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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From Apr. 09, 2012 to July. 10, 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study well documented, followed guideline and GLP

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report Date:
2013

Materials and methods

Test guideline
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 Good Laboratory Practice
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Type:
Constituent
Test material form:
solid: crystalline
Details on test material:
- Name of test material: C6-Pyrazole hemisulfate; 1-hexyl-1H-pyrazole-4,5-diamine hemisulfate- TSIN: Not reported- Substance type: Pure active substance- Physical state: White crystalline solid- Storage conditions: At room temperature, protected from light, and blanketed with nitrogen

Test animals

Species:
rat
Strain:
other: Hsd:Sprague Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS- Source: Male and female rats were received from Harlan Laboratories, Inc., Indianapolis, Indiana - Age at study initiation: 6 to 7 weeks old- Weight at study initiation: 197 to 241 g for males and 156 to 196 g for females- Housing: Male and female rats were housed individually in stainless steel cages - Diet: Certified Rodent Diet #2016C (Harlan Laboratories, Inc.); ad libitum- Water: ad libitum- Acclimation period: Not reportedENVIRONMENTAL CONDITIONS- Temperature: 16 - 22 °C - Relative humidity: 30 - 70 % - Air changes: 10 air changes per hour - Photoperiod: 12-hour light/12-hour dark cycle IN-LIFE STARTING DATE: Apr. 18, 2012IN-LIFE ENDING DATE: Aug. 01, 2012

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: 10% (w/v) propylene glycol and 0.1% (w/w) ascorbic acid in reverse osmosis water
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: Test and control substance formulations were prepared by Covance according to the mixing procedure once daily. The required amount of test substance was mixed with the appropriately pH-adjusted vehicle control substance and sonicated until dissolved. The pH of the test substance formulations was recorded with a target pH of 6.0 to 8.0. Prepared test substance formulations were stored at room temperature, protected from light, and blanketed with nitrogen and used within 4 hours of completion of formulation. VEHICLE- Concentration in vehicle: 0, 0.5, 1.0, 1.6 and 4 mg/mL at the dose levels of 0, 2.5, 5, 8 and 20 mg/kg bw/day- Amount of vehicle: 5 mL/kg- Adjustment of pH: The vehicle control was pH-adjusted as appropriate for each formulation concentration and apportioned for daily use by using Sodium hydroxide.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
- Method: High-performance liquid chromatography (HPLC). Dose preparations were analysed for stability and concentration.- Sampling: Two sets of duplicate samples (1.0 mL each) were taken from the vehicle control and test substance formulations prepared for administration on Days 1, 2, 3, 8, 15, 22, 50, 79, and 85 of the dosing phase. One set of duplicate samples was analyzed for test substance content. - Storage: Samples to be analyzed were stored at room temperature blanketed with nitrogen. The remaining set of duplicate samples was retained as a backup and stored blanketed with nitrogen and protected from light in a freezer, set to maintain -60 to -80ºC, and discarded after acceptable results were obtained and upon authorization from the sponsor.
Duration of treatment / exposure:
90 days
Frequency of treatment:
Once daily
Doses / concentrations
Remarks:
Doses / Concentrations:0 (Group 1-control), 2.5 (Group 2-low), 5 (Group 3-mid low) 8 (Group 4-mid high) and 20 (Group 5-high) mg/kg bw/day Basis:actual ingested
No. of animals per sex per dose:
10 animals/sex/dose in 2.5 (Group 2-low), 5 (Group 3-mid low) and 8 (Group 4-mid high) mg/kg bw/day dose groups.15 animals/sex/dose in 0 (Group 1-control) and 20 (Group 5-high) mg/kg bw/day dose groups. In these groups, 5 animals/sex/dose out of 15/sex/dose were observed for additional 14 days recovery period.
Control animals:
yes, concurrent vehicle
Details on study design:
- Rationale for route selection: The use of oral route is recommended based on the SCCS Notes of Guidance for the Testing of Cosmetic Ingredients and Their Safety Evaluation, 7th Revision, 14 December 2010 (SCCS/1416/11).
Positive control:
None

