Registration Dossier

Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 13. Nov. 1987 to 10. Dec. 1987
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1987
Report Date:
1988

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Version / remarks:
1981
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Version / remarks:
1984
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder

Test animals

Species:
rat
Strain:
Wistar
Details on species / strain selection:
KFM-Han, outbred, SPF quality. Recognised by international guidelines as the recommended teest system
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Kleintierfarm Madörin AG, 4414 Füllinsdorf, CH
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 8 weeks
- Weight at study initiation: males: 160 to 184 g; females: 142 to 166 g
- Fasting period before study: not specified
- Housing: individually in Makrolon type-3 cages with standard softwood bedding ('Lignocel', Schill AG, 4132 Muttenz, CH)
- Diet: ad libitum; pelleted standard Kliba no. 343, batch 82/87 rat maintenance diet ('Kliba', Klingentalmühle AG, 4303 Kaiseraugst, CH)
- Water: ad libitum
- Acclimation period: seven days under laboratory conditions, after veterinary examination

DETAILS OF FOOD AND WATER QUALITY:
- Water analysis: bacteriological and chemical assay performed for suitability
- Chemical analysis of feed: assay for contaminants performed for suitability

ENVIRONMENTAL CONDITIONS
- Temperature: 22 °C ± 3
- Humidity: 40 to 70 %
- Air changes: 10 to 15 per hour
- Photoperiod (hrs dark / hrs light): 12 / 12

Administration / exposure

Route of administration:
oral: gavage
Details on route of administration:
Oral by gavage, once daily, 7 days per week for 28 days. The control animals received the vehicle without test item.
Vehicle:
CMC (carboxymethyl cellulose)
Remarks:
Fluka AG, 9470 Buchs, CH
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
- The test item was weighed in a glass beaker on a tared Mettler PK 300 balance and the vehicle was added.
- The mixture was prepared daily prior to administration using a homogeniser and kept stable during application with a magnetic stirrer.

VEHICLE :
- Concentration in vehicle: 4 % in distilled water
- Amount of vehicle: 10 mL/kg bw
Analytical verification of doses or concentrations:
yes
Remarks:
Pretest (09. Nov. 1987) and at 3 weeks (30. Nov. 1987).
Details on analytical verification of doses or concentrations:
Concentration, homogeneity and stability of the test item / vehicle mixture was determined during pretest. Intercurrent sampling for analyses was additionally performed during week 3 of the test.
Duration of treatment / exposure:
28 days
Frequency of treatment:
daily
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Dose / conc.:
50 mg/kg bw/day (nominal)
Dose / conc.:
200 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
5 males and 5 females for each dose including control
Total: 40 animals
Control animals:
yes, concurrent vehicle
Details on study design:
HAEMATOLOGY
- The following anticoagulants were used during blood collection:
EDTA-K2 (haematology)
Sodium Citrate, 3.8 % (coagulation, 1:10)
- The following commercial reference controls were used to monitor the performance of the method:
Haematology:
Eightcheck (normal range)
Eightcheck-L (lower abnormal range)
(TOA International Corporation, Kobe, Japan)
Coagulation:
Ci-Trol-1 (normal range)
Ci-Trol-2 (high range)
(Merz & Dade AG, Duedingen, CH)

CLINICAL BIOCHEMISTRY
- The following anticoagulant was used during blood collection:
Lithium Heparin (140 45 U.S.P. Units)
- The following commercial reference controls were used to monitor the performance of the method:
Clinical Biochemistry:
Seronorm (normal range)
Pathonorm L (low range)
Pathonorm H (high range)
(Nyegaard & Co., Oslo, Norway)
Moni-Trol I-E (normal range)
Moni-Trol II-E (abnormal range)
(Merz& Dade AG, Duedingen, CH)

URINALYSES
- The following commercial reference control was used to monitor the performance of the method:
Chek-Stix Urinalysis Control Strips
(Ames Division, Miles Laboratories, Inc., Elkhard, Indiana, USA).
Positive control:
None

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: observations for mortality and viability were recorded daily .

