Registration Dossier

Endpoint:

extended one-generation reproductive toxicity - with F2 generation (Cohorts 1A, and 1B with extension)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Treatment of F0 animals commenced - 26 February 2019 Experimental completion date (Pathology) -12 March 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Justification for study design:

Purpose
The purpose of this study was to assess the influence of DAPSONE CAS 80-08-0 (an industrial chemical) on reproductive performance when administered continuously by oral gavage to Sprague-Dawley rats. Cohorts of F1 animals were used to assess the potential for systemic toxicity, immunotoxicity and potential effects on the sexual maturation, estrous cycles and reproductive performance

Animal Model
The rat was chosen as the test species because of the requirement for a rodent species by regulatory agencies. The Sprague-Dawley [Crl:CD(SD)] strain was used because of the historical control data available at this laboratory.

Specific details on test material used for the study:

Test item: DAPSONE CAS 80-08-0
CAS number: 80-08-0
Intended use: Industrial chemical
Appearance: Light yellow powder
Storage conditions: At ambient temperature (15 to 25C) in the dark
Supplier: Sponsor
Lot number: DH20170291
Expiry date: 02 July 2022
Purity: 99.8%
Supplier’s responsibilities: Characterization of the test item and the documentation of the methods of synthesis, fabrication or derivation and stability.
Archive sample: A 0.5 g representative sample was taken, placed in a well closed glass container and stored in the archives under the same conditions as the bulk material.

Species:

rat

Strain:

other: Crl:CD(SD)

Details on species / strain selection:

Number of animals
Males and females unrelated (no male/female siblings)
Supplied as 54 litters of identified litter mates
27 litters of four male siblings and 27 litters of four female siblings; no male/female sibling relationships at 22 to 28 days of age.

Duration of acclimatization Six days before commencement of treatment.

Age of the F0 animals at the start of the treatment
28 to 34 days old.

Weight range of the F0 animals at the start of the treatment
Males 69 to 125 g.
Females 59 to 106 g.

Sex:

male/female

Details on test animals or test system and environmental conditions:

Supplier Charles River (UK) Ltd.

Age of the F0 animals at the start of the treatment 28 to 34 days old.
Weight range of the F0 animals at the start of the treatment Males 69 to 125 g.
Females 59 to 106 g.

Animal Care and Husbandry
Environmental Control
Animal facility Limited access - to minimize entry of external biological and chemical agents.
Air supply Filtered fresh air which was passed to atmosphere and not recirculated.
Temperature and relative humidity Monitored and maintained within the range of 20-24°C and 40-70%.

There were no deviations from these ranges.

Lighting Artificial lighting, 12 hours light : 12 hours dark.
Electricity supply Public supply with automatic stand-by generators.

Animal Accommodation
Cages Cages comprised of a polycarbonate body with a stainless steel mesh lid; changed at appropriate intervals.
Solid (polycarbonate) bottom cages were used throughout the study except during pairing.
Grid bottomed cages were used during pairing. These were suspended above absorbent paper which was changed daily.

Cage distribution The cages were distributed on the racking to equalize, as far as possible, environmental influences amongst the groups.

Bedding Solid bottom cages contained softwood based bark-free fiber bedding, which was changed at appropriate intervals each week.
Study period Number of animals/cage
Male Female
Pre-pairing (acclimatization and after selection) Up to 4 Up to 4
Pairing 1 : 1
Males to termination Up to 4 -
Females after mating (from Day 0 after mating) - 1
Females during littering (from Day 20 after mating) - 1 + litter
Females to termination (after weaning) - Up to 4
Offspring maturation (from weaning until selection) Litter

Environmental Enrichment
Aspen chew block A soft white untreated wood product; provided to each cage throughout the study (except during late gestation and during lactation) and replaced when necessary.

For F0 and F1 Cohort 1B females, chew blocks were returned on Day 21 of lactation after weaning of offspring.

Plastic shelter Provided to each cage throughout the study (except during pairing, late gestation and during lactation) and replaced at the same time as the cages.

For F0 and F1 Cohort 1B females shelters were returned on Day 21 of lactation after weaning of offspring.

Paper shavings From Day 20 after mating and throughout lactation, approximately two handfuls of paper shavings were provided to each cage as nesting material; this nesting material was changed at the same frequency as the cage bedding.

Diet Supply
Diet SDS VRF1 Certified, pelleted diet.
The diet contained no added antibiotic or other chemotherapeutic or prophylactic agent.
Availability Non-restricted (diet was removed overnight before blood sampling for hematology or blood chemistry).

Water Supply
Supply Potable water from the public supply via polycarbonate bottles with sipper tubes. Bottles were changed at appropriate intervals.
Availability Non-restricted.

Supplier Certificates of Analysis
Certificates of analysis for the diet were scrutinized and approved before any batch of diet was released for use. Certificates of analysis were routinely provided by the water supplier.

Certificates of analysis were also received from the suppliers of the softwood based bark-free fiber bedding and Aspen chew blocks.

No specific contaminants were known that may have interfered with or prejudiced the outcome of the study and therefore no special assays were performed.

Route of administration:

oral: gavage

Details on exposure:

Vehicle CMC (carboxymethylcellulose) (0.5%).

Method of preparation
The required amount of test item was ground in a mortar using a pestle and mixed with some vehicle to form a paste. Further amounts of vehicle were gradually added and mixed to produce a smooth, pourable suspension. The suspension was quantitatively transferred and diluted to volume and finally mixed using a high-shear homogenizer.

A series of suspensions/formulations at the required concentrations were prepared by dilution of individual weighings of the test item.

Frequency of preparation Weekly, based on the storage stability detailed in Section 3.2.2

Storage of formulation Refrigerated storage (2-8C).

Test item accounting Detailed records of compound usage were maintained. The amount of test item necessary to prepare the formulations and the amount actually used were determined on each occasion. The difference between these amounts was checked before the formulations were dispensed.

Details on mating procedure:

Mating Procedure - F0 and F1 Cohort 1B Generation
F0 pairing commenced After ten weeks of treatment.
F1 Cohort 1B pairing commenced Approximately ten weeks after selection.
Male/female ratio 1:1 from within the same treatment groups (sibling pairing was not permitted).
Duration of pairing Up to two weeks.
Daily checks for evidence of mating Ejected copulation plugs in cage tray and sperm in the vaginal smear.
Day 0 of gestation When positive evidence of mating was detected.
Male/female separation Day when mating evidence was detected.
Pre-coital interval Calculated for each female as the time between first pairing and evidence of mating.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Formulation Analysis
Stability and homogeneity Before commencement of treatment, the suitability of the proposed mixing procedures was determined and specimen formulations were analyzed to assess the stability and homogeneity of the test item in the liquid matrix.
Formulations were found to be homogenous and stable for one day following ambient storage (15 to 25C) and for 20 days following refrigerated storage (2 to 8C).
Achieved concentration Samples of each formulation prepared for administration in first week of treatment for F0 and F1 generation and the last weeks of F1B gestation and for the F1B generation were analyzed for achieved concentration of the test item.

Duration of treatment / exposure:

Duration of Treatment
F0 animals For ten weeks before pairing until termination after litters were weaned.

F1 animals From weaning until termination of respective cohort.

Although direct exposure starts at weaning on Day 21 of age, all offspring had potential indirect exposure in utero and through the milk during lactation.

Unselected F1 offspring : Retention of limited tissues and organ weights recorded, no direct treatment, killed on Day 22 of age.
Cohort 1A : General toxicity and pathology of the tissues of the male and female reproductive systems.
Treated from weaning to 13 weeks of age.
Cohort 1B : Treated from weaning throughout pairing, gestation and lactation and up to either scheduled termination after weaning of F2 litters or completion of immunotoxicity investigations.

Frequency of treatment:

Once daily at approximately the same time each day. Animals were not dosed if parturition was in progress at the scheduled time of administration.

Details on study schedule:

Allocation to Treatment Groups (F0 Generation)
Allocation After a period of acclimatization.

By sex. After exclusion of animals showing signs of ill health, allocated so as to ensure not more than one offspring of each sex from each litter was present in each group. Animals at the extremes of the body weight range or litters showing large variation in individual weights were not selected if alternatives were available. Record of litter mate relationships was retained.

At commencement of the study the body weight of animals did not exceed +/-20% of the mean for each sex.

Selection of Offspring to Form F1 Generation
Selection Nominally Day 21 of age; selected animals receive direct treatment.

Allocation - formal start of F1 generation Formal commencement of the F1 generation was on a nominal Day 28 of age (28±2 days of age for selected F1 animals).

Method Where possible, two male and two female were selected from each selected litter and were allocated to each of the two cohorts. If more were required, up to three males and three females were selected from each selected litter.

Selected animals were microchipped on Day 18-21 of age and separated from littermates on Day 21 of age.

Up to two male and two female F1 offspring per group were retained as spares, to provide potential replacement in the event of any mortalities. These spares had body weights and clinical signs monitored weekly and were terminated after commencement of the F1 generation.

No. of animals per sex per dose:

F0 generation
25 males, 25 females per dose group

PLEASE REFER TO "ANY OTHER INFORMATION ON MATERIALS AND METHODS" FOR
FURTHER DETAILS REGARDING DOSE GROUPS

Control animals:

yes, concurrent no treatment

Details on study design:

Route of Administration
The oral gavage route of administration was chosen to simulate the conditions of possible human exposure.

Rationale for Dose Level Selection
Dose levels of 7.5, 15 and 30 mg/kg/day were selected in conjunction with the Sponsor based on the result of the Preliminary Study of Reproductive Performance in the Sprague-Dawley Rat by Oral Gavage Administration, where rats were dosed with 0, 7.5, 15 or 30 mg/kg bw/day.

In the preliminary study, effects at 30 mg/kg bw/day consisted of reduced body weight gain, an initial reduction in food consumption for males, and abnormally colored and enlarged spleens for males, indicating that 30 mg/kg/day represented the maximum threshold dose (MTD).

Based on these results, a high dose of 30 mg/kg/day was considered suitable for this study with low and intermediate dose levels of 7.5 and 15 mg/kg/day providing approximately 2 fold dose increments.

Administration
Route Oral gavage using a suitably graduated syringe and a rubber catheter inserted via the mouth.
Treated at Constant doses in mg/kg/day.
Volume dose 5 mL/kg body weight.
Individual dose volume Calculated from the most recently recorded scheduled body weight.
Control (Group 1) Vehicle at the same volume dose as treated groups.
Frequency Once daily at approximately the same time each day. Animals were not dosed if parturition was in progress at the scheduled time of administration.
Formulation A daily record of the usage of formulation was maintained based on weights. This balance was compared with the expected usage as a check of correct administration. No significant discrepancy was found.
Formulations were stirred using a magnetic stirrer before and throughout the dosing procedure.

Parental animals: Observations and examinations:

Serial Observations
Clinical Observations - F0 and F1 Generation
Animals were inspected visually at least twice daily for evidence of ill-health or reaction to treatment. Cages were inspected daily for evidence of animal ill-health amongst the occupant(s).

Any deviation from normal was recorded at the time in respect of nature and severity, date and time of onset, duration and progress of the observed condition, as appropriate.

During the acclimatization period, observations of the animals and their cages were recorded at least once per day.

Signs Associated with Dosing
Detailed observations were performed to establish and confirm a pattern of signs in association with dosing according to the following schedule:

F0 generation Week 1 - Daily.
Weeks 2 to 4 - twice weekly (middle and end of the week).
Week 5 onward - once each week (Days 0, 7, 14 and 20 after mating and Days 1, 7, 14 and 20 of lactation for F0 females).
F1 generation Week 1 - Daily.
Weeks 2 to 4 - twice weekly (middle and end of the week).
Week 5 onward - once each week (Days 0, 7, 14 and 20 after mating and Days 1, 7, 14 and 20 of lactation for F1B females).
For F1B females selected for TDAR investigation - once each week after weaning

Detailed observations were recorded at the following times in relation to dose administration:
Prior to dosing.
One to two hours after completion of dosing
As late as possible in the working day.

