N-butylbenzenesulphonamide

Endpoint summary

Description of key information

A subacute 28 -day oral toxicity with BBSA was conducted by oral daily gavage in Wistar rats at dose levels of 0, 50, 150 and 1000 mg/kg bw. The high dose of 1000 mg/kg/day resulted in the death or moribund state of all rats, with liver enlargement and hepatocyte hypertrophy, liver necrosis, hyaline droplets formation in the renal papillary collecting ducts, adrenal gland enlargement with fatty change or cortical hypertrophy and reduced size and/or atrophy of the thymus, spleen and male reproductive organs. At 150 mg/kg/day, post-mortem findings were confined to liver enlargement and hepatocyte hypertrophy, thymic atrophy and lymphocytolysis as relevant changes. At 50 mg/kg/day, there were no relevant changes, therefore a NOAEL of 50 mg/kg/day was established.

A dietary 14-day dose range finding study was conducted in Wistar rats to prepare for the 90-day toxicity study at dietary concentrations of 625, 1563, 5875 and 12500 ppm, corresponding with mean test item intake values of 52.4, 124.2, 392.7 and 543.7 mg/kg bw/day, respectively. The 5875 and 12500 ppm dose levels were concluded to be above the Maximum Tolerable Dose for the sub-chronic repeated dose toxicity study.

A key 90-day dietary study in Wistar rats was conducted at 0, 700, 1750 and 3750 ppm dose levels, corresponding with 51.9, 132.5 and 265.4 mg/kg bw/day respectively. Effects observed included decreased body weight (gain) in the animals of the High and Mid dose groups and food consumption which was significantly reduced by the test item administration in the High dose group; in the Mid dose group a transient effect in the first one or two weeks was seen. Food efficiency was statistically significantly lower at 1750 and 3750 ppm both in males and females. There were signs of slight anaemia in the High dose groups (up to 10% lower RBC, HCT and haemoglobin concentration) with a similar trend in the Mid dose. Compared to control, test item related increase in kidney weights (absolute and body related) were detected for High and Mid dose males, associated with increased kidney size and histopathology change. Increased adrenal weights were also noted for High dose males, but without any histopathology change. Test item related dark red foci on liver lobes were noted in some High and Mid dose animals at necropsy, but no Low dose animals were affected. The change corresponded with histopathology observations of necrosis (mild to moderate at Mid dose males and High dose female; marked in High dose male). Test item related alpha 2-microglobulin nephropathy was observed in male rats only. The observed microscopic changes were confirmed by special immunohistochemistry staining and not considered relevant for humans. Test item-related focal/multifocal hepatocellular necrosis was noted as adverse change but at low incidence in the liver of High and Mid dose males as well as in High dose females. No test item related effect was observed in the adrenals and thymus of male and female animals in any dose groups. Finally, there were no treatment-related effects at any dose levels on sperm morphology or enumeration. In conclusion. The NOAEL was 700 ppm dietary concentration (51.9 mg/kg bw/day).

A key repeated dose 28 day dermal toxicity study was conducted in Wistar Rats at dose levels of 0, 250, 500 and 1000 mg/kg bw (semi-occlusive dosing; no vehicle). On the basis of the results obtained in the present study, no mortality was observed in both sex of treated as well as control group of animals during entire experiment period. Although few changes occurred in hematology and clinical biochemistry parameters, those changes were not related to test substance and could be considered as individual animal normal biological variance. There was no test substance related macroscopic and microscopic findings observed. In the light of above observations, the NOEL of N-n- Butylbenzenesulphonamide for Wistar rats could be considered as >1000 mg/kg bw under the experimental conditions.

Inhalation testing was waived.

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

2002

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Qualifier:

according to guideline

Guideline:

EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))

Principles of method if other than guideline:

The test substance was administered daily for 28d by oral gavage to SPF-bred Wistar rats. One control group and three treated groups were tested,
each consisting of 5 males and 5 females.
The following parameters were evaluated: clinical signs daily; functional observation tests; body weight and food consumption weekly; clinical pathology and macroscopy at termination; organ weights and histopathology on a selection of tissues. No clinical laboratory parameters and no organ weights were determined from the high dose animals of each group underwent whole-body perfusion, and the cranial part of the brain of the last two animals of each group was frozen for possible future analysis on brain tissue.

GLP compliance:

yes (incl. QA statement)

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Age at study initiation: Approximately 6 weeks
- Weight at study initiation: 200-210g (males) / 160-170g (females
- Fasting period before study:
- Housing:Group housing of 5 animals per sex per cage in stainless steel suspended cages with wire mesh floors (55*34*21.5 cm height).
- Diet (e.g. ad libitum): Free acces to standard pelleted laboratory animal diet (from Altromin (code VRF-1, Lage, Germany). Each batch is analysed for nutrients and contaminants are analysed on a regulair basis. Results are examined and archived.
- Water (e.g. ad libitum):free access to tap water. Certificate of analysis (performed quaterly) were examined and archived.
- Acclimation period: acclimatisation period was at least 5d before start of treatment under laboratory conditions. During activity monitoring, animals were individually housed overnight in Macrolon plastic cages with sterilised sawdustprovided as bedding.


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 +- 3°c / Temporary deviations from the light/dark cycle occured (max. 1h) due to performance of functional observations in the room.
- Humidity (%): 30-70%
- Air changes (per hr): approximately 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12h artificial fluorescent light and 12h dark per day


IN-LIFE DATES: From: 20/06/2002 To: 18/07/2002

Route of administration:

oral: gavage

Vehicle:

propylene glycol

Details on oral exposure:

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


DIET PREPARATION
- Rate of preparation of diet (frequency):
- Mixing appropriate amounts with (Type of food): to standard pelleted laboratory animal diet (from Altromin (code VRF-1, Lage, Germany).
- Storage temperature of food:


VEHICLE
- Justification for use and choice of vehicle (if other than water):
- Concentration in vehicle:
- Amount of vehicle (if gavage):
- Lot/batch no. (if required):
- Purity:

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

0-50-150-1000 mg/kg/day

Samples of week 4 formulations were analysed to check homogenity (highest and lowest concentration) and
accuracy of preparation (all concentrations). Stability in vehicle over 4h was also determined (highest and lowest concentration).
The analytical method used was based on the result of a separate project for the development and validation of the analytical method
(NOTOX project 355027).

Duration of treatment / exposure:

28 days

Frequency of treatment:

Frequency: Once daily for at least 28d, 7d per week, approximately the same time each day with a max. of 4h difference between the
earliest and latest dose. Animals were dosed up to the day prior to necropsy.

Dose volume: 5ml/kg body weight. Actual dose volumes were calculated weekly according to the latest body weight.

Remarks:

Doses / Concentrations:
0-50-150-1000 mg/kg/day
Basis:
actual ingested

No. of animals per sex per dose:

ALLOCATION

Group Dose level mg/kg/d Number of animals
Males females
1 main group 0 (vehicle) 5 5
2 main group 50 5 5
3 main group 150 5 5
4 main group 1000 5 5

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Based on the results of a 5d range finding study.
- Rationale for animal assignment (if not random):
- Rationale for selecting satellite groups:
- Post-exposure recovery period in satellite groups:
- Section schedule rationale (if not random):

Observations and examinations performed and frequency:

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once daily, detailed clinical observations were made in all animals. Once prior to start of treatment and on a weekly basis thereafter, this was also performed outside the home cage in a standard arena.


BODY WEIGHT: Yes
- Time schedule for examinations: On day 1, 8, 15, 22 and 29 (females) or 30 (males)


FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes




WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: Subjective appraisal was maintained during the study, but no quantitative investigation introduced .
as no effect was suspected.


OPHTHALMOSCOPIC EXAMINATION: No
- Time schedule for examinations:
- Dose groups that were examined:


HAEMATOLOGY: Yes
- Time schedule for collection of blood: immediately prior to scheduled post mortem examination, between 7.30 and 9.30 a.m.
- Anaesthetic used for blood collection: Yes (identity) : iso-fluarane anaesthesia
- Animals fasted: No
- How many animals: all rats/sex/group
- Parameters checked in table [No.?] were examined:
Haematology: erythrocytes count/ haemoglobin/ haemotocrit/ mean corpuscular volume/ mean corpuscular haemoglobin/
mean corpuscular haemoglobin conc. / platelets count/ red cell distribution width/ total leucocytes count/ differential leucocytes count: neutrophils/eosinophils/ basophils/ lymphocytes/ monocytes.


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: immediately prior to scheduled post mortem examination, between 7.30 and 9.30 a.m.
- Animals fasted: No
- How many animals: all rats/sex/group
- Parameters checked in table [No.?] were examined:
alanine aminotransferase/ alkaline phosphatase/ asparate aminotransferase/ bilirubin, total/ chloride/ cholesterol, total/ creatinine/ glucose/
phosphorus/ protein, total/ protein, albumin/ urea/ calcium/ potassium/ sodium.


URINALYSIS: No
- Time schedule for collection of urine:
- Metabolism cages used for collection of urine: Yes / No / No data
- Animals fasted: Yes / No / No data
- Parameters checked in table [No.?] were examined.


NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: during week 4 of treatment
- Dose groups that were examined: all animals
- Battery of functions tested: sensory activity / grip strength / motor activity / other: hearing ability, pupillary reflex, static righting reflex and grip strength


OTHER:

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes

Statistics:

The following statistical methods were used to analyse the data:
- if the variables could be assumed to follow a normal distribution, the Dunett-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 test were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance. Group means were calculated for
constinous data and medians were calculated for discrete data (scores) in the summary tables. No statistical analysis was performed on
motor activity data. 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.

Clinical signs:

effects observed, treatment-related

Mortality:

mortality observed, treatment-related

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, treatment-related

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

no effects observed

Details on results:

The high dose of 1000 mg/kg/day resulted in the deth or moribund state of all rats. Clinical signs shown by these animals prior to death/sacrifice included lethargy, hunched posture, uncoordinated movements, abnormal gait, salivation, emaciation, laboured respiration, swelling of the abdomen or head and piloerection.
Examination revealed liver enlargement and hepatocyte hyperthrophy, liver necrosis, hyaline droplets formation in the renal papillary collecting ducts, adrenal gland enlargement with fatty change or cortical hypertrophy and reduced size and/or arophy of the thymus, spleen and male reproductive organs.
At 150 mg/kg/day, post-mortem findings were confined to liver enlargement and hepatocyte hypertrophy, thymic atrophy and lymphocytolysis.
Cortical hyaline were noted in the kidneys of most male rats dosed at 50 mg/kg/day and above. This is a specific male rat response and is not observed in female rats or higher species of either sex.
Degenerating nerve fibres were observed at low incidence and severity in the spinal cord and sciatic nerves at 150 and 1000 mg/kg/day.
At 50 and 150 mg/kg/day, there were no changes at performance of functional observations, body weight and food consumption measurements.
At 50 mg/kg/day there sere no treatment related macro- or microscopic findings.

Dose descriptor:

NOAEL

Effect level:

50 mg/kg diet

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Critical effects observed:

not specified

Conclusions:

The high dose of 1000 mg/kg/day resulted in the death or moribund state of all rats. Clinical signs shown by these animals prioe to death/sacrifice included lethargy, hunched posture, uncoordinated movements, abnormail gait, salivation, emaciation, laboured respiration, swelling of the abdomen or head and piloerection. Examination revealed liver enlargement and hepatocyte hyperthrophy, liver necrosis, hyaline droplets formation in the renal papillary collecting ducts, adrenal gland enlargement with fatty change or cortical hypertrophy and reduced size and/or arophy of the thymus, spleen and male reproductive organs. At 150 mg/kg/day, post-mortem findings were confined to liver enlargement and hepatocyte hypertrophy, thymic atrophy and lymphocytolysis. Cortical hyaline droplets noted in the kidneys of most rats dosed at 50 mg/kg/day and above were considered to represent alpha-2-microglobulin, a normal protein in male rats that undergoes reabsorption in the proximal cortical tubules. A range of chemicals is known to increase hyaline droplet formation beyond the physiological capacity of the tubular epithelium. This is a specific male rat response and is not observed female rats or higher species of either sex, including humans and is therefore considered to be of no risk to humans.
There were no functional or morphological disturbances supporting the reduced haemoglobin values seen at 50 mg/kg/day. also, this change was
slight and occured in the absence of a clear dose-releated response. The slightly reduced motoric activity shown by males at 50 mg/kg/day occured incidentally and was not supported by other signs of neurotoxicity. Therefore, excluding the prescence of hyaline droplets in male kidneys, a No Observed Adverse Effect Level (NOAEL) 50 mg/kg/day was established.

