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Repeated dose toxicity: oral

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Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2002
Report Date:
2002

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
GLP compliance:
yes (incl. certificate)
Limit test:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report):Boric Acid reaction product with Mea and Tea (Cas. No. 685 12-53-8)
- Molecular formula (if other than submission substance):
- Molecular weight (if other than submission substance):
- Smiles notation (if other than submission substance):
- InChl (if other than submission substance):
- Structural formula attached as image file (if other than submission substance): see Fig.
- Substance type:reaction product
- Physical state: colourless to slightly yellow, viscous liquid
- Analytical purity:
- Impurities (identity and concentrations):
- Composition of test material, percentage of components:19.3 % Boric acid
17.7 % Monoethanolamine (MEA)
63.0 % Triethanolamine (TEA)
- Isomers composition:
- Purity test date:2001-10-01
- Lot/batch No.:SXR037050 - 137305296
- Expiration date of the lot/batch:2002-03-27
- Radiochemical purity (if radiolabelling):
- Specific activity (if radiolabelling):
- Locations of the label (if radiolabelling):
- Expiration date of radiochemical substance (if radiolabelling):
- Stability under test conditions:is guaranteed for 4 hours in deionized water by the sponsor (dated 01-Oct-2001, archived with the raw data)
- Storage condition of test material:20 DEG c IN FUME CUPBOARD
- Other:

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source:Harlan Winkelmann GmbH Gartenstrasse 27 D-33 178 Borchen
- Age at study initiation:approximately 6 weeks
- Weight at study initiation:
- Fasting period before study:
- Housing:In transparent macrolon@ cages (type IV) on soft wood granulate in an air-conditioned rooms, 5 animals per cage, separated according to sex
- Diet (e.g. ad libitum):ssniff@ W - H (V 1534), exept fot the period in which the animals were kept in diuresis cages
- Water (e.g. ad libitum):tap water ad libitum except for the period in which the animals were kept in diuresis cages
- Acclimation period:5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C):22&3 "C (except short lasting deviations due to disturbances of air condition)
- Humidity (%):50+/-20% (except short lasting deviations due to disturbances of air condition)
- Air changes (per hr):
- Photoperiod (hrs dark / hrs light):12/12

IN-LIFE DATES: From: To:

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:

DIET PREPARATION
- Rate of preparation of diet (frequency):
- Mixing appropriate amounts with (Type of food):
- Storage temperature of food:

VEHICLE
- Justification for use and choice of vehicle (if other than water):
- Concentration in vehicle: 0.0; 12.5; 50.0; 200.0; % w/v
- Amount of vehicle (if gavage): 5 mg/kg BW
- Lot/batch no. (if required):
- Purity:
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
28 days
Frequency of treatment:
28 applications in 29 days
Doses / concentrations
Remarks:
Doses / Concentrations:
0.0; 62.5; 250; 1000; mg/kg BW
Basis:
nominal in water
No. of animals per sex per dose:
5
Control animals:
yes, concurrent no treatment
Details on study design:
- Dose selection rationale:Acute oral toxicity testing of the test substance showed that the median lethal dose (LD50) is
above 2000 mg/kg body weight in both male and female animals. At the first day of the study the
animals showed coat bristling, drawn in flanks, stilted gait and squatting posture.
In a dose range finding study 3 male and 3 female rats received the test substance at doses of 500
and 1000 mg/kg body weight per day over a period of 14days and were necropsied on day 15.
After administration of 500 and 1000 mg/kg body weight neither deaths nor symptoms occurred.
Development of body weight was not impaired. The animals killed at the end of the observation
period showed no macroscopically visible changes.
Based on these results, dose levels of 0, 62.5, 250 and 1000 mgkg body weight per day were
selected for the present study.

Examinations

Observations and examinations performed and frequency:
IN-LIFE OBSERVATIONS
Mortality
Survival control of the animals was examined twice daily (on weekends and public holidays once
dai 1 y).
Clincal observations
Behavior and general health condition of the animals was observed once daily.
Neurological examinations
Once before the first treatment and thereafter once a week detailed clinical observations were
performed in all animals outside the home cage in a standard arena ('open field'). Each animal was
assessed for changes in skin, fur, eyes, mucous membranes, occurrence of secretions and
excretions and autonomic activity such as lacrimation, salivation, nasal discharge, piloerection,
pupil size, and unusual respiratory pattern. Changes in gait, posture, and response to handling as
well as the presence of clonic or tonic movements, tremor, and any other abnormal motor
movements (such as excessive grooming, repetitive circling or other stereotypes) or bizarre
behavior (e.g. self-mutilation, walking backwards) were also recorded. In addition, defecation and
urination were evaluated.

