Registration Dossier

Administrative data

Description of key information

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).

Test substance was administered daily at dose levels of 0, 1, 4 and 20 mg/kg bw/day via oral gavage to rats for 91 d followed by 4 weeks recovery period.

No treatment-related clinical observations were noted during the treatment or recovery phase of the study. 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.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Referenceopen allclose all

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From Mar. 15, 2004 to Nov. 30, 2005
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study well documented, followed guideline, GLP
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Deviations:
no
GLP compliance:
yes
Remarks:
according to U.S. FDA and OECD principles of GLP
Limit test:
no
Species:
rat
Strain:
other: Crl:CD(SD)IGS BR
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Portage, Michigan.
- Age at study initiation: Approx. 6-7 Wk
- Weight at study initiation: 204-299 g (males) and 153-194 g (females)
- Fasting period before study: No
- Housing: Individually, in suspended stainless-steel cages. The animals were given various cage-enrichment devices as a form of environmental enrichment.
- Diet: Certified rodent diet (#8728C, Harlan Teklad); ad libitum (except when animals were fasted for clinical pathology or necropsy procedures).
- Water: Ad libitum
- Acclimation period: 7 d
- The manufacturer analyzed the diet for nutritional components and environmental contaminants. Water samples were routinely analyzed for specified microorganisms and environmental contaminants. No contaminants were known to be present in the diet or water at levels which might have interfered with result of this study.

ENVIRONMENTAL CONDITIONS
- Temperature: 19-25°C
- Humidity: 30-70%
- Air changes: 10 or greater air changes/h
- Photoperiod: 12 h light/12 h dark cycle per d

IN-LIFE DATES: From: Mar. 22, 2004 To: July. 19, 2004
Route of administration:
oral: gavage
Vehicle:
other: 0.2% w/v erythorbic acid in reverse osmosis water
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: For each dose formulation, the required amount of test substance was added to a volume of vehicle and mixed on a magnetic stirrer until the formulation appeared to be a solution. Additional vehicle was added to achieve the final volume. The formulations were transferred to individual amber glass containers for daily use.
All dose solutions were allowed to come to room temperature prior to dosing.
- Rate of preparation of formulation (frequency): Dose formulations were prepared approx. weekly
- Storage temperature of formulation: Stored in a refrigerator set to maintain 2 to 8°C, protected from light

