Sodium 6-diazo-5,6-dihydro-5-oxonaphthalene-1-sulphonate

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

other: CRL/CD(SD)

Details on species / strain selection:

- Age at study initiation of dosing: 9 weeks old
- Recovery: 0, 1000 mg/kg/day

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River, Japan
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 8 weeks
- Weight at study initiation:
326.1 – 376.3 g for males and 199.6 – 238.6 g for females

- Housing:
Animals were housed individually in a stainless cage with mesh floor (W260 mm x D380 mm x H180 mm) for males and (W165 mm x D300 mm x H150 mm) for females until 13 days of pregnant and during fasting on the day before necropsy. During mating a male and a female were housed together in male’s cage. After confirming mating, female was moved and housed in polycarbonate flat cage (W265 mm x D426 mm x H150 mm) with bedding (Sanflakes, Japan Charles River) between day 14 of gestation and the day before fasting. After birth, pup was housed together with their dam.

- Diet (e.g. ad libitum):
ad libitum
- Water (e.g. ad libitum):
ad libitum
- Acclimation period:
10 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C):
21-25 °C
- Humidity (%): 40-70%
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light):
12/12 h

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

- VEHICLE
- Concentration in vehicle: 250 mg/mL (high doese), lower doses were obtained by dilution
- Amount of vehicle (if gavage): 4 mL

Details on mating procedure:

- M/F ratio per cage: 1:1, avoiding sibling mating
- Length of cohabitation: until confirmed mating, for maximum 14 days
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 post-coitum
- After successful mating each pregnant female was caged individually in Macrolon cages

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The stability during the study period was confirmed by measurements of Infrared absorption spectrum of the test substance before and after the treatment period. High dose solution was prepared by pure water (Takasugi pharmaceutical Co.) adding to the test substance. Middle and low dose solutions were prepared by dilution from the high dose solution. The stability of the test substance in the test solutions for 14 days were confirmed by HPLC analysis. The test solutions were stored in cool and dark place until dosing and used within 14 days. The concentration of the test substance in the test solutions were measured prior to treatment.

Duration of treatment / exposure:

Male: 42 days
Female: 42 - 54 days (from 14 days before mating to day 4 of lactation)

Frequency of treatment:

daily

Dose / conc.:

0 mg/kg bw/day (actual dose received)

Dose / conc.:

40 mg/kg bw/day (actual dose received)

Dose / conc.:

200 mg/kg bw/day (actual dose received)

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

Males, 12 (5 for recovery); females, 12; satellite females, 5

Control animals:

yes, concurrent vehicle

Details on study design:

The test doses were selected based on the results of 14 days repeat dose toxicological study. Some changes which were induced by irritation of the test substance such as Soft stool were recorded in females treated at 250 mg/kg and above and males treated at 500 mg/kg and above, elevated limiting ridge in forestomach in males treated at 1000 mg/kg and enormous of cecum in males and females treated at 1000 mg/kg were observed in the 14 days repeated dose toxicity study when Crl:CD(SD) rats, 3/sex/group, were treated with 0, 25, 250, 500 and 1000 mg/kg/day. There was no abnormality in kidney weight, while one male treated at 1000 mg/kg had a bilateral swallow and unilateral decoloration of kidney. Furthermore, although brown urine was detected in all treated groups, no abnormalities were detected in body weight and organ weights. Therefore, 1000 mg/kg/day was selected as the highest dose, and lower 2 dosage of 200 and 40 mg/kg/day calculated by a factor of 5 were selected for the main study.

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
Males were observed twice a day between the start of treatment and the day before necropsy and once a day on the day of necropsy. Females in the recovery group were observed twice a day until the end of the treatment, and clinical signs including in delivery and lactation status in females in the main group were observed twice a day between the start of doing and day 4 post-partum and once a day on day 5 post-partum (necropsy). Males and females were observed once a day until the day before necropsy during the recovery period.

DETAILED CLINICAL OBSERVATIONS: Yes
All the males and females were observed by blind and scored once prior to treatment and once a week just after dosing thereafter. For blind observation, all animals were re-sorted by random number, and then a label for examination in order to unknown their groups were given. Although females were examine on day 4 post-partum, no blind examination was performed if only one female was examined.
For the reactions of removing from cage, easy removing from cage and vocalization were observed when animals were given external stimulation such as holding or approached a hand to hold animals. Detailed clinical observations during handling were checked muscle tone, hypothermia with or without piloerection, condition of fur (soiled, coarse), color of skin and mucosa (pale, Reddening, cyanosis). abnormal eyes (lacrimation, ophthalmocele, Pupil size), salivation and secretion. Observations of behavior in arena were checked position, activity, respiration, closed eyelid, abnormal gait, tremor, twitch, convulsion, stereotyped behavior and abnormal behavior when animals were stayed in arena for 1 minute. The number of excretion (the number of feces) and the number of urination (the number of urine pools) were recorded for 1 minute.

BODY WEIGHT: Yes
Body weight of males were weighted on 1, 3, 7, 14, 21, 28, 35, 42 and 43 days (fasted, at removal) post dosing, and females were weighed on 1, 3, 7 and 14 days post dosing, 0, 7, 14, 17 and 20 days post pregnant, and 0 (the day of delivery), 4 and 5 days (fasted, at removal) post-partum. Females in the satellite groups were measured at the same days with males. Body weight of males and females during the recovery period were measured on 1, 3, 7, 14 and 15 (fasted, at removal) days post recovery.

FOOD CONSUMPTION: yes
Food consumption of males were measured on 1, 3, 7, 14, 21, 28, 35 and 42 days post dosing, and females were measured on 1, 3, 7 and 14 days post dosing, 0, 7, 14, 17 and 20 days post pregnant, and 0 (the day of delivery) and 4 days post-partum. Females in the satellite groups were measured at the same days with males. Food consumption of males and females during the recovery period were measured on 1, 3, 7 and 14 days post recovery. Mean daily food consumption was calculated using amount of food consumption until next measurement period.


WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
Animals were fasted for overnight (16-20 hours) from afternoon of the last day of the treatment or recovery period to necropsy, but drinking water accessed ad libitum. Blood were drawn from abdominal aorta under Ether anesthesia. Males used 5 animals of the smallest animal number among surviving animals of each dose, and females used 5 animals near each delivery for each dose, and all males and females in the recovery groups were selected for hematology. EDTA-2K was used as an anti-coagulant and hematology was measured using automated hematology analyzer (CELL-DYN 3500, Abbott Laboratories) for red blood cells (RBC), white blood cells (WBC), hemoglobin (Hb), hematocrit (Ht), mean cell volume(MCV), mean cell hemoglobin (MCH), mean cell hemoglobin concentration (MCHC) and platelet (Platelet). In addition, reticulocyte ratio (Reticulo) and differentiation of leukocyte (Differentiation of leukocyte) were measured by General hematology analyzer (ADVIA 120, Siemens AG). Plasma was separated from blood samples withdrawn into 3.2% sodium citrate solution as an anti-coagulant and measured for prothrombin time (PT) and activated partly thromboplastin (APTT) using Automatic blood coagulation and fibrinolysis measuring device (STA Compact, Roche Diagnostics K.K).


CLINICAL CHEMISTRY: Yes
Blood serum from the same animals employed for hematology were used for blood chemistry. aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), gamma-glutamyl-transpeptidase (γ-GTP), total cholesterol (T-Cho), triglycerides (TG), glucose (Glucose), total protein (T-Protein), albumin (Albumin), urea nitrogen (BUN), creatinine (Creatinine), total bilirubin (T-Bil), calcium (Ca) and inorganic phosphorus (IP) were measured using Blood chemistry Autoanalyzer (7180, Hitachi). Furthermore, sodium (Na), potassium (K) and chlorine (Cl) were measured using electrode analyzer (PVA-EX II, A&T). A/G ratio (A/G ratio) was calculated based on the serum levels of total protein and albumin.


