Registration Dossier

Toxicological information

Repeated dose toxicity: oral

Currently viewing:

Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
June 2, 1989 - July 3, 1989
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1992
Report Date:
1992

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Deviations:
no
Qualifier:
according to
Guideline:
other: EPA-TSCA-Test Guideline "Functional Observational Battery"
Version / remarks:
Federal Register, Vol. 50, No. 188, September 27, 1985, pp. 39458 - 39461, amended on May 20, 1987
Deviations:
no
Qualifier:
according to
Guideline:
other: EPA-TSCA Guideline "Neuropathology"
Version / remarks:
Vol. 52, No. 97, pp. 19081 - 19082
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
Test item: (N-Cyclohexyl-diazeniumdioxy)-potassium
IUPAC name: Cyclohexylhydroxydiazene 1-oxide, potassium salt
Chemical name: Cyclohexylhydroxydiazene 1-oxide, potassium salt; synonyma: (N-Cyclohexyl-diazeniumdioxy)-potassium, K-HDO, K-NCH, Xyligen K powder, Xyligen K
Molecular formula: C6 H11 K N2 O2
Molecular mass: 182.27
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Reu E 7350e
- Purity test date: N-Cyclohexyldiazeniumdioxy-potassium-hydrate powder, purity 100 % calculated as hydrate

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: before start and during test: test room temperature
- Stability under test conditions: stable

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing:
The test substance was weighed out and thorougly mixed with a small amount of feed in a beaker. The premix was subsequently prepared in a household mixer. A corresponding amount of feed was then added to the premix to obtain the desired concentration, and mixing was carried out for about 10 minutes in a laboratory mixer. The test substance preparations were prepared once for the whole study period.

FORM AS APPLIED IN THE TEST (if different from that of starting material)
see above

Test animals

Species:
rat
Strain:
Wistar
Details on species / strain selection:
Male and female Wistar rats from Dr. Karl Thomae GmbH, D-W7950 Biberach/Riss, FRG, which were free of signs of disease, were used for the investigations. The rats were unambiguously identified by tattoo of the respective animal number into the left ear.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Dr. Karl Thomae GmbH, D-W7950 Biberach/Riss, FRG
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 42 days
- Weight at study initiation (mean body weight): 176 g (male), 140 g (female)
- Fasting period before study: The animals were daily fed with the test substance preparations until the start of the fasting period (withdrawal of feed) of about 16 - 20 hours before necropsy.
- Housing: During the study, the rats were housed singly in type DK III stainless steel wire cages from Becker & Co., D-W4620 Castrop-Rauxel, FRG (floor area about 800 cm²). The animals were housed in a completely air-conditioned room in which a central air-conditioner ensured temperatures in the range 20 - 24°C and relative humidities in the range 30 - 70%. The day/night rhythm was 12 hours (12 hours light from 06.00 - 18.00 h, 12 hours dark from 18.00 - 6.00 h). Deviations from these ranges did not occur. The room was completely disinfected before the start of the study. The walls were cleaned once a week and the floor twice a week, in each case using water containing 0.1 % Incidin perfect®.
- Diet: ad libitum throughout the study (acclimatisation and administration period)
- Water: ad libitum throughout the study (acclimatisation and administration period).
- Acclimation period: 7 days

DETAILS OF FOOD AND WATER QUALITY:
The feed received by the animals was Kliba rats/mice/hamsters maintenance diet, 343 meal, Klingentalmühle AG, CH-4303 Kaiseraugst, Switzerland. The feed batch used in the study was assayed for contaminants.
The drinking water was regularly assayed for contaminants by the municipal authorities of D-W6710 Frankenthal, FRG, and by the Department of Water Chemistry and Technical Services of BASF Aktiengesellschaft.


ENVIRONMENTAL CONDITIONS
- Temperature (°C): see above
- Humidity (%): see above
- Air changes (per hr): see above
- Photoperiod (hrs dark / hrs light): see above

Administration / exposure

Route of administration:
oral: feed
Details on route of administration:
Application via diet
Vehicle:
other: food
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The test substance was weighed out and thoroughly mixed with a small amount of feed in a beaker. The premix was subsequently prepared in a household mixer. A corresponding amount of feed was then added to the premix to obtain the desired concentration, and mixing was carried out for about 10 minutes in a laboratory mixer. The test substance preparations were prepared once for the whole study period.
The homogeneous distribution of the test substance in the feed was checked using 6 samples, which were taken at the beginning of the study, of the concentration used.

DIET PREPARATION
- Rate of preparation of diet (frequency): The test substance preparations were prepared once for the whole study period.

VEHICLE
- Justification for use and choice of vehicle (if other than water): feeding study
- Concentration in vehicle: 900 ppm corresponding to a mean daily substance intake of about 82 mg/kg body weight in the male rats and of about 90 mg/kg body weight in the female rats.



Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
ANALYSES OF THE TEST SUBSTANCE:
The analyses regarding the characterization of the test substance were carried out prior to the start of the study. The degree of purity of the test substance was 91% (calculated: free of water) or 100% (calculated as hydrate), respectively.