VEHICLE
- Justification for use and choice of vehicle: Not reported
- Concentration in vehicle: 0.1, 0.4 and 2 mg/mL for 1.0, 4.0 and 20 mg/kg bw/day respectively.
- Amount of vehicle: 10 mL/kg, individual dose volume was based on the most recently recorded body weight.
- Lot/batch no. (Erythorbic acid): Sigma-Aldrich, Lot# 122K0539
- Purity (Erythorbic acid): 99.9%
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analyses were performed using high-performance liquid chromatography. The method was developed by the sponsor and modified and validated by Covance-Madison (Covance method MA-PG61-2050). For concentration determination, acceptance criteria for results of mean values were ±15% of the theoretical concentration.
1. Homogeneity:
Homogeneity of the test substance in vehicle was established on the first preparation used in this study. Duplicate samples (5 mL each) were taken from the top, middle, and bottom of the highest and lowest concentration dose solutions. Samples were maintained at room temperature protected from light and analyzed on the day of preparation. Mean results of the homogeneity analyses of the top, middle, and bottom locations of the low and high dose concentrations (0.1 and 2.0 mg/mL, respectively) 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.
2. Concentration verification:
Analyses of the concentration verification were performed from the first preparation used for dosing during weeks 2, 5, 9 and 13. Duplicate samples (5 mL each) were taken from the middle of each solution for concentration determination. For the first preparation used on study, the mean concentration obtained in the homogeneity determination for the middle sample from the lowest and highest concentration dose solution served as the concentration verification value. 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 Wk 13 analysis of the 0.1-mg/mL concentration did not meet acceptance criteria (within 15% of theoretical).
3. Stability:
The stability of the test substance in the vehicle was established in Covance 6114-461 for the concentration range used in the study. Results of stability analyses indicated that the formulations were stable in the vehicle for up to 7 d under refrigerated storage conditions.
Duration of treatment / exposure:
91 d; 5 animals/sex in control and high dose group underwent 91 d of treatment followed by at least 4 weeks of recovery.
Frequency of treatment:
Daily
Dose / conc.:
1 mg/kg bw/day (actual dose received)
Dose / conc.:
4 mg/kg bw/day (actual dose received)
Dose / conc.:
20 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
15 animals/sex/group; additional 5 animals/sex were included in control and high dose group as recovery group animals.
-Five extra animals/sex received in the animal shipment but not assigned to the study underwent both the pre-study
Expanded Clinical Observations and Motor Activity testing to establish baseline values in case replacement animals were needed.
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).
- Rationale for animal assignment: Post acclimation, animals were randomized into four groups using a computerized blocking procedure designed to achieve body weight balance with respect to treatment groups. After assignment to groups, the mean body weight for each group of each sex was not statistically different at the 5.0% probability level. At randomization, the coefficient of variation of the mean weight for each sex and group did not exceed 10%.
- Rationale for selecting satellite groups: 5 animals/sex were included in control and high dose group to assess the reversibility of any effects after a recovery period.
- Post-exposure recovery period in satellite groups: 4 Wk
- Assignment of animals: The animals were assigned into following control and treatment groups:
Group 1(vehicle control): 0 mg/kg bw/day
Group 2 (Low dose): 1 mg/kg bw/day
Group 3(Mid dose): 4 mg/kg bw/day
Group 4 (High dose): 20 mg/kg bw/day
Positive control:
No
Observations and examinations performed and frequency:
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.
Sacrifice and pathology:
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.
Other examinations:
ABSOLUTE AND RELATIVE ORGAN WEIGHT: At scheduled sacrifice, the following organs (when present) were weighed; paired organs were weighed together: adrenal (2), brain, epididymis (2), heart, kidney (2), liver, lung, ovary (2), pituitary gland, prostate, salivary gland (mandibular (2)), seminal vesicle (2), spleen, testis (2), thymus, thyroid (2) with parathyroid, uterus. Organ-to-body weight percentages and organ-to-brain weight ratios were calculated.

VAGINAL CYTOLOGY: Daily vaginal smears were prepared and examined to evaluate the stage of the estrous cycle for each female.
- Time schedule for examinations: Daily for 21 consecutive days beginning after Wk 10 (Days 71 through 91). For each animal, the estrous stage was documented each day for the 21 consecutive d.
- How many animals: All surviving females were evaluated

MALE REPRODUCTIVE ASSESSMENT: At each scheduled sacrifice, male reproductive assessment (sperm motility, sperm morphology, and total sperm count) was done by Pathology Associates (PAI).
The details on male reproductive assessment procedures are provided in the study report.
Statistics:
1. 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 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 was used to analyze data.
- If the ANOVA was significant (p < 0.05), Dunnett’s t-test was used for control versus treated group comparisons.
2. 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. The mean estrus cycle duration (i.e., number of days) was calculated for each animal. Group means and standard deviations for estrus cycle duration were calculated and reported.
For estrus cycle evaluations, the total number of days in each phase, the number of combined days in proestrus and estrus, and the number of combined days in metestrus and diestrus were calculated for each animal. Group means and standard deviations for these parameters were calculated and reported.
3. The following statistical methods were used to analyze the sperm motility, total count, and sperm morphology data:
- The data were analyzed by the Kruskal-Wallis H-test (nonparametric ANOVA).
- If the Kruskal-Wallis H-test was significant, the Wilcoxon-Mann-Whitney two-sample rank sum test was used to compare each treated group with the control group.
For each sex, Groups 2 through 4 were compared with Group 1 (control) at the 5%, two-tailed probability level.
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
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Behaviour (functional findings):
no effects observed
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Neuropathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
not examined
Details on results:
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.
Key result
Dose descriptor:
NOAEL
Effect level:
20 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: overall effects
Key result
Critical effects observed:
no
Conclusions:
Administration of N,N-bis (2-hydroxyethyl)-p-phenylenediamine sulfate to Crl:CD(SD)IGS BR rats at dose levels of 0, 1, 4 and 20 mg/kg bw/day by oral gavage over a period of 91 d did not elicit any toxic response at any of the dose levels tested.
The No Observed Adverse Effect Level (NOAEL) for subchronic oral toxicity of test substance was determined to be higher than 20 mg/kg bw/day.
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).