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: signs of toxicity were recorded once daily. Descriptions of all abnormalities were recorded and the subsequent progress was monitored.

BODY WEIGHT: Yes
- Time schedule for examinations: weekly during the acclimation and treatment period using an online electronic recording system consisting of a Mettler PK 4800 balance connected to the RCC computer.

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg bw/day: yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: yes
- The food consumption was recorded once during the acclimation period and weekly thereafter using an online electronic recording system consisting of a Mettler PK 4800 balance connected to the RCC computer. It was then calculated per cage and per food consumption interval. It expresses the average food consumed per animal and per day for the cage considered and the observed food consumption interval.
- Formula (calculated for each cage): FC = C / (SUM (ND)), where:
FC = food consumption per animal and per day (grams);
C = food consumption measured over the consumption interval;
ND = number of consumption days for one animal. The date of death if applicable is considered;
SUM (ND)= total number of consumption days for all animals in the cage during the consumption interval.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: once at 4 weeks (08. Dec. 1987)
- Dose groups that were examined: all 4 groups
- Ophthalmoscopic examinations were performed on all animals. A description of any abnormalities was recorded. Pictures were taken from any abnormal findings observed. Examinations were performed at termination of treatment. Ten minutes after the application of a mydriatic solution (Dispersa AG, Winterthur, CH) the cornea, lens, anterior chamber, vitreous body and ocular fundus of both eyes were examined under dimmed light using a Heine Miroflex 2 Ophthalmoscope (Eisenhut Vit. AG, Allschwil, CH)

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at 4 weeks (10. Dec. 1987); between the hours of 07.00 and 09.30 a.m. to reduce the biological variation caused by circadian rhythms
- Anaesthetic used for blood collection: yes, light ether anaesthesia
- Animals fasted: yes, for 18 hours, water provided
- How many animals: all (40)
- Parameters checked in Table 1 were examined. The assays of blood parameters were performed under internal laboratory quality control conditions to assure reliable test results.

CLINICAL BIOCHEMISTRY: Yes
- Time schedule for collection of blood: at 4 weeks (10. Dec. 1987); between the hours of 07.00 and 09.30 a.m. to reduce the biological variation caused by circadian rhythms
- Animals fasted: yes, for 18 hours, water provided
- How many animals: all (40)
- Parameters checked in Table 2 were examined. The assays of blood parameters were performed under internal laboratory quality control conditions to assure reliable test results.

URINALYSIS: Yes
- Time schedule for collection of urine: at 4 weeks (10. Dec. 1987)
- Metabolism cages used for collection of urine: yes, over an 18-hour period into a specimen vial, during which time the animals were deprived of food but allowed access to water ad libitum
- Animals fasted: yes, during urine collection period of 18 hours
- Parameters checked in Table 3 were examined. The assays of urine parameters were performed under internal laboratory quality control conditions to assure reliable test results.

NEUROBEHAVIOURAL EXAMINATION: no

IMMUNOLOGY: no
Sacrifice and pathology:
NECROPSY:
- All animals were necropsied after 4 weeks (11. Dec. 1987) and descriptions of all macroscopic abnormalities were recorded. Necropsies were performed by experienced prosectors supervised by a pathologist. All animals were anaesthetised by intraperitoneal injection of sodium pentobarbitone and killed by exsanguination.

GROSS PATHOLOGY: yes
- Organ weights: the following organ weights were taken from all animals necropsied at termination of treatment: adrenals, kidneys, liver, testes.

