Registration Dossier

Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Referenceopen allclose all

Reference Type:
study report
Title:
Unnamed
Year:
1989
Report Date:
1989
Reference Type:
study report
Title:
Unnamed
Year:
1989
Report Date:
1989

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Version / remarks:
12 May, 1981
Deviations:
yes
Remarks:
test temperature and relative humidity were 18-21 °C and 35-80 %, respectively
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
rat
Strain:
Sprague-Dawley
Remarks:
outbred, SPF quality
Details on species / strain selection:
Recognised by international guidelines as a recommended test system (e.g. EPA, OECD, EEC).
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
-Source: Charles River GmbH Wiga, Sulzfeld, West Germany
-Age at start of treatment: approx. 6 weeks
-Body weight at start of treatment: males 167-212 g, females 124-157 g
-Identification: earmark and tattoo
-Randomisation: computer-generated random algorithm according to body weight
-Housing: 5 animals (same sex) to a stainless steel suspended cage with wire mesh floors
-Diet: standard pelleted laboratory animal diet (RMH-B from Hope Farms, Woerden, The Netherlands), ad libitum
-Water: tap water, ad libitum
-Acclimation: 14 d (7 d pre- and 7 d post randomisation). Clinical examination performed prior to commencement of treatment to ensure that the animals were in a good state of health

ENVIRONMENTAL CONDITIONS
-Temperature: 18-21 °C
-Humidity: 35-80 %
-Air changes: 7.5 air changes per hr with air-conditioned
-Photoperiod: 12 hrs artificial fluorescent light / 12 hrs dark.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Remarks:
Milli-RO
Details on oral exposure:
PREPARATION
-Method: the substance weighed into a glass flask on an analytical balance and the vehicle (w/w) added
-Frequency of formulation: daily immediately prior to dosing.

VEHICLE
-Amount of vehicle: 5 ml/kg bw
-Homogeneity of substance in vehicle: following stirring the formulation formed a homogenous suspension in water
-Appearance of test formulation: black solution
-Storage instructions for test substance formulation: at ambient temperature
-Stability of test substance in vehicle: stable for at least 2 hours at all concentrations used.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The concentrations of the substance in Milli-RO water were determined for the subacute 28-day oral toxicity study in the rat, using a spectrophotometric method.
CHEMICAL ANALYSIS OF DOSE FORMULATION
-Analysis of formulations: samples of formulations prepared during weeks 1 and 4 were analysed to check the accuracy of preparation
-Concentration of the test substance in vehicle: determined on days 2 and 29
-Actual concentrations of preparations: in agreement with the treatment levels as per protocol.
TEST SYSTEM
The substance was dissolved/suspended in Milli-Ro water (Millipore Corp., Bedford, Mass., USA).
-Nominal concentrations: 10, 40 and 200 mg/g.
SAMPLING PROCEDURE
-Accuracy: samples were taken at 50 % height from the glass flask.
-Homogeneity: three samples were taken: one at the top (at 90 % height), one at the middle (at 50 % height) and one at the bottom (at 10 % height).
All samples were weighed accurately using an analytical balance and were further diluted with Milli-Q water (Millipore Corp., Bedford, Mass., USA) to give suitable concentrations for analysis.
QUANTITATIVE ANALYSIS
-Calibration solutions: two independently prepared calibration solutions of the substance in Milli-Q water were used each day of analysis to calibrate the analytical method.
-Method of chemical analysis: spectrophotometric method
Spectrophotometer: Perkin Elmer Lambda 5 (double beam)
Bandpass: 1 nm
Detection wavelength: 578 nm
Cuvettes polystyrol (path length= 1.0 cm).
The instrument is calibrated on a six monthly basis using K2Cr2O7 (pro analysis; Merck, FRG) solutions for absorbance accuracy and linearity and holmium glass (Shimadzu, Japan) for wavelength accuracy.
RESULTS
-The test substance formed a homogeneous suspension in Milli-Ro water at all concentrations tested.
-The accuracy of preparation testing revealed that the concentrations analysed were in agreement with the concentrations prepared.
Duration of treatment / exposure:
28 d
Frequency of treatment:
Once daily, approx. at the same time each d, 7 d per week
Doses / concentrationsopen allclose all
Dose / conc.:
50 mg/kg bw/day (nominal)
Remarks:
the concentrations analysed were in agreement with the concentrations prepared
Dose / conc.:
200 mg/kg bw/day (nominal)
Remarks:
the concentrations analysed were in agreement with the concentrations prepared
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Remarks:
the concentrations analysed were in agreement with the concentrations prepared
No. of animals per sex per dose:
5 males and 5 females per dose (included the control)
Control animals:
yes, concurrent vehicle

