cis-2-(2,4-dichlorophenyl)-2-(1H-1,2,4-triazol-1-ylmethyl)-1,3-dioxolane-4-methanol

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 2000-10-23 to 2000-11-06

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

The study was performed similar to OECD as well as food and drug safety guidelines and in accordance with GLP principles. No deviations were recorded in the study report. However, the observation period was not according to OECD 407 (animals were sacrificed after two weeks of dosing instead of 28 days after study initiation).

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2001

Report date:

2001

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Deviations:

yes

Remarks:

observation period was not 28 days: animals were sacrificed after two weeks of dosing instead of 28 days after study initiation

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: Toxicological Principles for the Safety Assessment of Direct Food Additives and Color Additives in Food, "Redbook IT", D.S. FDA Washington DC 1993

Deviations:

not specified

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: The rules governing medicinal products in the European Community. Volume ill: guidelines on the quality, safety and efficacy of medicinal products for human use. Revised and completed edition, January 1996.

Deviations:

not specified

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: Guidelines for non clinical studies of drugs Manual 1995, Ministry of Health and Welfare - Japan

Deviations:

not specified

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

cis-2-(2,4-dichlorophenyl)-2-(1H-1,2,4-triazol-1-ylmethyl)-1,3-dioxolane-4-methanol

EC Number:

267-747-4

EC Name:

cis-2-(2,4-dichlorophenyl)-2-(1H-1,2,4-triazol-1-ylmethyl)-1,3-dioxolane-4-methanol

Cas Number:

67914-85-6

Molecular formula:

C13H13Cl2N3O3

IUPAC Name:

(+-)-cis-2-(2,4-dichlorophenyl)-2-(1H-1,2,4-triazol-1-ylmethyl)-1,3-dioxolane-4-methanol

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

- Name of test material (as cited in study report): T001207 (T001207 is a drug substance impurity of Itraconazole R051211)
- Substance type: powder
- Physical state: solid
- Analytical purity: no data
- Lot/batch No.: ZT001207PUA441
- Expiration date of the lot/batch: no data
- Storage condition of test material: at room temperature in a closed and labelled container
- Other: Stability in the different solutions has been tested on solutions prepared on Oct. 20 2000 by analysed on Oct. 25 2000 and Nov. 7 2000. Stability was guaranteed.
- Conversion factor: 1.00

Test animals

Species:

rat

Strain:

other: HanBrl:WIST (SPF)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River
- Age at study initiation: at arrival 4 weeks old
- Weight at study initiation: males: 98-100 grams; females 93-95 grams
- Housing: individually in numbered wire-mesh cages 20 x 20 x 20 cm consisting of stainless steel walls and a wire-mesh bottom, suspended in wheeled racks above disposable polystyrene plates for the collection of faeces and urine
- Diet (e.g. ad libitum): powdered rat feed, manufactured the basis of an open formula of Janssen Pharmaceutica N.V., diet was sterilised by 2.5 Megarad of gamma irradiation
- Water (e.g. ad libitum): water, administered in drinking bottles
- Acclimation period: approx. one week.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-23°C
- Humidity (%): 40 - 70%
- Air changes (per hr): air-conditioned room, no further information
- Photoperiod (hrs dark / hrs light): routine test conditions of illumination

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: aqueous solution wit 400 mg hydroxypropyl-beta-cyclodextrin/ per ml and 1,2-propanediol, HCl 12N, NaOH 1N sufficient quantity to achieve a pH of 3.8 (± 0. 1)

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
In the present study, R051211 was formulated as an aqueous solution at 2 concentrations: 10 and 9.8 mg/ml. The impurity compound T001207 was added to the 9.8 mg/ml solutions at a concentration of 0.2 mg/ml. The solutions contained 400 mg hydroxypropyl-beta-cyclodextrin/ml and thus a ratio R051211 ( +T001207)/HP-beta-CD of 1/40 was achieved (= identical to the clinical formulation). The ingredients of the solutions were:
- R051211 or R01211 + impurity compound T001207 2%
- HP-beta-CD
- purified water
- 1,2-propanediol
- HCl 12N
- NaOH IN sufficient quantity to achieve a pH of 3.8 (± 0. 1)

A vehicle solution was prepared containing 400 mg HP-beta-CD/ml and the same ingredients as the previous formulation, except for the test substances.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The concentration and stability of T001207 in the different solutions was checked on October 25, 2000 and on November 7, 2000 with method TOX-LC-T0012207-GLP.