URINALYSIS: Yes
Pooled urine for 15-17 hours were obtained from the same animals employed to hematology. Animals were housed into a metabolic cage individually in the afternoon of the last day of the treatment or recovery period. During urine collection, animals were fasted but drinking water accessed ad libitum. Urine volume, color and clearly were checked using the collected urine, osmolarity were measured using automatic osmometer (OM-6040, Arkrey Inc.), pH, protein, glucose and blood were measured using urinalysis paper (Hemacombisticks, Siemens AG). Urine residue was also measured. Regarding to urine residue, since no treatment-related changes were detected in males and females in the high dose group when it was measured in the control and high dose groups, urinalysis of middle and low dose groups and recovery group were not determined.


NEUROBEHAVIOURAL EXAMINATION: Yes
Five males selected in ascending order of animal number of each dose were examined after dosing of the last week of the treatment period. Five delivery females with close delivery day at each dose were examined before fasting on day 4 post-partum. Animals assigned to in the recovery group were examined after dosing during the last week of the treatment period. Since no abnormalities which seemed to be treatment-related were observed during the treatment period, the examination during the recovery period was not performed.
For examination of reaction, the reactions when suitable external stimulation gave to the target sensory organ were checked and scored. This examination was conducted by blind continued the detailed clinical observation. For vision (approach, sense of touch), the response when the sheath of ballpoint pen were held keeping for 3 cm distance from the face, and holding it for 4 seconds was recorded. For auditory sense, the reaction when fingers were rang on overhead were recorded. Pain reaction was recorded when sandwiching the one third ridge portion of the tail with scissors. Pupillary reflex recorded the reaction of the pupil when light was irradiated after blocking the light. Righting reflex in air was recorded as the reaction when dropping from the height of about 30 cm with the abdomen of the animal facing upward.
Grip strength were measured by blind using a grip strength meter (FGC-2, Metis). The measurement was performed each twice for forelimb and hindlimb, and mean values were calculated as the grip strength values of forelimb and hindlimb to individual animal.
Locomotor activity was measured as motor activity using a motor activity measuring device for rats (ACTIMO-10, Shintechno Corporation). The number of times across the infrared beam was counted as the measured value, and it was measured for 1 hour (6 times at 10 minute intervals).


IMMUNOLOGY: No


OTHER:

Oestrous cyclicity (parental animals):

Vaginal smears from all females were taken within every morning from 1 day post dosing to confirming pairing, and smears stained with Giemsa solution were examined under light microscope. Mean days of estrous cycles were calculated based on the days between the first estrus (to use the first day of estrus if it was continued longer) and next estrus examined for 15 days. Incidence of animals who had abnormal cycles (did not have regular cycles) was calculated.

Litter observations:

The number of pups born (the number of stillborn + the number of live born), the number of stillborn, the number of live born, sex, the number of live born on day 0, sex of live born on day 0, external abnormality of live born were observed on the birth day (set up as day 0 post-partum). After then, clinical observations, the number of live pups, the number of dead pups and sex were observed until day 4 post birth, and live birth index: (the number of live pups/the number of implantation sites) x 100, viability index on day 0: (the number of live pups/the number of pups born) x 100, sex ratio of pups born: the number of male pups/the number of pups born, sex ration of pups on day 0: the number of male pups on day 0/the number of live pups on day 0, external anomaly index: (the number of live pups with external anomalies/live pups) x 100, viability index on day 4: (the number of live pups on day 4/live pups) x 100, sex ratio on day 4: the number of male live pups on day 4/the number of live pups on day 4 were calculated. Body weights of male or females per litter were measured at birth and on day 4 post-partum.

Postmortem examinations (parental animals):

GROSS NECROPSY
- All animals were examined macroscopically for body surface, opening, subcutaneous, cranial cavity, thoracic cavity, abdominal cavity, pelvic cavity and contents on the day after the end of the treatment and recovery period. The number of pregnant corpus luteum in ovaries and the number of implantation sites in uterus after incision were recorded.

HISTOPATHOLOGY / ORGAN WEIGHTS
Trachea and lung, stomach, intestine (duodenum - rectum with Peyer's patches), liver, heart, kidneys, urinary bladder, testes, epididymis, prostate, seminal vesicle, ovary, uterus, vagina, brain (cerebrum, cerebellum with pons), spinal cord, sciatic nerve, bone marrow (femur), lymph node (axillary lymph node, mesenteric lymph node ), spleen, thymus, pituitary, thyroid (with parathyroid) and adrenal gland were collected from all animals. Organ weights of liver, heart, kidney, testes, epididymis, brain, spleen, thymus and adrenal gland were measured, and relative organ weight per final body weight were calculated. Collected organs/tissues were fixed with 10 % neutral buffered formalin solution except for testes and epididymis which were fixed with modified Davidson solution.

Histopathological examination for trachea, lung, stomach, intestine, liver, heart, kidney, urinary bladder, testes, epididymis, prostate, seminal vesicle, ovary, uterus, vagina, brain, spinal cord, sciatic nerve, bone marrow, axillary lymph node, mesenteric lymph node, spleen, thymus, pituitary, thyroid and adrenal gland from the control and 1000 mg/kg groups were carried out.

Histopathological slides which were prepared HE stain to paraffin embedded thin section were examined under light microscope. The organs or tissues from the other groups and recovery groups were examined if treatment-related changes were detected in the high dose group. In addition, all gross abnormal changes were examined. Ovary, uterus and vagina from infertility females were examined, and teste from paired two males were also examined.
Furthermore, since one male in the 40 mg/kg group had Brown pigment deposition in tubular epithelium in kidney at HE stained slide and it seemed to be hemosiderin pigmentation, Berlin blue staining was performed.

Statistics:

Standard statistical methods have been applied for data processing.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Brown urine was observed in 9 out of 12 males in the 40 mg/kg group, all 12 males in the 200 mg/kg group during the study period. In the 1000 mg/kg group, brown urine and soft stool were observed all 12 males, mucosal stool and staining of abdominal were observed each one male. In females, brown urine was noted 4 out of 12 in the 40 mg/kg group and all females in the 200 mg/kg and above dose groups throughout pre-mating, pregnant and lactation periods. In addition, 3 out of 12 females in the 1000 mg/kg group showed soft stool. Furthermore, all 5 females and 3 females treated at 1000 mg/kg in the satellite group showed brown urine and soft stool, respectively. Although each one females from the control and 40 mg/kg groups was not pregnant, no abnormal change was observed in clinical observations.
During the recovery period, all 5 males in the 1000 mg/kg group showed brown urine on day 1, and it was disappeared the next day and thereafter. Furthermore, one male at the same group showed staining of abdominal until the end of the recovery period. All 5 females in the 1000 mg/kg group showed brown urine on day 1 and it was disappeared the next day and thereafter.

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

In males during the treatment period, final body weight (day 42 after treatment) decreased to 93.7%, 95.1% and 96.7% of control values in the 1000 mg/kg, 200 mg/kg and 40 mg/kg, respectively, but these reductions were not statistically significant. Body weight in females was not significant in the treatment groups.
There were no statistically significant differences in males and female in the treatment groups during the recovery period.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Lower level of food consumption was recorded in males at 200 mg/kg and above dose levels on day 3 during the treatment period. High value was noted in the 1000 mg/kg on day 28. Food consumption in the 40 mg/kg group was not significant difference. In females low value was noted in the 200 mg/kg and above dose groups on day 3. In addition, high value was recorded in the 1000 mg/kg group on days 7 and 20 post-pregnant as well as on days 21, 35 and 42 in the satellite group. There was no statistically significant difference in the 40 mg/kg group. During the recovery period, there was no statistically significant difference in males in the 1000 mg/kg group. Females in the 1000 mg/kg group showed high value on day 3 in the recovery period.

Food efficiency:

no effects observed

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no changes seemed to be treatment-related in males and females at the end of treatment period. Although low reticulocyte ratio was noted in males treated at 200 mg/kg, this was decided to be incidental because dose related relationship was not clear.
At the end of recovery period, high ratio of reticulocyte of males in the 1000 mg/kg group which was not detected at the end of the treatment period was recorded. However, because no change in the number of red blood cells was noted and therefore it was not considered to be replacement change of anemia, this change of reticulocyte was decided to be incidental. Statistically significant changes were not recorded in females treated at 1000 mg/kg.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

In males low serum level of total protein was noted in the 1000 mg/kg group at the end of the treatment period. Furthermore, low serum level of urea nitrogen was recorded in the 40 mg/kg and above dose groups, but dose dependency was not clear. Although low serum level of total cholesterol was also recorded in the 40 mg/kg group, this change was decided to be incidental because dose related relationship was not clear. There was no statistically significant change in females in the treatment groups.
At the end of the recovery period, high level of urea nitrogen in males which was not noted at the end of the treatment period was recorded in the 1000 mg/kg group. In females low levels of γ-GTP and Na were recorded in the 1000 mg/kg group. However, these changes were considered to be incidental because the same changes were not detected at the end of the treatment period.

