4-nitrotoluene-2-sulphonic acid

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

June 10th, 2020 to January 19th, 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Reason / purpose for cross-reference:

other: Dose range finding study

Reference

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

June 15th to 29th, 2020

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study without detailed documentation

Remarks:

Dose range finding test of the main OECD 408 study

Reason / purpose for cross-reference:

other: main study OECD 408

Qualifier:

no guideline followed

Principles of method if other than guideline:

Dose range finding test of the main OECD 408 study

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

other: Wistar Han; Crl: WI(Han)

Sex:

female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Condition: Outbred, SPF-Quality.
- Source: Charles River Deutschland, Sulzfeld, Germany or Charles River Laboratories France, L'Arbresle Cedex, France.
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 6-7 weeks old
- Weight at study initiation: 113 - 141 g
- Housing: group housed (up to 5 animals of the same dosing group together) in polycarbonate cages (Macrolon, MIV type, height 18 cm).

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-20°C
- Humidity (%): 56-68%.

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

water (elix)

Details on oral exposure:

Dose pot identification via Provantis is used as additional check to verify the dosing procedure according to Standard Operating Procedures.
The dose formulations were stirred continuously during dosing. The doses were given using a plastic feeding tube.

PREPARATION OF DOSING SOLUTIONS:
- prepared on w/w basis.
- homogenized by vortexing to visually acceptable levels.
- continuously stirred gently until dosing.

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

14 days

Frequency of treatment:

once daily, 7 days a week

Dose / conc.:

150 mg/kg bw/day (nominal)

Dose / conc.:

700 mg/kg bw/day (nominal)

No. of animals per sex per dose:

five per dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels were selected based on information and were based
on acute oral toxicity (OECD 423) and Combination Repeated dose toxicity study with reproduction/ developmental toxicity screening study (OECD 422) studies previously performed. In the OECD 423, 300 mg/kg bw was observed to be safe with no mortality observed and minimal clinical signs in a single animal (impaired general state, piloerection and dyspnea). Mortality was observed for animals dosed at 2000 mg/kg body weight, with severe clinical signs and significant findings during necropsy. In the OECD 422, the NOAEL was observed to be 175 mg/kg body weight/day in both males and female Sprague-Dawley rats, and there were indications for toxicity at higher dose levels (350 and 700 mg/kg) in various organs, including liver, kidney, spleen and reproductive organs.

Observations and examinations performed and frequency:

MORTALITY
- Time schedule: Twice daily throughout the study.
- Groups: all animals
- Procedure: Animals are observed within their cage unless necessary for identification or confirmation of possible findings

CAGE SIDE OBSERVATIONS
- Time schedule: At least three times daily: at 1-15 and 45-75 minutes Post Dose and at 2.5-3.5 hours Post Dose
- Groups: all animals
- Procedure: Animals are observed within their cage unless necessary for identification or confirmation of possible findings. For observations that cannot be attributed to an individual animal due to social housing (e.g., watery feces), the observation will be noted to each animal in the socialized group.

DETAILED CLINICAL OBSERVATIONS
- Time schedule: Weekly and on the day of necropsy
- Groups: all animals
- Procedure: Animals are removed from the cage.

BODY WEIGHT
- Time schedule for examinations: Once daily
- Groups: all animals

FOOD CONSUMPTION AND COMPOUND INTAKE
- Time schedule: Once daily
- Groups: all animals
- Procedure: Quantitatively measured per cage

No clinical laboratory investigations were performed.

Sacrifice and pathology:

TERMINAL PROCEDURES
On Day 15, the animals were deepy anesthetized using isofurane and subsequently exsanguinated and subjected to an external, thoracic and abdominal examination. Animals were not deprived of food prior to necropsy.

ORGAN WEIGHTS
The liver and kidneys were weighed at necropsy. Paired organs were weighed together. Organ weight as a percent of body weight (using the terminal body weight) was calculated.

TISSUE COLLECTION AND PRESERVATION
Representative samples of the liver, kidneys, heart and stomach were collected and preserved in 10% neutral buffered formalin. Additional tissue samples were collected to elucidate abnormal findings. No histopathological examination was performed

Statistics:

Constructed Variables
Body Weight Gains: calculated between at least each scheduled interval
Organ Weight Relative to Body Weight: calculated against the Terminal body weight for scheduled intervals

The procedures of statistical analysis, computerized systems, amendments and deviations, and retention of records are identical as for the main study, if applicable

Description (incidence and severity):

No findings at 0 and 150 mg/kg bw/day.
Erected fur was observed in two animals on Day 13 at 700 mg/kg bw/day

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

No findings at 0 and 150 mg/kg bw/day.
An increase in absolute and relative liver weight was seen compared to control animals (12 and 8% respectively) at 700 mg/kg bw/day

Description (incidence and severity):

No findings at 0 and 150 mg/kg bw/day.
In 3/5 animals a dark red glandular foci in the stomach were observed at 700 mg/kg bw/day

Dose descriptor:

NOAEL

Remarks on result:

not measured/tested

Remarks:

Based on the results of this range finding study, dose levels selected for the main study were 100, 250 and 700 mg/kg body weight/day.

