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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
08 October 2014 -- 14 November 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Compliant to GLP and testing guidelines; adequate consistence between data, comments and conclusions.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report Date:
2015

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Test material form:
other: colourless liquid
Details on test material:
- Physical state: colourless liquid
- Lot/batch No.: 2A187468501VSP
- Expiry date: 31 December 2015
- Storage conditions: at room temperature and protected from light

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: breeder: Charles River Laboratories Italia, Calco, Italy.
- Age at study initiation: approximately 5 weeks old.
- Mean body weight at study initiation: 174 g (range: 158 g to 193 g) for the males and 145 g (range: 126 g to 168 g) for the females.
- Fasting period before study: no
- Housing: polycarbonate cages with stainless steel lids
- Diet: SSNIFF R/M-H pelleted diet (free access)
- Water: tap water filtered with a 0.22 µm filter (free access)
- Acclimation period: for a period of 8 days before the beginning of the treatment period.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2°C
- Humidity (%): 50 ± 20%
- Air changes (per hr): approximately 12 cycles/hour of filtered, non-recycled air
- Photoperiod (hrs dark / hrs light): 12 h/12 h.

IN-LIFE DATES: 17 October 2014 to 14 November 2014

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The type of formulation (visual observation) is a solution in the vehicle.
The preparation procedure is according to Study of analyse homogeneity and stability, describing the preparation procedure for a range of concentrations covering the lowest and highest used in this study.
The frequency of preparation (Test item dose formulations) is daily.
The delivery conditions are at room temperature, protected from light and sealed vials.

VEHICLE
- Justification for use and choice of vehicle: test item soluble in the vehicle and corn oil is commonly used for this type of study
- Concentration in vehicle: 5, 16 and 48 mg/mL
- Amount of vehicle (if gavage): 5mL/kg/day.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Type of method: HPLC-UV
Test item concentrations in the administered dose formulations analyzed in Weeks 1 and 4 were within the acceptable range of ± 10%.
Homogeneity: not assessed, dose formulation is a solution
Stability: not assessed, dose formulation prepared daily
Duration of treatment / exposure:
4 weeks
Frequency of treatment:
Daily
Doses / concentrations
Remarks:
Doses / Concentrations:
25, 80, and 240 mg/kg/day
Basis:
actual ingested
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:
The dose-levels were selected in agreement with the Sponsor based on the results of a previous toxicology study.

- Rationale for animal assignment: computerized stratification procedure
Positive control:
no (not required)

Examinations

Observations and examinations performed and frequency:
MORTALITY/MORBIDITY:
- Time schedule: once a day during the acclimation period and at least twice a day during the treatment period.

DETAILED CLINICAL OBSERVATIONS:
- Time schedule: at the beginning of the treatment period and then once a week until the end of the study.
Each animal was observed once a day, at approximately the same time, for the recording of clinical signs.

BODY WEIGHT:
- Time schedule: once before group allocation, then on the first day of treatment and once a week until the end of the study.

FOOD CONSUMPTION:
- Time schedule: once a week until the end of the study.

NEUROBEHAVIOURAL EXAMINATION
- Time schedule: once in Week 4.

HEMATOLOGY, CLINICAL CHEMISTRY, URINALYSIS:
- Time schedule: at the end of the treatment period.
Sacrifice and pathology:
ORGAN WEIGHTS: at the end of the treatment period (see table below).

GROSS PATHOLOGY:
A complete macroscopic post-mortem examination was performed on all study animals. This included examination of the external surfaces, all orifices, the cranial cavity, the external surfaces of the brain, the thoracic, abdominal and pelvic cavities with their associated organs and tissues and the neck with its associated organs and tissues.

HISTOPATHOLOGY:
A microscopic examination was performed on:
- all tissues listed in the Tissue Procedure Table for the control and high-dose animals (groups 1 and 4) sacrificed at the end of the treatment period,
- all macroscopic lesions from all low- and intermediate-dose animals (groups 2 and 3) sacrificed on completion of the treatment period.