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 fed on certified rodent diet (#8728C, Harlan Teklad); ad libitum and acclimated to laboratory condition for a period of 7 d.

Dose formulations were prepared weekly, stored in a refrigerator set to maintain 2 to 8°C and protected from light. For each dose level, the required amount of test substance was added to a volume of vehicle (0.2% w/v erythorbic acid in reverse osmosis water) and mixed on a magnetic stirrer until the formulation appeared to be a solution.

Post acclimation, animals were randomized into four treatment and one vehicle control group (containing 15 animal/sex/ group each) using a computerized blocking procedure designed to achieve body weight balance with respect to treatment groups. An additional 5 animals/sex were included in control and high dose groups to assess the reversibility of any effects after a 4 Wk recovery period.

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. Vaginal cytology data were collected once daily for 21 consecutive days, beginning after Wk 10. 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).

 

On Day 92, up to 15 animals/sex/group were anesthetized, weighed, and necropsied. On Day 120, all surviving animals (recovery group) were anesthetized, weighed, and necropsied. Each animal sacrificed, or died during the study was necropsied, selected organs were weighed and selected tissues collected and preserved. At each scheduled sacrifice, male reproductive assessment (sperm motility, morphology, and count) was done by Pathology Associates (PAI). Microscopic examination of tissues was performed, and a pathology peer review was conducted.

 

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.

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 greater than 20 mg/kg/day.

 

This repeated dose toxicity study is classified as acceptable, and satisfies the guideline requirements of OECD 408 method.

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From Jan. 19, 2004 to Oct. 21, 2004
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study well documented, followed method comparable to guideline with deviation, GLP
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Deviations:
yes
Remarks:
14 d range finding study, no histopathology examination was conducted
GLP compliance:
yes
Remarks:
according to U.S. FDA and OECD principles of GLP
Limit test:
no
Species:
rat
Strain:
other: Crl:CD(SD)IGS BR rats
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Portage, Michigan.
- Age at study initiation: 47-53 d
- Weight at study initiation: 211-281 g (males); 157-196 g (females)
- Fasting period before study: No
- Housing: Individually, in suspended stainless-steel cages.
- Diet: Certified rodent diet (#8728C, Harlan Teklad); ad libitum (except when animals were fasted for clinical pathology or necropsy procedures).
- Water: Ad libitum
- Acclimation period: 9 d
- The manufacturer analyzed the diet for nutritional components and environmental contaminants. Water samples were routinely analyzed for specified microorganisms and environmental contaminants. No contaminants were known to be present in the diet or water at levels which might have interfered with result of this study.

ENVIRONMENTAL CONDITIONS
- Temperature: 19-25°C
- Humidity: 30-70%
- Air changes: Not reported
- Photoperiod: 12 h light/12 h dark cycle per d
The animals were given various cage-enrichment devices as a form of environmental enrichment.

IN-LIFE DATES: From: Jan. 28, 2004 To: Feb. 12, 2004
Route of administration:
oral: gavage
Vehicle:
other: 0.2% w/v erythorbic acid in reverse osmosis water.
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: For each dose formulation, the required amount of test substance was added to a volume of vehicle and mixed on a magnetic stirrer until the formulation appeared to be a solution. Additional vehicle was added to achieve the final volume. The formulations were transferred to individual amber glass containers for daily use.
- Rate of preparation of formulation (frequency): Dose formulations were prepared three times for this study
- Storage temperature of formulation: Stored in a refrigerator set to maintain 2 to 8°C, protected from light

