Registration Dossier

Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
April 12, 1990 to May 28, 1990
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1991
Report Date:
1991

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Qualifier:
according to
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
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:
Standard test system for this kind of study
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Dr. Karl Thomae GmbH, Biberach/Riss, Germany
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 42 d
- Weight at study initiation: mean weight of
- 190 (100 - 201) g for the males
- 146 (140 - 155) g for the females.
- Fasting period before study: no
- Housing: single, type DK III stainless steel wire cages supplied by BECKER & Co., Castrop-Rauxel, Germany
- Diet (e.g. ad libitum): ground Kliba maintenance diet rat/mouse/hamster, 343 meal, supplied by Klingentalmühle AG, CH-4303 Kaiseraugst, Switzerland, ad libitum
- Water (e.g. ad libitum): tap water ad libitum
- Acclimation period: 10 d

DETAILS OF FOOD AND WATER QUALITY:

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 30-70
- Air changes (per hr):
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: April 12, 1990 to May 28, 1990

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on oral exposure:
- PREPARATION OF DOSING SOLUTIONS: dissolved in double distilled water
The test substance was weighed in for the dose group and made up to the measuring mark, by adding distilled water, and subsequently it was brought into solution using an Ultraturrax.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The stability of the test substance in the vehicle over a period of 4 hours was verified by Project No.: 10A0796/891270. The homogeneity of the test substance preparations were verified analytically by Project No. 1150796/89062.

One sample of each concentration obtained at the start of the study was sent to the analytical laboratory for determination of the correctness of the concentration of the test substance preparations.
The analyses were carried out in the analytical laboratories of BASF Aktiengesellschaft
Duration of treatment / exposure:
29 days
Frequency of treatment:
daily
Doses / concentrationsopen allclose all
Dose / conc.:
100 mg/kg bw/day (nominal)
Dose / conc.:
300 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
control and high dose: 10
low and mid-dose: 5
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The LD50 value for rats after oral administration was > 2200 mg/kg body weight.
The 2-week test administration by gavage (11 x p.o.) to male and female Wistar rats at a dose of 1000 mg/kg body weight in both sexes led to no disturbance of the general state of the animals. The body weight change and feed consumption were unremarkable. The hematological and clinicochemical examinations carried out at the end of administration period resulted, in addition to a slightly decreased triglyceride values in the females, in no values deviating from normal. At necropsy, the males of the 1000 mg/kg body weight group showed a slightly reduced absolute and relative liver weight. In the males and females of the test group, both the kidneys as well as the contents of the stomach and intestinal tract were colored by the test substance. There were other gross-pathological changes.
Therefore, the following doses were selected for the 4-week administration period:
100 mg/kg body weight: as the expectable no adverse effect level
300 mg/kg body weight: as the intermediate concentration
1000 mg/kg body weight: as the concentration with expectable toxic effects

- Rationale for animal assignment (if not random): The male and female animals, separated by sex, were dis-tributed according to weight among the individual test groups before the start of the administration period. The list of randomization instructions was compiled with a computer (laboratory data processing, Department of Toxicology, BASF Aktiengesellschaft)
- Rationale for selecting satellite groups: to determine whether effects seen are reversible
- Post-exposure recovery period in satellite groups: 14 days in control and high-dose groups
- Section schedule rationale (if not random): randomised

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice (Mondays to Fridays) and once a day (Saturdays, Sundays and on public holidays)

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION: Yes
- Time schedule for examinations: weekly

WATER CONSUMPTION: Yes
- Time schedule for examinations: daily by visual inspection

OPHTHALMOSCOPIC EXAMINATION: No
- Time schedule for examinations:
- Dose groups that were examined:

HAEMATOLOGY: Yes
- Time schedule for collection of blood: in the morning of day 26 or 43
- Anaesthetic used for blood collection: No
- Animals fasted: No
- How many animals: 5/sex/group
- Parameters examined.:
- leukocytes
- erythrocytes
- hemoglobin
- hematocrit
- mean corpuscular volume
- mean corpuscular hemoglobin
- mean corpuscular hemoglobin concentration
- platelets
- thromboplastin time

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: in the morning of day 26 or 43
- Anaesthetic used for blood collection: No
- Animals fasted: No
- How many animals: 5/sex/group
- Parameters examined.:
- alanine aminotransferase
- aspartate aminotransferase
- alkaline phosphatase
- serum-gamma-glutamyltransferase
- potassium
- chloride
- inorganic phosphate
- calcium
- urea
- creatinine
- glucose
- total bilirubin
- total protein
- albumin
- globulins
- triglycerides
- cholesterol
- magnesium


URINALYSIS: Yes
- Time schedule for collection of urine: the individual animals were transferred to metabolism cages and urine was collected overnight at 4°C on days 26 or 40. The urine samples were evaluated in a randomized sequence.
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters examined:
- volume
- appearance
- nitrite
- pH
- protein
- glucose
- ketones
- urobilinogen
- bilirubin
- blood
- specific gravity
- sediment


NEUROBEHAVIOURAL EXAMINATION: Not specified
- Time schedule for examinations:
- Dose groups that were examined:
- Battery of functions tested: sensory activity / grip strength / motor activity / other:

IMMUNOLOGY: No
- Time schedule for examinations:
- How many animals:
- Dose groups that were examined:
- Parameters checked in table [No.?] were examined.

