2-[(2-nitrophenyl)amino]ethanol

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 30 March 2004 to 11 August 2004

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:

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

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

2-[(2-nitrophenyl)amino]ethanol

EC Number:

225-555-8

EC Name:

2-[(2-nitrophenyl)amino]ethanol

Cas Number:

4926-55-0

Molecular formula:

C8H10N2O3

IUPAC Name:

2-[(2-nitrophenyl)amino]ethanol

Test material form:

solid: particulate/powder

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Supplied by the Sponsor, lot No. 17
- Expiration date of the lot/batch: 11 December 2005
- Purity test date: 11 December 2002

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature
- Stability: The test article preparation in PEG 400 was stable for at least 24 hours at room temperature and for at least 15 days at 5±3ºC. The values obtained were within -2.3 to 0.4% of nominal for the 1 and 10 mg/mL formulations.
- Homogeneity: The results of the homogeneity analyses indicated that the test article formulations were homogeneous. The values obtained were -4.2% and -2.3% average bias for the 1 and 10 mg/mL formulations, respectively.
- Concentration: The results of the concentration verification analyses conducted during study weeks 0, 1, 4, 8 and 12 demonstrated that target dose concentrations were achieved. The range of values obtained for each concentration during this study were -13.2% to 0.7% (1 mg/mL), -1.1% to 2.5% (4 mg/mL) and -2.5% to 0.9% (10 mg/mL).

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
For each test article dose group, a specified amount of the test article was weighed into a beaker. A sufficient quantity of the vehicle control material was added to the beaker and hand stirred. The mixture was then brought to the appropriate concentration and volume with the vehicle control
material and stirred overnight with stir bars at room temperature. The beaker was wrapped in aluminum foil to protect it from light. Dose materials were prepared weekly, dispensed into amber glass containers for seven days of dosing and stored refrigerated at approximately 2 to 8ºC. The dose preparations were removed from the refrigerator and stirred while equilibrating to room temperature prior to dosing. The dosing preparations were stirred continuously prior to and during dosing. The appearance of each test article dosing preparation was documented. The group 1 preparation was a clear colorless solution, group 2 was a yellow solution and groups 3 and 4 were orange solutions.

Test animals

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

The laboratory rat was selected as the animal model for this study since it is one of the species recommended by the regulatory agencies for toxicity testing.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories (Michigan)
- Females (if applicable) nulliparous and non-pregnant: not specified
- Age at study initiation: 6 weeks
- Weight at study initiation: 147 to 187 grams (males) and 140 to 164 grams (females)
- Fasting period before study:not specified
- Housing: suspended stainless steel cage
- Diet (e.g. ad libitum): PMI Nutrition International Certified Rodent Meal® #5002 was provided ad libitum
- Water (e.g. ad libitum): Municipal tap water was provided to the animals ad libitum
- Acclimation period: 7 days

DETAILS OF FOOD AND WATER QUALITY: The feed was analyzed by the supplier for nutritional components and environmental contaminants. Water supplying the facility is analyzed for contaminants according to the Testing Facility's Standard Operating Procedures. The results of the feed and water analyses are maintained at the Testing Facility. Within generally accepted limits, there were no contaminants in the diet or drinking water which would interfere with the conduct of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 25 deg Celsius
- Humidity (%): 30 to 70% Relative humidity
- Air changes (per hr): 10 to 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12-hour light/12-hour dark cycle

IN-LIFE DATES: From: 30 March 2004 To: 11 August 2004

Administration / exposure

Route of administration:

oral: gavage

Details on route of administration:

The oral route of exposure was selected to maximize the potential to identify potential health hazards which may be associated with repeated exposure.

Vehicle:

polyethylene glycol

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
For each test article dose group, a specified amount of the test article, GTS03975, was weighed into a beaker. A sufficient quantity of the vehicle control material was added to the beaker and hand stirred. The mixture was then brought to the appropriate concentration and volume with the vehicle control material and stirred overnight with stir bars at room temperature. The beaker was wrapped in aluminum foil to protect it from light. Dose materials were prepared weekly, dispensed into amber glass containers for seven days of dosing and stored refrigerated at approximately 2 to 8ºC. The dose preparations were removed from the refrigerator and stirred while equilibrating to room temperature prior to dosing. The dosing preparations were stirred continuously prior to and during dosing. The
appearance of each test article dosing preparation was documented. The group 1 preparation was a clear colorless solution, group 2 was a yellow solution and groups 3 and 4 were orange solutions.