Based upon the microscopic results of the high-dose group and after agreement of the Sponsor, the following tissues from the low- and intermediate-dose animals were examined:
- forestomach,
- stomach,
- trachea,
- cecum.
Other examinations:
no
Statistics:
yes

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Mortality:
mortality observed, treatment-related
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:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Behaviour (functional findings):
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
effects observed, treatment-related
Details on results:
MORTALITY:
No unscheduled deaths occurred during the study.

CLINICAL SIGNS:
There were no test item-related clinical signs in animals treated at 25 mg/kg/day during the study. Reflux at dosing was noted on Day 17 in one female. This was considered incidental since it was transient and observed in one animal without relationship to dose-levels.

At 80 mg/kg/day, ptyalism was transiently observed in 3/5 males and in 2/5 females along with loud breathing in 2/5 males.

At 240 mg/kg/day, ptyalism was occasionally observed in all animals and loud breathing along with hunched posture and piloerection was noted in 1/5 males. Loud breathing was also observed in 2/5 females along with dyspnea and thin appearance in one of them.
These clinical signs were considered test item-related with a minor toxicological significance since they were transient and observed with a low incidence.
Other clinical signs (scabs and soiled around the mouth) were considered incidental since they are often observed in laboratory housed animals of this species and was observed at a low incidence.

BODY WEIGHT:
There were no toxicologically relevant test item-related changes in mean body weight and mean body weight gain during the study.
At 240 mg/kg/day, statistically significant difference in body weight change observed between Days 1 and 8 in males, was considered incidental since it was transient and isolated.

FOOD CONSUMPTION:
There were no changes in food consumption during the study.

NEUROBEHAVIOURAL EXAMINATION:
There were no toxicologically relevant test item-related effects at the Functional Observation Battery including motor activity (horizontal movements and rearing) in both genders.

Grooming was noted in 1/5 males at 80 and 240 mg/kg/day. Slight hypotonia was also observed in 1/5 males at 240 mg/kg/day. In view of the slight severity and incidence and in absence of correlating clinical signs during the study, these findings were considered to be unrelated to the test item treatment. Other changes observed at 240 mg/kg/day, limited to scabs, piloerection, hunched posture and loud breathing, were noted in 1/5 males. Loud breathing was also noted in 1/5 males at 80 mg/kg/day. These changes correlated with clinical signs observed.

HEMATOLOGY:
There were no test item-related effects on hematology parameters in males.
Changes in hematology parameters were restricted to statistically significant and not dose-related prolonged prothrombin time in females at ≥ 25 mg/kg/day, when compared to controls. In view of the minimal amplitude and the absence of dose-relationship, this difference was considered not toxicologically relevant.

CLINICAL CHEMISTRY:
There were no test item-related effects on biochemistry parameters in females.
Changes in biochemistry parameters were restricted to a slightly higher chloride level at ≥ 25 mg/kg/day (statistically significant at 25 mg/kg/day), when compared to controls. These changes were considered to be of no toxicological importance since they were of low amplitude and not dose related.
Other changes in biochemistry values (aspartate aminotransferase), including some that resulted in statistically significant differences between groups, were considered incidental since they reflected the normal inter-animal variation in this species.

URINALYSIS:
There were no test item-related effects on urinary parameters in both genders.
A very high urinary volume associated with a lower specific gravity and a pH equal to 7 was noted at 80 mg/kg/day in 1/5 males and at 240 mg/kg/day in 2/5 males, compared to controls. In view of the direction of the changes and the very high urinary volume, these differences were considered not toxicologically relevant. Therefore a contamination of urinary samples with drinking water was suspected.

ORGAN WEIGHTS:
The test item administration did not induce any changes in organ weights.

Organ weight changes were not considered to be related to the test item as they were small in amplitude, had no gross or microscopic correlates, and/or were not dose-related in magnitude. The minimal, not dose related increases in the mean testis and epididymis weights in all test item-treated groups were due to low testis and epididymis weights of control male which showed severe testicular atrophy with no sperm in the epididymides.