Clinical Signs
A detailed physical examination was performed on each animal to monitor general health according to the following schedule:

Physical examination Once each week.
After mating of F0 and F1 Cohort 1B females: Days 0, 5, 12, 18 and 20 after mating and Days 1, 7, 14 and 21 of lactation.
For F1B females selected for TDAR investigation performed weekly following weaning up to termination.

Particular attention was paid to possible signs of neurotoxicity such as convulsions, tremor and abnormalities of gait or behavior.

Mortality - F0 and F1 Generation
A viability check was performed near the start and end of each working day. Animals were killed for reasons of animal welfare where necessary.

A complete necropsy was performed in all cases as described in Section 3.8.

Body Weight - F0 and F1 Generation
The weight of animals was recorded as follows:
F0 males Day that treatment commenced.
Each week.
Before necropsy.
F0 females Day that treatment commenced.
Each week until mating detected.
Days 0, 2, 4, 6, 8, 10, 12, 14, 16, 18 and 20 after mating.
Days 1, 4, 7, 14, 21 and 28 of post partum.
Before necropsy
F1 selected animals From nominal Week 4 of age, twice during Week 1 of the F1 generation and weekly thereafter.

F1 Cohort 1B females after mating Days 0, 2, 4, 6, 8, 10, 12, 14, 16, 18 and 20 after mating.
Days 1, 4, 7, 14, 21 and 28 post partum.

Selected F1B females for TDAR investigation Weekly from Day 28 post partum.

Food Consumption - F0 and F1 Generation
The weight of food supplied to each cage, that remaining and an estimate of any spilled was recorded as follows:

F0 males Weekly until paired for mating.
F0 females Weekly until paired for mating.
Days 0-1, 2-3, 4-5, 6-7, 8-9, 10-11, 12-13, 14-15, 16-17 and 18 20 after mating.
Days 1-3, 4-6, 7-13, and 14-20 of lactation.
F1 selected animals From nominal Week 4 of age, twice during Week 1 of the F1 generation and weekly thereafter.
F1B males Weekly following pairing up to termination of the males that were not subject to TDAR.
F1 Cohort 1B females after mating: Days 0-1, 2-3, 4-5, 6-7, 8-9, 10-11, 12-13, 14-15, 16-17, 18-19 after mating.
Days 1-3, 4-6, 7-13 and 14-20 of lactation.

Food consumption was not measured for F1B females selected for TDAR.

From these records the mean weekly or daily consumption per animal (g/animal/week or g/animal/day) was calculated for each relevant phase.

Mating Procedure - F0 and F1 Cohort 1B Generation
F0 pairing commenced After ten weeks of treatment.
F1 Cohort 1B pairing commenced Approximately ten weeks after selection.
Male/female ratio 1:1 from within the same treatment groups (sibling pairing was not permitted).

Duration of pairing Up to two weeks.
Daily checks for evidence of mating Ejected copulation plugs in cage tray and sperm in the vaginal smear.
Day 0 of gestation When positive evidence of mating was detected.
Male/female separation Day when mating evidence was detected.
Pre-coital interval Calculated for each female as the time between first pairing and evidence of mating.

Parturition Observations and Gestation Length - F0 and F1 Cohort 1B Generation
Duration of gestation Time that elapsed between mating and commencement of parturition.
Parturition observations From Day 20 after mating animals were checked three times daily for evidence of parturition. The progress and completion of parturition was monitored; numbers of live and dead offspring were recorded and any difficulties observed were noted.

Hematology, Peripheral Blood - F0 and F1 Cohort 1A Generation
Blood samples were collected after overnight withdrawal of food. Samples were collected at the following occasions:

Occasion Generation Animals
Termination F0 Adults Ten male and ten female animals per group
F1 Cohort 1A Ten male and ten female animals per group

Animals were held under light general anesthesia induced by isoflurane. Blood samples (nominally 0.5 mL) were withdrawn from the sublingual vein, collected into tubes containing EDTA anticoagulant and examined for the following characteristics using a Bayer Advia 120 analyzer:

Hematocrit (Hct)*
Hemoglobin concentration (Hb)
Erythrocyte count (RBC)
Mean cell hemoglobin (MCH)*
Mean cell hemoglobin concentration (MCHC)*
Mean cell volume (MCV)
Total leucocyte count (WBC)
Differential leucocyte count:
Neutrophils (N)
Lymphocytes (L)
Eosinophils (E)
Basophils (B)
Monocytes (M)
Large unstained cells (LUC)
Platelet count (Plt)

* Derived values calculated in ClinAxys

Blood film (prepared for all samples) - Romanowsky stain, examined for abnormalities by light microscopy, in the case of flags from the Advia 120 analyzer. Confirmation or a written description from the blood film was made where appropriate. A manual count of the differential white blood cell parameters was performed where necessary.

Additional blood samples (nominally 0.5 mL) were taken into tubes containing citrate anticoagulant and examined using a Stago STA Compact Max analyzer and appropriate reagent in respect of:
Prothrombin time (PT) - using IL PT Fibrinogen reagent.
Activated partial thromboplastin time (APTT) - using IL APTT reagent.

Blood Chemistry - F0 and F1 Cohort 1A Generation
Blood samples were collected after overnight withdrawal of food. Samples were collected at the following occasions:
Occasion Generation Animals
Termination F0 Adults Ten male and ten female animals per group
F1 Cohort 1A Ten male and ten female animals per group

Animals were held under light general anesthesia induced by isoflurane. Blood samples (nominally 0.7 mL) were withdrawn from the sublingual vein and collected into tubes containing lithium heparin as anticoagulant. After separation, the plasma was examined using a Roche Cobas 6000 Analyzer in respect of:

Alkaline phosphatase (ALP)
Alanine aminotransferase (ALT)
Aspartate aminotransferase (AST)
Gamma-glutamyl transpeptidase (gGT)
Total bilirubin (Bili)
Urea
Creatinine (Creat)
Glucose (Gluc)
Total cholesterol (Chol)
Sodium (Na)
Potassium (K)
Chloride (Cl)
Calcium (Ca)
Inorganic phosphorus (Phos)
Total protein (Total Prot)
Albumin (Alb)

Albumin/globulin ratio (A/G Ratio) was calculated from total protein concentration and analyzed albumin concentration.

Biomarkers - TSH and T4
Blood samples were collected at the following occasions:
Occasion Generation Animals
Termination F0 Adults Ten male and ten female animals per group
F1 Offspring Ten litters per group - pooled litter sample Day 4 of age (T4 only)
Ten male and ten female animals per group on Day 22 of age from as many litters as possible
F1 Adults - Cohort 1A Ten male and ten female animals per group (approx. 13 weeks of age)

Conditions Adults: Following overnight deprivation of food.
Offspring: No overnight deprivation of food.
Blood sample site Adults and offspring on Day 22 of age: Sublingual vein.
Offspring Day 4 of age: Decapitation.
Anaesthetic Adults and offspring on Day 22 of age: Isoflurane.
Offspring Day 4 of age: Not required.
Anticoagulant None.
Tubes Greiner Minicollect - with clot activator.
Blood volume Adults and offspring on Day 22 of age: 1 mL.
Offspring Day 4 of age: Maximum possible.
Treatment of samples Samples were kept at ambient temperature (15 to 25˚C) for a minimum of 30 minutes prior to centrifugation.
Centrifugation conditions At 2000 g for ten minutes at 4°C.
Number of aliquots Adults and offspring on Day 22 of age: Two per animal Aliquot 1: 0.2 mL serum for T4
Aliquot 2: residual serum for TSH.
Offspring Day 4 of age: Single aliquot for T4 analysis all available collected.
Final storage conditions Deep frozen (approximately -60°C to -90ºC).
Fate of samples Aliquot 1 (T4): dispatched to the Department of Bioanalysis, Covance.
Aliquot 2: dispatched to the Department of Biomarkers, Bioanalysis and Clinical Sciences, Covance.
T4 Performed by the Department of Department of Bioanalysis, Covance.

TSH Performed by the Department of Department of Biomarkers, Bioanalysis and Clinical Sciences, Covance.

Oestrous cyclicity (parental animals):

Estrous Cycle Monitoring - F0 and F1 Cohort 1B Generation
Dry and wet smears were taken as follows:
Dry smears - F0 females only For 15 days before pairing, using cotton swabs.
Wet smears - F0 and F1B females After pairing until evidence of mating confirmed.

For four days before scheduled termination (nominally Days 25 to 28 post partum).

Sperm parameters (parental animals):

Sperm Analysis - F0 and F1 Cohort 1A Generation
Immediately after scheduled sacrifice of each F0, F1 Cohort 1A and Cohort 1B male and collection of blood, the left vas deferens, epididymis and testis were removed and the epididymis and testis were weighed.

The following tests were performed:
F0 generation
Sperm motility: all groups
A sample of sperm was expressed from the left vas deferens into prewarmed (target 37°C) medium M199, which contained 0.5% w/v bovine serum albumin (BSA Fraction V). A sample for assessment was taken into a 100 µm depth cannula by capillary action and, at least 200 sperm per animal analysed using the Hamilton Thorne IVOS II Computer Assisted Sperm Analyser (CASA).

Sperm morphology: Groups 1 and 4 A 200 L aliquot of the sperm/medium mixture (described above) was diluted with 800 L of 10% neutral buffered formalin. After staining with nigrosine and eosin an air-dried smear was prepared. Slides were examined by light microscopy for the assessment of sperm morphology. At least 200 sperm were assessed for each male where possible.

Sperm morphology: Groups 2 and 3 Fixed samples retained for possible future assessment.
Sperm count: Groups 1 and 4 The left cauda epididymis of each male was weighed and then the tunica was removed, then homogenised for at least 30 seconds in 10 mL of a mixture of 0.9% saline and 0.01% merthiolate (SM). An aliquot of this mixture was added to a pre-prepared IDENT stain tube before being assessed for sperm count using CASA.

Sperm count: Groups 2 and 3 Samples frozen for possible future assessment.
Homogenization-resistant spermatid counts: Groups 1 and 4 After removal of the tunica, the left testis of each male was homogenised for at least 30 seconds in 25 mL of SM. An aliquot of this mixture was added to a pre-prepared IDENT stain tube before being assessed for homogenisation-resistant spermatid count using CASA.

Homogenization-resistant spermatid counts: Groups 2 and 3 Samples frozen for possible future assessment.

F1 generation Cohort 1A
Sperm motility: all groups
A sample of sperm was expressed from the left vas deferens into prewarmed (target 37C) medium M199, which contained 0.5% w/v bovine serum albumin (BSA Fraction V). A sample for assessment was taken into a 100 µm depth cannula by capillary action and evaluated by light microscopy.

Sperm morphology: Groups 1 and 4 A 200 L aliquot of the sperm/medium mixture (described above) was diluted with 800 L of 10% neutral buffered formalin. After staining with nigrosine and eosin an air-dried smear was prepared. Slides were examined by light microscopy for the assessment of sperm morphology. At least 200 sperm were assessed for each male, (Group 4 male 477 only 199 were examined in error).

Sperm morphology: Groups 2 and 3 Fixed samples retained for possible future assessment.
Sperm count: Groups 1 and 4 The left cauda epididymis of each male was weighed and then the tunica was removed. The portion obtained was weighed then frozen. Prior to analysis the cauda epididymis portions were allowed to thaw then homogenised for at least 30 seconds in 10 mL of a mixture of 0.9% saline and 0.01% merthiolate (SM). An aliquot of this mixture was added to a pre-prepared IDENT stain tube before being assessed for sperm count using CASA.

Sperm count: Groups 2 and 3 Samples frozen for possible future assessment.

Homogenization-resistant spermatid counts: Groups 1 and 4 After removal of the tunica, the left testis of each male was frozen. Prior to analysis, the testes were allowed to thaw then homogenised for at least 30 seconds in 25 mL of SM. An aliquot of this mixture was added to a pre-prepared IDENT stain tube before being assessed for homogenisation-resistant spermatid count using CASA.

Homogenization-resistant spermatid counts: Groups 2 and 3 Samples frozen for possible future assessment.

F1 generation Cohort 1B
Sperm motility: All groups A sample of sperm was expressed from the left vas deferens into prewarmed (target 37°C) medium M199, which contained 0.5% w/v bovine serum albumin (BSA Fraction V). A sample for assessment was taken into a 100 µm depth cannula by capillary action and assessed manually.