VEHICLE
- Justification for use and choice of vehicle (if other than water): No justification
- Concentration in vehicle: 1, 4, 10 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg
- Lot/batch no. (if required): Lot No. SP0092 Lot No. ST0645 from Spectrum Manufacturing Corp.
- Purity: not specified

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Each test article dosing mixture and the vehicle control material were analyzed for test article concentration during weeks 0, 1, 4, 8 and 12. Quadruplicate samples (5 mL each) were taken from the middle of each solution and stored refrigerated, protected from light. Two of the four samples from each solution were analyzed on the day after preparation. The unused set of samples from each solution was discarded at the end of the study.

The results of the concentration verification analyses conducted during study weeks 0, 1, 4, 8 and 12 demonstrated that target dose concentrations were achieved. The range of values obtained for each concentration during this study were -13.2% to 0.7% (1 mg/mL), -1.1% to 2.5% (4 mg/mL) and -2.5% to 0.9% (10 mg/mL).

Duration of treatment / exposure:

91 Days

Frequency of treatment:

Once daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

5 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

20 mg/kg bw/day (nominal)

Doses / concentrations 4

Dose / conc.:

50 mg/kg bw/day (nominal)

No. of animals per sex per dose:

15 animals per sex per dose were used. 5 animals per sex for control and high dose level were used as satellite group (28 days of recovery)

Control animals:

yes

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
Dosage levels were selected based on the results of a 14-day range-finding study in rats conducted (Study No. 3029.2264 ). The goal of the dose level selection was to establish satisfactory safety margins for various levels of potential human exposure to the test article. All dose levels were expected to produce no significant adverse effects.

- Rationale for animal assignment (if not random): Animals determined to be suitable test subjects were assigned to groups using a computer randomization program. The program
ranked the animals according to day -5 body weights and randomly assigned the rats to study groups. After assignment to study groups, the mean body weight for each group of each sex was not statistically different at the 5.0% probability level and the standard deviation of the mean weight for each sex and group did not exceed 15%.

- Rationale for selecting satellite groups: Not specified

- Post-exposure recovery period in satellite groups: Five animals/sex (the last five in each group) underwent at least 91 days of treatment followed by at least 28 days of recovery.

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: performed twice daily, in the
morning and afternoon. Cage-side observations were performed on all animals approximately 30 to 90 minutes after dosing.
- Cage side observations checked : General health/mortality and moribundity checks

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: -5, 0, 6, 13, 20, 27, 34, 41, 48, 55, 62, 69, 76 (females only), 77 (males only), 83 and 90

BODY WEIGHT: Yes
- Time schedule for examinations: Individual body weights were recorded prior to in-life initiation on day -5 for randomization purposes. During the study, individual body weights were recorded on days 0, 6, 13, 20, 27, 34, 41, 48, 55, 62, 69, 76, 83 and 90. For the recovery animals, individual body weights were recorded on days 97, 104, 111 and 118. A
final (fasted) body weight was obtained from each animal prior to scheduled euthanasia.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
During the study, individual food consumption was measured on days 0, 6, 13, 20, 27, 34, 41, 48, 55, 62, 69, 76, 83 and 90. For the recovery animals, individual food consumption was measured on days 97, 104, 111 and 118. Calculated as Grams of food/Animal/Day

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: once prior to in-life initiation (days -6/-7) and on days 84 (females) and 85 (males).
- Dose groups that were examined:on all animals

