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Description of key information

Male and female Han Wistar rats were exposed to the test substance for 5 days per week for 14 weeks in a non-GLP study, comparable to OECD Guideline 408. Based upon the following effect observed in animals initially dosed with 100 mg/kg bw/day and subsequently increased to 200 mg/kg bw/day in week 7:  Mucosal necrosis and purulent mucosal inflammation in the cutaneous stomach, single cell necroses and slightly increased single-cell fatty degeneration in the liver, increased hypoxic changes in the heart, progressive transformation of the adrenal cortex and activation of the lymph nodes, the NOAEL for this substance in Han Wistar rats has be determined to be 50 mg/kg bw/day.

Key value for chemical safety assessment

Toxic effect type:
dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Version / remarks:
The study was conducted in 1973 prior to establishment of OECD guidelines and GLP- all the significant parameters relating to potential toxicological events have been examined.
GLP compliance:
no
Remarks:
The study was conducted before the introduction of GLP
Limit test:
no
Specific details on test material used for the study:
The Bronidox is a crystalline substance. The test substance is dissolved in olive oil, under gentle stirring and heating
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Mus-Rattus, 8011 Brunntal
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: not given
- Weight at study initiation: Males 145g females 147g
- Fasting period before study: not stated
- Housing: males 2-3 animals /cage females 5 animals /cage
- Diet (e.g. ad libitum): Altromin R-Pressling diât no. 1324 ad libitum
- Water (e.g. ad libitum): municipal drinking water ad libitum
- Acclimation period: 4 weeks after delivery

DETAILS OF FOOD AND WATER QUALITY:

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 24
- Humidity (%): not given
- Air changes (per hr): not given
- Photoperiod (hrs dark / hrs light): natural light conditions

IN-LIFE DATES: From: To: 08-01-1973 to 14-05-1973
Route of administration:
oral: gavage
Vehicle:
olive oil
Details on oral exposure:
Test concentrations were prepared fresh 1-2 times a week
1ml preparation/100g bodyweight
Analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
14 weeks
Frequency of treatment:
5 days per week (not weekends)
Dose / conc.:
0 mg/kg bw/day (nominal)
Dose / conc.:
10 mg/kg bw/day (nominal)
Dose / conc.:
50 mg/kg bw/day (nominal)
Dose / conc.:
100 mg/kg bw/day (nominal)
Remarks:
increased to 200 mg/kg bw/day in week 7
Dose / conc.:
200 mg/kg bw/day (nominal)
Remarks:
from week 7 until sacrifice of group
No. of animals per sex per dose:
10 (ten)
Control animals:
yes, concurrent vehicle
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: weekly


FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No
- Time schedule for examinations:

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at the start and end of the study
- Anaesthetic used for blood collection: Not specified
- Animals fasted: Not specified
- How many animals: 10 male/ 10 female
- Parameters below were examined.:
Haemoglobin
Erythrocyte number
Leukocyte number
differential white blood cell count

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at the start and end of the study
- Animals fasted: Not specified
- How many animals: 10 male/ 10 female
- Parameters below were examined.:

Glutamate-Oxaloacetate Transaminase
Glutamate-Pyruvate Transaminase
Alkaline Phosphatase
Calcium
Glucose
Urea

Additonal parameters measured at sacrifice:
total albumin
Lactate dehydrogenase

URINALYSIS: Yes
- Time schedule for collection of urine: last day of study
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Not specified
- Parameters below were examined:
Urine output
pH
albumin
glucose
Urobilinogen
Ketone bodies
Density
blood
Urinary sediment: epithelia, leukocytes, erythrocytes, bacteria, Urate crystals, calcium oxalate, triphosphate

NEUROBEHAVIOURAL EXAMINATION: No


IMMUNOLOGY: No


OTHER:
Sacrifice and pathology:
At the end of the experiment, all rats were in anethetised in ether, exsanguinated by cardiac puncture and dissected. Internal
Organs were examined macroscopically. The most important
Organs were removed and the absolute wet weights determined.