Sperm morphology: Groups 1 and 4 A 200 L aliquot of the sperm/medium mixture (described above) was diluted with 800 L of 10% neutral buffered formalin. After staining with nigrosine and eosin an air-dried smear was prepared. Slides were examined by light microscopy for the assessment of sperm morphology. At least 200 sperm were assessed for each male.

Sperm morphology: Groups 2 and 3 Fixed samples retained for possible future assessment.

Sperm Count: Groups 1 and 4 The left cauda epididymis of each male was weighed and then the tunica was removed. The portion obtained was weighed then frozen. Prior to analysis the cauda epididymis portions were allowed to thaw then homogenised for at least 30 seconds in 10 mL of a mixture of 0.9% saline and 0.01% merthiolate (SM). An aliquot of this mixture was added to a pre-prepared IDENT stain tube before being assessed for sperm count using CASA.

Sperm count: Groups 2 and 3 Samples frozen for possible future assessment.
Homogenisation-resistant spermatid count: Groups 1 and 4 After removal of the tunica, the left testis of each male was frozen. Prior to analysis, the testes were allowed to thaw then homogenised for at least 30 seconds in 25 mL of SM. An aliquot of this mixture was added to a pre prepared IDENT stain tube before being assessed for homogenisation-resistant spermatid count using CASA.

Homogenisation-resistant spermatid count: Groups 2 and 3 Samples frozen for possible future assessment.

Terminal Investigations
Sperm Analysis - F0 and F1 Cohort 1A and 1B
Individual values were tabulated for the following:
Motility The percentages of motile and progressively motile sperm and sperm motion (F0 only) parameters were reported.
Morphology The number and percentages of normal and abnormal sperm were reported, including regions and specific types of abnormalities were reported.
Count (Testis and Cauda epididymis) The sperm concentration (Million/g) and total number were reported.

The following definitions relate to the sperm motion data:
VAP - the average path velocity, this is the average velocity of the smoothed cell path.
VSL - the progressive or straight line velocity, this is the average velocity measured in a straight line from the beginning to the end of the track.
VCL - the curvilinear velocity or track speed, this is the average velocity measured over the actual point to point track followed by the cell.
ALH - amplitude of lateral head displacement, this is the mean over all cell tracks of twice the maximum displacement between each sperm track and its average path. This corresponds to the average of the sperm track width.
BCF - beat cross frequency, this is the frequency with which the sperm head crosses the sperm average path.
STR - straightness, the average value of the ratio VSL/VAP which measures the departure of the cell path from a straight line.
LIN - linearity, the average value of the ratio VSL/VCL which measures the departure of the cell track from a straight line.
Rapid - the fraction of cells moving with VAP > progressive minimum VAP.
Medium - the fraction of cells moving with slow VAP cut-off < VAP < progressive minimum VAP.
Slow - the fraction of cells moving with VAP< slow VAP cut-off or VSL < slow VSL cut-off.
Static - the fraction of all cells which are not moving at all.

Litter observations:

Records Made During Littering Phase - F0 and F1 Cohort 1B Generation
The records maintained were as follows:

Clinical observations Observed approximately 24 hours after birth (Day 1 of age) and then daily for evidence of ill-health or reaction to treatment.

On Day 1 of age, all offspring received a qualitative assessment of body temperature, state of activity and reaction to handling.

Litter size Daily records were maintained of mortality and consequent changes in litter size from Days 1-21 of age.

On Day 4 of age, litters containing more than ten offspring were reduced to ten by random culling, leaving, whenever possible, five male and five female offspring in each litter.

Culled offspring were examined
Ratio of each litter Recorded on Days 1, 4 (before and after culling) and on Day 21 of age.
Individual offspring body weights Recorded on Days 1, 4 (before culling), 7, 14 and 21 of age.
Selected F1 generation: Days 23, 25, 27* and 29* of age.
* Only applicable before formal commencement of the F1 generation at nominal 4 weeks of age (Day 28 of age ± 2 days).

Weaning of offspring The dam was removed from the litter cage and offspring were weaned on Day 21 of age.
Ano-genital distance Day 1 of age - all offspring.
Nipple/areolae count Day 13 of age - male offspring.

Postmortem examinations (parental animals):

Time of Necropsy
F0 males After weaning of the F1 animals, after confirmation that no further mating required.
F0 females Day 28 post partum.
F0 and F1 Cohort 1B females failing to produce a viable litter and those with total litter loss Terminated with first cohort of females with live litters.
Unselected offspring Culled on Day 4 and Day 22 of age.
F1 Cohort 1A adult animals At approximately 13 weeks of age.
F1 Cohort 1B adult animals Ten males and ten females per group after weaning of F2 offspring - females on Day 28 post partum.
Ten males and ten females per group after immunotoxicity assessment.

All animals, including surplus offspring culled on Day 4 of age and Day 22 of age were subject to a complete macroscopic examination. Any abnormality in the appearance or size of any organ and tissue was recorded and the required tissue samples preserved in appropriate fixative. Decedent offspring ≤21 Days of age, (found dead or welfare kill), where possible, were examined and carcass retained.

For females of Cohort 1A, counts were performed for the number of ovarian follicles and corpora lutea

PLEASE REFER TO ATTACHED TABLE RE "PATHOLOGY PROCEDURES - F0 (PARENTAL)
ANIMALS

Postmortem examinations (offspring):

PLEASE REFER TO ATTACHED TABLES:
* Pathology Procedures - Cohort 1A
* Pathology Procedures - Cohort 1B
* Pathology Procedures - Unselected F1 and F2 offspring on day 22 of age

Statistics:

Data Evaluation
This report contains serial observations pertaining to all weeks of treatment completed, together with signs data collected during the necropsy period. No serial observations relating to the acclimatization period are included in this report.

Summary statistics (e.g. means and standard deviations) presented in this report were calculated from computer-stored individual raw data. The summary statistics and the individual data were stored in the computer to a certain number of decimal places, different for each parameter. For presentation purposes, however, they were usually rounded to fewer places. It is, therefore, not generally possible to reproduce the presented means and standard deviations exactly using the presented individual data.

Throughout the report the following abbreviations were used:
M Male
F Female
SD Standard deviation
N Number contributing to the mean (normally the number of animals/litters)

PLEASE ALSO REFER TO "ANY OTHER INFORMATION ON MATERIALS AND METHODS" BELOW

Reproductive indices:

Mating Performance and Fertility - F0 and F1 Cohort 1B Generation
Individual data was tabulated. Group values were calculated for males and females separately for the following:

Percentage mating (%) = (Number of animals mating/ Animals paired) x 100

Conception rate (%) = (Number of animals achieving pregnancy / Animals mated)

Fertility index (%) = (Number of animals achieving pregnancy / animals paired) x 100

Gestation Length and Gestation Index - F0 and F1 Cohort 1B Generation
Gestation length was calculated as the number of gestation days up to and including the day on which offspring were first observed, with Day 1 = day of mating for calculation purposes. Where parturition had started overnight, this value was adjusted by subtracting half of one day.

Gestation index was calculated for each group as:

Gestation index (%) = (Number of live litters born / animals paired) x 100

Offspring viability indices:

Litter Size – F1 and F2
Individual litter values were tabulated for the number of implantation sites, total at Day 1 and live at Days 1, 4 (before and after culling), 7, 14 and 21 of age. Group mean litter size and SD were calculated from the individual litter values.

Survival Indices - F0-F1 and F1 Cohort 1B – F2

The following were calculated for each litter:

Post implantation survival index (%) = (Total number of offspring born / Total number of uterine implantation sites) x 100

Post-implantation survival index was expressed as 100% where the number of offspring exceeded the number of implantation sites recorded.

Live birth index (%) = (Number of offspring on Day 1 after littering / Total number of offspring born) x 100

Viability index (%) = (Number of live offspring on Day 4 before culling/ Number of live offspring on Day 1 after littering) x 100

Lactation index (%) = (Number of live offspring on Day 21 after littering / Number of live offspring on Day 4 (after culling)) x 100

Group mean values were calculated from individual litter values.

Sex Ratio – F1 and F2
The percentage of male offspring in each litter was calculated at Day 1, and for live offspring on Days 1, 4 (before and after culling) and 21 of age.

Percentage males = (Number of males in litter/ Total number of offspring in litter) x 100

Group mean values were calculated from individual litter values.

Clinical signs:

no effects observed

Description (incidence and severity):

No signs were observed in association with dose administration and there were no signs at routine physical examination were considered to be related to treatment.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

There were two F0 decedents on this study. Control female No. 213 was found dead on Day 19 of treatment. Macroscopic findings observed were dark areas and a perforation in the esophagus, a small, pale spleen, edematous thymus and abnormal contents (fluid) in the thoracic cavity. Microscopic findings observed included slight inflammation and perforation of the esophagus, slight epicardial inflammation of the heart, slight inflammation in the pleura of the lungs, slight decreased cellularity of the red pulp of the spleen, minimal inflammation of the peri-thymic tissue where foreign material was noted. Macroscopic and microscopic findings were consistent with accidental perforation of the esophagus during dosing and this was the major factor resulting in the death of this animal.
Group 2 female No. 230 that received 7.5 mg/kg/day was found dead on Day 55 of treatment. A macroscopic finding of a depression on the left kidney was observed which correlated with a microscopic finding of a focal cortical scar. Other microscopic findings observed were minimal cortico-medullary mineralization in the kidneys and minimal axonal degeneration of the sciatic nerve. These findings are commonly observed in laboratory rats and were considered unrelated to treatment. No major factor contributing to death was identified. As this was a female from a low dose group and there were no other decedent animals from treated groups this death was considered unrelated to treatment.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

The overall bodyweight gain for males receiving 30 mg/kg/day during the 10 week pre pairing treatment period was slightly low at approximately 92% of Controls (p<0.05); this was not evident for females at this dose level.

There was no effect on body weight gain for males, or females before pairing, receiving 7.5 or 15 mg/kg/day.

There was no effect of treatment on body weight gain during gestation.

During Days 1 to 4 of lactation females receiving 15 mg/kg/day showed low weight gain (p<0.01) and females at 30 mg/kg/day showed body weight loss (p<0.01) when compared with Control animals and overall (LD1-21) body weight gain at 30 mg/kg/day was low at approximately 56% of Controls (p<0.01).

PLEASE REFER TO THE ATTACHED TABLES "BODYWEIGHT DATA - F0 GENERATION"

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

During the first three weeks of treatment food consumption for males receiving 15 or 30 mg/kg/day was statistically significantly low when compared with Controls and overall the total food consumption for males receiving 30 or 15 mg/kg/day was 95% of Controls for the 10 week pre-pairing phase.

Food consumption before pairing for males receiving 7.5 mg/kg/day and for females at dose levels up to and including 30 mg/kg//day was unaffected by treatment.

There was no effect of treatment on food consumption during gestation.

During lactation, at 30 mg/kg/day food consumption was low when compared with Controls, with the differences attaining statistical significance on lactation Days 1-4 (p<0.05) and Days 14-21 (p<0.01).

Up to Day 13 of lactation food consumption at 7.5 and 15 mg/kg/day was similar to Controls, however from Day 14-21 food consumption at these dose levels was slightly low when compared with Controls (p<0.01).

PLEASE REFER TO THE ATTACHED TABLES "FOOD CONSUMPTION DATA - F0 GENERATION"

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

Description (incidence and severity):

After 18 weeks of treatment males that received Dapsone had statistically significantly low mean haematocrit, haemoglobin concentration, erythrocyte count, mean cell haemoglobin, mean cell haemoglobin concentration and significantly high mean cell volume when compared with Controls; these differences were evident at all dose levels with limited evidence for a dose related response.

In addition males that received Dapsone up to and including 30 mg/kg/day had statistically significantly high neutrophil and platelet counts; there was no evidence for a dose response.

On Day 28 post partum the haematocrit value, haemoglobin concentration and erythrocyte count was statistically significantly low for females that received 30 mg/kg/day, haemoglobin was also significantly low at 7.5 and 15 mg/kg/day and the erythrocyte count was significantly low at 15 mg/kg/day. Females that received Dapsone at all dose levels also showed a significantly high lymphocyte count (and hence total white blood cell count); no dose response was evident.

