4,4'-oxydi(benzenesulphonohydrazide)

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3100 (90-Day Oral Toxicity in Rodents)

Deviations:

no

Principles of method if other than guideline:

NA

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

4,4'-oxydi(benzenesulphonohydrazide)

EC Number:

201-286-1

EC Name:

4,4'-oxydi(benzenesulphonohydrazide)

Cas Number:

80-51-3

Molecular formula:

C12H14N4O5S2

IUPAC Name:

4-[4-(hydrazinesulfonyl)phenoxy]benzene-1-sulfonohydrazide

Test material form:

solid: particulate/powder

Details on test material:

white fine powder

Specific details on test material used for the study:

Test item
Identity: 4,4’-Oxybis(benzenesulfonyl hydrazide) (OBSH)
CAS No.: 80-51-3
EC No.: 201-286-1
Test facility Code No.: K-5450
Batch/Lot No.: 201512001
Appearance: White fine powder
Purity: 99.5~100 %
Storage Conditions: Room temperature, Protected from light, Protected from moisture
Expiry Date: December, 2016

Vehicle
Identity: Corn oil
Batch/Lot No.: MKBS6944V, MKBV2080V
Expiry Date: March 09, 2021
Supplier: Sigma-Aaldrich
Storage Conditions: Room temperature

Test animals

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

Species: Rat (Specific Pathogen Free)
Strain/Substrain: Sprague-Dawley/Crl:CD (SD)
Number at receipt: 88 (Male 44, Female 44)
The females were nulliparous and nonpregnant
Number on study start: 80 (Male 40, Female 40)
Age range at receipt: Approximately 5 weeks
Age range on study start: Approximately 6 weeks
Body weight range: 196.1 to 217.5 g in males and 144.0 to 176.0 g in females at the start of dosing
Supplier: Orient Bio Inc. 322, Galmachi-ro, Jungwon-gu, Seongnam-si, Gyeonggi-do 462-807, Republic of Korea
Method of identification: Color marking, Tail tattoo, Cage card
Acclimation period: 5 days
Pre-treatment period: 3 days

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Rat (Specific Pathogen Free), Sprague-Dawley/Crl:CD (SD), Supplier: Orient Bio Inc. 322, Galmachi-ro, Jungwon-gu, Seongnam-si, Gyeonggi-do 462-807, Republic of Korea
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: Approximately 6 weeks
- Weight at study initiation: 96.1 to 217.5 g in males and 144.0 to 176.0 g in females
- Fasting period before study: 3 days pre-treatment period
- Housing: Three or less animals per cage were housed in suspended stainless-steel cages
- Diet (e.g. ad libitum): ad libitum - a standard rat and mouse pelleted diet (Lab Diet® #5053, Lot No. NOV 09 15 2, FEB 26 16 1, MAR 18 16 1 and MAR 28 16 2, PMI Nutrition International, USA; irradiated by gamma-ray). Microbial monitoring for diet was performed at KIT and a certificate of analysis for the diet was provided by the source.
- Water (e.g. ad libitum): ad libitum - filtered, ultraviolet light-irradiated municipal tap water at all times. The drinking water was analyzed every 6 months for specified contaminants in the Daejeon Regional Institute of Health and Environment (407, Daehak-ro, Yuseong-gu, Daejeon 34142, Republic of Korea ).
- Acclimation period: 5 days

DETAILS OF FOOD AND WATER QUALITY:

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23±3°C
- Humidity (%): 30 - 70%
- Air changes (per hr): 10 - 20 times/hour
- Photoperiod (hrs dark / hrs light): 12 hours light cycle with 150 - 300 Lux

IN-LIFE DATES: from April 20, 2016 to July 29, 2016

Administration / exposure

Route of administration:

oral: gavage

Details on route of administration:

Oral gavage was chosen as this has been used in previous studies

Vehicle:

corn oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:

DOSE FORMULATION
- OBSH was suspended in the vehicle to reach the target concentration of the highest dose group by weighing the necessary amount of test item in a container and slowly adding vehicle while mixing to be the concentration of 25 mg/mL of stock solution. Lower doses were prepared by diluting the high dose with the vehicle. The formulations were dispensed into light-protective tubes for use and then stirred on a magnetic stirrer at room temperature until the completion of formulation.
- Dose formulations were prepared once weekly and stored in room temperature until it is given to the animals.

