals and after exsanguinating the moribund animals and the scheduled sacrifice animals. The cranial, thoracic, and abdominal organs and tissues were observed in detail for the presence abnormalities and tumor.

Organ Weight:
For scheduled sacrifice animals, the brain, pituitary gland, thyroid, thymus, heart, lung, liver, spleen, kidneys, adrenal gland, testis, and ovary were excised to measure wet weight (absolute weight). Organ-body weight ratio (relative weight) was calculated based on the wet weight and body weights determined after overnight fasting.

HISTOPATHOLOGY: Yes
For the humane sacrifice animals and scheduled sacrifice animals, the brain, pituitary gland, thyroid, thymus, heart, lung, liver, spleen, kidneys, adrenal gland, testis, ovary, esophagus, stomach, duodenum, jejunum, colon, prostate, urine, submandibular gland, pancreas, spinal cord, sciatic nerve, eyes, skin, mammalian gland, urinary bladder, trachea, submandibular lymph node, sternum, sternal marrow, skeletal muscle, and abnormal regions were fixed with phosphate buffered 10% formalin, and hematoxylin-eosin stain specimens were prepared by the usual method followed by microscopy examination. Dead animal were treated in a similar manner as much as possible.
Other examinations:
Organ Accumulation Analysis:
At each sacrifice, the liver and kidneys were collected from 3 animals each per group to analyze organ accumulation of test article. Analysis was conducted at Japan Oil Chemicals, Inc.
Statistics:
For the study results which were converted to numerical value, group means and standard deviation were calculated by group for each test period, and a significance test was performed by t-test between the control group and each test-article dose group. If there was no normal distribution, asignificance test was conducted with t-test approximation method (Aspin-Welch method).
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:
no effects observed
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY:
No differences for clinical signs were observed between each test-article dose group and the control group. As to mortality conditions, no difference for time and number of death were observed between each test-article dose group and the control group.

BODY WEIGHT AND WEIGHT GAIN:
No difference for body weight change was observed for both male and female rats of the 30 and 100 ppm groups in comparison with the control group, while male and female rats of the 300 ppm group showed tendency of suppression of body weight gain.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
For food consumption, food efficiency, and water consumption, no effect of the test article administration was observed in both male and female rats of the test-article dose groups.

FOOD EFFICIENCY:
For food consumption, food efficiency, and water consumption, no effect of the test article administration was observed in both male and female rats of the test-article dose groups.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study):
For food consumption, food efficiency, and water consumption, no effect of the test article administration was observed in both male and female rats of the test-article dose groups.

OPHTHALMOSCOPIC EXAMINATION:
not examined

HAEMATOLOGY, Clinical Chemistry and Urinanalysis:
The results of urinalysis, and hematology and clinical chemistry examinations show significant differences in some parameters in male and female rats of test-article dose groups in comparison with the control group. These changes, however, were very mild in magnitude and had no correlation with sex, doses, and dose period was observed. All parameters indicated no effect of test article administration.

NEUROBEHAVIOUR:
not examined

ORGAN WEIGHTS:
Although liver weight of 100-ppm (male) and 300-ppm dose groups at 6 month, there were no histopathological findings which suggest body weight gain and no significant change was observed for body weight on and after 12 month. Although some organs exhibited significant differences between the test-article dose groups and the control group, the differences were very slight in magnitude and had no correlation with sex, doses, and dose period. Therefore, the administration of the test article did not affect the organ weight.

GROSS PATHOLOGY:
In both survived and dead animals, no specific changes which are considered to be caused by the test article administration were observed although neoplastic changes which are considered to be due to aging were observed in the pituitary, thyroid, lung, liver, spleen, adrenal gland, testis, uterine, lymph nodes, thorax, abdominal cavity, subcutaneous area, skin, and pancreas.

HISTOPATHOLOGY: NON-NEOPLASTIC:
In both survived and dead animals, no specific changes which were likely to be caused by test article administration were noted.

HISTOPATHOLOGY: NEOPLASTIC (if applicable):
No difference was observed for the types and incidence of neoplasm between each test-article dose group and the control group. The test article had neither carcinogenicity nor carcinogenesis promotion. With respect to the tumors which were observed in this study, tumor-like lesions were observed in the various organs in male and female rats in addition to the high incidence of interstitial cell tumor in the testis in male rats of all the groups including the control group and adenoma in the pituitary gland and thyroid in male and female rats. These lesions were, however, incidentaloma which has been observed in rats frequently, and there were no specific tendencies in types or incidences of the tumors and there was no correlation with the doses. Hence, we have thought that the test article has no effect of carcinogenesis or carcinogenesis promotion.

HISTORICAL CONTROL DATA (if applicable): no data

OTHER FINDINGS:
.Organ Accumulation
At the time of sacrifice every 6 months, the liver and kidneys were removed from 3 animals per group and were weighed. After portions of these organs was dissected for histopathological studies, the organs were homogenized to measure test article concentration in the organs. No accumulation of test article in the liver and kidneys was observed for both male and female rats of each group.
Dose descriptor:
NOAEL
Effect level:
4.83 - 5.72 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: tendency of suppression of body weight gain at 300 ppm
Critical effects observed:
not specified

For tables please refer to attached background material.

Body Weight Change (Table 1):

Although transient body weight gain was observed in male rats of the 100-ppm dose group at the early time of administration in male and female rats of the 30- and 100 ppm dose groups, both groups showed similar body weight gain with the control group.

In male and female rats of the 300-ppm dose group, tendency of suppression of body weight gain was observed and a significant difference was observed in comparison with the control group from Week 28 to the completion of study in male rats and from Week 8 to Week 64 in female rats.

 

Food Consumption (Table 2):

Male and female rats of each test-article dose group showed tendency of slight decrease of food consumption and significant differences were observed sporadically or frequently, while no relationship with the dose was observed. Female rats of the 30-ppm dose group exhibited significant increase during latter period of administration.

 

Food Efficiency (Table 3):

Male and female rats of each test-article dose group showed significant increase or decrease of food efficiency in comparison with the control group during administration period, while no tendency of constant change was observed.

 

Water Consumption (Table 5):

Although some test-article dose group sporadically exhibited significant increase or decrease water consumption in comparison with the control group during administration period, no tendency of constant change was observed for both male and female rats of each test-article dose group.