VEHICLE
- Justification for use and choice of vehicle: Not Reported
- Concentration in vehicle: 0, 5, 10, 20, and 40 mg/mL for 0, 50, 100, 200 and 400 mg/kg bw. However, on Day 4 the dose for 50 and 100 mg/kg bw were reduced to 5 and 10 mg/kg bw respectively. Therefore after Day 4 the concentration in vehicle were revised to:
0, 0.5, 2.5, 20 and 40 mg/mL
- Amount of vehicle: 10 mL/kg, individual dose volume was based on the most recently recorded body weight. Dose solutions were allowed to come to room temperature prior to dosing
- Lot/batch no (Erythorbic acid): Sigma-Aldrich, Lot# 122K0539
- Purity: 100% pure Erythorbic acid was dissolved in reverse osmosis water.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analyses were performed using high-performance liquid chromatography. The method was developed by the sponsor and modified and validated by Covance-Madison (Covance method MA-PG61-2050). For concentration determination, acceptance criteria for results of mean values were ±15% of the theoretical concentration.
- Homogeneity of the test article in vehicle was established on the first preparation used in this study. Duplicate samples (5 mL each) were taken from the top, middle, and bottom of the highest and lowest concentration dose solutions. Samples were maintained at room temperature protected from light and analyzed on the day of preparation. Mean results of the homogeneity analyses of the top, middle, and bottom locations of the low and high dose concentrations (0.5 and 40 mg/mL, respectively) ranged from 97.8 to 98.8% and from 93.0 to 93.4% of theoretical, respectively, indicating that the mixing procedure resulted in homogeneous preparations.
- Analyses of the concentration verification were performed for each dose preparation. Duplicate samples (5 mL each) were taken from the middle of each solution for concentration determination. For the first preparation used on study, the mean concentration obtained in the homogeneity determination for the middle sample from the lowest and highest concentration dose solution served as the concentration verification value. Results of the concentration verification assays from all concentrations indicated that animals received the intended doses.
- The stability of the test article in the vehicle was established in Covance 6114-461 for the concentration range used in the study. Results of stability analyses indicated that the formulations were stable in the vehicle for up to 7 days under refrigerated storage conditions.
Duration of treatment / exposure:
14 d
Frequency of treatment:
Daily (with the exception that no dosing was performed on Day 3, dosing resumed from Day 4 with reduction in dose levels)
Remarks:
Due to toxicity and mortality observed on Day 1 and 2 of treatment, dose levels were reduced for groups 50 and 100 mg/kg bw/day to 5 and 25 mg/kg/day, respectively
Remarks:
Due to toxicity and mortality observed on Day 1 and 2 of treatment, dose levels were reduced for groups 50 and 100 mg/kg bw/day to 5 and 25 mg/kg/day, respectively
Dose / conc.:
200 mg/kg bw/day (actual dose received)
Dose / conc.:
400 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
5 animals per sex per dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Not reported
- Rationale for animal assignment: Post acclimation, animals were randomized into five groups using a computerized blocking procedure designed to achieve body weight balance with respect to treatment groups. At randomization, the coefficient of variation of the mean weight for each sex and group did not exceed 10%.
- Rationale for selecting satellite groups: No satellite group were included in the study
- Assignment of animals: The animals were assigned into following groups in the study report:
Group 1(Vehicle control): 0 mg/kg bw/day
Group 2 (Low): 50 mg/kg bw/day
Group 3 (Mid-low): 100 mg/kg bw/day
Group 4(Mid-high): 200 mg/kg bw/day
Group 5 (High): 400 mg/kg bw/day
Due to toxicity and mortality in the two highest dose groups (group 4 and 5), animals were not dosed on Day 3. Dosing resumed on Day 4 with a reduction in dose levels in the two lowest dose groups. Dose for Groups 2 and 3 were reduced to 5 and 25 mg/kg/day, respectively.
Positive control:
No
Observations and examinations performed and frequency:
MORTALITY/MORBIDITY: Yes
- Time schedule: Twice daily (a.m. and p.m.)

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily (a.m. and p.m.)
- Cage side observation included: Abnormalities, and signs of pain or distress

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, stereotypies (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 and on Days 1, 3, 8, 10, and 15

BODY WEIGHT: Yes
- Time schedule: Prior to treatment and on Days 1, 3, 8, 10, and 15

FOOD CONSUMPTION: Yes, food consumption data were collected for Days 1 to 3, 3 to 8, 8 to 10, and 10 to 15
- Food consumption for each animal determined and mean daily diet consumption calculated as mg food/kg body weight/day: Yes

