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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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report Date:
2014

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.3100 (90-Day Oral Toxicity in Rodents)
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
Purity: 93.6%

Test animals

Species:
rat
Strain:
other: Hsd: Sprague Dawley®™SD®™
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Age at study initiation: 40-53 days
- Weight at study initiation: Males: 186.02 to 220.17 g; Females: 138.81 to 158.30 g
- Housing: two per sex per cage in clean, sterilized polycarbonate rat cages (size: approximately 41.0 L x 26.5 W x 20.0 H cm) covered with stainless steel grill tops. Enrichment items (nestlings) were placed in each cage. Autoclaved corncob was used as the bedding.
- Diet (e.g. ad libitum): Nutrilab Rodent Mash feed, ad libitum
- Water (e.g. ad libitum): Reverse osmosis-treated water, ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.4 to 24.8°C
- Humidity (%): 48 to 68%
- Air changes (per hr): 22
- Photoperiod (hrs dark / hrs light): 12 hour light/12 hour dark

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: 0.1% Tween-80 in an 0.5% methylcellulose aqueous solution
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: The required quantity of the test item was transferred into a volumetric flask and dispersed in a small quantity of vehicle (0.1% Tween-80 in 0.5% methylcellulose aqueous solution). The contents were vortexed for five minutes and the flask was filled to the appropriate volume with vehicle to form a suspension. The contents of the volumetric flask were sonicated, then vortexed further for five minutes and then transferred to a labelled amber coloured bottle with a magnetic stir bar. Formulation preparations for subsequent weeks were made based on the quantities required for dosing and was used within the stability period (15 days). After dosing, the formulations were stored at room temperature when they were not used. The volumes administered were calculated for individual animals based on the body weight taken prior to treatment on the first day, and were thereafter adjusted to the most recent individual body weights. Daily individual dose volumes were 10 mL/kg body weight. During dosing, the homogeneity of the formulations was maintained by constant stirring over the magnetic stirrer. Dose administrations were performed approximately the same time each day (± 2 hour), and the time of dosing was recorded.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The test item stability in the vehicle of the dosing formulation was determined by the Sponsor prior to the initiation of this study. The test item was determined to be stable in the vehicle (0.1% Tween-80 in 0.5% methylcellulose aqueous solution) at 1 and 200 mg/mL concentrations for a minimum of 15 days at room temperature. Homogeneity of the test item in the vehicle was carried out in a GLP method validation (25 mL volume) study. Gavage formulations prepared one day prior to initiation of treatment and subsequently on Weeks 7 and 13 were analysed for test item content and homogeneity. Triplicate aliquots (top, middle and bottom layers) from each concentration (1, 5, 25 and 100 mg/mL) were sent for analysis.
Duration of treatment / exposure:
90 days males, 91 days females
Frequency of treatment:
daily
Doses / concentrationsopen allclose all
Dose / conc.:
10 mg/kg bw/day (actual dose received)
Dose / conc.:
50 mg/kg bw/day (actual dose received)
Dose / conc.:
250 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
10/sex/dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dosage levels were selected based on a 14-day dose range finding study and in consultation with the Sponsor. The high dose, 1000 mg/kg/day, was expected to produce minimal toxicity. The other doses were selected to assess a dose response for any observed effects and to establish a No-Observed-Adverse-Effect-Level (NOAEL).

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily to detect moribund or dead animals and abnormal behaviour and/or appearance among animals. An additional cage-site evaluation was conducted daily at approximately 2 hours (± 1 hour) post-dosing to detect acute clinical signs of systemic toxicity.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Detailed clinical examinations were performed for all animals on Day 1 (prior to the initiation of treatment) and weekly thereafter, excluding on the days of necropsy.

BODY WEIGHT: Yes
- Time schedule for examinations: on the first day of treatment prior to exposure and weekly thereafter. Terminal (fasting) body weights were taken on the day of scheduled necropsy. The body weight changes were calculated and reported weekly and monthly along with the body weight data.

FOOD CONSUMPTION: Yes
- Time schedule for examinations: Weekly (on the days that body weights are recorded) from the day of initiation of treatment until sacrifice. There was no food spillage observed in the cages in this study, hence, it was not considered for food consumption calculations.