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

In males yellow-red urine in the 200 mg/kg and above dose groups and brown urine in the 1000 mg/kg group were noted at the end of the treatment period. No abnormal change in the 40 mg/kg group was detected. In females yellow-red urine or brown urine was noted in the 1000 mg/kg group at the end of the treatment period. Incidence of the number of animals have pH 6.0 was a tendency to increase at 200 mg/kg and above dose levels. No abnormal change was noted in the 40 mg/kg group.
At the end of the recovery period, there were no abnormal changes in males and females in the 1000 mg/kg group

Behaviour (functional findings):

no effects observed

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

At the end of the treatment period, hyperplasia of the squamous epithelium in the limiting ridge of the forestomach which was corresponded to the elevated limiting ridge observed at necropsy was observed in 5 out of 7 males in the 1000 mg/kg group. Furthermore, hyperplasia of the surface epithelial cells in the glandular stomach was observed in 3 males at the same dose group. For cecum, although enormous was noted at necropsy, no histopathological abnormality was detected. No abnormal changes in liver which was detected low relative weight in the 1000 mg/kg group were observed. In the 40 mg/kg group, necrosis of fundic mucosa in fundus gland area of glandular stomach and brown pigment deposition in tubular epithelium in kidney in one male and diverticulum in jejunum and unilateral spermatic granuloma in epididymis in another male were observed and these changes were corresponded to the changes observed at necropsy. Some spontaneous findings such as mineralization in Peyer’s patches in jejunum, focal myocarditis in heart, solitary cyst in medulla and subcapsular solitary cyst in kidney were observed individual one male in the 1000 mg/kg group. In addition, focal necrosis of hepatocytes in liver and bilateral degeneration of seminiferous tubules in testes were observed each 1 out of 6 males in the control group. Abnormal changes in kidney was not reproduced in this study, while enlargement and faded color in kidney were detected in the dose-range finding study.
No abnormal changes in testes from one male who was partner of infertility female in the control or 40 mg/kg group were detected.
There was no positive substance in tubular epithelium of the kidney with Berlin blue staining which was conducted to confirm brown pigment deposition in tubular epithelium observed in one male treated at 40 mg/kg.
In females hyperplasia of the squamous epithelium in the limiting ridge of the forestomach which was corresponded to the elevated limiting ridge observed at necropsy was observed in 6 out of 12 females in the 1000 mg/kg group. Furthermore, edema in submucosal layer and hyperplasia of surface epithelial cells in the glandular stomach were observed in each at the same dose group. For cecum, although enormous was noted at necropsy, no histopathological abnormality was detected.
In the control group, necrosis of fundic mucosa in glandular stomach and diverticulum in jejunum in each, and cyst formation in pars nervosa of pituitary gland in 2 females were observed and they were a correspondence to macroscopic findings. Some spontaneous findings such as mineralization in Peyer’s patches in duodenum and jejunum, solitary cyst in medulla of kidney in each of the 1000 mg/kg group were observed. In addition, mineralization in Peyer’s patches in jejunum in 4 out of 11 females, focal necrosis of hepatocytes in liver, cyst formation in pars intermedia of pituitary gland and unilateral focal necrosis of cortex in adrenal were observed in each of the control group.
No abnormal changes in ovary, uterus and vagina in one infertility female from the control and 40 mg/kg groups were detected.
At the end of the recovery period, there were no changes in forestomach and glandular stomach in males and females. Abnormal changes in liver was not detected in females in the 1000 mg/kg group, though relative liver weight showed high value.

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

No animals showed abnormal estrous cycles in the treated groups. Also there were no statistically significant difference in mean estrous cycle days in the treated groups.

Reproductive function: sperm measures:

not examined

Reproductive performance:

effects observed, non-treatment-related

Description (incidence and severity):

All animals in the control and treated groups were mated successfully. Since infertility female was detected in each of the control and 40 mg/kg groups, the final number of pregnant females in the control, 40, 200 and 1000 mg/kg were at 11, 11, 12 and 12 animals, respectively. Fertilization index and conception index were 91.7% for the control and 40 mg/kg groups. However, dose-relationship was not clear and these changes were considered to be incidental. No abnormalities in the number of days until mating confirmed, gestational period, the number of pregnant corpus luteum, the number of implantation site, implantation index, pre-implantation loss, post-implantation loss, delivery index and parturition index were found in the treated groups.

Key result

Dose descriptor:

NOAEL

Remarks:

systemic toxicity

Effect level:

200 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

food consumption and compound intake

histopathology: non-neoplastic

Key result

Dose descriptor:

NOEL

Remarks:

reproductive performance

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no effects on reproductive performance were observed

Key result

Critical effects observed:

no

Lowest effective dose / conc.:

1 000 mg/kg bw/day (actual dose received)

System:

gastrointestinal tract

Organ:

stomach

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

Mortality / viability:

no mortality observed

Body weight and weight changes:

not examined

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

not examined

Histopathological findings:

not examined

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Key result

Dose descriptor:

NOEL

Generation:

F1

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no effects observed

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Critical effects observed:

no

Key result

Reproductive effects observed:

no

Conclusions:

The NOAEL (systemic toxicity) of the test item was determined to be 200 mg/kg bw/day for parental male and females rats. The NOEL for the reproductive performance were 1000 mg/kg/day for parental animals as no effects were observed.
The NOEL for general toxicity was determined to be 1000 mg/kg bw/day for F1-generation as no effects occurred.

Executive summary:

Since there were no treatment-related effects on parents and pups, it was considered to be no reproductive and developmental toxicity under the study conditions.

Based on the above results, it was concluded that the No-Observed-Effect-Level (NOAEL) for the repeated dose toxicity of sodium 6-diazo-5-oxo-5,6-dihydronaphthalene-1-sulfonate was 200 mg/kg/day for males and females based on hyperplasia of the squamous epithelium in the limiting ridge of the forestomach and hyperplasia of the surface epithelial cells in the glandular stomach in males and females treated at 1000 mg/kg, and decrease of food consumption in males treated at 1000 mg/kg. The No-Observed-Effect-Level (NOEL) was 40 mg/kg/day for males and females based on decreased food consumption in males and females treated at 200 mg/kg and above.

The NOEL for the reproductive performance were 1000 mg/kg/day for parental animals as no effects were observed. The NOEL for general toxicity was determined to be 1000 mg/kg bw/day for F1-generation as no effects occurred.

Cross-reference 2

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

other: CRL/CD(SD)

Details on species / strain selection:

- Age at study initiation of dosing: 9 weeks old
- Recovery: 0, 1000 mg/kg/day

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River, Japan
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 8 weeks
- Weight at study initiation: 326.1 – 376.3 g for males and 199.6 – 238.6 g for females

- Housing: Animals were housed individually in a stainless cage with mesh floor (W260 mm x D380 mm x H180 mm) for males and (W165 mm x D300 mm x H150 mm) for females until 13 days of pregnant and during fasting on the day before necropsy. During mating a male and a female were housed together in male’s cage. After confirming mating, female was moved and housed in polycarbonate flat cage (W265 mm x D426 mm x H150 mm) with bedding (Sanflakes, Japan Charles River) between day 14 of gestation and the day before fasting. After birth, pup was housed together with their dam.

- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 10 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21-25 °C
- Humidity (%): 40-70%
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12/12 h

Route of administration:

oral: gavage

Details on route of administration:

Test volume of 4 mL/kg of the test solutions were treated to the test animals using syringe (Terumo Corporation) connected to Nelaton catheter (Terumo Corporation) once a day (7 days per week) and at the same time each day.