Examinations

Observations and examinations performed and frequency:
MORTALITIES/VIABILITY: Yes- Time schedule: Twice dailyCLINICAL SIGNS: Yes- Time schedule: Once daily cage side observations were done during the dosing and recovery phases, except on days when detailed observations and expanded clinical signs were conducted. DETAILED CLINICAL OBSERVATIONS: Yes- Time schedule for examinations: Once the pre-dose phase, prior to dosing on Day 1, and weekly (based on Day 1) throughout the dosing phase on the day of terminal sacrifice; and on Days 1, 8, 14, and 15 of the recovery phase. Unscheduled observations were recorded.EXPANDED CLINICAL SIGNS: Yes- Time schedule for examinations: Recorded once during the pre-dose phase and Day 86 of the dosing phase on the last 10 animals/sex/group. Observations were performed on all animals in random order and with the group blinded to the observer.LOCMOTOR ACTIVITY: Yes- Time schedule for examinations: Measured once during the pre-dose phase and Day 86 of the dosing phase on the last 10 animals/sex/group. Animals were placed in an automated photocell activity recording device, and activity was recorded for 40 minutes. Testing was done to include an approximately equal distribution of animals/group/device.BODY WEIGHT: YesTime schedule for examinations: Recorded once during the pre- dose phase; before dosing onDays 1; weekly thereafter through the dosing phase (based on Day 1); on the day prior to dosing phase necropsy; and on Days 1, 8, and 14 of the recovery phase.FOOD CONSUMPTION: Yes Time schedule for examinations: Measured weekly during Weeks 1 to 13 of the dosing phase, for Days 85 to 90 of the dosing phase, and from Days 1 to 8 and 8 to 14 of the recovery phase.WATER CONSUMPTION AND COMPOUND INTAKE: NoOPHTHALMOLOGICAL EXAMINATIONS: Yes - Time schedule for examinations: Performed once during the pre-dose phase and on Day 85/89 (males/females) of the dosing phase, and Day 9/8 (males/females) of the recovery phase by a veterinarian using an indirect ophthalmoscope. The eyes were dilated with a mydriatic agent prior to examination.CLINICAL PATHOLOGY: Yes- Time schedule for collection of blood: On the day of scheduled sacrifices. - Site of blood collection: Blood samples were collected for hematology, coagulation, and clinical chemistry from fasted animals via a jugular vein on the day of scheduled sacrifices. - Animals fasted: Yes, animals were fasted overnight- Time schedule for collection of urine: Urine samples were collected overnight from animals fasted overnight for urinalysis.
Sacrifice and pathology:
GROSS PATHOLOGY: Yesa) Dosing Phase sacrifice: After 90 days of dosing, 10 animals/sex/group were sacrificed and necropsied. Animals were fasted overnight, anesthetized with sodium pentobarbital, and exsanguinated. Terminal body weights were recorded.b) Recovery Phase sacrifice: After 90 days of dosing followed by 14 days of recovery, all surviving animals were fasted overnight, anesthetized with sodium pentobarbital, exsanguinated, and necropsied.An examination of the external features of the carcass; external body orifices; abdominal, thoracic, and cranial cavities; organs; and tissues was performed.- Tissue preservation: The following tissues from each animal were preserved in 10% neutral-buffered formalin, with the exception of the eyes, Harderian gland, optic nerves, and testes, which were preserved in modified Davidson’s fixative and stored in 10% neutral-buffered formalin. Adrenal (2), aorta, brain, cecum, cervix, colon, duodenum, epididymis (2), esophagus, eye (2), femur with bone marrow (articular surface, of the distal end), gut-associated lymphoid tissue (GALT), Harderian gland, heart, ileum, jejunum, kidney (2), lesions, liver, lung with large bronchi, lymph node (mandibular), lymph node (mesenteric), mammary gland (females), muscle, biceps femoris, optic nerve (2), ovary (2), pancreas, pituitary gland, prostate, rectum, salivary gland [mandibular (2)], sciatic nerve, seminal vesicle, skin/subcutis, spinal cord (cervical, thoracic, and lumbar), spleen, sternum with bone marrow, stomach, testis (2), thymus, thyroid (2 lobes) with parathyroid, tongue, trachea, urinary bladder, uterus, vagina.ORGAN WEIGHTS: The following organ weights were recorded at each scheduled sacrifice. 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, spleen, testis (2), thymus, thyroid (2 lobes) with parathyroid, uterus.HISTOPATHOLOGY: YesAll tissues mentioned in the tissue preservation section from all animals were processed and examined microscopically. In addition to the hematoxylin-and-eosin-stained slides, additional slides containing the liver, spleen, femur with bone marrow, and sternum with bone marrow from all animals were stained with Prussian Blue and examined microscopically.
Statistics:
The following statistical methods were used to analyze the body weight, body weight change, food consumption, continuous clinical pathology, motor activity, grip strength, nociceptive reflex, 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, 1971)] 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. For data that exhibited heterogeneous variances after the series of transformations, Dunnett’s t-test for unequal variances with Welch’s degrees of freedom (Welch, 1947) was employed.For each sex, Groups 2 through 5 were compared with Group 1 (control) at the 5%, two-tailed probability level. If the ANOVA was not significant (p > 0.05), no further analyses were conducted. Only data collected on or after the first day of dosing were analyzed statistically.