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Yes

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Prior to initiation of treatment and during the last week of treatment (Day 87).

HAEMATOLOGY: Yes
- Time schedule for collection of blood: At the end of the treatment period
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals: All animals
- Parameters checked in Table No. 1 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: At the end of the treatment period
- Animals fasted: Yes
- How many animals: All animals
- Parameters checked in Table No. 2 were examined.

URINALYSIS: Yes
- Time schedule for collection of urine: At the end of the treatment period
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters checked in Table No. 2 were examined.

NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
GROSS PATHOLOGY: At the end of treatment period, all the animals were euthanized in stratified order by exsanguination under deep anaesthesia with isoflurane. All animals were examined for gross pathological changes. See Table 3 for organs collected, weighed, and preserved. Adherent adipose tissue from the organs was removed and the organ weights were recorded. Paired organs were weighed together. Terminal body weight on the day of necropsy was used to calculate organ weights relative to body weight. Organ weights relative to brain weights were also calculated. Organs were preserved in 10% neutral buffered formalin, except the testes, which were preserved in modified Davidson’s fixative, and the epididymides and eyes were preserved in Davidson’s fixative for 48 hours, and then preserved in 10% neutral buffered formalin.

HISTOPATHOLOGY: Histopathologic examinations were performed on all preserved organs of the control and high dose group animals. In addition, histopathological evaluation of the liver and kidney was performed in the low, intermediate, and high intermediate dose groups, as they were identified as target organs based on findings in the high dose group. Gross lesions from all the groups were subjected to histopathological evaluation. The tissues were processed and embedded in paraffin, then sectioned and stained with haematoxylin and eosin (See Table 3).
Statistics:
Statistical analysis was performed using validated statistical software (Graph Pad Prism 5.02). Analyses were conducted using two-tailed tests for a minimum significance level of 5%, comparing each test item treated group to the control group for each sex. Each mean was presented with the standard deviation (SD) and the number of animals (N) used to calculate the mean. A 'p' value ≤ 0.05 was considered as statistically significant. Numerical data, including body weights, body weight gain, food consumption, clinical pathology (haematology, clinical chemistry, and urinalysis) and organ weights were subjected to a statistical analysis. When an individual observation was recorded as being less than a certain value, calculations were performed on half the recorded value. When an individual observation was recorded as being greater than a certain value, calculations were performed on the recorded value. The data was examined for outliers and when required was subjected to Grubb's test. The significant values in Grubb's test were excluded from statistical analysis. Initially data was subjected to a Shapiro-Wilk normality test to check for normal distribution of the data and Bartlett's test for homogeneity of variance. When Bartlett's test or the Shapiro-Wilk normality tests were non-significant, Dunnett‘s post hoc test was used to compare the control group with test item treated groups. When Bartlett's test or Shapiro-Wilk normality tests were significant, data was subjected to a Kruskal-Wallis ANOVA followed by Dunn‘s post hoc test.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Description (incidence and severity):
No test item related clinical signs were observed in males or females at any dose level. One male rat receiving 50 mg/kg/day had soft stool on Day 71 of treatment. This clinical sign was not considered test item related due to the absence of a dose response and the transient nature of the effect.
Mortality:
no mortality observed
Description (incidence):
All the animals survived until the end of the study. No unscheduled deaths were observed in males or females at any dose level.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
There were no test item related changes in body weight parameters in male or female rats at any dose level. No statistically significant changes were observed in mean body weight in treated groups as compared to controls during the treatment period in male or female rats. Isolated incidences of statistically significant changes observed in body weight gain of males or females were not considered test item related due to the small magnitude of change and the lack of a dose response.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
