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Repeated dose toxicity: oral

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short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
December 19 1991 to February 14 1992
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guideline
according to guideline
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)
GLP compliance:
Limit test:

Test material

Constituent 1
Test material form:
Specific details on test material used for the study:
Not specified

Test animals

other: Crl :CD ISO) BR
Details on species / strain selection:
As per guideline at the time of report production.
Details on test animals or test system and environmental conditions:
Animal husbandry
Supply, acceptance and selection of animals for the experiment.
The 60 (30M + 30F) Sprague Dawley Crl:CD (SD) BR rats, selected for this study from a larger group (35M + 35F) than that required. were purchased from Charles River Italia S.p.A., Via Indipendenza 11 - 22050 CALCO (Como) - (received on November 29, 1991 - Shipping slip no. 09184, dated November 29. 1991L.
When received. the rats were about 4 weeks old \27-29 days); the males weighed about 75-85 g and the females about 60-70 g.
On arrival at RBM, all animals were clinically observed and 20% of them were weighed: their weight conformed to that required. During the acclimatization period of about two weeks the rats were housed in a Quarantine room (BOSA) and their health status was assessed by daily clinical observations.
Before dosing commenced, all the animals received were weighed. The body weight increase proved to be within the limits of normal variability of this strain.
In view of the normal health of all the animals of the batch received. The entire group was deemed fit for the experiment.

Accommodation of the animals
During the study period the rats were housed in room BI6A, limited access, barriered rodent facility. Animal room conditioning controls were set to maintain temperature and relative humidity at 22 +/- 2 °C and 55 % +/- 10, respectively. There were approximately 20 air changes per hour filtered on HEPA 99.97%. The rooms were illuminated by artificial lighting with a 12-hour circadian cycle (7 a.m. - 7 p. m. ).An automatic standby power was brought into operation should the main supply fail.
The rats were kept for the entire duration of the study in grill cages (40.5x38.5xISh), with stainless feeder. The waste that drops through the grill bottom on a removable paper was periodically disposed of.
Animals of group 1 and 4 were caged in 4 cages/group/sex of 3 or 2 animals/cage alternately.
Animals of groups 2 and 3 were caged in 2 cages/group/sex of 3 or 2 animals.

The distribution of the cages in the animal room was designed to minimize possible environmental effects on the test animals.
The position of the cages in the rack was alternated by groups horizontally and vertically.
The diagram of the cage location in the animal room is kept in the study file.

Diet and water supply
The rat s were fed a diet coded 4 RF 21 GLP, produced by the Charles River
Italia's feed licensee Mucedola S.r.l., Settimo Milanese.
On the label. the contents declared by the producer, were:
Moisture 12.0 %
Crude protein 18.5 %
Crude fat 3.0 %
Crude fiber 6.0 %
Ash 7.0 %

The diet was supplemented by the Producer with vitamins and trace elements. According to the analytical certificates provided by the Supplier, the contents of the batches of diet used in this study were within +- 5ï. of the declared values and contaminants were within the limits proposed by EPA-TSCA (44FR:44053-44093. July26,1979).
The animal feed. in compliance with RBM SOP's, is analysed twice a year for bacterial contamination.
The diet ",as available to the animals "ad libitum".
Filtered water was distributed by means of an automatic watering valve system. The drinking water offered to the animals came from the municipal water main. The water is periodically analysed for microbiological count, heavy metals, other contaminants (e. g. solvents. pesticides) and other physical and chemical properties.
The acceptance Directive 80/778. limits of quality of drinking water were those defined in EEC
Contaminants that might interfere with the purpose of the study were not expected to be present either in the diet or in the water.
The analytical certificates of the animals' feed and water are filed at RBM premises.

Allocation to groups and identification system.
Before commencement of treatment all the animals were weighed; animals at the extremes of the body weight range were discarded. The required number of rats (30M + 30F) was allocated to the dosage groups by means of a computerized randomisation program. Each rat was numbered and individually identified by an ear-tag.
Numbering of the animals went from 1130 to 1169.
Each single cage bore a tag on which experiment number, dosage group, sex, progressive cage and animal numbers were indelibly indicated.
The colour of the tag indicated the dosage group to which the cage belonged. All experimental materials (cages, beakers, trays) belonging to each group were colour-tagged according to the following scheme.