HAEMATOLOGY: Yes
- Time schedule for collection of blood: days 91 and 92 for males, day 92 for females and day 119 for recovery
animals
- Anaesthetic used for blood collection: Yes (light isoflurane inhalation)
- Animals fasted: Yes
- How many animals: All animals (140)
- Parameters checked:
Erythrocyte count (RBC)
Hematocrit (HCT)
Hemoglobin concentration (Hgb)
Mean corpuscular hemoglobin (MCH)
Mean corpuscular hemoglobin concentration (MCHC)
Mean corpuscular volume (MCV)
Platelet count
Reticulocyte count
Total and differential leukocyte counts (including red blood cell morphology)
Activated partial thromboplastin time (APTT)
Prothrombin time (PT)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: days 91 and 92 for males, day 92 for females and day 119 for recovery
animals
- Anaesthetic used for blood collection: Yes (light isoflurane inhalation)
- Animals fasted: Yes
- How many animals: All animals (140)
- Parameters checked:
Alanine aminotransferase (ALT)
Albumin
Albumin/globulin ratio (calculated)
Alkaline phosphatase (ALP)
Aspartate aminotransferase (AST)
Blood creatinine
Blood urea nitrogen (BUN)
Calcium
Cholesterol
Electrolytes (sodium, potassium and chloride)
Gamma glutamyltransferase (GGT)
Globulin (calculated)
Glucose
Phosphorus
Total bilirubin
Total serum protein

URINALYSIS: Yes
- Time schedule for collection of urine: days 91 and 92 for males, day 92 for females and day 119 for recovery
animals
- Metabolism cages used for collection of urine: No : During urine collection, each rat was individually housed in a stainless steel urine collection cage equipped with a water bottle with a ball bearing sipper tube.
- Animals fasted: Yes
- Parameters checked :Bilirubin (qualitative)
Blood (qualitative)
Glucose (semi-qualitative)
Gross appearance (qualitative)
Ketones (semi-quantitative)
Leukocytes (qualitative)
Microscopic examination of spun deposit (semi-quantitative)
Nitrites (qualitative)
pH (semi-quantitative)
Protein (semi-quantitative)
Specific gravity (quantitative)
Urobilinogen (semi-quantitative)
Total volume (quantitative)

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: performed prior to dosing on days 26 and 40
- Dose groups that were examined: 10 rats/sex/group
- Battery of functions tested: sensory activity / grip strength / motor activity

IMMUNOLOGY: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes

HISTOPATHOLOGY: Yes

Fresh organ weights were obtained at scheduled euthanasia for the adrenal glands, brain, epididymides, heart, kidneys, liver, lungs, ovaries, pituitary gland, prostate, salivary gland (mandibular), seminal vesicles, spleen, testes, thymus, thyroid (with parathyroid) and uterus. Paired organs were weighed together. With the exception of the bone marrow smear, the following organs and tissues obtained at necropsy were preserved in 10% neutral buffered formalin for possible histopathological examination:

Accessory genital organs (epididymides, seminal vesicles and prostate or uterus
with uterine horns and vagina)
Adrenals
All gross lesions
Aorta
Axillary lymph node
Bone marrow smear (femur)
Brain (including sections of medulla/pons, cerebellar cortex and cerebral cortex)
Cecum
Cervix
Colon (proximal and distal)
Duodenum
Esophagus
CRL-Ohio Study No. 3029.2266 (25)
Sponsor Study Reference No. 2747-53907
Exorbital lachrymal glands
Eyes
Femur (including articular surface of distal end) and bone marrow
Harderian gland
Heart
Ileum
Jejunum
Kidneys
Liver (three sections collected)
Lungs (infused with formalin) with bronchi
Mammary gland
Mandibular lymph node
Mesenteric lymph node
Nasal turbinates
Ovaries
Pancreas
Pituitary
Peyer’s patches (3 separate sections from ileum)
Rectum
Salivary gland (mandibular)
Sciatic nerve
Skeletal muscle (thigh)
Skin
Spinal cord (cervical, midthoracic and lumbar)
Spleen
Stomach (glandular and nonglandular)
Sternum with bone marrow
Testes (preserved in Bouin’s fixative)
Thymus
Thyroids/parathyroids
Tongue
Trachea
Urinary bladder
Zymbal’s gland

Other examinations:

OTHER:
- Estrous Cycle Determinations and Analysis
Estrous cycle length and normality were evaluated by daily vaginal smears from all females on days 63 through 83. The evaluations were performed at approximately the same time each day and were performed following FOB assessments. 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 were calculated and reported.