 

Test Article Intake (Table 4):

The total test article intake during 104 weeks were 0.42, 1.40, and 4.21 g/rat in male and 0.31, 1.02, and 3.06 g/rat in female of the 30, 100, and 300-ppm dose groups, respectively. These values corresponded to 1.45, 4.83, and 14.93 mg/kg/day in male rats and 1.75, 5.72, and 17.59 mg/kg/day in female rats, respectively.

Result of Urinalysis (Table 6):

Although there was no significant difference between each test-article dose group and the control group in the qualitative examination on each test period, all groups resulted in the increase of urine protein over time.

Specific gravity decreased in male rats of the 300-ppm dose group at 6 months and in female rats of the 30- and 100-ppm dose groups at 18 months, and increased in male rats of the 30- and 100-ppm dose groups at 24 months.

 

Results of Hematology Examination (Table 7):

RBC decreased in male rats of the 100- and 300-ppm dose groups and in female rats of the 100-ppm dose group at 12 months.

 

Hemoglobin decreased in male rats of each test-article dose group at 12 months.

 

Hematocrit decreased in male rats of the 100-ppm dose groups at 12 months.

 

Reticulocyte rate increased in female rats of the 300-ppm dose group at 6 months and decreased in female rats of the 300-ppm dose group at 12 months and in male rats of the 300-ppm dose group at 18 months.

 

WBC showed no difference for both male and female rats between each test-article dose group and control group on all examination period.

 

For differential blood count, the ratio of lymphocytes increased in male rats of the 30-ppm dose group at 6 months and in female rats of the 30 and 300-ppm dose groups at 12 months, the ratio of neutrophils decreased in male rats of the 30-ppm dose group at 6 months (segmented), in female rats of the 300-ppm dose group at 12 months (stab, segmented), and in female rats of the 100-ppm dose group at 24 months (stab), and increased in female rats of the 100-ppm dose group at 24 months (segmented) and ratio of monocytes increased in female rats of the 300-ppm dose group at 18 months and in female rats of the 30 ppm at 24 months. Regarding the ratios of eosinophils and of basophils, there was no difference in male and female rats of each test-article dose group from those of the control group at all examination period.

 

Platelet increased in female rats of the 100-ppm dose group at 6 months and decreased in male rats of the 100- and 300-ppm dose groups at 12 months.

 

Results of Clinical Chemistry examination (Table 8):

GOT increased in female rats of the 100-ppm dose group at 12 months and decreased in male rats of the 300 ppm at 12 months and in male rats of the 100- and 300-ppm dose groups and in female rats of the 300-ppm dose group at 18 months.

Page 13

GPT increased in male rats of the 100-ppm dose group at 6 months and in female rats of the 30-ppm dose group at 12 months, and decreased in female rats of the 300-ppm dose group at 18 months.

 

ALP decreased in male rats of the 100 ppm and in female rats of all the test-article dose groups at 6 months, in female rats of the 100 and 300-ppm dose groups at 18 months, and in female rats of the 100-ppm dose group at 24 months, and increased in male rats of all the test-article dose groups at 12 months.

 

There was not observed any difference in LDH between all the test-article dose groups and the control group at all examination periods.

 

Cholinesterase decreased in male rats of the 100-ppm dose group at 6 months and in female rats of the 300-ppm dose group at 12 months, and increased in male rats of the 100-ppm dose group at 18 months and in female rats of the 100-ppm dose group at 24 months.

 

Leucine aminopeptidase decreased in male rats of the 30- and 100-ppm dose groups at 6 months and increased in male rats of the 100-ppm dose group from 12 months and in female rats of all the test-article dose groups at 12 months.

 

Total protein decreased in male rats of the 30-ppm dose group at 6 months.

 

Albumin increased in male rats of the 30- and 100-ppm dose groups at 12 months and A/G ratio increased concurrently. A/G ratio also increased in female rats of the 300-ppm dose group at 18 months.

 

Urea nitrogen increased in male rats of the 100- and 300-ppm dose groups at 6 months and in female rats of all the test-article dose groups at 12 months, and decreased in female rats of the 30-ppm dose groups at 18 months.

 

Creatinine decreased in female rats of the 300-ppm dose group at 24 months.

 

Uric acid decreased in male and female rats of the 100- and 300-ppm dose groups at 6 months and in male rats of the 30 ppm at 18 months.

 

Blood glucose level decreased in male rats of the 300-ppm dose group at 18 months.

 

Total cholesterol increased in male rats of all the treatment groups at 6 months and in male rats of the 100-ppm dose group at 18 months.

 

Triglyceride increased in male rats of the 100-ppm dose group at 6 months.

 

Free fatty acid increased in male rats of the 30-ppm dose group at 6 months and in male rats of the 300-ppm dose group at 12 months.

 

Regarding total fat, there was no difference between the test-article dose groups and the control group at all examination periods.

 

Total bilirubin increased in male rats of the 100 ppm at 12 months.

 

Sodium increased in male rats of the 300-ppm dose group at 6 months and decreased in female rats of the 100-ppm dose group at 18 months and in female rats of all the test-article dose groups at 24 months.

 

Potassium decreased in female rats of the 100- and 300-ppm dose groups at 6 months and increased in male rats of the 30-ppm dose group at 18 months and in female rats of the 300-ppm dose group at 24 months.

 

Chlorine increased in male rats of the 300 ppm at 6 months and decreased in female rats of the 100-ppm dose group at 12 months.

 

Calcium decreased in female rats of the 100 and 300-ppm dose groups at 6 months and increased in male rats of the 100-ppm dose group at 18 months.

 

Necropsy Findings (Table 9):

At 6 and 12 months, hematoma of the pituitary, swelling of the thyroid, hypertrophy of the adrenal gland, hypertrophy and softening of the testis, nodule in the abdominal cavity, nodule and tumor mass in the subcutaneous region, and tumor mass around the nasal cavity were sporadically observed without relationship with dose.