Duration of treatment / exposure:

2 weeks

Frequency of treatment:

daily

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
group V: 0 + 0 + 800 R051211 +T001207 + HP-beta-CD mg/kg body weight
Basis:
other: nominal based on solutions prepared

Doses / concentrations 2

Remarks:

Doses / Concentrations:
group A: 5 + 0 + 200 R051211 +T001207 + HP-beta-CD mg/kg body weight
Basis:
other: nominal based on solutions prepared

Doses / concentrations 3

Remarks:

Doses / Concentrations:
group B: 4.9 +0.1 + 200 R051211 +T001207 + HP-beta-CD mg/kg body weight
Basis:
other: nominal based on solutions prepared

Doses / concentrations 4

Remarks:

Doses / Concentrations:
group D: 20 + 0 + 800 R051211 +T001207 + HP-beta-CD mg/kg body weight
Basis:
other: nominal based on solutions prepared

Doses / concentrations 5

Remarks:

Doses / Concentrations:
group E: 19.6 + 0.4 + 800 R051211 +T001207 + HP-beta-CD mg/kg body weight
Basis:
other: nominal based on solutions prepared

No. of animals per sex per dose:

5 groups including one vehicle group each consisting of 5 males and 5 females, in total 27 males and 27 females

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose selection for the present study was based upon information of previously conducted two 1-month toxicity studies with R051211, administered orally by gavage, in the Wistar rat.

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: see detailed clinical observations

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily: for signs of waning health, abnormal behaviour or unusual appearance, occurrence of untoward clinical effects and manifestations of toxic and pharmacological response, moribund state and mortality.

BODY WEIGHT: Yes
- Time schedule: rats were weighed daily for dosing volume adjustments, but records were maintained on a weekly basis (initially, after one week and at the end of the study).

ABSOLUTE AND RELATIVE ORGAN WEIGHTS
-After removing adherent adipose tissue, the following organs of all rats killed at the end of the dosing period were weighed: lungs, spleen, liver, heart, pancreas, kidneys, brain, thymus, adrenal glands, thyroid glands (including parathyroid glands) and gonads (testes, ovaries).

FOOD CONSUMPTION:
- Food consumption was measured in all rats and at weekly intervals during the dosing period. The measurements were carried out by an automated weighing procedure by use of a calibrated balance and on-line recorded.

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: No

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
Blood sampling: towards the end of the study
Individual blood samples were obtained by puncture of the orbital venous plexus.
The following parameters were determined:
- haematocrit
- haemoglobin
- red and white blood cell count
- red blood cell indices: mean cell volume, mean cell haemoglobin and mean cell haemoglobin concentration
- thrombocyte count
- differential white blood cell count


CLINICAL CHEMISTRY: Yes (Serum Analysis)
- Time schedule for collection of blood: towards the end of the study
- The following parameters were determined with a Hitachi Modular Analyser: - sodium - triglycerides- potassium - phospholipids - chloride - blood urea nitrogen - calcium - creatinine - inorganic phosphate - total bilirubin - total protein - alkaline phosphatase - albumin - aspartate aminotransferase - glucose - alanine aminotransferase - cholesterol

URINALYSIS: YES
- Time schedule for collection of urine: towards the end of the study
- The following parameters were determined: - volume - urobilinogen, pH, proteins, glucose, ketones, bilirubin, occult blood (1) - specific gravity (2)
- sediment (microscopic)
Methods:
(1) Combur 10M dipstick, Boehringer
(2) refractometric

