(p-ammoniophenyl)bis(2-hydroxyethyl)ammonium sulphate

Toxicity to reproduction

Administrative data

Endpoint:

fertility, other

Remarks:

90 days repeated toxicity study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 12 March 2004 To 30 November 2005

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2005

Report date:

2005

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 408 Repeated Dose 90-Day Oral Toxicity Study in Rodents

Version / remarks:

In this Repeated dose -90 day oral toxicity study, some reproductive parameters as fertility were evaluated

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

(p-ammoniophenyl)bis(2-hydroxyethyl)ammonium sulphate

EC Number:

259-134-5

EC Name:

(p-ammoniophenyl)bis(2-hydroxyethyl)ammonium sulphate

Cas Number:

54381-16-7

Molecular formula:

C10H16N2O2.H2O4S

IUPAC Name:

(p-ammoniophenyl)bis(2-hydroxyethyl)ammonium sulphate

Constituent 2

Reference substance name:

N,N-bis (2-hydroxyethyl)-p-phenylenediamine sulfate

IUPAC Name:

N,N-bis (2-hydroxyethyl)-p-phenylenediamine sulfate

Details on test material:

- Name of test material: N,N-bis (2-hydroxyethyl)-p-phenylenediamine sulfate
- TSIN: GTS03849
- Substance type: Pure active substance
- Physical state: White powder
- Stability under test conditions: Not reported
- Storage condition of test material: At room temperature, Protected from light.

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Lot No. A203G157
- Expiration date of the lot/batch: not specified
- Purity test date: 20 November 2003

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature
- Stability under test conditions: not specified
- Solubility and stability of the test substance in the solvent/vehicle: homogeneity : Mean results of the homogeneity analyses from the top, middle, and bottom locations of the 0.1- and 2.0-mg/mL concentrations ranged from 96.6 to 97.0% and 98.5 to 99.5% of theoretical, respectively, indicating that the mixing procedure resulted in homogeneous preparations.

Stability : Dose confirmation mean results for all concentrations ranged from 96.6 to 98.5%, 96.8 to 99.5%, 97.7 to 99.7%, 98.6 to 99.5%, and 74.7 to 90.8% of theoretical for Weeks 1 (initial), 2, 5, 9, and 13, respectively. Results of the Week 13 analysis of the 0.1-mg/mL concentration did not meet acceptance criteria (within 15% of theoretical).

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
For each dose formulation, a volume of vehicle was added to the required amount of GTS03849 and mixed on a magnetic stir plate and stir bar until the formulation appeared to be a uniform suspension. Additional vehicle was added to achieve the final volume. The formulations were sampled (if required), then transferred to individual amber glass containers for daily use and stored in a refrigerator set to maintain 2 to 8°C, protected from light.

Test animals

Species:

rat

Strain:

Crj: CD(SD)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories
- Females (if applicable) nulliparous and non-pregnant: not specified
- Age at study initiation: the animals were approximately 6 to 7 weeks old,
- Weight at study initiation:their body weights were ranged from 204 to 299 g for the males and 153 to 194 g for the females
- Fasting period before study: not specified
- Housing: The animals were housed individually in suspended, stainless-steel cages.
- Diet (e.g. ad libitum):Certified Rodent Diet (#8728C, Harlan Teklad) was available ad libitum
- Water (e.g. ad libitum):Water was available ad libitum. Water samples are routinely analyzed for specified microorganisms and environmental contaminants
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 25°C
- Humidity (%):30 to 70% Relative humidity
- Air changes (per hr): 10 or greater air changes/hour
- Photoperiod (hrs dark / hrs light):12-hour light/12-hour dark cycle

IN-LIFE DATES: From: 15 March 2004 To: 19 July 2004

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: 0.2% erythorbic acid in reverse osmosis water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Vehicle formulations were prepared weekly for this study. The vehicle components were reported to be 100% pure for the purpose of dosage calculations. For the preparation of the
vehicle, a portion of reverse osmosis (RO) water was transferred to a labeled container. The required amount of erythorbic acid was transferred to the RO water while mixing using a
magnetic stir plate and stir bar until the erythorbic acid was dissolved. The preparation was then brought to final volume with RO water.
Test article formulations were prepared approximately weekly for this study. The test article was 100% pure for the purpose of dosage calculations. For each dose formulation, a volume of vehicle was added to the required amount of registered substance and mixed on a magnetic stir plate and stir bar until the formulation appeared to be a uniform suspension. Additional vehicle was added to achieve the final volume. The formulations were sampled (if required), then transferred to individual amber glass containers for daily use and stored in a refrigerator set to maintain 2 to 8°C, protected from light.