ANALYSES OF THE DOSES:
The method used for analyses of doses in the feed (extraction out of the carrier and colorimetric determination) resulted in recovery rates of less than 80% due to the poor extractability of the test substance. The analytical results for potassium hydrate can be extrapolated from the analytically verified copper values (Project No. 20C0124/88078). Therefore, all values obtained with the colorimetric determination have to be calculated with the recovery rate. The following results were obtained: The homogeneity analyses of the 900 ppm dose revealed that the test substance was homogeneously distributed in the feed and the concentration was correct. The stability analyses carried out over a period of 10 and 32 days showed that the test substance is stable for these periods: calculated with the recovery rate of the starting value, the values obtained correspond to 93% (after 10 days) and 91% (after 32 days) of the expected values. As some animals received the test substance for 36 days due to technical reasons, it was assumed, based upon the above mentioned findings, that the test substance would be stable also for 36 days in the feed.

FEED ANALYSES: :
On the basis of duration of use and the analytical findings the feed was found to be suitable. Fed. Reg. Vol. 44, No. 91 of Hay 9, 1979, p. 27354 (EPA) served as a guideline for maximum tolerable contaminants.

Duration of treatment / exposure:
28 days
Frequency of treatment:
daily by food
Doses / concentrationsopen allclose all
Dose / conc.:
900 ppm
Remarks:
corresponding to a mean daily substance intake of about 82 mg/kg body weight in the male rats and of about 90 mg/kg body weight in the female rats.
Dose / conc.:
0 ppm
Remarks:
The control group (5 male and 5 female Wistar rats) of a study running in parallel(Project No. 20C0124/88078) was used.
No. of animals per sex per dose:
5
Control animals:
yes, concurrent no treatment
yes, plain diet
Details on study design:
- Dose selection rationale: The dose was chosen on the basis of the following investigations (1, 2, 3):
1) Study of the acute toxicity of N-cyclohexyldiazeniumdioxy-potassium-hydrate on rats (BASF Aktiengesellschaft, Project No. XXV/210)
2) Study of the subchronic oral toxicity (3 months) of N-cyclohexyldiazeniumdioxy-potassium-hydrate on rats (Laboratorium fur Pharmakologie und Toxikologie, Prof. Dr.med. F. Leuschner, 1978)
3) Study of the palatability of N-cyclohexyldiazenium dioxy-potassium-hydrate on rats BASF Aktiengesellschaft

- Rationale for animal assignment (if not random): Before the start of the administration period the animals were distributed according to weight among the test group separated by sex. The randomization list was drawn up by a computer (laboratory data processing, Department of Toxicology, BASF Aktiengesellschaft).
Positive control:
No

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: A check was made for dead or moribund animals twice a day (Mondays to Fridays) and once a day (Saturdays, Sundays and public holidays)

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: The animals were examined twice daily (Mondays to Fridays) or once a day (Saturdays, Sundays and public holidays) for any evident signs of toxicity. Once weekly they underwent an additional detailed clinical examination.

BODY WEIGHT: Yes
- Time schedule for examinations: the body weight was determined once a week and at the start of the study (day 0)

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- The feed consumption was determined once a week during the period of administration.

FOOD EFFICIENCY: Yes
- Body weight gain: has been determined

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: once a week

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: end of study (three days prior to necropsy )
- Anaesthetic used for blood collection: Not specified
- Animals fasted: No
- How many animals: 5 animals per sex and test group
- Parameters tested: Leukocytes, erythrocytes, haemoglobin, haematocrit, mean corpuscular volume, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration, platelets, differential blood count, reticulocytes, clotting analysis (thromboplastin time), haemoglobin derivatives (total haemoglobin, oxyhaemoglobin, carboxyhaemoglobin, methaemoglobin, content of oxygen bound to haemoglobin, oxygen saturation, reduced haemoglobin, oxygen capacity), clotting analysis (thromboplastin time)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: end of study (three days prior to necropsy )
- Animals fasted: Yes / No / Not specified
- How many animals: 5 animals per sex and test group
- Parameters tested: Enzymes: alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, serum-gamma-glutamyltransferase
Blood chemistry: Sodium, 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:
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Not specified
- Parameters tested: Appearance, nitrite, pH, protein, glucose, ketones, urobilinogen, bilirubin, blood, sediment

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: The observation of the neurofunction was made on all animals once prior to the start of the test substance administration, 24 hours after the first administration and on days 7, 14 and on day 27.
- Dose groups that were examined: all dose groups were examined
- Battery of functions tested: The examination of the neural functions was performed using a "functional observational battery"· which includes various parameters of sensoric and motoric functions as follows: general appearance, tremors, convulsions, piloerection, lacrimation/secretion of pigmented tears, salivation, pupil size, diarrhea, vocalization, paresis, paralysis, ataxia, body tone, posture, animal body (appearance), locomotor activity, respiration, urination, skin colour, righting reflex, behaviour, grip strength, pupillary reflex, winking reflex, vision, audition, olfaction, sensitivity of the body surface, pain perception (hot plate test), tail pinch, toe pinch, visual placing response, miscellaneous: all other visible clinical signs.