 

At 18 and 24 months, hypertrophy (atrophy for some animals) and softening which were considered to be due to aging were observed in almost all the animals. Hematoma of the pituitary gland, swelling and nodule on the thyroid, hypertrophy of the adrenal gland, nodule in the abdominal cavity, and nodule and tumor mass in the subcutaneous region were slight in severity but tended to increase over time. In addition, neoplastic change which was thought to be attributable to aging including hypertrophy of the pituitary gland, tumor mass and nodule of the lung, tumor mass and nodule of the liver, hypertrophy and blackening of the spleen, nodule of the uterine, hypertrophy of the lymph node, ulcer of the skin were observed sporadically without relationship with dose.

 

In the dead animals, unspecific neoplastic change which was thought to be attributable to aging as with changes observed at 18 and 24 months.

 

Organ Weight (Table 10):

For the brain, the absolute weight decreased in female rats of the 300-ppm dose group at 24 months and the relative weight increased in male rats of the 300-ppm dose group at 18 and 24 months.

 

For the pituitary gland, the relative weight increased in male rats of the 300-ppm dose group at 6 months, and the absolute and relative weights decreased in female rats of the 30-ppm dose group at 18 months.

 

Regarding the thyroid, the absolute and relative weights decreased in female rats of the 100-ppm dose group at 6 months and in male rats of the 30-ppm dose group at 12 months, and the absolute and relative weights increased in female rats of the 30-ppm dose group at 12 months.

 

Although the thymus was not weighed at 24 months due to severe atrophy, there was no difference in the values at 6, 12, and 18 months between the control group and each test-article dose group.

 

For the heart, the absolute weight decreased in male rats of the 100- and 300 ppm dose groups at 24 months.

 

For the lung, the absolute weight decreased in female rats of the 300-ppm dose group at 12 months.

 

For the liver, the absolute weight in male rats of the 100-ppm dose group at 6 months and the relative weight in the 300-ppm dose group increased.

 

For the spleen, no difference was observed in male and female rats between all the test-article dose groups and the control group at all examination period.

 

Regarding the kidneys, the absolute and relative weights in male rats of the 100- and 300 ppm at 6 months, the relative weight in female rats of the 300-ppm dose group at 16 and 24 months, the absolute weight in male rats of the 100-ppm dose group at 18 months, and the relative weight in male rats of the 300-ppm dose group at 18 months increased, and the absolute weight in female rats of the 300-ppm dose group at 12 months decreased.

 

For the adrenal gland, the absolute weights decreased in female rats of the 30-ppm dose group and male and female rats of the 300-ppm dose group at 18 months.

 

For the testis, the relative weights decreased in the 100-ppm dose group at 6 months, while increase was observed in the relative weights in the 300-ppm dose group at 12 months, and in the absolute and relative weights in all of the test groups at 24 months.

 

For the ovary, absolute and relative weights increased in the 300-ppm dose group at 6 month and absolute weight decreased in the 300-ppm dose group at 12 months.

 

Organ Accumulation (Table 11):

Throughout the examination periods, no accumulation of test article was observed in the liver and kidneys in all the groups.

 

Histopathological Findings (Table 12):

The major histopathological findings were described by necropsy period in the following order: a) 6-month dose group, b) 12-month dose group, c) 18-month dose group, d) 24-month dose group I, e) 24-month dose group-II, f) Unscheduled death.

 

a)      6-month dose group table (12 -1)

Spleen:          Mild extramedullary hematopoiesis increased was observed in 1 female rat of the 300-ppm dose group.

Thymus:        Minimal atrophy was observed in small numbers or approximately half of male and female animals of all groups including the control group.

Heart:            Minimal or mild interstitial inflammatory cell infiltration was observed in small numbers of male rats of all the groups including the control group.

Liver:            With respect to the liver, there were the following findings: minimal lymphocyte infiltration in Glisson’s sheath in small numbers of male and female rats in all the groups including the control (except female rats of the 100-ppm dose group), minimal or moderate granuloma in small numbers of animals in the 30- and 300-ppm dose groups, minimal small bile duct proliferation in small numbers of male rats of all the groups including the control and in female rats of the 100-ppm dose group, and minimal fatty change in approximately half of male rats of all the groups including the control group.

 

Pancreas:       Minimal interstitial lymphocyte infiltration was observed in small numbers or approximately half of male and female rats of all the test groups.

Kidney:         Minimal cortical lymphocyte infiltration was observed in small numbers of male and female rats of the control and the 30-ppm dose group, in male rats of the 100- and 300-ppm dose groups, minimal urinary cast in small numbers or approximately half of male rats of all the test groups, and minimal or mild renal tubular calcification in approximately half or all females of all the groups including the control group.

Adrenal Gland:      Minimal or mild cortical vacuolation was observed in approximately half or large number of male rats of all the groups including the control group.

Prostate:               Minimal interstitial inflammatory cell infiltration was observed in one animal of the 30-ppm dose group.

 

The brain, the pituitary gland, thyroid, lung, testis, esophagus, stomach, duodenum, jejunum, colon, uterus, submandibular gland, spinal cord, sciatic nerve, eyes, skin, mammalian gland, urinary bladder, trachea, submandibular lymph node, sternum, sternal marrow, and skeletal muscle:

                           There were no specific histopathological findings in these tissues and organs.

 

Discussion and Conclusion:

                           Since the findings in the various organs showed no relationship with the doses and were found in the control group similarly, these findings were considered to be incidental and, no effects of the test article on each organ examined were noticed.

 

b) 12 month dose group (Table 12-2)

Spleen:                 Minimal or mild extramedullary hematopoiesis was observed in small numbers of female rats of the control group, 30- and 100-ppm dose groups.

Thymus:               Minimal or mild atrophy was observed in all male and female rats.

Lung:                   Minimal pulmonary vascular calcification was observed in small numbers or approximately half of female rats of the control group and male and female rats of all the test groups, and adenoma in 1 female rat of the 300-ppm dose group.

 

Heart:                   There were observed minimal interstitial inflammatory cell infiltration in small numbers of male and female rats of the control group and male rats of all the test groups, and minimal fibrosis in small numbers or approximately half of male rats of the control and male and female rats of all the test groups.

Liver:                   There were observed minimal lymphocyte infiltration in Glisson’s sheath in small numbers or approximately half of female rats of the control group and male and female rats of all the test groups, minimal granuloma in 1 female rat of the control group, minimal or mild small bile duct proliferation in all the male rats and small number or approximately half of female rats of all the groups including the control group, and minimal or mild fatty change in small or large numbers of male and female in all the groups including the control group.

