[2-(Isopropoxycarbonyloxy)-benzoyl]-benzoylperoxide

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

2012-01-30 to 2012-08-13

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Qualifier:

no guideline followed

Principles of method if other than guideline:

- Principle of test: The objectives of this study were the following: [1] to assess the potential systemic toxicity of CD08467 formulated in vehicle (CMC 0.1% PG 4%/0.2 % Poloxamer 124) in male and female Göttingen® minipigs upon repeated daily oral administration (gavage) at 300, 600 or 1200 mg/kg/day for at least two consecutive weeks and [2] to determine the concentrations of CD08467 main metabolites, CD0345 (salicylic acid) ,and CD08465 (carbonate moiety) in plasma samples (Proof of exposure) from male and female Göttingen® minipigs after multiple oral administration for approximately two consecutive weeks.

- Short description of test conditions: In each group one male and one female naïve Göttingen® minipigs received single daily oral doses of CD08467 at 300, 600 or 1200 mg/kg/day for two weeks at the dosing volume of 10 mL/kg/day.

- Parameters analysed / observed: Parameters evaluated included mortality/morbidity, clinical observations (at least twice a day), bodyweight and food consumption (once a week). After a 14-day treatment period, blood samples were collected at 0.5, 1, 4 and 8 hours after treatment for determination of plasma drug levels. Plasma CD08465 and CD0345 concentrations were analysed in collected samples using a HPLC method with TIS-MS/MS detection (Limit of Quantification: 0.5 ng/mL and 150 ng/mL for CD08465 and CD0345, respectively). At the end of the dosing period, all animals were subjected to necropsy procedure. Selected organs and tissues were sampled and weighed, and histopathological evaluation was performed.

GLP compliance:

no

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

[2-(Isopropoxycarbonyloxy)-benzoyl]-benzoylperoxide

EC Number:

814-835-0

Cas Number:

1310672-91-3

Molecular formula:

C18H16O7

IUPAC Name:

[2-(Isopropoxycarbonyloxy)-benzoyl]-benzoylperoxide

Specific details on test material used for the study:

- Name: CD08467
- Appearance: Off-White Powder
- Purity: 98.3%LC

SOURCE OF TEST MATERIAL
- Source and lot/batch No. of test material: Source: Unité de Gestion de la Librairie Chimique (UGLC); Batch No.: 11.00664
- Expiration date of the lot/batch: 2012-02-29

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Stability of the test item in vehicle between 1 mg/mL and 100 mg/mL at ambient temperature, under magnetic agitation and protected from light was demonstrated stable at 1 mg/mL and 100 mg/mL during ten days. The dosage form preparation was manufactured six times and delivered as ready-to-use formulations.
- Storage conditions: During the study, the dosage form preparations were stored in the study room dedicated to test items storage (Room # C.1.3.10), according to the instruction from the supplying department. Except during dispatching, the dosage form preparations were kept under continuous agitation (magnetic stirrer) during the preparation, storage and dosing procedures.

Test animals

Species:

pig

Strain:

other: Göttingen® minipigs

Details on species / strain selection:

The Göttingen® minipig was selected as test system because it is a non-rodent species commonly used for conducting research in pharmacology and toxicology; hence, sufficient background data exist to support interpretation of results.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Animals were delivered from Ellegaard, Dalmose, Denmark.
- Females (if applicable) nulliparous and non-pregnant: not specified
- Age at study initiation: 3 to 4 months
- Weight at study initiation: 6.7 to 7.5 kg for both males and females
- Housing: Animals were housed in a conventional “minipig unit” with restricted entry in pens containing wood shavings (FB8/10, Anibed, Pontvallain, France) for bedding material. Each 2 m2 pen contained one minipig.
- Diet: The feeding rations of pelleted complete diet (SDS, Saint Gratien, France) were calculated as specified by the breeder (Ellegaard) according to the animal’s age. The rations were given twice a day as equal amounts; animals had access to their first daily ration approximately 1 hour after dosing. The feeding ration was adjusted according to the oldest animal included in the study.
- Water: ad libitum, tap water
- Acclimation period: 2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 °C ± 3 °C
- Humidity (%): 50 ± 20%
- Air changes (per hr): 15 to 20
- Photoperiod (hrs dark / hrs light): 12/12

OTHER: Before their arrival, the animals were subjected to a biological follow-up against the principal diseases found in the (mini)pig species, following the FELASA recommendations (viral – bacterial – fungal and parasitological infections). Upon their arrival at Galderma R&D, each animal was given a complete clinical examination including the general appearance (posture, behaviour, head, teguments, anogenital region, tail etc.) and a palpation of the abdominal region under the supervision of a veterinarian. In addition, the animals were treated against parasitic arthropods and helminths using an appropriate prophylactic treatment (Ivermectine).

Administration / exposure

Route of administration:

oral: gavage

Details on route of administration:

Animals were treated orally by gavage using a plastic syringe fitted with an esophageal cannula. Immediately after dosing, tap water was used for flushing the cannula (approximately 5 mL, equivalent to the cannula dead volume) in order to administer entirely the test item.