Examinations

Observations and examinations performed and frequency:
OBSERVATIONS
-Signs: at least once daily
-Mortality / Viability: twice daily
-Food consumption: weekly
-Body weights: weekly and on the day preceding termination, prior to overnight fasting

CLINICAL LABORATORY INVESTIGATIONS
-Blood samples: collected under light ether anaesthesia immediately prior to post mortem examination, between 9.00 and 10.20 am. The animals were fasted overnight before blood sampling, but water was provided. Blood samples were drawn from the retro-orbital sinus of all rats/sex/group and collected into tubes prepared with EDTA for haematological parameters (0.6 ml) and untreated tubes for clinical biochemistry parameters (>2.0 ml). All samples were despatched to "Bergschot Centrum voor Onderzoek" (BCO), Breda, The Netherlands for analysis.

HAEMATOLOGY
The following haematology parameters were determined from blood containing EDTA as an anti-coagulant (Parameter/Abbreviation (Unit), Instrumentation/Method):
-Erythrocyte count/RBC( T/l), Technicon – H1 Light scatter
-Haemoglobin/HB (mmol/l) Technicon – H1 Cyaanmethemoglobin
-Haematocrit/HCT (l/l) Technicon – H1 Calculation [MCVxRBC]
-Mean corpuscular volume/MCV (fl) Technicon – H1 two angle light scatter
-Mean corpuscular haemoglobin/MCH (fmol) Technicon – H1 Calculation [Hb/RBC]
-Mean corpuscular haemoglobin concentration/MCHC (mol/l) Technicon – H1 Calculation [Hb/(MCVxRBC)]
-Red cell distribution width/RDW Technicon – H1 Calculation
-Platelet count/PLATELETS (G/l) Technicon – H1 Light scatter
-Total leucocyte count / WBC (G/l) Technicon – H1 Cytochemistry (peroxidase activity + scatter)
-Differential leucocyte count/(Neutrophils/SEG, Eosinophils/EO, Basophils/BASO, Lymphocytes/LYMPH Monocytes/MONO, Large Unstained Cells/L.UNST.CEL) ( l(rel)) Technicon – H1 Cytochemistry (peroxidase activity + scatter)
Key to quantitative parameters and scores in haematology:
a) OPM, OIFF. (Remark. Differentiation)(Code/Differentiation):
343/No Abnormalities
58/Platelet aggregates
40/A-typical lymphocytes
b) OPM. GRAO. (Remark.Grading) (Code/Description):
0000/Not Applicable
2063/Occasional
2055/Many
C) THROMBOCYTES (Score/Description)
0/See remark
1/Low
2/Normal
3/Increased

CLINICAL BIOCHEMISTRY
The following clinical biochemistry parameters were determined from serum samples after clotting and centrifugation (Parameter/Abbreviation (Unit), Method/Instrumentation):
-Glucose/GLUCOSE (mmol/I), American Monitor - parallel - GOD/POD
-Urea/UREA (mmol/l ), American Monitor - parallel -Diacetyl-monoxim
-Creatinine/CREATININE (umol/l ), American Monitor - parallel - Jaffre
-Bilirubin, total/ BLI T. (umol/l), American Monitor - parallel - Jendrassik Grof
-Aspartate aminotransferase/(ASAT/GOT) (ukat/l), American Monitor - parallel - standard method (NVKC)
-Alanine aminotransferase/(ALAT/GPT) (ukat/l ), American Monitor - parallel - standard method (NVKC)
-Gamma-glutamyltransferase/G-GT (nkat/l), American Monitor - parallel - y-glutamyl 3-carboxy-4-nitro anilide
-Sodium/SODIUM (mmol/l), American Monitor - parallel - ion selective electrode
-Potassium/POTASSIUM (mmol/l), American Monitor - parallel - ion selective electrode
-Chloride/CHLORIDE (mmol/l), American Monitor - parallel - T.P.T.Z.
-Calcium/CALCIUM (mmol/l), American Monitor - parallel - o-cresol phtalein-complexone
-Phosphorus/INORG PHOSPH (mmol/l), American Monitor - parallel - ammonium molybdate
-Protein, total/PROTEIN T. (g/l ), American Monitor - parallel - biuret
-Protein, albumin/ALBUMIN (g/l), American Monitor - parallel - bromide cresol green
Key to quantitative scores in biochemistry
a) ASPEKT (appearance of sample): 7/Normal
Sacrifice and pathology:
NECROPSY
All animals were necropsied and descriptions of all macroscopic abnormalities recorded. All animals survived to the end of the observation period (day 29) and were anaesthetised with pentobarbitone and killed by exsanguination. Samples of the following tissues (and any noted gross abnormalities) and organs were collected from all animals at necropsy and fixed in neutral phosphate buffered 4% formaldehyde solution:
-Adrenal glands
-Heart
-Kidneys
-Liver
-Spleen
-Stomach
-Testes

ORGAN WEIGHTS
The following organ weights (and terminal body weight) were recorded at termination on the scheduled dates of necropsy:
-Adrenal glands
-Kidneys
-Liver
-Spleen
-Testes

HISTOTECHNOLOGY
All organ and tissue samples, as defined under Histopathology (following), were processed, embedded and were cut at a thickness of 2-4 micrometers and stained with haematoxylin and eosin.