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION AND COMPOUND INTAKE: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes, blood samples were collected from a jugular vein of animals.
- Time schedule for collection of blood: Day 16, prior to the scheduled sacrifice
- Anaesthetic used for blood collection: No, the animals were unanesthetized
- Anticoagulants used: Yes (potassium EDTA) and sodium citrate for hematology and coagulation samples respectively
- Animals fasted: Yes (overnight)
- How many animals: All surviving animals of group 1, 2 and 3. Blood was also collected from one animal sacrificed at an unscheduled interval.
- 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 were collected from a jugular vein of unanesthetized animals.
- Time schedule for collection of blood: Day 16, prior to the scheduled sacrifice
- Animals fasted: Yes (overnight)- How many animals: All surviving animals of group 1, 2 and 3. Blood was also collected from one animal sacrificed at an unscheduled interval.
- 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: Day 16, Prior to each clinical sampling
- Metabolism cages used for collection of urine: Yes, individual urine collection cages were used
- Animals fasted: Yes (overnight)
- How many animals: All surviving animals of group 1, 2 and 3
- Parameters examined: Specific gravity, pH, protein, glucose, ketones, bilirubin blood, urobilinogen, volume, microscopic examination of sediment and appearance/color

NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
SACRIFICE: On Day 16, all surviving animals were weighed, anesthetized with sodium pentobarbital, and exsanguinated.

GROSS PATHOLOGY: Yes, all animals found dead or sacrificed during the study or at scheduled necropsy were subjected to a gross postmortem examination. Animals were necropsied in random order to eliminate temporal bias. The necropsy included an examination of the external features of the carcass; external body orifices; the abdominal, thoracic, and cranial cavities; organs; and tissues.

HISTOPATHOLOGY: No histopathology examination was conducted.
Other examinations:
Absolute and relative organ weight: At scheduled sacrifice, the following organs (when present) were weighed; paired organs were weighed together: adrenal (2), brain, epididymis (2), heart, kidney (2), liver, spleen, testis (2), and thymus.
Organ-to-body weight percentages and organ-to-brain weight ratios were calculated.
Statistics:
The observed values for body weights, body weight changes, food consumption, continuous clinical pathology data, and organ weight data were evaluated statistically.
If Levene’s test for variance homogeneity was not significant (p > 0.05), one-way analysis of variance (ANOVA) was performed on the observed values.
If Levene’s test was significant (p < 0.05), ANOVA was done on the rank transformed data. Post hoc Dunnett’s t-test was used for control versus treated group mean comparison, incorporating transformations when necessary.
Controls versus treated group comparisons (Groups 2 through 5 versus Group 1) were evaluated at the 5.0%, two-tailed probability level. Only data collected on or after the first day of treatment were analyzed statistically.
Data for each sex were analyzed separately.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Groups 3, 4 and 5
Mortality:
mortality observed, treatment-related
Description (incidence):
Groups 3, 4 and 5
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Groups 3
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Groups 3
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
Groups 3
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Group 3
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Details on results:
CLINICAL SIGNS AND MORTALITY: All animals from dose levels 200 or 400 mg/kg/day died or were sacrificed in a moribund condition on Day 2 or 3. The death or moribund condition of the animals was attributed to test substance effects. Clinical observations for these animals included hunched and thin appearance, tremors, ataxia, hypoactivity, vocalization, sternal recumbency, low carriage, tiptoe and swaying gate, oral and nasal discharge, discolored urine, few feces, ocular discharge, dilated pupils, protruding eyes, periorbital squinting, ocular fasciculation, altered respiration, pale, red haircoat, rough haircoat, and cold to touch.
- One male given 100 mg/kg/day was found dead on Day 3 and one male given 100/25 mg/kg/day was sacrificed in a moribund condition on Day 5. The overt toxicity for the latter animal was attributed to effects of test substance administration at 100 mg/kg. There were no remarkable clinical observations for the 100 mg/kg animal that died on Day 3. Clinical observations for the animal sacrificed in a moribund condition included hunched appearance, tremors, hypoactivity, few feces, altered respiration, and red haircoat.
- All control animals, males and females given 50/5 mg/kg/day, and females given 100/25 mg/kg/day survived until scheduled sacrifice.
- Test substance-related clinical observations noted on Day 3 for surviving animals given 100 mg/kg/day included few feces for males and females and thin appearance, mild tremors, and ocular fasciculation for females. Following a reduction in dose level to 25 mg/kg/day, test article-related clinical observations included thin appearance, few feces, and rough haircoat for males and females; tremors, hunched appearance, hypoactivity, altered respiration and red haircoat for males only; and swaying gait for females only.
- There were no remarkable clinical observations for animals given 50/5 mg/kg/day.