PLEASE REFER TO THE ATTACHED TABLES - "HAEMATOLOGY AND BLOOD CHEMISTRY DATA, F0 GENERATION"

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Plasma concentrations of bilirubin were statistically significantly high, whilst creatinine levels were significantly low for males that received Dapsone at all dose levels. At 15 and 30 mg/kg/day males showed significantly low cholesterol and high albumin/globulin ratio.

Both males and females that received 30 mg/kg/day also showed a slightly but significantly high mean alanine amino transferase activity when compared with Controls.

There were no other differences that were considered to be related to treatment.

PLEASE REFER TO THE ATTACHED TABLES - "HAEMATOLOGY AND BLOOD CHEMISTRY DATA, F0 GENERATION"

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

effects observed, treatment-related

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

F0 Males after 18 Weeks of Treatment and F0 Females on Day 28 Post-partum
Treatment Related Findings
Findings related to treatment with DAPSONE CAS 80-08-0 were observed in the spleen and bone marrow of males and females, in the liver, kidneys, testes and epididymides of males and in the mammary of females.

Spleen
Minimal to severe congestion, minimal to moderate increased extramedullary hematopoiesis and minimal to moderate increased pigmented macrophages were observed in all males that received 7.5, 15 or 30 mg/kg/day. Minimal to marked decreased marginal zone lymphocytes were observed in all males that received 15 or 30 mg/kg/day and in some males that received 7.5 mg/kg/day. Minimal to marked capsular fibrosis was observed in the majority of males that received 30 mg/kg/day and in some males that received 15 mg/kg/day. Minimal to slight congestion was observed in the majority of females that received 30 mg/kg/day and in some females that received 7.5 or 15 mg/kg/day. Minimal to moderate increased pigmented macrophages were observed in all females that received 30 mg/kg/day and the majority of females that received 7.5 or 15 mg/kg/day. Minimal decreased marginal zone lymphocytes were observed in the majority of females that received 30 mg/kg/day and minimal increased extramedullary hematopoiesis in a low number of females that received 30 mg/kg/day.

PLEASE REFER TO THE TABLE IN "ANY OTHER INFORMATION ON RESULTS" SECTION: "SUMMARY OF FINDING IN THE SPLEEN FOR F0 ANIMALS AT SCHEDULED TERMINATION"

Bone Marrow (Sternum)
Minimal to slight increased hematopoietic cells were observed in the bone marrow of the sternum in all males that received 15 or 30 mg/kg/day and in the majority of males that received 7.5 mg/kg/day. Minimal or slight decreased hematopoietic cells were observed in the bone marrow of the sternum in all females that received 30 mg/kg/day and in some females that received 7.5 or 15 mg/kg/day.

PLEASE REFER TO THE TABLE IN "ANY OTHER INFORMATION ON RESULTS" SECTION: "SUMMARY OF FINDINGS IN THE BONE MARROW FOR F0 ANIMALS AT SCHEDULED TERMINATION"

Testes and Epididymides
Minimal to slight germ cell degeneration was observed in males that received 7.5, 15 or 30 mg/kg/day. Minimal spermatid retention was observed in some males that received 15 or 30 mg/kg/day. Associated minimal luminal cell debris was observed in the epididymides in a low number of males that received 7.5, 15 or 30 mg/kg/day. These changes did not affect fertility. Group 2 male (No. 33) that received 7.5 mg/kg/day was not included in the table data below as this male had testicular/epididymal pathology that was not considered test-item related.

PLEASE REFER TO THE TABLE IN "ANY OTHER INFORMATION ON RESULTS" SECTION: "SUMMARY OF FINDINGS IN THE TESTES AND EPIDIDYMIDES FOR F0 MALES AT SCHEDULED TERMINATION"

Liver
Minimal pigmented Kupffer cells were observed in the majority of males that received 15 or 30 mg/kg/day and in some males that received 7.5 mg/kg/day. Minimal extramedullary hematopoiesis was observed in the majority of males that received 30 mg/kg/day and in a low number of males that received 7.5 or 15 mg/kg/day.

PLEASE REFER TO THE TABLE IN "ANY OTHER INFORMATION ON RESULTS" SECTION: "SUMMARY OF FINDINGS IN THE LIVER FOR F0 ANIMALS AT SCHEDULED TERMINATION"

Kidneys
Minimal to moderate hyaline droplet accumulation was observed in the majority of males that received 7.5, 15 or 30 mg/kg/day. Minimal to slight pigment was observed in tubules in the majority of males that received 30 mg/kg/day and in one male that received 15 mg/kg/day.

PLEASE REFER TO THE TABLE IN "ANY OTHER INFORMATION ON RESULTS" SECTION: "SUMMARY OF FINDINGS IN THE KIDNEYS FOR F0 ANIMALS AT SCHEDULED TERMINATION"

Mammary
Minimal to moderate ductal/alveolar dilation was observed in some females that received 7.5, 15 or 30 mg/kg/day. Inactive mammary glands were observed in a low number of females that received 30 mg/kg/day.

PLEASE REFER TO THE TABLE IN "ANY OTHER INFORMATION ON RESULTS" SECTION: "SUMMARY OF FINDINGS IN THE MAMMARY FOR F0 ANIMALS AT SCHEDULED TERMINATION"

Incidental Findings
An incidental finding of a benign hemangioma was observed in the right testis of male No. 33 that received 7.5 mg/kg/day, with associated slight luminal cell debris and minimal reduced luminal sperm in the epididymides. As this was a single incidence in a low dose male it was considered unrelated to treatment.
The incidence and distribution of all other microscopic findings were considered unrelated to treatment.

Histopathological findings: neoplastic:

not examined

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

Estrous cycles before pairing were unaffected by treatment. All females except for one female in the 30 mg/kg/day group showed estrus before termination, and treatment did not affect the cycle stage at termination.

Reproductive function: sperm measures:

effects observed, treatment-related

Description (incidence and severity):

Following treatment with Dapsone CAS 80-08-0, reductions were seen on motile % and progressive % along with associated motion parameters (all statistically significant), across all treated groups.

A correlating effect on morphology was also observed at 30 mg/kg/day, affecting the head, neck and tail regions. These were specifically decapitate and flat heads; broken necks; folded and coiled tails (all statistically significant), along with a statistically significant decrease in cauda weight. All remaining cauda and testis parameters were unaffected.

In conclusion the sperm structure has been affected by treatment with Dapsone CAS 80-08-0 at doses up to 30 mg/kg/day, which in turn has affected the sperm motility, however these effects did not affect fertility.

PLEASE REFER TO THE ATTACHED TABLES "SPERM DATA - F0 GENERATION"

Reproductive performance:

no effects observed

Description (incidence and severity):

Pre-Coital Interval
Pre coital interval was unaffected by treatment.

Mating Performance and Fertility
Mating performance and fertility were unaffected by treatment.

Gestation Length and Gestation Index
Treatment did not affect the length of gestation, as at least 87% of females in all groups had 22 to 23 day gestation lengths; one Control female and two females at 30 mg/kg/day had a 24 day gestation length but no effect of treatment was inferred.

Treatment did not affect the gestation index.

Clinical signs:

no effects observed

Description (incidence and severity):

Treatment did not affect the clinical condition of the offspring.

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Offspring body weight on Day 1 of age was similar across the groups and considered unaffected by parental treatment.
During Days 1 to 4 of age offspring body weight gain at 30 mg/kg/day for both male and female offspring was significantly high when compared with Controls (p<0.01 and p<0.05, respectively); however the overall weight gain from Day 1 of age up to weaning on Day 21 of age did not show any differences that could be associated to administration of Dapsone.
Body weight gain for offspring at 7.5 or 15 mg/kg/day was unaffected by parental administration of Dapsone.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

There was considered to be no effect of treatment on the weight of the brain, spleen and thymus for F2 offspring at Day 22 of age.

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no findings considered to be related to treatment with Dapsone.

Neuropathological findings:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

effects observed, treatment-related

Description (incidence and severity):

The mean number of implantation sites was low at all dose levels when compared with Controls (p<0.05/p<0.01); a dose response was apparent at 15 and 30 mg/kg/day. As a consequence, total born and number of live offspring on Day 1 and Day 4 of age at all dose levels was significantly low when compared with Controls (p<0.05/p<0.01).
Offspring survival and sex ratio were unaffected by treatment at all dose levels.

Ano-Genital Distance
Treatment did not affect ano-genital distance of either the male or female offspring.

Nipple counts
On Day 13 of age areolae/nipples were not evident in any of the male offspring examined.

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

not examined

Clinical signs:

no effects observed

Description (incidence and severity):

There were no signs at routine physical examination that are considered related to treatment and no signs were observed in assentation with dose administration

Treatment did not affect the clinical condition of the offspring.

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

There was one decedent in Cohort 1A:
Group 2 male No. 427 that received 7.5 mg/kg/day was euthanized on Day 36 of treatment due to poor clinical condition. Macroscopic findings observed included a perforated esophagus, edematous thymus, abnormal contents (fluid) and adhesions in the thoracic cavity. Microscopic findings observed included minimal submucosal inflammation of the esophagus, minimal epicardial inflammation of the heart, minimal inflammatory cell infiltrate in the pleura of the lungs were foreign material was noted, minimally increased apoptosis of thymic lymphocytes and peri-thymic inflammation, moderate inflammation in the thoracic cavity and thoracic cavity adhesions. These macroscopic and microscopic findings were consistent with accidental perforation of the esophagus during dosing and this was the major factor resulting in the death of this animal. Other microscopic findings observed included congestion, increased extramedullary hematopoiesis and slight decreased marginal zone lymphocytes in the spleen. Findings in the spleen were considered treatment-related.
There was two decedents in Cohort 1B:
There were two decedents in this generation. A control male No. 485 died at bleed on Day 131 of treatment and had no significant macroscopic or microscopic findings. This death was considered accidental. Male No. 533 that received 15 mg/kg/day was found dead on Day 95 of treatment. Macroscopic findings observed were depressions on the kidneys, which correlated with a microscopic finding of minimal cortical scars, and an abnormally colored (dark) spleen, which correlated with a microscopic finding of marked congestion. Other microscopic findings observed in the spleen were slight increased pigmented macrophages, slight increased extramedullary hematopoiesis and minimal decreased marginal zone lymphocytes, and in the bone marrow of the sternum minimal increased hemopoietic cells were observed. Findings in the spleen and bone marrow of this male were considered treatment related. There were no findings attributable to death and the cause of this death was undetermined.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

From Day 1 of the F1 generation at nominal Day 28 of age, absolute mean body weight for selected F1 male and F1 female animals at 30 mg/kg/day was low when compared with Controls (p<0.01); absolute mean body weights for males at 7.5 or 15 mg/kg/days were also low from Day 22 of the F1 generation (p<0.05/0.01). The overall weight gain up to approximately 13 weeks of nominal age (Day 57 of the F1 generation) for males at all dose level was low at approximately 90% of Controls (p<0.01); however the overall weight gain for females showed no effect to treatment.

During gestation the mean absolute body weights remained low for females receiving 30 mg/kg/day. From GD0 to GD10 body weight gain for females at 30 mg/kg/day was essentially similar to Controls. However, from GD10 up to GD20 body weight gain at 30 mg/kg/day was low when compared with Controls with the difference attaining statistical significance from GD14; overall body weight gain during gestation (GD0-20) was significantly low when compared with Controls (p<0.01).

During lactation the mean absolute body weight for females receiving 30 mg/kg/day were significantly low when compared with Controls (LD1-21: p< 0.01). On Day 28 post partum the mean body weight at 30 mg/kg/day remained low (p<0.05) but for those animals selected for TDAR examinations mean body weight was not significantly different from Controls (Day 28 to 42 post partum). Bodyweight change during lactation was unaffected by treatment however after weaning on LD21 up to Day 28 post partum the body weight loss for females receiving 30 mg/kg/day was significantly less than the Controls (p<0.01). Subsequent body weight change from Day 28 to Day 42 post partum for the F1B females selected for the TDAR investigation was similar to Controls and considered unaffected by treatment.

