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Repeated dose toxicity: oral

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Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Remarks:
combined repeated dose and reproduction / developmental screening
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
From January to September 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was conducted according to standard guidelines in compliance with GLP.
Cross-referenceopen allclose all
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
yes
Remarks:
During the acclimation period, the animals were observed twice daily for mortality and general changes in appearance and behavior instead of once. The cage-board was changed at least 3 times per week . Body weights recorded on gestation day 14 were used t
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): PETIA
- Physical state: Clear colourless viscous liquid
- Lot/batch No.: JBGK0072T
- Expiration date of the lot/batch: 10 June 2010

Test animals

Species:
rat
Strain:
other: Crl:CD(SD)]
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Inc., Raleigh, NC, USA
- Age at study initiation: ca. 63 d
- Housing: stainless steel wire mesh cages suspended above cage board
- Diet (e.g. ad libitum): PMI Nutrition International, LLC Certified Rodent LabDiet® 5002
- Water (e.g. ad libitum): yes
- Acclimation period: 17 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.8 - 22.7°C
- Humidity (%): 38.2 - 45.1%
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 29 January 2010 To: 19 May 2010

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:

VEHICLE
- Justification for use and choice of vehicle (if other than water): corn oil
- Concentration in vehicle: 0, 5, 15 or 40 mg/ml
- Amount of vehicle (if gavage): 5ml/kg bw
- Lot/batch no. (if required): YF0793, YR1134, and YJ0917
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Fifteen-day room temperature resuspension homogeneity and stability of the test substance formulated in the vehicle at concentrations of 10 and 200 mg/mL were established in a previous study (Stump, Draft, WIL-738003). Therefore, resuspension homogeneity and stability were not assessed for the 15 and 40 mg/mL formulations prepared for the current study.

Prior to the initiation of dose administration, quadruplicate samples for homogeneity and concentration determination were collected from the top, middle, and bottom strata of the 5 mg/mL non-dosing formulation. However, because the analytical results of the initial samples as well as the back-up samples did not meet the WIL Research Laboratories, LLC’s SOP requirements, a new 5 mg/mL non-dosing formulation was prepared. Quadruplicate samples for homogeneity and concentration determination were collected from the top, middle, and bottom strata of the new 5 mg/mL non-dosing formulation. In addition, quadruplicate samples for resuspension homogeneity and stability determinations were collected from aliquots prepared from this same non-dosing formulation following room temperature storage for 5 and 13 days; the aliquots were stirred for at least 60 minutes prior to sampling. Quadruplicate samples for homogeneity and concentration analyses were collected from the top, middle, and bottom of the test substance formulations prepared for the first week of dose administration; samples were also collected from the middle stratum of the vehicle control formulation. Additionally, quadruplicate samples for concentration analysis were collected from the middle stratum of each dosing formulation and vehicle control formulation prepared for the remainder of the study. One set of duplicate samples from each collection was subjected to the appropriate analyses. The remaining set of duplicate samples was stored frozen (approximately -70°C ± 5°C) as back-up. All analyses were conducted by the Analytical Chemistry Department at WIL Research Laboratories, LLC using a validated high performance liquid chromatography method with ultraviolet absorbance detection.
Duration of treatment / exposure:
The males were dosed once daily during study Days 0-27 (14 days prior to pairing through 1 day prior to scheduled euthanasia) for a total of 28 doses. The females were dosed once daily during study Days 0 through the day prior to euthanasia (14 days prior to pairing through lactation day 4) for a total of 40-47 doses. Females that failed to deliver were dosed through the day prior to euthanasia (post-mating day 25) for a total of 41 doses.
Frequency of treatment:
Daily
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 25, 75 or 200 mg/kg bw
Basis:
actual ingested
No. of animals per sex per dose:
12 males and 12 females
Control animals:
yes
Details on study design:
- Dose selection rationale: Dosage levels were selected based on the results of previous studies and were provided by the Sponsor after consultation with the Study Director. In a previous 14-day toxicity study in Crl:CD(SD) rats (Stump, Draft, WIL-738003), the maximum tolerated dose was exceeded at 1000 mg/kg/day. At 300 mg/kg/day, lower mean body weight gains, reduced food consumption, adverse clinical signs, and/or changes in mean organ weights were noted for males and females. Based on these results, dosage levels of 25, 75, and 200 mg/kg/day were selected to be evaluated in the current study.

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All rats were observed twice daily, once in the morning and once in the afternoon, for moribundity and mortality. A detailed physical examination was conducted weekly on each animal beginning approximately 1 week prior to the initiation of dose administration, including on the day of necropsy.

BODY WEIGHT: Yes
- Time schedule for examinations: Individual male body weights were recorded beginning approximately 1 week prior to the initiation of dose administration, on the first day of dose administration and weekly thereafter until the day prior to scheduled euthanasia. Individual female body weights were recorded beginning approximately 1 week prior to the initiation of dose administration, on the first day of dose administration and weekly thereafter until the day evidence of copulation was observed.