Pancreas:              Minimal or mild interstitial lymphocyte infiltration was observed in small numbers or approximately half of male and female rats of all the groups including the control group.

Kidney:                There were observed minimal cortical lymphocyte infiltration in small numbers or approximately half of male rats of the control group, male and female rats of the 30- and 100-ppm dose groups, and male rats of the 300-ppm dose group, minimal urinary cast in small or large numbers of male and female rats of all the groups including the control group, minimal or mild renal tubular calcification in almost all the female rats of all the groups including the control group, and moderate solitary renal cyst in 1 female rat of the control group.

Pituitary:              Anterior pituitary adenoma was observed in small numbers of male and female rats of the control group, 100- and 300-ppm dose groups.

Thyroid:               Adenoma was observed in 2 male rats of the 300-ppm dose group.

Adrenal Gland:      Minimal cortical vacuolation was observed in large numbers of male rats of all the groups including the control and 1 female rat of the 300-ppm dose group, and phaeochromocytoma was noted in 1 male rat of the control group.

Testis:                   There were observed minimal or mild interstitial cell proliferation in small numbers or approximately half of animals of all the groups including the control group, minimal or moderate atrophy in small numbers of animals of the control and 300-ppm dose groups, and interstitial cell tumor in one animal of the 300-ppm dose group.

Prostate:               Minimal interstitial inflammatory cell infiltration was observed in small numbers of animals of the 30- and 100-ppm dose groups.

Skin/Subcutaneous:    

There were observed keratotic papilloma was observed in 1 male rat each of the 30- and 300-ppm dose groups, mild calcified epithelioma in 1 male rat of the 300-ppm dose group, mild epidermal cyst in 1 female rat of the 30-ppm dose group, and fibroma in 1 female rat of the 300-ppm dose group.

Eyes:                    There were observed minimal or moderate retinal degeneration in small numbers of female rats of the 30- and 300-ppm dose groups, and minimal or severe lens degeneration in small numbers of male and female rats of all the test groups.

Abdominal Cavity:        Lipoma was observed in small numbers of male rats of the 30-ppm dose group and female rats of the 100-ppm dose group.

The brain, ovary, esophagus, stomach, duodenum, jejunum, colon, uterus, submandibular gland, spinal cord, sciatic nerve, mammalian gland, urinary bladder, trachea, submandibular lymph node, sternum, sternal marrow, and skeletal muscle:

                           There were no specific histopathological findings.

Discussion and Conclusion:

                                   Although adenoma in the lung, pituitary gland and thyroid and interstitial cell tumor in the testis were observed in small numbers of animals, no relationship with doses was observed; hence, these events were considered to be incidentaloma. Since there was no relationships between other findings in the various organs and doses and those events were found in the control group similarly, any of these were considered to be incidental.

 

c) 18 month dose group (Table 12-3, 12-4)

Spleen:                 Minimal increased extramedullary hematopoiesis was observed in small numbers of female rats of all the groups including the control and male rats of the 30- and 300-ppm dose groups.

Thymus:               Minimal or mild atrophy was observed in all male and female rats of all the groups including the control group.

Lung:                   Minimal pulmonary vascular calcification was observed in small numbers of male and female rats of all the groups including the control (except female rats of the 300-ppm dose group).

Heart:                   There were observed minimal interstitial inflammatory cell infiltration in 1 female rat of the control group and 1 male rat of the 300-ppm dose group, and minimal or mild fibrosis in small numbers or all male and female rats of all the groups including the control group.

Liver:                   There were observed minimal or mild lymphocyte infiltration in Glisson’s sheath and granuloma with various severities in small numbers or approximately half of male and female rats of all the groups including the control group, minimal or mild small bile duct proliferation, and fatty change in small numbers or all male and female rats of all the groups including the control group.

Pancreas:              There were observed minimal or mild interstitial lymphocyte infiltration in small numbers or approximately half of male and female rats of all the groups including the control group, and islet cell adenoma in 1 male rat of the 100-ppm dose group.

Kidney:                There were observed minimal cortical lymphocyte infiltration in 1 male rat of the control and 1 female rat of the 30-ppm dose group, minimal urinary cast in small numbers or approximately half of male and female rats of the control group and female rats of all the test groups, minimal or mild chronic nephropathy in approximately half or all male and female rats of all the groups including the control group, and minimal or mild renal tubular calcification in small or large numbers of male of the 100-ppm dose group and female rats of all the groups including control.

Urinary Bladder:   Minimal transitional cell hyperplasia was observed in 1 female rat of the 100-ppm dose group.

Pituitary Gland:     There were observed mild chromophobe cell hyperplasia in small numbers or approximately half of male rats of the control group, 30- and 100-ppm dose groups, anterior pituitary adenoma in approximately half of male and female rats of all the groups including the control group, and intermediate lobe adenoma in 1 male rat of the control group.

Thyroid:               There were observed minimal or mild follicular cell hyperplasia in 1 male rat each of the 30- and 300-ppm dose groups, minimal or mild parafollicular cell hyperplasia in small numbers of male rats of the control group, 100- and 300-ppm dose groups, and parafollicular cell adenoma in 1 male rat of the 300-ppm dose group.

Adrenal Gland:      Minimal or mild cortical vacuolation was observed in small numbers of male rats of the control group, 30 and 300-ppm dose group, cortical adenoma in 1 male rat of the control group, and phaeochromocytoma in 1 male rat of the 30-ppm dose group.

Testis:                   There were observed mild or severe atrophy in small numbers of animals of the control group, 100- and 300-ppm dose groups, and interstitial cell tumor in almost all the animals of all the groups including the control group.

Prostate:               There were observed minimal or mild interstitial inflammatory cell infiltration in small numbers or approximately half of animals of the control group, 100- and 300 ppm dose groups, and mild partial hyperplasia of glandular epithelium in one animal each of the 100- and 300-ppm dose groups.

Uterus:                 There were observed minimal or mild endometrium hyperplasia in small numbers of animals of the 30- and 100-ppm dose groups, endometrium interstitial cell adenoma in one animal of the 30 ppm dose group, and uterine cervix fibroma in one animal of the 100-ppm dose group.