Conclusions:

Based on the results of this range finding study, dose levels selected for the main study were 100, 250 and 700 mg/kg body weight/day.

Executive summary:

A dose range finding test was conducted in order to determine the doses for the main study (OECD 408).

Five female rats were exposed to the substance at 0, 150 and 700 mg/kg bw/day for 14 days. Mortality, cage side and clinical observations, body weight and food consumption were examined. Also organ weight and macroscopic examinations took place.

No effects were observed in the control and 150 mg/kg bw/day dose group. Instead at the highest dose group (700 mg/kg bw/day) the following were observed:

- Erected fur was observed in two animals on Day 13 

- In 3/5 animals a dark red glandular foci in the stomach were observed

- An increase in absolute and relative liver weight was seen  compared to control animals (12 and 8% respectively).

Based on the results of this range finding study, dose levels selected for the main study were 100, 250 and 700 mg/kg body weight/day.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

EPA 712-C-98-199

Deviations:

yes

Remarks:

None of the deviations were considered to have impacted the overall integrity of the study or the interpretation of the study results and conclusions.

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)

Version / remarks:

EC No 440/2008

Deviations:

yes

Remarks:

None of the deviations were considered to have impacted the overall integrity of the study or the interpretation of the study results and conclusions.

Test guideline 3

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2018

Deviations:

yes

Remarks:

None of the deviations were considered to have impacted the overall integrity of the study or the interpretation of the study results and conclusions.

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

4-nitrotoluene-2-sulphonic acid

EC Number:

204-445-3

EC Name:

4-nitrotoluene-2-sulphonic acid

Cas Number:

121-03-9

Molecular formula:

C7H7NO5S

IUPAC Name:

2-methyl-5-nitrobenzenesulfonic acid

Test animals

Species:

rat

Strain:

other: Wistar Han; Crl: WI(Han)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Condition: Outbred, SPF-Quality.
- Source: Charles River Deutschland, Sulzfeld, Germany or Charles River Laboratories France, l'Arbresle Cedex, France.
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 6-7 weeks old
- Weight at study initiation: Males weighed between 153 and 202 g and females weighed between 121 and 158 g
- Fasting period before study:
- Housing: Polycarbonate cages containing sterilized wooden fibers as bedding material equipped with water bottles. Up to 5 animals of the same sex and same dosing group together. During locomotor activity monitoring, animals were housed individually in a Hi-temp polycarbonate cage without cage-enrichment, bedding material, food and water. Cages were arranged on the racks according to a Latinsquare model.
- Diet: Ad libitum, except during designated procedures. type: pellets. SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany. During motor activity measurements, animals did not have access to food for a maximum of 2 hours
- Water: Freely available to each animal via water bottles. Municipal tap water.
- Acclimation period: at least 5 days before the commencement of dosing. Veterinary care was available throughout the course of the study, and animals were examined by the veterinary staff once during the Acclimate Period, due to a temporary change in the environmental conditions.
- Other: Animals were socially housed for psychological/environmental enrichment and were provided with paper and /or objects for chewing, except when interrupted by study procedures/activities.

DETAILS OF FOOD AND WATER QUALITY:
- Food quality: Results of analysis for nutritional components and environmental contaminants were provided by the supplier. It is considered that there were no known contaminants in the feed that would interfere with the objectives of the study.
- Water quality: Periodic analysis of the water was performed. It is considered that there were no known contaminants in the water that could interfere with the outcome of the study.

ENVIRONMENTAL CONDITIONS
- Temperature : Targeted: 18 to 24°C; Actual mean: 22 to 24°C
- Humidity: Targeted: 40 to 70%; Actual mean: 45 to 72%. The values that were outside the targeted humidity range occurred for 4 out of 108 days with a maximum of 72% and were without a noticeable effect on the clinical condition of the animals or on the outcome of the study.
- Air changes: Ten or more air changes per hour
- Photoperiod: 12 hours light and 12 hours dark (except during designated procedures)

Administration / exposure

Route of administration:

oral: gavage

Details on route of administration:

The oral route of exposure was selected because this is a possible route of human exposure during manufacture, handling or use of the test item.