FOOD CONSUMPTION:
- Individual food consumption was recorded on the corresponding weekly body weight days until pairing. Food intake was not recorded during the mating period. Once evidence of mating was observed, female food consumption was recorded on gestation days 0, 4, 7, 11, 14, 17, and 20 and on lactation days 1 and 4. Following mating, food consumption for the female with no evidence of mating was measured on a weekly basis until parturition. When food consumption could not be determined for an animal during a given interval (due to an unscheduled death, as females enter gestation/lactation, weighing error, food spillage, obvious erroneous value, etc.), group mean values were calculated for that interval using the available data.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at necropsy (Study Day 28 for males and Lactation Day 5 for females)
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals: 6 animals/sex/group

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at necropsy (Study Day 28 for males and Lactation Day 5 for females)
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals: 6 animals/sex/group

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- FOB assessments were recorded for 6 animals/sex/group prior to dose administration and fasting for clinical pathology sampling on study day 27 (males) and lactation day 4 (females).
- Locomotor activity counts were recorded for 6 animals/sex/group prior to dose administration on study day 27 (males) and on lactation day 4 (females); the same animals evaluated for FOB were selected for locomotor activity assessment.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Mortality:
mortality observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
not examined

Effect levels

Dose descriptor:
NOAEL
Effect level:
75 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Test substance-related mortality and/or moribundity were noted in the 75 and 200 mg/kg bw/day group males and females during the treatment period. Two males in the 75 mg/kg bw/day group and 2 males and 3 females in the 200 mg/kg bw/day group were found dead or euthanizedin extremis. Although an exact cause of death was not determined, these deaths were attributed to test substance administration. Test substance-related clinical findings were noted in a dose-related manner across all dosage levels and included salivation or evidence thereof, yellow and red material primarily around the mouth, and wiping of mouth in the bedding (females only). Incidences of rales were also noted in the 75 and 200 mg/kg bw/day group males and females sporadically during the treatment period. All of the aforementioned clinical findings were primarily noted at the time of dose administration and/or approximately 1 hour following dose administration, were attributed to the irritative properties of the test substance, and therefore, were not considered adverse.

In the 200 mg/kg bw/day group males, test substance-related lower mean body weight gains were noted when the overall pre-mating (Days 0-13) and treatment (Days 0‑27) periods were evaluated; correspondingly lower mean food consumption was noted during the pre-mating period. As a result, mean male body weight in this group was 8.4% lower than the control group on Day 27. For the 75 mg/kg bw/day group males, slightly lower mean body weight gains were noted throughout the treatment period, with the most severe reduction occurring during study days 21-27 primarily due to 2 males with body weight losses and evidence of irritation in the non-glandular portion of the stomach at necropsy. Based on these results, the lower mean body weight gains in the 75 mg/kg bw/day group males were attributed to test substance administration. However, the magnitude of these differences was not of a sufficient magnitude to affect mean body weights when compared to the control group and mean food consumption in this group was similar to the control group during the pre-mating period (Days 0-13). Mean body weights, body weight gains, and food consumption were unaffected by test substance administration in the 25 mg/kg bw/day group males throughout the study and in the 25, 75, and 200 mg/kg bw/day group females during the pre-mating, gestation, and lactation periods. 

No test substance-related effects were noted during the FOB or locomotor activity evaluations at any dosage level.

At the scheduled necropsy, higher mean absolute neutrophil counts were noted for the 75 and 200 mg/kg bw/day group males and females, which were consistent with the test substance‑related ulceration and associated inflammation in the non‑glandular stomach. Gross observations consisted of thickened non-glandular stomach in the 75 and 200 mg/kg/day group males and females and adhesions to the liver or spleen in males at 200 mg/kg bw/day. Test substance‑related histopathologic alterations included ulceration, epithelial hyperplasia and hyperkeratosis, and/or chronic-active inflammation in the non‑glandular stomach in the 75 and 200 mg/kg bw/day group males and females. Organ weight changes included higher mean adrenal gland weights in the 75 mg/kg bw/day group males and 200 mg/kg/day group males and females and lower mean thymus weight in the 200 mg/kg bw/day group females. Hypertrophy of the zona fasciculata of the adrenal cortex and lymphoid depletion was also noted in the 200 mg/kg bw/day group males and females, respectively. Mean numbers of corpora lutea, unaccounted-for sites, and implantation sites in the 25, 75, and 200 mg/kg bw/day groups were similar to the control group values.

Applicant's summary and conclusion

Conclusions:
Under the conditions of this screening study, the NOAEL for systemic toxicity of the test substance to rat was considered to be 75 mg/kg bw/day.
Executive summary:

A study was conducted to determine the repeated dose oral toxicity of PETIA to rat after 28 days of exposure.

Under the conditions of this screening study, based on test substance-related lower mean body weight gains during the overall study period for the 200 mg/kg bw/day males as well as a lower mean body weight Day 27, the NOAEL for systemic toxicity was considered to be 75 mg/kg bw/day.

Test substance-related findings at 75 and 200 mg/kg bw/day relating to mortality/morbidity, adrenal gland weights, neutrophil counts, macroscopic and/or microscopic findings of the stomach, adrenal cortex and thymus were attributed to the irritative properties of the test substance and corresponding stress, rather than systemic toxicity.