Sternal marrow:    There were observed minimal or mild myeloid reticulosis in 1 female rat each of the control and 30-ppm dose groups, and moderate myelofibrosis in 1 male rat of the 30-ppm dose group.

Skin/Subcutaneous:  There were observed basal cell epithelioma in 1 male rat of the control group, mild epidermal cyst in 1 male rat of the 300-ppm dose group, and fibroma in 1 male rat of the 100-ppm dose group.

Eyes:                      There were observed mild retinal degeneration in small numbers of male and female rats of the control and 300-ppm dose groups, minimal or mild lens degeneration in small numbers of male and female rats of the control group, 100- and 300-ppm dose groups, and minimal or mild corneal epithelium hyperplasia in 1 female rat of the control and 1 male rat of the 30-ppm dose group.

Abdominal Cavity:   Lipoma was observed in small numbers of male and female rats of the control and male rats of the 30- and 100-ppm dose groups.

The brain, ovary, esophagus, stomach, duodenum, jejunum, colon, submandibular gland, spinal cord, sciatic nerve, mammalian gland, trachea, submandibular lymph node, sternum, and skeletal muscle:

                           No specific histopathological findings were observed.

Discussion and Conclusion:

                                   There were observed islet cell adenoma in the pancreas, anterior pituitary adenoma, and intermediate lobe adenoma in the pituitary gland, parafollicular cell adenoma in the thyroid, cortical adenoma and phaeochromocytoma in the adrenal gland, interstitial cell tumor in the testis, endometrium interstitial cell adenoma and uterine cervix fibroma in the uterus, basal cell epithelioma and fibroma in the skin/subcutis, and lipoma in a the abdominal cavity; there was, however, no relationship with the doses and occurred in the control group similarly. Hence, we have considered that these were incidentaloma. Since no relationship between other findings in the various organs and the doses and the findings were found in the control group similarly, these findings were considered to be incidental.

 

d) 24 month dose group-I (Table 12-5, 12-6)

Cerebrum:               Glioblastoma was observed in 1 female rat of the 30-ppm dose group.

Cerebellum:             Minimal gliosis was observed in 1 male rat of the control group.

Spinal Cord:            Minimal gliosis was observed in 1 male rat of the control group.

Spleen:                   There were observed minimal or mild increased extramedullary hematopoiesis in large numbers or all male and female rats of all the groups including the control group, minimal or moderate infarct in 1 male rat each of the control and 30-ppm dose groups, and moderate multiple cyst in 1 male rat of the 30-ppm dose group.

Thymus:                  Minimal or moderate atrophy was observed in approximately half or all male and female rats of all the groups including the control group.

Lung:                      There were observed minimal or mild pulmonary vascular calcification in small numbers or approximately half of male rats of all the groups including the control and female rats of the 30- and 300-ppm groups, mild alveolar cell hyperplasia in 1 female rat of the 30-ppm dose group, adenoma in small numbers of male rats of the 30- and 100-ppm dose groups and female rats of the 300-ppm dose group, and mild pulmonary emphysema in 1 male rat of the 100-ppm dose group.

Heart:                     There were observed minimal or mild interstitial inflammatory cell infiltration in small numbers or approximately half of male rats of the control group, 30- and 300-ppm dose groups and female rats of all the test groups, minimal or mild fibrosis in small or large numbers of male and female rats of all the groups including the control group, and myxoma in 1 male rat of each the 30- and 300-ppm dose groups.

Small Intestine:        Adenocarcinoma was observed in 1 female rat of the control group.

Liver:                     There were observed minimal lymphocyte infiltration in Glisson’s sheath in small numbers of female rats of all the groups including the control group, minimal or mild granuloma in small numbers of male rats of the control group and female rats of the 30-ppm dose group, minimal or mild small bile duct proliferation in small numbers or all of male rats of the control group and male and female rats of all the test groups, minimal or mild fatty change in small numbers or approximately half of male and female rats of the control and 300-ppm dose groups and female rats of the 30- and 100-ppm dose groups, hepatocellular carcinoma in 1 male rat in the 100-ppm dose group, and hemangioendothelioma in 1 female rat of the 100-ppm dose group.

Pancreas:                 There were observed minimal or moderate focal atrophy in small numbers or approximately half of male and female rats of the control and 30-ppm dose groups and male rats of the 100- and 300-ppm dose groups, and islet cell adenoma in small numbers or approximately half of male rats of the control group, 30- and 100-ppm dose groups and female rats of the 300-ppm dose group.

Kidney:                   There were observed minimal urinary cast in small numbers of female rats of the control group and male rats of the 100- and 300-ppm dose groups, minimal or moderate chronic nephropathy in approximately half or all male and female rats of all the groups including the control group, minimal renal tubular calcification in small numbers or approximately half of female rats of all the groups including the control group, and mild transitional cell hyperplasia in 1 female rats in the control group.

Urinary Bladder:      There were observed minimal transitional cell hyperplasia in 1 female rat of the 30-ppm dose group, transitional cell carcinoma in 1 female rat of the 30-ppm dose group, and papillary adenoma in 1 male rat of the control group.

Pituitary Gland:       There were observed minimal anterior pituitary cell hyperplasia in 1 male rat of the control group, anterior pituitary adenoma in small or large numbers of male and female rats of all the groups including the control group, anterior pituitary adenocarcinoma in small numbers of female rats of the control group, 30- and 100 ppm dose groups and male rats of the 300-ppm dose group.

Thyroid:                  There were observed minimal or mild parafollicular cell hyperplasia in small numbers or approximately half of male and female rats of the control and 300-ppm dose groups and female rats of the 30-ppm dose group, parafollicular cell adenoma in small numbers of male and female rats of the control and 300-ppm dose groups and female rats of the 100-ppm dose group, follicular cell adenoma in 4 male rats of the 100-ppm dose group, and follicular cell adenocarcinoma in 1 male rat of the 30-ppm dose group.

Adrenal Gland:        There were observed minimal or mild cortical vacuolation in small or large numbers of male and female rats of all the groups including the control group, cortical cell adenoma in 1 male rat each of the control and 100-ppm dose groups, mild medullary cell hyperplasia in 1 male rat of the control group, and phaeochromocytoma in small numbers of male rats of the control and male and female rats of the 100- and 300-ppm dose groups.