BODY WEIGHT AND WEIGHT GAIN: Due to mortality, body weight data could not be evaluated for animals of dose levels 200 and 400 mg/kg bw/day (Groups 4 and 5). Mean body of males and females weights of Group 3 (100 mg/kg bw/day) were significantly decreased on Day 3. Following the reduction in dose level to 25 mg/kg/day, there was a notable increase in body weight, indicating partial recovery. However, due to the lack of any test article-related effects on body weight or body weight gains for animals given 50 mg/kg/day (Group 2), effects on Group 3 animals were attributed to test article administration at 100 mg/kg/day.

FOOD CONSUMPTION: A decrease in mean food consumption was observed during Days 1 to 3 for Group 3 animals given 100 mg/kg/day and for Days 3 to 8 for Group 3 animals given 25 mg/kg/day. Following the decrease in dose level on Day 4, mean food consumption for Days 8 to 10 and 10 to 15 were similar to controls.

HAEMATOLOGY: There were relatively few statistically significant or otherwise notable differences for clinical pathology test results at Day 16 between the control and treated animals. Statistically significant differences included mildly lower red blood cell count, hemoglobin, hematocrit, and glucose and moderately higher absolute reticulocyte count for males given 100/25 mg/kg/day. Females given 100/25 mg/kg/day were not similarly affected.
- There were no differences considered test substance-related for animals given 50/5 mg/kg/day. Higher absolute reticulocyte count for males given 100/25 mg/kg/day was considered a normal regenerative response to the reduced circulating red blood cell mass, but a mechanism for the reduced red blood cell mass was undetermined.

CLINICAL CHEMISTRY: There were no clinical chemistry observations attributable to the treatment with test substance.

URINALYSIS: There were no urinalysis observations attributable to the treatment with test substance.

ORGAN WEIGHTS: Among females given 100/25 mg/kg/day, absolute and relative kidney weights were increased. In males given the same dose, the kidney-to-body weight percentage (but not the other comparisons) was increased. The increased kidney weights were regarded as test article-related in females and likely test substance-related in males. All other statistically significant changes were not considered test substance-related.

GROSS PATHOLOGY: There were no macroscopic observations attributed to the test substance among any of the animals.

HISTORICAL CONTROL DATA: No historical control data were reported.
Dose descriptor:
NOAEL
Effect level:
5 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Mortality, clinical findings, Body weight food consumption and organ weight
Critical effects observed:
not specified
Conclusions:
14 d oral range finding study of N,N-BIS (2HYDROXYETHYL)-PPD SULF to Crl:CD(SD)IGS BR rats revealed a NOAEL of 5 mg/kg bw/day (based on revised dose levels).
The dose levels initially tested were 0, 50, 100, 200 and 400 mg/kg bw/day. Due to toxicity and mortality in the two highest dose groups (400 and 200 mg/kg bw/day) during first 2 days of treatment , the dose levels of two lowest dose groups were reduced to 5 and 25 mg/kg bw from 50 and 100 mg/kg bw respectively.
Executive summary:

14-Day Oral Gavage Range Finding Toxicity study of N,N-BIS (2HYDROXYETHYL)-PPD SULF was performed by following method comparable to the OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents).

The study was designed to evaluate the repeated dose toxicity of the test substance, when administered daily via oral gavage at dose levels of 0, 50, 100 200 and 400 mg/kg bw/day to Crl:CD(SD)IGS BR rats for at least 14 d.

 

Due to toxicity and mortality in the two highest dose groups (400 and 200 mg/kg bw/day) during first 2 days of treatment, animals were not dosed on Day 3. Dosing resumed on Day 4 with a reduction in dose levels in the two lowest dose groups from 50 to 5 mg/kg bw/day and 100 to 25 mg/kg bw/day respectively. The animals received the following dose levels:

Group 1: 0 mg/kg bw/day (vehicle control)

Group 2: 50 or 5 mg/kg/day

Group 3: 100 or 25 mg/kg/day

Group 4: 200 mg/kg bw/day

Group 5: 400 mg/kg bw/day.

