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Toxicological information

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)
Rationale for reliability incl. deficiencies:
other: according to OECD guideline, GLP

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1994
Report Date:
1994

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Version / remarks:
adopted 1981-05-12
Deviations:
not specified
Qualifier:
according to
Guideline:
other: EC Guideline 84/449/EC, J. of Eur. Publ. L251, 1984-09-19
Deviations:
not specified
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Kerobit BPD, N,N'-Di-sec.-butyl-p-phenylene diamine
- Physical state: reddish liquid
- Analytical purity: 98.8%
- Lot/batch No.: from continuous production of 1990-12-20, 13.30h
- Expiration date of the lot/batch: no limit when stored under nitrogen
- Storage condition of test material: room temperature, absence of light and under nitrogen

The test substance was administered as its hydrochloride salt. See section "preparation of dosing solutions".

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Wiga GmbH, Sulzfeld, Germany
- Age at study initiation: 6 weeks
- Weight at study initiation: average 186.75 g for males, average 140.7 for females
- Housing: suspended stainless steel cages fitted with wire mesh floor and front, in groups of 5, separated by sex
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 10 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 40-70
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12 / 12

IN-LIFE DATES: From: 1992-03-31 To: 1992-05-10

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: diluted aqueous HCl, adjusted to pH c. 3.8 using NaOH
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The top dose dilution was prepared by dissolving 0.6 g Kerobit BPD in 30 mL 0.2N HCl. Then 160 mL demineralized water was added and the pH was adjusted to 3.8 using 4N NaOH. Finally the dilution was made up to 200 mL with demineralized water. The top dose thus contained 0.6 g Kerobit BPD per 200 mL diluted HCl. The lower dose dilutions were prepared by diluting the top dose solution with demineralized water. If necessary, the pH was adjusted to the same pH value as the top dose dilution with either 4N NaOH or 2N HCl. The demineralized water and the 0.3N HCl solutions were saturated with argon prior to use to supersede oxygen.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Spectra of solutions of Kerobit BPD were run on a Varian Cary 219 UV spectrometer from 700 nm to 200 nm.

The actual concentrations of the Kerobit BPD solutions are close to the nominal concentrations.
Duration of treatment / exposure:
28 days
Frequency of treatment:
daily
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 3, 10 and 30 mg/kg
Basis:
other: nominal, dosed by gavage as a solution in diluted aqueous HCl.
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: dose levels were selected on the basis of a preceding oral (5-day) range-finding study with Kerobit BPD in rats (TNO-report v91.572, dated November, 1992 and amendment I to report v91.572, dated March, 1994)

data owner: BG Chemie (Berufsgenossenschaft Chemie)

data submitter has letter of access

Doses of 0, 10, 30 and 100 mg / kg body weight were administered daily for 5 consecutive days to groups of 5 male and 5 female rats. The test substances induced a number of changes in the top-dose group like a higher incidence of alopecia, growth retardation, in males associated with decreased food intake and food conversion efficiency and effects on red blood cell system, lymphocytes, clotting parameters and plasma clinical chemistry variables. Moreover in both sexes the relative liver weight was increased whereas the relative weight of the thymus was decreased in males. A number of these changes viz. effect on clotting parameters, clinical chemistry parameters and organ weights were also observed in the mid-dose group. Whether the intercurrent death of one female of the top-dose group has to be considered of toxicological significance is unclear since it can not be excluded that the cause of death is due to faulty dosing. Pathological examination of the animal (lungs) could not be performed anymore due to cannibalism.



Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least daily (2x on working days, 1x on weekend days)

CLINICAL OBSERVATIONS: All abnormalities, signs of ill health or reaction to treatment were recorded

BODY WEIGHT: Yes
- Time schedule for examinations: initially before the experiment and weekly thereafter

FOOD CONSUMPTION: Yes
- Food consumption for each cage was determined and mean daily diet consumption calculated as g food/rat/day

FOOD EFFICIENCY: Yes
- The efficiency of food utilization was calculated and expressed as gram weight gain per gram food consumed

WATER CONSUMPTION: Yes
- Time schedule for examinations: water bottles were weighted daily during day 1-7 (week 1) and during day 15-21 (week 3)

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: day 25
- Animals fasted: Yes
- How many animals: 27
- Examined parameters: haemoglobin concentration, percentage methaemoglobin, packed cell volume, red blood cell count, red blood cell distribution width, total white blood cell count, differential white blood cell count, prothrombin time, thrombocyte count, reticulocyte count, Heinz body count, mean platelet volume, platelet distribution width, mean corpuscular volume, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: day 25 and day 28
- Animals fasted: Yes
- How many animals: 27
- Examined parameters: alkaline phosphatase activity, aspartate aminotransferase activity, alanine aminotransferase acitivity, gamma glutamyl transferase activity, total protein, albumin, albumin/globulin ratio, urea, creatinine, bilirubin total, sodium (Na), potassium (K), calcium (Ca), chloride (Cl), inorganic phosphate, total cholesterol, triglycerides