Testis:                     There were observed interstitial cell tumor in almost all the animals in all the groups including the control group, and mesothelioma in one animal of the 300-ppm dose group.

Prostate:                  Minimal or mild interstitial inflammatory cell infiltration was observed in small numbers or approximately half of animals of the control group, 30- and 100-ppm dose groups.

Uterus:                    There were observed polyp in small numbers or approximately half of animals of the control and test groups, and uterine cancer in one animal of the 100-ppm dose group.

Mammalian Gland:   There were observed minimal mammary gland hyperplasia in 1 male of the 100-ppm dose group, adenoma in 1 female rat of the 300-ppm dose group, fibroadenoma in 1 male rat of the control group, and adenocarcinoma in 1 male of the 100-ppm dose group and 1 female rat of the 300-ppm dose group.

Sternal Marrow:       There were observed minimal or moderate bone marrow hypoplasia in small or large numbers of male and female rats of all the groups including the control group, minimal or mild bone marrow hyperplasia in small numbers of male and female of the 100-ppm dose group, minimal or mild myelofibrosis in 1 female rat each of the 30- and 100-ppm dose groups, and leukemia in small numbers of female rats of the control and 300-ppm dose groups and male rats of the 30- and 100-ppm dose groups.

Skin/Subcutaneous:  There were observed basal cell carcinoma in 1 male of the 100-ppm dose group, moderate epidermal cyst in 1 female of the 100-ppm dose group, moderate skin ulcer in 1 female rat of the 300-ppm dose group, squamous cell carcinoma in 1 male rat each of the control and 300-ppm dose groups, leiomyoma in 1 female rat of the control group and 1 male of the 100-ppm dose group, and mesothelioma in 1 male rat each of the 100- and 300-ppm dose groups.

Eyes:                      There were observed minimal or moderate scleral calcification was observed in small numbers or approximately half of female rats of the control and 300-ppm dose groups and male rats of all the test groups, and minimal or moderate retinal edema in small or large numbers of female rats of the 30-ppm dose group and male and female rats of the 100 and 300-ppm dose groups.

Abdominal Cavity:   There were observed liposarcoma in 1 female rat of the 100-ppm dose group, and lipoma in 1 male rat of the control and 1 female rat of the 100-ppm dose group.

The ovary, esophagus, stomach, colon, submandibular gland, sciatic nerve, trachea, submandibular lymph node, sternum, and skeletal muscle:

                           There were no specific histopathological findings.

Discussion and Conclusion:

                                   Although various lesions including neoplastic change were observed in various organs, there was no relationship between these findings and doses; as a result we have thought that those findings described above is incidentaloma or incidental lesion.

 

e) 24 month dose group-I (Table 12-7 - 12-14)

Cerebrum:               Astrocytoma was observed in 1 female rat of the control group.

Trachea:                  Polyp was observed in 1 male rat of the 30-ppm dose group.

Lung:                      There were observed adenoma in small numbers of male and female rats of the control and 100-ppm dose groups and male rats of the 30- and 300-ppm dose groups, and adenocarcinoma in small numbers of male and female rats of the 30-ppm dose group and male rats of the 100-ppm dose group.

Stomach/Small Intestine:

                              Leiomyoma was observed in 1 male rat of the 300-ppm dose group.

Liver:                     There were observed hemangioendothelioma in 1 male rat of the control group, and hepatocellular carcinoma in small numbers of male rats of the 30- and 100-ppm dose group.

Pancreas:                 There were observed islet cell adenoma in small numbers of male and female rats of all the groups including the control (except female rats of the 100-ppm dose group), and acinic cell tumor in 1 male rat of the 30-ppm dose group.

Submandibular Gland:

                              Fibroma was observed in 1 male rat of the 300-ppm dose group.

Pituitary Gland:       There were observed adenoma in approximately half of male and female rats of all the test groups, and adenocarcinoma in small numbers of male and female rats of all the groups including the control (except male rats of the 30-ppm dose group).

Thyroid:                  There were observed adenoma in small numbers or approximately half of male and female rats of all the groups including the control group, and adenocarcinoma in small numbers of female rats of the control and male rats of the 300-ppm dose group.

 

Adrenal Gland:        Phaeochromocytoma was observed in small numbers of male and female rats of the control group and male rats of all the test groups.

Urinary Bladder:      Polyp was observed in 1 female rat of the control group.

Testis:                     Interstitial cell tumor was observed in almost all the animals of all the groups including the control group.

Uterus:                    There were observed polyp in small numbers of animals of all the groups including the control group, leiomyoma in one animal of the 100-ppm dose group, and uterine cancer in one animal each of the control and 300-ppm dose groups.

Mammalian Gland:   There were observed adenoma in small numbers of female rats of all the test groups and male rats of the 300-ppm dose group, fibroadenoma in small numbers of male and female rats of the 100-ppm dose group, adenocarcinoma in small numbers of male rats of the 30- and 100-ppm dose groups and female rats of the 30- and 300-ppm dose groups, and cystosarcoma phyllodes in small numbers of female rats of the 300-ppm dose group.

Skin/Subcutaneous:  There were observed lipoma in 1 male rat of the 30-ppm dose group and 1 male and female rats each of the 100-ppm dose group, fibroma in small numbers of male rats of the 30- and 100-ppm dose groups and female rats of 100 and 300-ppm dose groups, squamous cell carcinoma in 1 male and female rat each of all the groups including the control (except female rats of the 300-ppm dose group), basal cell carcinoma in 1 female rat in the 300-ppm dose group, malignant perithelioma and rhabdomyosarcoma in 1 male each in the 30-ppm dose group, fibrosarcoma in 1 female rat of the control group, and leiomyoma in 1 male rat of the control group.

Muscle:                   Rhabdomyosarcoma was observed in 1 male rat of the 30-ppm dose group.

Abdominal Cavity:   There were observed mesothelioma in 1 male rat each of the 30- and 300-ppm dose groups, lipoma in 1 female rat of the control group, and leiomyoma in 1 male rat of the control group and 1 female of the 100-ppm dose group.