VEHICLE
- Justification for use and choice of vehicle (if other than water): not specified
- Concentration in vehicle: 0, 0.1, 0.4, 2.0 mg/mL
- Amount of vehicle (if gavage): 10mL/kg
- Lot/batch no. (if required): 11719CA Sigma-Aldrich Co.
- Purity: 99.9%

Details on mating procedure:

not applicable

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Mean results of the homogeneity analyses from the top, middle, and bottom locations of the 0.1- and 2.0-mg/mL concentrations ranged from 96.6 to 97.0% and 98.5 to 99.5% of theoretical, respectively, indicating that the mixing procedure resulted in homogeneous preparations. Stability analyses generated in Covance 6114-461 indicated that the formulations were stable for the concentration range used and under conditions of use for the study.

Dose confirmation mean results for all concentrations ranged from 96.6 to 98.5%, 96.8 to 99.5%, 97.7 to 99.7%, 98.6 to 99.5%, and 74.7 to 90.8% of theoretical for Weeks 1 (initial), 2, 5, 9, and 13, respectively. Results of the Week 13 analysis of the 0.1-mg/mL concentration did not meet acceptance criteria (within 15% of theoretical).

Duration of treatment / exposure:

91 days

Frequency of treatment:

Once daily

Details on study schedule:

no mating was performed

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

1 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

4 mg/kg bw/day (nominal)

Doses / concentrations 4

Dose / conc.:

20 mg/kg bw/day (nominal)

No. of animals per sex per dose:

20 animals per sex were sued for control and high dose group, 15 animals per sex were used for low and mid dose level group

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The doses selected for this study were based on the results of a 14-day range-finding study (Covance 6114-469). The goal of the dose level selection was to establish satisfactory safety margins for various levels of potential human exposure to the test article. Ideally, none of the dose levels should produce significant adverse effects. The oral route was chosen to maximize the potential to identify possible health hazards that may be associated with repeated exposure.
- Rationale for animal assignment (if not random): Animals were assigned to treatment groups using a computerized blocking procedure designed to achieve body weight balance with respect to treatment groups.

Examinations

Parental animals: Observations and examinations:

CLINICAL OBSERVATIONS: Yes, each animal was observed for mortality, abnormalities and signs of pain or distress; findings were recorded as they were observed.
- Time schedule: Twice daily (a.m. and p.m.)

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Once daily
- Cage side observations included: Abnormal findings were recorded

DETAILED CLINICAL OBSERVATIONS: Yes, these observations were made outside the home cage and included, but were not limited to, changes in skin, fur, eyes, and mucous membranes; occurrences of secretions and excretions; and autonomic activity (e.g., lacrimation, piloerection, pupil size, unusual respiratory pattern). Changes in posture and reactivity to handling and the presence of clonic or tonic movements, stereotypes (e.g., excessive grooming, circling), or bizarre behavior (e.g., self-mutilation, walking backwards) were also recorded. Changes in gait were assessed by allowing the animal to walk freely to allow evaluation of gait. Additional findings were recorded as they were observed.
- Time schedule: Prior to treatment, on Day 1, weekly thereafter, and on the day of each scheduled sacrifice

BODY WEIGHT: Yes
- Time schedule for examinations: Individual body weights were recorded twice prior to treatment, on the first day of treatment, and weekly thereafter.