HISTOPATHOLOGY: yes
- Samples of the following organs and tissues were collected from all animals at necropsy and fixed in phosphate buffered neutral 4 % formaldehyde solution: adrenals, aorta, brain, caecum, colon, cervix, duodenum, epididymides, oesophagus, eyes with optic nerve and Harderian gland, female mammary glad area, femur including joint, heart, ileum, jejunum, kidneys, larynx, exorbital lacrimal gland, liver, lung infused with formalin, mandibular lymph nodes, mesenteric lymph nodes, nasopharynx, ovaries, pancreas, pituitary gland, prostate gland, rectum, mandibular salivary gland, sublingual salivary gland, seminal vesicles, sciatic nerve, skeletal muscle, skin, cervical spinal cord, spleen, sternum with marrow, stomach, testes, thymus, thyroid gland, tongue, trachea, urinary bladder infused with formalin, uterus, gross lesions.
- All organ and tissue samples were processed, embedded and cut at a thickness of 2 to 4 μm and stained with haematoxylin.
- Slides of adrenals, heart, kidneys, liver, spleen and gross lesions of all animals were collected at terminal sacrifice from the animals of the control and high-dose groups and were examined by a pathologist. Upon detection of treatment-related morphologic changes in the organs of any high-dose animals, histologic evaluation of the same organs in all dose groups were performed. All abnormalities were described and included in the report.
Statistics:
The following statistical methods were used to analyse the body weights, food consumption, organ weights and clinical laboratory data:
- Univariate one-way analysis of variance was used to assess the significance of intergroup differences.
- If 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 between the treated groups and the control groups.
- The Steel-test (many-one rank test) was applied when the data could not be assumed to follow a normal distribution.
- For the overall spontaneous mortality data, the Fisher's exact test for 2x2 tables was applied
- Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables.
- Individual values, means, standard deviations and statistics were rounded off before printing. For example, test statistics were calculated on the basis of exact values for means and pooled variances and then rounded off to two decimal places. 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:
effects observed, treatment-related
Description (incidence and severity):
The faeces of the animals of groups 3 and 4 (both males and females) was black discoloured between days 7 and 28 (termination) of the study period.
No other clinical signs were observed which could be related to test item treatment.
Mortality:
no mortality observed
Description (incidence):
All animals were sacrificed at date of terminal necropsy.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
No statistically, test item-related differences in absolute body weight and body weight gain were observed when the figures of the control animals were compared to those of the animals of the test item-treated groups 2 to 4.
Treatment-unrelated: the statistically significant increase in absolute body weight gain of group 3 (males) (day 22) and groups 3 and 4 (females) (day 29) were considered to be incidental and of normal biologic variation known for this species and age.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
No differences which could be related to test item treatment were observed.
Treatment-unrelated: increased food consumption was observed in group 4 (females) between days 15 and 28 of treatment. This finding was considered to be an effect of food wastage, well known for this species and age.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
effects observed, non-treatment-related
Description (incidence and severity):
No treatment-related findings were observed.
Treatment-unrelated: male animal no. 10 of group 2 (50 mg/kg) showed a posterior cataract in the right eye. This finding was considered to be of spontaneous nature and not related to the test item treatment.
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
No changes of toxicological significance at the end of the study period. However, a slight decrease in the erythrocyte count (by 7 %), haemoglobin concentration (by 6 %) and haematocrit value (by 5 %) was observed in group 4 (females) when compared to the controls.
Treatment-unrelated: all other statistical differences in the results were considered to be incidental and of normal biological variation.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
A slight increase in the phosphorus level (by 17 %) and potassium level (by 17 %) was observed in group 4 (males) when compared to the controls. These findings were considered to be of an adaptive nature and therefore not significant in toxicological terms.
Treatment-unrelated: all other statistical differences were considered to be incidental and of normal biological variation.
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
No changes of toxicological significance were seen, however, a moderate increase in urobilinogen was observed in group 4 (males and females). This is not considered an effect of treatment but related to direct interference (brown-black urine dicolouration) with the substance.
Treatment-unrelated: all other differences in the results were considered to be incidental and of normal biological variation.
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):
A significant increase in absolute kidney weights was observed in group 4 (males) and in group 3 (females), whereas the kidney weight ratios were increased in groups 2 and 3 (females). These findings were considered to be of a spontaneous nature and not related to the test item treatment. They were not supported by any other diagnosis.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Black brown discolouration of the gastrointestinal contents and mucosa was recorded in most rats of groups 3 and 4. In the lungs, black-brown foci were seen in two rats of group 2, four rats of group 3 and one rat of group 4. Black-brown discolouration of the mesenteric lymph nodes was recorded in one rat of group 3 and seven rats of group 4.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
No treatment-related histopathic findings were recorded.
Treatment-unrelated: a spurious amound of brownish pigment was found within macrophages in the lung and mesenteric lymph nodes of some rats. This pigmentation results from physiological phagocytosis of the test item which, most likely, entered the airways by accidental inhalation. The discolouration of the gastrointestinal mucosa could not be substantiated microscopially.
A few spontaneous microscopic findings were recorded in both control and treated rats. They are within the range of background pathology encountered in rats of this strain and age.
Histopathological findings: neoplastic:
no effects observed