GROSS PATHOLOGY:
Test item-related macroscopic changes were seen in the forestomach at 240 mg/kg/day.
Many white, often raised foci, with occasional thickening were noted on the wall of the forestomach in 4/5 males and 3/5 females at 240 mg/kg/day. These foci correlated with squamous hyperplasia and hyperkeratosis of the mucosa at microscopic examination.
The other macroscopic findings had no histologic correlates or correlated with common histologic findings in control rats, and were considered to be incidental.

HISTOPATHOLOGY: NON-NEOPLASTIC (see Table 1):
The main test item-related microscopic changes were found in the forestomach at 240 mg/kg/day in both sexes, males being more affected than females. Additional changes at 240 mg/kg/day were noted in the glandular stomach in both sexes, and in the trachea and bone marrow in males.

Forestomach and stomach
Minimal to marked, multifocal to diffuse hyperplasia (i.e. increased thickness) of the squamous epithelium was observed in all animals at 240 mg/kg/day. This was associated in most animals with minimal or slight hyperkeratosis (i.e. increased thickness of the keratin layer) and slight or moderate subacute inflammation. In some males, there were minimal focal or multifocal erosions. Inflammation was mainly located in the submucosa and was characterized by mixed inflammatory cell infiltrates with eosinophils and edema.
In the glandular stomach, minimal multifocal hyperplasia of the glands was noted in a few animals with inflammation in a single animal.
There were no significant microscopic changes in the forestomach and stomach at 25 and 80 mg/kg/day.

Trachea
Minimal epithelial degeneration/regeneration characterized by loss of cilia, flattening and horizontal orientation of surface epithelial cells was noted in 2/5 males which both showed hyperplasia and inflammation in the forestomach at 240 mg/kg/day.
There were no significant microscopic changes in the trachea at 25 and 80 mg/kg/day.

Bone marrow
In 2/5 males which both showed hyperplasia and inflammation in the forestomach at 240 mg/kg/day, the myeloid cell numbers were minimally increased.
Other microscopic findings noted in treated animals were considered incidental changes, as they also occurred in controls, were of low incidence, had no dose-relationship in incidence or severity, and/or are common background findings for the rat. Among them was minimal mucosal inflammation in the cecum which was seen with higher incidence in animals given the test item at all doses (respectively 1 3 4 4 males and 1-2-0-2 females at 0-25-80-240 mg/kg/day). This inflammation consisted of mixed inflammatory cell infiltrate including eosinophils in the lamina propria of the mucosa, with occasional elongation of the crypts. In the absence of dose-relationship, given its occasional occurrence in controls, and in the absence of inflammation in the small intestine at the highest dose-level, this finding was considered as incidental and unrelated to the test item administration.

Effect levels

Dose descriptor:
NOAEL
Effect level:
80 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see 'Remark'

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Table 1

Incidence and severity of test item-related microscopic findings in the forestomach and stomach (n = 5)

 

Sex

Male

Female

Group

1

2

3

4

1

2

3

4

Dose-level (mg/kg/day)

0

25

80

240

0

25

80

240

Forestomach

 

 

 

 

 

 

 

 

- Hyperplasia; squamous cell

 

 

 

 

 

 

 

 

Minimal (grade 1)

-

-

-

-

-

-

-

2

Slight (grade 2)

-

-

-

1

-

-

-

1

Moderate (grade 3)

-

-

-

4

-

-

-

1

Marked (grade 4)

-

-

-

-

-

-

-

1

- Hyperkeratosis

 

 

 

 

 

 

 

 

Minimal (grade 1)

-

-

-

2

-

-

-

2

Slight (grade 2)

-

-

-

3

-

-

-

1

- Inflammation

 

 

 

 

 

 

 

 

Slight (grade 2)

-

-

-

4

-

-

-

2

Moderate (grade 3)

-

-

-

1

-

-

-

-

- Erosion

 

 

 

 

 

 

 

 

Minimal (grade 1)

-

-

-

3

-

-

-

-

Stomach

 

 

 

 

 

 

 

 

- Hyperplasia; gland

 

 

 

 

 

 

 

 

Minimal (grade 1)

-

-

-

3

-

-

-

1

- Inflammation

 

 

 

 

 

 

 

 

Minimal (grade 1)

-

-

-

1

-

-

-

-

-: no findings.