-Male Reproductive Assessment
On days 91, 92 and 119, sperm was collected from the vas deferens of each male rat following euthanasia for assessment of sperm enumeration, motility and morphology. Sperm analysis was performed with the aid of a computer-assisted sperm analyzer (Hamilton-Thorne IVOS 10). For motility and morphology assessment, the right vas deferense 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 was allowed for the sperm to swim out. Following the swim-out period, a sample of sperm was collected using a 100-micron-deep cannula and immediately loaded into a prewarmed stage of the Hamilton-Thorne IVOS 10 automated sperm analyzer. Five fields/animal were selected and stored as digital images. These images were analyzed for percent
motility. Sperm morphology was assessed with two slides of sperm stained with eosin for each male. A minimum of 200 sperm cells were evaluated.

Statistics:

Body weights, body weight gain, food consumption and parametric and count FOB data were analyzed by One-Way Analysis of Variance (ANOVA) . If significance was detected with ANOVA (p<0.05), pair-wise group comparison was performed using the Tukey-Kramer test. Ranked FOB data were analyzed by Kruskal-Wallis non-parametric ANOVA, followed by Dunn’s test, when p<0.05. Descriptive (categorical) and quanta FOB data were analyzed using a multiple Fisher Exact test for group by group comparison to the control group. Absolute and relative organ weights and clinical pathology data were analyzed for homogeneity of variance using Levene’s test. If significance was detected with Levene’s test (p<0.01), multiple group comparison was performed using the Kruskal-Wallis non-parametric ANOVA, followed by Dunn’s test, when p<0.05. If significance was not detected with Levene’s test, parametric procedures were used to analyze the data, i.e., ANOVA followed by Tukey-Kramer test when p<0.05. The number of females cycling normally, as well as the number of females with all stages of estrus, were compared among the groups by R x C chi-square test, followed by Fisher’s Exact test for group-wise comparisons to the control group, when appropriate. Estrus cyclicity was analyzed by ANOVA followed by Dunnett’s test for group-wise comparisons to the control group, when appropriate. Summary data tables present group mean and standard deviation (S.D.) values, where appropriate. Statistical ignificance was reported at three levels of alpha: p<0.05, p<0.01 and p<0.001.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Clinical findings in the treated animals were consistent with oral administration of a chemical dye and generally included dark yellow staining on tail; dark yellow/orange colored urine; dark yellow colored urine on litter paper; and yellow staining on nose/mouth
area, forelimbs, ventral region and anogenital/urogenital area.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

One group 4 male (#665) was euthanized moribund on day 87. Notable clinical signs prior to death included cool to touch, extremities pale in color, eye(s) appear pale, dark material around nose, yellow staining on entire body, dark yellow staining on tail, urine stain, dark yellow colored urine on litter paper, slight fecal stain, no feces, prostration, tremors, labored breathing and dehydration. Gross necropsy findings revealed the presence of calculi in the bladder which suggested that this animal’s condition was secondary to a bladder infection and/or blockage. Clinical pathology and histopathological findings supported this conclusion.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There were no statistically significant or toxicologically meaningful differences in mean body weights or body weight changes throughout the main and recovery phases of this study.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

There were no statistically significant or toxicologically meaningful differences in mean food consumption throughout the main and recovery phases of this study.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

no effects observed

Description (incidence and severity):

No test article-related ocular findings were noted in any of the animals towards the end of the treatment period or towards the end of the recovery period. Slight corneal crystals were observed in both eyes of several animals in all groups towards the end
of the treatment period and in groups 1 and 4 towards the end of the recovery period. However, these changes are typical for this age and strain of rat and therefore were not considered treatment related.

