N-ethyl-o(or p)-toluenesulphonamide

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

11 August 2005 to 11 November 2005

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2006

Report date:

2006

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)

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)

Test guideline 3

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

N-ethyl-o(or p)-toluenesulphonamide

EC Number:

232-465-2

EC Name:

N-ethyl-o(or p)-toluenesulphonamide

Cas Number:

8047-99-2

Molecular formula:

C9H13NO2S

IUPAC Name:

N-ethyl-4-methylbenzene-1-sulfonamide

Test material form:

liquid: viscous

Remarks:

Colour: light yellow

Details on test material:

Ketjenflex 8 (NETSA)
Chemical name in report: N-ethyl-o/p-toluenesulfonamide (or N-substituted toluene sulphonamide)
Description: light yellow viscous liquid
Purity 100% (or 90%)

Test substance storage: at room temperature in the dark
Stability under storage conditions: stable

Test animals

Species:

rat

Strain:

Wistar

Remarks:

Cri:(WI) BR

Details on species / strain selection:

Recognised by international guidelines as the recommended test system (e.g. EPA, FDA, OECD, EEC).

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 6 weeks
- Weight at study initiation:
Males: Ranges of 189 - 212, 193 - 221, 192 - 212, 191 - 212g in the control, 25 mg/kg, 100 mg/kg, and 400 mg/kg groups respectively.
Females: Ranges of 163 - 181, 152 - 176, 153 - 179, 157 - 176g in the control, 25 mg/kg, 100 mg/kg, and 400 mg/kg groups respectively.
- Fasting period before study: no
- Housing: Group housing of 5 animals per sex in Macrolon plastic cages (MIV type, height 18 em; during overnight activity monitoring individual housing in Mill type; height 15 em.) with sterilised sawdust as bedding material (Woody-Clean type 3/4, Tecnilab-BMI BV, Someren, The Netherlands) and paper as cage-enrichment (Enviro-dri, Tecnilab·-BMI BV, Someren, The Netherlands). No cage-enrichment was provided during overnight activity monitoring.
- Diet: standard pelleted laboratory animal diet (from Altromin (code VRF-1, Lage,Germany), ad libitum.
- Water: tap water, ad libitum
- Acclimation period: at least 5 days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.0 ± 3.0 (actual range: 19.2- 22.9)
- Humidity (%): 30-70 (actual range: 37- 98)
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Details on route of administration:

Oral gavage, using a stainless steel stomach tube. Formulations were placed on a magnetic stirrer during dosing. 5 ml/kg body weight Actual dose volumes were calculated weekly according to the latest body weight

Vehicle:

propylene glycol

Remarks:

(Merck, Darmstadt, Germany) specific gravity 1.036

Details on oral exposure:

- PREPARATION OF DOSING SOLUTIONS:
Formulations (w/w) were prepared daily within 4 hours prior to dosing from days 1 to 28 (and on day 92), and on a weekly basis from day 29 onwards (except for day 92) after stability of the formulations over 8 days was confirmed. The formulations were homogenised to visually acceptable levels. Adjustment was made for density of the test substance and specific gravity of the vehicle.

- VEHICLE
- Justification for use and choice of vehicle: Based on trial formulations performed at NOTOX and based on information from the sponsor

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples of formulations prepared for use on days 1, 36 and 85 were analysed to check homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations). Stability in vehicle over 8 days was also determined (highest and lowest concentration). The analytical method used was based on the results of a separate project for the development and validation of the analytical method (NOTOX project 441293).

Duration of treatment / exposure:

90-days

Frequency of treatment:

Once daily, 7 days per week, approximately the same time each day with a maximum of 5.5 hours difference between the earliest and latest dose. Animals were dosed up to the day prior to necropsy.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Doses / concentrations 2

Dose / conc.:

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

Doses / concentrations 3

Dose / conc.:

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

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Dose levels were based on results of a 14-day oral range finding study.
- Rationale for animal assignment: By computer-generated random algorithm according to body weight, with all animals within ± 20% of the sex mean. A health inspection was performed prior to commencement of treatment to ensure that the animals were in a good state of health.

Positive control:

n.a.

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: At least twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once daily, detailed clinical observations were made in all animals. Once prior to start of treatment and at weekly intervals, this was also performed outside the home cage in a standard arena. The time of onset, degree and duration were recorded. All symptoms were recorded and graded according to fixed scales: Maximum grade 1: grade 0 = absent, grade 1 = present Maximum grade 3 or 4: grade 1 = slight, grade 2 = moderate, grade 3 = severe, grade 4 = very severe

BODY WEIGHT: Yes
- Time schedule for examinations: Weekly and on the day prior to scheduled necropsy.