At the end of the 4-week dosing period, 5 animals/sex in each of the groups 1 and 4 (those with higher identification numbers) were kept off treatment and subjected to a 4-week recovery period.

Administration / exposure

Route of administration:
oral: gavage
Details on route of administration:
As per guideline at the time of conducting the study.
corn oil
Details on oral exposure:
Every day an exact amount of test article was dissolved with the vehicle in a graduated glass container in order to obtain the concentration of 500 mg/ml. Part of this solution was administered to group 4 animals and part was serially diluted 1: 10 with the vehicle in order to obtain the concentrations of 50 mg/ml and 5 mg/ml, administered respectively to group 3 and group 2 animals.
Analytical verification of doses or concentrations:
Details on analytical verification of doses or concentrations:
Stability of test article formulates and concentration checks were performed by RBM according to the analytical method supplied by the Sponsor. Minor modifications, recorded in the raw data, were introduced to adapt these method to RBM's procedures.
Duration of treatment / exposure:
4 Weeks
Frequency of treatment:
Once a day. seven days a week for 4 consecutive weeks
Doses / concentrationsopen allclose all
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
10 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
30M + 30F (10 animals/sex for each groups 1 and 4 and 5 animals/sex for each groups 2 and 3)
Control animals:
yes, concurrent vehicle
Details on study design:
RBM - Experiment No. : 910A15
Test article :ANOX BF batch no. 0004
Control article: Corn oil
Dose levels (* )
Group 1 : 0 mg/Kg/day (Control article)
Group 2: 10 mg/Kg/day of ANOX BF
Group 3 ; 100 mg/Kglday of ANOX BF
Group 4 : 1000 mglKg/day of ANOX BF
(*) The dose levels administered in this study were established with the Sponsor.
- The study was conducted in compliance with "Organization for Economic Cooperation and Development Guidelines-, section 4, subpart 407 Paris 1981 as well as to the EEC Guidelines (EEC Directive 34/4A9-Annex 5 to EEC Directive 79/831) .
Administration volume; 2 ml/kg b.w.
Concentration of test article in vehicle: 5, 50, 500 mg/ml at the doses of 10, 100 and 1000 mg/kg/day respectively.

Administration route oral (by gavage).
Reason for selection of the administration route- possible route- of human exposure
Dosing regimen once a day. seven days a week
Total duration of dosing 4 consecutive weeks (treatment lasted until the day before killing or until the last day of week 4 for animals subdued to recovery).
Recovery period at the end of the 4 weeks of dosing 5 animals/sex from groups and 4 (those with the higher identification numbers) were kept off treatment and killed after 4 weeks, for recovery evaluation.
Species, strain and substrain of the test system Sprague Dawl ey Crl: CD(SD) BR rat
Justification for selection of the test system' the Sprague Dawley rat was chosen as rodent species, since it is widely accepted by Health Authorities as an appropriate experiment ai model with documented susceptibility to a wide range of toxic substances.
- Number and sex of animals: 30M ~ 30F (10 animals/sex for each groups and 4 and 5 animals/sex for each groups 2 and 3)
- Beginning of treatment: Males December 19, 1991
Females December 20. 1991
- Killing of the animals: at the end of the 4-week treatment period:
Males January 16, 1992
Females January 17 1992

- At the end of the 4-week recovery period: Males and Females: February 14, 1992

Test article characterization
Test article ANOX BF batch 0004 was supplied by the Sponsor in the form of a pale yellow liquid.
The batch used in this study was produced on January 15 1991 and it was declared stable until January 1993 if stored at room temperature (test article information sheet)

Characteristics of the control article and vehicle
The vehicle used in this study consisted of Maya Corn oil, batch 2K, expiry date July 1992.
Positive control:
Not specified


Observations and examinations performed and frequency:
Clinical signs
Physical appearance, behaviour and clinical signs of the rats were observed daily .
Any deviation from normality was recorded.

Throughout the study, inspections for mortality were made twice a day (early in the morning and late in the afternoon). Animals found dead before the end of the trial period were subjected to autopsy and the organs listed under the "Gross Pathology Examination" paragraph were removed and histologically examined.