NEUROBEHAVIOURAL EXAMINATION: NO

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
Sacrifice: at the end of the dosing period
Samples of the following tissues from all rats were preserved in 10% buffered formalin, with exception of the eyes and testes which were preserved in a mixture of 2.5% buffered formalin and 1.25% glutaraldehyde solution:
- adrenal glands
- aorta
- bone (stifle joint, rib, sternum) with bone marrow
- brain
- brown adipose tissue
- epididymides
- esophagus
- extraorbital lacrimal glands
- external ears with parotid glands
- eyes
- heart
- kidneys
- large intestine (cecum, colon, rectum)
- liver
- lungs
- lymph node(s), mesenteric
- mammary gland
- nose
- ovanes
- pancreas
- peripheral nerve (sciatic nerve)
- pharynx - larynx
- pituitary gland
- prostate
- salivary glands (submandibular)
- seminal vesicles and coagulating glands
- skeletal muscle (diaphragm, psoas muscle)
- skin
- small intestine (duodenum, jejunum, ileum)
- spinal cord (thoracic, lumbar)
- spleen
- stomach
- testes
- thymus
- thyroid glands with parathyroid gland(s)
- tongue
- trachea
- urinary bladder
- uterus with cervix
- vagma
- all tissues showing gross changes

HISTOPATHOLOGY: NO

Statistics:

The significance of differences between each dosage group and the vehicle group was assessed by the following statistical methods:
1. Chi-Square test for pairwise comparison with vehicle according to Siegel (*) (two-tailed, with Yates' correction for continuity).
2. Fisher exact probability test (two tailed)
3. Mann-Whitney U test for pairwise comparison with vehicle according to Siegel (*) (two-tailed with correction for ties).

(*) Siegel S., Nonparametric Statistics for the behavioral Sciences, McGraw-Hill, New York, 1956

mortality: Fisher exact probability test
clinical observations: Fisher exact probability test
body weight: Mann-Whitney U test
weight gain: Mann-Whitney U test
food consumption: Mann-Whitney U test
haematology: Mann-Whitney U test
serum analysis: Mann-Whitney U test
urinalysis: Mann-Whitney U test
organ weights (absolute-relative): Mann-Whitney U test
gross pathology: Chi-square test

In addition to this comparison, records for the vehicle- and test article dosed rats were compared with the historical control data.

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 examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

no effects observed

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Details on results:

CLINICAL SIGNS AND MORTALITY
Oral administration of R051211 and of R051211 spiked with 2% T001207 during 2 weeks at a dose up to 20 mg/kg body weight, did not result in mortality.
Oral administration of R051211 and of R051211 spiked with 2% T001207 during 2 weeks at a dose up to 20 mg/kg body weight, did not result in clinical findings.

BODY WEIGHT
Oral administration of R051211 and of R051211 spiked with 2% T001207 during 2 weeks at a dose up to 20 mg/kg body weight, did not result in relevant body weight or weight gain changes.

FOOD CONSUMTPION:
Oral administration of R051211 and of R051211 spiked with 2% T001207 during 2 weeks at a dose up to 20 mg/kg body weight, did not result in changes in food consumption.

HAEMATOLOGY
Oral administration of R051211 to male and female rats at 5 mg/kg (with or without 2% T001207) had no adverse effects on haematological parameters. In males dosed at 20 mg/kg body weight (with or without 2% T001207), a marginal decrease was noted in white blood cells, neutrophils and eosinophils. In females dosed at 20 mg/kg body weight (with or without 2% T001207), slight decreases were noted in white blood cells, thrombocytes and eosinophils. Changes noted in spiked and non-spiked groups were comparable.

CLINICAL CHEMISTRY
No changes were noted in male rats dosed at 5 mg/kg (with or without 2% T001207). In female rats dosed at 5 mg/kg (with or without 2% T001207) slight increases were noted in cholesterol and phospholipids. In males and females dosed at 20 mg/kg (with or without 2% T001207), increases in glucose, cholesterol and phospholipids were noted. In females dosed at 20 mg/kg (with or without 2% T001207), a slight decrease in triglycerides was observed. Changes noted in spiked and non-spiked groups were comparable.