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

The mean concentration of lymphocytes in control males was higher than expected but not significantly different from the treatment groups. The high mean concentration was caused by a very high lymphocyte concentration in one rat (#676). This animal also had anemia and thrombocytopenia. Despite these hematology findings, male #676 was still utilized in the calculation of means for all clinical pathology parameters. There were no apparent toxicologically meaningful differences in the hematology and coagulation parameters evaluated at the end of the treatment or recovery phases of this study. Statistically decreased MCHC along with statistically increased hematocrit and MCV were observed in the group 4 females on day 92. In addition, statistically decreased RBC’s were noted in the group 4 females on day 119. These changes were not considered to be toxicologically significant due to the absence of microscopic changes in the bone marrow or spleen, as well as the lack of correlative changes in the other hematology parameters.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

In males, there were no statistically significant or toxicologically meaningful differences in mean clinical chemistry parameters evaluated at the end of the treatment period. At the end of the recovery period, statistically increased potassium was noted in the group 4 males. This change was not considered to be toxicologically significant since it was within the historical control range for this parameter and it occurred at the end of the recovery phase. There were several statistically significant differences noted in the females at the end of the treatment period. These included decreased chloride and GGT (group 2), decreased creatinine and increased potassium (groups 2 and 3), increased calcium and phosphorus (groups 2, 3 and 4), decreased urea nitrogen (groups 2 and 4) and increased albumin (group 4). There were no statistically significant or toxicologically meaningful differences in female clinical chemistry parameters evaluated at the end of the recovery period. Decreased GGT in group 2 animals was not considered biologically relevant since there were no correlative findings and no changes in animals from groups 3 and 4 receiving higher doses. Decreased BUN and creatinine were also not considered to be toxicologically meaningful since the magnitude of the change was small, there was no abnormal renal histopathology and the changes were not dose related. The toxicological significance of the increased albumin, calcium, phosphorus and the decreased chloride in females is unclear, but in the absence of any notable changes in kidney weights, renal histopathology or urinalysis parameters, these findings are not considered adverse.

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

There were no toxicologically meaningful differences noted in any urinalysis parameter at the end of the treatment or recovery phases of this study. Statistically significant differences in urinalysis parameters evaluated at the end of the treatment period included a decreased pH for group 4 males on days 91 and 92 and a decreased specific gravity for group 2 females on day 92. These changes were not considered to be toxicologically significant due to the absence of changes in the renal histopathology and/or the absence of a dose-response relationship. There were no statistically significant or toxicologically meaningful differences noted in any urinalysis parameter at the end of the recovery period for males or females.

Behaviour (functional findings):

effects observed, treatment-related

Description (incidence and severity):

There were no toxicologically meaningful FOB abnormalities in the main or recovery phase animals. Body posture (more animals sitting/standing vs. laying) was noted in the group 3 males on day 40. However, this finding was not considered to be relevant since this change is within the normal range of behavior for this species and there was no dose-response relationship. Forelimb grip strength was statistically increased in the group 4 males on days 26 and 40. This finding was not considered to be toxicologically significant since the changes were within the historical control range for this parameter, there were not similar findings at the day 89 interval and there were no comparable changes in the other parameters of this functional domain. Finally, motor activity was statistically decreased in the group 4 males on day 89. This finding was also not considered to be toxicologically significant since the changes were within the historical control range for this parameter and there were no comparable changes in the other parameters of this functional domain.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

There were no clear toxicologically meaningful changes in the organ weight data collected on this study. Statistically significant differences at the end of the treatment period were limited to increased absolute kidney weight for group 2 males, a decreased brain-to-body weight ratio for group 2 males and increased kidney-to-brain weight ratio for group 3 males. These changes were not considered to be toxicologically significant due to the lack of a dose-response effect and the absence of notable renal or brain histopathology. Statistically significant differences at the end of the recovery period were limited to the group 4 males and included increased spleen-to-body weight ratio, increased spleen-to-brain weight ratio and increased pituitary-to-brain weight ratio. These changes were also not considered to be toxicologically significant due to the lack of any organ weight changes at the end of the treatment period in these animals and the absence of notable spleen, brain or pituitary histopathology.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Gross necropsy observations at the end of the treatment period were consistent with oral administration of a chemical dye and included abnormal colored contents in the cecum, large intestine and colon and stained tail/haircoat. Notable gross necropsy observations at the end of the recovery period were limited to stained tail/haircoat. Gross necropsy observations for group 4 male #665 euthanized moribund on day 87 included pale carcass; staining and dried matted material on haircoat; abnormal content in cecum, duodenum, ileum, jejunum and stomach; enlarged kidney; dilated pelvis in kidney; small thymus; distended ureter; enlarged and thickened urinary bladder; calculi on urinary bladder; and edematous prostate.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

There were no test article-related microscopic findings in this study. All microscopic findings were considered incidental and consistent with spontaneous changes in this species. This absence of treatment-related microscopic changes on this study was confirmed following peer review. Prominent microscopic findings in the group 4 male (#665) euthanized moribund on day 87 were moderate renal pelvis dilation and inflammation associated with tissues of the urogenital tract.