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes

All surviving rats were killed on day 28 in such a sequence that on the average the time of killing was about the same for each group. The animals were anaesthetized by ether, bled to death by cannulating the abdominal aorta and then examined grossly for pathological changes. Samples of the following tissues and organs of all animals were preserved in a neutral, aqueous, phosphate-buffered, 4% solution of formaldehyde: all gross lesions, adrenals, heart, kidneys, liver, ovaries, spleen, testes, thymus, thyroid (with parathyroids), urinary bladder, oesophagus and stomach.
The following organs of all surviving animals were weighed: adrenals, kidneys, liver, spleen, testes and thymus.
The tissues required for microscopic examination were embedded in paraffin wax, sectioned at 5 microm and stained with haematoxylin and eosin. Histological examination was performed on the liver, kidneys, spleen, adrenals and heart in all rats of the control group and the top-dose group.
Histopathological examination of the liver was extended to rats of mid- and low-dose groups because treatment-related microscopic changes were found in the top-dose group.
Statistics:
Body weights were evaluated by one-way analysis of co-variance followed by Dunnett's multiple comparison tests. Haematological variables (except for those mentioned below), clinical chemistry values and organ weights were evaluated by one-way analysis of variance (ANOVA), followed by Dunnett's multiple comparison tests. Differential white blood cell count, methaemoglobin and Heinz body and reticulocyte counts were analysed by Kruskal-Wallis nonparametric ANOVA followed by the Mann-Whitney U-test (for percentages). Histopathological changes were evaluated by Fisher's exact probability test.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
increased incidence of focal alopecic areas and sparsely haired regions
Mortality:
mortality observed, treatment-related
Description (incidence):
increased incidence of focal alopecic areas and sparsely haired regions
Body weight and weight changes:
no effects observed
Food efficiency:
no effects observed
Ophthalmological findings:
not specified
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
increase in total white blood cell count, not dose-related and considered to be of no toxicological relevance
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
females of the mid and top-dose group: increased plasma concentrations of glucose and albumine
Urinalysis findings:
not specified
Behaviour (functional findings):
not specified
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
liver, kidney
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
liver
Histopathological findings: neoplastic:
not specified
Details on results:
CLINICAL SIGNS AND MORTALITY:
Female rats of the top-dose group: increased incidence of focal alopecic areas and sparsely haired regions


FOOD CONSUMPTION:
Not significantly changed.


FOOD EFFICIENCY:
Not significantly changed.


WATER CONSUMPTION:
Top-dose group (male and female): slight, but consistent increase in water consumption


HAEMATOLOGY:
Females of the low and top-dose group: increase in total white blood cell count, not dose-related and considered to be of no toxicological relevance


CLINICAL CHEMISTRY:
Females of the mid and top-dose group: increased plasma concentrations of glucose and albumine
Females of the top-dose group: CGT activity and the concentrations of the total protein, bilirubin, cholesterol and calcium were increased
Males of the low-dose group showed an isolated decrease in bilirubin concentration. This was considered to be fortuitous because it was not found in the top-dose group.


ORGAN WEIGHTS:
Liver: absolute and relative weight was increased in males and females of the top-dose group; kidney: relative weight increased in females in the top dose group


HISTOPATHOLOGY: NON-NEOPLASTIC:
Microscopic examination revealed clear histopathological changes in the liver of the mid- and top-dose rats. These changes consisted of too homogenous cytoplasm (ground-glass cytoplasm) in the periportal hepatocytes. The incidence and the degree of severity were dose-related and the difference with the controls were statistically significant in the top-dose group.

Effect levels

Dose descriptor:
NOAEL
Effect level:
3 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: The no-toxic effect level, as mentioned in the study report, is considered as the NOAEL.

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
Because no treatment-related changes were observed at the low-dose level, the no-toxic effect level of Kerobit BPD in this study was 3 mg/kg body weight/day.

Executive summary:

1. The oral toxicity of Kerobit BPD was examined in a 28-day oral toxicity study. The test substance was dissolved in a HCl solution and further diluted in demineralized water. The Kerobit BPD solutions were administered daily by gavage to groups of 5 male and 5 female rats for 28 consecutive days at dose levels of 0 (control), 3, 10 and 30 mg/kg body weight.

2. One male of the mid-dose group died on day 24 of the study, probably due to a dosing error and/or regurgitation and aspiration of the dosing solution. The death of this animal was not ascribed to any toxicity of the test substance. An increased incidence of focal alopecic areas was observed in females of the top-dose group. Otherwise, there were no relevant differences in appearance or behaviour between the treated rats and the controls.

3. Body weights and food and water intake were not significantly affected by the treatment.

4. The total number of white blood cells was increased in females of the low and top-dose group, which was mainly due to an increase in lymphocytes. This effect was not dose-related.

5. Clinical chemistry showed increased glucose and albumine levels in the plasma of females in the mid- and top-dose groups. Plasma gamma-glutamyltransferase (GGT) activity and concentrations of the total protein, bilirubin, cholesterol and calcium were significantly increased in females of the top-dose group.

6. At the end of the treatment the absolute and relative weight of the liver were increased in the top-dose group of both sexes. In addition, the relative weight of the kidney was increased in females in the top dose group.

7. There were no treatment-related gross abnormalities. Upon microscopy, hepatocellular alterations in the periportal area of the liver (ground glass cytoplasm) was observed in the mid- and top-dose rats in both sexes. The incidence and severity of these alterations was treatment-related.

8. Because no treatment-related changes were observed at the low-dose level, the no-toxic effect level of Kerobit BPD in this study was 3 mg/kg body weight / day.