VEHICLE
Identity: Corn oil
Batch/Lot No.: MKBS6944V, MKBV2080V
Expiry Date: March 09, 2021
Supplier: Sigma-aldrich
Storage Conditions: Room temperature

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

OBSH was suspended in the vehicle (corn oil) to reach the target concentration of the highest dose group by weighing the necessary amount of test item in a container and slowly adding vehicle while mixing to be the concentration of 25 mg/mL of stock solution. Lower doses were prepared by diluting the high dose with the vehicle. The formulations were dispensed into light-protective tubes for use and then stirred on a magnetic stirrer at room temperature until the completion of formulation.

Dose formulations were prepared once weekly and stored at room temperature until it was given to the animals.

Dose formulation analyses were conducted according to validated method (KIT Study No.: G115156). Dose formulations in the range 0.5-150 mg/mL have shown to be stable for 7 days when stored at room temperature. Homogeneity of dose formulations for dosing start date (Day 1), and last week (Week 13) were determined prior to dosing with formulations. Samples (approximately 5 mL/each sample) were taken from the top, middle and bottom of all dose formulations. Dose formulations were considered homogeneous if Coefficient Variation (CV) results were within 5 % of the mean of the top, middle and bottom results (0.4-2.5% on Day 1 and 1.6-4.4% on Week 13).

Concentration of all dose formulations for dosing start date (Day 1), Week 4 and last week (Week 13) were measured prior to dosing. Samples (approximately 5 mL/each sample) were taken from the middle of each dose formulation. Results for dose formulations were 101.7-103.9% on Day 1, 97.4-100.9% on Week 4, and 90.5-102.2% on Week 13. They were acceptable as the mean concentration was ±10% of the nominal concentration.

Duration of treatment / exposure:

Dose formulations were administered by oral gavage one time per day at approximately the same time each day (08:30 - 12:00) for 90 days. Dose was based on the most recently measured body weight. Animals were dosed at a volume of 2 mL/kg. Treatment was continued through the day prior to terminal sacrifice. Dose formulation was continuously stirred by magnetic stirrer in the animal room (before and throughout the dosing procedure).

Frequency of treatment:

One time per day at approximately the same time each day (08:30 - 12:00) for 90 days.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Remarks:

The dose levels were selected based on available data from a public available OECD SIDS document (SIDS Initial Assessment Report For SIAM 22, Paris, France, 18 - 21 April 2006) on repeated dose toxicity of OBSH (OECD TG 407 and TG 422). In the OECD TG 407 study (28 days), dose levels of 10, 30, 100, 200 mg/kg bw/day were used. A NOAEL of 10 mg/kg bw/day was found for both sexes, whereas the dose level of 200 mg/kg bw/day was above the maximum tolerable dose level, as all animals died or had to be killed. At 100 mg/kg bw/day adverse effects in the kidneys was observed in all animals. In the OECD 422 screening study (42-54 days), dose levels of 5, 15, 45 mg/kg bw/day was used. The NOAEL for females was 15 mg/kg bw/day whereas no NOAEL could be found for males i.e. a LOAEL of 5 mg/kg bw/day due to increased salivation. Therefore, 2, 10 and 50 mg/kg were selected for this study. Control group was administered with vehicle.

Doses / concentrations 2

Dose / conc.:

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

Remarks:

The dose levels were selected based on available data from a public available OECD SIDS document (SIDS Initial Assessment Report For SIAM 22, Paris, France, 18 - 21 April 2006) on repeated dose toxicity of OBSH (OECD TG 407 and TG 422). In the OECD TG 407 study (28 days), dose levels of 10, 30, 100, 200 mg/kg bw/day were used. A NOAEL of 10 mg/kg bw/day was found for both sexes, whereas the dose level of 200 mg/kg bw/day was above the maximum tolerable dose level, as all animals died or had to be killed. At 100 mg/kg bw/day adverse effects in the kidneys was observed in all animals. In the OECD 422 screening study (42-54 days), dose levels of 5, 15, 45 mg/kg bw/day was used. The NOAEL for females was 15 mg/kg bw/day whereas no NOAEL could be found for males i.e. a LOAEL of 5 mg/kg bw/day due to increased salivation. Therefore, 2, 10 and 50 mg/kg were selected for this study. Control group was administered with vehicle.