Vehicle:

water

Details on oral exposure:

- VEHICLE
- Justification for use and choice of vehicle (if other than water): water was used
- Concentration in vehicle: 250 mg/mL (high doese), lower doses were obtained by dilution
- Amount of vehicle (if gavage): 4 mL
- Lot/batch no. (if required): -
- Purity: -

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The stability during the study period was confirmed by measurements of Infrared absorption spectrum of the test substance before and after the treatment period. High dose solution was prepared by pure water (Takasugi pharmaceutical Co.) adding to the test substance. Middle and low dose solutions were prepared by dilution from the high dose solution. The stability of the test substance in the test solutions for 14 days were confirmed by HPLC analysis. The test solutions were stored in cool and dark place until dosing and used within 14 days. The concentration of the test substance in the test solutions were measured prior to treatment.

Duration of treatment / exposure:

Male: 42 days
Female: 42 - 54 days (from 14 days before mating to day 4 of lactation)

Frequency of treatment:

daily

Dose / conc.:

0 mg/kg bw/day (actual dose received)

Dose / conc.:

40 mg/kg bw/day (actual dose received)

Dose / conc.:

200 mg/kg bw/day (actual dose received)

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

Males, 12 (5 for recovery); females, 12; satellite females, 5

Control animals:

yes, concurrent vehicle

Details on study design:

The test doses were selected based on the results of 14 days repeat dose toxicological study. Some changes which were induced by irritation of the test substance such as Soft stool were recorded in females treated at 250 mg/kg and above and males treated at 500 mg/kg and above, elevated limiting ridge in forestomach in males treated at 1000 mg/kg and enormous of cecum in males and females treated at 1000 mg/kg were observed in the 14 days repeated dose toxicity study when Crl:CD(SD) rats, 3/sex/group, were treated with 0, 25, 250, 500 and 1000 mg/kg/day. There was no abnormality in kidney weight, while one male treated at 1000 mg/kg had a bilateral swallow and unilateral decoloration of kidney. Furthermore, although brown urine was detected in all treated groups, no abnormalities were detected in body weight and organ weights. Therefore, 1000 mg/kg/day was selected as the highest dose, and lower 2 dosage of 200 and 40 mg/kg/day calculated by a factor of 5 were selected for the main study.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
Males were observed twice a day between the start of treatment and the day before necropsy and once a day on the day of necropsy. Females in the recovery group were observed twice a day until the end of the treatment, and clinical signs including in delivery and lactation status in females in the main group were observed twice a day between the start of doing and day 4 post-partum and once a day on day 5 post-partum (necropsy). Males and females were observed once a day until the day before necropsy during the recovery period.


DETAILED CLINICAL OBSERVATIONS: Yes
All the males and females were observed by blind and scored once prior to treatment and once a week just after dosing thereafter. For blind observation, all animals were re-sorted by random number, and then a label for examination in order to unknown their groups were given. Although females were examine on day 4 post-partum, no blind examination was performed if only one female was examined.
For the reactions of removing from cage, easy removing from cage and vocalization were observed when animals were given external stimulation such as holding or approached a hand to hold animals. Detailed clinical observations during handling were checked muscle tone, hypothermia with or without piloerection, condition of fur (soiled, coarse), color of skin and mucosa (pale, Reddening, cyanosis). abnormal eyes (lacrimation, ophthalmocele, Pupil size), salivation and secretion. Observations of behavior in arena were checked position, activity, respiration, closed eyelid, abnormal gait, tremor, twitch, convulsion, stereotyped behavior and abnormal behavior when animals were stayed in arena for 1 minute. The number of excretion (the number of feces) and the number of urination (the number of urine pools) were recorded for 1 minute.

BODY WEIGHT: Yes
Body weight of males were weighted on 1, 3, 7, 14, 21, 28, 35, 42 and 43 days (fasted, at removal) post dosing, and females were weighed on 1, 3, 7 and 14 days post dosing, 0, 7, 14, 17 and 20 days post pregnant, and 0 (the day of delivery), 4 and 5 days (fasted, at removal) post-partum. Females in the satellite groups were measured at the same days with males. Body weight of males and females during the recovery period were measured on 1, 3, 7, 14 and 15 (fasted, at removal) days post recovery.

FOOD CONSUMPTION: yes
Food consumption of males were measured on 1, 3, 7, 14, 21, 28, 35 and 42 days post dosing, and females were measured on 1, 3, 7 and 14 days post dosing, 0, 7, 14, 17 and 20 days post pregnant, and 0 (the day of delivery) and 4 days post-partum. Females in the satellite groups were measured at the same days with males. Food consumption of males and females during the recovery period were measured on 1, 3, 7 and 14 days post recovery. Mean daily food consumption was calculated using amount of food consumption until next measurement period.


WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
Animals were fasted for overnight (16-20 hours) from afternoon of the last day of the treatment or recovery period to necropsy, but drinking water accessed ad libitum. Blood were drawn from abdominal aorta under Ether anesthesia. Males used 5 animals of the smallest animal number among surviving animals of each dose, and females used 5 animals near each delivery for each dose, and all males and females in the recovery groups were selected for hematology. EDTA-2K was used as an anti-coagulant and hematology was measured using automated hematology analyzer (CELL-DYN 3500, Abbott Laboratories) for red blood cells (RBC), white blood cells (WBC), hemoglobin (Hb), hematocrit (Ht), mean cell volume(MCV), mean cell hemoglobin (MCH), mean cell hemoglobin concentration (MCHC) and platelet (Platelet). In addition, reticulocyte ratio (Reticulo) and differentiation of leukocyte (Differentiation of leukocyte) were measured by General hematology analyzer (ADVIA 120, Siemens AG). Plasma was separated from blood samples withdrawn into 3.2% sodium citrate solution as an anti-coagulant and measured for prothrombin time (PT) and activated partly thromboplastin (APTT) using Automatic blood coagulation and fibrinolysis measuring device (STA Compact, Roche Diagnostics K.K).


CLINICAL CHEMISTRY: Yes
Blood serum from the same animals employed for hematology were used for blood chemistry. aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), gamma-glutamyl-transpeptidase (γ-GTP), total cholesterol (T-Cho), triglycerides (TG), glucose (Glucose), total protein (T-Protein), albumin (Albumin), urea nitrogen (BUN), creatinine (Creatinine), total bilirubin (T-Bil), calcium (Ca) and inorganic phosphorus (IP) were measured using Blood chemistry Autoanalyzer (7180, Hitachi). Furthermore, sodium (Na), potassium (K) and chlorine (Cl) were measured using electrode analyzer (PVA-EX II, A&T). A/G ratio (A/G ratio) was calculated based on the serum levels of total protein and albumin.


URINALYSIS: Yes
Pooled urine for 15-17 hours were obtained from the same animals employed to hematology. Animals were housed into a metabolic cage individually in the afternoon of the last day of the treatment or recovery period. During urine collection, animals were fasted but drinking water accessed ad libitum. Urine volume, color and clearly were checked using the collected urine, osmolarity were measured using automatic osmometer (OM-6040, Arkrey Inc.), pH, protein, glucose and blood were measured using urinalysis paper (Hemacombisticks, Siemens AG). Urine residue was also measured. Regarding to urine residue, since no treatment-related changes were detected in males and females in the high dose group when it was measured in the control and high dose groups, urinalysis of middle and low dose groups and recovery group were not determined.


NEUROBEHAVIOURAL EXAMINATION: Yes
Five males selected in ascending order of animal number of each dose were examined after dosing of the last week of the treatment period. Five delivery females with close delivery day at each dose were examined before fasting on day 4 post-partum. Animals assigned to in the recovery group were examined after dosing during the last week of the treatment period. Since no abnormalities which seemed to be treatment-related were observed during the treatment period, the examination during the recovery period was not performed.
For examination of reaction, the reactions when suitable external stimulation gave to the target sensory organ were checked and scored. This examination was conducted by blind continued the detailed clinical observation. For vision (approach, sense of touch), the response when the sheath of ballpoint pen were held keeping for 3 cm distance from the face, and holding it for 4 seconds was recorded. For auditory sense, the reaction when fingers were rang on overhead were recorded. Pain reaction was recorded when sandwiching the one third ridge portion of the tail with scissors. Pupillary reflex recorded the reaction of the pupil when light was irradiated after blocking the light. Righting reflex in air was recorded as the reaction when dropping from the height of about 30 cm with the abdomen of the animal facing upward.
Grip strength were measured by blind using a grip strength meter (FGC-2, Metis). The measurement was performed each twice for forelimb and hindlimb, and mean values were calculated as the grip strength values of forelimb and hindlimb to individual animal.
Locomotor activity was measured as motor activity using a motor activity measuring device for rats (ACTIMO-10, Shintechno Corporation). The number of times across the infrared beam was counted as the measured value, and it was measured for 1 hour (6 times at 10 minute intervals).