The first day of dosing was designated as Day 1. The dose formulations were stirred continuously during dosing. The doses were given using a plastic feeding tube.
Dose pot identification via Provantis was used as additional check to verify the dosing procedure according to Standard Operating Procedures.
Animal No. 36 (700 mg/kg/day) had reflux when dosed on Day 68 and therefore the actual dose volume was unable to be confirmed.

Vehicle:

water

Remarks:

Water (Elix) - Trial preparations were performed to select the suitable vehicle and to establish a suitable formulation procedure.

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
- prepared on w/w basis.
- homogenized by vortexing to visually acceptable levels.
- continuously stirred gently until dosing.

VEHICLE
- Justification for use and choice of vehicle: Stability analyses performed previously in conjunction with the method development and validation study demonstrated that the test item is stable in the vehicle

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Dose formulation samples were collected for analysis
- of concentration: week 1-6-13: from all groups 2 x approximately 500 mg. Sample collected from approximately middle
- of homogeneity: week 1-6-13 from Groups 2 and 4 2 x approximately 500 mg. Sample collected from approximately Top, Middle, Bottom

Analysis was performed using a validated analytical procedure.

Concentration and Homogeneity Analysis
- Storage Conditions: Temperature set to maintain 2-8 °C.
- Acceptance Criteria: For concentration: mean sample concentration results within or equal to ± 10% of theoretical concentration.
- For homogeneity, relative standard deviation (RSD) of concentrations of ≤ 5% for each group.

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

Once daily, 7 days a week

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

100 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

250 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

700 mg/kg bw/day (nominal)

No. of animals per sex per dose:

10 per sex per dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels for the 90-day study were selected based on results of a 14-day dose range finder with the test item in rats and in an attempt to produce graded responses to the test item. The high-dose level should produce some toxic effects, but not death or severe suffering that would prevent meaningful evaluation. The low-dose level should produce no observable indications of toxicity

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS
- Population: all animals
- Time schedule: At least once daily; from Day 1 at 0 to 1 hours postdose
- Animals were observed within their cage unless necessary for identification or confirmation of
possible findings. For observations that could not be attributed to an individual animal due to social housing (e.g., watery feces), the observation was recorded to each animal in the socialized group

MORTALITY
- Population: all animals
- Time schedule: At least twice daily beginning upon arrival through termination. Except on days of receipt and necropsy where frequency was at least once daily
- Animals were observed within their cage unless necessary for identification or confirmation of possible findings.

DETAILED CLINICAL OBSERVATIONS:
- Population: all animals
- Time schedule: Once pretreatment and weekly; from Week 1 and throughout the study, and on the day of necropsy.
- Animals were removed from the cage.

ARENA OBSERVATIONS:
- Population: all animals
- Time schedule: Once before the first administration of the test item and weekly during the treatment Period.
- Animals were observed for clinical signs outside the home cage in a standard arena. The time of onset, grade and duration of any observed signs was recorded.

INDIVIDUAL BODY WEIGHT
- Population: all animals
- Time schedule for examinations: Weekly; from at least Day 1 and throughout the study. In order to monitor the health status, male Nos. 35 and 36 and female No. 72(700 mg/kg/day) were weighed more often on Days 9 and 11, Day 53, and Day 45, respectively.
- Fasted weight on the day of necropsy.

FOOD CONSUMPTION
- Population: all animals
- Time schedule: Weekly; from at least Day 1 and throughout the study
- Quantitatively measured per cage.

WATER CONSUMPTION
- Population: all animals
- Time schedule: Regular basis throughout the study.
- Water consumption was monitored by visual inspection of the water bottles.

OPHTHALMOSCOPIC EXAMINATION
- Time schedule for examinations: Pretreatment Period - All Study animals once (including spare animals). Dosing Period - All Group 1 and 4 Study animals during Week 13. Since no test item-related findings were noted, the other animals were not examined.
- Procedure: The eyes were examined using an ophthalmoscope after application of a mydriatic agent (Tropicol 5 mg/mL solution, THEA Pharma, Wetteren, Belgium).

FUNCTIONAL TESTS
- Time schedule: Once during the Dosing Period. The first 5 animals per sex per group during Weeks 12-13. These tests were performed after clinical observations.
The following tests were performed:
- hearing ability, pupillary reflex and static righting reflex (score 0 = normal/present, score 1 = abnormal/absent).
- fore- and hind-limb grip strength were recorded as the mean of three measurements.
- locomotor activity (recording period: 1 hour under normal laboratory light conditions, using a computerized monitoring system). Total movements and ambulations were reported. Ambulations represent movements characterized by a relocation of the entire body position like walking, whereas total movements represent all movements made by the animals, including ambulations but also smaller or finer movements like grooming, weaving or movements of the head.