Doses / concentrations 3

Dose / conc.:

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

Remarks:

The dose levels were selected based on available data from a public available OECD SIDS document (SIDS Initial Assessment Report For SIAM 22, Paris, France, 18 - 21 April 2006) on repeated dose toxicity of OBSH (OECD TG 407 and TG 422). In the OECD TG 407 study (28 days), dose levels of 10, 30, 100, 200 mg/kg bw/day were used. A NOAEL of 10 mg/kg bw/day was found for both sexes, whereas the dose level of 200 mg/kg bw/day was above the maximum tolerable dose level, as all animals died or had to be killed. At 100 mg/kg bw/day adverse effects in the kidneys was observed in all animals. In the OECD 422 screening study (42-54 days), dose levels of 5, 15, 45 mg/kg bw/day was used. The NOAEL for females was 15 mg/kg bw/day whereas no NOAEL could be found for males i.e. a LOAEL of 5 mg/kg bw/day due to increased salivation. Therefore, 2, 10 and 50 mg/kg were selected for this study. Control group was administered with vehicle.

No. of animals per sex per dose:

10 male/female/dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels were selected based on available data from a public available OECD SIDS document (SIDS Initial Assessment Report For SIAM 22, Paris, France, 18 - 21 April 2006) on repeated dose toxicity of OBSH (OECD TG 407 and TG 422). In the OECD TG 407 study (28 days), dose levels of 10, 30, 100, 200 mg/kg bw/day were used. A NOAEL of 10 mg/kg bw/day was found for both sexes, whereas the dose level of 200 mg/kg bw/day was above the maximum tolerable dose level, as all animals died or had to be killed. At 100 mg/kg bw/day adverse effects in the kidneys was observed in all animals. In the OECD 422 screening study (42-54 days), dose levels of 5, 15, 45 mg/kg bw/day was used. The NOAEL for females was 15 mg/kg bw/day whereas no NOAEL could be found for males i.e. a LOAEL of 5 mg/kg bw/day due to increased salivation. Therefore, 2, 10 and 50 mg/kg were selected for this study. Control group was administered with vehicle.

- Rationale for animal assignment (if not random): Random. Animals were selected for use in study on the bases of adequate body weight and absence of clinical signs of disease or injuries. They were assigned to treatment groups as a stratified manner using the Pristima System based on the most recent body weight.

- Rationale for selecting satellite groups: NA (no satellite group included in study design).

- Post-exposure recovery period in satellite groups: NA (no satellite group included in study design).

- Section schedule rationale (if not random): Random.

Positive control:

No

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Two times per day, before and after dosing on the day of dosing

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Detailed clinical signs were conducted in all toxicology animals individually for abnormalities once a week. Signs noted were observed to fur, skin, eyes, mucous membranes, occurrence of secretions and excretions, autonomic nervous system activity (lacrimation, piloerection, and unusual respiratory pattern), changes in gait, posture, stereotypical and bizarre behavior etc.

BODY WEIGHT: Yes
- Time schedule for examinations: One time per week

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

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: once weekly

OPHTHALMOSCOPIC EXAMINATION: Yes
Ophthalmologic examination was conducted by a veterinarian with competency in ophthalmological evaluation as designated by KIT Management. Opthalmological examinations was performed on all animals using binocular indirect ophthalmoscopy (Vantage Plus Digital, Keeler Ltd., England) after the animals were treated with mydriatic (Mydrin-P, Santen Pharmaceutical Co., Japan).