During gestation, lactation and the post weaning phase body weight and body weight change for F1B females at 7.5 or 15 mg/kg/day were unaffected by treatment.

Offspring Body Weight
Offspring body weight on Day 1 of age and weight gain up to Day 4 of age was similar across the groups and considered unaffected by parental treatment. During Days 4 to 7 of age body weight gain for both male and female offspring at 30 mg/kg/day was low when compared with Controls (p<0.01). During Week 2 of lactation body weight gain at all dose levels was similar to Controls but then during the final week of lactation (LD14-21) offspring at 30 mg/kg/day showed low weight gain when compared with Controls [males (p<0.05); females (p<0.01)].
At 30 mg/kg/day, the overall body weight gain for offspring from Day 1 of age up to weaning was slightly low for males at approximately 93% of Controls and for females at approximately 91% of Controls, with the differences attaining statistical significance (p<0.05).
Body weight gain for offspring at 7.5 or 15 mg/kg/day was unaffected by parental administration of Dapsone.

PLEASE REFER TO ATTACHED TABLES "BODYWEIGHT DATA F1 GENERATION"

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

F1 male animals at all dose levels showed low food consumption when compared with Controls commencing from Day 1 of the F1 generation at 30 mg/kg/day (p<0.01) and from Day 8 at 7.5 and 15 mg/kg/day (p<0.05/0.01).

F1 female animals receiving 30 mg/kg/day showed slightly low food consumption during Week 1 of the F1 generation (p<0.01), thereafter consumption was similar to Control values; food consumption for females at 7.5 or 15 mg/kg/day was unaffected by administration of Dapsone.

Food consumption for F1B females during gestation was essentially similar to Controls and considered to be unaffected by treatment. During lactation however food consumption at 30 mg/kg/day was statistically significantly lower than Controls (p<0.05-0.01); food consumption at 7.5 or 15 mg/kg/day was unaffected by treatment.

PLEASE REFER TO ATTACHED TABLES "FOOD CONSUMPTION DATA F1 GENERATION"

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

Description (incidence and severity):

Hematology - Cohort 1A
At approximately 13 weeks of age F1A males that received Dapsone had low mean haemoglobin concentration, erythrocyte count, mean cell haemoglobin concentration and high mean cell volume when compared with Controls (p<0.01); these differences were evident at all dose levels with limited evidence for a dose response. In addition males at 30 mg/kg/day had a low hematocrit (p<0.05) and males at 15 or 30 mg/kg/day had high mean cell hemaglobin (p<0.01).

Males that received Dapsone had high neutrophil and lymphocyte counts (p<0.05-0.01); there was evidence for a dose response for mean lymphocyte counts but not the neutrophil counts. At 30 mg/kg/day monocyte counts were also high when compared with Controls (p<0.05).

F1A females at 30 mg/kg/day showed low haemoglobin concentration (p<0.05), low erythrocyte count (p<0.01), high mean cell haemoglobin (p<0.05), low mean cell haemoglobin concentration (p<0.01) and high mean cell volume (p<0.01); at 15 mg/kg/day the erythrocyte count was also low (p<0.05) and mean cell volume high (p<0.01) when compared with Controls. In addition females that received Dapsone at 30 mg/kg/day also showed a slightly high lymphocyte, basophil, monocyte and large unstained cell counts (p<0.05).

Blood Chemistry - Cohort 1A
Plasma concentrations of bilirubin were high for males that received Dapsone at all dose levels (p<0.01), at 15 and 30 mg/kg/day males showed low cholesterol (p<0.01) and at 30 mg/kg/day urea levels were high (p<0.01) when compared with Controls. Males receiving Dapsone showed slightly high albumin concentrations (p<0.05) and the albumin/globulin ratio at 30 mg/kg/day was high when compared with Controls (p<0.01).

Females that received Dapsone at 30 mg/kg/day showed high alanine amino transferase activity with high plasma levels of glucose, sodium, potassium, chloride and calcium when compared with Controls (p<0.01). At all dose levels, females showed slightly but significantly low phosphorous levels with no evidence of a dose response.

There were no other differences that were considered to be related to treatment.

PLEASE REFER TO THE ATTACHED TABLES - "HAEMATOLOGY AND BLOOD CHEMISTRY DATA, F01GENERATION"

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Plasma concentrations of bilirubin were high for males that received Dapsone at all dose levels (p<0.01), at 15 and 30 mg/kg/day males showed low cholesterol (p<0.01) and at 30 mg/kg/day urea levels were high (p<0.01) when compared with Controls. Males receiving Dapsone showed slightly high albumin concentrations (p<0.05) and the albumin/globulin ratio at 30 mg/kg/day was high when compared with Controls (p<0.01).

Females that received Dapsone at 30 mg/kg/day showed high alanine amino transferase activity with high plasma levels of glucose, sodium, potassium, chloride and calcium when compared with Controls (p<0.01). At all dose levels, females showed slightly but significantly low phosphorous levels with no evidence of a dose response.

There were no other differences that were considered to be related to treatment.

PLEASE REFER TO THE ATTACHED TABLES - "HAEMATOLOGY AND BLOOD CHEMISTRY DATA, F1 GENERATION"

Urinalysis findings:

not examined

Sexual maturation:

effects observed, treatment-related

Description (incidence and severity):

There was no effect of treatment of females on the mean age at vaginal opening, although females at 30 mg/kg/day had a lower mean body weight at attainment when compared with Controls (p<0.01).
Males receiving 30 mg/kg/day, showed balano preputial separation 2 days later than in Controls (p<0.01) and the other study groups; males at 15 or 30 mg/kg/day groups attained preputial separation at a lower mean body weight than in Controls (p<0.05 at both doses).

Anogenital distance (AGD):

no effects observed

Description (incidence and severity):

Treatment did not affect ano-genital distance of either the male or female offspring.

Nipple retention in male pups:

no effects observed

Description (incidence and severity):

On Day 13 of age areolae/nipples were not evident in any of the male offspring examined.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

There was considered to be no effect of treatment on the weight of the brain, spleen and thymus in F1 offspring at Day 22 of age.
In female offspring in the 30 mg/kg/day group, as expected the mean absolute brain weight was similar to controls despite a lower body weight; this resulted in a slightly but statistically significantly higher body weight relative brain weight when compared with Controls (p<0.05).

6.7.14 Organ Weights
Table 66 to Table 75, Appendix 52 to 57
Cohort 1A
At approximately 13 weeks of age, a dose related increase in body weight relative mean spleen weights for males at all dose levels (p<0.01); high body weight relative spleen weights were also observed for females at 30 mg/kg/day (p<0.01 ). Males that received Dapsone also showed significantly high body weight relative mean kidney and heart weights at all dose levels when compared with Controls.
At 15 and 30 mg/kg/day body weight relative seminal vesicle weights, body weight relative epididymal weights and absolute testes weights were high when compared with Controls at 15 and 30 mg/kg/day (p<0.05-0.01) and for females that received 30 mg/kg/day the absolute and body weight relative uterine weight was high when compared with Controls (p<0.05 and p<0.01, respectively).
There were no other differences in organ weights that could be attributed to administration of Dapsone.
Cohort 1B
The termination of F1B animals was split over two occasions the first phase of animals were terminated after weaning of the F2 offspring and the second phase after completion of the TDAR investigation.
On each occasion males that received Dapsone at all dose levels had high body weight relative spleen weights when compared with Controls (p<0.01); this was also evident for females after the TDAR investigation. After weaning females that received 15 or 30 mg/kg/day showed high body weight relative spleen weights (p<0.05).
Absolute mean testes weight was high at 30 mg/kg/day on both occasions (p>0.05-0.01) and at 15 mg/kg/day on the occasion after weaning (p<0.01).
After weaning the mean body weight relative uterine weight at 15 or 30 mg/kg/day was high when compared with Controls (p<0.05).
After the TDAR investigation males that received 30 mg/kg/day had low mean pituitary weight and females at 30mg/kg/day had high absolute and body weight relative (ovarian weight when compared with Controls (p<0.05 and p<0.01, respectively)

PLEASE REFER TP ATTACHED TABLES "ORGAN WEIGHT DATA F1 GENERATION"

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Cohort 1A
The macroscopic examination performed after after treatment from weaning to 13 weeks of age revealed findings in the spleen.
Abnormal coloration (dark) was observed in the majority of males that received 7.5, 15 or 30 mg/kg/day, in the majority of females that received 30 mg/kg/day and in some females that received 15 mg/kg/day.

Abnormal coloration (dark) of the kidneys was observed in a low number of males that received 30 mg/kg/day for which there was no histopathological correlate. In this well-vascularized organ, this was likely related to test item-induced methemoglobinemia which results in dark colored blood.
The incidence and distribution of all other macroscopic findings were considered to be unrelated to treatment.
Cohort 1B
The macroscopic examination performed after at least 18 weeks of treatment revealed findings in the spleen.
Abnormal coloration (dark) was observed in the majority of males that received 7.5, 15 or 30 mg/kg/day, in the majority of females that received 30 mg/kg/day and in some females that received 15 mg/kg/day. Marked enlargement was observed in the majority of males that received 30 mg/kg/day and some males that received 15 mg/kg/day. Capsular thickening was observed in some males that received 7.5, 15 or 30 mg/kg/day.
Abnormal coloration (dark) of the kidneys was observed in the majority of males that received 30 mg/kg/day and in a low number of males that received 15 mg/kg/day. Abnormal coloration (dark) was also observed in the liver of one male that received 30 mg/kg/day. There was no histopathological correlate for the abnormal coloration observed. In these well vascularized organs, this was likely related to test item induced methemoglobinemia which results in dark colored blood.
The incidence and distribution of all other macroscopic findings were considered to be unrelated to treatment.

PLEASE REFER TO THE TABLES IN "ANY OTHER INFORMATION ON RESULTS": "SUMMARY OF FINDINGS IN THE SPLEEN FOR F1A AND F1B ANIMALS AT SCHEDULED TERMINATION"

Histopathological findings:

effects observed, treatment-related

Description (incidence and severity):

Findings related to treatment with DAPSONE CAS 80-08-0 were observed in the spleen of males and females and in the liver, kidneys, bone marrow, testes and epididymides of males.
Spleen
Minimal to severe congestion and minimal to moderate increased extramedullary hematopoiesis were observed in all males that received 7.5, 15 or 30 mg/kg/day. Minimal to moderate increased pigmented macrophages and minimal to marked decreased marginal zone lymphocytes were observed in all males that received 15 or 30 mg/kg/day and the majority of males that received 7.5 mg/kg/day. An increased incidence of minimal capsular fibrosis was also observed in a low number of males that received 15 or 30 mg/kg/day. Minimal to slight congestion and minimal to slight increased extramedullary hematopoiesis were observed in the majority of females that received 30 mg/kg/day and some females that received 7.5 or 15 mg/kg/day. Minimal to moderate increased pigmented macrophages were observed in the majority of females that received 7.5, 15 or 30 mg/kg/day. Minimal to slight decreased marginal zone lymphocytes were observed in the majority of females that received 30 mg/kg/day and in one female that received 15 mg/kg/day.

PLEASE REFER TO THE TABLE IN "ANY OTHER INFORMATION IN RESULTS": "SUMMARY OF FINDINGS IN THE SPLEEN FOR FA1 ANIMALS AT SCHEDULED TERMINATION"

Bone Marrow (Sternum)
Minimal increased hematopoietic cells were observed in the majority of males that received 30 mg/kg/day.

PLEASE REFER TO THE TABLE IN "ANY OTHER INFORMATION ON RESULTS": "SUMMARY OF FINDINGS IN THE BONE MARROW FOR F1A ANIMALS AT SCHEDULED TERMINATION"

Testes and Epididymides
Minimal germ cell degeneration observed in a low number of males that received 7.5, 15 or 30 mg/kg/day. This was associated with minimal luminal cell debris in the epididymides in a low number of males that received 30 mg/kg/day and two males that received 7.5 mg/kg/day.