FOOD EFFICIENCY: YES
- Food consumption in absolute terms or when related to body weight:

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Following instillation of tropicamide solution (5 mg/ml, THEA Pharma, Ukkel, Belgium) both eyes were examined by means of an ophthalmoscope (Heine Beta 200): at pretest: All animals
at week 13 : Groups 1 and 4 (Main and Recovery group animals)
Since no treatment-related ophthalmologic findings were noted at week 13, the eyes of the rats of groups 2 and 3 were not examined, and no ophthalmoscopic examination was performed at the end of the recovery period.
- Dose groups that were examined: group 1 (vehicle) group 4 (400 mg/kg bw day).

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Blood samples were collected immediately prior to scheduled post mortem examination.
- Anaesthetic used for blood collection: Yes, iso-flurane anaesthesia
- Animals fasted: Yes, with a maximum of 20 hours
- How many animals: all animals
- Parameters checked: White blood cells, Differential leucocyte count, Red blood cells, Red blood cell distribution width, Haemoglobin, Haematocrit, Mean corpuscular volume, Mean corpuscular haemoglobin, concentration Platelets, Prothrombin time, Activated Partial thromboplastin time.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Blood samples were collected immediately prior to scheduled post mortem examination.
- Animals fasted: Yes, with a maximum of 20 hours
- How many animals: all animals
- Parameters checked: Alanine aminotransferase, Aspartate aminotransferase, Alkaline phosphatase, Total Protein, Albumin, Total Bilirubin, Urea, Creatinine, Glucose, Cholesterol, Sodium, Potassium, Chloride, Calcium, Inorganic Phosphate.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: During week 12-13 of treatment.
- Battery of functions tested: The following tests were performed on all animals (abbreviations mentioned in the respective tables indicated between brackets): hearing ability (HEARING), pupillary reflex (PUPIL L/R), static righting reflex (STATIC R) and grip strength (GRIP) (Score 0 = normal/present, score 1 = abnormal/absent). motor activity test (recording period: 12 hours during overnight for individual animals, using a computerised monitoring system, Pearson Technical Services, Debenham, Stowmarket, England).

IMMUNOLOGY: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
All animals surviving to the end of the observation period were deeply anaesthetised using iso­ flurane vapour (Abbott Laboratories Ltd., Zwolle, The Netherlands) and subsequently exsanguinated. All animals assigned to the study were necropsied and descriptions of all macroscopic abnormalities recorded. Samples of the following tissues and organs were collected from all animals at necropsy and fixed in a neutral phosphate buffered 4% formaldehyde solution (Kiinipath, Duiven, The Netherlands):

Adrenal glands, Aorta, Brain [cerebellum, mid-brain, cortex] Caecum, Cervix, (Clitoral gland) Colon Duodenum Epididymides, (Eyes with optic nerve [if detectable] and Harderian gland), Female mammary gland area (Femur including joint), Heart Ileum, Jejunum Kidneys (Larynx), (Lacrimal gland, exorbital), Liver, Lung, infused with formalin, Lymph nodes - mandibular, mesenteric (Nasopharynx), Oesophagus, Ovaries Pancreas, Peyer's patches [jejunum, ileum] if detectable Pituitary gland, (Preputial gland), Prostate, gland Rectum, Salivary glands - mandibular, sublingual Sciatic nerve, (Seminal vesicles),(Skeletal muscle) (Skin), Spinal cord -cervical, midthoracic, lumbar Spleen, Sternum with bone marrow Stomach, Testes, Thymus, Thyroid including parathyroid [if detectable] (Tongue), Trachea, Urinary bladder Uterus, Vagina, All gross lesions.

The following organ weights and terminal body weight were recorded from the surviving animals on the scheduled day of necropsy:
Adrenal glands, Brain, Epididymides, Heart, Kidneys, Liver, Ovaries, Spleen, Testes, Thymus, Uterus.