 

A total of 50 Crl:CD(SD)IGS BR rats (25/sex) of 47-53 d age (source: Charles River Laboratories, Portage, Michigan), weighing 211-281 g (males); 157-196 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%, 12 h light/12 h dark cycle per d). The animals were fed on certified rodent diet (#8728C, Harlan Teklad); ad libitum and acclimated to laboratory condition for a period of 9 d.

Dose formulations were prepared three times for this study and stored in a refrigerator set to maintain 2 to 8°C and protected from light. For each dose level, the required amount of test substance was added to a volume of vehicle (0.2% w/v erythorbic acid in reverse osmosis water) and mixed on a magnetic stirrer until the formulation appeared to be a solution. Post acclimation, animals were randomized into four treatment and one vehicle control group (containing 5 animal/sex/ group each) using a computerized blocking procedure designed to achieve body weight balance with respect to treatment groups.

During study period animals were observed for mortality, clinical signs of toxicity, detailed clinical observations, body weights, and food consumption data.

 

Blood and urine samples for hematology, coagulation, clinical chemistry, and urinalysis were collected at the scheduled sacrifice. On Day 16, surviving animals were anesthetized, weighed, exsanguinated, and necropsied. At necropsy, macroscopic observations were recorded, selected organs weighed, and selected tissues collected and preserved. Animals that died during test or were sacrificed at unscheduled intervals were also necropsied, but organ weights were not recorded.

 

All animals given 200 or 400 mg/kg/day died or were sacrificed in a moribund condition on Day 2 or 3. One male given 100 mg/kg/day was found dead on Day 3 and a second male was sacrificed in a moribund condition on Day 5. The death or moribund condition of the animals was attributed to test substance effects. Test substance-related clinical observations for surviving animals given 100 or 25 mg/kg/day included thin appearance, mild tremors, few feces, and rough haircoat for males and females and ocular fasciculation and swaying gait for females only. There were no remarkable clinical observations for control animals or animals given 50 or 5 mg/kg/day.

 

Due to mortality, body weight data could not be evaluated for animals of dose levels 200 and 400 mg/kg bw/day. Treatment related decreases in body weight/body weight gain and food consumption were observed in 100 or 25 mg/kg bw/day animals. Among females given 100 or 25 mg/kg/day, absolute and relative kidney weights were increased. In males given the same dose, the kidney-to-body weight percentage (but not the other comparisons) was increased.

 

There were no macroscopic observations attributed to the test substance among any of the animals.

Doses of 100 mg/kg/day and higher exceeded the maximum-tolerated dose based on mortality and moribundity for all animals given 200 or 400 mg/kg/day and two males given 100 mg/kg/day.

 

Based on the revised dose groups, the administration of N,N-BIS (2HYDROXYETHYL)-PPD SULF to Crl:CD(SD)IGS BR rats at dose levels of 0, 5, 25, 200 and 400 mg/kg bw/day by oral gavage over a period of 14 d revealed a NOAEL of 5 mg/kg bw/day.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
20 mg/kg bw/day
Study duration:
subchronic
Species:
rat

Additional information

A ninety-one day oral gavage subchronic study with a twenty-eight day recovery period was conducted in Crl:CD®(SD)IGS BR rats. Following an appropriate fourteen day range finding study involving oral gavage administration of N,N-Bis(2-hydroxyethyl)-p-phenylenediamine sulfate at doses of 50 mg/kg/d (reduced to 5 mg/kg/d on day 4), 100 mg/kg/d (reduced to 25 mg/kg/d on day 4), 200 and 400 mg/kg/d, N,N-Bis(2-hydroxyethyl)-p-phenylenediamine sulfate was administered daily at doses of 1, 4 and 20 mg/kg/d by oral gavage in the subchronic study. No treatment related effects were noted in any dose group at the end of the treatment and recovery phases and as such N,N-Bis(2-hydroxyethyl)-p-phenylenediamine sulfate was concluded to have a no observed effect level (NOEL) of up to 20 mg/kg/d.

Justification for classification or non-classification

Based on the results of the key 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. Hence, based on the results of the key studies and according to CLP criteria, the registered substance was not classified for STOT-RE.