Executive summary:

A subacute 28-day oral toxicity with BBSA was conducted by oral daily gavage in the rat at dose levels of 0, 50, 150 and 1000 mg/kg bw. The high dose of 1000 mg/kg/day resulted in the death or moribund state of all rats. Clinical signs shown by these animals prior to death/sacrifice included lethargy, hunched posture, uncoordinated movements, abnormail gait, salivation, emaciation, laboured respiration, swelling of the abdomen or head and piloerection. Examination revealed liver enlargement and hepatocyte hyperthrophy, liver necrosis, hyaline droplets formation in the renal papillary collecting ducts, adrenal gland enlargement with fatty change or cortical hypertrophy and reduced size and/or arophy of the thymus, spleen and male reproductive organs. At 150 mg/kg/day, post-mortem findings were confined to liver enlargement and hepatocyte hypertrophy, thymic atrophy and lymphocytolysis. Cortical hyaline droplets noted in the kidneys of most rats dosed at 50 mg/kg/day and above were considered to represent alpha-2-microglobulin,a normal protein in male rats that undergoes reabsorption in the proximal cortical tubules. A range of chemicals is known to increase hyaline droplet formation beyond the physiological capacity of the tubular epithelium. This is a specific male rat response and is not observed female rats or higher species of either sex, including humans and is therefore considered to be of no risk to humans. There were no functional or morphological disturbances supporting the reduced haemoglobin values seen at 50 mg/kg/day. also, this change was slight and occured in the absence of a clear dose-releated response. The slightly reduced motoric activity shown by males at 50 mg/kg/day occured incidentally and was not supported by other signs of neurotoxicity. Therefore, excluding the prescence of hyaline droplets in male kidneys, a No Observed Adverse Effect Level (NOAEL) 50 mg/kg/day was established.

Reference 2

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2018-2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

25 June 2018

Deviations:

yes

Remarks:

no thyroid hormone analysis was performed. Thyroid hormone analysis will be part of the upcoming Extended One Generation Reproductive Toxicity Study.

Principles of method if other than guideline:

- Principle of test:
The objective of this study was to obtain information on toxicity of the test item (N-n-Butylbenzenesulphonamide) when administered by diet to Wistar rats for 90 consecutive days. The study has been designed for the characterization of the test item toxicity, for an indication of the dose relationship and the determination of the No Observed Adverse Effect Level (NOAEL). The dose levels and dietary concentrations were selected by the Sponsor and Study Monitor.
The results of the study will also provide data for dose selection of the upcoming Extended One-Generation Reproductive Toxicity Study.

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 201705080023
- Expiration date of the lot/batch: 08 May 2019
- Purity test date: 99.93%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Controlled room temperature (15-25°C, <70% relative humidity, protected from humidity (stored in a tightly closed container)
- Stability under test conditions: Stability in diet (625 and 12500 mg/kg) at -20°C for 5 months: 104 and 101% (cfr. method validation in diet)
- Solubility and stability of the test substance in the solvent/vehicle: homogenous mixture in diet (cfr. method validation in diet)

Species:

rat

Strain:

Wistar

Remarks:

Crl:WI

Details on species / strain selection:

Wistar rat as a rodent is one of the standard strains for repeat-dose toxicity studies

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH, Sandhofer Weg 7, D-97633, Sulzfeld, Germany
- Females were nulliparous and non-pregnant.
- Age at study initiation: Young adult rats, approx. 7 weeks old at the start of the experiment, approx. 8 weeks old at the start of the treatment. The age range within the study was kept to the minimum practicable; minor variations were acceptable for practical reasons.
- Weight at study initiation: Males: 292-332 g, females: 204-243 g. The actual body weights did not exceed ± 20% of the mean weight for each sex at onset of treatment.
- Fasting period before study:not specified
- Housing: Animal room 523. Cage type: Type II and/or III polycarbonate. Rodents were housed as 2 animals of the same sex and group/cage. Group housing allowed social interaction and the deep wood sawdust bedding allowed digging and other normal rodent activities. Cages were arranged in such a way that possible effects due to cage placement are minimized (cage rotation was performed in every month).
Bedding and nesting: Lignocel® 3/4-S Hygienic Animal Bedding ((Batch Number: 03018180508, Expiry date: 08 May 2021), produced by J. Rettenmaier & Söhne GmbH+Co.KG (Holzmühle 1, D-73494 Rosenberg, Germany), were used for the study. Nest building material (Arbocel® crinklets natural (Batch Number: 05072180406 / 05072180528, Expiry date: 06 April 2021 / 28 May 2021), produced by J. Rettenmaier & Söhne GmbH + Co.KG, Germany, were also added to the cages. Copies of the Certificate of Analysis were filed in the raw data and reported.
- Diet (e.g. ad libitum): The animals received ssniff® SM R/M-Z+H “Complete Diet for Rats and Mice – Breeding and Maintenance” produced by ssniff Spezialdiäten GmbH (Ferdinand-Gabriel-Weg 16, D-59494 Soest, Germany), with or without N-n-Butylbenzenesulphonamide (treated or control groups, respectively) ad libitum. The food was removed overnight prior to necropsy.
- Water (e.g. ad libitum): The animals received tap water from municipal supply, as for human consumption, from 500 mL bottles, ad libitum.
- Acclimation period: At least 12 days. Before the onset of treatment, all animals received the study control diet for 7 days.

DETAILS OF FOOD AND WATER QUALITY:
For contents of the standard diet, see Appendix 2. The supplier provided analytical certificate for each batch used, which will be archived with the study raw data.
Water quality control analysis was performed at least once every three months and microbiological assessment was performed monthly by Veszprém County Institute of State Public Health and Medical Officer Service (ÁNTSZ, H-8201 Veszprém, József A.u.36., Hungary). The quality control results will be archived with the study raw data.
The food and water are considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.0 – 24.7 °C (target range: 22 ± 3°C)
- Humidity (%): 34 – 77% (target range: 30-70%)
- Air changes (per hr): 15-20 air exchanges/hour
- Photoperiod (hrs dark / hrs light) 12 hours light daily, from 6 a.m. to 6 p.m.
Environmental parameters (temperature and relative humidity) were continuously measured, minimum and maximum values were recorded twice a day during the study.

IN-LIFE DATES:
Start of experiment: 07 / 09 August 2018 (males/females)
Start of treatment: 14 / 16 August 2018 (males/females)
End of treatment: 11 / 13 November 2018 (males/females)
Scheduled necropsy: 12 / 14 November 2018 (males/females)
End of experiment: 14 November 2018 (last scheduled necropsy)

Route of administration:

oral: feed

Details on route of administration:

The oral dietary route was selected as it is the most relevant route of human exposure.

Vehicle:

unchanged (no vehicle)

Remarks:

in the diet

Details on oral exposure:

DIET PREPARATION
- Rate of preparation of diet (frequency): Preparation of two lots of diet for each concentration level and control was scheduled. The first lot was used for a suitable period based on extended stability results, the second batch was ordered for safety reason, it was intended to be used only for a very short period, but finally was not used in the study.
- Mixing appropriate amounts with (Type of food):
The test item was incorporated into ssniff® SM R/M-Z+H “Complete Feed for Rats and Mice-maintenance” by ssniff Spezialdiäten GmbH (Address: Ferdinand-Gabriel-Weg 16, D-59494 Soest, Germany) to generate the test concentrations required. The proper performance of the method use for diet preparation was confirmed during the analytical method validation study (Citoxlab study code: 17/150-316ANP).
The pelleting process was considered not to induce an "unmixing" of the diets or particle segregation, and prevented the potential settling out of the fine-heavy particles that could occur in handling/transport of powder diets. The test item was incorporated into the diet and mixed for up to approximately 16 minutes (approximately 8 minutes for premix preparation, and approximately 8 minutes for preparation of the complete diets; minor variations were acceptable as practical).
Water for dampening the diet was used as practical. Following mixing, pellets were prepared by simple compression; no binding agents, steam, external heat, any other process or substance were used that might affect the test item or the quality of the diets. Similar diet preparation procedures were used to generate study control diet (0 mg N-n-Butylbenzenesulphonamide/kg diet).
- Storage temperature of food: The prepared diets were stored at room temperature, in sewed bags pending and during transport to the Test Facility. At Citoxlab Hungary Ltd., the prepared diets were stored also at controlled room temperature. The stability of the test item in the diet has been verified for a period of at least 6 months at room temperature (Citoxlab study code: 17/150-316ANP).

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The concentration and homogeneity of the test item in the diet was determined by using a validated HPLC-UV method four times during the study (three times – once before start of feeding, in the middle and at the end of use for the first batch, and once before use for the second batch (although finally that batch was not used for feeding).
No test item was detected in the control samples at any occasion.
The mean N-n-Butylbenzenesulphonamide concentrations evaluated for the test item containing diet samples were in the range of 93-106 % of the nominal concentration (individual values of the replicate samples varied in the range of 85-112% of the nominal value). These results were within the acceptable range (85% - 115% of the nominal concentration) and were considered to be suitable for the study purposes.
The test item was homogenously distributed in the diet (six representative samples were taken and analysed for each test item containing diet at each occasion). The relative standard deviation (RSD%) of the replicates was in the range 1.7 -5.1%, which met the acceptance criteria of being <10%.
Diets were considered to be adequately stable under the study conditions (test item containing diet samples were shown to have 6 months of stability when stored at room temperature, the batches used in the study were stored under adequate conditions and used within 4 months after preparation).

Duration of treatment / exposure:

A constant concentration of test item in diet was administered to treated groups for a 90-day treatment period.

Frequency of treatment:

continuous in the diet

Dose / conc.:

0 ppm

Remarks:

in the diet (control)

Dose / conc.:

700 ppm

Remarks:

in the diet; mean achieved dose level (combined for males and females) was 51.9 mg/kg bw/day

Dose / conc.:

1 750 ppm

Remarks:

in the diet; mean achieved dose level (combined for males and females) was 132.5 mg/kg bw/day

Dose / conc.:

3 750 ppm

Remarks:

in the diet; mean achieved dose level (combined for males and females) was 265.4 mg/kg bw/day

No. of animals per sex per dose:

10

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: The dietary concentrations were set by the Sponsor and Study Monitor, based on the available data and information from previous experimental work, including the results of a 14-day Dose Range Finding and palatability study (Citoxlab study code 17/150-031P). The aim was to induce toxic effects but ideally no death or suffering at the highest dose, and a NOAEL at the lowest dose.
- Rationale for animal assignment (if not random): At the end of the acclimatisation period, the animals were assigned to their respective dose groups by randomisation based on body weights. Animals were randomly allocated to the control and dose groups based on the most recent actual body weight; PROVANTIS v.9 software was used in order to verify homogeneity/variation among/within groups. Males and females were randomised separately. The software checking process was run additionally when the 7-day study control diet feeding period was ended (before the start of the test item treatment).
- Fasting period before blood sampling for clinical biochemistry: Blood samples for clinical pathology evaluation (haematology, coagulation and clinical chemistry) were collected immediately prior to the scheduled necropsy, by heart puncture under pentobarbital anaesthesia on Day 90. After an overnight period of food deprivation of animals, 3 blood samples were collected by heart puncture under pentobarbital anaesthesia, for haematology (in tubes with K3-EDTA as anticoagulant, 1.6 mg/mL blood), for blood clotting times (in tubes with sodium citrate as anticoagulant) and one to obtain serum (in tubes with no anticoagulant) for clinical chemistry.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule:
Animals were inspected for signs of morbidity and mortality twice daily (at the beginning and end of each day). The principles and criteria summarized in the OECD Humane Endpoints Guidance Document No. 19 were taken into consideration.
General (routine) clinical observations were made at least once a day at approximately the same time with minor variations as practical. No general clinical observations were made on those days when detailed clinical observations were made.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Detailed clinical observations were made on all animals outside the home cage in a standard arena at the start of the study, prior to the first treatment (to allow for within-subject comparisons), weekly thereafter and on necropsy day. Those observations were conducted in the morning hours (am).
Observation were performed on the skin, fur, eyes, eyeballs and mucous membranes, autonomic activity (lachrymation, piloerection, pupil size, respiratory pattern, occurrence of secretions and excretions), circulatory and central nervous system (tremor, convulsion, muscular contractions, etc.), somatomotor activity and behaviour pattern (changes in exploratory behaviour, ordinary behaviour including changes in grooming, headshaking, gyration, etc., abnormal behaviour such as autophagia/self-mutilation, backward motion, abnormal vocalization, etc., aggression, etc.), motor coordination, ambulatory abnormalities, changes in body position and posture (hunchback posture, etc.), gait, posture and response to handling and to environmental stimulation. Particular attention was directed to observation of tremors, convulsions, salivation, diarrhoea, lethargy, sleep and coma.