PLEASE REFER TO THE TABLE IN "AY OTHER INFORMATION IN RESULTS": "SUMMARY OF FINDINGS IN THE TESTES AND EPIDIDYMIDES FOR F1A MALES AT SCHEDULED TERMINATION"

Liver
Minimal extramedullary hematopoiesis was observed in the majority of males that received 30 mg/kg/day and some males that received 15 mg/kg. Minimal pigmented Kupffer cells were observed in half of the males that received 30 mg/kg/day.

PLEASE REFER TO THE TABLE IN "ANY OTHER INFORMATION ON RESULTS": "SUMMARY OF FINDINGS IN THE LIVER FOR F1A ANIMALS AT SCHEDULED TERMINATION"

Kidneys
Minimal to moderate hyaline droplet accumulation was observed in the majority of males that received 7.5, 15 or 30 mg/kg/day. Minimal pigment was observed in tubules in a low number of males that received 30 mg/kg/day.

PLEASE REFER TO THE TABLE IN "ANY OTHER INFORMATION ON RESULTS": "SUMMARY OF FINDINGS IN THE KIDNEY S FOR F1A ANIMALS AT SCHEDULED TERMINATION"

Incidental Findings
The incidence and distribution of all other microscopic findings were considered to be unrelated to treatment.

Histopathology - Cohort 1B
Only tissues observed as abnormal on macroscopic examination were examined microscopically.

Treatment Related Findings
Findings related to treatment with DAPSONE were observed in the spleen of males and females and in the liver and kidneys of males.

Spleen
Minimal to severe congestion, minimal to moderate increased pigmented macrophages and minimal to moderate increased extramedullary hematopoiesis were observed in all examined males that received 7.5, 15 or 30 mg/kg/day. Minimal to marked decreased marginal zone lymphocytes were observed in all examined males that received 15 or 30 mg/kg/day and in the majority of examined males that received 7.5 mg/kg/day. Minimal to slight capsular fibrosis was observed in the majority of examined males that received 30 mg/kg/day, in some examined males that received 15 mg/kg/day and in one male that received 7.5 mg/kg/day. Minimal congestion and minimal to moderate increased pigmented macrophages were observed in all examined females that received 7.5, 15 or 30 mg/kg/day. Minimal to slight increased extramedullary hematopoiesis was observed in some examined females that received 7.5, 15 or 30 mg/kg/day and minimal decreased marginal zone lymphocytes were observed in some examined females that received 15 or 30 mg/kg/day.

PLEASE REFER TO THE TABLE IN "ANY OTHER INFORMATION ON RESULTS": "SUMMARY OF FINDINGS IN THE SPLEEN FOR F1B ANIMALS AT SCHEDULED TERMINATION"

Kidneys
Minimal to moderate hyaline droplet accumulation hyaline droplet accumulation was observed in the majority of examined males that received 15 or 30 mg/kg/day and in all examined males that received 7.5 mg/kg/day. Minimal to slight pigment was observed in tubules in the majority of males that received 30 mg/kg/day and in one male that received 15 mg/kg/day.

PLEASE REFER TO THE TABLE IN "ANY OTHER INFORMATION ON RESULTS": SUMMARY OF FINDINGS IN THE KIDNEYS FOR F1B ANIMALS AT SCHEDULED TERMINATION"

Liver
The liver examined from male No. 554 that received 30 mg/kg/day was observed to have minimal extramedullary hematopoiesis and minimal pigmented Kupffer cells.
Incidental Findings
A finding of a malignant mammary adenocarcinoma was observed in female No. 735 that received 15 mg/kg/day. As this was a single incidence and no associated hyperplastic changes were observed in any other animals this was considered a spontaneous finding.

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Formulation Analysis

The analytical procedure was successfully validated with respect to specificity of chromatographic analysis, limits of detection and quantification, linearity of detector response, repeatability, method accuracy and precision, calibration standard stability and extract stability.

The homogeneity and stability was confirmed for Dapsone in CMC (carboxymethylcellulose) (0.5%) formulations at nominal concentrations of 1 mg/mL and 100 mg/mL during distribution between the bottles, during magnetic stirring for 2 hours, ambient temperature storage (15 to 25ºC) for up to 24 hours and refrigerated storage (2 to 8ºC) for up to 20 days.

The mean concentrations of Dapsone in test formulations analyzed for the study were within +10%/-15% of nominal concentrations, confirming accurate formulation. With one exception of the Group 2 samples for Week 1 F1 which was -18.0% from nominal. The difference from mean and coefficient of variation values remained within 5%, confirming precise analysis. There is one exception from this which was final week F1 Group 3, which had a high coefficient of variation following re-work of 8.69%, however as the relative mean error was within acceptance limits, it was considered that this formulation was prepared correctly.

Summary of findings in the spleen for F0 animals at scheduled termination

Group/sex	1M	2M	3M	4M	1F	2F	3F	4F
Dose (mg/kg/day)	0	7.5	15	30	0	7.5	15	30
Marked Enlargement	0	0	5	22	0	0	0	0
Abnormal Color (Dark)	0	19	24	25	0	1	4	16#
Capsule Thickened	0	0	5	18	0	0	0	0
Number of tissues examined	25	25	25	25	22	22	22	23#

# Includes females with litters that died prematurely

Summary of findings in the spleen for F0 animals at scheduled termination

Group/sex	1M	2M	3M	4M	1F	2F	3F	4F
Dose (mg/kg/day)	0	7.5	15	30	0	7.5	15	30
Congestion
Minimal	1	8	1	0	1	4	8	18
Slight	0	12	10	0	0	0	0	2
Moderate	0	5	8	9	0	0	0	0
Marked	0	0	4	8	0	0	0	0
Severe	0	0	2	8	0	0	0	0
Total	1	25	25	25	1	4	8	20
Pigmented Macrophages, Increased
Minimal	0	8	3	5	0	13	12	12
Slight	0	14	16	17	0	0	6	10
Moderate	0	3	6	3	0	0	0	1
Total	0	25	25	25	0	13	18	23
Fibrosis, Capsule
Minimal	0	0	5	8	0	0	0	0
Slight	0	0	1	3	0	0	0	0
Moderate	0	0	0	1	0	0	0	0
Marked	0	0	0	1	0	0	0	0
Total	0	0	6	13	0	0	0	0
Extramedullary Hematopoiesis, Increased
Minimal	2	15	9	4	0	0	0	4
Slight	0	10	16	14	0	0	0	1
Moderate	0	0	0	7	0	0	0	0
Total	2	25	25	25	0	0	0	5
Lymphocytes, Decreased, Marginal Zone
Minimal	0	6	10	0	0	0	0	15
Slight	0	2	11	7	0	0	0	0
Moderate	0	0	3	14	0	0	0	0
Marked	0	0	1	4	0	0	0	0
Total	0	8	25	25	0	0	0	15
Number of tissues examined	25	25	25	25	21	22	21	23

Summary of findings in the bone marrow for F0 animals at scheduled termination

Group/sex	1M	2M	3M	4M	1F	2F	3F	4F
Dose (mg/kg/day)	0	7.5	15	30	0	7.5	15	30
Hematopoietic Cells, Increased
Minimal	0	20	21	12	0	0	0	0
Slight	0	0	4	13	0	0	0	0
Total	0	20	25	25	0	0	0	0
Hematopoietic Cells, Decreased
Minimal	0	0	0	0	0	7	8	15
Slight	0	0	0	0	0	0	0	8
Total	0	0	0	0	0	7	8	23
Number of tissues examined	25	25	25	25	22	22	22	23

Summary of findings in the testes and epididymides for F0 males at scheduled termination

Group/sex	1M	2M	3M	4M
Dose (mg/kg/day)	0	7.5	15	30
Testes
Degeneration, Germ Cell
Minimal	0	6	7	12
Slight	0	0	1	2
Total	0	6	8	14
Spermatid Retention
Minimal	0	0	3	7
Total	0	0	3	7
Number of tissues examined	25	24	25	25
Group/sex	1M	2M	3M	4M
Dose (mg/kg/day)	0	7.5	15	30
Epididymides
Cell Debris, Luminal
Minimal	0	2	2	3
Total	0	2	2	3
Number of tissues examined	25	24	25	25

Summary of findings in the liver for F0 animals at scheduled termination

Group/sex	1M	2M	3M	4M	1F	2F	3F	4F
Dose (mg/kg/day)	0	7.5	15	30	0	7.5	15	30
Pigment, Kupffer Cell
Minimal	0	10	17	21	0	0	0	0
Total	0	10	17	21	0	0	0	0
Extramedullary Hematopoiesis
Minimal	0	3	5	16	0	0	0	0
Total	0	3	5	16	0	0	0	0
Number of tissues examined	25	25	25	25	22	0	0	23

Summary of findings in the kidneys for F0 animals at scheduled termination

Group/sex	1M	2M	3M	4M	1F	2F	3F	4F
Dose (mg/kg/day)	0	7.5	15	30	0	7.5	15	30
Accumulation, Hyaline Droplets
Minimal	1	14	15	11	0	0	0	0
Slight	0	5	3	8	0	0	0	0
Moderate	0	0	2	4	0	0	0	0
Total	1	19	20	23	0	0	0	0
Pigment, Tubules
Minimal	0	0	1	19	0	0	0	0
Slight	0	0	0	2	0	0	0	0
Total	0	0	1	21	0	0	0	0
Number of tissues examined	25	25	25	25	22	1	3	23

Summary of findings in the mammary for F0 animals at scheduled termination

Group/sex	1M	2M	3M	4M	1F	2F	3F	4F
Dose (mg/kg/day)	0	7.5	15	30	0	7.5	15	30
Dilation, Duct/Alveolus
Minimal	0	0	0	0	0	5	8	5
Slight	0	0	0	0	0	1	1	2
Moderate	0	0	0	0	0	0	0	1
Total	0	0	0	0	0	6	9	8
Number of tissues examined	25	0	0	25	22	22	22	23

F1 Litter Responses

General Condition of Offspring

Treatment did not affect the clinical condition of the offspring.

Litter Size, Survival Indices and Sex Ratio

The mean number of implantation sites was low at all dose levels when compared with Controls (p<0.01/p<0.05); a dose response was apparent. As a consequence, total born and number of live offspring on Day 1 at all dose levels was significantly low when compared with Controls (p<0.05/p<0.01).

At 30 mg/kg/day the live birth index was slightly low at 89.8% compared with 98.6% in the Control group (with four litters dying before Day 1 of age) and the viability index significantly low at 81.5% compared with 99.1% in the Controls (p<0.05). Subsequent survival following litter standardisation on Day 4 of age was similar to the Controls.

Offspring survival at 7.5 or 15 mg/kg/day was unaffected by parental treatment.

Sex ratio was unaffected by treatment at all dose levels.

Estrous cycles

For F1A females the interval between attainment of vaginal opening to the first estrus vaginal smear showed no clear effect of treatment.

Assessment of F1A estrous cycles from approximately Day 25 of age for a period of 2 weeks showed a slight increase in the incidence of acyclic females or females with irregular cycles in the groups receiving Dapsone, however there was no evidence of a dose response and differences did not attain statistical significance.

The majority of F1A females and all F1B females showed estrus in the four days prior to scheduled termination.

Pre-Coital Interval

The majority of animals mated within the first four days of pairing, with the exception of one pairing at 15 mg/kg/day and three at 30 mg/kg/day

Mating Performance and Fertility

Mating performance and fertility were unaffected by treatment.

Gestation Length and Gestation Index

Treatment did not affect the length of gestation, with the majority of females in all groups with a 22 to 23 day gestation length; one female at 7.5 mg/kg/day and one female at 15 mg/kg/day had a 23.5 day gestation length but no effect of treatment was inferred.

Treatment did not affect the gestation index.

Ovarian follicle and Corpora lutea count – Cohort 1A

Administration of Daponse at 30 mg/kg/day had no adverse effect on either the mean follicle or corpora lutea count when compared with the concurrent control group.

Sperm Assessment

Cohort 1A

Males that received Dapsone at 15 and 30 mg/kg/day showed low sperm motility (p<0.05‑0.01). At 30 mg/kg/day the testicular sperm count in terms of millions/g was slightly low (p<0.05) when compared with Controls; however the total number of sperm in the high dose group was similar to Controls.