IMMUNOLOGY: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
Organs: Duodenum, jejunum, ileum, cecum, colon, rectum, liver, kidneys, brain, all gross lesions

HISTOPATHOLOGY: Yes
Organs: Duodenum, jejunum, ileum
Statistics:
STATISTICAL EVALUATION
Clinical examinations:
The parameter substance intake was determined using the following formula:
substance intake: (FC • D) ⁄ BWX
FC: mean daily feed consumption (in g) from day X - 7 to day X
D: Dosage in ppm
BWX: Body weight on day x of the study (in g)
For the statistical evaluation of the study, means and standard deviations were calculated for the variables feed consumption, water consumption, body weight, substance intake, grip strength (fore and hind limbs) and hot-plate test for the animals, and printed out in the form of tables.
The statistical significance of the clinical data was determined by the MANN-WHITNEY-U-test.
Significances (p-markers: * for p ≤ 0.05, ** for p ≤ 0.02, *** for p ≤ 0.002) resulting from the test have been shown in the tables.

Clinical chemistry and hematology:
Mean and standard deviation were calculated for each test group and tabulated together with the individual values. Except for the differential blood count, a statistic analyses is done via the Mann-Whitney-U-test. If the results of this test are significant, p-markers (* for p ≤ 0.05, ** for p ≤ 0.02, *** for p ≤ 0.002) were printed together with the group mean in the tables.

Urinalyses:
The assessment to whether certain characteristics differed in degree in the control and test groups was carried out using the chi² test in appropriate two-by two contingency tables.
Significances which resulted from this chi² test have been indicated in the tables (* for p ≤ 0.05, ** for p ≤ 0.01).

Results and discussion

Results of examinations

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
The only clinical finding was a discoloration of the faeces at a dose of 1500 ppm. This finding was considered to represent a chemical reaction of the test substance in the digestive tract rather than being the consequence of a toxic effect of the animals.
Mortality:
no mortality observed
Description (incidence):
No animal died intercurrently during the test period.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
The body weight gain of the male and female animals of the 900 ppm group was not affected by the test substance administration when compared to the control group.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
When compared to the untreated controls (Project No.: 20C0124/88078), no substance-related effects were seen regarding the amount of feed consumed daily.
Food efficiency:
no effects observed
Description (incidence and severity):
The amount of test substance intake (in mg) was calculated based upon the weekly determined feed consumption and the week1y determined body weight. The average daily test substance intake was for the male rats (900 ppm) about 82 mg/kg body weight. The female rats (900 ppm) had a mean daily substance intake of about 90 mg/kg body weight.
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
There were no statistically significant differences between the water consumption of the male or female animals of the 900 ppm group and the corresponding control group.
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Description (incidence and severity):
No substance related effects were observed
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
Throughout the administration period, male and female animals (900 ppm) showed no abnormal clinical symptoms.
Urinalysis findings:
no effects observed
Description (incidence and severity):
No substance related effects were observed
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
Neurofunctional tests:
No substance related changes of grip-strength of forelimbs, grip strength of hind limbs, or in the Hot Plate Test were seen in male and female animals of the three dose groups in comparison to the control group. The statistically significantly reduced value in the hot plate test of test group 2 (females) on day 1 can be assessed as an outlier. All other results of the “functional observational battery” also revealed no evidence that the test substance might be neurotoxic under the chosen conditions.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
The mean organ weights (absolute and relative) showed slight fluctuations, which did not give any indication of a relationship to the substance.
Gross pathological findings:
no effects observed
Neuropathological findings:
no effects observed
Description (incidence and severity):
The grip strength of the fore limbs of the males was statistically significantly increased on day 14, and the grip strength of the hind limbs of the females was significantly reduced on day 1.
Both findings were assessed as being incidental and not related to the test substance administration. All other results of the “functional observational battery" also revealed no evidence that the test substance might be neurotoxic under the chosen conditions
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed

Effect levels

Key result
Dose descriptor:
NOAEL
Effect level:
> 90 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs

Target system / organ toxicity

Key result
Critical effects observed:
no

Applicant's summary and conclusion

Conclusions:
NOAEL for K-HDO > 90 mg/kg bw/day or
NOAEL for 30% K-HDO > 300 mg/kg bw day
Executive summary:

The aim of the study was to investigate the mechanistic effect of N-Cyclohexyldiazeniumdioxy-potassium-hydrate on the digestive tract after 4 weeks administration via the diet. Particular attention was paid to possible neurotoxic effects.

The oral administration of 900 ppm (mean daily substance intake of about 82 mg/kg body weight in the male rats and of about 90 mg/kg body weight in the female rats) for a period of 4 weeks caused the following substance induced changes:

-increase in magnesium in both sexes

-increase in inorganic phosphatase and calcium in the females.

-decrease in glucose and triglycerides in the females

The results of clinical examinations, neurofunctional tests, or pathological investigations did not show any substance-related changes.

There were no indications of any damage to the intestinal mucosa. In particular, there were no signs of any irritant effect.