HISTOPATHOLOGY: Yes
Following fixation, organs (except those listed in brackets) from group 1 and 4 rats, the one unscheduled death as well as liver, lungs and kidneys and all organs with macroscopic abnormalities from all rats, were trimmed, processed and embedded in paraffin wax. Sections were cut at a thickness of 2-4 micrometers and stained with hematoxylin and eosin. Numbers of sections made are given in parentheses.
Adrenal glands (2), aorta (1), bone - sternum (1) [and femur including joint]; bone marrow - sternal (1), brain (3), [clitoral glands], epididymides (4), esophagus (1), [eyes with optic nerve and Harderian glands]; heart (1), [identification marks], kidneys (2), [lacrimal glands - exorbital], large intestine (3) - cecum, colon and rectum; [larynx], liver (2), lungs (2), lymph nodes - mandibular (2) and mesenteric (1); female mammary gland area (1), [nasopharynx], ovaries (2), pancreas (1), pituitary gland (1), [preputial glands], prostate gland (1), [salivary glands mandibular and sublingual]; sciatic nerve (1), [seminal vesicles], [skeletal muscle], [skin], small intestine (3)- duodenum, jejunum and ileum with Peyer's patches; spinal cord (3)­ cervical, midthoracic and lumbar; spleen (1), stomach (1), testes (2), thymus (1), thyroid glands (2) with parathyroid glands (2), [tongue], trachea (1), urinary bladder (1), uterus with uterine cervix (3), vagina (1) and all organs or tissues with macroscopic abnormalities.

Other examinations:

From the last five animals per group, bone marrow was collected from the right femur at scheduled necropsy. Bone marrow collected from each animal was further processed, and bone marrow slides were prepared for evaluation for presence of micronuclei.

Statistics:

The following statistical methods were used to analyse the data:
- If the variables could be assumed to follow a normal distribution, the Dunnett-test (many-to­ one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
- The Steel-test (many-to-one rank test) was applied when the data could not be assumed to follow a normal distribution.
- The exact Fisher-test was applied to frequency data.

All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance. No statistical analysis was performed on histopathology findings. Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables. Test statistics were calculated on the basis of exact values for means and pooled variances. Individual values, means and standard deviations may have been rounded off before printing. Therefore, two groups may display the same printed means for a given parameter, yet display different test statistics values.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Lethargy, flat posture, uncoordinated movements, and salivation were observed among all animals at 400 mg/kg/day from week 1 of treatment onwards, and at lower incidence also at 100 mg/kg/day. These signs (with the exception of salivation) were also incidentally noted at 25 mg/kg/day. These symptoms showed a dose-related occurrence with regard to incidence and time of appearance. However, within the same dose group no relation of frequency/severity of the symptoms to increasing duration of treatment was apparent, and these symptoms were not consistently noted in all animals during treatment. Symptoms generally occurred 5-10 minutes after dosing and lasted approximately one hour. Generally, these symptoms were of a minor nature and not overtly present In general, animals appeared in a good health condition throughout the study period.
In addition, it was frequently noted that animals secluded from their cage mates after dosing (general observation; no specification of which animal on which days). In most cages containing animals at 400 mg/kg/day, dry faeces was recorded on most days from week 4 of treatment onwards. In one instance this was recorded as pale faeces for high dose females (day 22).

The male at 25 mg/kg/day that was found dead on day 89 (no. 12) showed swelling of the abdomen (from week 11 onwards), and incidentally hunched/flat posture, piloerection and a pale appearance. These findings were considered to be related to the presence of a nephroblastoma in the right kidney found histopathologically, and not related to treatment with the test substance.
Incidental findings that were noted included alopecia, scales and scabs on various body parts, brown discolouration on the head, rales, scraping, quick breathing, chromodacryorrhoea, and a broken tail apex. These findings are occasionally noted in rats of this age and strain which are housed and treated under the conditions in this study. At the incidence observed, these were considered signs of no toxicological significance.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

No mortality occurred during the study period that was considered to be related to treatment with the test substance.
The male at 25 mg/kg/day (no.12) found dead on day 89 was diagnosed with a nephroblastoma in the right kidney, which correlated with findings at necropsy. This death was considered unrelated to treatment with the test substance. No further mortality occurred.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Body weights and body weight gain of treated animals remained in the same range as controls over the study period.
The apparent increase of body weight on day 29 and of body weight gain on days 15 and 29 of males at 25 mg/kg/day was considered to have achieved a level of statistical significance by chance as no dose or time-related response was noted.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

no effects observed

Description (incidence and severity):

Food consumption (in absolute terms or when related to body weight) was unaffected by treatment with the test substance.

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

not examined

Ophthalmological findings:

no effects observed

Description (incidence and severity):

There were no ophthalmology findings at pre-dose and in week 13.

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Haematological parameters of treated rats were considered not to have been affected by treatment
Any (minor) statistically significant differences arising between controls and treated animals were considered to have arisen by chance and not to represent a change of toxicological significance. These changes consisted of lower relative neutrophil and monocyte counts in males at 25 mg/kg/day, lower relative basophil counts in males at 100 mg/kg/day, lower mean corpuscular haemoglobin concentration (MCHC) in males and females at 100 mg/kg/day, and higher partial thromboplastin time (APTT) in females at 25 mg/kg/day.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

The following statistically significant changes in clinical biochemistry parameters distinguished animals at 400 mg/kg/day from control animals:
Increased total protein and albumin levels in males and females; Increased cholesterol levels in males and females;
Increased potassium level in females; Increased calcium level in females.