At the termination of the study sensory reactivity to stimuli of different types (auditory, visual,
and proprioceptive) was evaluated including startle reflex (click response), response to approach
with the finger to the nose of the animal, and righting reflex. The presence and absence of
pupillary constriction was assessed using a pen flashlight directed into the eye. Assessments of
motor function were performed including measurement of motor activity, and forelimb and
hindlimb grip strength. The animals were evaluated for motor activity during a 60-minute period
in a 16-station automated motor activity-monitoring device (FMI, Fohr Medical Instruments
GmbH). Activity counts were recorded by the interruption of photocells in 3-minute-intervals to
give a total of 20 intervals. A strain gauge device (FMI, Fohr Medical Instruments GmbH)
measured fore- and hindlimb grip strength.

Body weight
The body weights of all animals were determinated before the start of the study and then twice
weekly throughout the study.

Food consumption
Food consumption was determined continuously (two times per week),

CLINICAL PATHOLOGY
Hematological investigations
At the termination of the study, hematological examinations were performed on all animals
without previous withdrawal of food. Blood samples were taken from the retrobulbar venous
plexus in narcosis (intraperitoneal injection of 67 + 6.7 mgkg body weight Ketamine-
Hydrochloride + Xylazine). In order to prevent systematic errors, blood sampling was conducted
in a randomized order.
The abbreviations, units, instrumentation, and methodologies used for these tests are presented in
the tables (30).
Hematology parameters evaluated consisted of the following:

Red Cell Counts parameters evaluated
Erythrocyte counts (RBC) Mean corpuscular hemoglobin Mean corpuscular volume (MCV)
Heinz Body Counts'
Mean corpuscular hemoglobin
Hematocrit (packed cell volume) concentration (MCHC)
Hemoglobin
This paramenter was only evaluated in the animals from the control and high dose group.
(MCW Reticulocyte counts

White Cell Counts parameters evaluated
Differential leukocyte counts Leukocyte counts (WBC)

Coagulation parameters evaluated P,
Coagulation time (clotting time) Thrombocyte counts (platelets)

Clinical Chemistry -
After blood sampling for hematological testing, the animals were killed by section of the vena
cava cranialis in deep narcosis and exsanguinated. In order to prevent systematic errors,
exsanguination was conducted in a randomized order.
The abbreviations, units, instrumentation, and methodologies used for these tests are presented in
the tables (32).
Clinical chemistry parameters evaluated consisted of the following:

Clinical Chemistry parameters evaluated
y-Glutamyltranspeptidase Bilirubin direct Inorganic Phosphorous
Alanine Aminotransferase Bilirubin total Potassium (K+)
(AUT or GPT) Calcium Sodium (Na+)
Albumin Chloride (Cl-) Total Protein

Albumin I Globulin ratio
(calculated) Cholesterol
Creatinine
Globulin (calculated)
Alkaline Phosphatase
Aspartate Aminotransferase
Triglycerides
Urea
Uric Acid
(ASAT or GOT) Glucose

Urinalysis
Urine analysis was performed in all animals a few days before termination of the study. For this
purpose, the urine was collected, using metabolism cages (overnight from day 26 to day 27). Food
was withdrawn during this period.
The abbreviations, units, instrumentation, and methodologies used for these tests are presented in
the tables (33).
Urinalysis consisted of the following:

Urinalysis - Semiquantitative parameters evaluated
i Appearence
Bilirubin
Blood
Color
Protein # Glucose
Ketone bodies Urobilinogen
Microscopic Examination
(Sediment)'

PH
* This parameter was only evaluated in the animals from the control and high dose group.
Table 10: Urinalysis - Quantitative Parameters evaluated
Specific Weight Volume

ANATOMIC PATHOLOGY
Necropsy and macroscopic examination
After exsanguination, all animals were necropsied and checked for macroscopically visible
abnormalities. The autopsy included macroscopic examination of the skin, orifices, eyes, teeth,
oral mucosa and internal organs.
All abnormal findings were recorded.
Endotracheal fixation of the lunvs: The lungs, including part of the trachea, were removed. The
lungs were then fixed endotracheally with a formalin solution using a needle inserted into the
trachea. The instillation pressure was between 20 and 30cm water column. Following completion
of the endotracheal fixation the lungs were fixed, together with the other organs, in formalin
solution.

Organ weights
The following organs were weighed and the organ to body weight ratios calculated:
List of weighed organs
Adrenals
Brain
Epidymides
Heart
Kidneys
Liver
Spleen
Testes
Thymus

Macroscopic and microscopic observations
The following tissues or organs (or pieces of them) were preserved in a suitable fixative and
processed for histopathological investigations:

List of tissues fixed and submitted for histological examinations
Adrenals
Bone marrow I sternum
Brain with medulla oblongata
Epididymides
Heart
Small Intestine 2 jejunum
Large intestine 2 colcrn
Kidneys
Liver
Lungs
lymph nodes 1 mandibular
Lymph nodes 2 iliac
Nerve sciatic nerve
Ovaries with oviducts
Prostate
Seminal vesicle
Spinal cord 1 cervical
Spleen
Stomach
Testes
Thymus
Thyroid gland with parathyroids
Trachea
Urinary bladder
Uterus
All other gross lesions
Histopathological examinations were carried out of the control and high dose animals.
Samples of organs mentioned above were embedded by conventional histological technique in
Paraplast. All organs were stained with hematoxylin-eosin.
Sacrifice and pathology:
See above