IMMUNOLOGY: No


OTHER:

Sacrifice and pathology:

All animals were examined macroscopically for body surface, opening, subcutaneous, cranial cavity, thoracic cavity, abdominal cavity, pelvic cavity and contents on the day after the end of the treatment and recovery period. The number of pregnant corpus luteum in ovaries and the number of implantation sites in uterus after incision were recorded.

Other examinations:

Trachea and lung, stomach, intestine (duodenum - rectum with Peyer's patches), liver, heart, kidneys, urinary bladder, testes, epididymis, prostate, seminal vesicle, ovary, uterus, vagina, brain (cerebrum, cerebellum with pons), spinal cord, sciatic nerve, bone marrow (femur), lymph node (axillary lymph node, mesenteric lymph node ), spleen, thymus, pituitary, thyroid (with parathyroid) and adrenal gland were collected from all animals. Organ weights of liver, heart, kidney, testes, epididymis, brain, spleen, thymus and adrenal gland were measured, and relative organ weight per final body weight were calculated. Collected organs/tissues were fixed with 10 % neutral buffered formalin solution except for testes and epididymis which were fixed with modified Davidson solution.

Histopathological examination for trachea, lung, stomach, intestine, liver, heart, kidney, urinary bladder, testes, epididymis, prostate, seminal vesicle, ovary, uterus, vagina, brain, spinal cord, sciatic nerve, bone marrow, axillary lymph node, mesenteric lymph node, spleen, thymus, pituitary, thyroid and adrenal gland from the control and 1000 mg/kg groups were carried out.

Histopathological slides which were prepared HE stain to paraffin embedded thin section were examined under light microscope. The organs or tissues from the other groups and recovery groups were examined if treatment-related changes were detected in the high dose group. In addition, all gross abnormal changes were examined. Ovary, uterus and vagina from infertility females were examined, and teste from paired two males were also examined.
Furthermore, since one male in the 40 mg/kg group had Brown pigment deposition in tubular epithelium in kidney at HE stained slide and it seemed to be hemosiderin pigmentation, Berlin blue staining was performed.

Statistics:

Standard statistical methods have been applied for data processing.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Brown urine was observed in 9 out of 12 males in the 40 mg/kg group, all 12 males in the 200 mg/kg group during the study period. In the 1000 mg/kg group, brown urine and soft stool were observed all 12 males, mucosal stool and staining of abdominal were observed each one male. In females, brown urine was noted 4 out of 12 in the 40 mg/kg group and all females in the 200 mg/kg and above dose groups throughout pre-mating, pregnant and lactation periods. In addition, 3 out of 12 females in the 1000 mg/kg group showed soft stool. Furthermore, all 5 females and 3 females treated at 1000 mg/kg in the satellite group showed brown urine and soft stool, respectively. Although each one females from the control and 40 mg/kg groups was not pregnant, no abnormal change was observed in clinical observations.
During the recovery period, all 5 males in the 1000 mg/kg group showed brown urine on day 1, and it was disappeared the next day and thereafter. Furthermore, one male at the same group showed staining of abdominal until the end of the recovery period. All 5 females in the 1000 mg/kg group showed brown urine on day 1 and it was disappeared the next day and thereafter.

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

In males during the treatment period, final body weight (day 42 after treatment) decreased to 93.7%, 95.1% and 96.7% of control values in the 1000 mg/kg, 200 mg/kg and 40 mg/kg, respectively, but these reductions were not statistically significant. Body weight in females was not significant in the treatment groups.
There were no statistically significant differences in males and female in the treatment groups during the recovery period.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Lower level of food consumption was recorded in males at 200 mg/kg and above dose levels on day 3 during the treatment period. High value was noted in the 1000 mg/kg on day 28. Food consumption in the 40 mg/kg group was not significant difference. In females low value was noted in the 200 mg/kg and above dose groups on day 3. In addition, high value was recorded in the 1000 mg/kg group on days 7 and 20 post-pregnant as well as on days 21, 35 and 42 in the satellite group. There was no statistically significant difference in the 40 mg/kg group. During the recovery period, there was no statistically significant difference in males in the 1000 mg/kg group. Females in the 1000 mg/kg group showed high value on day 3 in the recovery period.

Food efficiency:

no effects observed

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no changes seemed to be treatment-related in males and females at the end of treatment period. Although low reticulocyte ratio was noted in males treated at 200 mg/kg, this was decided to be incidental because dose related relationship was not clear.
At the end of recovery period, high ratio of reticulocyte of males in the 1000 mg/kg group which was not detected at the end of the treatment period was recorded. However, because no change in the number of red blood cells was noted and therefore it was not considered to be replacement change of anemia, this change of reticulocyte was decided to be incidental. Statistically significant changes were not recorded in females treated at 1000 mg/kg.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

In males low serum level of total protein was noted in the 1000 mg/kg group at the end of the treatment period. Furthermore, low serum level of urea nitrogen was recorded in the 40 mg/kg and above dose groups, but dose dependency was not clear. Although low serum level of total cholesterol was also recorded in the 40 mg/kg group, this change was decided to be incidental because dose related relationship was not clear. There was no statistically significant change in females in the treatment groups.
At the end of the recovery period, high level of urea nitrogen in males which was not noted at the end of the treatment period was recorded in the 1000 mg/kg group. In females low levels of γ-GTP and Na were recorded in the 1000 mg/kg group. However, these changes were considered to be incidental because the same changes were not detected at the end of the treatment period.

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

In males yellow-red urine in the 200 mg/kg and above dose groups and brown urine in the 1000 mg/kg group were noted at the end of the treatment period. No abnormal change in the 40 mg/kg group was detected. In females yellow-red urine or brown urine was noted in the 1000 mg/kg group at the end of the treatment period. Incidence of the number of animals have pH 6.0 was a tendency to increase at 200 mg/kg and above dose levels. No abnormal change was noted in the 40 mg/kg group.
At the end of the recovery period, there were no abnormal changes in males and females in the 1000 mg/kg group.

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

There was no abnormal changes in males and females treated with the test substance during the treatment period.
Functional observations were not examined to males and females within the recovery period because treatment-related changes were not detected at examination of functional observations during the treatment period.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

At the end of the treatment period, low value of absolute liver weight in males treated at 200 mg/kg and above, low value of relative liver weight in males at 1000 mg/kg were recorded. Statistically significant difference was not detected in the 40 mg/kg group. In females there was no significant change in all treated groups.
At the end of the recovery period no statistically significant difference in males at 1000 mg/kg were noted. In females, high value of absolute kidney weight and relative liver weight were recorded in the 1000 mg/kg group, while these changed were not noted at the end of the treatment period.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

In male at the end of the treatment period, elevated limiting ridge in forestomach and enormous of cecum were observed in 2 out of 7 males and all males, respectively, in the 1000 mg/kg group. In addition, some spontaneous changes such as black colored membrane in glandular stomach and dark browning of kidney which were observed one male, diverticulum in jejunum and white area in epididymis that both findings were detected in one male were observed in the 40 mg/kg group. There was no abnormal change in the 200 mg/kg group. In females, elevated limiting ridge in forestomach and enormous of cecum were observed in 2 out of 12 females and 11 females, respectively, in the 1000 mg/kg group. Some spontaneous changes such as black colored membrane in glandular stomach in one female, diverticulum in jejunum in another one female, and cyst in pituitary gland in 2 females were detected in the control group. There were no abnormal changes in the 40 and 200 mg/kg groups.
At the end of the recovery period no abnormal changes were observed in males and females in the 1000 mg/kg group.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