No toxicological significance was ascribed to the lower aspartate aminotransferase activity (ASAT) of females at 100 and 400 mg/kg/day, as the opposite effect would be expected in case of target organ toxicity and since the change was of a slight nature. No dose-response relationship was apparent for the statistically significant reduction of chloride levels in males at 400 mg/kg/day, the mean of which was similar to the next lower dose level and within the range expected for rats used in this type of study. Other statistically significant changes observed also occurred in the absence of a dose-related response. These changes included a higher glucose level in males at 25 mg/kg/day, a higher sodium level in males at 100 mg/kg/day and a lower inorganic phosphate level in females at 25 mg/kg/day. These changes were therefore considered to be of no toxicological significance.

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

Hearing ability, pupillary reflex, static righting reflex and grip strength were normal in all animals. The variation in motor activity did not indicate a relation with treatment.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

The following statistically significant changes in organ weights and organ to body weight ratios distinguished treated from control animals:
Increased absolute liver weight in males at 400 mg/kg/day and in females at 100 and 400 mg/kg/day;
Increased liver to body weight ratio in males at 400 mg/kg/day and in females at 25 mg/kg/day and higher;
Increased kidney weights in females at 400 mg/kg/day;
Increased kidney to body weight ratio in males at 400 mg/kg/day and in females at 100 and 400 mg/kg/day.

No dose-related change in absolute spleen weights was noted that would support the slightly higher spleen to body weight ratio in males at 400 mg/kg/day. In the absence of morphological correlates this slight change was considered to have achieved a level of statistical significance by chance. The lower thymus weight and thymus to body weight ratio of females at 100 mg/kg/day was considered to have achieved a level of statistical significance by chance as no dose-response relationship was apparent and the mean remained within the range expected for rats of this age and strain.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Macroscopic observations at necropsy did not reveal any alterations that were considered to have arisen as a result of treatment with the test substance.
In the male at 25 mg/kg/day (no. 12) found dead on day 89, the right kidney was enlarged and showed a large gray-white/black soft nodule (diameter 7.5 x 4.5 cm). This finding correlated to a nephroblastoma in the right kidney. Other findings in this animal included red contents in the urinary bladder, enlargement of the spleen and red discolouration of the thymus. Incidental findings among control and/or treated animals included red discolouration of the lungs, thymus or mandibular lymph node, liver grown together with the diaphragm, red foci on the thymus or kidneys, enlarged mandibular lymph node, alopecia, a tail fracture and fluid in the uterus. These findings are occasionally seen among rats used in these types of studies. In the absence of a treatment-related distribution they were considered changes of no toxicological significance.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

The following microscopic abnormalities were considered to be related to treatment:
Increased incidence and severity of cortical hyaline droplets in the kidneys in 9/10 males at 400 mg/kg/day (seven instances at a slight degree and two at moderate) and 9/10 males at 100 mg/kg/day (six minimal and three slight).
Increased incidence of hyaline casts (minimal or slight degrees) in 7/10 males at 400 mg/kg/day
Increased incidence of corticomedullary tubular basophilia in the kidneys (at minimal to moderate degree) in 8/10 males at 400 mg/kg/day.
Increased incidence and severity of diffuse midzonal/centrilobular hypertrophy in the liver in 7/10 females at 400 mg/kg/day (at a minimal or slight degree).
All other microscopic findings were within the range of background pathology encountered in Wistar rats of this age and strain and occurred at similar incidences and severity in both control and treated rats

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Description (incidence and severity):

No increase in the mean frequency of micronucleated polychromatic erythrocytes was observed in the polychromatic erythrocytes of the bone marrow of animals at 400 mg/kg/day compared to control animals.

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male

Basis for effect level:

histopathology: non-neoplastic

Target system / organ toxicity

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

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

System:

hepatobiliary

Organ:

liver

Treatment related:

yes

Dose response relationship:

not specified

Relevant for humans:

not specified

Applicant's summary and conclusion

Conclusions:

Under the conditions of the test histopathological findings in male kidneys 400 mg/kg/day were considered to represent an adverse effect of the test substance therefore, No Observed Adverse Effect Level (NOAEL) for NETSA of 100 mg/kg/day was established.