OTHER:
Sacrifice and pathology:
GROSS PATHOLOGY: Yes

HISTOPATHOLOGY: Yes
Statistics:
The KRUSKAL-WALLIS one way analysis of variance by ranks.
In: SIEGEL, S. (1956): Non-parametric Statistics for the behavioral sciences, pp. 184-194, McGraw-Hill Book Company, New York, Toronto, London

The MANN-WHITNEY U test
In: SIEGEL, S. (1956): Non-parametric Statistics for the behavioral sciences, pp. 116-127, McGraw-Hill Book Company, New York, Toronto, London

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Water consumption and compound intake (if drinking water study):
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Details on results:
Neither premature mortalities nor clinical signs of toxicity occurred. Faeces were discoloured blue among the animals from the medium and high-dose groups. This effect proved to be reversible during the recovery phase.
Haematologically and clinico-chemically, there were no toxicologically relevant changes.
Morphologically, there was blue discolouration of the mucosa in the gastro-intestinal tract of the animals from the medium and high-dose groups. In addition, among the female animals, the relative kidney weights were elevated as were some of the absolute kidney weights in the follow-on observation period. Macroscopically, the kidneys of the animals from the high-dose group were coloured due to the dye. This observation also resulted for the mesenteric lymph nodes at the end of the follow-on observation period.
Pathohistologically, there were no changes. The changes which were detected were assessed as being exclusively deposition and excretion or temporary deposition (lymphogenic drainage) of the dye and were not regarded as toxicologically relevant.

Effect levels

Key result
Dose descriptor:
NOAEL
Effect level:
>= 1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects

Target system / organ toxicity

Critical effects observed:
no

Applicant's summary and conclusion

Conclusions:
Based on the results obtained during this oral toxicity study, the "no adverse effect level" of Reaktivblau 1463 is above 1000 mg/kg body weight.
Executive summary:

Reaktivblau 1463 was administered by gavage to 3 groups, each of 5 male and 5 female Wistar rats, for 29 consecutive days, at 3 dose levels. A control group of 5 male and 5 female rats was dosed with vehicle alone. Two satellite groups, each of 5 males and 5 females, were treated with the high dose (dose of test group 3) or the vehicle alone also for 29 consecutive applications and then maintained without treatment for a further 14 days.

The doses were 100 mg/kg body weight, 300 mg/kg body weight and 1000 mg/kg body weight.

Feed consumption and body weight were determined weekly. Water consumption and the state of health was checked each day and once a week the Wistar rats were inspected and palpated. During the study period, the appearance of clinical signs was checked.

Toward the end of the administration period, a urine examination, a clinicochemical and hematological examination were carried out. Toward the end of the recovery period these examinations were also performed for the satellite groups.

All animals were assessed by gross pathology, followed by a histopathological examination.

Results:

Feed consumption: No treatment-related differences in feed consumption were noted during the study.

Body weight: Body weight in test animals was comparable with that seen in controls.

Water consumption: No overt differences were detected.

 Clinical signs:          No clinically observable signs of toxicity were detected in test or control animals. Blue faeces were seen 300 mg/kg body weight and more marked (deep blue) in test at 1000 mg/kg body weight; but this regressed during the 14-day treatment-free period.

Mortality:      There were no deaths during the study period.

Clinical chemistry and haematology:          There were no changes in the clinical chemistry and haematology parameters measured which could be considered toxicologically significant.

Urinalysis:     No treatment-related changes were detected.

The application of the test substance led to blue-green discoloration of the contents of the stomach and the intestinal tract of most rats in groups 2 (300 mg/kg body weight) and 3 (1000 mg/kg body weight).

Blue-green discoloration was observed in the kidneys of male and female rats of group 3 and most females of group 2. The discoloration of the kidneys persisted after a recovery period of 2 weeks; however, the colour had changed to green-brown. At the end of the recovery period the mesenteric lymph nodes of the animals of the 1000 mg/kg body weight group were discoloured. In the latter organ, no discoloration was seen at the animals of the main groups.

The relative weights of kidneys (related to body weight) of the females of main groups 2 and 3 showed significant increases. At the end of the recovery period the absolute kidney weight was significantly increased.

Histopathology:       No treatment-related changes were detected.

 

CONCLUSION

The observed statistically significant increases of the organ weights in the main group and in the recovery groups do not represent a treatment-related effect as no clear dose-response relationship was observed.

The discolorations in the kidneys and in the mesenteric lymph nodes did not result in microscopic organ lesions relatable to it, so that it is interpreted only as a physical property of the absorbed test article.

Based on the results obtained during this oral toxicity study, the "no adverse effect level" of Reaktivblau 1463 is above 1000 mg/kg body weight.