Registration Dossier

Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
03 January 2018 - 31 October 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report Date:
2018

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
GLP compliance:
yes (incl. certificate)
Limit test:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid
Specific details on test material used for the study:
Batch number: 0727/16
Storage conditions: At room temperature (20 ± 5 ºC) in the dark

Test animals

Species:
rat
Strain:
Wistar
Details on species / strain selection:
Species: Recognized by international guidelines as a recommended test system.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Reputable commercial supplier
- Females (if applicable) nulliparous and non-pregnant: no
- Age at study initiation:
Males: 9 to 13 weeks
Females: 11 to 13 weeks

- Weight at study initiation:
Males: 289-415 g
Females: 190-263 g

- Fasting period before study: none
- Housing:
Cages with standard, granulated, S8-15 sawdust bedding
Premating period (maximum 5 animals/cage) Makrolon type IV cages
Mating period (one male and one female/cage) Makrolon type III cages
Postmating, gestation and lactation periods (individual) Makrolon type III cages

- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: Five days after arrival and pre-treatment start. After acclimatization period, the animals were subjected to a pre-test period.

DETAILS OF FOOD AND WATER QUALITY: Pelleted standard Teklad 2014C rat/mouse maintenance diet ad libitum. Pelleted standard Teklad 2018C rat/mouse maintenance diet ad libitum, for lactating females and pups (until sacrifice).
Tap water in bottles ad libitum.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24 ºC
- Humidity (%): 30 and 70%
- Air changes (per hr): 15-20
- Photoperiod (hrs dark / hrs light): 12 hours fluorescent light/12 hours dark.

Administration / exposure

Route of administration:
oral: gavage
Details on route of administration:
Oral gavage was used as the route of administration as recommended by the OECD 422 guideline, in order to deliver accurate doses. In addition, oral ingestion is a possible route for human exposure to the test item.
Vehicle:
other: 0.5 % Tween 80 + 1% CMCNa (medium viscosity) in water
Details on oral exposure:
- PREPARATION OF DOSING SOLUTIONS:
Formulation for Group 1 (Vehicle)
The necessary volume of the vehicle was added into a suitable container (stock solution) and the formulation was aliquoted for each day of administration, when required.
Formulation for Groups 2, 3 and 4
1. The required quantity of test item was weighed in a single-use container.
2. The test item was transferred to a sufficiently large mortar and a pestle was used to pulverize it.
3. Small amounts of vehicle were added and mixed with the pestle. Any lumps were broken up at this point, resulting in a homogeneous suspension.
4. The suspension was transferred to a volumetric flask or graduated measuring cylinder that was previously moistened with vehicle. The mortar and the single-use container were rinsed completely with vehicle to ensure that there were no remnants of the test item. This vehicle was added to the volumetric container and make up to the mark.
5. If it was necessary to adjust volume, first the volume was added into volumetric vessel and after the remainder passed at final container.
6. The suspension was transferred to a suitable container and mixed until the suspension was homogenized. The necessary time to obtain a homogenized formulation was visually checked and documented in the raw data.
7. After mixing the formulation for five minutes, samples for analysis were taken, and the formulation was aliquoted for each day of administration, when required.

- DIET PREPARATION
Pelleted standard Teklad 2014C rat/mouse maintenance diet ad libitum (supplied by Envigo RMS, S.L.)

- VEHICLE
- Justification for use and choice of vehicle (if other than water): not specified
- Concentration in vehicle: 0.5-100mg/mL
- Amount of vehicle (if gavage): 10 mL/kg/day

- Lot/batch no. (if required):
Tween 80 - BCBG4547V
CMCNa (medium viscosity) - BCBN1690V
Water: 17382411