HAEMATOLOGY: Yes
Blood and urine samples for clinical pathology were taken from all surviving animals prior to terminal sacrifice. Blood samples were collected from caudal vena cava. Approximately 1.5 mL of blood samples was collected from all toxicology animals. For hematological test, about 0.5 ml of blood was put into tubes containing potassium salt of EDTA. About 1.0 mL of blood was put into tubes containing 3.2% sodium citrate and then centrifugation (approximate 3000 rpm, 10 min, at room temperature) for clotting potential test. Blood smears were prepared from all available animals but not examined. The following parameters were evaluated: Total leukocyte count (WBC), Mean corpuscular volume (MCV), Total red blood cell count (RBC), Mean corpuscular hemoglobin (MCH), Red cell distribution width (RDW), Mean corpuscular hemoglobin concentration (MCHC), Hemoglobin (HGB), Platelet count (PLT), Hemoglobin distribution width (HDW), Reticulocyte counta, Hematocrit (HCT), WBC differential counta,
Immature reticulocyte fraction (IRF), Activated partial thromboplastin time (APTT), Prothrombin time (PT).


CLINICAL CHEMISTRY: Yes
The animals were fasted more than 16 hours prior to blood collection. Drinking water was provided at libitum. Approximately 1.5 mL of blood samples were collected from all toxicology animals and put into tubes without anticoagulant for serum separation. The tubes were kept at room temperature for a minimum of 90 min and then centrifuged (approximate 3000 rpm, 10 min, at room temperature) to obtain serum. The following parameters were evaluated: Glucose (GLU), Alanine aminotransferase (ALT), Blood urea nitrogen (BUN), Total bilirubin (TBIL), Creatinine (CREA) Alkaline phosphatase (ALP), Total protein (TP), Gamma glutamyl transpeptidase (GGT), Albumin (ALB), Creatine phosphokinase (CK), Albumin/globulin ratio (A/G), Calcium (Ca), Total cholesterol (TCHO), Inorganic phosphorus (IP), Triglyceride (TG), Sodium (Na), Phospholipid (PL), Potassium (K), Lactate dehydrogenase (LDH), Total bile acid (TBA), Aspartate aminotransferase (AST), Chloride (Cl).


URINALYSIS: Yes
On the day before terminal sacrifice, urine was collected overnight (approximate 16 hours) all surviving animals for each scheduled sacrifice using metabolism cages. Each animal was housed in an individual metabolism cage. Food was withdrawn overnight during urine collection but drinking water was available. The following parameters were evaluated: Volume (VOL), Nitrite (NIT), Color (COL) / Clarity (CLA), Glucose (GLUu), pH, Erythrocyte (ERY), Specific gravity (SG), Ketone (KETu),
Bilirubin (BILu), Leukocytes (LEU), Protein (PRO), Urine sediments, Urobilinogen (URO).

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: prior to sacrifice
- Dose groups that were examined: all dose groups
- Battery of functions tested: sensory activity / grip strength / motor activity / other: An abbreviated neurobehavioral test battery, consisting of selected functional observational battery assessments (FOB) and motor activity (MA), was conducted on all animals per each group in sequential order (20-27 animals/day).
FOB and MA parameters were evaluated according to the KIT’s SOP once prior to the final sacrifice (Week 13). Grip strength was measured using a grip strength meter (Model GS3, BIOSEB, France). The amount of spontaneous movement of the animals in motor activity was measured using an apparatus (Etho Vision XT Version 8.0, Noldus Internation Technology B.V., Netherlands). FOB analyses included home cage observations, outside the home cage observations, open field observations, sensory function assessments, forelimb and hindlimb grip strength (grip strength was measured using a grip strength meter (Model GS3, BIOSEB, France). Measurement was conducted three times each for the forelimb and hind limb and the mean values were regarded as the data.

IMMUNOLOGY: No

OTHER:

Sacrifice and pathology:

GROSS PATHOLOGY: Yes, complete necropsy examinations were performed under the direct supervision of a veterinary pathologist on all animals at terminal sacrifice. After blood sampling, the animals was sacrificed by exsanguinations from the vena cava and aorta. The animals were examined carefully for external abnormalities. The abdominal, thoracic and cranial cavities were examined for abnormalities and the organs were removed and examined. The following organ weights were captured: Brain, kidneys, Pituitary gland, Adrenal glands, Liver, Testes, Spleen, Epididymides, Heart, Lung (with bronchi/bronchioles), Thymus, Thyroids (with parathyroids), Salivary glands (submandibular and sublingual), Uterus (with cervix), Seminal vesicles (with coagulation gland), Ovaries (with oviduct), Prostate.