Results and discussion

Results of examinations

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 specified
Ophthalmological findings:
no effects observed
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
at 20 mg/kg bw/day in males and at ≥8 mg/kg bw/day in females
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
at 20 mg/kg bw/day in females
Urinalysis findings:
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
at 20 mg/kg bw/day in females
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
at 20 mg/kg bw/day in males and at ≥8 mg/kg bw/day in females
Details on results:
MORTALITYNo test substance-related mortality occurred.CLINICAL SIGNS, EXPANDED CLINICAL SIGNS AND LOCOMOTOR ACTIVITYNo test substance-related effects were observed in clinical signs, expanded clinical observations and locomotor activity.BODY WEIGHT AND WEIGHT GAINNo test substance related effects were observed on mean body weight and mean body weight changes.FOOD CONSUMPTION AND COMPOUND INTAKENo test substance related effects were observed on mean food consumption.OPHTHALMOSCOPIC EXAMINATIONNo test substance related effects were observed during ophthalmic observations.CLINICAL PATHOLOGY (HEMOTOLOGY, CLINICAL CHEMISTRY AND URINALYSIS)Test substance-related effects on clinical pathology test results were minimal to mild in severity, generally reversible, and observed for males given 20.0 mg/kg/day or females given >8.0 mg/kg/day. Prominent effects were limited to hematology test results and included decreased red blood cell mass [i.e., red blood cell count, hemoglobin, and hematocrit (females only)], higher mean corpuscular volume (females only), higher mean corpuscular hemoglobin (females only), lower mean corpuscular hemoglobin concentration, and higher reticulocyte count. These effects were consistent with an appropriate regenerative response to decreased red blood cell mass. Other effects were less pronounced; found only in animals given 20.0 mg/kg/day; and included minimally higher absolute neutrophil count (females only) and bilirubin (females only). The exact cause of the bilirubin finding was not determined; however, increased bilirubin can be associated with hemolysis, which would correlate with hematology findings and pigment-laden macrophages observed in the liver.ORGAN WEIGHTSTest substance-related mean absolute and relative spleen weights were increased at the dosing phase necropsy in females given 20.0 mg/kg/day. These increases correlated with the microscopic observation of increased extramedullary hematopoiesis. No C6 pyrazole hemisulfate-related changes in spleen weights were noted at the recovery phase necropsy. GROSS PATHOLOGYNo test substance-related macroscopic findings were noted at the dosing or recovery phase necropsy. HISTOPATHOLOGY: NON-NEOPLASTICAt the dosing phase necropsy, test substance-related microscopic findings in the liver included minimal infiltrates of pigment-laden macrophages/Kupffer cells in females given 20.0 mg/kg/day and minimally increased Prussian blue staining in males and females given 20.0 mg/kg/day. In the spleen, test substance-related findings included minimal infiltrates of pigment-laden macrophages in males given 20.0 mg/kg/day, minimally increased extramedullary hematopoiesis in males and females given 20.0 mg/kg/day, and minimally increased Prussian blue staining in males given 20.0 mg/kg/day and females given 8.0 or 20.0 mg/kg/day. At the recovery phase necropsy, minimal infiltrates of pigment-laden macrophages and minimally increased Prussian blue staining persisted in the liver of females given 20.0 mg/kg/day. These findings were considered of toxicological relevance. No delayed effects were noted at the end of the recovery phase.