- Purity: not specified
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The formulations prepared at three different concentrations were analyzed twice over the course of the study to verify its correct preparation.
Control (vehicle) formulations were analyzed to confirm the absence of test item or other substances at the retention time of the test item.
The test item was used as analytical standard.
Results were within ±20% of nominal value and the coefficient of variation (CV) was ≤10%.
Other parameters were also tested, meeting the following acceptance criteria:
• System Suitability Test (SST) --- CV ≤ 2%.
• Calibration curve --- R2 ≥ 0.99; deviation of the calibration standards ≤ 10% (75% complies).
Duration of treatment / exposure:
Males - 2 weeks before pairing up to necropsy after 5-6 weeks
Females - 2 weeks before pairing, then throughout pairing and gestation until days 13 15 of lactation (until the day before sacrifice)
Frequency of treatment:
Once daily
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Dose / conc.:
100 mg/kg bw/day (nominal)
Dose / conc.:
300 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Dose / conc.:
5 mg/kg bw/day (nominal)
Dose / conc.:
25 mg/kg bw/day (nominal)
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
- Rationale for animal assignment (if not random): Randomized
- Rationale for selecting satellite groups: none specified
- Post-exposure recovery period in satellite groups: not applicable
- Section schedule rationale (if not random): not specified
Positive control:
none specified

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Visually inspected twice daily for evidence of reaction to treatment or ill-health.

BODY WEIGHT: Yes
- Time schedule for examinations: See table 1
F1 Individual offspring body weights: Days 1, 4, 7 and 13 of age

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
See table 2
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Not specified
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Not specified

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at termination
- Anaesthetic used for blood collection: Yes - Isoflurane
- Animals fasted: Yes
- How many animals: all animals that died or were sacrificed (table 3)
- The following parameters were examined:
Hematocrit (Hct), Hemoglobin concentration (Hb), Erythrocyte count (RBC), Absolute reticulocyte count (Retic) , Total leucocyte count (WBC), Differential leucocyte count (N: neutrophils, L: lymphocytes, E: eosinophils, B: basophils, M: monocytes, LUC: large unstained cells), Platelet count (Plt), Mean cell hemoglobin (MCH), Mean cell volume (MCV), Mean cell hemoglobin concentration (MCHC), Red cell distribution width (RDW), Using citrate as anticoagulant, Prothrombin time (SPT), Activated partial thromboplastin time (SAPT), Fibrinogen (SFIB).

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at termination
- Animals fasted: Yes
- How many animals: Table 3
- The following parameters were examined:
Alkaline phosphatase (ALP), Alanine amino-transferase (ALT), Aspartate amino-transferase (AST), Glucose (Gluc), Bilirubin – total (Bili), Bilirubin – direct (BILD), Bilirubin indirect* (INDC), Cholesterol – total (Chol), HDL, LDL, Triglycerides (Trig), Creatinine (Creat), Creatine kinase (CK), Lactate dehydrogenase (LDH), Urea, Total protein (Total prot), Albumin (Alb), Albumin/globulin ratio (A/G Ratio), Protein electrophoretogram , Sodium (Na), Potassium (K), Chloride (Cl), Calcium (Ca), Phosphorus (Phos), Bile acids (Bi Ac)
*Indirect bilirubin was calculated from total & direct bilirubin.

URINALYSIS: No
- Time schedule for collection of urine: n/a
- Metabolism cages used for collection of urine:n/a
- Animals fasted: n/a
- Parameters checked: n/a

NEUROBEHAVIOURAL EXAMINATION: Yes - Table 4
- Time schedule for examinations:
- Dose groups that were examined:
- The following measurements, reflexes and responses were recorded:
- Blink reflex
- Pinna reflex
- Iridic light / Pupil closure reflex - Proprioception (right leg) / push-off (hind legs)
- Pain response / Tail pinch response - Startle / hearing
- Righting reflex in the air
Grip strength was quantitatively measured with equipment for measuring the force of grip from BIOSEB, GT-3 model. The grip strength of the forelimbs was measured 3 times, as well as that of the hind limbs, to analyze the average value of the three occasions for hind- and forelimbs separately.
Motor activity was quantitatively measured with an AMS from Medical Instruments GmbH (FMI) and DeMeTec-Ams. Activity of the animals was recorded in 10-minute intervals over a 60-minute period. For testing, designated animals were placed singly into observation cages.

IMMUNOLOGY: No
- Time schedule for examinations: n/a
- How many animals: n/a
- Dose groups that were examined: n/a
- Parameters checked in table [No.?] were examined. n/a

OTHER: n/a
Sacrifice and pathology:
GROSS PATHOLOGY: Yes (see table 5)

HISTOPATHOLOGY: Yes (see table 5)
Statistics:
The following comparisons were performed: Group 1 vs. 2, 3, 4, 5 and 6 (separately for males and females).
Statistics:
Continuous data - a parametric analysis was performed if Bartlett's test for variance homogeneity was not significant at the 1% level.
Treated groups - Williams' test, unless there was evidence against a monotonic dose-response when Dunnett's test was performed instead.
A non-parametric analysis was performed if Bartlett's test was still significant at the 1% level.
Treated groups - Shirley's test, unless there was evidence against a monotonic dose response when Steel's test was performed instead.
For organ weight data, analysis of covariance was performed using terminal bodyweight as covariate, unless non-parametric methods were applied.
For estrous cycles an exact one-tailed (upper-tail) Linear-by-linear test was applied to all groups.