Histopathological findings: neoplastic:

not examined

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Estrous Cycle Determinations
No statistically significant or toxicologically meaningful changes were observed in the number of estrous cycles/animal, cycle length, or the number of animals cycling normally. In addition, there were no perceivable differences between the various phases of estrous.

Male Reproductive Assessment

A statistically significant, treatment-related reduction in the percent motility was observed in groups 3 and 4 at the end of the treatment period. However, the change was not dose related and no effects were noted on the percent motile sperm at the end of the recovery period. No apparent treatment-related or biologically meaningful differences were observed in the number of sperm per gram of cauda epididymis at the end of the treatment or recovery periods.
No apparent treatment-related or biologically meaningful differences were observed in the sperm morphology at the end of the treatment or recovery periods. A low incidence of head abnormalities was observed for animals in all treatment groups for both sacrifice phases. However, group mean values were comparable between the study groups and no treatment related differences were observed.

Details on results:

Oral administration of the test article preparation was not associated with any test-article related mortalities. In addition, test article treatment did not produce toxicologically meaningful clinical abnormalities, neurological changes, variations in estrous cyclicity, body weight effects or food consumption changes during this study. There were a variety of statistically significant changes in the female clinical chemistry parameters examined on this study at the end of the treatment phase. These included decreased chloride and GGT (group 2), decreased creatinine and increased potassium (groups 2 and 3), increased calcium and phosphorus (groups 2, 3 and 4), decreased urea nitrogen (groups 2 and 4), and increased albumin (group 4). Decreased GGT in group 2 animals was not considered biologically relevant since there were no correlative findings and no changes in animals from groups 3 and 4 receiving higher doses. Decreased BUN and creatinine were also not considered to be toxicologically meaningful since the magnitude of the change was small, there was no abnormal renal histopathology and the changes were not dose related. The toxicological significance of the increased albumin, calcium, phosphorus and the decreased chloride in females is unclear, but in the absence of any notable changes in kidney weights, renal histopathology or urinalysis parameters, these findings are not considered adverse. There were no toxicologically meaningful changes in the ophthalmology, gross necropsy or organ weight data collected on this study. In addition, there were no test article-related microscopic findings in this study. All microscopic findings were considered incidental and consistent with spontaneous changes in this species. The most notable change on this study was a statistically significant reduction in the percent motility of the sperm in groups 3 and 4 at the end of the treatment period. However, the change was not dose related and no effects were noted on the percent motile sperm at the end of the recovery period. In addition, these changes did not appear to impact sperm production or sperm morphology as the number of sperm per gram of cauda epididymis and the general appearance of the sperm at the end of the treatment or recovery periods was similar among control and treated groups.

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

ca. 50 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

gross pathology

haematology

histopathology: non-neoplastic

mortality

ophthalmological examination

Target system / organ toxicity

Critical effects observed:

no

Applicant's summary and conclusion

Conclusions:

Based on the results of this study, oral administration of HC Yellow n° 2 up to 50 mg/kg bw/d in the rat for 91 consecutive days produced no mortality or notable signs of systemic toxicity. In addition, there were no significant microscopic lesions observed following histopathological evaluation of selected organs/tissues. A variety of statistically significant changes were observed in the 5, 20 and 50 mg/kg bw/d animals, but these were deemed of no toxicological significance. Based on the findings in this study, the no-observed-adverseeffect level (NOAEL) for oral administration of HC Yellow n° 2 to rats for at least 91 consecutive days was 50 mg/kg bw/d.

Executive summary:

The purpose of this GLP-compliant study was to evaluate the potential toxicity of the test item when administered to rats via oral gavage for at least 91 consecutive days and to assess the reversibility of any effects after a 28-day recovery phase according to OECD Guideline 408 method.