 

Applicant's summary and conclusion

Conclusions:
Once daily oral administration of the test item to Sprague-Dawley rats at dose-levels of 25, 80 or 240 mg/kg/day for 4 weeks, resulted in the absence of premature deaths or mortalities, no relevant adverse clinical signs, no adverse findings in in vivo parameters and in laboratory parameters at any dose-levels.
At 240 mg/kg/day, adverse inflammatory, erosive and/or hyperplasic changes in the forestomach and to a lesser extent in the stomach and trachea were recorded in the microscopic examination. These observations are considered to be adverse and were indicative of irritant properties of the test item.
Consequently, under the experimental conditions of this study, the No Observable Adverse Effect Level (NOAEL) was considered to be 80 mg/kg/day.
Executive summary:

The objective of this study was to evaluate the potential toxicity of the test item following daily oral administration to rats for 4 weeks.

 

Methods

Three groups of five male and female Sprague-Dawley rats received the test item daily by oral administration for 4 weeks at dose-levels of 25, 80 or 240 mg/kg/day. The test item was administered as a solution in the vehicle (corn oil). A control group of five male and female Sprague-Dawley rats received the vehicle alone under the same experimental conditions.

Mortality and morbidity were checked once a day during the acclimation period and at least twice a day during the treatment period. Clinical signs were recorded once a day during the study. Body weight was recorded once before the beginning of the treatment period, and then at least once a week during the study as food consumption. Towards the end of the dosing period, a Functional Observation Battery including motor activity measurement, and hematology, blood biochemistry and urinalysis investigations were performed on all animals. On completion of the treatment period, all animals were euthanized and submitted to a full macroscopic post-mortem examination. Designated organs were weighed and selected tissues were preserved. A microscopic examination was performed on selected tissues and on macroscopic lesions.

 

Results

No clinical signs were observed at 25 mg/kg/day andno toxicologically significant effects on mean body weight, mean body weight change and mean food consumption were noted at ≥ 25 mg/kg/day. In addition, there were no relevant effects at the Functional Observation Battery, on motor activity and in hematology, blood chemistry and urinary parameters at ≥ 25 mg/kg/day.

At 80 mg/kg/day, treatment-related clinical signs were limited to transient ptyalism (3 males, 2 females) and loud breathing (2 males). At 240 mg/kg/day the same clinical signs were occasionally noted in males and females along with hunched posture and piloerection in one male, dyspnea and thin appearance in one female.

The test item administration did not induce any changes in organ weights. The main histopathological changes induced by the test item administration at 240 mg/kg/day were seen in the forestomach and were indicative of irritant properties of the test item. They consisted of squamous cell hyperplasia and hyperkeratosis (correlated with macroscopic white discoloration), subacute inflammation and low grade erosions. Low grade inflammation and hyperplasia were additionally noted in the glandular stomach in a few animals at this dose-level. Minimal epithelial degeneration/regeneration was noted in the trachea in two males at this dose-level and may have been related to irritation consecutive to aspiration of the test item during the gavage procedure. Increased myeloid cell numbers seen in the bone marrow of two males at 240 mg/kg/day were considered to be related to the inflammation noted in the forestomach. There were no significant macroscopic or microscopic changes at 25 and 80 mg/kg/day.

Conclusion

Once daily oral administration of the test item to Sprague-Dawley rats at dose-levels of 25, 80 or 240 mg/kg/day for 4 weeks, resulted in the absence of premature deaths or mortalities, no relevant adverse clinical signs, no adverse findings in in vivo parameters and in laboratory parameters at any dose-levels.

At 240 mg/kg/day, adverse inflammatory, erosive and/or hyperplasic changes in the forestomach and to a lesser extent in the stomach and trachea were recorded in the microscopic examination. These observations are considered to be adverse and were indicative of irritant properties of the test item. Consequently, under the experimental conditions of this study, the No Observable Adverse Effect Level (NOAEL) was considered to be 80 mg/kg/day.