BODY WEIGHT: Yes
- Time schedule for examinations: Body weights were recorded with a precision of 1 g at start of the control diet feeding (Day -7), on the first day of treatment (Day 0, prior to start of treatment), then at least weekly, on Day 89 and also prior to necropsy (fasted if scheduled euthanasia).
Body weights and feed (food) intake was at least once a week* recorded simultaneous on the same day to generate more precise dose intake data.
*Note: Animals were measured usually once per week. However, in case of females, an extra measurement was inserted on Day 87 after a sudden drop was noted on their body weight profile (all groups including control) on Day 84, during the period when FOB and locomotor activity measurements were carried out. It was concluded that there was no issue of concern, so subsequent body weights were weekly as scheduled.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
Food consumption was measured with a precision of 1 g twice during the study control diet feeding period (Day -7 and Day -3), before the start of treatment (Day 0), then at least twice weekly during the treatment period. Food hoppers were replenished more frequently if required.
Animal food consumption per cage was measured at least twice weekly and the mean weekly food consumption and daily feed intake per rat were calculated. Based on food consumption data, the mean test item intake of each group was calculated for reporting purposes on the basis of mg/kg body weight/day, in addition food conversion efficiency (g/g) was calculated as [weekly body weight gain (g)/weekly food consumption (g)]. The data are reported on a cage basis (two animals per cage, hence n=5 per sex/group).

WATER CONSUMPTION AND COMPOUND INTAKE
- Time schedule for examinations: As agreed by the Sponsor, water consumption was measured daily for a short period during the study (Days 42-45 for males and Days 40-43 for females) for animal welfare reason (to monitor the water consumption of the animals as marked body weight loss was detected in the High dose group). It was performed by measuring the weight of the water bottle before and after use. The actual volume of the consumed water was calculated using the density of water.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Ophthalmoscopic examination was conducted in all animals before the start of the treatment (on Day -7), and in the Control (Group 1) and High dose (Group 4) animals during Week 13 (on Day 84). As no treatment related alterations were found, no additional animals of the Mid and Low dose groups were examined.
Mydriasis was produced after instillation of “Cicloplegicedol” (10 mg/mL cyclopentolate hydrochloride, Lot No: 170966, Expiry date: 20 September 2022) eye drops into the conjunctival sac. The evaluation was performed by external examination using an OMEGA 500 (Manufacturer: Heine) or WelchAllyn®TM ophthalmoscope.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: At the end of the treatment period, prior to scheduled necropsy on Day 90.
- Anaesthetic used for blood collection: Yes. 3 Blood samples were collected by heart puncture under pentobarbital anaesthesia: for haematology (in tubes with K3-EDTA as anticoagulant, 1.6 mg/mL blood), for blood clotting times (in tubes with sodium citrate as anticoagulant) and one to obtain serum (in tubes with no anticoagulant) for clinical chemistry.
- Animals fasted: Yes overnight
- How many animals: In all animals. Blood smears were also prepared for all animals but not examined, as the minor changes in the haematology parameters were considered to be of no toxicological significance.
- Parameters examined:
RBC Red Blood Cell (erythrocyte) count
WBC White Blood Cell (leukocyte) count
Hgb Haemoglobin concentration
Hct Haematocrit (relative volume of erythrocytes)
MCV Mean Corpuscular (erythrocyte) Volume
MCH Mean Corpuscular (erythrocyte) Haemoglobin,
MCHC Mean Corpuscular (erythrocyte) Haemoglobin Concentration
RDW Red Cell (erythrocyte) volume
Plt Platelet (thrombocyte) count
MPV Mean Platelet Thrombocyte volume
RETIC % Reticulocyte count
NE % Neutrophil
LY % Lymphocyte
MO % Monocyte
BA % Basophil
EO % Eosinophil
LUC % Large Unstained Cells
APTT Activated Partial Thromboplastin Time
PTT Prothrombin Time

CLINICAL CHEMISTRY: Yes.
- Time schedule for collection of blood: At the end of the treatment period, prior to scheduled necropsy on Day 90.
- Animals fasted: Yes overnight
- How many animals: all surviving animals
- Parameters examined.:
Glucose Blood sugar concentration
T-BIL Total Bilirubin concentration
Urea Urea concentration
Chol. Cholesterol concentration
HDL / High Density Lipoprotein
LDL / Low Density Lipoprotein
Creat. Creatinine concentration
Phos. Phosphorus concentration
Na+ Sodium concentration
K+ Potassium concentration
Ca++Calcium concentration
Cl- Chloride concentration
Tot. Prot. Total Protein concentration
Alb. Albumin concentration
A/G Alb/glob ration
AST/GOT Aspartate Aminotransferase activity
ALT/GPT Alanine Aminotransferase activity
ALKP Alkaline Phosphatase activity
GGT Gamma-Glutamyl transferase activity
Bile acids

URINALYSIS: Yes
- Time schedule for collection of urine: At the end of the treatment period, prior to scheduled necropsy on Day 90..
- Metabolism cages used for collection of urine: Yes. Urine samples were collected for approximately 16 hours, during an overnight period of food deprivation after the end of the treatment period by placing the animals in individual metabolic cages. The evaluation of the urine samples was performed by observation (e.g. appearance, colour) or test strips as applicable
- Animals fasted: Yes, urine collection during an overnight period of food deprivation
- Parameters examined:
LEU / Leukocyte
NIT / Nitrite
pH
PRO / Protein
GLU / Glucose
UBG / Urobilinogen
BIL / Bilirubin
KET / Ketones
ERY / Erythrocytes (blood)
SG / Specific Gravity
SED / Sediment
VOL / Volume
Colour / Appearance

NEUROBEHAVIOURAL EXAMINATION: Yes, Functional Observation Battery and SMART
- Time schedule for examinations:
During Week 13, each animal was subjected to the functional observation battery, including measurements of the landing foot splay, fore/hind grip strength and motor activity assessment.
Sensory reactivity to different type of stimuli (e.g. auditory, visual and proprioceptive), assessment of grip strength and motor activity were conducted and the general physical condition and behaviour of animals were tested.
A modified Irwin test was performed on Day 83 as detailed assessment for neurotoxicity effects. Parameters such as, but not limited to body position, locomotor activity, respiration rate, respiration type, piloerection, head searching, compulsive biting or licking, circling, upright walking, retropulsion, jumping, exophthalmos, twitches, clonic convulsions, tonic convulsions, tremor, startle, transfer arousal, spatial locomotion, gait, posture, limb position, finger approach, finger withdrawal, touch escape response, diarrhoea, diuresis, visual placing, grip strength, body tone, corneal reflex, pinna reflex, toe pinch, grasping reflex, positional struggle, skin, mucous membrane colour, salivation, palpebral closure, lachrymation, limb tone, abdominal tone, tail pinch, righting reflex, and/or vocalisation were evaluated.
To measure the landing foot splay, the fore/hind paws of the rat were painted with ink and the rat was dropped from a horizontal position onto the appropriate record sheet covering the examination table. The distance between the two resulting ink spots of the hind limbs was measured at the first instance. Based on the results, the distance of the two forelimbs was also measured as it was deemed necessary by the Study Director.
Fore/hind grip strength was measured using a grip strength meter (Model GS3, Bioseb, Chaville, France), an instrument designed to objectively quantify rodent muscular strength, in order to identify and assess quantitatively any potential effect of test item. The rats were held appropriately such that the fore limbs were allowed to grip the support bar and gently pulled back until they release the bar; the device measured the maximum grip strength. This step was performed 3 times for each animal on the test day. The procedure was repeated with the hind limbs with the appropriate grip support. The results were tabulated with individual and mean data.
Motor activity assessment was conducted* using Automatic Monitoring System of rat locomotor activity SMART v. 2.5 (Harvard Apparatus, Germany). Locomotor activity was monitored by placing each animal individually into an open-field (45 x 45 cm, by 40 cm high) for 1-hour observation time. A continuous DVD recording was made for a duration of 60 minutes, under dim-light and undisturbed conditions. The DVD was analysed with “SMART” software after all recordings are made to produce the appropriate parameters. Data from all dose groups and the control group were evaluated for distance travelled in twelve 5-minute segments. The data from the 5-minute segments were presented graphically with the intention of showing initial exploring activity then plateau activity in controls, and comparing the treatment groups.
*Note: During the scheduled measurement, two High dose females (#4503 and #4508) jumped over the wall of the open-field area, thus the recording could not be evaluated (nor for the Control, Low dose or Mid dose animals of the same table ). The measurements for all animals were repeated to obtain analysable data. Female #4503 did not produce analysable data even after three attempts (the animal repeatedly jumped out of the dedicated area). Based on the observed results, this fact did not adversely affect the results or integrity of the study.
- Dose groups that were examined: each animal was subjected to the functional observation battery.
- Battery of functions tested: sensory activity / grip strength / motor activity / other: landing foot splay, modified Irwin test

IMMUNOLOGY: No
- Immunological evaluation however was included in hematological evaluation and macroscopy & microscopy of immunological tissues.

OTHER:
-Examination of vaginal smears:
Prior to necropsy, the oestrus cycle of all females was determined by taking vaginal smears, which was prepared and stained with 1% aqueous methylene blue solution. The smears were examined with a light microscope, in order to provide information regarding the stage of oestrus cycle at the time of sacrifice and assist in histological evaluation of oestrogen sensitive tissues.
-Other measurements:
In order to provide adequate data, other measurement like sperm analysis was conducted as requested by the Sponsor.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
Necropsy and macroscopic examination (gross necropsy) was performed on all animals at the end of treatment period, on Day 90 (after the sample collection for clinical pathology evaluation). All animals were euthanized under pentobarbital anaesthesia by exsanguination.
-Macroscopic evaluation: After exsanguination, the external appearance was examined, cranium, thoracic and abdominal cavities were opened and the appearance of the tissues and organs was observed macroscopically. Any abnormality was recorded with details of the location, colour, shape and size, as appropriate.
In addition, bone marrow smears from the femur of each animal was prepared at necropsy. The smears were fixed, then stained, but not analysed. They were stored at room temperature and will be archived for later possible analysis.
-Organ weight measurements: The following organs were weighed in all animals:
With a preecision of at least 0.01g: Brain, Epididymides, Heart, Kidneys, Liver, Prostate, Seminal vesicles with coagulating glands, Spleen, Testes, Thymus, Uterus including cervix.
With a preecision of at least 0.001g: Adrenal glands, Ovaries, Thyroid with parathyroid glands, Pituitary.
Paired organs were weighed together (except of testis and epididymis, where individual weights were measured and paired weights were calculated and reported in the summary tables). Absolute organ weights were measured, and relative organ weights to the body and brain weights were calculated and reported.

SPERM ANALYSIS: Yes.
At the scheduled termination, after the organ-weight measurement of testis and epididymis, one of each organ was collected for sperm analysis as described below.
First, an approx. 0.5 cm piece of ductus epididymis of each male rat was collected into sterile plastic centrifuge tubes containing PBS (phosphate buffered saline) + 10% (v/v) FBS (Foetal Bovine Serum).
Sperm motility (from the cauda epididymis) was evaluated immediately after sacrifice. The percentage of progressively motile sperms was determined subjectively for 200 sperms for each male of each test group.
For measurement of sperm morphology (from the cauda epididymis), at least 200 sperm per sample (each male from each test group) were evaluated immediately after sacrifice from wet preparations and classified as either normal or abnormal (fusion, isolated heads, misshapen heads and/or tails). At the same time, smears for morphology were prepared and fixed, and stored at room temperature for later additional analysis (the fixed slides were retained).
Testes and remaining part of epididymides were preserved (frozen) for counting. Enumeration of homogenisation-resistant testicular spermatids and cauda epididymal sperm reserves was performed after the in life phase ended. Analysis was made on these parameters for the Control and High dose males then the Mid and Low dose groups.