Body weight
Each animal was weighed prior to the beginning of the treatment period (on day 0, the day before the first administration) and then at weekly intervals throughout the study period.
At the beginning of the study, the body weight variation of the test animals did not exceed +/- 20% or the mean weight.

Food and water consumption.
Food consumption was recorded, for each cage, at weekly intervals throughout the study period.
Consumption was calculated as the difference between the known offered amount per cage and the remainder recorded after 7 days. Individual food intake was then calculated, in g/an/day.
Water consumption was not measured.
Ophthalmoscopic examination
Ophthalmoscopic examination was performed on both eyes of all the animals prior to the start of treatment (week -1/day -S for males and day -6 for females) by the end of the dosing period (week 4/day 23 for males and day 22 for females) and at the end of the recovery period (week S/day 54 for males and day 53 for females). Eyes were examined with a direct ophthalmoscope (Mlroflex - Heine), after instillation of 0.5% Tropicamide (visumidpiatic - MSD).

At the end of treatment period (week 5; day 29). the haematological examinations, blood chemistry tests and urinalyses listed below were performed on all the animals of all groups.
The same examinations were also performed at the end of the recovery period (week 9; days 57 and 58) on the recovered animals.
In order to collect urine samples, on the day before the scheduled analysis the animals received 10 ml/Kg of tap water (by gavage), as water load; subsequently they were Kept in metabolism cages for about 16 hours without food and water.
On the scheduled analysis day, blood was sampled from one of the sublingual veins, while the fasted animals were slightly anesthetized with ether.
Sacrifice and pathology:
The killing method was incision of the femoral arteries after having been pentobarbital at the with an i.p. injection of an overdosage of sodium dose of 50 mg/kg. Each animal was subjected to a detailed completely anesthetized.

gross necropsy.
The following organs were removed and those underscored were also trimmed and weighed. Individual organ weight/fasted body weight ratio was calculated.
Skin and mammary gland
urinary bladder
seminal ves icles
intestine: duodenum, jejunum, ileum, cecum, colon, rectum
mesenteric lymph nodes
submandibular salivary glands and lymph nodes
sternum with bone marrow
thyroid with parathyroids-if present in the thyroid section
exorbital lacrimal glands
brain - coronal sections at three levels
Skeletal muscle: biceps femoris
peripheral nerve: sciatic nerve
spinal cord: thoracic, cervical and lumbar
femur, including articular surface
gross lesions

All or part of each of the organs were fixed in 10% buffered neutral formalin.
Histology of animals was carried out on the organs and tissues sacrificed at the end of treatment in the çontrol and high dosage groups.
Gross lesions and liver (target organ) were also examined in the intermediate and low dosage groups killed at the end of the treatment period and in the animals that underwent recovery.
The organs to be histologically examined were post-fixed for about half an hour in Carnoy's fluid, embedded in paraffin blocks, sectioned .and stained with hematoxylin and eosin.