HISTOPATHOLOGY: Yes, The following tissues from each animal were preserved in 10% neutral buffered formalin, except the eyes (with optic nerve) which were fixed in Davidson’s fixative and the testes and epididymides which were fixed in Bouin’s fixative. The tissues was placed in the appropriate fixative for approximate 48 hours, and then transferred to 70% ethanol. Formalin was infused into lung via the trachea and into the urinary bladder. All tissues collected from animals at terminal sacrifice, in the high-dose and vehicle control groups, and abnormal lesions were further processed to slides, stained with hematoxylin and eosin, and examined microscopically. Test item-related microscopic findings were observed in the liver, spleen, kidney, femur/marrow and sternum/marrow, so they were additionally evaluated in animals in the low- and intermediate-dose groups. The microscopic findings and pathology report were conducted an internal peer review according to the SOP.

Tissues preserved:
Abnormal lesion
Adrenal glands
Animal IDa
Aorta (thoracic)
Brain
Bone marrow smear
Cecum
Colon
Duodenum
Epididymides
Esophagus
Eyes (with optic nerveb)
Femur with marrow (F-T joint)
Heart
Ileum
Jejunum
Kidneys
Larynx
Liver
Lung (with bronchi)
Mammary gland (female only)
Mandibular lymph node
Mesenteric lymph node
Ovaries (with oviduct)
Pancreas
Peyer’s patches
Prostate
Pituitary gland
Rectum
Salivary glands (submandibular and sublingual)
Sciatic nerve
Seminal vesicle (with coagulation gland)
Skeletal muscle
Skin (inguinal)
Spinal cord (cervical, thoracic, lumbar)
Spleen
Sternum (with marrow)
Stomach
Testes
Thymus
Thyroids (with parathyroidsb)
Tongue
Trachea
Urinary bladder
Uterus with cervix
Ureter
Vagina

Statistics:

Mean values and standard deviations were calculated in final report. Statistical analyses for comparisons of the various dose groups with the vehicle control group were conducted using Pristima System (Version 6.4 and 7.x Xybion Medical Systems Corporation, USA) or Statistical Analysis Systems (SAS/STAT Version 9.2, USA).

Multiple comparison tests for different dose groups were conducted. Variance of homogeneity was examined using the Bartlett’s Test. Homogeneous data were analyzed using the Analysis of Variance (ANOVA) and the significance of inter-group differences were analyzed using Dunnett’s Test. Heterogeneous data were analyzed using Kruskal-Wallis Test and the significance of inter-group differences between the control and treated groups were assessed using Dunn’s Rank Sum Test.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Test item-related salivation was observed at ≥10 mg/kg in both sexes but resolved before the next dosing. Other clinical findings such as scratch wound and loss of fur were not considered test item-related since there were no dose-dependency or they were observed sporadically.

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There were no test item-related changes in body weights and body weight gains during the study.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

There were no test item-related changes in food consumption during the study.

Food efficiency:

no effects observed

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

no effects observed

Description (incidence and severity):

There were no test item-related changes in water consumption during the study. Statistically significant changes observed during the study were considered incidental as there was no dose-dependency.

Ophthalmological findings:

no effects observed

Description (incidence and severity):

There were no test item-related abnormalities in ophthalmologic examination.

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Absolute (RETA; 1.39 and 1.46-folds over control in males and females, respectively) and relative (RET%; 1.44 and 1.49-folds over control in males and females, respectively) reticulocyte counts were increased in both sexes at 50 mg/kg/day. These changes were associated with the presence of increased extramedullary hematopoiesis in the spleen microscopically. Absolute (NEUA; 1.94-fold over control) and relative (NEU%; 1.93-fold over control) neutrophil counts were increased in females at 50 mg/kg/day. It was considered non-adverse as there were no microscopic correlates.