URINALYSIS
Oral administration of R051211 and of R051211 spiked with 2% T001207 during 2 weeks at a dose up to 20 mg/kg body weight, did not result in changes in urinary variables.

ORGAN WEIGHTS
No changes in organ weights were noted in male and female rats dosed at 5 mg R051211/kg body weight with or without 2% T001207. In male and female rats dosed at 20 mg R051211 / kg body weight with or without 2% T001207, marginal ( males) to slight (females) increases were observed in the absolute and relative weight of the adrenals. In male rats dosed at 20 mg/kg body weight with or without 2% T001207 a slight increase was noted in the absolute and relative weight of the lungs.

GROSS PATHOLOGY
Oral administration of R051211 and of R051211 spiked with 2% T001207 during 2 weeks at a dose up to 20 mg/kg body weight, did not result in relevant gross pathological changes.

Effect levels

Dose descriptor:

NOAEL

Remarks on result:

not determinable

Remarks:

no NOAEL identified

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

Results of previously conducted two 1-month toxicity studies:

In one study rats were dosed orally at 5 and 40 mg R051211 / kg body weight/day combined with HP-beta-CD at a ratio R051211 / HP-beta-CD of 1 / 40 (in that specific study, an old and a new batch were compared). Based upon this study it was concluded that oral administration of itraconazole as a HP-beta-CD formulation at doses of 5 and 40 mg/kg b.w. was well tolerated and did not lead to drug-related mortality. No relevant effects on behaviour and body weight were seen in any dosage group. Dosing at 5 mg/kg b.w. for one month resulted in the presence of intra-alveolar foamy macrophages in the lungs in some males, while in females, no relevant organ and/or tissue changes were noted. Dosing at 40 mg/kg b.w. resulted in increased swelling of the adrenocortical cells. In females, the swelling was more prominent than in males. Intra-alveolar macrophages were also seen in some males and in all females. In several males and females, perivascular exudate with granulocytes was observed in the lungs.

In the other study, the effect of the storage conditions (4 - 40°C) on the subchronic toxicity potential of itraconazole in Wistar rats was checked at 5 and 20 mg/kg b.w. combined with HP-beta-CD at 200 and 800 mg/kg b.w., respectively. Dosing at 5 mg/kg b.w. did not result in any changes and was considered to be the non-toxic dose level. In males dosed at 20 mg/kg b.w., slight changes in serum parameters (increased cholesterol and phospholipids) occurred and a slight increase in the weight of the adrenals was noted. In females, changes consisted of increases in serum cholesterol and phospholipids, an increase in adrenal weight together with a swollen aspect of the adrenals in a few animals. Vehicle-related changes consisted of the presence of dilated kidneys in a few males in all groups.

Results of analytical verification:

This chemical analysis determined concentration and stability of R051211 and T001207 in the solutions.

-date of preparation: Oct. 20 2000

R051211 in solutions:

analysis on Oct. 25 2000:

nominal concentration 10 mg/ml -> 102 % measured versus nominal concentration

nominal concentration 9.8 mg/ml -> 101 % measured versus nominal concentration

analysis on Nov. 7 2000:

nominal concentration 10 mg/ml -> 102 % measured versus nominal concentration

nominal concentration 9.8 mg/ml -> 103 % measured versus nominal concentration

T001207 in the solutions

analysis on Oct. 25 2000:

nominal concentration 0.2 mg/ml -> 95 % measured versus nominal concentration

analysis on Nov. 7 2000:

nominal concentration 0.2 mg/ml -> 97 % measured versus nominal concentration

As can be deduced from these results, the concentrations fell within the predefined range (90- 110%) and stability was guaranteed.

Applicant's summary and conclusion

Conclusions:

No differences in toxicological profile were detected between rats dosed with itraconazole (R051211) and in rats dosed with itraconazole spiked with the impurity at 2 % (T001207). Based on this finding it can be concluded that the substance T001207 does not contribute to subacute toxicological effects under the conditions in the study. No NOAEL was stated in the study report.