There were no test item related changes in food consumption parameters in male or female rats at any dose level. Statistically significant changes observed occasionally in food consumption or food conversion efficiency in treated male and female rats were not considered test item related due to the small magnitude of change and the lack of a dose response.
Food efficiency:
effects observed, non-treatment-related
Description (incidence and severity):
There were no test item related changes in food consumption parameters in male or female rats at any dose level. Statistically significant changes observed occasionally in food consumption or food conversion efficiency in treated male and female rats were not considered test item related due to the small magnitude of change and the lack of a dose response.
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Description (incidence and severity):
No ophthalmological lesions were observed in males or females at any concentration.
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
There were no adverse changes in haematology or coagulation parameters in males or female rats at any dose level. Test item related, but non-adverse decreases in reticulocyte counts, MCV and MCH were observed in females at 1000 mg/kg/day. Statistically significant, but minimal decreases in MCH (5%) and APTT (25%), as well as increases in RDW (5%), monocytes (61%) and platelets (26%) were observed in male rats at 1000 mg/kg/day. These changes were not associated with changes in other haematology parameters or changes in bone marrow cellularity. Therefore, these findings in males were considered not related to treatment. Statistically significant but minimally increased lymphocytes (31%) at 50 mg/kg/day, decreased prothrombin time (12%) at 250 mg/kg/day in males and increased RBC counts (<4%) at >50 mg/kg/day and hematocrit at 250 mg/kg/day in females were not considered treatment related due to the small magnitude of change and the lack of a dose response. Statistically significant, but minimal decreases in MCV (2%) and MCH (3%) were associated with a decrease (statistically non-significant) in reticulocytes (23%) in females at the 1000 mg/kg/day. The decrease in reticulocytes was not accompanied by a reduction in RBC count or hematocrit or microscopic changes in bone marrow cellularity. Hence, the minimal decrease observed in these parameters may have been test item related, but considered as secondary and non-adverse findings. There were no other statistically significant changes in other haematology or coagulation parameters.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were no treatment-related or adverse changes in clinical chemistry parameters in males or female rats at any dose level. Statistically significant decrease in glucose (20%) and creatinine (-26%) at 250 mg/kg/day and increase in triglycerides (54%) at 10 mg/kg/day were observed in male rats. As there was neither a clear dose response nor any correlated findings in this case, the mild changes in these parameters were considered spurious. Statistically significant but minimal decrease in creatinine (-18%) and increase in albumin (7%) at 1000 mg/kg/day in females were not considered as treatment related findings.
Urinalysis findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were no treatment related changes in group mean urinalysis parameters or microscopic findings in male or female rats compared to the control group at any dose tested. Statistically increased urine volume at 1000 mg/kg/day (57%) and decreased urobilinogen (statistically non-significant) at >10 mg/kg/day in male rats were not considered treatment related due to the small magnitude of change or the lack of a dose response.
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Test item-related, organ weight changes were present in the liver and kidneys of male and female rats at 1000 mg/kg/day dose level. Statistically significant increased absolute and relative (to body and brain) liver weights were observed in males and females at 1000 mg/kg/day. Mean liver weight relative to body weight increases were 16% and 10% above controls in males and females at 1000 mg/kg/day, respectively. Increased liver weight was correlated with increased hepatocellular hypertrophy at 1000 mg/kg/day in both males and females. The test item associated liver weight increases were interpreted to be adaptive and non-adverse.

Statistically significant increased relative (to body) kidney weights in males and absolute and relative (to body and brain) kidney weights in females were observed at 1000 mg/kg/day. Mean kidney weight relative to body weight was increased 9% and 10% above controls in males and females at 1000 mg/kg/day, respectively. Increased kidney weight was correlated with tubular cell vacuolation at 1000 mg/kg/day in both males and females and was considered adverse. At 250 mg/kg/day increases in relative (to brain) kidney weight in female rats was considered of unclear relationship to treatment, as there were no significant changes in other kidney weight parameters for either sex at this dose level and because it was not associated with other significant microscopic changes (except for a single case of minimal tubular cell vacuolation).

