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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:
From the 20th of December 2001 to the 17th of January, 2002
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference Type:
study report
Report Date:

Materials and methods

Test guidelineopen allclose all
according to
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
according to
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Principles of method if other than guideline:
The Dutch Act on Animal Experimentation (February 1997), the study protocol was reviewed and agreed by the Article 14-functionary and the Ethical Committee of NOTOX (DEC NOTOX 97-O3-15).
GLP compliance:
yes (incl. certificate)

Test material

Test material form:
solid: particulate/powder
Details on test material:
Expiry date: 29 August 2002
Storage condition: at room temperature in the dark
Specific details on test material used for the study:
Vehicle Propylene glycol, specific gravity.
Method of formulation Formulations (w/w) were prepared daily within 4 hours prior to dosing and were homogenised to a visually acceptable level. Adjustment was made for specific gravity of the vehicle. Storage conditions: at ambient temperature

Test animals

Details on species / strain selection:
Crl:(Wl) BR
Details on test animals and environmental conditions:
Source: Charles River Deutschland, Sulzfeld, Germany.
Age at Start of Treatment: Approximately 6 weeks.
Number of animals: 20 males, 20 females (females were nulliparous and non-pregnant).
Randomisation: prior to commencement of treatment, by computer-generated random algorithm according to body weight, with all animals within 120% of the sex meant
Identification: earmark and tattoo.
The dose levels were selected on the basis of a 5-day dose range finding study (NOTOX Project 337916).
Conditions: a controlled environment was maintained in the room with optimal conditions considered as being approximately 15 air changes per hour, a temperature of 21±3°C, a relative humidity of 30-70% and 12 hours artificial fluorescent light and 12 hours dark per day.
Temporary deviations from the light/dark cycle (with a maximum of 1 hour; main study) occurred due to performance of functional obsenrations in the room. Based on laboratory historical data these deviations were considered not to have affected the study integrity.
Accommodation: group housing of 5 animals per sex per cage in stainless steel suspended cages with wire mesh floors (55 x 34 x 21.5 cm height). Acclimatisatlon period was at least 5 days before start of treatment under laboratory conditions. During activity monitoring, animals were individually housed overnight in Macrolon plastic cages (type III, height 15 cm.) with sterilised sawdust provided as bedding. Results of bedding analyses for contaminants are examined and archived.
Diet: free access to standard pelleted laboratory animal diet (from Altromin (code VRF-1, Lage, Germany). Each batch is analysed for nutrients and contaminants are analysed on a regular basis.
Water: free access to tap water. Certificates of analysis (performed quarterly) were examined and archived.
Analysis of bedding, diet and water did not reveal any findings that were considered to have affected study integrity.

Administration / exposure

Route of administration:
oral: gavage
Details on route of administration:
stainless steel stomach tube.
propylene glycol
Details on oral exposure:
Formulations were placed on a magnetic stirrer during dosing. Frequency Once daily for at least 28 days, 7 days per week, approximately the
same time each day with a maximum of 4 hours difference between the earliest and latest dose. Animals were dosed up to the day prior to
Dose volume 5 ml/kg body weight. Actual dose volumes were calculated weekly according to the latest body weight.
Duration of treatment / exposure:
28 days
Frequency of treatment:
Doses / concentrationsopen allclose all
Dose / conc.:
50 mg/kg bw/day (actual dose received)
Main group 2
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Main group 3
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Main group 4
No. of animals per sex per dose:
5 male
5 female
Main group 1, 2, 3,4
Control animals:
yes, concurrent vehicle
Details on study design:
A health inspection was performed prior to commencement of treatment to ensure that the animals were in a good state of health.

Frequency Once daily for at least 28 days, 7 days per week, approximately the
same time each day with a maximum of 4 hours difference between
the earliest and latest dose. Animals were dosed up to the day prior to
Dose volume 5 ml/kg body weight. Actual dose volumes were calculated weekly
according to the latest body weight.