For pre-coital intervals an exact one-tailed (upper-tail) Linear-by-linear test was applied to all groups.
For gestation length an exact two-tailed Linear-by-linear test, with equally spaced scores, was applied to all groups.
When the exact version of the Linear-by-linear test could not be calculated, then the asymptotic version was used instead.
For number mating, number conceiving, number fertile, number live and terminal smear status an exact one-tailed (lower-tail) Cochran-Armitage test was applied to all groups. If the test was statistically significant (p<0.05), the highest dose group was excluded and the test re-applied. This ‘step down’ process was repeated until the test was no longer statistically significant (p≥0.05).
Significant differences between the groups compared were expressed at the 5% (p<0.05) or 1% (p<0.01) level.

Results and discussion

Results of examinations

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Adverse clinical signs were observed from day 8 of treatment and onwards at the doses of 300 and 1000 mg/kg/day (shown as thinness, piloerection, hunched posture, pallor, loss of pigmentation, low body temperature). Some of these clinical signs were also observed in females at 100 mg/kg/day during late gestation. On some occasions and at all abovementioned doses it was decided to apply the final endpoint and the animals were killed for welfare reasons (mainly females during late gestation).
At 25 mg/kg/day, hunched posture, abnormal gait, piloerection and/or pallor were observed occasionally in some males and females during the administration period.
At 5 mg/kg/day, pallor was occasionally recorded in one male on day 15 post-mating, and piloerection, hunched posture and pallor were observed in some females during late gestation/lactation.
Mortality:
no mortality observed
Description (incidence):
No mortality was recorded in the males or females administered at 5 or 25 mg/kg/day.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
In males and females, groups administered at 100, 300 and 1000 mg/kg/day showed lower mean body weights than control during post-mating and gestation/lactation periods, generally in line with a dose-response relationship.
Male mean body weights at treatment start were higher compared to control. The same profile was seen at 5 mg/kg/day. At 25 mg/kg/day, however, there was no mean body weight recovery until the end of the study period.
Females showed the same trend in control and at 5 and 25 mg/kg/day; however, at 25 mg/kg/day mean body weight was always lower when compared to control (from the beginning of gestation until sacrifice).
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
Test item administration at 100, 300 and 1000 mg/kg/day caused a significant decrease in food consumption with respect to control.
No relevant differences were observed between control and 5 mg/kg/day among females or between control and 5 mg/kg/day among males.
In males, significantly lower mean values with respect to control were observed in all test tem administered groups between days 1-8 post-mating, as well as between days 8-15 at 25, 100, 300 and 1000 mg/kg/day.
Significantly lower mean values were observed during gestation and lactation at 100, 300 and 1000 mg/kg/day when compared to control as well as at 25 mg/kg/day between gestation days 0-7 and during lactation.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Statistically significant differences observed in coagulation (prothrombin time and activated partial thromboplastin time in males and females and in fibrinogen in males) were considered not relevant given their magnitude and the absence of a dose-response relationship.
At the end of the administration period, males and females showed lower than control hematocrit, hemoglobin, red blood cells, reticulocytes (only in males), white blood cells and lymphocytes at 25, 100, 300 mg/kg/day and in males at 1000 mg/kg/day. Most of these mean values are outside the range commonly recorded in rats under the same conditions.
At 5 mg/kg/day, mean hematocrit and hemoglobin in males and females and white blood cells and lymphocytes in males were lower with respect to Control, although the difference was minimal and the values remained within the range of historical control values.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
Blood chemistry in males after 5 weeks of treatment revealed significantly higher aspartate aminotransferase, bile acids and urea values mainly at 25, 100, 300 and 1000 mg/kg/day when compared to control. Although no dose-response relationship was observed and mean values were similar to those recorded in Control, lower than Control mean values were observed in the test item administered groups in males and at 100 mg/kg/day in females.
At the end of their corresponding treatment periods, lower mean triglyceride values were recorded in all test-item-administered groups with respect to control. However, mean values at 5 mg/kg/day were close to those recorded in control and within the historical control data.
Clinical biochemistry reveals higher than control mean creatinine values at 100 and 300 mg/kg/day in females and at 25, 100, 300 and 1000 mg/kg/day in males.
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
Treatment with Triazolone during 5-8 weeks caused an increase in thyroids and parathyroids in all test-item-administered groups.
In addition, a decrease in some of the evaluated organs in the test-item-administered groups (adrenals, spleen, prostate, seminal vesicles and coagulating glands, epididymides, liver, thymus, heart and kidneys), mainly at 100, 300 and 1000 mg/kg/day was also observed.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
The macroscopic examination performed 5-8 weeks after treatment revealed the following changes in the thyroids and thymus of both sexes and in the male accessory sex organs:
- Bilateral enlargement of the thyroids was observed in all treated male groups, and in treated female groups down to 25 mg/kg/day.
- A small thymus was seen in a few males and females receiving > 100 mg/kg/day, with the highest incidence found at 1000 mg/kg for both sexes.
- Accessory sex organs (prostate, seminal vesicles and coagulating glands) were noted to be smaller in males given > 100 mg/kg compared to controls, with a dose-dependent increase in incidence.
All the other gross findings were considered to be incidental and unrelated to the test item.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Changes that were considered related to treatment with the test item were seen in the thyroids (all administered doses), sternal bone marrow (all dose levels) and thymus of both males and females (at 25, 100, 300 and 1000 mg/kg/day); testes, accessory sex organs (all administered doses), liver (100 mg/kg/day) and skin of males (at 25 and 100 mg/kg/day); and in the spleen of females (at 25 mg/kg/day).
Histopathological findings: neoplastic:
not examined
Other effects:
not examined