Effect levels

open allclose all
Dose descriptor:
NOAEL
Effect level:
200 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
mortality
body weight and weight gain
food consumption and compound intake
ophthalmological examination
haematology
clinical biochemistry
urinalysis
organ weights and organ / body weight ratios
gross pathology
histopathology: non-neoplastic
Dose descriptor:
LOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
mortality
body weight and weight gain
food consumption and compound intake
ophthalmological examination
haematology
clinical biochemistry
urinalysis
organ weights and organ / body weight ratios
gross pathology
histopathology: non-neoplastic

Target system / organ toxicity

Critical effects observed:
no

Applicant's summary and conclusion

Conclusions:
The NOAEL and the LOAEL (oral, rat, 28 days) of the substance were found to be 200 and 1000 mg/kg bw/day, respectively.
Executive summary:

The repeated dose toxicity of the substance was evaluated in a subacute 28-day oral toxicity study according to the OECD Guideline 407 (1981) and method B.7 of the Directive 84/449 EEC. The substance was administered daily by oral gavage to SPF-bred Wistar rats of both sexes for a period of 28 days. Test animals were randomly assigned to dose levels of 50, 200 and 1000 mg/kg bw/day and a control group which received only the vehicle (carboxymethyl cellulose (4 %) in distilled water) (5 males and 5 females per group).
Observations for mortality and clinical signs were recorded daily; food consumption and body weights were measured weekly. At four weeks, ophthalmic examinations, blood samples for haematology and clinical biochemistry, and urinalyses were performed. All test animals were sacrificed at four weeks; at necropsy, organ weights were recorded and macroscopic and microscopic histopathologic examinations were performed.
Females administered the highest dose recorded a slight decrease in erythrocyte count, haemoglobin concentration and haematocrit value; males administered the highest dose recorded a slight increase in phosphorus and potassium. Both males and females administered the highest dose recorded a moderate increase in urobilinogen which was attributed to direct interaction with urine discolouration; however this finding was considered adaptive and not of toxicological significance. 
A significant increase in absolute kidney weights was observed among males administered with 1000 mg/kg bw/day and females administered 200 mg/kg bw/day; and increased kidney to body weight ratios were observed among females administered 50 and 200 mg/kg bw/day, however these findings were considered to be of a spontaneous nature and not related to treatment.
Macroscopic pathology findings included discolouration. Faeces and gastrointestinal contents of most test animals in groups administered 200 and 1000 mg/kg bw/day were discoloured black between day 7 and the end of treatment period. Black-brown foci in the lungs were observed in 2, 4 and 1 test animals from dosage levels 50, 200 and 1000 mg/kg bw/day, respectively, however a dose-response relationship cannot be established as incidence did not increase with dose. Mesenteric lymph nodes displayed black-brown discolouration in one test animal which received 200 mg/kg bw/day, and 7 which received 1000 mg/kg bw/day, however these findings were considered to be of a spontaneous nature and common among rats of this strain and age.
No treatment-related histopathological findings were recorded, however a spurious amount of brownish pigment was found within macrophages in the lung and mesenteric lymph nodes of some rates which was attributed to accidental inhalation of the substance.
Based on the results observed, the NOAEL for the substance in male and female rats is 200 mg/kg bw/day. Therefore, the lowest adverse effect level (LOAEL) can be considered to be 1000 mg/kg bw/day.