At the end of the treatment period, hyperplasia of the squamous epithelium in the limiting ridge of the forestomach which was corresponded to the elevated limiting ridge observed at necropsy was observed in 5 out of 7 males in the 1000 mg/kg group. Furthermore, hyperplasia of the surface epithelial cells in the glandular stomach was observed in 3 males at the same dose group. For cecum, although enormous was noted at necropsy, no histopathological abnormality was detected. No abnormal changes in liver which was detected low relative weight in the 1000 mg/kg group were observed. In the 40 mg/kg group, necrosis of fundic mucosa in fundus gland area of glandular stomach and brown pigment deposition in tubular epithelium in kidney in one male and diverticulum in jejunum and unilateral spermatic granuloma in epididymis in another male were observed and these changes were corresponded to the changes observed at necropsy. Some spontaneous findings such as mineralization in Peyer’s patches in jejunum, focal myocarditis in heart, solitary cyst in medulla and subcapsular solitary cyst in kidney were observed individual one male in the 1000 mg/kg group. In addition, focal necrosis of hepatocytes in liver and bilateral degeneration of seminiferous tubules in testes were observed each 1 out of 6 males in the control group. Abnormal changes in kidney was not reproduced in this study, while enlargement and faded color in kidney were detected in the dose-range finding study.
No abnormal changes in testes from one male who was partner of infertility female in the control or 40 mg/kg group were detected.
There was no positive substance in tubular epithelium of the kidney with Berlin blue staining which was conducted to confirm brown pigment deposition in tubular epithelium observed in one male treated at 40 mg/kg.
In females hyperplasia of the squamous epithelium in the limiting ridge of the forestomach which was corresponded to the elevated limiting ridge observed at necropsy was observed in 6 out of 12 females in the 1000 mg/kg group. Furthermore, edema in submucosal layer and hyperplasia of surface epithelial cells in the glandular stomach were observed in each at the same dose group. For cecum, although enormous was noted at necropsy, no histopathological abnormality was detected.
In the control group, necrosis of fundic mucosa in glandular stomach and diverticulum in jejunum in each, and cyst formation in pars nervosa of pituitary gland in 2 females were observed and they were a correspondence to macroscopic findings. Some spontaneous findings such as mineralization in Peyer’s patches in duodenum and jejunum, solitary cyst in medulla of kidney in each of the 1000 mg/kg group were observed. In addition, mineralization in Peyer’s patches in jejunum in 4 out of 11 females, focal necrosis of hepatocytes in liver, cyst formation in pars intermedia of pituitary gland and unilateral focal necrosis of cortex in adrenal were observed in each of the control group.
No abnormal changes in ovary, uterus and vagina in one infertility female from the control and 40 mg/kg groups were detected.
At the end of the recovery period, there were no changes in forestomach and glandular stomach in males and females. Abnormal changes in liver was not detected in females in the 1000 mg/kg group, though relative liver weight showed high value.

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Key result

Dose descriptor:

NOAEL

Effect level:

200 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

food efficiency

histopathology: non-neoplastic

Key result

Critical effects observed:

no

Lowest effective dose / conc.:

1 000 mg/kg bw/day (actual dose received)

System:

gastrointestinal tract

Organ:

stomach

Conclusions:

The NOAEL of the test item was determined to be 200 mg/kg bw/day for male and females rats.

Executive summary:

A Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test according to OECD 422 under GLP conditions was conducted with the test item. Each five females and five males per dose group were treated with the test item daily for 54 days or 42 days, respectively. The test item was administered in following concentrations: 40, 200, and 2000 mg/kg bw/d.

The No Observed Adverse Effect Level was determined to be 200 mg/kg bw/day for males and females based the histopathological findings and changes in food consumption.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: OECD 422 (Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test)

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Sodium 6-diazo-5,6-dihydro-5-oxonaphthalene-1-sulphonate

EC Number:

220-189-5

EC Name:

Sodium 6-diazo-5,6-dihydro-5-oxonaphthalene-1-sulphonate

Cas Number:

2657-00-3

Molecular formula:

C10H6N2O4S.Na

IUPAC Name:

sodium 6-diazo-5,6-dihydro-5-oxonaphthalene-1-sulphonate

Test material form:

solid

Test animals

Species:

rat

Strain:

other: CRL/CD(SD)

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River, Japan
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 8 weeks
- Weight at study initiation:
326.1 – 376.3 g for males and 199.6 – 238.6 g for females

- Housing:
Animals were housed individually in a stainless cage with mesh floor (W260 mm x D380 mm x H180 mm) for males and (W165 mm x D300 mm x H150 mm) for females until 13 days of pregnant and during fasting on the day before necropsy. During mating a male and a female were housed together in male’s cage. After confirming mating, female was moved and housed in polycarbonate flat cage (W265 mm x D426 mm x H150 mm) with bedding (Sanflakes, Japan Charles River) between day 14 of gestation and the day before fasting. After birth, pup was housed together with their dam.

- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 10 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21-25 °C
- Humidity (%): 40-70%
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12/12 h

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

- VEHICLE
- Concentration in vehicle: 250 mg/mL (high doese), lower doses were obtained by dilution
- Amount of vehicle (if gavage): 4 mL

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The stability during the study period was confirmed by measurements of Infrared absorption spectrum of the test substance before and after the treatment period. High dose solution was prepared by pure water (Takasugi pharmaceutical Co.) adding to the test substance. Middle and low dose solutions were prepared by dilution from the high dose solution. The stability of the test substance in the test solutions for 14 days were confirmed by HPLC analysis. The test solutions were stored in cool and dark place until dosing and used within 14 days. The concentration of the test substance in the test solutions were measured prior to treatment.

Details on mating procedure:

- M/F ratio per cage: 1:1, avoiding sibling mating
- Length of cohabitation: until confirmed mating, for maximum 14 days
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 post-coitum
- After successful mating each pregnant female was caged individually in Macrolon cages

Duration of treatment / exposure:

Male: 42 days
Female: 42 - 54 days (from 14 days before mating to day 4 of lactation)

Frequency of treatment:

daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (actual dose received)

Doses / concentrations 2

Dose / conc.:

40 mg/kg bw/day (actual dose received)

Doses / concentrations 3

Dose / conc.:

200 mg/kg bw/day (actual dose received)

Doses / concentrations 4

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

Males, 12 (5 for recovery); females, 12; satellite females, 5

Control animals:

yes, concurrent vehicle

Details on study design:

The test doses were selected based on the results of 14 days repeat dose toxicological study. Some changes which were induced by irritation of the test substance such as Soft stool were recorded in females treated at 250 mg/kg and above and males treated at 500 mg/kg and above, elevated limiting ridge in forestomach in males treated at 1000 mg/kg and enormous of cecum in males and females treated at 1000 mg/kg were observed in the 14 days repeated dose toxicity study when Crl:CD(SD) rats, 3/sex/group, were treated with 0, 25, 250, 500 and 1000 mg/kg/day. There was no abnormality in kidney weight, while one male treated at 1000 mg/kg had a bilateral swallow and unilateral decoloration of kidney. Furthermore, although brown urine was detected in all treated groups, no abnormalities were detected in body weight and organ weights. Therefore, 1000 mg/kg/day was selected as the highest dose, and lower 2 dosage of 200 and 40 mg/kg/day calculated by a factor of 5 were selected for the main study.

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
Males were observed twice a day between the start of treatment and the day before necropsy and once a day on the day of necropsy. Females in the recovery group were observed twice a day until the end of the treatment, and clinical signs including in delivery and lactation status in females in the main group were observed twice a day between the start of doing and day 4 post-partum and once a day on day 5 post-partum (necropsy). Males and females were observed once a day until the day before necropsy during the recovery period.