ESTROUS STAGE CYCLE
- Frequency: End of Treatment - on the day of necropsy, a vaginal smear was taken to determine the stage of estrus from all Study animals. This was done for all females, except for the female that died spontaneously.
- Procedure: Estrous stage was evaluated by examining the vaginal cytology of samples obtained by vaginal smears procedures.

HAEMATOLOGY
- Time schedule for collection of blood: Sampled between 7.00 and 10.30 from the retro-orbital
sinus under anesthesia using isoflurane (Abbott B.V., Hoofddorp, The Netherlands). at the end of the treatment
- Volume: 0.5 ml
- Anti-coagulant: (K3) EDTA
- Animals fasted: Yes (overnight with a maximum of 24 hours)
- How many animals: all animals
- Processing: A blood smear was prepared from each hematology sample. Blood smears were labeled, stained, and stored. One blood smear was evaluated as required to confirm analyzer results
- Parameters examined. White blood cell (WBC), Neutrophils (absolute), Lymphocytes (absolute)
Monocytes (absolute), Eosinophils (absolute), Basophils (absolute), Large unstained cells (LUC) (absolute), Red blood cell (RBC), Reticulocytes (absolute), Red blood cell distribution width (RDW), Hemoglobin, Hematocrit, Mean corpuscular volume (MCV), Mean corpuscular hemoglobin (MCH), Mean corpuscular hemoglobin concentration (MCHC), Platelets

COAGULATION
- Time schedule for collection of blood: Sampled between 7.00 and 10.30 from the retro-orbital
sinus under anesthesia using isoflurane (Abbott B.V., Hoofddorp, The Netherlands). at the end of the treatment
- Volume: 0.9 ml
- Anti-coagulant: Citrate
- Processing: plasma
- Animals fasted: Yes (overnight with a maximum of 24 hours)
- How many animals: all animals
- Parameters examined: Prothrombin time (PT), Activated partial thromboplastin time (APTT)

CLINICAL CHEMISTRY
- Time schedule for collection of blood: Sampled between 7.00 and 10.30 from the retro-orbital
sinus under anesthesia using isoflurane (Abbott B.V., Hoofddorp, The Netherlands). at the end of the treatment
- Volume: 0.5 mL (plasma) and 1.0 mL (serum; thyroid hormone measurement).
- Anti-coagulant: Li-Heparin (not for serum tubes)
- Processing: plasma
- Animals fasted: Yes (overnight with a maximum of 24 hours)
- How many animals: all animals
- Parameters examined: Alanine aminotransferase (ALT), Triglycerides, Aspartate aminotransferase (AST), HDL and LDL Cholesterol, Alkaline phosphatase (ALP), Sodium, Total protein, Potassium, Albumin, Chloride, Total bilirubin, Calcium, Urea, Inorganic phosphate (Inorg. Phos), Creatinine, Triiodothyronine (T3), ,Glucose, Thyroxine (T4), Cholesterol, Thyroid-stimulating hormone (TSH)

URINALYSIS: Yes / No / Not specified
- Time schedule for collection of urine: Urine was collected into a specimen vial from animals
housed in individual metabolism cages overnight (approximately 15-20 hours).
- Animals fasted: Yes (overnight with a maximum of 24 hours)
- How many animals: all animals
- Volume: As available, up to 10 mL
- Parameters examined: Volume, Specific gravity, Clarity, Color, pH, Blood, Leukocyte esterase, Bilirubin, Urobilinogen, Protein, Ketones, Glucose, Nitrite

THYROID HORMONES
After receipt of the serum for T3, T4 and TSH analysis it was divided in two aliquots. One aliquot was used for measurement of thyroid hormones using the Immulite (TSH). These samples were stored in an ultra-low freezer (≤ -75°C) until analysis. Any remaining sample was discarded. The other aliquot was used for measurement of T3 and T4 using LC-MS.
These samples were stored in a freezer (≤ -15°C) until analysis. Measurement was performed according to the bioanalytical method validated. Any remaining samples were returned to storage for the retention period.
The LC-MS analysis was based on the following guidelines:
- European Medicines Agency (EMA). Guideline on Bioanalytical Method Validation. EMEA/CHMP /EWP/192217/2009, 01 February 2012
Guidance for industry: Bioanalytical Method Validation, U.S. Department of Health and Human Services, Food and Drug Administration, Center for Drug Evaluation and Research (CDER) and Center for Veterinary Medicine (CVM), May 2018.