Results and discussion

Results of examinations

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Throughout the either sex which study period no clnical Changes were observed in animals of received the low or the intermediate dosages Of ANOX BF (10 or 100 mg/kg/day).
With the high dose of the test article (1000 mg/kg/day) episodes of salivation were observed just after treatment in some rats of both sexes starting from the third week of the study onwards. This symptom, which generally lasted a few minutes after each dosing, was no longer observed after discontinuation of treatment.
Female no. 1189 of this group showed incidental fur loss (weeks 2/3).
No other clinical changes were seen in any groups including the controls.
mortality observed, non-treatment-related
Description (incidence):
Group 2 female no. 1159 and group 4 male no. 117& died before the end of the treatment period in consequence of incidental wrong gavage into the lungs. In fact, foamy fluid in the trachea and/or congested lungs were seen at autopsy. concomitant with presence of alveolar edema and/or fibrin noted at histology.
No drug-related modifications were noted in any of the examined organs.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Body weights of both males and females were found to have been unaffected by ANOX BF oral administration up to and including the highest dosage administered mg/kg/day. At the statistical analysis of body weight data no significant variations merged between the treated and control groups at any dose.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
No treatment-related changes in food consumption were seen during the study period in any group of either sex. Statistical analysis only revealed an incidental statically significant decrease controls, in males of the lowest dosage group; in food intake as compared to this change, which was slight in degree, appeared to be confined to he 2nd week of dosing. No other significant variations were seen at any time.
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
no effects observed
Description (incidence and severity):
No eye changes were seen in any of the animals tested.
Haematological findings:
no effects observed
Description (incidence and severity):
No variations of toxicological relevance were seen at hematology tests in either sex at any dose, after 4 weeks of treatment.
from evaluation of mean hematological data the only slight variations which emerged between treated and control groups were:
- a trend towards a decrease in the Leukocyte number, observed in males from all treated groups (not dosage-related) and in females from the highest dosage group.
This change never reached the statistical relevance
- a statistically significant increase in HCH without changes in hemoglobin and erythrocyte noted for males which received the highest dose. This variation was considered incidental.
On discontinuation of treatment, the Leukocyte number in recovered animals of both sexes (group 4) was still found to be reduced as compared to the controls and in females this change reached the statistical significancy. An incidental statistically significant increase in platelet number was observed for the recovered males.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
No treatment-related changes in blood chemistry parameters were seen at the end of the dosing period in animals of either sex which received the low and intermediate dosage of the test article.
For these groups only inçidental slight variations from control data i.e. increase in urea serum level for males of group 3 and decrease in Alkaline phosphatase activity for females of group 2 were highlighed by the statistical analysis.
At the highest in the dosage tested the only slight effect was a trend towards a decrease glucose serum levels which involved both sexes. This change did not achieve the statistical significancy. No other variations were seen.
At the end of the withdrawal period the above modification in the glucose serum levels were no longer observed in the recovered groups.
An incidental statistically significant increase in creatinine values was seen in the recovered females.
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
Evaluation of urine parameters statistically examined (i.e.: diuresis, specific gravity and pH) performed at the end of the administration period did not reveal major variations between the treated and control groups of either sex. Only a slight increase in the specific gravity, as compared to control data, was noted in ANOX dosed males of all group (with indication of statistical significancy for groups 3 and 4).
At the semiquantative analysis and microscopic examination of the sediment a slight increase in frequency of Leukocytes, compared to controls, was observed in individual males and females of the high dosed group.
On disçontinuation of treatment a slight increase in frequency of Leukocytes was still evident at the semiquantitative analyses in some males of the recovered group.
No notable changes were seen for females.
Behaviour (functional findings):
not specified
Immunological findings:
not specified
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
The only observed modification of compound-related origin was a slight increase in absolute and relative mean weights of liver in group 4 males and females killed at the end of the treatment period, statistically significant in males. A very slight trend to increase was also present in group 3 animals. However all these changes were characterized by individual values comprised in the range of control rats of the same age and strain used in other experiments.
Group 4 females killed at the end of the recovery period had mean relative liver weights significantly higher than controls: this change was considered of incidental origin because all individual values fell within the normal variability of control Sprague Dawley rats of this age. and was considered mainly caused by the final fasting body weight in group 4 that was slightly. lower than that of the
No changes were noted in males at the end of the recovery period.
No other differences induced by treatment were noted at either periods.
In particular, the statistically significant decrease in mean relative weight of pituitary seen in group 3 males at the final killing was of incidental origin because it was unrelated to the dosage.
Gross pathological findings:
no effects observed
Description (incidence and severity):
No changes induced by treatment were noted at either killings.
All the observed modifications were of incidental origin, not infrequently present in Sprague Dawley control rats of this age. They were slight aspects of incidental spontaneous pathology, as gastric glandular mucosa erosion, congestion in various organs and increased size of mandibular lymph nodes, or aspects of various estrual phases in females (ovarian cyst, fluid within the uterine lumen, with increased size).
Neuropathological findings:
not specified
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Treatment induced hepatic centrilobular hypertrophy in males and females of group 4 and in males of group 3. This change was generally slight and was dosage related in males for degree (in one male of group 4 it was moderate) and diffusion (in group 4 it appeared generally diffuse, while in group 3 centrilobular hypertrophy was multifocal).
Complete recovery was achieved after the withdrawal period.
No other compound-related modifications were observed.
All other variations were generally seen with similar frequency and degree in treated and control rats, and/or are not infrequently seen in Sprague Dawley rats.
of the same age used as controls in other trials in our Laboratory.
Among the most frequent spontaneous pathology were inflammatory aspects of various organs and tissues, vacuolation consistent with fatty change in the liver and renal tubules, hematopoiesis in the spleen, basophilia of renal tubules, foci of degeneration with or without inflammation in the heart and gastric glandular mucosa erosicns .
Focus of hemorrhage and edema, concomitant with hepatocyte degeneration in the liver of group 4 female no. 1182 was consequent to needle trauma incidentally induced during the intraperitoneal injection of barbiturate performed at sacrifice to anesthetize the animal.
Histopathological findings: neoplastic:
not specified
Other effects:
not specified