There were no other treatment-related or statistically significant organ weight changes.
Gross pathological findings:
no effects observed
Description (incidence and severity):
There were no treatment-related gross pathology findings observed in this study. All findings observed were incidental in nature.
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Test item-related microscopic findings were observed in liver and kidney of both sexes. Centrilobular hepatocellular hypertrophy was observed in rats at 1000 mg/kg/day in male (7/10) and female (4/10) rats. Hepatocellular hypertrophy was characterized by enlarged hepatocytes with a homogeneous cytoplasm. This microscopic observation was correlated with an increase in liver weight parameters at this dose. As this observation was not associated with alterations in clinical pathology parameters indicative of liver toxicity, it was considered test item related, adaptive and non-adverse. Minimal to mild hepatocellular single cell necrosis was observed at 50 (females: 5/10), 250 (males: 4/10, females: 10/10) and 1000 (males: 7/10, females: 10/10) mg/kg/day dose levels. This lesion was characterized by single hepatocytes or clusters of hepatocytes that were shrunken with eosinophilic cytoplasm and pyknotic nuclei. This change was mainly seen in hepatocytes around central veins and was often associated with small numbers of mononuclear cells. This observation was not associated with alterations in clinical chemistry parameters. This finding was considered test item related and potentially adverse. The severity and incidence of hepatocellular single cell necrosis was greater in females compared to males.

Pigmentation observed at 50 (females: 3/10), 250 (females: 10/10) and 1000 (males: 3/10, females: 10/10) mg/kg/day dose levels was considered a treatment-related, non-adverse change. Pigmentation was observed in hepatocytes and Kupffer cells around the central vein. The colour varied from pale yellow to deep granular brown. The severity and incidence of pigmentation was greater in females compared to males.

Tubular cell vacuolation was observed in kidneys at the following incidences in both sexes. Tubular cell vacuolation observed at 250 (males: 1/10, females: 1/10) and 1000 (males: 9/10, females: 8/10) mg/kg/day was considered a treatment-related, adverse change. Tubular cell vacuolation was mainly observed in the outer strip of the outer medulla (OSOM) and was characterised by vacuolar degeneration of epithelial cells lining the tubules. Coarse and/or fine cytoplasmic vacuoles were observed in affected cells. Affected tubules occasionally contained scattered necrotic cells.

Suppurative inflammation and associated changes observed in the nasal mucosa of three high dose male rats and one control female rat was considered likely due to accidental aspiration of dose formulation owing to reflux related to gavage dosing. All other microscopic findings observed in this study were either spontaneous or incidental in nature as the lesions were distributed randomly across the groups in low incidences and were consistent with gavage trauma or with common background lesions in rats of this strain and age.
Histopathological findings: neoplastic:
no effects observed

Effect levels

open allclose all
Key result
Dose descriptor:
NOAEL
Effect level:
50 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: hepatocellular single cell necrosis and tubular cell vacuolation in kidneys at ≥250 mg/kg/day dose
Key result
Dose descriptor:
NOAEL
Effect level:
10 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: hepatocellular single cell necrosis at ≥50 mg/kg/day

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

 

Organ Weight (and % Change from Controls)

 

Parameter

Male (Dose in mg/kg/day)

Female (Dose in mg/kg/day)

0 (control)

10

50

250

1000

0 (control)

10

50

250

1000

Liver weight - absolute

10.542

11.568

(10)

11.009

(4)

11.012

(4)

12.050*

(14)

6.139

6.317

(3)

6.211

(1)

6.604

(8)

6.839 *

(11)

Liver weight – relative to bw

2.810

3.036

(8)

2.881

(3)

2.981

(6)

3.258*

(16)

2.708

2.691

(-1)

2.732

(1)

2.829

(4)

2.975*

(10)

Liver weight – relative to brain weight

539.032

597.562

(11)

562.534

(4)

565.596

(5)

619.187*

(15)

339.067

341.750

(1)

337.904

(0)

366.953

(8)

376.154*

(11)

Kidneys weight - absolute

2.682

2.861

(7)

2.670

(0)

2.734

(2)

2.884

(8)

1.589

1.627

(2)

1.666

(5)

1.729

(9)

1.770*

(11)

Kidneys weight – relative to bw

0.715

0.753

(5)

0.699

(-2)

0.741

(4)

0.779 *

(9)

0.700

0.692

(-1)

0.732

(5)

0.741

(6)

0.768*

(10)

Kidneys weight – relative to brain weight

137.131

147.761

(8)

136.508

(0)

140.616

(3)

148.128

(8)

87.661

88.006

(0)

90.559

(3)

96.037*

(10)

97.501*

(11)

* Indicates statistically significant as compared to controlusing Dunnett’s test or Dunn’s test.
  Values in parenthesis are % change from control

  bw: Body weight

 