Sacrifice and pathology:

SCHEDULED EUTHANASIA
Scheduled euthanasia day: at the end of treatment
Study animals surviving until scheduled euthanasia were weighed, and euthanized using isoflurane, followed by exsanguination. Animals were fasted (overnight with a maximum of 24 hours) before their scheduled necropsy

Unscheduled Deaths
A necropsy was conducted for the female (No. 73, 700 mg/kg/day) that died on study, and specified tissues were saved.

NECROPSY
Animals were subjected to a complete necropsy examination, which included evaluation of
the carcass and musculoskeletal system; all external surfaces and orifices; cranial cavity and external surfaces of the brain; and thoracic, abdominal, and pelvic cavities with their associated organs and tissues.
Necropsy procedures were performed by qualified personnel with appropriate training and experience in animal anatomy and gross pathology. A veterinary pathologist, or other suitably qualified person, was available.

ORGAN WEIGHTS
The organs identified below were weighed at necropsy for all scheduled euthanasia animals. Organ weights were not recorded for the animal found dead. Paired organs were weighed together. Organ to body weight ratio (using the terminal body weight) were calculated.
Brain; Epididymis; Gland, adrenal; Gland, Pituitary; Gland, prostate; Gland, seminal vesicle; Gland, thyroid; Heart; Kidney; Liver; Ovary; Spleen; Testis; Thymus; Uterus/cervix

TISSUE COLLECTION AND PRESERVATION
Representative samples of the tissues identified below were collected from all animals and preserved in 10% neutral buffered formalin (neutral phosphate buffered 4% formaldehyde solution, Klinipath, Duiven, The Netherlands), unless otherwise indicated
Animal identification; Artery, aorta; Body cavity, nasal; Bone, femur; Bone marrow, sternum; Bone, sternum; Brain; Epididymis*; Esophagus; Eye*; Gland, adrenal; Gland, clitoral; Gland, harderian*; Gland, lacrimal; Gland, mammary; Gland, parathyroid**; Gland, pituitary; Gland, preputial; Gland, prostate; Gland, salivary, submandibular; Gland, salivary, parotid; Gland, seminal vesicle (including coagulation gland); Gland, thyroid; Gross lesions/masses; Gut-associated lymphoid tissue***; Heart; Kidney; Large intestine, cecum; Large intestine, colon; Large intestine, rectum; Larynx; Liver
Lung****; Lymph node, mandibular; Lymph node, mesenteric; Muscle, skeletal; Nerve, optica*****; Nerve, sciatic; Nerve, tibial; Ovary; Pancreas; Skin; Small intestine, duodenum; Small intestine, ileum; Small intestine, jejunum; Spinal cord; Spleen; Stomach; Testis*,******; Thymus; Tongue; Trachea; Urinary bladder; Uterus/Cervix; Vagina
Macroscopic abnormalities in the organs listed and in other organs were sampled at necropsy, processed for histology and examined microscopically.
*Preserved in Modified Davidson’s fixative. Tissues were transferred to formalin after fixation for at least 24 hours.
** Examined only if detectable in the routine section of thyroid. Weighed with gland, thyroid.
*** Examined only if detectable in routine section of intestine.
**** Infused with formalin.
***** Examined only if detectable in the routine section of the eye. Part of the optic nerve remained attached to the eye and fixed in Modified Davidsons’s fixative. The remaining part of the optic nerve was placed in formalin.
****** From all males of Group 1 and 4 the hematoxylin and eosin stained slides of the testes were specifically evaluated for the presence of all stages as an indication for normal spermatogenesis.

HISTOLOGY - HISTOPATHOLOGY
Tissues identified (except animal identification, bone marrow smears, femur, clitoral gland, harderian gland, lacrimal gland, preputial gland, parotid salivary gland, larynx, tibial nerve, and tongue) were embedded in paraffin, sectioned, mounted on glass slides, and stained with hematoxylin and eosin.
All tissues as defined were examined by a board-certified toxicological pathologist with training and experience in laboratory animal pathology. Target tissues identified by the study pathologist during microscopic evaluation were communicated to the Study Director; tissues were evaluated and reported.

Optional endpoint(s):

Constructed Variables
- Body Weight Gains: Calculated between at least each scheduled interval.
- Food Consumption: Calculated between at least each scheduled interval.
- Organ Weight Relative to Body Weight: Calculated against the terminal body weight.