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Description (incidence and severity):
There were no deaths throughout the study. Behavior and state of health remained uneffected by the administration of the test compound in all dose groups. No opacity of the refracting media of the eyes, changes of the oral mucosa, or impairment of dental growth was observed in all study groups
Mortality:
no mortality observed
Description (incidence):
There were no deaths throughout the study. Behavior and state of health remained uneffected by the administration of the test compound in all dose groups. No opacity of the refracting media of the eyes, changes of the oral mucosa, or impairment of dental growth was observed in all study groups
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Body weight gains were not influenced by the administration of the test compound in all groups.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Food consumption remained unaffected by the administration of the test compound throughout the study in all dose groups.
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Haematological findings:
no effects observed
Description (incidence and severity):
Hematological examination did not reveal any adverse substance-related findings. The slightly higher number of platelets in high dose males was not confirmed by the females and hence was considered to be incidental.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
As a substance-related non-adverse finding, serum mean total bilirubin, cholesterol and tryglycerides were decreased in high dose males and females. All other findings were within the range of historical control data, and not considered to be incidental
Urinalysis findings:
no effects observed
Description (incidence and severity):
Urine analysis including sediment was not adversely influenced by the test compound at the end of the study period in any of all group animals.
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
Neurotoxicological measurements including 'open field' observations, assessment, of sensory function, and forelimb and hindlimb grip strength, were not influenced by the administration of the test compound in all groups.
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
There were no differences in mean absolute organ weights between all treatment groups and the controls at the end of the study treatment period.
Gross pathological findings:
no effects observed
Description (incidence and severity):
No gross pathology findings could be observed in the animals of all experimental groups.
Histopathological findings: non-neoplastic:
not specified
Histopathological findings: neoplastic:
not specified

Effect levels

open allclose all
Dose descriptor:
NOEL
Effect level:
250 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Clinical pathology
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Clinical pathology

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
In conclusion, repeated administration of 68512-53-8 at dose levels of 62.5, 250 and 1000 mg/kg body weight and day did not cause any adverse substance related alterations. With regard to the present study the 'No Observed Effect Level' (NOEL) is 250 mg/kg body weight. The 'No Observed Adverse Effect Level' (NOAEL) is 1000 mg/kg body weight per day.
Executive summary:

1.1 OBSERVATIONS AND MEASUREMENTS Groups of male and female rats received 68512-53-8 by oral gavage at dose levels of 0, 62.5, 250 or 1000 mg/kg body weight per day for a period of 28 days. On day 29 all males and all females from each group were killed and necropsied Behavior and state of health were observed daily in all groups. Body weights and food consumption were recorded twice weekly. Once before the first treatment and once a week thereafter, detaiIed clinical observations were performed in all animals outside the home cage in a standard arena ('open field'). Additionally, the animals were examined for opacity of the refracting media of the eyes, damage to the oral mucosa, and impairment of dental growth. Neurotoxicological measurements, including assessment of sensory function, motor activity, forelimb and hindlimb grip strength, were conducted at the end of the treatment period. I. Hematological examinations, clinical chemistry and urine analyses were carried out at the end of the treatment period. During necropsy the animals were examined for macroscopically visible abnormalities, the main organs were weighed and the organ to body weight ratios calculated. Organs and tissues were processed for histopathological examination and checked for microscopically visible changes. Body weights, hematological and clinical chemistry data, urine data (volume, specific weight), absolute and relative organ weights and neurotoxicological measurements (motor activity, forelimb and hindlimb grip strength) were analyzed with the aid of a statistical program to show differences compared to the controls.

1.2 RESULTS

- No deaths occurred throughout the study. Behavior, state of health, body weight development and food consumption, were not affected by administration of the test substance. No treatment-related clinical signs were observed in all animals. No changes of the oral mucosa or impairment of dental growth was observed in all study groups. There were no substance related adverse findings either in hematological examinations, clinical chemistry or urine analyses throughout the study period. As a non-adverse finding, the test substance induced slightly lower total bilirubin, cholesterol and triglycerides in high dose group rats (1000 mgkg body weight) and possibly marginally lower hemoglobinhematocrit content when compared to the controls at the end of the study period. These changes were at the lower range of the historical control data for this rat strain and age and hence, not considered to be of toxicological significance. The test substance did not influence any of the neurotoxicological parameters. No test article related changes in organ weights were observed. Necropsy at terminal sacrifice revealed no gross pathology findings in the animals of all experimental groups. All microscopic findings sporadically observed in different organs of single animals were interpreted as not compound related. I

1.3 CONCLUSION

In conclusion, repeated administration of 68512-53-8 at dose levels of 62.5, 250 and 1000 mg/kg body weight and day did not cause any adverse substance related alterations. With regard to the present study the 'No Observed Effect Level' (NOEL) is 250 mg/kg body weight. The 'No Observed Adverse Effect Level' (NOAEL) is 1000 mg/kg body weight per day.