 

Incidence of Microscopic Findings – Liver

Microscopic findings

Male Dose (mg/kg/day)

Female Dose (mg/kg/day)

0

10

50

250

1000

0

10

50

250

1000

Number examined

10

10

10

10

10

10

10

10

10

10

Hepatocellular hypertrophy, Centrilobular

Minimal

-

-

-

-

7

-

-

-

-

4

Total

0

0

0

0

7

0

0

0

0

4

Hepatocellular single cell necrosis

Minimal

-

-

-

2

6

-

-

5

10

6

Mild

-

-

-

0

0

-

-

0

0

4

Total

0

0

0

2

6

0

0

5

10

10

Pigmentation

Minimal

-

-

-

-

2

-

-

3

10

5

Mild

-

-

-

-

1

-

-

0

0

4

 Moderate

-

-

-

-

0

-

-

0

0

1

Total

0

0

0

0

3

0

0

3

10

10

 

 

Incidence of Microscopic Findings – Kidneys

Microscopic findings

Male Dose (mg/kg/day)

Female Dose (mg/kg/day)

0

10

50

250

1000

0

10

50

250

1000

Number examined

10

10

10

10

10

10

10

10

10

10

Tubular cell vacuolation

Minimal

-

-

-

1

6

-

-

-

1

6

Mild

-

-

-

0

3

-

-

-

0

2

Total

0

0

0

1

9

0

0

0

1

8

 

Applicant's summary and conclusion

Conclusions:
Male NOAEL = 50 mg/kg bw/day
Female NOAEL = 10 mg/kg bw/day
Executive summary:

The objective of the study was to determine the subchronic toxicity of the test substance when administered by oral gavage in Sprague Dawley rats. Groups of 10 male and 10 female rats received daily oral doses of 0, 10, 50, 250, or 1000 mg/kg of the test item for a minimum 90 consecutive days (90 days for males and 91 days for females). The test item was shown to be homogeneous and at the targeted concentrations in the dose formulations. Stability of 15 days at room temperature was established for the test item prior to the study start. Parameters evaluated in this study included twice daily mortality checks, daily checks for clinical signs, weekly detailed clinical examinations, weekly measurement of body weight and food consumption and ophthalmological examinations before the start and at the end of the treatment period. At the end of the treatment period, haematology, clinical chemistry, urinalysis, gross pathology and organ weights were evaluated. All organs preserved from the control and high dose groups, all gross lesions, liver, and kidneys in both sexes at all dose levels were subjected to histopathology examinations.

No mortality, clinical signs or ophthalmological lesions were observed in males or females at any dose level. There were no test item related changes in body weight or food consumption parameters. There were no adverse changes in haematology, coagulation, clinical chemistry, and urinalysis parameters. Non-adverse test item-related changes included decreased MCV (2%), MCH (3%), and reticulocyte counts (23%) at 1000 mg/kg/day dose in female rats. There were no treatment related gross pathology findings observed in males or females. At 1000 mg/kg/day, test item related increased relative (to body and brain) liver weights in both sexes and relative kidney (to body) weights in males and absolute and relative (to body and brain) kidney weights in females were observed. Test item related potentially adverse histopathological findings included minimal to mild hepatocellular single cell necrosis (≥250 mg/kg/day in males and ≥50 mg/kg/day in females) and minimal to mild tubular cell vacuolation in kidneys (≥250 mg/kg/day in males and females). Test item related, non-adverse findings included minimal centrilobular hepatocellular hypertrophy (1000 mg/kg/day in males and females) and minimal to moderate pigmentation in hepatocytes and Kupffer cells (1000 mg/kg/day in males and ≥50 mg/kg/day in females). Under the conditions of the study, the No-Observed-Adverse-Effect-Level (NOAEL) of the test substance is 10 mg/kg bw/day in the female and 50 mg/kg bw/day in male rats. The NOAEL in female rats is based on hepatocellular single cell necrosis at ≥50 mg/kg/day. The NOAEL in male rats is based on the hepatocellular single cell necrosis and tubular cell vacuolation in kidneys at ≥250 mg/kg/day dose.