Vehicle:

other:

Remarks:

Carboxymethyl cellulose (CMC) 0.1% / PG 4% / 0.2% Poloxamer 124

Details on oral exposure:

- PREPARATION OF DOSING SOLUTIONS: CD08467 was supplied as weighed raw material (Drug Substance). The drug substance was reconstituted in vehicle to obtain final dosage forms at required concentrations (30, 60 and 120 mg/mL) by the supplying department (UGLC).

- VEHICLE
- Justification for use and choice of vehicle (if other than water): CD08467 formulated in vehicle containing carboxymethyl cellulose (CMC) 0.1% / PG 4% / 0.2% Poloxamer 124 was demonstrated to be stable at 1 and 100 mg/mL.
- Concentration in vehicle:
- Purity: CMC 0.1% / PG 4% / 0.2% Poloxamer 124

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Homogeneity (2 samples) and concentrations were checked before the beginning of the study with a range of acceptability of 85-115%. These checks were conducted twice (first and second preparations) out of six.

Duration of treatment / exposure:

at least 2 consecutive weeks

Frequency of treatment:

once daily, at approximately the same time each day, 7 days a week

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

300 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

600 mg/kg bw/day (nominal)

Doses / concentrations 4

Dose / conc.:

1 200 mg/kg bw/day (nominal)

No. of animals per sex per dose:

1

Control animals:

yes, concurrent vehicle

Positive control:

Not applicable

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All animals were observed at least twice daily during both pre-dosing and dosing periods (including during non-working days) for mortality or signs of morbidity.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: The animals were regularly observed at least once daily during both pre-dosing and dosing period (after treatment) to detect any behavioural and physical abnormalities. The nature, onset, severity and duration of clinical signs were recorded.

BODY WEIGHT: Yes
- Time schedule for examinations: Body weights of each animal were recorded weekly during the pre-dosing period, on Day -1, once a week during the dosing period and prior to necropsy.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
The amount of food consumed by each animal was estimated by noting the difference between the quantities given per day (twice daily) and that remaining before the following ration distribution. This was performed daily during both pre-dosing and dosing periods. Food consumption was expressed as a percentage of the quantity given. Whenever fasting was required, the food was removed at the end of the day and the estimation of food consumed was made at that time.

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: Not specified

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

OPHTHALMOSCOPIC EXAMINATION: Not specified

HAEMATOLOGY: Not specified

CLINICAL CHEMISTRY: Not specified

URINALYSIS: Not specified

NEUROBEHAVIOURAL EXAMINATION: Not specified

IMMUNOLOGY: Not specified

OTHER: Determination of plasma drug levels
Time schedule: Blood samples were taken from all animals at the following time points: 0.5 h, 1 h, 4 h, and 8 h post-dosing (four time points per animal). Animals were fasted overnight (maximum 20 hours) before the blood sampling procedure for
clinical pathology investigations, any anaesthesia procedure (skin biopsy) and before the final sacrifice.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (see Table 1 in box "Any other information on materials & methods incl. tables")

HISTOPATHOLOGY: Yes (see Table 1 in box "Any other information on materials & methods incl. tables")

Other examinations:

Organ weights: At scheduled necropsy, selected organs, as listed in the Table 1 in "Any other information on materials & methods incl. tables" section, were weighed (fresh). Paired organs were weighed together (except if a difference in size was observed macroscopically). Organ weights were expressed as absolute values (g) and relative values (g per 1000 g of body weight).

Statistics:

Owing to the small sample size (n = 1 per group per sex), no statistical analysis could be performed. An animal-by-animal analysis was done and data from treated animals were compared with control animal data.

Results and discussion

Results of examinations

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

No clinical signs were observed during the two weeks of treatment with CD08467 except vomiting in the high dose group.
The female (8F) vomited on Day 4 and on Day 14 (about 10 minutes after treatment) as well as male (7M) on Day 14.
A relationship with the high dose level administered cannot be ruled out.

Mortality:

no mortality observed

Description (incidence):

No mortality occurred during the study.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Compared to control values, there were notable changes in the body weight for female from high dose group and in the body weight gain for both sexes from high dose group. This effect is considered linked with the high dose of CD08467 administered. In both male and female, when compared to body weight gain in control, decrease in body weight gain (-62.5% and -137%, respectively) was observed at the high dose of 1200 mg/kg/day. This effect was particularly marked during the second week of treatment. Consequently, this dose was considered to exceed the maximum tolerated dose. By contrast, no clear effect was observed at the 300 and the 600 mg/kg/day in male and female.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

At 1200 mg/kg/day, there was a reduction in food consumption during the second week of the study in the female and from Day 13 for the male. In the high-dose group, the male ate 75% of the given feeding ration on Day 13 (AM), 25% on Day 13 (PM) and 25% on Day 14 (AM). This male did not eat on the Day 14 (PM). Female did not eat her entire feeding ration during the 2nd week of the study in the high-dose group.

Males and females of the control and other dose groups ate their entire feeding rations (i.e. 100%).