After weighing the following organs were preserved in 20% Formalin :
Salivary glands
Lungs
Heart
Lymph nodes
spleen
liver
Pancreas
kidney
stomach
Small intestine
large intestine
testicles
Uterus
ovary
adrenals
Thyroid gland
secondary gland
Cerebellum
brain

Statistics:
The determination of the means, standard deviations, the
upper and the lower confidence limits were determined using statistical software (not specified)
Test for significance:
parametric: t-test
non-parametric: U test
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Animals treated with 200 mg/kg bw/day following week 7 demonstrated apathy, piloerection and haunch squatting
Mortality:
mortality observed, treatment-related
Description (incidence):
2 animals died following the first dosing at 200 mg/kg bw/day and a third following the second dosing. Treatment suspended.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
No dose dependent decreases in body weight changes observed
Food consumption and compound intake (if feeding study):
not examined
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):
female 10 mg rats:Reduction of hemoglobin and increase in the number of white blood cells.
The male 50 mg animals had a significant increase in hemoglobin content compared to controls
as well as a lowering of the leukocyte number. In Female rats of this group the hemoglobin content was also increased as well as
significant increases in leukocyte count
Despite the valuses being significantly different from controls, they are in the normal range for studies conducted in this species.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
10 mg males- slight reduction in serum protein
50 mg females- increases in Glutamate-Oxaloacetate Transaminase and glucose.
These findings were not dose dependent and are considered incidental findings.
Urinalysis findings:
effects observed, non-treatment-related
Description (incidence and severity):
Reduction in specific gravity of urine in 10 mg/kg/day males and 50 mg/kg/day females.
These findings were not dose dependent and are considered incidental findings.
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
The male animals of the 10 mg group showed decreased absolute organ weights of brain, kidneys,
Gonads and liver; in the female animals of this group slightly lower weights of thyroid. The lowered absolute organ weights of the brain were also observed in
male and female rats of the 50 mg group. The discrepancies in terms of significance between absolute and relative organ weights indicate that the different end-body weights
here were relevant to the differences. Histologically there are no conspicuous, substance-related changes in the above organs.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
In the prematurely killed rats of the highest dose, local findings in the stomach were ablation of the mucus
membrane, necrotic changes in the fundus of the stomach as well
slight enlargement of the regional lymph nodes. The enlarged adrenal glands at
the lower dosages were not susbstance related.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
In group I (Bronindox 200 mg / kg / day):
Mucosal necrosis and purulent mucosal inflammation
in the cutaneous stomach, single cell necroses and slightly increased single-cell fatty degeneration in the liver, increased hypoxotic changes in the heart, progressive transformation of the adrenal cortex and easy activation of the lymph nodes.
In group 2 (Bronindox 10 mg / kg / day) found in 2 of 2 animals a leukocyte inflammation in the
Mucous membrane of the cutaneous stomach. The other organs showed no significant differences from the controls.
Histopathological findings: neoplastic:
not examined
Other effects:
not examined
Key result
Dose descriptor:
NOAEL
Effect level:
ca. 50 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
gross pathology
mortality
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
100 mg/kg bw/day (nominal)
System:
gastrointestinal tract
Organ:
stomach
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
yes
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
100 mg/kg bw/day (nominal)
System:
hepatobiliary
Organ:
liver
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
yes
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
100 mg/kg bw/day (nominal)
System:
cardiovascular
Organ:
heart
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
yes
Conclusions:
Based upon the folowing effect observed in animals initially dosed with 100 mg/kg bw/day and subsequently increased to 200 mg/kg bw/day in week 7: Mucosal necrosis and purulent mucosal inflammation in the cutaneous stomach, single cell necroses and slightly increased single-cell fatty degeneration in the liver, increased hypoxic changes in the heart, progressive transformation of the adrenal cortex and activation of the lymph nodes, the NOAEL for this substance in Han Wistar rats has be determined to be 50 mg/kg bw/day.
Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
50 mg/kg bw/day
Study duration:
subchronic
Experimental exposure time per week (hours/week):
120
Species:
rat
System:
hepatobiliary
Organ:
heart
liver
stomach

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Justification for classification or non-classification

Because the adverse changes were observed in animals dosed at 100 mg/kg bw/day for 7 weeks and then increased to 200 mg/kg bw/day until study termination, it is not possible to determine whether the observed changes were as a result of the exposure to the lower dose. In addition , many of the adverse changes observed are not of sufficient severity to warrant classification for Specific Target Organ Toxicity Categroy 2 in accordance with the criteria set out in 1272/2008/EC.

Mortality observed in P1 females dosed at 45 mg/kg bw/day in an OECD 414 study for 3 days before dosing was terminated (4 /30 dead/sacrificed and 26 with symptoms) is sufficient to classify the test substance for Specific Target Organ Toxicity -Repeat Dose Category 2 in accordance with paragraph 3.9.2.7.3a of Annex I to 1272/2008/EC.

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