Results and discussion

Results of examinations

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

No test item-related clinical signs were noted for males and females dosed at 100 mg/kg/day.
Labored breathing was noted in 1/10 males at 700 mg/kg/day between Days 8-11. Shallow breathing was noted in 1/9 females at 700 mg/kg/day between Days 42-63. In addition, abnormal breathing sounds were observed in 4/10 males and 3/9 females at 700 mg/kg/day and 1/10 males and 1/10 females at 250 mg/kg/day. The start and duration of these clinical signs varied between animals and did not seem to be related to a specific time point during the study. The first observation of abnormal breathing sounds was made on Day 7 and the last observation of this clinical sign was made on Day 95.
Hunched posture was noted in 2/10 males and 1/10 females at 700 mg/kg/day between Days 50-58 and 74-94 (males) and 42-66 (females), respectively. Furthermore, erected fur was observed in 2/10 males dosed at 700 mg/kg/day between Days 8-9 and 31-34, respectively.
Salivation (slight) noted in 8/10 males and 7/9 females at 700 mg/kg/day on one or more days during the treatment period was considered to be not toxicologically relevant, taking into account the nature and minor severity of the effect and its time of occurrence (i.e. after dosing). This sign was regarded as a physiological response rather than a sign of systemic toxicity.
Other findings (e.g., scabs and lesions (skin) and discharge from the eye) noted during the Dosing Period occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study and were observed in a control animal only or did not show any apparent dose-related trend. At the incidence observed, these were considered to be unrelated to treatment with the test item.

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 item.
At 700 mg/kg/day, Female No. 73 was found dead on Day 28 prior to dosing. Salivation (slight) was noted between Days 3 and 8 and abnormal breathing sounds were noted on Days 12-15. Body weight gain remained normal during the observation period. Macroscopic red fluid accumulation in the thoracic cavity was seen. Several organs showed a variable degree of autolytic changes. A finding of note was present in the lung and consisted of moderate mixed peribronchial/perivascular cell infiltration, mainly localized in one lobe, which likely attributed to the death of this animal. This might be the result of aspiration or spilling of test item in the lung. Therefore, this death was regarded to be gavage procedure related.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

No toxicologically relevant changes in body weight and body weight gain were observed in males and females up to 700 mg/kg/day.
A slightly reduced mean body weight gain was observed in males at 700 mg/kg/day over Days 1-8 and 43-50 when compared to the control group (not statistically significant). These changes could be attributed mainly to male Nos. 35 and 36, respectively, which presented with temporary body weight loss (No. 35: -17%; No. 36: -38%) in the mentioned periods.
Also, in females at 700 mg/kg/day, a slight decrease in body weight gain was noted between Days 36-43 (statistically significant). This change could be attributed mainly to the temporary body weight loss (-26%) in female No. 72. No toxicological relevance was attached to these findings as they occurred incidentally, and complete recovery was observed thereafter.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

No toxicologically relevant changes in food consumption were recorded.
A minimal decreased food consumption was observed in both males and females at 700 mg/kg/day during Week 7 and 6 respectively (0.92x and 0.91x, respectively, compared to controls). Since these differences are only minimal and temporary, this is considered to be not toxicologically relevant.

Food efficiency:

not examined

Ophthalmological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No ophthalmology findings were noted that were considered to be related to treatment with the test item.
The nature and incidence of ophthalmology findings noted during the Pretreatment Period and in Week 13 was similar among the groups and occurred within the range considered normal for rats of this age and strain. These findings were therefore considered to be unrelated to treatment with the test item.

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

No test item-related hematology parameter changes were observed in males and females up to 250 mg/kg/day.
At 700 mg/kg/day, increased reticulocytes count in males (1.22x of control), decreased hemoglobin concentration in males (0.94x of control), increased platelet counts in males and females (1.17x and 1.20x of control, respectively), and increased red blood distribution width in males and females (1.10x and 1.12x of control, respectively) were noted.
Remaining differences in hematology parameters, regardless of statistical significance, were considered not test item-related based on the absence of a dose response and general overlap of individual values with the range of control values.
Coagulation parameters of treated rats were considered not to have been affected by treatment with the test item.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

No test item-related changes in clinical chemistry parameters were observed in males and females up to 250 mg/kg/day.
At 700 mg/kg/day, a decrease in total protein concentration in males and females (0.92x and 0.94x of controls, respectively), albumin concentration in males (0.93x of controls), and an increase in concentrations of cholesterol and HDL cholesterol in males (1.22x and 1.20x of control, respectively) were observed.
Remaining differences in clinical chemistry parameters, regardless of statistical significance, were considered not test item-related based on the absence of a dose response, general overlap of individual values with the range of control values, and/or were of a magnitude of change commonly observed in rats under similar study conditions.