FOOD CONSUMPTION: Yes
- Time schedule for examinations: Weekly

FOOD EFFICIENCY: No

WATER CONSUMPTION AND COMPOUND INTAKE: No

OPHTHALMOSCOPIC EXAMINATION: Yes, Animals were examined by a board-certified veterinary ophthalmologist using an indirect ophthalmoscope and a slit lamp. The eyes were dilated with a mydriatic agent prior to examination.
- Time schedule for examinations: Prior to treatment in a sequential manner and during Wk 13 in random order. The ophthalmic examinations were done on a day other than that scheduled for the expanded clinical observations.
- Dose groups that were examined: All animals

HAEMATOLOGY: Yes, blood samples for were collected from a jugular vein of animals.
- Time schedule for collection of blood: At each scheduled sacrifice (on Day 92 and on Day 120)
- Anaesthetic used for blood collection: No
- Anticoagulants used: Yes potassium EDTA (for hematology) and sodium citrate (for coagulation parameters)
- Animals fasted: Yes (overnight)
- How many animals: All surviving animals
- Parameters examined: Red blood cell (erythrocyte) count, hemoglobin, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, platelet count, white blood cell (leukocyte) count, differential blood cell count, blood smear, reticulocyte count, prothrombin time and activated partial thromboplastin time.

CLINICAL CHEMISTRY: Yes, blood samples for were collected from a jugular vein of unanesthetized animals.
- Time schedule for collection of blood: At each scheduled sacrifice (on Day 92 and on Day 120)
- Animals fasted: Yes (overnight)
- How many animals: All surviving animals
- Parameters examined: Glucose, urea nitrogen, creatinine, total protein, albumin, globulin, albumin/globulin ratio, cholesterol, total bilirubin, alanine aminotransferase, alkaline phosphatase, gamma glutamyl transferase, aspartate amino transferase, calcium, inorganic phosphorus, sodium, potassium and chloride.

URINALYSIS: Yes, urine specimens were collected on wet ice
- Time schedule for collection of urine: At each scheduled sacrifice (on Day 92 and on Day 120).
- Metabolism cages used for collection of urine: Yes, individual urine collection cages were used
- Animals fasted: Yes (overnight)
- Parameters examined: Specific gravity, pH, protein, glucose, ketones, bilirubin blood, urobilinogen, volume, microscopic examination of sediment, appearance/color and urine chemistry (Sodium, potassium, chloride, sodium excretion, potassium excretion and chloride excretion).

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Hand-held and open-field expanded clinical observations were done once prestudy and during Wk 4, 8, and 13 (in random order); elicited behavior observations were done once prestudy and during Wk 13 (in random order); and motor activity data were collected prestudy and during Wk 13. Expanded clinical observations and motor activity testing were done on a day other than that scheduled for the weekly detailed clinical observations
- Dose groups that were examined: 10 animals/sex/group (same animals were tested for all the three battery of functions)
- Battery of functions tested: Hand-held and open-field expanded clinical observations, elicited behavior observations, and motor activity. Additional findings were recorded as they were observed.

Oestrous cyclicity (parental animals):

Vaginal Cytology
All surviving females were evaluated daily for 21 consecutive days beginning after Week 10 (Days 71 through 91). Daily vaginal smears were prepared and examined to evaluate the stage of the estrous cycle for each female. For each animal, the estrous stage was documented each day for the 21 consecutive days. Examinations were done at approximately the same time each day and at a time corresponding to one that was after the completion of the expanded clinical observations.

Sperm parameters (parental animals):

Male Reproductive Assessment

Sample Collection
For motility and morphology assessment, the right vas deferens was excised and immediately placed in a petri dish containing 10 mL of a 1% bovine serum albumin dissolved in phosphate-buffered saline. The solution was prewarmed to approximately 38°C. A 3 to 4-minute period (minimum to maximum period, respectively) was allowed for the sperm to swim out.
The right epididymis was removed and divided in half by cross-sectioning through the middle. The tail end caudal section was placed on dry ice, transferred to PAI, and stored frozen at approximately -60 to -80°C for total sperm count. The left epididymis and remaining portion of the right epididymis were collected as described under Tissue Preservation.

Sperm Motility Evaluation
Following the swim-out period, a sperm sample was collected using a 100-micron-deep cannula and immediately loaded into a prewarmed stage of the Hamilton Thorne IVOS automated sperm analyzer. Five fields/animal were selected and stored as digital images. These images were analyzed for percent motility and transferred to optical media for permanent storage.

