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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
Study period:
From 1989-09-28 to 1989-10-26
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Guideline study with acceptable restrictions. No analytical measures of the concentration administered and histopathology limited heart, lungs, liver, kidneys, spleen, stomach, jejunum, colon, thymus, testicles, adrenal glands and bone marrow (femur).

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1990
Report Date:
1990

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Type:
Constituent

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Hoechst AG, Kastengrung, SPF breed
- Age at study initiation: 6 weeks old
- Weight at study initiation: males:106-110 g, females: 105-111 g
- Housing: in Makrolon Type 4 cages, with woodchip bedding, in groups of 5 animals
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22±3°C
- Humidity (%): 50±20%
- Photoperiod: 12 hour light/dark cycle

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: sesame oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
- Justification for use and choice of vehicle : sesame oil, fully solluble.
- Concentration in vehicle: 0, 0.25, 1.25 and 6.25 % v/v
- Amount of vehicle (if gavage): 8ml/kg bw
The volume of substance preparation to administer was recalculated each time body weight was determined (twice weekly)
- Dosing solutions were prepared daily immediately prior to dosing.


Analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
29 days (28 applications)
Frequency of treatment:
Daily, 7 days/week for 28 days
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 20, 100 and 500 mg/kg bw/day
Basis:
other: nominal concentration
No. of animals per sex per dose:
5 rats/sex/dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
A preliminary 14 day toxicity study was conducted using doses of 200 and 1000 mg/kg (3 rats/sex/dose). In animals given 1000 mg/kg, autopsy revealed a gas filled gastrointestinal tract and effects on the liver and spleen. No clinical signs, no effect on body weight gain and no macroscopic changes were seen in the 200 mg/kg group. Therefore, doses of 0, 20, 100 and 500 mg/kg bw were selected for the main experiment.
- Rationale for animal assignment (if not random): random
- Rationale for selecting satellite groups: n.a.
- Post-exposure recovery period in satellite groups: n.a.
Positive control:
no.

Examinations

Observations and examinations performed and frequency:
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: behaviour and general health of all animals was observed twice daily (once daily on weekends and public holidays).

BODY WEIGHT: Yes
- Time schedule for examinations: Rats were weighed at the start of the study, and twice weekly thereafter.

FOOD CONSUMPTION AND FOOD EFFICIENCY: Yes
- Food consumption was measured continuously, based on twice weekly weighings of remaining food. The values were converted to food consumption per 100 g body weight per 24 hour period.

WATER CONSUMPTION : Yes
- Time schedule for examinations: Water consumption was determined weekly, and was expressed as consumption per 100 g body weight in a 16 hour period (15:15 pm to 07:15 am)

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: once a week

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at the study termination
- Anaesthetic used for blood collection: no
- Animals fasted: no
- How many animals: all animals of the study.
- Parameters checked : erythrocyte count, haemoglobin, haematocrit, leukocyte count, platelet count, differential white cell count, reticulocyte count*, Heinz bodies* and clotting time. Parameters marked * were measured in females in the control and high dose group only. MCV, MCH and MCHC were calculated.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: following the collection of blood samples
- Anaesthetic used for blood collection: yes
- Animals fasted: no
- How many animals: all animals of the study.
- Parameters checked: sodium, potassium, inorganic phosphate, uric acid, total bilirubin, creatinine, serum glucose, blood urea nitrogen, calcium, chloride, ASAT, ALAT, alkaline phosphatase, gamma-glutamyltranspepitdase, total protein, and the albumin:globulin ratio.

URINALYSIS: Yes
- Time schedule for collection of urine: overnight (ca. 16 hours) on day 26-27 of the study
- Metabolism cages used for collection of urine: Yes
- Animals fasted: no
- Parameters checked: appearance, colour, pH, occult blood, protein, glucose, ketone bodies, bilirubin, urobilinogen, density and sediment.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: each week
- Dose groups that were examined: all for neurological disorders
- Battery of functions tested: not performed
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
After sacrifice the animals were examined externally, then subject to gross necropsy. Skin, orifices, eyes, teeth, oral mucous membranes and internal organs were assessed at the macroscopic level.

ORGAN WEIGHT: Yes
The following organs were weighed: heart, lung, liver, kidneys, spleen, testicles and adrenals.