Thymus:                  Malignant thymoma was observed in 1 female rat of the 30-ppm dose group.

Lymph Node/Bone marrow:

                              Leukemia was observed in small numbers of male and female rats of all the groups including the control group.

Sternum:                 There were observed osteochondroma in 1 female rat of the 30-ppm dose group, and osteosarcoma in 1 male rat of the control group.

Others:                    Adenocarcinoma (Site unknown) was observed in 1 male rat of the 300-ppm dose group.

 

Discussion and Conclusion:

                                   Although various neoplastic lesions were observed in the various organs. Since no relationship was observed between doses and the neoplastic lesion and similar findings were obtained in the control group, we have thought that they were incidentaloma associated with aging.

Conclusions:
According to the study results, the maximum no- effect level and the minimum effective dose of Nisseki Hisol SAS were 100 ppm {4.83~5.72 mg/kg/day) and 300 ppm (14.93~ 17.59 mg/kg/day) respectively.
Executive summary:

Summary

 

In the implementation of the chronic toxicity and oncogenicity study, Nisseki Hisol SAS was mixed with powder food (CRF-1: 95%, Olive oil: 3% and Cellulose: 2%) at concentrations of the 30, 100, and 300 ppm, and the mixture feed was given ad libitum to male and female Fischer rats for 24 months. Animals were examined for clinical signs, body weight, food consumption, and water consumption during administration period, and 10 animals per group were sacrificed at 6, 12, 18, and 24 month of administration and subjected to laboratory examinations and pathology. All survived animals were sacrificed at 24 month for a pathology test. The following results were obtained.

 

1. Clinical Signs, Mortality Conditions, and Body Weight Change

No differences for clinical signs were observed between each test-article dose group and the control group.

 

As to mortality conditions, no difference for time and number of death were observed between each test-article dose group and the control group as shown below.

 

Dosage

Male

Female

Total

ppm

Week 0 - 52

Week 0 - 104

Week 0 - 52

Week 0 - 104

 

Control

0

15

0

9

24

30

0

17

1

13

30

100

0

12

0

8

20

300

0

14

0

11

25

 

No difference for body weight change was observed for both male and female rats of the 30 and 100 ppm groups in comparison with the control group, while male and female rats of the 300 ppm group showed tendency of suppression of body weight gain.

2. Food Consumption, Food Efficiency, Test Article Intake, and Water Consumption

For food consumption, food efficiency, and water consumption, no effect of the test article administration was observed in both male and female rats of the test-article dose groups.

 

Amounts of test article intake were 1.45 mg/kg/day (male) and 1.75 mg/kg/day (female) for 30 ppm group, 4.83 mg/kg/day (male) and 5.72 mg/kg/day (female) for 100 ppm group, and 14.93 mg/kg/day (male) and 17.59 mg/kg/day (female) for 300 ppm group.

 

3. Urinalysis, Hematology and Clinical Chemistry Findings

The results of urinalysis, and hematology and clinical chemistry examinations show significant differences in some parameters in male and female rats of test-article dose groups in comparison with the control group. These changes, however, were very mild in magnitude and had no correlation with sex, doses, and dose period was observed. All parameters indicated no effect of test article administration.

 

4. Necropsy Findings

In both survived and dead animals, no specific changes which are considered to be caused by the test article administration were observed although neoplastic changes which are considered to be due to aging were observed in the pituitary, thyroid, lung, liver, spleen, adrenal gland, testis, uterine, lymph nodes, thorax, abdominal cavity, subcutaneous area, skin, and pancreas.

 

5. Organ Weight

Although liver weight of 100-ppm (male) and 300-ppm dose groups at 6 month, there were no histopathological findings which suggest body weight gain and no significant change was observed for body weight on and after 12 month. Although some organs exhibited significant differences between the test-article dose groups and the control group, the differences were very slight in magnitude and had no correlation with sex, doses, and dose period. Therefore, the administration of the test article did not affect the organ weight.

6. Organ Accumulation

At the time of sacrifice every 6 months, the liver and kidneys were removed from 3 animals per group and were weighed. After portions of these organs was dissected forhistopathological[t1] studies, the organs were homogenized to measure test article concentration in the organs. No accumulation of test article in the liver and kidneys was observed for both male and female rats of each group.

 

7. Histopathological Findings

In both survived and dead animals, no specific changes which were likely to be caused by test article administration were noted.

 

No difference was observed for the types and incidence of neoplasm between each test-article dose group and the control group. The test article had neither carcinogenicity nor carcinogenesis promotion. With respect to the tumors which were observed in this study, tumor-like lesions were observed in the various organs in male and female rats in addition to the high incidence of interstitial cell tumor in the testis in male rats of all the groups including the control group and adenoma in the pituitary gland and thyroid in male and female rats. These lesions were, however, incidentaloma which has been observed in rats frequently, and there were no specific tendencies in types or incidences of the tumors and there was no correlation with the doses. Hence, we have thought that the test article has no effect of carcinogenesis or carcinogenesis promotion.

Conclusions:

According to the above results, the maximum no- effect level and the minimum effective dose of Nisseki Hisol SAS were 100 ppm (4.83~5.72 mg/kg/day) and 300 ppm (14.93 ~17.59 mg/kg/day) respectively.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
4.83 mg/kg bw/day
Study duration:
chronic
Species:
rat
Quality of whole database:
The study has Klimisch score 2

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Available information:

Subacute toxicity:

SAS-40:

In a 28-day oral repeated dose toxicity study according to OECD guideline 407 under GLP, rats were exposed daily to 0, 50, 250 or 1000 mg SAS-40/kg bodyweight by oral gavage. No mortality occurred during the study, but rats in the highest dose group showed signs of toxicity (salivation, hunched back, pilo erection). Salivation was also seen in rats in the intermediate dose group. At 28 days, rats in the highest dose group showed enlarged livers and kidneys, deviant haematology and clinical chemistry and weight loss. Some of these effects were also seen in the intermediate dose group. No effects were seen in the lowest dose group. Therefore, the NOAEL was determined to be 50 mg/kg bw/day based on subacute effects seen at 250 and 1000 mg/kg bw/day.