Urinalysis findings:

effects observed, non-treatment-related

Description (incidence and severity):

Urine parameters of treated rats were considered not to have been affected by the treatment with the test item.
An unrealistic high urine volume (29 mL) was observed in one animal at 700 mg/kg/day (male Group 4, No. 37). This was likely to be caused by a leaky water bottle in the metabolic cage and therefore considered to be not test item-related.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Test item-related lower prostate gland weights (absolute and relative to body weights) were noted in males treated at 700 mg/kg/day.
In one male, mild multifocal atrophy was recorded, correlating to the lower prostate gland weight. There was no clear microscopic correlate for the lower mean prostate gland weight in the remaining males at 700 mg/kg/day.
The statistically significant lower liver weight recorded in males at 100 mg/kg/day (relative to body weight) and at 250 mg/kg/day (absolute and relative to body weight) lacked a dose response and was regarded unrelated to the treatment with the test item.
There were no other test item-related organ weight changes.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Test item-related dark red foci in the glandular part of the stomach were recorded in
2/10 males and 3/9 females at 700 mg/kg/day. The microscopic correlate for this finding was mixed cell infiltration of the glandular mucosa, mucosal hemorrhage and/or erosions of the glandular mucosa. A thickened limiting ridge of the stomach was recorded in 3/9 females at 700 mg/kg/day (without microscopic correlate).
The remainder of the recorded macroscopic findings were within the range of background gross observations encountered in rats of this age and strain.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Test item-related microscopic findings after treatment with 4-nitrotoluene-2-sulphonic acid were noted in the stomach of males and females and in the prostate gland of males.
At the end of the 90-Day treatment period at 700 mg/kg/day a combination of test item related findings was seen in the glandular stomach:
 Diffuse infiltration of mixed cells in 8/10 males and 9/9 females at minimal or mild degree.
 Focal/multifocal infiltration of lymphocytes in 9/10 males and 8/9 females at minimal or mild degree.
 Mucosal hemorrhage at minimal degree in 3/9 females.
 Erosions in a single male (mild degree) and a single female (minimal degree).
In males at 100 and 250 mg/kg/day a slightly higher incidence of minimal focal lymphocytic infiltrates in the glandular stomach was recorded (4/10 and 3/10, respectively), which was regarded to be test item-related.
Remaining alterations recorded for the stomach were considered to be within background.
An increased incidence and severity (minimal-mild) of lymphocytic infiltration in the
prostrate gland was recorded at 700 mg/kg/day. In addition, multifocal atrophy of the prostate gland at mild degree was recorded in a single male at 700 mg/kg/day.
The lung and trachea were examined from all animals, because of perivascular/ peribronchial inflammatory infiltrates (eosinophilic or mixed) in the lungs at a slightly higher incidence and/or severity compared to control animals. In a few animals at 700 mg/kg/day hypertrophy/hyperplasia of the trachea epithelium was recorded. These findings were regarded to have attributed to the abnormal breathing sounds recorded for several animals at 250 and 700 mg/kg/day during the course of this study. These findings were likely the result of incidental aspiration of test item and not a systemic test item effect.
The spleen of males showed a high incidence and severity of extramedullary hematopoiesis.
This was seen at minimal to moderate degree, at comparable incidence and severity in males of both the control and the 700 mg/kg/day treated animals. This was most likely a regenerative effect after the blood sampling procedure which was performed two days prior to necropsy and therefore considered to be not test item-related.
The remainder of the recorded microscopic findings were within the range of background pathology encountered in rats of this age and strain. There was no test item-related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.

Description (incidence and severity):

- Functional Observations
No test item-related effects on functional observation was observed.
The total number of ambulations during the motor activity measurement in males dosed at 700 mg/kg/day was lower compared to controls (0.46x), although this is not statistically significant. As all groups showed a similar motor activity habituation profile with a decreasing trend in activity over the duration of the test period, this is considered to be not toxicologically relevant.
The mean grip strength in the forelegs of females dosed at 700 mg/kg/day was slightly lower compared to control animals (0.82x), although this is not statistically significant and caused by a single animal (No. 72). As all other animals in this group have grip strength in the forelegs comparable to controls, and hindleg strength is normal in animal No. 72, the lower grip strength in the forelegs is considered to be not test item-related.

Effect levels

Dose descriptor:

NOAEL

Effect level:

250 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

gross pathology

Remarks on result:

other: .