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

The female animal at the high-dose group showed a high liver weight (absolute and relative) when compared to the other females. This was correlated with the pale appearance noted at necropsy and the diffuse hepatocytes vacuolation observed at microscopic examination. Other individual variations were observed with no dose-effect relationship trend or microscopic correlation. Considering the low number of animals in the study, no consistent trend could be identified on organ weight changes.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

With the female animal at the high-dose group, a pale appearance of the liver was observed in correlation with a higher liver weight and with the microscopic findings (hepatocytes vacuolation). Other macroscopic findings in the same animal were observed including: dark abnormal colour in the cardia region of the stomach (correlated with the presence of multifocal ulcers / erosions), brown deposits at the surface of the tongue (in correlation with diffuse erosion with bacterial clusters). In addition, a diffuse dark red abnormal colour was noted in the abdominal cavity (no sample done). A possible relationship with the gavage procedure and/or with the administration of CD08467 cannot be excluded.

A dark abnormal colour was observed in the thoracic cavity of several animals including the control animals (male and female in group 1 (control), female in group 3, male and female in group 4). This finding was associated in some animals with a dark abnormal colour of the thymus, which was correlated with the presence of hemorrhagic tissue surrounding the thymus. Considering the absence of dose-effect relationship and the anatomical location, these findings were considered to be related to the blood sampling procedure. The few other macroscopic findings observed in this study did not show dose-effect relationship and were considered to be part of the normal background of minipigs of this age and strain.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

The female animal at the high-dose group showed microscopic findings in several organs/tissues:
- Slight but diffuse hepatocytes vacuolation of the liver. This finding was correlated with the pale appearance and a higher liver weight.
- Moderate 'starry sky' pattern in the thymus.
- Multifocal ulcers/ erosions in the cardia region of the stomach and diffuse erosion with bacterial clusters at the surface of the tongue in correlation with the macroscopic observations.

The few other microscopic findings observed in this study were considered to be part of the normal background of minipigs of this age and this strain.

Histopathological findings: neoplastic:

not specified

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Plasma concentrations: All treated animals had detectable levels of both key metabolites of CD08467 (i.e. CD08465 and CD0345) at all time points measured. Concentrations of both metabolites increased with the dose. There were no differences between the sexes. CD0345 was by far the predominant metabolite with maximum values of 189,954 ng/mL, 296,830 ng/mL and 609,370 ng/mL at 8 h found in the males treated with 300, 600 and 1200 mg/kg/day, respectively, and 148,973 ng/mL at 4 h, 254,781 ng /mL at 8 h, 853,005 ng/mL at 4 h in females treated with 300, 600 or 1200 mg/kg/day, respectively.

Details on results:

The systemic exposure to CD08467 metabolites (CD0345 and CD08465) was demonstrated in all treated animals. CD0345 (salicylic acid) was the predominant metabolite with maximum values of 609,370 ng/mL in males and 853,005 ng/mL in females treated with 1200 mg/kg/day. No treatment-related mortality occurred during the study and there were no notable treatment-related effects at 300 and 600 mg/kg/day.

At 1200 mg/kg/day notable effects on physiological parameters, such as reduced body weight and food consumption were noticed. These signs were associated with microscopic findings in the female of high dose level group such as multifocal ulcers/ erosions in the cardia region of the stomach and slight but diffuse hepatocytes vacuolation of the liver. These microscopic findings associated with effects on physiological parameters could be related to the high dose level.

Effect levels

Remarks on result:

not determinable because of methodological limitations

Target system / organ toxicity

Critical effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

The oral administration of CD08467 at 300 and 600 mg/kg/day to male and female Göttingen® minipigs for fourteen consecutives days was well tolerated and not associated with any toxicological findings. At the high dose level of 1200 mg/kg/day, slight treatment-related effects were observed on physiological parameters such as body weight loss, decreased food consumption and few slight microscopic findings in the liver and stomach. No NOAEL could be determined due to methodological limitations (only 1 animal per sex per dose used; non-GLP).

Executive summary:

In a 14-day repeated dose toxicity study, the test item CD08467 (purity: 98.3%LC) was applied orally by gavage to Göttingen® minipigs (1/sex/dose) at dose levels of 0, 300, 600 and 1200 mg/kg bw/day for 14 days. Animals were observed for mortality/morbidity, clinical observations (at least twice a day), body weight and food consumption. After 14 days, blood samples were collected for determination of plasma drug levels. Plasma CD08465 and CD0345 concentrations were analysed, and at the end of the dosing period, all animals were subjected to necropsy procedure. Selected organs and tissues were sampled and weighed, and histopathological evaluation was performed.

At 1200 mg/kg/day, notable effects on physiological parameters, such as reduced body weight and food consumption, were noticed. These signs were associated with microscopic findings in the female of high dose level group such as multifocal ulcers/erosions in the cardia region of the stomach and slight but diffuse hepatocytes vacuolation of the liver. These microscopic findings associated with effects on physiological parameters could be related to the high dose level.

However, no NOAEL could be determined due to methodological limitations (only 1 animal per dose used; non-GLP).