SAS-305:

In a 28-day oral repeated dose toxicity study according to OECD guideline 407 under GLP, rats were exposed daily to 0, 15, 150 or 1000 mg SAS-305/kg bodyweight by oral gavage. No mortality occurred during the study, but rats in the highest dose group showed signs of toxicity (salivation, fur loss, red/brown staining of external body surface, incidental hunched posture and/or lethargy). No signs of toxicity were observed in the other groups. In the third week weight loss and/or food efficiency was reduced in arats exposed to 1000 mg/kg/day. At 28 days, rats in the highest dose group showed enlarged livers and kidneys, and significant increase in plasma bilirubin. Hepatocyte enlargement for all rats in the highest dose group and for male rats dosed with 150 mg/kg/day were observed. One male dosed at 15 mg/kg/day was similarly affected. Almost complete regression of the condition was seen amongst 1000 mg/kg/day animals following an additional fourteen days without treatment.

As there is no evidence for mechanism of action for the observed SAS-305 liver effects, the effects are considered potentially adverse, and need to be considered for risk assessment. Therefore, based on the effects observed on the liver in mid and high dose animals, the oral NOAEL for sub-acute toxicity for SAS-305 is 15 mg/kg bw/day.

Phenyl-tolyl-ethane:

In a 28-day oral repeated dose toxicity study with the reproduction/developmental toxicity screening test according to OECD guideline 422 under GLP, rats were exposed daily to 0, 10, 30 or 100 mg Phenyl-tolyl-ethane/kg bodyweight by oral gavage followed by a 14-day recovery period.

Parental toxicity was evident at 100 mg/kg bw/day.

At 100 mg/kg bw/day, five females had hunched posture and/or piloerection on several days during treatment. Toxicologically relevant changes in haematology parameters were noted for (paired) Main females at 100 mg/kg only. These changes included slightly increased red blood cells with corresponding slightly increased haemoglobin. In addition, decreased reticulocytes with corresponding decreased red blood cell distribution width (RDW), and decreased platelets were noted. These latter changes are in line with the microscopic observation of decreased hemopoietic foci in the spleen of Main females at 100 mg/kg bw/day. It should be noted that for (nulliparous) Recovery females, there were no haematology changes. Moreover, no difference was noted in hemopoietic foci in the spleen from control Recovery females (3/5 minimal) and Recovery females at 100 mg/kg bw/day (2/5 minimal, 1/5 slight). Pregnancy normally leads to an increase in hemopoietic foci in the spleens of female rats as seen in Main Groups 1 to 3. The hemopoietic foci seen in Main females at 100 mg/kg bw/day more closely resembles the amount present in nulliparous females. This may indicate the normal increase seen at pregnancy is blocked by the test item. Since the Recovery Group animals were not mated, the possible recovery from this could not be investigated.

No parental toxicity was observed at 10 and 30 mg/kg bw/day.

Based on these results, the following No Observed Adverse Effect Level (NOAEL) was derived: Parental NOAEL : 30 mg/kg bw/day.

SAS-296 (for endpoint study record see section 7.8, toxicity to reproduction):

The study was conducted according to an OECD combined repeated dose toxicity study with the reproduction/developmental toxicity screening test guideline [OECD 422] under GLP. Based on the pathological findings in the

adrenals in males and in the liver in females, no NOAEL could be derived in males for repeated dose toxicity and the LOAEL for repeated dose toxicity was considered to be 12.5 mg/kg bw/day in male rats. In female rats, the LOAEL for repeated dose toxicity was 200 mg/kg bw/day and the NOAEL for repeated dose toxicity was considered to be 50 mg/kg bw/day. The overall NOAEL is

lower than 12.5 mg/kg bw/day and the LOAEL is 12.5 mg/kg bw/day.

Chronic toxicity:

SAS-296:

In a combined chronic and carcinogenicity feeding study similar to OECD guideline 453, rats were exposed daily to 0, 30, 100 or 300 ppm SAS-296/kg bodyweight for 24 months.

No differences for clinical signs were observed between each test-article dose group and the control group.

As to mortality conditions, no difference for time and number of death were observed between each test-article dose group and the control group.

No difference for body weight change was observed for both male and female rats of the 30 and 100 ppm groups in comparison with the control group, while male and female rats of the 300 ppm group showed tendency of suppression of body weight gain.

For food consumption, food efficiency, and water consumption, no effect of the test article administration was observed in both male and female rats of the test-article dose groups.

The results of urinalysis, and hematology and clinical chemistry examinations show significant differences in some parameters in male and female rats of test-article dose groups in comparison with the control group. These changes, however, were very mild in magnitude and had no correlation with sex, doses, and dose period was observed. All parameters indicated no effect of test article administration.

In both survived and dead animals, no specific changes which are considered to be caused by the test article administration were observed although neoplastic changes which are considered to be due to aging were observed in the pituitary, thyroid, lung, liver, spleen, adrenal gland, testis, uterine, lymph nodes, thorax, abdominal cavity, subcutaneous area, skin, and pancreas.

Although liver weight of 100-ppm (male) and 300-ppm dose groups at 6 month, there were no histopathological findings which suggest body weight gain and no significant change was observed for body weight on and after 12 month. Although some organs exhibited significant differences between the test-article dose groups and the control group, the differences were very slight in magnitude and had no correlation with sex, doses, and dose period. Therefore, the administration of the test article did not affect the organ weight.

At the time of sacrifice every 6 months, the liver and kidneys were removed from 3 animals per group and were weighed. After portions of these organs was dissected for histopathological studies, the organs were homogenized to measure test article concentration in the organs. No accumulation of test article in the liver and kidneys was observed for both male and female rats of each group.

In both survived and dead animals, no specific changes which were likely to be caused by test article administration were noted.

No difference was observed for the types and incidence of neoplasm between each test-article dose group and the control group. The test article had neither carcinogenicity nor carcinogenesis promotion.

According to the above results, the maximum no- effect level and the minimum effective dose of Nisseki Hisol SAS were 100 ppm (4.83~5.72 mg/kg/day) and 300 ppm (14.93 ~17.59 mg/kg/day) respectively.


Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
The study with the lowest NOAEL is choosen.

Repeated dose toxicity: via oral route - systemic effects (target organ) digestive: liver; urogenital: kidneys

Justification for classification or non-classification