Other statistically significant changes were not considered test item-related changes, as there were no dose-dependency or degree of changes were relatively small compared to vehicle control group.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Aspartate aminotransferase (AST; 28% and 33% of control in males and females, respectively) at 50 mg/kg/day and alanine aminotransferase (ALT, down to 6% and 10% of control in males and females, respectively) at 10 and 50 mg/kg/day were decreased in both sexes. These changes were not considered toxicologically significant. Increases in total bilirubin (TBIL, 1.21-fold over control), triglyceride (TG, 2.04-fold over control), inorganic phosphorus (IP, 1.21-fold over control) and potassium (K, 1.30-fold over control) and a decrease in glucose (GLU; 0.67% of control) were observed in females at 50 mg/kg/day. These changes were considered non-adverse as there were no microscopic correlates.

Other statistically significant changes were not considered test item-related changes, as there were no dose-dependency or degree of changes were relatively small compared to vehicle control group.

Urinalysis findings:

no effects observed

Description (incidence and severity):

There were no test item-related changes in treatment group. Noted statistically significant changes were not considered test item-related changes, as there were no dose-dependency.

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

There were no test item-related changes in abbreviated neurobehavioral evaluation (FOB analyses). Statistically significant change observed during the observations were considered incidental as there was no dose-dependency.

Immunological findings:

effects observed, treatment-related

Description (incidence and severity):

Test item-related changes were observed in the spleen. In the spleen, minimally increased extramedullary hematopoiesis (EMH) was observed in both sexes at 50 mg/kg/day (see table 1). EMH was characterized by increased haemopoietic cells in the red pulp. This change was associated with the increase of absolute and relative reticulocyte in both sexes at 50 mg/kg/day. It was considered test item-related change, but non-adverse as the change was minimal and there were no other microscopic alteration in the spleen.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Absolute and relative (to body weights and to brain weights) liver (up to 1.16 and 1.17-fold over control in males and females, respectively) and kidneys (up to 1.21 and 1.18-fold over control, in males and females, respectively) weights were increased in both sexes at 50 mg/kg/day. These changes were considered non-adverse as there were no microscopic correlates.

Other statistically significant changes were not considered test item-related changes, as there was no dose-dependency.

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no test item-related changes in males or females.

All macroscopic findings were considered to be incidental or spontaneous changes that often occur among SD rats of the same age.

Neuropathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Test item-related changes were observed in the spleen (see under Immunological findings). All other histopathology findings not specifically discussed were not considered to be associated with test item-related changes, as they were incidental or spontaneous changes and/or infrequent, generally of low severity, and/or similarly distributed among treated and control groups.

Histopathological findings: neoplastic:

no effects observed

Description (incidence and severity):

C-cell adenoma in the thyroid glands was observed in a male (animal No. 24) at 10 mg/kg/day. This change was considered to be incidental as there was no test-item related change of thyroids in the any other groups.

Other effects:

not specified

Details on results:

Overall, no test item-related mortality, body weight and food consumption changes, abnormalities in ophthalmology, urinalysis/urine chemistry and abbreviated neurobehavioral test battery and macroscopic observations were observed in this study.

Test item-related salivation was observed at ≥10 mg/kg in both sexes but resolved before the next dosing.

In hematology, absolute and relative reticulocytes in both sexes and absolute and relative neutrophil counts in females were increased at 50 mg/kg/day. In clinical chemistry, aspartate aminotransferase (AST) at 50 mg/kg/day and alanine aminotransferase (ALT) at 10 and 50 mg/kg/day were decreased in both sexes. Increases in total bilirubin (TBIL), triglyceride (TG), inorganic phosphorus (IP) and potassium (K) and a decrease in glucose (GLU) were observed in females at 50 mg/kg/day.

In organ weights, increased kidneys and liver weights were observed in both sexes at 50 mg/kg/day. In microscopic examination, minimally increased extramedullary hematopoiesis (EMH) in the spleen was observed in both sexes at 50 mg/kg/day. This change was associated with an increase of reticulocyte. It was considered test item-related changes, but not considered adverse.