HISTOPATHOLOGY: Yes
On completion of the macroscopic examination the following tissues and organs were retained from all animals:
Gross findings, Adrenals, Animal identification (1), Aorta (2), Brain (3), Epididymis, Eye with the optic nerve (4), Oesophagus, Femur with marrow, Heart (5), Kidney, Large intestine (6), Extraorbital lachrymal gland, Harderian gland, Liver (7), Lungs with bronchi (8), Lymph node (9), Ovary, Oviduct, Pancreas, Pituitary, Prostate, Salivary gland (including mandibular, sublingual and parotid glands), Sciatic nerve, Seminal vesicle with coagulating gland, Skin, subcutis with mammary gland (inguinal), Skeletal muscle (quadriceps), Small intestine (10), Spinal cord (11), Spleen, Sternum with marrow, Stomach, Testis, Thymus, Thyroid with parathyroid gland (4), Tongue, Trachea, Urinary bladder, Uterus (12), Vagina.
(1) Fixation and preservation only.
(2) Aorta thoracic and abdominal.
(3) 7 sections according to the STP recommendations.
(4) If applicable, parathyroids and optic nerves will be examined histologically only if present in routine sections.
(5) Section including both ventricles and atria, septum with papillary muscle.
(6) Caecum, colon and rectum.
(7) Liver: 3 lobes, left lateral, right medial, caudate
(8) Lungs of euthanized animals were infused with formalin; 3 lobes, left, right cranial, right caudal.
(9) Mandibular and mesenteric.
(10) Duodenum, ileum and jejunum with Peyer’s patches.
(11) Transverse sections, 3 levels -cervical, thoracic and lumbar.
(12) Horns, body and cervix.
The eyes with the optic nerves and testes with epididymides were retained in modified Davidson’s fixative, all other organs in 10% buffered formalin solution.

Full histopathology was performed in all animals assigned to Control (Group 1) and High dose (Group 4) groups, as well as livers (as target organ) of all animals. Histopathology evaluation of any macroscopic lesion was also performed. The standard stain kidney samples of all remaining Mid and Low dose males were analysed additionally. All male kidneys were further examined after special staining (see 4.6.7. below). Furthermore, the adrenals and thymus of all Mid and Low dose animals (males and females) were evaluated additionally.For standard histopathology evaluation, the retained tissues and organs were embedded in paraffin wax, sections were cut at 4-6 µm by microtome and transferred to slides. Tissue sections were stained with haematoxylin-eosin/phloxine and examined by light microscope by the Study Pathologist.

Immunohistochemistry (IHC) staining of kidney samples for alpha 2-microglobulin in all male rats were performed and slides evaluated at a designated Test Site (Citoxlab North America) to provide more data for study interpretation.
Kidney samples of all male animals of the study (a total of 40 samples) were trimmed, embedded in paraffin wax, sectioned at a thickness of approximately 4 microns, and slides (two replicates per sample; total number of slides = 80) were sent to the Test Site to the attention of:
Johan Ferrari
Scientist I, Immunohistochemistry, Pathology
Citoxlab North America
445 Boul. Armand-Frappier, Laval (Quebec) Canada H7V 4B3
Phone: [phone] ext. 2066
E-mail: [e-mail]
At the Test Site, slides of all male rats (a total of 40 samples) were stained for alpha 2-microglobulin. Due to an inconsistency of the staining those slides could not be evaluated, therefore in a second shipment blocks of all males (a total of 40 samples) were sent to the Test Site. Slides from the blocks were generated at the Test Site stained for alpha 2-microglobulin and evaluated.Haematoxylin and Eosin (H&E) slides of the kidneys of the same animals were also sent to the Citoxlab North America for reference purposes only and will be returned after completion of the IHC phase.The phase conducted at the Test Site was performed under the control of the Principal Investigator in accordance with the Study Plan, its amendments and the relevant SOPs of the Test Site

Statistics:

See under "Any other information on material and methods incl. tables".

Clinical signs:

no effects observed

Description (incidence and severity):

No morbidity was observed and no clinical signs were recorded during the study.

Mortality:

no mortality observed

Description (incidence):

No mortality was observed during the study.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Test item related adverse effect was noted on body weight and/or body weight gain in the animals of the High and Mid dose groups, no such an effect was observed in the Low dose group.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

The food consumption was significantly reduced by the test item administration in the High dose group, in the Mid dose group a transient effect in the first one or two weeks was seen; there were no food intake effects in the Low dose group.
The test item intake values (mg/kg bw/day) were calculated from the weekly individual body weight, weekly mean food intake and nominal diet concentration. The mean achieved dose levels (combined for males and females) were 265.4, 132.5 and 51.9 mg/kg bw/day in the High (3750 ppm), Mid (1750 ppm) and Low (700 ppm) dose groups, respectively. For all dose groups, the target dose levels were close to the actual achieved test item intake (within 6.5% for the combined result in all groups).

Food efficiency:

effects observed, treatment-related

Description (incidence and severity):

The food efficiency was significantly reduced by the test item administration in the Mid and High dose group both in male and female animals.

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

no effects observed

Description (incidence and severity):

There was no test item related effect on the water consumption identified in any dose group. The water consumption of all animals was measured for a short period of 4 days about Week 7. After the water consumption of the High dose animals was proven to be the same as controls, the water consumption measurement was not continued for the rest of the in life phase.

Ophthalmological findings:

no effects observed

Description (incidence and severity):

No test item related changes were detected in the ophthalmology evaluation during the study

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

There were signs of slight anaemia in the High dose groups (up to 10% lower RBC, HCT and haemoglobin concentration) with a similar trend in the Mid dose. These changes in both groups were considered to be minor but adverse. There was no effect of the test item on blood clotting parameters in any dose groups.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no significant changes or adverse effects on the serum chemistry that could be clearly ascribed to the test item administration. Increased bile acid values were recorded in High and Mid dose males and High and Mid dose females (by 42% and 31% in males and by 47% and 23% in females, respectively). The difference compared to control gained statistical significance (at p<0.01 level) in case of High and Mid dose males and High dose females. The observed mean values in the High and Mid dose groups were right in the middle of the historical control range. However, effect relationship to the treatment is uncertain.

Urinalysis findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no adverse test item related effects noted during urine analysis.

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

There were no toxicologically significant changes in the animal behaviour, general physical condition, or in the reactions to different type of stimuli in the test item treated groups when compared to control at the evaluation performed on Week 13.

Immunological findings:

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Compared to control, test item related increase in kidney weights (absolute and body related) were detected for High and Mid dose males, while no effect was noted in the Low dose males or the females.
Increased adrenal weights were also noted for High dose males, but without any histopathology change.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Test item related effects (bilateral enlargement of kidney) were noted in High and Mid dose males (3750 and 1750 ppm, respectively) at necropsy. No such a finding was observed in Low dose males (700 ppm) or in any test item treated females.
Test item related liver findings (many dark red focus on all liver lobes) were noted in some High and Mid dose animals, but no Low dose animals were affected.

Neuropathological findings:

no effects observed

Description (incidence and severity):

There was no indication of test item related effects on the neurological assessment in any test item treated dose groups.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Test item related alpha 2-microglobulin nephropathy was observed in male rats only (accumulation of cortical tubules hyaline droplets, focal/multifocal tubular basophilia, granular or hyaline casts and chronic progressive nephropathy). The kidney of High and Mid dose were all affected, 2/10 Low dose had some evidence of change (hyaline droplets). The observed microscopic changes were confirmed by special immunohistochemistry staining. These changes are not considered relevant for humans.
Test item-related focal/multifocal hepatocellular necrosis was noted during microscopic evaluation in the liver of High (1/10)and Mid dose males (4/10) as well as in High dose females (1/10). These changes were correlated with necropsy findings in most of the affected animals. ). In the affected animals, slight increase in AST aspartate Aminotransferase) and ALT (Alanine Aminotransferase) activities were observed, this fact is in line with the scientific literature. The difference was statistically significant (at p<0.05) when the resulted values of the subgroup of the Mid dose males (animals with confirmed hepatocellular necrosis) were compared to the Control males. Despite the low incidence it was considered as adverse change.
No test item related effect was observed in the adrenals and thymus of male and female animals in any dose group.

Histopathological findings: neoplastic:

no effects observed

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

It was concluded that there were no treatment-related effects at any dose levels on sperm morphology or enumeration (a statistically lower sperm motility in the High dose group was not ascribed to treatment).

Details on results:

No mortality was observed during the study.

No clinical signs were recorded during the study; all animals were symptom free during the entire observation period.

Test item related adverse effect was noted on body weight and/or body weight gain in the animals (males and females) of the High and Mid dose groups (3750 and 1750 ppm, respectively); no such an effect was observed in the Low dose group (700 ppm). More detailed results were provided under 'Attached Background Material', and attached full study report.
In the pre-treatment period when study control diet was supplied, the body weight profile of all males and females was similar. Reduced body weights were recorded in High dose males and females and in Mid dose females when compared to control throughout the entire study: the reduction on Day 89 was 15.0% in case of High dose males and females (statistically significant at p<0.01) and 9.2% in case of Mid dose females (statistically significant at p<0.05).The weight gain difference was statistically significant for Mid and High dose males in the first week, thereafter although usually lower the difference to controls was only sporadically statistically significant. In females during the first week the High dose lost about 3% of their body weight, and the Mid and Low groups had slightly lower (p<0.05) gains than controls; thereafter there were no differences in weekly body weight gain between groups. The overall body weight gain (D0-89) was statistically significantly lower (at p<0.01 or p<0.05) in High and Mid dose animals respectively when compared to control (decrease of 32.0% and 16.2% and in High and Mid dose males; the decreases were 23.3% and 45.1% (p<0.01) in High and Mid dose females, respectively, relative to controls).
No clearly adverse test item related effect was noted on body weight or body weight gain of Low dose animals (males and females). The transient statistical difference in weight gain in the first week was small enough to be considered as not adverse.

The food consumption was significantly reduced by the test item administration in the High dose (3750 ppm) males and females in most weeks. A transient initial effect was seen in the Mid dose group (1750 ppm, males and females). The food consumption was not affected by the test item administration in the Low dose group (700 ppm). More detailed results were provided under 'Attached Background Material', and attached full study report.
In the pre-treatment period when study control diet was supplied, the food consumption of all males and females was similar (with occasional statistical difference (p<0.05) in Mid dose males when compared to control). At the beginning of the treatment (Days 0-7), significantly reduced food consumption was noted in High and Mid dose animals (males and females) when compared to control (the difference from control value was approximately 31% in High dose males and females, 9% in Mid dose males and 12% in Mid dose females, respectively; all those cases were statistically significant at p<0.01).
When calculated for the entire treatment period (Days 0-89), reduced daily food consumption was observed in High dose males and females compared to control animals (the difference of approx. 16% was statistically significant at p<0.01 in both cases). No toxicologically significant variations were recorded in the Mid and Low dose groups (the interim drop in the first week of the treatment in Mid dose males and females was fully recovered later).

The test item intake values (mg/kg bw/day) were calculated from the weekly individual body weight, weekly mean food intake and nominal diet concentration. The mean achieved dose levels (combined for males and females) were 265.4, 132.5 and 51.9 mg/kg bw/day in the High (3750 ppm), Mid (1750 ppm) and Low (700 ppm) dose groups, respectively. The mean achieved dose levels fwere 248.38, 122.91 and 47.57 mg/kg bw/day in males and 282.37, 142.01 and 56.20 mg/kg bw/day in females of the High (3750 ppm), Mid (1750 ppm) and Low (700 ppm) dose groups, respectively. For all dose groups, the target dose levels were close to the actual achieved test item intake (within 6.5% for the combined result in all groups). More detailed results were provided under 'Attached Background Material', and attached full study report.

There was no test item related effect on the water consumption during the short 4-day period it was investigated. More detailed results were provided under 'Attached Background Material'.
The daily water consumption of the High, Mid and Low dose animals (3750, 1750 and 700 ppm, respectively) was not affected by the test item treatment when compared to the Control group. Minor changes without statistical significance and/or without dose response were considered as toxicologically not relevant.