Sperm Morphology Evaluation
Sperm morphology was assessed with two slides of sperm stained with eosin for each male. A minimum of 200 sperm cells were evaluated.

Total Sperm Count Determination
At Pathology Associates, each frozen epididymis was thawed, and the caudal section was trimmed, weighed, and homogenized. A 100-microliter sample of the suspension was stained
with a dye that uniquely stains the sperm heads. A sample of the stained sperm was placed into a20-micron-deep glass slide and loaded into the analyzer. Twenty fields/animal were counted and reported, and adjusted for caudal epididymidal weight (taken following thawing at PAI).

Postmortem examinations (parental animals):

SACRIFICE: On Day 92, up to 15 animals/sex/group were fasted overnight, anesthetized with carbon dioxide, weighed, and exsanguinated. On Day 120, (after 4 Wk recovery), all surviving animals in Groups 1 and 4 were fasted overnight, anesthetized with carbon dioxide, weighed, and exsanguinated.

GROSS PATHOLOGY: Yes, each animal sacrificed, or died during the study was necropsied. The necropsy included an examination of the external features of the carcass; external body orifices; the abdominal, thoracic, and cranial cavities; organs; and tissues.
- Tissues from each animal (when present) were preserved in 10% neutral-buffered (except testis-preserved in Bouin’s fixative): Adrenal (2), aorta, brain (cerebrum, cerebellum, and medulla), cecum, cervix, colon (proximal and distal (2)), duodenum, epididymis (2), esophagus, eye (2), femur with bone marrow (articular surface, of the distal end), Harderian gland, heart, ileum (including Peyer’s patch), jejunum, kidney (2), lacrimal gland (exorbital), liver, lung with mainstem bronchi, lymph node (mandibular and mesenteric), mammary gland (females), nasal turbinates, ovary (2), pancreas, pituitary gland, prostate, rectum, salivary gland (mandibular (2)), sciatic nerve, seminal vesicle (2), skeletal muscle (thigh), Skin, spinal cord (cervical, thoracic, and lumbar), spleen, sternum with bone marrow, stomach (nonglandular and glandular), testis (2), thymus, thyroid with parathyroid, tissues with macroscopic changes, or alterations (i.e., gross lesions), tongue, trachea, urinary bladder, uterus with uterine horns vagina and zymbal’s gland.

HISTOPATHOLOGY: Yes, preserved tissues from each animal in the control and high-dose groups at the terminal sacrifice and those that died during the study were embedded in paraffin, sectioned, stained with hematoxylin and eosin, and examined microscopically. Macroscopic lesions were also examined microscopically from each animal in the low- and mid-dose groups.
Testes and epididymides from each terminal sacrifice male in the control and high-dose groups were examined microscopically by a board-certified veterinary pathologist:
- Following collection from each male at each scheduled sacrifice, the epididymis was separated from the testes. After adequate fixation in 10% neutral buffered formalin, each testis was sectioned transversely in half, and the cranial pole was sectioned for routine embedding in paraffin, histologic processing, and staining with hematoxylin and eosin. The remaining testicular tissue was stored in 70% ethyl alcohol.
- The epididymides were sectioned longitudinally and processed routinely for histologic evaluation. This longitudinal section should ensure that the head and mid portion of the epididymides were evaluated histologically. The remaining portions of the epididymides were stored in 10% neutral-buffered formalin.

PATHOLOGY PEER REVIEW:
A peer review of microscopic findings was performed by a board-certified veterinary pathologist at Pathology Associates (PAI). The peer review was performed according to the reviewer’s standard operating procedures.