The mean caudal epididymal weight at 30 mg/kg/day was significantly low when compared with Controls (p<0.05), whilst the mean testis weight at this dose level was significantly high when compared with Controls (p<0.01).

There were also several abnormalities observed at 30 mg/kg/day relating to the sperm head, neck and tail morphology including decapitated and flat head, broken neck, folded midpiece and bent/folded tail. The percentage of abnormal sperm examined in the high dose group was significantly high when compared with Controls (p<0.01).

Cohort 1B

Following treatment with Dapsone, males that received 15 or 30 mg/kg/day showed statistically significantly low motility when compared with the concurrent control (p<0.01).

At 30 mg/kg/day the caudal epididymal sperm count and concentration were low (p<0.05), and the testicular weight high, whilst the spermatid count was low (p<0.01) when compared with the concurrent Controls.

A correlating effect on morphology was also observed at 30 mg/kg/day, affecting all regions, with the exception of midpiece. These were specifically decapitate and flat heads, bent/kinked necks and broken necks, all statistically significant (individual tail abnormalities were not statistically significant, just collectively).

Summary of findings in the spleen for F1A animals at scheduled termination:

Group/sex	1M	2M	3M	4M	1F	2F	3F	4F
Dose (mg/kg/day)	0	7.5	15	30	0	7.5	15	30
Abnormal Color (Dark)	2	18	17	20	0	0	5	13
Number of tissues examined	20	19	20	20	20	20	20	20

Summary of findings in the spleen for F1B animals at scheduled termination:

Group/sex	1M	2M	3M	4M	1F	2F	3F	4F
Dose (mg/kg/day)	0	7.5	15	30	0	7.5	15	30
Marked Enlargement	0	0	7	17	0	0	0	0
Abnormal Color (Dark)	0	15	13	16	1	3	10	9
Capsule Thickened	0	2	1	8	0	0	0	0
Number of tissues examined	19	20	19	20	20	19	20	19

Summary of findings in the spleen for F1A animals at scheduled termination

Group/sex	1M	2M	3M	4M	1F	2F	3F	4F
Dose (mg/kg/day)	0	7.5	15	30	0	7.5	15	30
Congestion
Minimal	0	5	0	0	0	2	5	12
Slight	0	11	4	0	0	0	1	2
Moderate	0	3	10	7	0	0	0	0
Marked	0	0	5	10	0	0	0	0
Severe	0	0	1	3	0	0	0	0
Total	0	19	20	20	0	2	6	14
Group/sex	1M	2M	3M	4M	1F	2F	3F	4F
Dose (mg/kg/day)	0	7.5	15	30	0	7.5	15	30
Pigmented Macrophages, Increased
Minimal	0	15	12	3	0	11	13	8
Slight	0	3	5	14	0	0	2	10
Moderate	0	0	3	3	0	0	0	1
Total	0	18	20	20	0	11	15	19
Fibrosis, Capsule
Minimal	0	0	4	5	0	0	0	0
Slight	0	0	0	0	0	0	0	0
Moderate	1	0	0	0	0	0	0	0
Total	1	0	4	5	0	0	0	0
Extramedullary Hematopoiesis, Increased
Minimal	2	9	8	3	1	2	8	10
Slight	2	9	12	13	0	0	1	4
Moderate	0	1	0	4	0	0	0	0
Total	4	19	20	20	1	2	9	14
Group/sex	1M	2M	3M	4M	1F	2F	3F	4F
Dose (mg/kg/day)	0	7.5	15	30	0	7.5	15	30
Lymphocytes, Decreased, Marginal Zone
Minimal	0	11	4	0	0	0	1	11
Slight	0	1	13	9	0	0	0	1
Moderate	0	0	3	10	0	0	0	0
Marked	0	0	0	1	0	0	0	0
Total	0	12	20	20	0	0	1	12
Number of tissues examined	20	19	20	20	20	20	20	20

Summary of findings in the bone marrow for F1A animals at scheduled termination

Group/sex	1M	2M	3M	4M	1F	2F	3F	4F
Dose (mg/kg/day)	0	7.5	15	30	0	7.5	15	30
Hematopoietic Cells, Increased
Minimal	0	0	0	11	0	0	0	0
Total	0	0	0	11	0	0	0	0
Number of tissues examined	20	19	20	20	19	0	0	20

Summary of findings in the testes and epididymides for F1A males at scheduled termination

Group/sex	1M	2M		3M	4M
Dose (mg/kg/day)	0	7.5		15	30
Testes
Degeneration, Germ Cell
Minimal	0	3		4	5
Total	0	3		4	5
Number of tissues examined	20	19		20	20
Epididymides
Cell Debris, Luminal
Minimal	0		2	0	7
Total	0		2	0	7
Number of tissues examined	20		19	20	20

Summary of findings in the liver for F1A animals at scheduled termination

Group/sex	1M	2M	3M	4M	1F	2F	3F	4F
Dose (mg/kg/day)	0	7.5	15	30	0	7.5	15	30
Pigment, Kupffer Cell
Minimal	0	0	0	10	0	0	0	0
Total	0	0	0	10	0	0	0	0
Extramedullary Hematopoiesis
Minimal	0	0	7	15	0	0	0	0
Total	0	0	7	15	0	0	0	0
Number of tissues examined	20	20	20	20	20	0	0	20

Summary of findings in the kidneys for F1A animals at scheduled termination

Group/sex	1M	2M	3M	4M	1F	2F	3F	4F
Dose (mg/kg/day)	0	7.5	15	30	0	7.5	15	30
Accumulation, Hyaline Droplets
Minimal	5	12	9	6	0	0	0	0
Slight	0	1	5	8	0	0	0	0
Moderate	0	0	2	4	0	0	0	0
Total	5	13	16	18	0	0	0	0
Pigment, Tubules
Minimal	0	0	0	3	0	0	0	0
Total	0	0	0	3	0	0	0	0
Number of tissues examined	20	19	20	20	20	1	7	20

Summary of findings in the spleen for F1B animals at scheduled termination

Group/sex	1M	2M	3M	4M	1F	2F	3F	4F
Dose (mg/kg/day)	0	7.5	15	30	0	7.5	15	30
Congestion
Minimal	0	3	0	0	0	3	10	9
Slight	0	12	1	1	0	0	0	0
Moderate	0	0	8	4	0	0	0	0
Marked	0	0	6	10	0	0	0	0
Severe	0	0	1	5	0	0	0	0
Total	0	15	16	20	0	3	10	9
Pigmented Macrophages, Increased
Minimal	0	3	3	4	0	3	6	2
Slight	0	9	12	9	0	0	4	5
Moderate	0	3	1	7	0	0	0	2
Total	0	15	16	20	0	3	10	9
Fibrosis, Capsule
Minimal	0	1	8	11	0	0	0	0
Slight	0	0	0	5	0	0	0	0
Total	0	1	8	16	0	0	0	0
Group/sex	1M	2M	3M	4M	1F	2F	3F	4F
Dose (mg/kg/day)	0	7.5	15	30	0	7.5	15	30
Extramedullary Hematopoiesis, Increased
Minimal	0	13	10	6	0	2	5	2
Slight	0	2	3	8	0	0	0	2
Moderate	0	0	3	6	0	0	0	0
Total	0	15	16	20	0	2	5	4
Lymphocytes, Decreased, Marginal Zone
Minimal	0	11	7	2	0	0	2	4
Slight	0	1	7	4	0	0	0	0
Moderate	0	0	2	10	0	0	0	0
Marked	0	0	0	4	0	0	0	0
Total	0	12	16	20	0	0	2	4
Number of tissues examined	0	15	16	20	1	3	10	9

Summary of findings in the kidneys for F1B animals at scheduled termination

Group/sex	1M	2M	3M	4M	1F	2F	3F	4F
Dose (mg/kg/day)	0	7.5	15	30	0	7.5	15	30
Accumulation, Hyaline Droplets
Minimal	0	4	3	9	0	0	0	0
Slight	0	0	3	2	0	0	0	0
Moderate	0	0	2	2	0	0	0	0
Total	0	4	8	13	0	0	0	0
Pigment, Tubules
Minimal	0	0	1	9	0	0	0	0
Slight	0	0	0	2	0	0	0	0
Total	0	0	1	11	0	0	0	0
Number of tissues examined	4	4	9	17	2	2	1	0

Conclusions:

Based on the results of this study, it is concluded that a No-Observed-Adverse-Effect-Level (NOAEL) for systemic toxicity in Sprague Dawley rats was not established and lies below 7.5 mg/kg/day since test item related germ cell degeneration in the testes was detected at all doses in Dapsone F0 and F1A males.
The NOAEL for Reproductive toxicity also lies below 7.5 mg/kg/day, since reduced F1 and F2B litter size was seen at all doses of Dapsone.
The NOAEL for Developmental immunotoxicity was 30 mg/kg/day.

Executive summary:

The purpose of this study was to assess the influence of DAPSONE CAS 80-08-0 (an industrial chemical) on reproductive performance when administered continuously by oral gavage to Sprague-Dawley rats. Cohorts of F1 animals were used to assess the potential for systemic toxicity, immunotoxicity and potential effects on the sexual maturation, estrous cycles and reproductive performance

In the F0 generation, three groups of 25 male and 25 female rats received DAPSONE CAS 80-08-0 at dose levels of 7.5, 15 or 30 mg/kg/day at a volume dose of 5 mL/kg/day. Males were treated for ten weeks before pairing, up to necropsy after litters were weaned. Females were treated for ten weeks before pairing, throughout pairing up to necropsy on Day 28 post partum. In the F1 generation, 40 males and 40 females were treated from weaning to their scheduled termination (relevant to each cohort) at the same dose levels and volume-dose as the F0 generation. A similarly constituted Control group received the vehicle, CMC (carboxymethylcellulose) (0.5%) at the same volume dose.

Clinical observations, body weight, food consumption and macropathology examinations were performed on all animals for signs of toxicity, with special emphasis on the integrity and performance of the male and female reproductive system and the health (including thyroid hormone analysis), growth, development and function of the offspring.

At weaning, two cohorts of selected males and females were assigned for further investigations, including sexual maturation, reproductive organ integrity and function and immune functions. The cohorts were as follows:

Cohort	Designation	Animals/Cohort	Sexual maturation assessment	Approximate age at necropsy
1A	Reproductive & general toxicity	20M + 20F	Yes	13 weeks
1B	Reproductive & Immunotoxicity	20M + 20F	Yes	20-22 weeks

Results

F0 responses

There was dose related increase in serum TSH concentrations in F0 adult female rats that received 30 mg/kg/day but with a high level of variation between animals. Mean T4 concentrations for F0 adult animals were similar to Controls, showing no effects of treatment.

There were no deaths considered to be related to treatment and there was no effect on clinical condition

The overall bodyweight gain for males receiving 30 mg/kg/day during the 10 week pre‑pairing treatment period was slightly but statistically significantly low at approximately 92% of Controls; this was not evident for females at this dose level. There was no effect of treatment on body weight gain during gestation, but during lactation the overall body weight gain at 30 mg/kg/day was low at approximately 56% of Controls and this difference was statistically significant.

During the first three weeks of treatment food consumption for males receiving 15 or 30 mg/kg/day was statistically significantly low. There was no effect of treatment on food consumption during gestation. During lactation food consumption at 30 mg/kg/day was statistically significantly low, from LD 14-21 food consumption at 7.5 or 15 mg/kg/day was also slightly but statistically significantly low.

Oestrus cycles, mating performance, pre coital interval, fertility, gestation length and index were unaffected by treatment.

Haematology investigations revealed that after 18 weeks of treatment males that received Dapsone at all dose levels had statistically significantly low mean haematocrit, haemoglobin concentration, erythrocyte count, mean cell haemoglobin, mean cell haemoglobin concentration and significantly high mean cell volume. In addition males that received Dapsone up to and including 30 mg/kg/day had significantly high neutrophil and platelet counts. On Day 28 post partum the haematocrit value, haemoglobin concentration and erythrocyte count was statistically significantly low for females that received 30 mg/kg/day, haemoglobin was also low at 7.5 and 15 mg/kg/day and the erythrocyte count was low at 15 mg/kg/day. Females that received Dapsone at all dose levels also showed a significantly high lymphocyte count (and hence total white blood cell count); no dose response was evident.