Executive summary:

A repeated dose 90-Day oral toxicity study with NETSA by daily gavage in the rat was performed according to OECD TG 408.

 

Based on a 14-day oral range finding study, the dose levels for this 90-day oral gavage study were selected to be 0, 25, 100 and 400 mg/kg/day. The test substance was administered daily for at least 90 days by oral gavage to SPF-bred Wistar rats. One control group and three treated groups were tested, each consisting of 10 males and 10 females. The following parameters were evaluated: Clinical signs, functional observations, body weight, food consumption and ophthalmoscopy. At termination: clinical pathology, macroscopy, organ weights and histopathology on a selection of tissues.  Accuracy, homogeneity and stability over 8 days of formulations of test substance in propylene glycol were demonstrated by analyses.

The following results were reported per dose group:

 

25 mg/kg/day:

- Incidental observations of lethargy, flat posture and uncoordinated movements (males/females)

- Increased liver to body weight ratio (females).

 

100 mg/kg/day:

- Lethargy, flat posture and uncoordinated movements (males/females)

- Increased absolute liver weight and liver to body weight ratio (females), and kidney to body weight ratio (females) .

- Increased incidence and severity of cortical hyaline droplets in the kidneys (9/10 males)

 

400 mg/kg/day:

- Lethargy, flat posture, uncoordinated movements, dry/pale faeces (males/females)

- Increased total protein, albumin and cholesterol levels (males/females).

- Increased absolute liver weight (males/females) and liver to body weight ratio (males),

increased kidney weights (females) and kidney to body weight ratio (males/females).

-Increased incidence and/or severity of cortical hyaline droplets (9/10 males), hyaline casts (7/10 males) and corticomedullary tubular basophilia (8/10 males)in the kidneys, and increased incidence and severity of diffuse midzonal/centrilobular hypertrophy in the liver (7/10 females).

 

Clinical signs observed after dosing (including lethargy, flat posture and uncoordinated movements) showed a dose-related occurrence with regard to incidence and time of appearance. In addition, it was frequently noted that animals secluded from their cage mates after dosing. Functional observations, motor activity measurements and histopathological assessment of neuronal tissues revealed no abnormalities that would support these clinical observations. In addition, the symptoms were of a minor and temporary nature (i.e. occurring only a short period after dosing, and not overtly and consistently present within the same dose group for the duration of treatment). With the exception of these signs, animals appeared in a good health condition throughout the study period. Therefore, although a relation to treatment with the test substance could not be excluded these findings were regarded to be of no toxicological significance.

 

In most cages containing animals at 400 mg/kg/day, dry faeces was recorded on most days from week 4 of treatment onwards. On a single day this was recorded as pale faeces for high dose females.

 

Cortical hyaline droplets representing alpha2 globulin were recorded at increased incidence and severity in the kidneys of males at 400 mg/kg/day. This was accompanied by an increase in the incidence of minor degrees of hyaline casts and increased incidence and severity of corticomedullary tubular basophilia. These latter findings were considered as indicators of renal tubular damage due to the increase in hyaline droplets. Although the incidence of hyaline droplets was increased also at 100 mg/kg/day compared to controls, the alteration was not accompanied by an increase in the abovementioned indicators of tubular damage and the severity grades of minimal or slight were within the range which may be seen in untreated male rats. This specific male rat response is not observed in normal female rats, and higher species of either sex, including humans.

 

In females at 400 mg/kg/day diffuse midzonal/centrilobular hypertrophy in the liver was present in seven cases at minor degrees of severity. In these females this hepatocellular hypertrophy correlated to increased liver weights, while the higher absolute or relative liver weights in males at 400 mg/kg/day and in females at 25 and 100 mg/kg/day occurred in the absence of any morphological correlates. In none of the dose groups there was microscopic evidence of any degenerative changes that would be indicative of hepatocytotoxicity. Therefore, these alterations were considered to be an adaptive physiological response following xenobiotic administration. The higher total protein and albumin and cholesterol levels in males and females at 400 mg/kg/day were considered to be related to these liver findings, and means of these parameters were largely within the normal range to be expected for rats of this age and strain.

 

No toxicological significance was ascribed to the higher absolute and/or relative kidney weights of females at 100 and 400 mg/kg/day since no morphological correlate was found.

 

Histopathological findings in male kidneys at 400 mg/kg/day were considered to represent an adverse effect of the test substance. Findings at 25 or 100 mg/kg/day occurred in the absence of supportive functional or morphological disturbances indicative of organ dysfunction.

Therefore, from the results presented in this report a definitive No Observed Adverse Effect Level (NOAEL) for NETSA of 100 mg/kg/day was established.