Remarks:

Adverse morphological changes were observed in the glandular stomach and prostate gland at 700 mg/kg/day. In the glandular stomach a combination of focal/multifocal lymphocytic infiltrates, diffuse mixed infiltrates, erosions and/or hemorrhage, correlating to macroscopic dark red foci were recorded in males and females. In the prostate gland a single atrophy was recorded. The other morphological and also clinical pathology findings observed in males and females at 100, 250 and 700 mg/kg/day were considered to be non-adverse.

Target system / organ toxicity

Critical effects observed:

yes

Lowest effective dose / conc.:

700 mg/kg bw/day (nominal)

System:

gastrointestinal tract

Organ:

stomach

Treatment related:

yes

Dose response relationship:

not specified

Relevant for humans:

not specified

Any other information on results incl. tables

Dose Formulation Analyses
- Accuracy: The concentrations analyzed in the formulations of Groups 2, 3 and 4 were in agreement with target concentrations (i.e. mean accuracies between 90% and 110%). No test item was detected in the Group 1 formulations. 

- Homogeneity: The formulations of Group 2 and Group 4 were homogeneous (i.e. coefficient of variation ≤ 5%).

Applicant's summary and conclusion

Conclusions:

NOAEL (rat, male/female) = 250 mg/kg bw/day

Executive summary:

The objective of this study was to determine the potential toxicity of 4-nitrotoluene-2-sulphonic acid, when given orally by gavage for 90 days to Wistar Han rats. In addition, a No Observed Adverse Effect Level (NOAEL) was evaluated.

Ten male and ten female Wistar Han rats were exposed to 100, 250 and 700 mg/kg bw/day of the substance for 90 days. A concurrent vehicle control (water) run.

Chemical analyses of formulations were conducted in Weeks 1, 6 and 13 to assess accuracy and homogeneity.
The following parameters and end points were evaluated in this study: clinical signs, functional observation tests, body weights, food consumption, ophthalmology, estrous stage determination, clinical pathology parameters (hematology, coagulation, clinical chemistry, and urinalysis), gross necropsy findings, organ weights, and histopathologic examinations.
Formulation analyses confirmed that formulations of test item in water were prepared accurately and homogenously.

Results

No mortality occurred during the study period that was considered to be related to treatment with the test item. One female at 700 mg/kg/day was found dead on Day 28, which was regarded to be gavage-related error.
In males at 100 and 250 mg/kg/day a slightly higher incidence of minimal focal lymphocytic infiltrates in the glandular stomach was recorded, which was regarded to be test item-related, but in absence of any degenerative lesions considered to be non-adverse.
At 700 mg/kg/day, test item-related increased reticulocytes count and decreased hemoglobin concentration in males, and increased platelet counts and red blood distribution width in males and females were noted. Furthermore, a decrease in albumin levels and an increased level of cholesterol and HDL cholesterol in males, and a decreased total protein concentration in males and females was noted. In absence of a histopathological correlation and at the magnitude of change, these findings were considered to be not adverse.
At histopathological examination, test item-related morphological alterations were recorded in the stomach (glandular stomach) and consisted of inflammatory changes at minimal to mild degree and in a few animals consisted of mucosal hemorrhage and erosions and macroscopic dark red foci. These alterations were considered to be related to the irritating properties of the test item and regarded local test item-related findings. The combination of erosions with increased incidence and severity of lymphocytic and mixed cell infiltrates were considered to be adverse.
In the prostate gland, atrophy was recorded for a single male for which a relation to the treatment could not be excluded, but it was in line with the lower prostate gland weights recorded in males and females at 700 mg/kg/day. Atrophy of the prostate gland is a degenerative finding and therefore regarded to be adverse. In addition, lymphocytic infiltrates in the prostate gland were recorded at a slightly higher incidence and severity in males at 700 mg/kg/day, which was regarded to be non-adverse.
In conclusion, administration of 4-nitrotoluene-2-sulphonic acid by once daily oral gavage was well tolerated in Wistar Han rats at levels of up to 700 mg/kg/day. Adverse morphological changes were observed in the glandular stomach and prostate gland at 700 mg/kg/day. In the glandular stomach a combination of focal/multifocal lymphocytic infiltrates, diffuse mixed infiltrates, erosions and/or hemorrhage, correlating to macroscopic dark red foci were recorded in males and females. In the prostate gland a single atrophy was recorded. The other morphological and also clinical pathology findings observed in males and females at 100, 250 and 700 mg/kg/day were considered to be non-adverse.
Based on these results in this study, the No Observed Adverse Effect Level (NOAEL) for 4-nitrotoluene-2-sulphonic acid was considered to be 250 mg/kg/day.