Effect levels

open allclose all
Dose descriptor:
NOAEL
Effect level:
8 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: Adverse effects observed during clinical pathology (minimal to mild decreases in red blood cell mass) at 20 mg/kg bw/day; microscopic findings in the liver and spleen at 20 mg/kg bw/day.
Dose descriptor:
NOAEL
Effect level:
5 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: see 'Remark'

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
C6-Pyrazole hemisulfate when administered by oral gavage daily for 90 days to male and female Hsd:Sprague Dawley rats at dose levels of 0, 2.5, 5, 8 and 20 mg/kg bw/day, revealed a No adverse effect level (NOAEL) at 8 mg/kg bw/day for males and at 5 mg/kg bw/day for female rats respectively. (Based on the adverse effects observed in clinical pathology, organ weights, microscopic examinations).
Executive summary:

The 90-day repeated dose oral toxicity study of C6-Pyrazole hemisulfate was performed by following the OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents).

Male and female Wistar rats (source: Harlan Laboratories, Inc., Indianapolis, Indiana) were used in the study. Animals were individually housed in stainless steel cages and maintained under controlled environmental conditions (temperature: 16 - 22 °C, humidity: 30 - 70 %, air changes: 10 times per hour and 12 hours light /12 hours dark). Certified Rodent Diet and tap water were providedad libitum.

Animals were assigned to the following dose groups:

Group 1 (vehicle control): 0 mg/kg bw/day (10% (w/v) propylene glycol and 0.1% (w/w) ascorbic acid in reverse osmosis water)

Group 2 (low dose): 2.5 mg/kg bw/day

Group 3 (mid low dose): 5 mg/kg bw/day

Group 4 (mid high dose): 8 mg/kg bw/day

Group 5 (high dose): 20 mg/kg bw/day

10 animals/sex/dose were used in 2.5 (Group 2-low), 5 (Group 3-mid low) and 8 (Group 4-mid high) mg/kg bw/day dose groups. 15 animals/sex/dose were used in 0 (Group 1-control) and 20 (Group 5-high) mg/kg bw/day dose groups. In the control and high dose groups, 5 animals/sex/dose out of 15/sex/dose were observed for additional 14 days recovery period.

The test substance was administered to the treatment groups daily by oral gavage for 90 days.

Mortalities/viability were observed twice daily. Once daily cage side observations were done during the dosing and recovery phases. Detailed observations were done once the pre-dose phase, prior to dosing on Day 1, and weekly throughout the dosing phase on the day of terminal sacrifice; and on Days 1, 8, 14, and 15 of the recovery phase. Expanded clinical signs and locomotor activity were performed during the pre-dose phase and on Day 86 of the dosing phase.