DETAILED CLINICAL OBSERVATIONS: Yes
All the males and females were observed by blind and scored once prior to treatment and once a week just after dosing thereafter. For blind observation, all animals were re-sorted by random number, and then a label for examination in order to unknown their groups were given. Although females were examine on day 4 post-partum, no blind examination was performed if only one female was examined.
For the reactions of removing from cage, easy removing from cage and vocalization were observed when animals were given external stimulation such as holding or approached a hand to hold animals. Detailed clinical observations during handling were checked muscle tone, hypothermia with or without piloerection, condition of fur (soiled, coarse), color of skin and mucosa (pale, Reddening, cyanosis). abnormal eyes (lacrimation, ophthalmocele, Pupil size), salivation and secretion. Observations of behavior in arena were checked position, activity, respiration, closed eyelid, abnormal gait, tremor, twitch, convulsion, stereotyped behavior and abnormal behavior when animals were stayed in arena for 1 minute. The number of excretion (the number of feces) and the number of urination (the number of urine pools) were recorded for 1 minute.

BODY WEIGHT: Yes
Body weight of males were weighted on 1, 3, 7, 14, 21, 28, 35, 42 and 43 days (fasted, at removal) post dosing, and females were weighed on 1, 3, 7 and 14 days post dosing, 0, 7, 14, 17 and 20 days post pregnant, and 0 (the day of delivery), 4 and 5 days (fasted, at removal) post-partum. Females in the satellite groups were measured at the same days with males. Body weight of males and females during the recovery period were measured on 1, 3, 7, 14 and 15 (fasted, at removal) days post recovery.

FOOD CONSUMPTION: yes
Food consumption of males were measured on 1, 3, 7, 14, 21, 28, 35 and 42 days post dosing, and females were measured on 1, 3, 7 and 14 days post dosing, 0, 7, 14, 17 and 20 days post pregnant, and 0 (the day of delivery) and 4 days post-partum. Females in the satellite groups were measured at the same days with males. Food consumption of males and females during the recovery period were measured on 1, 3, 7 and 14 days post recovery. Mean daily food consumption was calculated using amount of food consumption until next measurement period.


WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
Animals were fasted for overnight (16-20 hours) from afternoon of the last day of the treatment or recovery period to necropsy, but drinking water accessed ad libitum. Blood were drawn from abdominal aorta under Ether anesthesia. Males used 5 animals of the smallest animal number among surviving animals of each dose, and females used 5 animals near each delivery for each dose, and all males and females in the recovery groups were selected for hematology. EDTA-2K was used as an anti-coagulant and hematology was measured using automated hematology analyzer (CELL-DYN 3500, Abbott Laboratories) for red blood cells (RBC), white blood cells (WBC), hemoglobin (Hb), hematocrit (Ht), mean cell volume(MCV), mean cell hemoglobin (MCH), mean cell hemoglobin concentration (MCHC) and platelet (Platelet). In addition, reticulocyte ratio (Reticulo) and differentiation of leukocyte (Differentiation of leukocyte) were measured by General hematology analyzer (ADVIA 120, Siemens AG). Plasma was separated from blood samples withdrawn into 3.2% sodium citrate solution as an anti-coagulant and measured for prothrombin time (PT) and activated partly thromboplastin (APTT) using Automatic blood coagulation and fibrinolysis measuring device (STA Compact, Roche Diagnostics K.K).


CLINICAL CHEMISTRY: Yes
Blood serum from the same animals employed for hematology were used for blood chemistry. aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), gamma-glutamyl-transpeptidase (γ-GTP), total cholesterol (T-Cho), triglycerides (TG), glucose (Glucose), total protein (T-Protein), albumin (Albumin), urea nitrogen (BUN), creatinine (Creatinine), total bilirubin (T-Bil), calcium (Ca) and inorganic phosphorus (IP) were measured using Blood chemistry Autoanalyzer (7180, Hitachi). Furthermore, sodium (Na), potassium (K) and chlorine (Cl) were measured using electrode analyzer (PVA-EX II, A&T). A/G ratio (A/G ratio) was calculated based on the serum levels of total protein and albumin.


URINALYSIS: Yes
Pooled urine for 15-17 hours were obtained from the same animals employed to hematology. Animals were housed into a metabolic cage individually in the afternoon of the last day of the treatment or recovery period. During urine collection, animals were fasted but drinking water accessed ad libitum. Urine volume, color and clearly were checked using the collected urine, osmolarity were measured using automatic osmometer (OM-6040, Arkrey Inc.), pH, protein, glucose and blood were measured using urinalysis paper (Hemacombisticks, Siemens AG). Urine residue was also measured. Regarding to urine residue, since no treatment-related changes were detected in males and females in the high dose group when it was measured in the control and high dose groups, urinalysis of middle and low dose groups and recovery group were not determined.


NEUROBEHAVIOURAL EXAMINATION: Yes
Five males selected in ascending order of animal number of each dose were examined after dosing of the last week of the treatment period. Five delivery females with close delivery day at each dose were examined before fasting on day 4 post-partum. Animals assigned to in the recovery group were examined after dosing during the last week of the treatment period. Since no abnormalities which seemed to be treatment-related were observed during the treatment period, the examination during the recovery period was not performed.
For examination of reaction, the reactions when suitable external stimulation gave to the target sensory organ were checked and scored. This examination was conducted by blind continued the detailed clinical observation. For vision (approach, sense of touch), the response when the sheath of ballpoint pen were held keeping for 3 cm distance from the face, and holding it for 4 seconds was recorded. For auditory sense, the reaction when fingers were rang on overhead were recorded. Pain reaction was recorded when sandwiching the one third ridge portion of the tail with scissors. Pupillary reflex recorded the reaction of the pupil when light was irradiated after blocking the light. Righting reflex in air was recorded as the reaction when dropping from the height of about 30 cm with the abdomen of the animal facing upward.
Grip strength were measured by blind using a grip strength meter (FGC-2, Metis). The measurement was performed each twice for forelimb and hindlimb, and mean values were calculated as the grip strength values of forelimb and hindlimb to individual animal.
Locomotor activity was measured as motor activity using a motor activity measuring device for rats (ACTIMO-10, Shintechno Corporation). The number of times across the infrared beam was counted as the measured value, and it was measured for 1 hour (6 times at 10 minute intervals).


IMMUNOLOGY: No


OTHER:

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other:

Fetal examinations:

The number of pups born (the number of stillborn + the number of live born), the number of stillborn, the number of live born, sex, the number of live born on day 0, sex of live born on day 0, external abnormality of live born were observed on the birth day (set up as day 0 post-partum). After then, clinical observations, the number of live pups, the number of dead pups and sex were observed until day 4 post birth, and live birth index: (the number of live pups/the number of implantation sites) x 100, viability index on day 0: (the number of live pups/the number of pups born) x 100, sex ratio of pups born: the number of male pups/the number of pups born, sex ration of pups on day 0: the number of male pups on day 0/the number of live pups on day 0, external anomaly index: (the number of live pups with external anomalies/live pups) x 100, viability index on day 4: (the number of live pups on day 4/live pups) x 100, sex ratio on day 4: the number of male live pups on day 4/the number of live pups on day 4 were calculated. Body weights of male or females per litter were measured at birth and on day 4 post-partum.

Statistics:

Standard statistical methods have been applied for data processing.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Brown urine was observed in 9 out of 12 males in the 40 mg/kg group, all 12 males in the 200 mg/kg group during the study period. In the 1000 mg/kg group, brown urine and soft stool were observed all 12 males, mucosal stool and staining of abdominal were observed each one male. In females, brown urine was noted 4 out of 12 in the 40 mg/kg group and all females in the 200 mg/kg and above dose groups throughout pre-mating, pregnant and lactation periods. In addition, 3 out of 12 females in the 1000 mg/kg group showed soft stool. Furthermore, all 5 females and 3 females treated at 1000 mg/kg in the satellite group showed brown urine and soft stool, respectively. Although each one females from the control and 40 mg/kg groups was not pregnant, no abnormal change was observed in clinical observations.
During the recovery period, all 5 males in the 1000 mg/kg group showed brown urine on day 1, and it was disappeared the next day and thereafter. Furthermore, one male at the same group showed staining of abdominal until the end of the recovery period. All 5 females in the 1000 mg/kg group showed brown urine on day 1 and it was disappeared the next day and thereafter.