Effect levels

1

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

2

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical biochemistry

3

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

organ weights and organ / body weight ratios

Target system / organ toxicity

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

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

System:

other: urinary and hepatic

Organ:

kidney

liver

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

Any other information on results incl. tables

Table 1 Summary of microscopic findings
	Male
Group	V.C	T1	T2	T3
Dose of OBSH (mg/kg/day)	0	2	10	50
Number of Animals	10	10	10	10
Spleen
Extramedullary hematopoiesis	(2)^a	(1)	(0)	(6)
Minimal	2	1	0	6
	Female
Group	V.C	T1	T2	T3
Dose of OBSH (mg/kg/day)	0	2	10	50
Number of Animals	10	10	10	10
Spleen
Extramedullary hematopoiesis	(0)	(0)	(0)	(2)
Minimal	0	0	0	2

^aTotal finding incidence

Applicant's summary and conclusion

Conclusions:

The toxicity of 4,4’-Oxybis(benzenesulfonyl hydrazide) (OBSH) was investigated in Sprague-Dawley (SD) rats following 13 weeks (appromximately 90D) of treatment by oral gavage administration at dose levels of 0, 2, 10 and 50 mg/kg. Organ weight changes (kidney and liver), histopathological changes (EHM) in the spleen and changes in haematological and clinical chemistry parameters were seen at 50 mg/kg bw/d. Based on these results, NOAEL was considered to be 10 mg/kg in both sexes.

Executive summary:

This study was conducted to investigate the potential toxicity of 4,4’-Oxybis(benzenesulfonyl hydrazide) (OBSH) in Sprague-Dawley (SD) rats following 13 weeks of treatment by oral gavage administration. OBSH was administered once daily to SD rats for 13 weeks (approximately 90-days). The animals were assigned to 4 groups (10 animals/sex/group) receiving test item at dose levels of 0, 2, 10 and 50 mg/kg. Following final dosing on Day 91 (male) or 92 (female), all animals were necropsied on Day 92 (male) or 93 (female).

Observations for mortality, morbidity, general appearance and behaviour changes were recorded twice daily for all animals during the treatment period. Body weight and food consumption were measured once weekly until terminal sacrifice. Terminal body weight was measured before necropsy. Ophthalmic examinations were conducted once prior to the initiation of treatment and then prior to the terminal necropsy. Also an abbreviated neurobehavioral test battery, consisting of selected functional observational battery assessments (FOB) and motor activity (MA), was conducted prior to the terminal necropsy. Blood samples for the hematology, clinical chemistry and urinalysis were collected from all animals at the terminal necropsy. At the sacrifice, macroscopic examinations were performed; organ weights recorded and collected tissues for microscopic examination.

No test item-related mortality, body weight and food consumption changes, abnormalities in ophthalmology, urinalysis/urine chemistry and abbreviated neurobehavioral test battery and macroscopic observations were observed in this study. Test item-related salivation was observed at≥10 mg/kg in both sexes but resolved before the next dosing.

In hematology, absolute and relative reticulocytes in both sexes and absolute and relative neutrophil counts in females were increased at 50 mg/kg/day. In clinical chemistry, aspartate aminotransferase (AST) at 50 mg/kg/day and alanine aminotransferase (ALT) at 10 and 50 mg/kg/day were decreased in both sexes. Increases in total bilirubin (TBIL), triglyceride (TG), inorganic phosphorus (IP) and potassium (K) and a decrease in glucose (GLU) were observed in females at 50 mg/kg/day.

In organ weights, statistical and significant increased kidneys and liver weights, both absolute and relative, were observed in both sexes at 50 mg/kg/day. These organ weight changes were not supported with histopathological changes. In spleen, minimally increased extramedullary hematopoiesis (EMH) was observed in both sexes at 50 mg/kg/day. This change was associated with an increase of reticulocyte. It was considered test item-related changes, but not considered adverse.

In conclusion, daily oral administration of 4,4’-Oxybis(benzenesulfonyl hydrazide) (OBSH) to SD rats at 0, 2, 10 and 50 mg/kg bw/d for 13 weeks resulted in organ weight changes (kidney and liver), histopathological changes (EHM) in the spleen and changes in haematological and clinical chemistry parameters at 50 mg/kg bw/d. Based on these results, NOAEL was considered to be 10 mg/kg in both sexes.