HISTOPATHOLOGY: Yes
The following organs were examined histopathologically: heart, lungs, liver, kidneys, spleen, stomach, jejunum, colon, thymus, testicles, adrenal glands and bone marrow (femur).
Other examinations:
No other examinations reported.
Statistics:
Mean values were compared between groups using standard statistical methods.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Mortality:
mortality observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
no effects observed
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
no effects observed
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Details on results:
CLINICAL SIGNS AND MORTALITY:
- There were no mortalities during the study. Animals from the 500 mg/kg group exhibited abnormal gait, irregular breathing and respiratory sounds. In the females there was also reduced spontaneous activity, withdrawn flanks and abdominal distension. Clinical signs were not observed in the other treatment groups.

BODY WEIGHT AND WEIGHT GAIN:
- Body weight gain of the female animals in the 500 mg/kg bw group was affected slightly from the 12th test day and in the males in this group from the 22nd test day. There were no effects on body weight gain in the other treated groups.

FOOD CONSUMPTION AND FOOD EFFICIENCY:
-Absolute and relative food consumption in the treated groups over the entire test period was not affected by the test substance.

WATER CONSUMPTION :
- Relative drinking water consumption was also not impaired by the test substance.

OPHTHALMOSCOPIC EXAMINATION
- no effects observed.

HAEMATOLOGY:
- A significantly reduced reticulocyte count was seen in the 500 mg/kg males, however values were still within the range considered normal for this strain therefore the result was not considered to be of toxicological significance. The differential white cell count was reduced in 2 males and 3 females in the 500 mg/kg group, and this was considered to be a treatment-related effect. No other haematological effects were observed.

CLINICAL CHEMISTRY:
- ALAT was significantly increased in 500 mg/kg males, whilst alkaline phosphatase and blood-urea nitrogen levels were significantly decreased in this group. Glucose levels were elevated in the 500 mg/kg females. Lower concentrations of albumin and the albumin-globulin ratio, higher gamma-globulin values were observed in males of the high dose group (all changes were significant).

URINALYSIS:
-There were no abnormal findings in urinalysis.

NEUROBEHAVIOUR:
- There were no treatment related findings when animals were examined for neurological disorders but no FOB was performed.

ORGAN WEIGHTS:
- The absolute and relative adrenal weights were increased in 500 mg/kg males. The absolute liver weights of the 500 mg/kg males were slightly reduced, but this was thought to be due to the lower body weights in this group.

GROSS PATHOLOGY:
- Isolated renal pelvic dilation was noted in some animals. 3 females of the 500 mg/kg group had larger and/or whitish discoloured adrenals, however no correlation was found histopathologically.

HISTOPATHOLOGY: NON-NEOPLASTIC
-There were two 500 mg/kg females with focal necrosis of the adrenal cortex with granulocyte infiltration. In one animal, this condition was associated with severe haemorrhage and ulceration of the stomach lining.

Effect levels

Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
Based on the findings in this subacute (28 day) oral toxicity study, a NOAEL was established at 100 mg/kg body weight per day.
Executive summary:

In a 28-day oral toxicity study, dioctadecyldimethylammonium chloride (90% active in 5% isopropanol 5% water) was administered by gavage in sesame oil at doses of 0, 20, 100, and 500 mg/kg to Wistar rats. The study was performed according to OECD guideline 407 in compliance with the principles of Good Laboratory Practices.Dosages were calculated on nominal concentration values.

Beginning at the 8th day (females) and 14th day (males) of treatment some high dose animals showed squatting position, abnormal gait, disregular and noisy breathing. Reduced spontaneous activity, retracted flanks and distended abdomen were also seen in some high dose females.

Body weight gain was slightly (non-significantly) lower in high dose males and females compared to the control values.

Hematology revealed significantly reduced reticulocyte counts in high dose males which were within the normal range for this species, sex and age, whereas no other red cell parameter was changed. Mean values for segmented neutrophile ratio were increased in high dose males and females due to abnormal rates in two males and three females. Lower concentrations of albumin and the albumin-globulin ratio and higher gamma-globulin values were observed in males of the high dose group (all changes were significant).

Absolute and relative organ weights of the adrenals were determined to be significantly increased in high dose males. Adrenal weights of high dose females were also higher than controls, however adrenals from three females only were weighted. Macroscopically, enlargement of the adrenals was seen in one female and discoloration of the adrenal surface was evident in three females of the high dose group. Corresponding to these observations in the adrenals, two females had cortical necrosis with peripheral granulocytic infiltration, one of this was hemorrhagic. Furthermore in a single high dose female ulceration of the forestomach was found.

The NOAEL from this study was considered to be 100 mg/kg bw/d.