Dosage levels were selected based on the results of a 14-day range-finding study. The test article and vehicle control material were administered once daily, by oral gavage, for 91 consecutive days at 0, 5, 20 and 50 mg/kg/day. Control animals received the vehicle control material at a dosage volume comparable to that received by the test animals. The last five animals in the control group and the high dose group remained on study following the dosing phase for a 28-day recovery phase. General health/mortality and moribundity checks were performed twice daily, in the morning and afternoon. Cage-side observations were performed on all animals approximately 30 to 90 minutes after dosing. Detailed clinical observations were performed weekly on all animals. Functional observation battery (FOB) observations were conducted on 10 rats/sex/group on days -1, 26, 40 and 89. Individual body weights and food consumption were recorded weekly. Ophthalmological examinations were performed on all animals once prior to treatment (days -6/-7) and on days 84 (females) and 85 (males). Recovery animals received ophthalmological examinations on days 114 (females) and 115 (males). Blood and urine samples were collected from all animals for analysis of selected clinical pathology parameters on the day of scheduled euthanasia. All animals were subjected to a complete gross necropsy examination upon death or scheduled euthanasia. Sperm was collected from each male rat following euthanasia for assessment of sperm enumeration, motility and morphology. Fresh organ weights were obtained at scheduled euthanasia from selected organs.

Oral administration of the test article preparation was not associated with any test-article related mortalities. In addition, test article treatment did not produce toxicologically meaningful clinical abnormalities, neurological changes, variations in oestrous cyclicity, body weight effects or food consumption changes during this study. There were a variety of statistically significant changes in the female clinical chemistry parameters examined on this study at the end of the treatment phase. These included decreased chloride and GGT (5 mg/kg bw group), decreased creatinine and increased potassium (5 and 20 mg/kg bw group), increased calcium and phosphorus (5, 20 and 50 mg/kg bw group), decreased urea nitrogen (5 and 50 mg/kg bw group), and increased albumin (50 mg/kg bw group). Decreased GGT in group 5 mg/kg bw animals was not considered biologically relevant since there were no correlative findings and no changes in animals from groups 20 and 50 mg/kg bw receiving higher doses. Decreased BUN and creatinine were also not considered to be toxicologically meaningful since the magnitude of the change was small, there was no abnormal renal histopathology and the changes were not dose related. The toxicological significance of the increased albumin, calcium, phosphorus and the decreased chloride in females is unclear, but in the absence of any notable changes in kidney weights, renal histopathology or urinalysis parameters, these findings are not considered adverse.Statistically decreased MCHC along with statistically increased haematocrit and MCV were observed in 4 females of the 50 mg/kg bw group on day 92. In addition, statistically decreased RBC’s were noted in this group with 4 females on day 119. These changes were not considered to be toxicologically significant due to the absence of microscopic changes in the bone marrow or spleen, as well as the lack of correlative changes in the other haematology parameters. There were no toxicologically meaningful changes in the ophthalmology, gross necropsy or organ weight data collected on this study. In addition, there were no test article-related microscopic findings in this study. All microscopic findings were considered incidental and consistent with spontaneous changes in this species. The most notable change on this study was a statistically significant reduction in the percent motility of the sperm in groups 20 and 50 mg/kg bw at the end of the treatment period. However, the change was not dose related and no effects were noted on the percent motile sperm at the end of the recovery period. In addition, these changes did not appear to impact sperm production or sperm morphology as the number of sperm per gram of cauda epididymis and the general appearance of the sperm at the end of the treatment or recovery periods was similar among control and treated groups.

Conclusion

Based on the results of this study, oral administration of HC Yellow n° 2 up to 50 mg/kg bw/d in the rat for 91 consecutive days produced no mortality or notable signs of systemic toxicity. In addition, there were no significant microscopic lesions observed following histopathological evaluation of selected organs/tissues. A variety of statistically significant changes were observed in the 5, 20 and 50 mg/kg bw/d animals, but these were deemed of no toxicological significance. Based on the findings in this study, the no-observed-adverseeffect level (NOAEL) for oral administration of HC Yellow n° 2 to rats for at least 91 consecutive days is 50 mg/kg bw/d.