There was no indication of any test item related effects on the neurological assessment in any test item treated dose group.
There were no toxicologically significant changes in the animal behaviour, general physical condition, or in the reactions to different type of stimuli in the test item treated groups when compared to control at the evaluation performed on Week 13.
When compared to the control, statistically lower values were noted in grip strength tests for forelimb in High dose females (3750 ppm). The difference compared to control (-12.3%) was statistically significant (p<0.05). However, the observed mean value was well within the historical control range, and an opposite trend (increase of 17.3%) was noted for the same animals for the hind limb. Taking into account historic data, this statistical decrease was considered as biologically not relevant, and not being a test item related effect. No similar changes were observed in the Mid or Low dose groups (1750 and 700 ppm, respectively). More detailed results were provided under 'Attached Background Material', and attached full study report.
In the landing foot splay test, statistically significantly (p<0.05) reduced values (by 30-35%) were recorded in the hind limbs of High dose males and females compared to control. However, the observed values were within the historical control range (although close to the lower limit), furthermore in case of a test item related adverse effect, higher values would have been recorded. Therefore, these differences were not considered as a test item related effect. No similar trends were observed in Mid and Low dose groups. No similar significant differences to control were noted in the forelimbs of any of the test item treated dose groups. More detailed results were provided under 'Attached Background Material', and attached full study report.
No toxicologically relevant changes were detected in the locomotor activity of the test item treated groups when compared to the control animals. Minor differences were noted in some cases, but there was no statistical significance and no dose response, thus the observed differences were concluded as not being a test item related effect.

No test item related ophtalmic changes were detected during the study.
No observations were found during ophthalmoscopy in the animals of the Control and High dose groups at the start (Day -7) and at end of the study (Day 84).

No major toxicologically important changes in the haematology parameters were concluded in any dose groups, although signs of slight anaemia were noted in High and Mid dose groups (3750 and 1750 ppm, respectively). There was no adverse effect of the test item on blood clotting parameters in any dose group.
Signs of slight anaemia (decreased (by 4-10%) red blood cell count, haemoglobin concentrations and haematocrit values) were noted in High dose (3750 ppm) males and females, and to a lesser extent in Mid dose males and females. The differences were statistically significant (generally at p<0.05 in the Mid and p<0.01 in the High dose group) when compared to control. Statistically significant decrease (p<0.05) in red blood cell distribution width was also observed for High dose males. The differences were considered to be treatment related but relatively minor, all the mean values were within the historical control range. Therefore, these changes in both dose groups were considered to be minor but adverse.
No relevant statistically significant changes were recorded for any other haematology parameters in the test item treated males and females (sporadic statistical differences were not considered to be treatment related). Statistical significances (p<0.05 or p<0.01) in the relative monocytes in males of all dose groups was considered incidental, due to the lack of dose response, being well within the historic range and lack of similar trend in females.
As for coagulation, statistically significant decrease (p<0.01) in prothrombin time (PTT) was detected in High dose animals (males and females) and in Mid dose males compared to control animals. However, the differences were less than 5% and all the individual and mean values were within the historical control range. Furthermore, no dose response was seen in the males. Therefore, these differences observed between the control and treated groups for PTT were considered to be incidental findings, which were not related to test item treatment. More detailed results were provided under 'Attached Background Material', and attached full study report.

There were no significant adverse effects on the serum chemistry that could be ascribed to the test item administration. More detailed results were provided under 'Attached Background Material', and attached full study report.
When compared to the controls, the following tendency was observed after 90 days of treatment: sodium and chloride concentration was slightly higher than control in High dose males, while calcium was slightly higher than control in High dose females (the differences gained statistical significance at p<0.01 in case of sodium and chloride, and at p<0.05 in case of calcium). However, the differences were minor (in the range of
1.6-3.7%), the observed values were within the historical control range and there were no similar trends in the other sex. Therefore, these differences were considered as biological variability, not related to the test item administration.
Increased bile acid values were recorded in High and Mid dose males and High and Mid dose females (by 42% and 31% in males and by 47% and 23% in females, respectively). The difference compared to control gained statistical significance (at p<0.01 level) in case of High and Mid dose males and High dose females. The observed mean values in the High and Mid dose groups were right in the middle of the historical control range. However relationship to the treatment is uncertain.
Statistically significant decrease in triglyceride concentration was noted in High and Mid dose males compared to control (by 53% at p<0.01 and by 42% at p<0.05, respectively). However, the observed values were within the historical control range and opposite trend was noted in female animals (by 19% increase in the High dose group, without statistical significance). Therefore, those values were considered to have no biological relevance and not being a test item related effect.
Statistically significant increase (at p<0.01) was observed in cholesterol concentration in High dose females (by 77%), the observed mean values was slightly higher than the upper limit of the historical control range. However, no similar trend (slightly increased values without dose response and without statistical significance) were noted in male animals, thus the toxicological significance of this difference is unclear.
Statistically significant increase (at p<0.01) was observed in HDL concentration in High dose females (by 53%) when compared to the control group. However, no similar trend was noted in males, furthermore for this parameter the decreased values have biological relevance, thus this increase was considered as having no toxicological relevance, and not being a test item related adverse effect.
No statistically significant and biologically relevant changes were recorded for any other parameters in the test item treated males and females*.
*Note: In case of GGT (Gamma Glutamyltransferase) activity, all the values were below the Limit of Quantification (LOQ = 5 U/L), the LOQ value was used for tabulating the mean value for this parameter. Similarly, in case of some individual total bilirubin and cholesterol values below the LOQ, the LOQ value was used for calculation of the mean value (LOQ = 1.7 µmol/L for total bilirubin and LOQ = 1.16 mmol/L for cholesterol).

No test item related effects were noted in any dose groups on the urinalysis parameters evaluated.
The statistically significant increases in the urinary volume of High, Mid and Low dose males compared to the controls were considered as unrelated to the treatment due to the lack of dose response and as no similar changes were observed in test item treated females. Furthermore, all the observed individual values were in the historical control range.
Higher amounts of urinary bacteria were presented in the samples of the High dose males, but no similar trend was observed in the High dose females. This fact might be related to the progressive neuropathy seen in the kidney of High dose males.

Compared to control, test item related increase in kidney weights (absolute and body related) were detected for High and Mid dose males (3750 and 1750 respectively), while no effect was noted in the Low dose males (700 ppm). Increased adrenal weights were also noted for High dose males. More detailed results were provided under 'Attached Background Material'., and attached full study report.
Terminal body weights of the High dose animals were significantly decreased (by 16.1% in males and 16.3% in females). Terminal body weights of the Mid dose females were also significantly decreased (by 10.0%). Consequently, significant changes in several body related weight of High dose animals and Mid dose females were seen, but those differences had no toxicological significance unless the absolute or brain related weight showed confirmatory significant differences.
Statistically significant (p<0.01) increases in the absolute and body related kidney weights of High and Mid dose males compared to control were noted (by 29% and 18% in High and Mid dose absolute weights, respectively). The brain related weight was also significantly (p<0.01) increased in High dose males (by 29%). Although no similar finding was seen in females, those differences were considered as being a test item related effect and were in line with the presence of hyaline droplets in kidneys noted during histopathology evaluation.
Statistically significant (p<0.01) increase in the absolute and body/brain related adrenal glands weights of High dose males compared to control were noted (by 26% in case of absolute weights). Similar trend was noted in High dose females (by 10%) although no statistical significance in the absolute weight was gained. The observed values were within the historical control range and there were no associated histopathology changes in the adrenals*. Therefore, the weight change was considered as probably having no toxicological relevance not to reflect a significant adverse effect although it may be treatment related in the High dose males.
*Note: 5/10 High dose males had hypertrophy noted in the adrenals, but there was no correlation between these 5 cases and their individual adrenal weights.
Statistically significant decrease (by 25%, significant at p<0.01) in absolute thymus weight was noted in High dose males and statistically significant decrease (by 5%, significant at p<0.05) in absolute brain weight of High dose females were recorded. However, as no statistically significant differences and/or no dose response were seen in the other sex in both cases, the mean values were within the historical control range and there were no histopathological findings, those changes were considered as not related to the test item.

Test item related effects (bilateral enlargement of kidney) were noted in High and Mid dose males (3750 and 1750 ppm, respectively) at necropsy. No such a finding was observed in Low dose males (700 ppm) or in any test item treated females. This correlated with histopathology findings. More detailed results were provided under 'Attached Background Material', and attached full study report.
Test item related liver findings (many dark red focus on all liver lobes) were noted in some High and Mid dose animals, but no Low dose animals were affected. This correlated with histopathology findings.
Bilateral enlargement of kidney was recorded in 7/10 High dose males (#4001, #4002, #4004, #4005, #4006, #4009 and #4010) and in 4/10 Mid dose males (#3003, #3005, #3006 and #3007). No such a finding was observed in Low dose males or in any test item treated females. These findings were considered as being a test item related effect.
Many dark red focus on all liver lobes in 1/10 High dose male (#4003), 1/10 High dose female (#4503) as well as in 2/10 Mid dose males (#3007 and #3010); these findings were considered as attributed to the test item administration.
Other changes such as diffuse bilateral pale discoloration of adrenal glands of one High dose male (#4003); bilateral enlargement of thyroid and parathyroid gland of one High dose male (#4001) and one Mid dose male (#3003); and small soft testis in one Control male (#1010) were considered as incidental or background findings.
Furthermore, the dilatation of uterine body and horns in one Control (#1509), one Low dose (#2508), one Mid dose (#3504) and two High dose (#4504 and #4505) females was considered as a normal physiological change according to the regular oestrus cycle.

The stage of oestrus was determined using the vaginal smears prepared at the time of necropsy. The results did not indicate any test item effect on the oestrus cycle but remained in the normal range of distribution of female animals. More detailed results were provided under 'Attached Background Material', and attached full study report.

Test item related accumulation of hyaline droplets in the cortical tubules, multifocal unilateral/bilateral tubular basophilia, granular or hyaline casts and chronic progressive nephropathy (CPN) were observed in the kidney of test item treated males. Detailed results for kidney were selected in Table 8 below under 'Any other information incl. tables'. Test item-related focal/multifocal hepatocellular necrosis was noted in the liver of High and Mid dose males (3750 and 1750 ppm, respectively) as well as in High dose females. These changes were correlated with necropsy findings in most of the affected animals. More detailed results were provided under 'Attached Background Material' and attached full study report.
Hyaline droplets in the cortical tubules (to be confirmed by special staining), multifocal/focal, unilateral/bilateral tubular basophilia, granular or hyaline casts were found in all High and Mid dose males, plus 2/10 Low dose males. Chronic progressive nephropathy (CPN) was noted in the kidney samples of the all High dose males and 3/10 Mid dose males. The changes in the kidney including hyaline droplet accumulation in the cortical tubules, tubular basophilia, hyaline and granular casts located at the junction with outer and inner stripes of outer medulla, are considered most probably caused by alpha 2-microglobulin, which is a low molecular weight protein produced by the liver and excreted through kidney in male rats only. The spectrum of changes observed in the kidney is considered to be alpha 2-microglobulin nephropathy. CPN is a spontaneous finding which can be exacerbated following hyaline droplet nephropathy, as it was seen in High dose males. Similar changes were not observed in High dose females. Since alpha 2-microglobulin nephropathy can only exist in male rats and is irrelevant for human exposure, all the kidney-related effects, although are adverse for male rats, are not considered to be an adverse effect in the context of a study for human safety. More detailed results were provided under 'Attached Background Material'.,and attached full study report.
Focal/multifocal hepatocellular necrosis were noted in the liver of the High dose female, and High and Mid dose males with low incidence, but was considered as adverse change. The appearance was mild to moderate in Mid dose males (#3003 and #3010: mild; #3005 and 3007: moderate) and High dose female (#4503), and marked in High dosed male (#4003). In the affected animals, slight increase in AST aspartate Aminotransferase) and ALT (Alanine Aminotransferase) activities were observed (as detailed in Table 15), this fact is in line with the scientific literature (Giannini. E., Testa, R., and Savarino, V., “Liver enzyme alteration: a guide for clinicians” CMAJ, 2005 (172 (3), p367-379). The difference was statistically significant (at p<0.05) when the resulted values of the subgroup of the Mid dose males (animals with confirmed hepatocellular necrosis) were compared to the Control males. More detailed results were provided under 'Attached Background Material',and attached full study report.
Other alterations such as cortical vacuolation and hypertrophy of zona glomerulosa in the adrenals; mineralisation and pyelonephritis in the kidney; degeneration/necrosis of cardiomyocytes; Harderian metaplasia in the lacrimal glands; congestion or haemorrhages in the thymus, tubular degeneration/atrophy and tubular dilatation in the testes; reduced sperm content in the epididymis, and oestrus, based on low incidence, the occurrence and/or distribution in control and dosed groups, were considered as incidental or background. More detailed results were provided under 'Attached Background Material',and attached full study report.