Statistics:

The following statistical methods were used to analyze the body weight, body weight change, food consumption, motor activity, grip strength, nociceptive reflex, continuous clinical pathology, and organ weight data.
• Levene’s test (Levene, 1960; Draper and Hunter, 1969) was done to test for variance homogeneity. In the case of heterogeneity of variance at p < 0.05, rank transformation was used to stabilize the variance. Comparison tests took variance heterogeneity into consideration.
• One-way analysis of variance [(ANOVA, (Winer, 1971a)] was used to analyze data.
• If the ANOVA was significant (p < 0.05), Dunnett’s t-test (Dunnett, 1955, 1964) was used for control versus treated group comparisons.
The length of an estrus cycle for individual animals was determined by counting the number of days from a recording of estrus (including this first day of estrus) up to (but not including) the next recording of estrus that occurs after metestrus, diestrus, or proestrus are recorded. If estrus was not observed in an apparent cycle, then a metestrus recording that was preceded by either a diesturs or proestrus was considered the beginning of the cycle or a diestrus recording that was preceded by a proestrus was considered the beginning of the cycle. A full cycle ended with the day prior to a recording of estrus, with the exception that a recording of proestrus on the last (21st) day of estrus evaluations was considered the end a full cycle. This last proestrus recording was included as the final day of the last estrus cycle. The mean estrus cycle duration

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Other effects:

no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

The mean number of days in each stage and the mean estrus cycle duration were comparable
across groups.

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

The mean percent sperm motility, caudal epididymal sperm count, and sperm morphology were
not affected by treatment with GTS03849 at dose levels of 1, 4, and 20 mg/kg/day. No
biologically meaningful differences were observed between the study groups.

Reproductive performance:

no effects observed

Details on results (P0)

CLINICAL SIGNS AND MORTALITY: One Group 1 (vehicle control) male died on Day 72 from causes unrelated to treatment (severe, acute pyelonephritis). All other animals survived in apparent good health until scheduled sacrifice.
- No treatment-related clinical observations were noted during the treatment or recovery phase of the study. The few clinical observations noted for surviving animals (malocclusion, missing teeth, barbering, oral discharge, few feces, red haircoat, and sores or scabs) were unrelated to dose, were of small incidence and short duration, and therefore, were not considered treatment related.

BODY WEIGHT AND WEIGHT GAIN: Mean body weights and body weight changes of treated animals were comparable with controls throughout the treatment and recovery periods.

FOOD CONSUMPTION: No treatment-related effects on food consumption were noted during treatment and recovery.

OPHTHALMOSCOPIC EXAMINATION: There were no treatment-related ophthalmic observations

HAEMATOLOGY: Very few statistically significant or otherwise notable differences for clinical pathology test results were observed between the control and treated animals. None of the differences in the haematology parameters were attributable to treatment with test substance.

CLINICAL CHEMISTRY: Statistically significantly higher triglyceride concentration was observed in mid dose females at Wk 14. However, this variation was considered incidental, as the difference was extremely small (i.e., only 6 mg/dL), males were unaffected, and there were no correlative findings. The difference for triglyceride concentration was consistent with normal biological variation.

URINALYSIS: None of the differences in the urinalysis parameters were attributable to treatment with test substance.

NEUROBEHAVIOUR: No test substance-related effects on neurobehavioral assessment tests were apparent during treatment. Motor activity was significantly decreased at the 30-40 and 0-40 minute intervals for high dose group females. However, because values recorded during acclimation were also lower for high dose group females compared with controls and because a similar effect was not seen in males, these decreases were not considered to be toxicologically important.

ORGAN WEIGHTS: Terminal organ weight and organ to body weight ratio were not affected by treatment. Decreased mean absolute testicular and thymic weight values were observed in high dose group recovery males. However, there was no histological correlate to support this finding and were not attributed to the treatment.

GROSS PATHOLOGY: No treatment related differences were observed in macroscopic findings.

HISTOPATHOLOGY: NON-NEOPLASTIC: All histological findings were of the kind commonly found in laboratory rats of age and strain used in the study or were of singular occurrence or distributed (scattered) randomly among the groups without relation to dosage.

OTHER FINDINGS:
- Vaginal Cytology: The mean number of days in each stage and the mean estrus cycle duration was comparable across groups.
- Male Reproductive Assessment: The mean percent sperm motility, caudal epididymal sperm count, and sperm morphology were not affected by treatment. No biologically meaningful differences were observed between the study groups.