Observations and examinations performed and frequency:
Mortality: twice daily
Clinical signs Once daily, detailed clinical observations were made in all animals.
Once prior to start of treatment and on days 8, 15, 22 and 28, this was also performed outside the home cage in a standard arena. The
symptoms were graded according to fixed scales and the time of onset, degree and duration were recorded:
Maximum grade 4: grading slight (1) to very severe (4).
Maximum grade 3: grading slight (1) to severe (3).
Maximum grade 1: presence is scored (1).
Functional Observations During week 4 of treatment, the following tests were performed on all animals:
- hearing ability, pupillary reflex, static righting reflex and grip strength (Score 0 = normal/present, score 1 = abnormal/absent).
- motor activity test (recording period: 12 hours during overnight for individual animals, using a computerised monitoring system,
Body weights On days 1,8, 15, 22 and 28.
Food consumption Weekly.
Water consumption Subjective appraisal was maintained during the study, but no quantitative investigation introduced as no effect was suspected.
Sacrifice and pathology:
All animals surviving to the end of the observation period were deeply anaesthetised using isoflurane and subsequently exsanguinated. All animals assigned to the study were necropsied and descriptions of all macroscopic abnormalities recorded, Samples of the following tissues
and organs were collected from all animals at necropsy and fixed in a 4% formaldehyde solution.
The following organ weights (and terminal body weight) were recorded from the animals on the scheduled day of necropsy:
Adrenal glands Liver
Brain Spleen
Epididymldes Testes
Heart Thymus

Other examinations:
Blood samples were collected under iso-flurane anaesthesia immediately prior to scheduled post mortem examination, between 7.30 and 9.30 a.m. The animals were fasted overnight (with a maximum of 20 hours) before blood sampling, but water was provided. Blood samples were
drawn from the retroorbitaI sinus of all rats/sex/group and collected into tubes prepared with EDTA for haematological parameters (0.25 mL), with citrate for clotting tests (1.0 mL) and Li-heparin treated tubes for clinical biochemistry parameters (1.0 mL).

All organ and tissue samples, as defined under Histopathology (following), were processed, embedded and cut at a thickness of 2-4 micrometers and stained with haematoxylin and eosin.
The following slides were examined by a pathologist:
- all tissues and organs collected at the scheduled sacriuce from all animals of the control and the highest dose group;
·- all gross lesions of all animals.
Based on the treatment related morphologic changes, mesenterial lymph nodes, liver, spleen and testes were also examined from all rats of the intermediate dose groups. Based on findings in the testes, staging of spermatogenesis was performed on PAS·stained testes slides of all males. All abnormalities were described and included in the report. Tissues mentioned within brackets were not examined as there were no signs of toxicity or target organ involvement.

The following statistical methods were used to analyse the data:
- lf the variables could be assumed to follow a normal distribution, the Dunnett-test (many-to-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
- The Steel-test (many-to-one rank test) was applied when the data could not be assumed to follow a normal distribution.
-The exact Fisher test was applied to frequency data.
All tests were two»sided and in all cases p < 0.05 was accepted as the lowest level of significance. Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables. No statistical analysis was performed on motor activity data. Test statistics were calculated on the basis of exact values for means and pooled variances. individual values, means and standard deviations may have been rounded off before
printing. Therefore, two groups may display the same printed means for a given parameter, yet display different test statistics values.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Description (incidence and severity):
Incidental findings that were noted included salivation, diarrhoea, brown/red/orange staining of the fur and/or alopecia of various body parts, piloerection, regurgitation, scabs and wounds. These findings are commonly noted in rats of this age and strain which are housed and treated
under the conditions in this study. At the incidence observed, these were considered signs of no toxicological significance.
no mortality observed
Description (incidence):
No mortality occurred during the study period.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Body weights and body weight gain of treated animals did not show changes that were considered to be related to treatment.
The statistically significant higher body weight of group 2 and 3 males in week 4 and at termination was related to the slightly low control body weight (gain) values in that week. No explanation could be given for these sligtly deviating control values. Slight weight loss was noted for one control and one group 3 female (nos. 25 and 33 respectively). Other females within this group showed normal weight gain and the incidence of these reductions was not related to the dose.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
There were no differences in food consumption before or after allowance for body weight between treated and control animals that were considered to be an effcet of treatment.
Food consumption of control males was considered to be slightly low based on similar studies.
This was less apparent when food intake was corrected for body weight. No explanation could be given for these slightly low control values.
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):
Haematological parameters of treated rats were considered not to have been affected by treatment.
Mean erythrocyte counts showed an apparent reduction among group 3 males. However, mean erythrocyte counts of control males were considered to be slightly high when compared to values of similar studies and the change did not attain a dose-related response. The apparent
increase of the mean partial thromboplastin time value for group 3 males was related to slightly low mean control values. Therefore, this finding was considered not to represent a change of toxicological significance.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
There were no differences noted between control and treated rats that were considered to be
related to treatment with the substance.
Potassium values of high dose males appeared increased in group 4 males but the mean value remained comparable to controls of similar studies. Similarly, the reduced mean ASAT values of group 2 and 3 males were related to slightly high mean control values. No dose-related
response was obtained for the increased creatinine value of group 3 females. Other individual increases (i.e. a slightly high alkaline phosphatase activity of one control male (no. 3)) were not related to the dose. No supportive microscopic correlate was found for the
notably increased alanine aminotransferase activity value for one group 4 female (no. 38), and other values within this group were comparable to controls. These changes were therefore not considered to be of toxicological significance.
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
No changes were obsenred in hearing ability, pupillary reflex, static righting reflex and grip strength in the animals treated, when compared to control animals. The variation in motor activity did not indicate a relation with treatment since these occurred in the absence of a dose-related response.

Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
Organ weights and organzbody weight ratios of treated animals were considered not to have
been affected by treatment with the test substance.
Statistically significant changes between treated and control males were considered to be related to the slightly low body weights of control males. No toxicological importance was ascribed to these changes.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Dark red discolouration of the mandibular lymph nodes was noted among all females and one male of the high dose group.
No (treatmenl-related) supportive microscopic findings were found for the thickened limiting ridge and glandular mucosa of the stomach, an accentuated Iobular pattern of the liver and an enlarged spleen in the high dose. Other incidental findings among control and/or treated animals included dark red foci on the thymus or lungs, dark red discolouration of the mesenteric lymph nodes, alopecia or sores on the skin, fluid in the uterus and a reduced size of the adrenal glands. These Endings are occasionally seen among rats used in these types of studies and in
the absence of a dose-related distribution they were considered changes of no toxicological significance.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
Microscopic Examination
A minimal to slight amount of yellow-brown pigment was recorded in the macrophages located in the mesenteric lymph nodes of all high dose animals. This pigment was also present as yellow-brown particulate matter in the lumen of ileum, cecum and colon in three high dose males and females.
Seminiferous atrophy of the testes was seen in four group 2 males (moderate), in two group 3 males (minimal/slight) and in one high dose male (moderate). This was considered to be an incidental finding since there was no effect on the stages of the spermatogenic cycle, lesions
were of low severity and focal in nature (i.e. unaffected lubules were also present in all animals), and the incidence and severity were unrelated to the dose.
All other microscopic findings were within the range of background pathology encountered in Wistar rats of this age and strain and occurred at similar incidences and severity in both control and treated rats.
Histopathological findings: neoplastic:
not examined
Other effects:
not examined

Effect levels

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

Critical effects observed:
not specified

Applicant's summary and conclusion

All high dose animals displayed the presence of yellow-brown pigment in macrophages ofthe mesenteric lymph nodes. Macroscopically, this was supported by dark red discolouration of these lymph nodes. The presence of this pigment was considered to have resulted from normal phagocytosis of the test substance, which is a brown powder. Also, yellow-brown particulate matter was present in the lumen of some parts of the intestines. Since there were no other (pathological) alterations in the mesenteric lymph nodes or gut lumen, no toxicological significance was ascribed to these findings. There were no changes at determination of clinical appearance, performance of functional observations, body weight and food consumption measurements, or alterations during clinical laboratory investigations and organ weight determination that were considered to be an effect of
From the results presented in this report a No Observed Adverse Effect Level (NOAEL) of 1000 mg/kg/day was established.
Executive summary:

The study was based on the following guidelines EEC Directive 96/54/EEC, B.7 Repeated Dose (28 days) Toxicity (oral), 1996 and OECD 407, Repeated Dose 28-day Oral Toxicity Study in Rodents, 1995.

Based on the results of a 5-day range finding study, the dose levels for the 28-day toxicity studywere selected to be 0, 50, 150 and 1000 mg/kg/day.

The test substance was administered daily for 28 days by oral gavage to SPF-bred Wistar rats.

One control group and three treated groups were tested, each consisting of 5 males and 5 females.

The following parameters were evaluated: clinical signs daily; functional observation tests; body weight and food consumption weekly; clinical pathology and macroscopy at termination; organ weights and histopathology on a selection of tissues.

Formulations: Accuracy, homogeneity and stability over 4 hours of formulations of test substance in propylene glycol were demonstrated by analyses.

Treatment-related findings observed were as follows:

50 mg/kg/day; No treatment-reIated findings.

150 mg/kg/day: No treatmenbrelated findings.

1000 mg/kg/days No treatment-related findings.

From the results presented in this report a No Observed Adverse Effect Level (NOAEL) of 1000 mg/kg/day was established.