Effect levels

Key result
Dose descriptor:
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
clinical signs

Target system / organ toxicity

Key result
Critical effects observed:

Applicant's summary and conclusion

The NOAEL for 28 -day repeat administrations in rats was 1000 mg/kg/day.
Executive summary:

A 28 day repeat dose oral toxicity study was carried out in rats treated with test article ANOX – BF,  according to OECD test guideline 407, in compliance with GLP.

In this study the effects of repeated administration of the test article ANOX BF on Sprague Dawley rats were evaluated.
ANOX BF was administered by oral route as a suspension in corn oil once a day for 4 consecutive weeks to groups of Sprague Dawley Crl:CD (SD) BR rats at the doses of 10, 100 and 1000 mg/kg/day (groups 2,3 and 4. respectively).
Control animals (group 1) received 2 ml/kg/day of corn oil (control article).
The administered volume was kept constant at 2 ml/kg/day in all the treated groups, the test substance concentration in the vehicle being varied accordingly.
Experimental groups 1 and 4 consisted of 10 males and 10 females, while groups
2 and 3 consisted of 5 males and S females.
At the end of the 4-week dosing period. 5 animals/sex in each of the groups 1 and 4 (those with the higher identification numbers) were kept off treatment for a period of 4 weeks for recovery evaluation while the remaining rats were sacrificed for pathology studies (organ weight, gross pathology and histology).

Clinical observations
No deaths caused by effects of the test compound were observed.

Clinical signs
No clinical changes were seen at the two lowest dosages.
At the highest dose, episodes of salivation were observed just after treatment in some rats of both sexes starting from the third week of the study up to the end of the administration period.
No clinical changes were seen during the recovery period.

Body weight and food intake
No treatment-related changes were seen in either sex at any dose.

Ophthalmoscopic examination
No eye changes were observed.

Laboratory investigations

Haematology and blood chemistry
No changes of toxicological relevance were observed in either sex at any dose.
Only slight variations suggestive of tendencies were observed at the end of the
treatment period. They were:
- a decrease in the Leukocyte number in males of all treated groups and in females of the high dosage group, noted at haematology
- a decrease in glucose serum levels in both males and females receiving the high dose, seen at blood chemistry.
On discontinuation of treatment the Leukocyte number in recovered animals of both sexes was still found to be slightly reduced in comparison to the controls.

Urin analysis
Minor urinary modifications were seen at the end of the administrations period.
They included: a slight increase in the specific gravity of urine in treated males and a slight increase in frequency of Leukocytes (semiquantitative analysis and microscopic examination of the sediment) in individual males and females of the high dosed group. The latter change was still observed in some recovered males at the end of the withdrawal period.

Post -mortem examinations
Organ weight, Gross pathology and Histology
Slight increase in mean absolute and relative liver weights in males in females of the highest dosage group was seen at the end of treatment period. A slight trend to increase was also noted in rats of the intermediate dosage group.
These increases were consistent with generally slight hepatic centrilobular hypertrophy seen at histology in rats of the 1000 mg/kg/day group and in males treated with 100 mg/kg/day. In this sex hypertrophy was dosage-related in degree and degree.
Recovery was complete.
Centrilobular hypertrophy of the liver is regarded as a metabolic functional change, rather than a toxic change.


ANOX BF, when daily administered to Spragur Daewley rats by oral route for 4 weeks, was on the whole well tolerated up and including the highest dose administered because no signs of toxicity were observed.

The most important change, which proved reversible, was a slight hypertrophy of hepatocytes in rats treated with 1000 and 100 mg/kg/day, which was considered of adaptative origin. At 100 mg/kg/day, these signs were mostly limited to males. The NOAEL for 28 -day repeat administrations in rats was 1000 mg/kg/day.