Effect levels (P0)

Key result

Dose descriptor:

NOAEL

Effect level:

20 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reproductive function (oestrous cycle)

reproductive function (sperm measures)

reproductive performance

Target system / organ toxicity (P0)

Critical effects observed:

no

Results: P1 (second parental generation)

Effect levels (P1)

Remarks on result:

not measured/tested

Target system / organ toxicity (P1)

Critical effects observed:

no

Results: F1 generation

Effect levels (F1)

Remarks on result:

not measured/tested

Target system / organ toxicity (F1)

Critical effects observed:

no

Overall reproductive toxicity

Reproductive effects observed:

no

Any other information on results incl. tables

Table1 :Sperm analysis parameters

	Group	1	2	3	4	Group (Recovery )	1	4
	Dose	0	1	4	20	Dose	0	20
Motility	Mean	83	78	87	82	Mean	85	80
	SD	18	19	10	11	SD	8	27
	N	14	14	15	15	N	5	5
Epididymal count (million sperm/gram)	Mean	904	833.2	965.8	918.6	Mean	464.4	390.6
	SD	110.6	323.7	205.1	162.1	SD	90.3	65.3
	N	14	15	15	15	N	5	5
SpermMorphology(%abnormal)	Mean	0.5	0.5	0.4	0.5	Mean	0.5	0.2
	SD	0.6	0.8	0.7	0.7	SD	0.7	0.3
	N	14	15	15	15	N	14	15

 

Applicant's summary and conclusion

Conclusions:

Based on the results of the study, the No Observed Adverse Effect Level (NOAEL) following oral gavage administration of N,N-bis (2-hydroxyethyl)-PPD Sulf to rats at doses of 0, 1, 4, or 20 mg/kg/day for 91 d was determined to be 20 mg/kg/day. No toxicity on reproductive apparatus or fertily was observed.
 

Executive summary:

Subchronic toxicity of N,N-bis (2-hydroxyethyl)-p-phenylenediamine sulfate was performed following OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents). Analysis on fertility parameters as sperm motility on male and vaginal cytology. Hence, this study was considered to be relevant for toxicity to reproduction assessment.

The study was designed to evaluate the toxicity of the test substance, when administered daily at dose levels of 0, 1, 4 and 20 mg/kg bw/day via oral gavage to Crl:CD(SD)IGS BR rats for 91 d and to assess the reversibility of any effects after a 4 Wk recovery period. A total of 140 Crl:CD(SD)IGS BR rats (70/sex) of 6-7 Wk age (source: Charles River Laboratories, Portage, Michigan), weighing 204-299 g (males) and 153-194 g (females) were housed individually, in suspended stainless-steel cages. The animals were maintained under standard laboratory conditions (temperature: 19-25°C, relative humidity: 30-70%, 10 or greater air changes/h, 12 h light/12 h dark cycle per d).

The animals were observed twice daily (a.m. and p.m.) for mortality, abnormalities, and signs of pain or distress. Cage side observations were made for each animal once daily. Detailed clinical observations were performed prior to treatment, on Day 1, weekly thereafter, and on the day of each scheduled sacrifice. Neurobehavioral observations were performed weekly; hand-held and open-field expanded clinical observations were done pre-study and during Weeks 4, 8, and 13; elicited behavior observations were done prestudy and during Wk 13; and motor activity data were collected pre-study and during Wk 13. Ophthalmic examinations were done prior to treatment and during Wk 13. Body weights were collected twice prior to treatment, on Day 1, and weekly thereafter. Food consumption data were measured weekly. Blood and urine samples for hematology, coagulation, clinical chemistry, urinalysis, and urine chemistry were collected at each scheduled sacrifice (on Day 92 and Day 120).

Furthermore, vaginal cytology and sperm analysis were performed.

There were no test substance-related effects on ophthalmic observations; effects on neurobehavioral assessment tests; effects on body weights or body weight changes; effects on food consumption; or effects on vaginal cytology. Test substance had no effect on clinical pathology test results.

The mean percent sperm motility, caudal epididymal sperm count, and sperm morphology were not affected by treatment.No biologically meaningful differences were observed between the study groups.

 

Based on the results of the study, the No Observed Adverse Effect Level (NOAEL) following oral gavage administration of N,N-bis (2-hydroxyethyl)-PPD Sulf to rats at doses of 0, 1, 4, or 20 mg/kg/day for 91 d was determined to be 20 mg/kg/day. No toxicity on reproductive apparatus or fertily was observed.