Effect levels

open allclose all
Key result
Dose descriptor:
NOAEL
Effect level:
ca. 5 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
clinical signs
body weight and weight gain
food consumption and compound intake
clinical biochemistry
behaviour (functional findings)
organ weights and organ / body weight ratios
gross pathology
histopathology: non-neoplastic
Key result
Dose descriptor:
other: NOAEL not determined
Effect level:
ca. 0 other: not determinable
Based on:
test mat.
Sex:
male
Basis for effect level:
clinical signs
mortality
body weight and weight gain
food consumption and compound intake
clinical biochemistry
behaviour (functional findings)
organ weights and organ / body weight ratios
gross pathology
histopathology: non-neoplastic
Remarks on result:
not determinable because of methodological limitations

Target system / organ toxicity

Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
5 mg/kg bw/day (nominal)
System:
endocrine system
Organ:
coagulating gland
dorsolateral prostate gland
parathyroid gland
seminal vesicle
thymus
thyroid gland
ventral prostate gland
other: See table 6 for full results
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
yes

Any other information on results incl. tables

Table 6. Target system / organ toxicity

Group

Enlarged Thyroids and Parathyroids

Small Thymus

Small Accessory sex organs

 

Males

Pregnant Females

Males

Pregnant Females

Males

0 mg/kg/day

0/10

0/10

1/10

0/10

0/10

5 mg/kg/day

3/10

0/9

0/10

0/9

0/10

25 mg/kg/day

9/10***

4/10*

0/10

0/10

0/10

100 mg/kg/day

10/10***

9/9***

2/10

2/9

1/10

300 mg/kg/day

10/10***

9/9***

2/10

1/9

3/10

1000 mg/kg/day

10/10***

5/5***

5/10*

4/5*

5/10**

Applicant's summary and conclusion

Conclusions:
In conclusion, the effects of oral (gavage) administration of Triazolone to Wistar rats receiving 5, 25, 100, 300 or 1000 mg/kg/day for 14 days prior to mating and until sacrifice can be summarized as follows:
Repeated dose toxicity - Based on the histopathological findings observed in thyroids (bilateral hypertrophy/hyperplasia of follicular cells) and the corresponding decrement in T4 values at all dose levels administered that caused morphological and physiological changes, a NOAEL could not be established within the confines of this study for males; and based on the data obtained, the NOAEL for females could be considered to be 5 mg/kg/day.