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

In males during the treatment period, final body weight (day 42 after treatment) decreased to 93.7%, 95.1% and 96.7% of control values in the 1000 mg/kg, 200 mg/kg and 40 mg/kg, respectively, but these reductions were not statistically significant. Body weight in females was not significant in the treatment groups.
There were no statistically significant differences in males and female in the treatment groups during the recovery period.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Lower level of food consumption was recorded in males at 200 mg/kg and above dose levels on day 3 during the treatment period. High value was noted in the 1000 mg/kg on day 28. Food consumption in the 40 mg/kg group was not significant difference. In females low value was noted in the 200 mg/kg and above dose groups on day 3. In addition, high value was recorded in the 1000 mg/kg group on days 7 and 20 post-pregnant as well as on days 21, 35 and 42 in the satellite group. There was no statistically significant difference in the 40 mg/kg group. During the recovery period, there was no statistically significant difference in males in the 1000 mg/kg group. Females in the 1000 mg/kg group showed high value on day 3 in the recovery period.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no changes seemed to be treatment-related in males and females at the end of treatment period. Although low reticulocyte ratio was noted in males treated at 200 mg/kg, this was decided to be incidental because dose related relationship was not clear.
At the end of recovery period, high ratio of reticulocyte of males in the 1000 mg/kg group which was not detected at the end of the treatment period was recorded. However, because no change in the number of red blood cells was noted and therefore it was not considered to be replacement change of anemia, this change of reticulocyte was decided to be incidental. Statistically significant changes were not recorded in females treated at 1000 mg/kg.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

In males low serum level of total protein was noted in the 1000 mg/kg group at the end of the treatment period. Furthermore, low serum level of urea nitrogen was recorded in the 40 mg/kg and above dose groups, but dose dependency was not clear. Although low serum level of total cholesterol was also recorded in the 40 mg/kg group, this change was decided to be incidental because dose related relationship was not clear. There was no statistically significant change in females in the treatment groups.
At the end of the recovery period, high level of urea nitrogen in males which was not noted at the end of the treatment period was recorded in the 1000 mg/kg group. In females low levels of γ-GTP and Na were recorded in the 1000 mg/kg group. However, these changes were considered to be incidental because the same changes were not detected at the end of the treatment period.

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

In males yellow-red urine in the 200 mg/kg and above dose groups and brown urine in the 1000 mg/kg group were noted at the end of the treatment period. No abnormal change in the 40 mg/kg group was detected. In females yellow-red urine or brown urine was noted in the 1000 mg/kg group at the end of the treatment period. Incidence of the number of animals have pH 6.0 was a tendency to increase at 200 mg/kg and above dose levels. No abnormal change was noted in the 40 mg/kg group.
At the end of the recovery period, there were no abnormal changes in males and females in the 1000 mg/kg group

Behaviour (functional findings):

no effects observed

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

At the end of the treatment period, low value of absolute liver weight in males treated at 200 mg/kg and above, low value of relative liver weight in males at 1000 mg/kg were recorded. Statistically significant difference was not detected in the 40 mg/kg group. In females there was no significant change in all treated groups.
At the end of the recovery period no statistically significant difference in males at 1000 mg/kg were noted. In females, high value of absolute kidney weight and relative liver weight were recorded in the 1000 mg/kg group, while these changed were not noted at the end of the treatment period.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

In male at the end of the treatment period, elevated limiting ridge in forestomach and enormous of cecum were observed in 2 out of 7 males and all males, respectively, in the 1000 mg/kg group. In addition, some spontaneous changes such as black colored membrane in glandular stomach and dark browning of kidney which were observed one male, diverticulum in jejunum and white area in epididymis that both findings were detected in one male were observed in the 40 mg/kg group. There was no abnormal change in the 200 mg/kg group. In females, elevated limiting ridge in forestomach and enormous of cecum were observed in 2 out of 12 females and 11 females, respectively, in the 1000 mg/kg group. Some spontaneous changes such as black colored membrane in glandular stomach in one female, diverticulum in jejunum in another one female, and cyst in pituitary gland in 2 females were detected in the control group. There were no abnormal changes in the 40 and 200 mg/kg groups.
At the end of the recovery period no abnormal changes were observed in males and females in the 1000 mg/kg group.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

At the end of the treatment period, hyperplasia of the squamous epithelium in the limiting ridge of the forestomach which was corresponded to the elevated limiting ridge observed at necropsy was observed in 5 out of 7 males in the 1000 mg/kg group. Furthermore, hyperplasia of the surface epithelial cells in the glandular stomach was observed in 3 males at the same dose group. For cecum, although enormous was noted at necropsy, no histopathological abnormality was detected. No abnormal changes in liver which was detected low relative weight in the 1000 mg/kg group were observed. In the 40 mg/kg group, necrosis of fundic mucosa in fundus gland area of glandular stomach and brown pigment deposition in tubular epithelium in kidney in one male and diverticulum in jejunum and unilateral spermatic granuloma in epididymis in another male were observed and these changes were corresponded to the changes observed at necropsy. Some spontaneous findings such as mineralization in Peyer’s patches in jejunum, focal myocarditis in heart, solitary cyst in medulla and subcapsular solitary cyst in kidney were observed individual one male in the 1000 mg/kg group. In addition, focal necrosis of hepatocytes in liver and bilateral degeneration of seminiferous tubules in testes were observed each 1 out of 6 males in the control group. Abnormal changes in kidney was not reproduced in this study, while enlargement and faded color in kidney were detected in the dose-range finding study.
No abnormal changes in testes from one male who was partner of infertility female in the control or 40 mg/kg group were detected.
There was no positive substance in tubular epithelium of the kidney with Berlin blue staining which was conducted to confirm brown pigment deposition in tubular epithelium observed in one male treated at 40 mg/kg.
In females hyperplasia of the squamous epithelium in the limiting ridge of the forestomach which was corresponded to the elevated limiting ridge observed at necropsy was observed in 6 out of 12 females in the 1000 mg/kg group. Furthermore, edema in submucosal layer and hyperplasia of surface epithelial cells in the glandular stomach were observed in each at the same dose group. For cecum, although enormous was noted at necropsy, no histopathological abnormality was detected.
In the control group, necrosis of fundic mucosa in glandular stomach and diverticulum in jejunum in each, and cyst formation in pars nervosa of pituitary gland in 2 females were observed and they were a correspondence to macroscopic findings. Some spontaneous findings such as mineralization in Peyer’s patches in duodenum and jejunum, solitary cyst in medulla of kidney in each of the 1000 mg/kg group were observed. In addition, mineralization in Peyer’s patches in jejunum in 4 out of 11 females, focal necrosis of hepatocytes in liver, cyst formation in pars intermedia of pituitary gland and unilateral focal necrosis of cortex in adrenal were observed in each of the control group.
No abnormal changes in ovary, uterus and vagina in one infertility female from the control and 40 mg/kg groups were detected.
At the end of the recovery period, there were no changes in forestomach and glandular stomach in males and females. Abnormal changes in liver was not detected in females in the 1000 mg/kg group, though relative liver weight showed high value.

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Maternal developmental toxicity

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Changes in pregnancy duration:

no effects observed

Changes in number of pregnant:

no effects observed

Other effects:

no effects observed

Effect levels (maternal animals)

1

Key result

Dose descriptor:

NOAEL

Remarks:

systemic toxicity

Effect level:

200 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

food consumption and compound intake

histopathology: non-neoplastic

2

Key result

Dose descriptor:

NOEL

Remarks:

maternal developmental toxicity

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

other: no effects were observed

Maternal abnormalities

Key result

Abnormalities:

no effects observed

Results (fetuses)

Fetal body weight changes:

no effects observed

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

no effects observed

Changes in postnatal survival:

no effects observed

External malformations:

no effects observed

Skeletal malformations:

not examined

Visceral malformations:

not examined

Effect levels (fetuses)

Key result

Dose descriptor:

NOEL

Remarks:

developmental toxicity

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no effects were observed

Remarks on result:

not determinable due to absence of adverse toxic effects

Fetal abnormalities

Key result

Abnormalities:

no effects observed

Overall developmental toxicity

Key result

Developmental effects observed:

no

Applicant's summary and conclusion

Conclusions:

The NOAEL for systemic toxicity was determined to be 200 mg/kg bw/d for parental animals based on histopathological findings and changes in food consumption.
The NOEL for the developmental toxicity were 1000 mg/kg/day for parental animals and pups based on there were no treatment-related effects.