HISTOPATHOLOGY
Slides of adrenals, heart, kidneys, liver and spleen, collected at termination from all animals of the control and high dose group and slides of kidneys from the intermediate groups and any gross abnormalities were examined by a pathologist. All abnormalities were described.
Statistics:
The following statistical methods were used to analyse the body weight, 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 of 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.
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).
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, non-treatment-related
Description (incidence and severity):
There were no clinical signs noted that were considered related to treatment with the test substance.
Incidental findings that were noted included one incidence of diarrhoea and one rat with alopecia and encrustations.
Mortality:
no mortality observed
Description (incidence):
There was no mortality during the course of treatment.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
There were no statistically significant differences in the body weights or body weight gain of treated rats compared to controls.
Food efficiency:
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Description (incidence and severity):
There were no differences that were considered to have arisen as a result of treatment between control and treated rats. Any difference that did achieve a level of statistical significance was considered to have arisen fortuitously.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
Males receiving 1000 mg/kg had statistically significantly decreased levels of alanine aminotransferase (ALAT/GPT) and aspartate aminotransferase (ASAT/GOT) when compared to controls. However, the toxicological significance of a decrease in serum enzyme levels with no corresponding pathological change must be considered dubious.
Chloride levels of females receiving 1000 mg/kg were statistically significantly increased when compared to control values, but it was considered that this difference had arisen fortuitously as there was no corresponding alteration in the other electrolyte parameters.
There were no other differences of possible toxicological significance noted in blood chemistry parameters measured between control and treated rats. Minor statistically significant differences occurring were not considered to be related to treatment as all values remained within biologically normal limits for rats of this age and strain.
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):
Spleen weights of females receiving 50 mg/kg (and of females receiving 200 mg/kg after adjustment for body weight) were statistically significantly low when compared to control values. As there was no evidence of a significant decrease in females receiving 1000 mg/kg, it was considered that this statistical difference had arisen by chance due to a slightly higher than usual control value. Organ weights of treated males and females receiving 1000 mg/kg were similar to those of controls before and after allowance for body weight.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Dark red discolouration to the kidneys was noted in 1/5 females receiving 200 mg/kg and in 5/5 males and 4/5 females receiving 1000 mg/kg. This observation was not considered to be of toxicological significance, but to be related to the nature of the test substance.
Other macroscopic observations that were noted at necropsy (pelvic dilation of the kidneys and enlarged cervical lymph nodes) were not considered related to treatment, but within the normal background range for animals of this age and strain.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
No treatment related findings were recorded. In particular, those kidneys which were discoloured showed no histological abnormality to account for this appearance.
The small numbers of microscopic lesions identified were those of spontaneous origin commonly seen in rats of this age and strain, the incidence and severity of which bore no relationship to treatment.
Details on results:
The decrease in levels of serum enzymes noted among males receiving 1000 mg/kg/day was not substantiated by a similar effect in females or supported by corroborative pathological evidence. The toxicological significance of this finding is therefore doubtful.

Effect levels

Key result
Dose descriptor:
NOEL
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
haematology
clinical biochemistry
organ weights and organ / body weight ratios
gross pathology
histopathology: non-neoplastic

Target system / organ toxicity

Key result
Critical effects observed:
no

Any other information on results incl. tables

There were no differences in the food consumption of treated rats before and after correction for body weight compared to controls.

Applicant's summary and conclusion

Conclusions:
NOEL (oral, 28 d) = 1000 mg/kg/day
Executive summary:

The repeated dose toxicity of the substance was evaluated in a subacute 28-day toxicity study, according to the OECD Guideline 407 (1981). The substance was administered daily by gavage to SPF-bred Sprague-Dawley rats. The number of rats assigned to toxicity testing per group as well as the dose levels administered were as follows: 10 animals (5 females and 5 males) for concentrations (0, 50, 200, 1000 mg/kg bw). There were no signs of a toxic reaction to treatment noted in animals and a NOEL of 1000 mg/kg/day was established.