Sperm motility and morphology as well as number of sperms were examined in the study for all males. Increased number of immotile sperm cells was noted in the High dose group (3750 ppm), however no clear test item effect could be concluded; the results of the Mid and Low dose males (1750 and 700 ppm, respectively) were comparable with the control level. There was no treatment-related effect at any dose levels on sperm morphology or enumeration. Careful histopathological examination of the testes and epididymis of High dose animals confirmed the lack of an effect on sperm.
In case of sperm motility, the number of non-motile sperm was significantly (p<0.01) increased in the High dose males compared to control, while the observed values in the Mid and Low dose groups were comparable with the control level. However, there was no clear dose response, and approximately 25% of the animals in other recent studies had shown to have a result of similar level, therefore no clear test item related effect can be concluded on this parameter.
There were no biologically relevant changes in sperm morphology. The ratio of abnormal sperm was slightly increased in the High dose group, but without statistical significance. Thus, these values were considered not to be a test item-related effect.
No effect was seen on the number of sperm (either in testis or in cauda epididymis) in the animals of any test item treated dose groups when compared to the control. Careful histopathological examination of the testes and epididymis of High dose males confirmed the lack of an effect on sperm. More detailed results were provided under 'Attached Background Material', and attached full study report.
It is noted that in the subsequent Extended One Generation Reproductive Toxicity Study, this parameter will be examined in detail.

Key result

Dose descriptor:

NOAEL

Effect level:

700 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

food consumption and compound intake

food efficiency

gross pathology

haematology

histopathology: non-neoplastic

organ weights and organ / body weight ratios

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

food consumption and compound intake

food efficiency

gross pathology

haematology

histopathology: non-neoplastic

organ weights and organ / body weight ratios

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

1 750 ppm

System:

haematopoietic

Organ:

blood

liver

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

Study 17/150-316ANP was performed in order to validate the HPLC-UV method for the analysis of N-n-butylbenzenesulphonamide in formulation samples in diet.

The procedure was found to be suitable for the analysis. All validation parameters were within acceptation ranges. Summary of the method parameters is presented in Table 1.

 

Table 1. Results of the Method Validation

Selectivity	No interfering component was observed
Reinjection repeatability (7 injections)	CV% ≤ 0.5%
Linear range	0.5 – 50 µg/mL
Limit of Quantification	0.5 µg/mL
Recovery from diet (625 and 12500 mg/kg)	97% (range: 93-101%)
Inaccuracy in diet	3.0 and 3.7%
Imprecision in diet at low and high concentration	2.4 and 2.8%
Stability in diet (625 and 12500 mg/kg) at room temperature for 6 months	105 109 and 100% versus start
Stability in diet (625 and 12500 mg/kg) at -20^○C for 5 months	104 and 101% versus start
Stability of the samples in the autosampler	At least 29 hours
Stock solution stability at 5±3 °C	At least 2 days

Conclusions:

Under the conditions of this 90-day repeated dose toxicity study, the No-Observed-Adverse-Effect Level (NOAEL) for N-n-Butylbenzenesulphonamide was considered to be the 700 ppm dietary concentration (51.9 mg/kg bw/day).

Executive summary:

The objective of this study was to obtain information on toxicity of the test item (N-n-Butylbenzenesulphonamide) when administered by diet to Wistar rats for 90 consecutive days according to OECD No. 408 guideline.

Ten male and ten female Wistar rats/group were fed by diet containing N-n-Butylbenzenesulphonamide at 3 concentrations (ppm) for 90 consecutive days ad libitum. The control group received study control diet ad libitum. Before the onset of the 90-day treatment period, all animals received the study control diet for 7 days.

 

Group. No.	Group Designation	Target dose level (mg/kg/BW)	Test item concentration in the diet (ppm)	Number of animal (sex/group)	Animal Identity numbers (IDs)
					Males	Females
1	Control	0	0	10	1001 to 1010	1501 to 1510
2	Low dose	50	700	10	2001 to 2010	2501 to 2510
3	Mid dose	125	1750	10	3001 to 3010	3501 to 3510
4	High dose	250	3750	10	4001 to 4010	4501 to 4510

 

The first day of treatment of each animal was regarded as Day 0. All animals underwent necropsy upon completion of the 90-day treatment period (Day 90).

During the study, observations for mortality were performed twice a day, clinical signs and detailed clinical observations were also performed daily and weekly, respectively. Body weight were measured weekly intervals; food consumption measurements were performed in twice per week. Water consumption was measured for a short period (4 days) during the study.Ophthalmoscopy was performed before start and on the last week of the treatment. Functional Observation Battery (FOB) and measurement of locomotor activity was performed on the last week of treatment. Blood and urine samples for Clinical pathology(haematology, clinical chemistry, coagulation and urinalysis)were collected prior to necropsy.Sperm motility and morphology as well as number of sperms were examined in the study for all males.Full histopathology was performed for Group 1 (Control) and Group 4 (High dose) and liver samples (as target organ) of all animals were also histopathologically examined. Furthermore, kidney samples of all Mid and Low dose males were evaluated. Immunohistochemistry analysis foralpha 2-microglobulinfor kidney samples of all males is being performed at the designated Test Site.

Dietary analysis forN-n-Butylbenzenesulphonamideconcentration and homogeneity was performed in the Analytical Laboratory of Citoxlab Hungary Ltd.

 

Results

No test item was detected in the control diet samples. All test item containing diets were shown to be homogeneous. Test item concentration in the individual diet samples were found to be in the range of 93 to 106% of the nominal concentrations.

No morbidity or mortality was observed and no clinical signs were recorded during the study.

Test item related adverse effect was noted on body weight and/or body weight gain in the animals of the High and Mid dose groups, no such an effect was observed in the Low dose group.

The food consumption was significantly reduced by the test item administration in the High dose group, in the Mid dose group a transient effect in the first one or two weeks was seen; there were no food intake effects in the Low dose group. Food efficiency was statistically significantly lower at 1750 and 3750 ppm both in males and females. There was no test item related effect on the water consumption identified in any dose group.

No test item related changes were detected in the ophthalmology evaluation during the study.

There was no indication of test item related effects on the neurological assessment in any test item treated dose groups.

There were signs of slight anaemia in the High dose groups (up to 10% lower RBC, HCT and haemoglobin concentration) with a similar trend in the Mid dose. There was no effect of the test item on blood clotting parameters in any dose groups.

There were no significant changes or adverse effects on the serum chemistry that could be clearly ascribed to the test item administration.

There were no adverse test item related effects noted during urine analysis.

Compared to control, test item related increase in kidney weights (absolute and body related) were detected for High and Mid dose males, while no effect was noted in the Low dose males or the females. The increased kidney size was also seen at necropsy as a macroscopic observation; histopathology changes confirmed the observations. Increased adrenal weights were also noted for High dose males, but without any histopathology change.

Test item related dark red foci on liver lobes were noted in some High and Mid dose animals at necropsy, but no Low dose animals were affected. The change corresponded with histopathology observations of necrosis (mild to moderate at Mid dose males and High dose female; marked in High dose male).

Test item related alpha 2-microglobulin nephropathy was observed in male rats only (accumulation of cortical tubules hyaline droplets, multifocal tubular basophilia, granular or hyaline casts and chronic progressive nephropathy). The kidney of High and Mid dose were all affected, 2/10 Low dose had some evidence of change.

Test item-related focal/multifocal hepatocellular necrosis was noted in the liver of High (1/10) and Mid dose (4/10) males as well as in High dose (1/10) females. These changes were correlated with necropsy findings in most of the affected animals. Despite the low incidence it was considered as adverse change.

No test item related effect was observed in the adrenals and thymus of male and female animals in any dose groups.

It was concluded that there were no treatment-related effects at any dose levels on sperm morphology or enumeration (a statistically lower sperm motility in the High dose group was not ascribed to treatment).

The test item intake values were calculated from the body weight, food intake and nominal diet concentration. The mean achieved dose levels (combined for males and females) were 265.4, 132.5 and 51.9 mg/kg bw/day in the High (3750 ppm), Mid (1750 ppm) and Low (700 ppm) dose groups, respectively.

In conclusion, under the conditions of this 90-day repeated dose toxicity study, the No-Observed-Adverse-Effect Level (NOAEL) for N-n-Butylbenzenesulphonamide was considered to be the 700 ppm dietary concentration (51.9 mg/kg bw/day).

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

51.9 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

Reliable

Organ:

blood

kidney

liver

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: dermal

Type of information:

experimental study

Adequacy of study:

key study

Study period:

27/10/2011-03/01/2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

OECD Guideline 410 (Repeated Dose Dermal Toxicity: 21/28-Day Study)

Principles of method if other than guideline:

/

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River, USA
- Age at study initiation:
- Weight at study initiation: Males: 224-285g, Females: 221-237g
- Fasting period before study: no
- Housing: Standard polypropylene rat cages with stainless steel top grills supplied by M/s. Vishnu Traders, UP, India was used to house the animals. The cages were autoclaved. Gamma irradiated corn cobs supplied by M/s. Ceutics Pharma Pvt. Ltd., Bangalore, India was used as the bedding material.
- Diet (e.g. ad libitum):ad libitum
- Water (e.g. ad libitum):ad libitum
- Acclimation period: Five days prior to the range finding and main study in the test room.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.5-22.9°C
- Humidity (%): 53-65%
- Photoperiod (hrs dark / hrs light): 12hrs dark/12 hrs light

IN-LIFE DATES: From: 17/11/2011 To: 03/01/2012

Type of coverage:

semiocclusive

Vehicle:

unchanged (no vehicle)

Details on exposure:

TEST SITE
- Area of exposure: dorsal area
- % coverage: +/- 10%
- Type of wrap if used: a porous gauze dressing and a non irritating tape
- Time intervals for shavings or clipplings: Initially, 24 hours prior to the first application of the test substance, there after clipping was done at weekly intervals, before 24 hours of the application.

REMOVAL OF TEST SUBSTANCE
- Washing (if done): After the exposure period, the residual test substance was wiped gently from the skin using cotton soaked in water.
- Time after start of exposure: 6h

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): not specified
- Constant volume or concentration used: yes


USE OF RESTRAINERS FOR PREVENTING INGESTION: yes

Analytical verification of doses or concentrations:

no

Details on analytical verification of doses or concentrations:

/

Duration of treatment / exposure:

6 hours

Frequency of treatment:

5 days per week

Remarks:

Doses / Concentrations:
250, 500 and 1000 mg/kg bw
Basis:
nominal per unit body weight

No. of animals per sex per dose:

5 males, 5 females

Control animals:

yes, concurrent no treatment

Details on study design:

- Dose selection rationale: Based on a range finding study
- Rationale for selecting satellite groups: not specified
- Post-exposure recovery period in satellite groups: 14 days

Positive control:

/

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Range finding animals were observed for a period of 7 days. In main study, all animals were observed individually, daily, for 28 days
- Cage side observations: various end-points of toxicity

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule:In main study, during the treatment period, all animals were observed daily soon after the application for clinical signs of toxicity. During treatment period all groups were observed twice daily, and during the post-treatment observation period satellite groups of animals were observed once daily.
- Clinical signs of toxicity: changes in skin, fur, eyes and mucous membranes, occurrence of secretions and excretions.

DERMAL IRRITATION (if dermal study): No

BODY WEIGHT: Yes
- Time schedule for examinations:Body weight of each animal was recorded just prior to the application of the test substance (day 0) and then on a weekly basis upto termination of test.