 

Body weights were recorded once during the pre-dose phase; before dosing on Days 1; weekly thereafter through the dosing phase; on the day prior to dosing phase necropsy; and on Days 1, 8, and 14 of the recovery phase. Food consumption was measured weekly during Weeks 1 to 13 of the dosing phase, for Days 85 to 90 of the dosing phase, and from Days 1 to 8 and 8 to 14 of the recovery phase. Ophthalmic examinations were performed once during the pre-dose phase and on Day 85/89 (males/females) of the dosing phase, and Day 9/8 (males/females) of the recovery phase.

 

Blood and urine samples were collected prior to the scheduled necropsy and clinical pathology was conducted. After 90 days of dosing, 10 animals/sex/group were necropsied in all groups. In the control and high dose groups, the additional 5 animals/sex/dose were necropsied after 14 days of dosing phase necropsy. Organ weights were taken during necropsy. Tissues specified in the protocol from all animals were processed and examined microscopically. In addition to the hematoxylin-and-eosin-stained slides, additional slides containing the liver, spleen, femur with bone marrow, and sternum with bone marrow from all animals were stained with Prussian Blue and examined microscopically.

 

No test substance-related mortality occurred, and no test substance-related clinical signs were observed. Test substance administration had no effect on mean body weight, mean body weight change, mean food consumption, ophthalmic findings, expanded clinical observations, or locomotor activity.

 

Test substance-related effects on clinical pathology test results were minimal to mild in severity, generally reversible, and observed for males given 20.0 mg/kg/day or females given >8.0 mg/kg/day. Prominent effects were limited to hematology test results and included decreased red blood cell mass [i.e., red blood cell count, hemoglobin, and hematocrit (females only)], higher mean corpuscular volume (females only), higher mean corpuscular hemoglobin (females only), lower mean corpuscular hemoglobin concentration, and higher reticulocyte count. These effects were consistent with an appropriate regenerative response to decreased red blood cell mass. Other effects were less pronounced; found only in animals given 20.0 mg/kg/day; and included minimally higher absolute neutrophil count (females only) and bilirubin (females only). 

 

Test substance-related mean absolute and relative spleen weights were increased at the dosing phase necropsy in females given 20.0 mg/kg/day. These increases correlated with the microscopic observation of increased extramedullary hematopoiesis. No test substance-related changes in spleen weights were noted at the recovery phase necropsy.

 

No test substance-related macroscopic findings were noted at the dosing or recovery phase necropsy.

 

At the dosing phase necropsy, test substance-related microscopic findings in the liver included minimal infiltrates of pigment-laden macrophages/Kupffer cells in females given 20.0 mg/kg/day and minimally increased Prussian blue staining in males and females given 20.0 mg/kg/day. In the spleen, test substance-related findings included minimal infiltrates of pigment-laden macrophages in males given 20.0 mg/kg/day, minimally increased extramedullary hematopoiesis in males and females given 20.0 mg/kg/day, and minimally increased Prussian blue staining in males given 20.0 mg/kg/day and females given 8.0 or 20.0 mg/kg/day. At the recovery phase necropsy, minimal infiltrates of pigment-laden macrophages and minimally increased Prussian blue staining persisted in the liver of females given 20.0 mg/kg/day. These findings were considered of toxicological relevance. No delayed effects were noted at the end of the recovery phase.

 

Based on the above, C6-Pyrazole hemisulfate when administered by oral gavage daily for 90 days to male and female Hsd:Sprague Dawley rats at dose levels of 0, 2.5, 5, 8 and 20 mg/kg bw/day, revealed a No adverse effect level (NOAEL) at 8 mg/kg bw/day for males and at 5 mg/kg bw/day for female rats respectively based on the adverse effects observed in clinical pathology, organ weights, microscopic examinations).

This repeated dose oral 90-day toxicity study is classified as acceptable, and satisfies the guideline requirements of OECD 408 method.