Plasma concentrations of bilirubin were statistically significantly high, whilst creatinine levels were significantly low for males that received Dapsone at all dose levels. At 15 and 30 mg/kg/day males showed significantly low cholesterol and high albumin/globulin ratio. Both males and females that received 30 mg/kg/day also showed a slightly but statistically significantly high mean alanine amino transferase activity when compared with Controls.

Sperm assessments revealed statistically significant reductions in motile % and progressive % along with associated motion parameters, across all treated groups. A correlating effect on morphology was also observed at 30 mg/kg/day, affecting the head, neck and tail regions. These were specifically decapitate and flat heads; broken necks; folded and coiled tails (all statistically significant), along with a statistically significant decrease in cauda epididymis weight.

At scheduled termination there was a dose related increase in body weight relative mean spleen weights; these differences attained statistical significance in both sexes at all dose levels and the effect was greater in the males. Males that received Dapsone also showed statistically significantly high body weight adjusted kidney weights at all dose levels and significantly high mean body weight relative heart weight at 30 mg/kg/day. On Day 28 post-partum body weight relative adrenal weights for females that received 15 or 30 mg/kg/day were significantly high when compared with Controls.

Macroscopic examination revealed dose-related abnormal coloration (dark) in the spleen of males and females that received 7.5, 15 or 30 mg/kg/day. Marked enlargement and capsular thickening were observed in the majority of males that received 30 mg/kg/day and some males that received 15 mg/kg/day. In addition, abnormal coloration (dark) of the liver and/or kidneys was observed in a low number of males that received 30 mg/kg/day for which there was no histopathological correlate.

Microscopic examination of a full list of tissues revealed the following test item related findings:

Spleen: congestion, increased pigmented macrophages, decreased marginal zone lymphocytes, increased extramedullary hematopoiesis in males and females and capsular fibrosis in males only, some changes at all doses.

Bone marrow: increased hematopoietic cells in males and decreased hematopoietic cells in females at all dose levels.

Liver: pigmented Kupffer cells and extramedullary hematopoiesis in males at all dose levels

Kidneys: hyaline droplet accumulation in males at all doses and pigment in tubules in males at 30 mg/kg/day and one male at 15 mg/kg/day

Testes: germ cell degeneration in males at all doses and spermatid retention at 15/30 mg/kg/day.

Epididymides: luminal cell debris in male at all doses.

Mammary: ductal/alveolar dilation at all dose levels

F1 Litter responses

No effect was observed on serum TSH concentrations for F1 offspring on Day 22 of age. Mean T4 concentrations for F1 offspring (Day 4 and 22 of age) were similar to Controls, showing no effects of treatment.

Offspring clinical condition, ano-genital distance, sex ratio, nipple counts, macropathology and organ weights were all unaffected by treatment.

The mean number of implantation sites was statistically significantly low at all dose levels; a dose response was apparent. As a consequence, total born and number of live offspring on Day 1 at all dose levels was significantly low when compared with Controls.

At 30 mg/kg/day the live birth index was slightly low and the viability index statistically significantly low compared with Controls. Subsequent survival following litter standardisation on Day 4 of age was similar to the Controls.

Offspring body weight on Day 1 of age and weight gain up to Day 4 of age was similar across the groups. During Days 4 to 7 of age body weight gain for both male and female offspring at 30 mg/kg/day was statistically significantly low when compared with Controls. During Week 2 of lactation body weight gain at all dose levels was similar to Controls but then during the final week of lactation (LD14-21) offspring at 30 mg/kg/day showed statistically significantly low weight gain when compared with Controls.

F1 animals – Cohort 1A and 1B

There were no deaths considered to be related to treatment and there was no effect on clinical condition

From Day 1 of the F1 generation at nominal Day 28 of age, absolute mean body weight for F1 male and F1 female animals at 30 mg/kg/day was statistically significantly low and absolute mean body weights for males at 7.5 or 15 mg/kg/day were also significantly low from Day 22 of the F1 generation. The overall weight gain (approximately 13 weeks of nominal age) of males at all dose levels was significantly low at approximately 90% of controls; however the overall weight gain of females showed no effect to treatment.

F1 male animals at all dose levels showed statistically significantly low food consumption compared with Controls commencing from Day 1 of the F1 generation at 30 mg/kg/day and from Day 8 at 7.5 and 15 mg/kg/day. F1 female animals receiving 30 mg/kg/day showed slightly but significantly low food consumption during Week 1 of the F1 generation, thereafter consumption was similar to Control values; food consumption for females at 7.5 or 15 mg/kg/day was unaffected by administration of Dapsone.

There was no effect of treatment of females on the mean age at vaginal opening. However, males receiving 30 mg/kg/day, showed balano preputial separation 2 days later than in Controls and the other study groups and the difference attained statistical significance.

Cohort 1A

There was a dose related increase in serum TSH concentrations in F1 adult female rats that received 30 mg/kg/day. Mean T4 concentrations for selected F1 adult animals were similar to Controls, showing no effects of treatment.

Spleen immuno-phenotyping following administration of Dapsone to selected F1 animals (Cohort A) resulted in a dose‑dependent increase in male neutrophil percentage and cells/spleen; this was not apparent in female animals.

At approximately 13 weeks of age males that received Dapsone had statistically significantly low mean haemoglobin concentration, erythrocyte count, mean cell haemoglobin concentration and high mean cell volume; these differences were evident at all dose levels with limited evidence for a dose response. In addition males at 30 mg/kg/day had a significantly low haematocrit and males at 15 or 30 mg/kg/day had significantly high mean cell haemoglobin. Males that received Dapsone had significantly high neutrophil and lymphocyte counts and at 30 mg/kg/day monocyte counts were also significantly high.

Females at 30 mg/kg/day showed significantly low haemoglobin concentration, low erythrocyte count, high mean cell haemoglobin, low mean cell haemoglobin concentration and significantly high mean cell volume; at 15 mg/kg/day the erythrocyte count was significantly low and mean cell volume significantly high. Females that received 30 mg/kg/day also showed a slightly but significantly high lymphocyte, basophil, monocyte and large unstained cell counts.

Plasma concentrations of bilirubin were statistically significantly high for males that received Dapsone at all dose levels, at 15 and 30 mg/kg/day males showed significantly low cholesterol and at 30 mg/kg/day urea levels were significantly high. Males receiving Dapsone showed slightly but significantly high albumin concentrations and the albumin/globulin ratio at 30 mg/kg/day was significantly high. Females that received Dapsone at 30 mg/kg/day showed significantly high alanine amino transferase activity with significantly high plasma levels of glucose, sodium, potassium, chloride and calcium. At all dose levels, females showed slightly but significantly low phosphorous levels.

Sperm assessments revealed that males that received 15 or 30 mg/kg/day had statistically significantly low sperm motility. At 30 mg/kg/day the testicular sperm count in terms of millions/g was slightly but significantly low; however the total number of sperm was similar to Controls. There were also several morphological abnormalities observed at 30 mg/kg/day relating to the sperm head, neck and tail morphology.

There was a dose related statistically significant increase in body weight adjusted mean spleen weights for males at all dose levels; significantly high body weight relative spleen weights were also observed for females at 30 mg/kg/day. Males that received Dapsone showed significantly high body weight adjusted mean kidney and heart weights. At 15 and 30 mg/kg/day adjusted seminal vesicle weights and absolute testes weights were significantly high and for females at 30 mg/kg/day the adjusted uterine weight was significantly high.

Macroscopic examination revealed abnormal coloration (dark) in the spleen for the majority of males that received 7.5, 15 or 30 mg/kg/day and for the majority of females at 30 mg/kg/day and in some females at 15 mg/kg/day. In addition, abnormal coloration (dark) of the kidneys was observed in a low number of males at 30 mg/kg/day for which there was no histopathological correlate.

Microscopic examination of a full list of tissues revealed the following test item related findings:

Spleen: congestion, increased pigmented macrophages, decreased marginal zone lymphocytes, increased extramedullary hematopoiesis in males and females and capsular fibrosis in males only, some changes at all doses.

Bone marrow: increased hematopoietic cells in males at 30 mg/kg/day.

Liver: pigmented Kupffer cells in males at 30 mg/kg/day and extramedullary hematopoiesis in males at 15/30 mg/kg/day

Kidneys: hyaline droplet accumulation in males at all dose levels and pigment in tubules in males at 30 mg/kg/day

Testes: germ cell degeneration in males at all dose levels

Epididymides: luminal cell debris in males at 7.5/30 mg/kg/day.

Cohort 1B

The T-cell dependent antibody response (TDAR) in selected F1B animals following weaning of the F2 offspring was unaffected by treatment with Dapsone.

At 30 mg/kg/day body weights and body weight gain during gestation were statistically significantly low and during lactation absolute weights remained low; however body weight change during lactation was similar to Controls.

Food consumption during gestation was unaffected by treatment; however during lactation females receiving 30 mg/kg/day showed significantly low food consumption.

Males that received 15 or 30 mg/kg/day showed statistically significantly low sperm motility. At 30 mg/kg/day the caudal epididymal sperm count and concentration were significantly low and the testicular weight high, whilst the spermatid count was significantly low. A correlating effect on morphology was also observed at 30 mg/kg/day, affecting all regions, with the exception of midpiece.

F1B animals showed significantly high body weight relative spleen weights at all dose levels. At 15 or 30 mg/kg/day animals had significantly high testes weight and body weight relative uterine weights. At 30 mg/kg/day males had low mean pituitary weight and females significantly high ovarian weight.

The macroscopic examination revealed abnormal dark coloration of the spleen was observed in the majority of males that received 7.5, 15 or 30 mg/kg/day, in the majority of females that received 30 mg/kg/day and in some females that received 15 mg/kg/day. Marked enlargement of the spleen was observed in the majority of males that received 30 mg/kg/day and some males that received 15 mg/kg/day. Capsular thickening was also observed in some males that received 7.5, 15 or 30 mg/kg/day.

Abnormal coloration (dark) of the kidneys was observed in the majority of males that received 30 mg/kg/day and in a low number of males that received 15 mg/kg/day. Abnormal coloration (dark) was also observed in the liver of one male that received 30 mg/kg/day.

Microscopic examination was limited to tissues with macroscopic abnormality:

Spleen: congestion (males only), increased pigmented macrophages, decreased marginal zone lymphocytes, increased extramedullary hematopoiesis and capsular fibrosis (males only), some changes at all doses.

Liver: pigmented Kupffer cells and extramedullary hematopoiesis in one male 30 mg/kg/day.

Kidneys: hyaline droplet accumulation in males at all dose levels and pigment in tubules for males at 15/30 mg/kg/day

F2 responses

Clinical condition of offspring was unaffected by treatment.

The mean number of implantation sites, total born and the number of live offspring on Day 1 and Day 4 of age was statistically significantly low at all dose levels.

Offspring survival, sex ratio and ano-genital distance were unaffected by treatment at all dose levels.

On Day 13 of age areolae/nipples were not evident in any of the male offspring examined.

Offspring body weight on Day 1 of age was similar across the groups. During Days 1 to 4 of age offspring body weight gain at 30 mg/kg/day for both male and female offspring was significantly high; however the overall weight gain from Day 1 of age up to weaning on Day 21 of age did not show any differences that could be associated to administration of Dapsone.

Body weight gain for offspring at 7.5 or 15 mg/kg/day was unaffected by parental administration of Dapsone.

There was no effect of treatment on the weight of the brain, spleen and thymus, or the macropathology for F2 offspring at Day 22 of age.

Conclusion

Based on the results of this study, it is concluded that a No-Observed-Adverse-Effect-Level (NOAEL) for systemic toxicity in Sprague Dawley rats was not established and lies below 7.5 mg/kg/day since test item related germ cell degeneration in the testes was detected at all doses in Dapsone F0 and F1A males.

The NOAEL for Reproductive toxicity also lies below 7.5 mg/kg/day, since reduced F1 and F2B litter size was seen at all doses of Dapsone.

The NOAEL for Developmental immunotoxicity was 30 mg/kg/day.