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: 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: No
- Time schedule for examinations:

OPHTHALMOSCOPIC EXAMINATION: No
- Time schedule for examinations:
- Dose groups that were examined:

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Blood was collected through orbital sinus from main group animals (overnight fasted) at the end of treatment period (day 28). Blood was collected through orbital sinus from all the groups of satellite animals (overnight fasted) at the end of treatment period (day 28) and at the end of the post treatment period (day 42).
- Anaesthetic used for blood collection: No
- Animals fasted: Yes, overnight
- How many animals: all
- Parameters checked: Erythrocyte count, Hemoglobin (Hb), Haematocrit (HCT), MCV, MCH, MCHC, Platelet count, Leucocyte count, Differential count (5 part) using Hematology Bayer Advia 120 fully automated analyzer. Prothrombin time was done by Thromborel-S method. Clotting time was done by capillary tube method.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood:Blood was collected through orbital sinus from main group animals (overnight fasted) at the end of treatment period (day 28). Blood was collected through orbital sinus from all the groups of satellite animals (overnight fasted) at the end of treatment period (day 28) and at the end of the post treatment period (day 42).
- Animals fasted: Yes
- How many animals:all
- Parameters checked: Glucose, Total cholesterol, Triglycerides, Total Bilirubin, Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Alkaline phosphatase (ALP), Gamma glutamyl transpeptidase, Blood urea nitrogen (BUN), Creatinine, Total proteins, Albumin, Globulin, Calcium and Phosphorus using Seimens dimension Xpand plus fully automated biochemistry analyzer.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No


OTHER:

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
At the end of the observation period (day 28/42), all test animals were humanely sacrificed by using CO2 asphyxiation method. A detailed gross necropsy was carried out on all animals of all groups after examination of the external surfaces of the body including all orifices. The cranial, thoracic and abdominal cavities were opened and the appearance of the tissues and organs were observed. All gross pathological findings were recorded individually for each animal.
The following organ wet weights were determined from all the animals during the time of necropsy.
1. Liver 2. Kidneys 3. Adrenals 4. Testes (males)
Normal and treated skin, liver, kidneys were collected and kept in 10% neutral buffered formalin for fixation. Organs with gross pathological lesions were also collected and kept in the appropriate fixative and processed for histopathologic examinations.

HISTOPATHOLOGY: Yes
Histopathological examination was performed on the preserved organs and tissues of the high dose group, control group and on the organs showing gross pathological lesions.

Other examinations:

/

Statistics:

The data was subjected to Modified-levene equal-variance test for homogeneity. Homogenous data was submitted to Analysis of Variance (ANOVA) followed by Student-Newman-Keul’s test for post hoc comparison in NCSS, 2007. In the case of heterogeneous data it was subjected to kruskal-wallis multiple-comparison Z-value test.

Clinical signs:

no effects observed

Dermal irritation:

not examined

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Details on results:

CLINICAL SIGNS AND MORTALITY: None of the animals from treated as well as control group showed any clinical signs of toxicity during entire experimental period. No morbidity/mortality was observed in both treated and control groups of rats during the entire experiment period.

BODY WEIGHT AND WEIGHT GAIN:
Day-28
No statistical significant changes were observed in body weights of both sex belong to G2 - G4 groups when compared with G1 (Control) group of animals
Day-42
No statistical significant changes were observed in body weights of both sex belong to G6 group of animals when compared with G5 (Control - satellite) group of animals

FOOD CONSUMPTION:
Day-28
No statistical significant changes were observed in feed weight of both sex belong to G2 - G4 groups when compared with G1 (Control) group of animals
Day-42
No statistical significant changes were observed in feed weight of both sex belong to G6 group of animals when compared with G5 (Control - satellite) group of animals


HAEMATOLOGY
Day-28
Statistical significant difference was exhibited by G3 and G4 group of males in MCV when compared with G1 group of males. G6 males exhibited statistical difference in platelet and basophil count when compared with G5 males. No statistical significant changes were observed in hematology parameters of females of G2 -G4 and G6 group animals when compared with G1 and G5 groups respectively.
Day-42
No statistical significant changes were observed in hematology parameters of G6 group males when compared with G5 group males. Statistical significant difference in basophil count was observed in G6 females when compared with G5 group females

CLINICAL CHEMISTRY
Day- 28
Total Protein in G3 males and albumin in G4 males showed statistical significant difference when compared with G1 group males. Globulin in G3 females exhibited statistical significant difference when compared with G1 group females. No statistical significant changes were observed in males of G6 when compared with G5 group. Statistical significant difference in chlorides was observed in G6 females when compared with G5 group females.
Day-42
No statistical significant changes were observed in G6 males when compared with G5 group males. Statistical significant difference was noted in albumin of G6 females when compared with G5 female group
The above changes (day 28 & 42) were not observed in dose dependent manner, hence these changes could be considered as individual animal normal biological variance and not attributed to the application of test substance.

ORGAN WEIGHTS
Day-28
No statistical significant values were observed in relative as well as absolute organ weights of G2- G4 groups of both sex when compared with G1 group of animals
Day - 42
No statistical significant values were observed in relative as well as absolute organ weights of G6 of both sex when compared with G5 group of animals

GROSS PATHOLOGY: No test substance related gross pathological findings were observed. The gross necropsy findings observed were either related to physiological, agonal, to spontaneous, or were incidental and of the type routinely observed in Wistar rats of this age

HISTOPATHOLOGY: No test substance related histopathological findings were observed. The histopathologic findings observed were either related to agonal, to spontaneous, or were incidental and of the type routinely observed in Wistar rats of this age

Key result

Dose descriptor:

NOEL

Effect level:

> 1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: overall effects

Critical effects observed:

not specified

/

Conclusions:

On the basis of the results obtained in the present study, no mortality was observed in both sex of treated as well as control group of animals during entire experiment period. Although few changes occurred in hematology and clinical biochemistry parameters, those changes were not related to test substance and could be considered as individual animal normal biological variance. There was no test substance related macroscopic and microscopic findings observed. In the light of above observations, the NOEL of N-n- Butylbenzenesulphonamide (PROVIPLAST 024) for Wistar rats could be considered as> 1000 mg/kg b.w. under the experimental conditions.

Executive summary:

A repeated dose - 28 day dermal toxicity study was conducted with N-n-butylbenzenesulphonamide in Wistar Rats at dose levels of 0, 250, 500 and 1000 mg/kg bw (semi-occlusive dosing; no vehicle). On the basis of the results obtained in the present study, no mortality was observed in both sex of treated as well as control group of animals during entire experiment period. Although few changes occurred in hematology and clinical biochemistry parameters, those changes were not related to test substance and could be considered as individual animal normal biological variance. There was no test substance related macroscopic and microscopic findings observed. In the light of above observations, the NOEL of N-n- Butylbenzenesulphonamide for Wistar rats could be considered as >1000 mg/kg b.w. under the experimental conditions.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

Reliable

Repeated dose toxicity: dermal - local effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: dermal

Type of information:

experimental study

Adequacy of study:

key study

Study period:

27/10/2011-03/01/2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

OECD Guideline 410 (Repeated Dose Dermal Toxicity: 21/28-Day Study)

Principles of method if other than guideline:

/

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River, USA
- Age at study initiation:
- Weight at study initiation: Males: 224-285g, Females: 221-237g
- Fasting period before study: no
- Housing: Standard polypropylene rat cages with stainless steel top grills supplied by M/s. Vishnu Traders, UP, India was used to house the animals. The cages were autoclaved. Gamma irradiated corn cobs supplied by M/s. Ceutics Pharma Pvt. Ltd., Bangalore, India was used as the bedding material.
- Diet (e.g. ad libitum):ad libitum
- Water (e.g. ad libitum):ad libitum
- Acclimation period: Five days prior to the range finding and main study in the test room.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.5-22.9°C
- Humidity (%): 53-65%
- Photoperiod (hrs dark / hrs light): 12hrs dark/12 hrs light

IN-LIFE DATES: From: 17/11/2011 To: 03/01/2012

Type of coverage:

semiocclusive

Vehicle:

unchanged (no vehicle)

Details on exposure:

TEST SITE
- Area of exposure: dorsal area
- % coverage: +/- 10%
- Type of wrap if used: a porous gauze dressing and a non irritating tape
- Time intervals for shavings or clipplings: Initially, 24 hours prior to the first application of the test substance, there after clipping was done at weekly intervals, before 24 hours of the application.

REMOVAL OF TEST SUBSTANCE
- Washing (if done): After the exposure period, the residual test substance was wiped gently from the skin using cotton soaked in water.
- Time after start of exposure: 6h

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): not specified
- Constant volume or concentration used: yes


USE OF RESTRAINERS FOR PREVENTING INGESTION: yes

Analytical verification of doses or concentrations:

no

Details on analytical verification of doses or concentrations:

/

Duration of treatment / exposure:

6 hours

Frequency of treatment:

5 days per week

Remarks:

Doses / Concentrations:
250, 500 and 1000 mg/kg bw
Basis:
nominal per unit body weight

No. of animals per sex per dose:

5 males, 5 females

Control animals:

yes, concurrent no treatment

Details on study design:

- Dose selection rationale: Based on a range finding study
- Rationale for selecting satellite groups: not specified
- Post-exposure recovery period in satellite groups: 14 days

Positive control:

/

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Range finding animals were observed for a period of 7 days. In main study, all animals were observed individually, daily, for 28 days
- Cage side observations: various end-points of toxicity

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule:In main study, during the treatment period, all animals were observed daily soon after the application for clinical signs of toxicity. During treatment period all groups were observed twice daily, and during the post-treatment observation period satellite groups of animals were observed once daily.
- Clinical signs of toxicity: changes in skin, fur, eyes and mucous membranes, occurrence of secretions and excretions.

DERMAL IRRITATION (if dermal study): No

BODY WEIGHT: Yes
- Time schedule for examinations:Body weight of each animal was recorded just prior to the application of the test substance (day 0) and then on a weekly basis upto termination of test.

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: 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: No
- Time schedule for examinations:

OPHTHALMOSCOPIC EXAMINATION: No
- Time schedule for examinations:
- Dose groups that were examined:

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Blood was collected through orbital sinus from main group animals (overnight fasted) at the end of treatment period (day 28). Blood was collected through orbital sinus from all the groups of satellite animals (overnight fasted) at the end of treatment period (day 28) and at the end of the post treatment period (day 42).
- Anaesthetic used for blood collection: No
- Animals fasted: Yes, overnight
- How many animals: all
- Parameters checked: Erythrocyte count, Hemoglobin (Hb), Haematocrit (HCT), MCV, MCH, MCHC, Platelet count, Leucocyte count, Differential count (5 part) using Hematology Bayer Advia 120 fully automated analyzer. Prothrombin time was done by Thromborel-S method. Clotting time was done by capillary tube method.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood:Blood was collected through orbital sinus from main group animals (overnight fasted) at the end of treatment period (day 28). Blood was collected through orbital sinus from all the groups of satellite animals (overnight fasted) at the end of treatment period (day 28) and at the end of the post treatment period (day 42).
- Animals fasted: Yes
- How many animals:all
- Parameters checked: Glucose, Total cholesterol, Triglycerides, Total Bilirubin, Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Alkaline phosphatase (ALP), Gamma glutamyl transpeptidase, Blood urea nitrogen (BUN), Creatinine, Total proteins, Albumin, Globulin, Calcium and Phosphorus using Seimens dimension Xpand plus fully automated biochemistry analyzer.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No


OTHER:

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
At the end of the observation period (day 28/42), all test animals were humanely sacrificed by using CO2 asphyxiation method. A detailed gross necropsy was carried out on all animals of all groups after examination of the external surfaces of the body including all orifices. The cranial, thoracic and abdominal cavities were opened and the appearance of the tissues and organs were observed. All gross pathological findings were recorded individually for each animal.
The following organ wet weights were determined from all the animals during the time of necropsy.
1. Liver 2. Kidneys 3. Adrenals 4. Testes (males)
Normal and treated skin, liver, kidneys were collected and kept in 10% neutral buffered formalin for fixation. Organs with gross pathological lesions were also collected and kept in the appropriate fixative and processed for histopathologic examinations.

HISTOPATHOLOGY: Yes
Histopathological examination was performed on the preserved organs and tissues of the high dose group, control group and on the organs showing gross pathological lesions.

Other examinations:

/

Statistics:

The data was subjected to Modified-levene equal-variance test for homogeneity. Homogenous data was submitted to Analysis of Variance (ANOVA) followed by Student-Newman-Keul’s test for post hoc comparison in NCSS, 2007. In the case of heterogeneous data it was subjected to kruskal-wallis multiple-comparison Z-value test.

Clinical signs:

no effects observed

Dermal irritation:

not examined

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Details on results:

CLINICAL SIGNS AND MORTALITY: None of the animals from treated as well as control group showed any clinical signs of toxicity during entire experimental period. No morbidity/mortality was observed in both treated and control groups of rats during the entire experiment period.

BODY WEIGHT AND WEIGHT GAIN:
Day-28
No statistical significant changes were observed in body weights of both sex belong to G2 - G4 groups when compared with G1 (Control) group of animals
Day-42
No statistical significant changes were observed in body weights of both sex belong to G6 group of animals when compared with G5 (Control - satellite) group of animals

FOOD CONSUMPTION:
Day-28
No statistical significant changes were observed in feed weight of both sex belong to G2 - G4 groups when compared with G1 (Control) group of animals
Day-42
No statistical significant changes were observed in feed weight of both sex belong to G6 group of animals when compared with G5 (Control - satellite) group of animals


HAEMATOLOGY
Day-28
Statistical significant difference was exhibited by G3 and G4 group of males in MCV when compared with G1 group of males. G6 males exhibited statistical difference in platelet and basophil count when compared with G5 males. No statistical significant changes were observed in hematology parameters of females of G2 -G4 and G6 group animals when compared with G1 and G5 groups respectively.
Day-42
No statistical significant changes were observed in hematology parameters of G6 group males when compared with G5 group males. Statistical significant difference in basophil count was observed in G6 females when compared with G5 group females

CLINICAL CHEMISTRY
Day- 28
Total Protein in G3 males and albumin in G4 males showed statistical significant difference when compared with G1 group males. Globulin in G3 females exhibited statistical significant difference when compared with G1 group females. No statistical significant changes were observed in males of G6 when compared with G5 group. Statistical significant difference in chlorides was observed in G6 females when compared with G5 group females.
Day-42
No statistical significant changes were observed in G6 males when compared with G5 group males. Statistical significant difference was noted in albumin of G6 females when compared with G5 female group
The above changes (day 28 & 42) were not observed in dose dependent manner, hence these changes could be considered as individual animal normal biological variance and not attributed to the application of test substance.

ORGAN WEIGHTS
Day-28
No statistical significant values were observed in relative as well as absolute organ weights of G2- G4 groups of both sex when compared with G1 group of animals
Day - 42
No statistical significant values were observed in relative as well as absolute organ weights of G6 of both sex when compared with G5 group of animals

GROSS PATHOLOGY: No test substance related gross pathological findings were observed. The gross necropsy findings observed were either related to physiological, agonal, to spontaneous, or were incidental and of the type routinely observed in Wistar rats of this age

HISTOPATHOLOGY: No test substance related histopathological findings were observed. The histopathologic findings observed were either related to agonal, to spontaneous, or were incidental and of the type routinely observed in Wistar rats of this age

Key result

Dose descriptor:

NOEL

Effect level:

> 1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: overall effects

Critical effects observed:

not specified

/

Conclusions:

On the basis of the results obtained in the present study, no mortality was observed in both sex of treated as well as control group of animals during entire experiment period. Although few changes occurred in hematology and clinical biochemistry parameters, those changes were not related to test substance and could be considered as individual animal normal biological variance. There was no test substance related macroscopic and microscopic findings observed. In the light of above observations, the NOEL of N-n- Butylbenzenesulphonamide (PROVIPLAST 024) for Wistar rats could be considered as> 1000 mg/kg b.w. under the experimental conditions.

Executive summary:

A repeated dose - 28 day dermal toxicity study was conducted with N-n-butylbenzenesulphonamide in Wistar Rats at dose levels of 0, 250, 500 and 1000 mg/kg bw (semi-occlusive dosing; no vehicle). On the basis of the results obtained in the present study, no mortality was observed in both sex of treated as well as control group of animals during entire experiment period. Although few changes occurred in hematology and clinical biochemistry parameters, those changes were not related to test substance and could be considered as individual animal normal biological variance. There was no test substance related macroscopic and microscopic findings observed. In the light of above observations, the NOEL of N-n- Butylbenzenesulphonamide for Wistar rats could be considered as >1000 mg/kg b.w. under the experimental conditions.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Additional information

A subacute 28-day oral toxicity with BBSA was conducted by oral daily gavage in the rat at dose levels of 0, 50, 150 and 1000 mg/kg bw (Notox, 2002). The high dose of 1000 mg/kg/day resulted in the death or moribund state of all rats. Clinical signs shown by these animals prior to death/sacrifice included lethargy, hunched posture, uncoordinated movements, abnormal gait, salivation, emaciation, laboured respiration, swelling of the abdomen or head and piloerection. Examination revealed liver enlargement and hepatocyte hypertrophy, liver necrosis, hyaline droplets formation in the renal papillary collecting ducts, adrenal gland enlargement with fatty change or cortical hypertrophy and reduced size and/or atrophy of the thymus, spleen and male reproductive organs. At 150 mg/kg/day, post-mortem findings were confined to liver enlargement and hepatocyte hypertrophy, thymic atrophy and lymphocytolysis. Cortical hyaline droplets noted in the kidneys of most rats dosed at 50 mg/kg/day and above were considered to represent alpha-2-microglobulin,a normal protein in male rats that undergoes reabsorption in the proximal cortical tubules. A range of chemicals is known to increase hyaline droplet formation beyond the physiological capacity of the tubular epithelium. This is a specific male rat response and is not observed female rats or higher species of either sex, including humans and is therefore considered to be of no risk to humans. There were no functional or morphological disturbances supporting the reduced haemoglobin values seen at 50 mg/kg/day. Also, this change was slight and occurred in the absence of a clear dose-related response. The slightly reduced motoric activity shown by males at 50 mg/kg/day occurred incidentally and was not supported by other signs of neurotoxicity. Therefore, excluding the presence of hyaline droplets in male kidneys, a No Observed Adverse Effect Level (NOAEL) of 50 mg/kg/day was established.

A second subacute 27-day study (NTP, 2012) was conducted by oral daily gavage in the male rat at dose levels of 0,100,200,400 mg/kg/d. The 400 mg/kg dose resulted in lethality in 3 rats after 1 week of dosing and the dose was lowered to 300 mg/kg with no further incidence of morbidity. This study was conducted to examine Sprague-Dawley rats for hindlimb dysfunction, sciatic nerve histopathology, gliosis in the hippocampus and cerebellum, male reproductive organ weight and histopathology as well as pathological changes in the liver. In this study there was no evidence of decreased male reproductive organ weights up to 400 mg/kg/d. No significant changes in the liver were observed in the 100 and 200 mg/kg/d BBSA dose group as compared to the controls. However all rats of the high BBSA dose group displayed some indication of minimal centrilobular hepatocyte hypertrophy. There was no evidence of dose dependent or treatment–related peripheral nerve degeneration following BBSA exposure. Also no elevations were noticed in the hippocampus for mRNA levels of related genes. Levels of mRNA for GFAP, IFN-gamma, CXCR-3 and CD11b were not altered by BBSA exposure and remained within constitutive levels seen in the control hippocampus. Daily oral gavage for 5d/week for 4 weeks did not produce clinical signs of hindlimb dysfunction, altered gait, rearing behavior, and ambulatory motor activity. Upon examination of the H&E staining for GFAP in the hippocampus and cerebellum, no increased of GFAP or morphological indicators of astrocyte or microglial reactivity in the hippocampus or cerebellum were noticed. Overall, these finding did not offer support for effects previously reported in the NOTOX 2002 28 day study. And these data also do not support neurotoxicity in male rats for BBSA within a 4 week exposure regime.

A supporting dietary 14-day dose range finding study was conducted in Wistar rats to prepare for the 90-day toxicity study, with target dose levels of 1000, 470, 125 and 50 mg/kg bw/day (dietary concentrations of 12500, 5875, 1563 and 625 ppm, respectively). There was no mortality, however hunched back and/or piloerection were recorded for High and Mid-High dose animals. Reduction of the body weight, body weight gain and food consumption was seen in High, Mid-High and Mid dose males and females. No clear test item related effect was observed on water consumption of male and female animals. Test item related haematological and clinical chemistry parameters were observed from the High dose onwards. The spleen weight was statistically significantly lower than control in High dose males and females as well as in Mid-High dose males. Test item related macroscopic findings (small spleen and thymus) were recorded for some High dose animals of the study. Test item intake values were 52.4, 124.2, 392.7 and 543.7 mg/kg bw/day in the Low (625 ppm), Mid (1563 ppm), Mid-High (5875 ppm) and High (12500 ppm) dose groups when calculated for the entire study period. In conclusion, the High dose and Mid-High dose levels (12500 ppm and 5875 ppm, respectively) used in this Dose Range finder study are considered to be above the Maximum Tolerable Dose for a definitive guideline compliant sub-chronic repeated dose toxicity study.

A key 90-day dietary study in Wistar rats was conducted at 0, 700, 1750 and 3750 ppm dose levels. No morbidity or mortality was observed and no clinical signs were recorded during the study. Test item related adverse effect was noted on body weight and/or body weight gain in the animals of the High and Mid dose groups, no such an effect was observed in the Low dose group. The food consumption was significantly reduced by the test item administration in the High dose group, in the Mid dose group a transient effect in the first one or two weeks was seen; there were no food intake effects in the Low dose group. Food efficiency was statistically significantly lower at 1750 and 3750 ppm both in males and females. There was no test item related effect on the water consumption identified in any dose group. No test item related changes were detected in the ophthalmology evaluation during the study. There was no indication of test item related effects on the neurological assessment in any test item treated dose groups. There were signs of slight anaemia in the High dose groups (up to 10% lower RBC, HCT and haemoglobin concentration) with a similar trend in the Mid dose. There was no effect of the test item on blood clotting parameters in any dose groups. There were no significant changes or adverse effects on the serum chemistry that could be clearly ascribed to the test item administration. There were no adverse test item related effects noted during urine analysis. Compared to control, test item related increase in kidney weights (absolute and body related) were detected for High and Mid dose males, while no effect was noted in the Low dose males or the females. The increased kidney size was also seen at necropsy as a macroscopic observation; histopathology changes confirmed the observations. Increased adrenal weights were also noted for High dose males, but without any histopathology change. Test item related dark red foci on liver lobes were noted in some High and Mid dose animals at necropsy, but no Low dose animals were affected. The change corresponded with histopathology observations of necrosis (mild to moderate at Mid dose males and High dose female; marked in High dose male). Test item related alpha 2-microglobulin nephropathy was observed in male rats only (accumulation of cortical tubules hyaline droplets, multifocal tubular basophilia, granular or hyaline casts and chronic progressive nephropathy). The kidney of High and Mid dose were all affected, 2/10 Low dose had some evidence of change (hyaline droplets). The observed microscopic changes were confirmed by special immunohistochemistry staining. These changes are not considered relevant for humans. Test item-related focal/multifocal hepatocellular necrosis was noted in the liver of High (1/10) and Mid dose (4/10) males as well as in High dose (1/10) females. These changes were correlated with necropsy findings in most of the affected animals. Despite the low incidence it was considered as adverse change. No test item related effect was observed in the adrenals and thymus of male and female animals in any dose groups.

It was concluded that there were no treatment-related effects at any dose levels on sperm morphology or enumeration (a statistically lower sperm motility in the High dose group was not ascribed to treatment). The test item intake values were calculated from the body weight, food intake and nominal diet concentration. The mean achieved dose levels (combined for males and females) were 265.4, 132.5 and 51.9 mg/kg bw/day in the High (3750 ppm), Mid (1750 ppm) and Low (700 ppm) dose groups, respectively. In conclusion, the NOAEL was 700 ppm dietary concentration (51.9 mg/kg bw/day).

A key repeated dose 28 day dermal toxicity study was conducted with N-n-butylbenzenesulphonamide in Wistar Rats at dose levels of 0, 250, 500 and 1000 mg/kg bw (semi-occlusive dosing; no vehicle) (IIBAT, 2012). On the basis of the results obtained in the present study, no mortality was observed in both sex of treated as well as control group of animals during entire experiment period. Although few changes occurred in hematology and clinical biochemistry parameters, those changes were not related to test substance and could be considered as individual animal normal biological variance. There was no test substance related macroscopic and microscopic findings observed. In the light of above observations, the NOEL of N-Butylbenzenesulphonamide for Wistar rats could be considered as >1000 mg/kg bw under the experimental conditions.

Testing for the inhalation route was waived.

Justification for classification or non-classification

In the key oral 90-day study test item related effects were reported only at and above 132.5 mg/kg/day; no observations were reported at 51,9 mg/kg/day. Classification is not warranted since no effects are reported at doses <100 mg/kg/day and the effects observed on the liver at doses above 100 mg/kg/day are seen only in males (not in females), without dose trend and necrosis was only mild to moderate.

Based on the results and according to the EC criteria for classification and labelling requirements for dangerous substances and preparations (Guidelines in Commission Directive 93/21/EEC) and CLP regulation (EC No. 1272/2008 of 16 December 2008), a classification as STOT RE is not justified for N-butylbenzenesulphonamide.