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Repeated dose toxicity: oral

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Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
March 1996 - January 1997
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2000
Report date:
2000

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.3150 (90-Day Oral Toxicity in Non-rodents)
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Reference substance name:
67359-57-3
EC Number:
614-052-2
Cas Number:
67359-57-3
IUPAC Name:
67359-57-3
Details on test material:
- Chemical name: mixture of N,N-Dimethydecan-1-amide and N,N-Dimethyloctan-1-amide
- Physical state: liquid
- Storage condition of test material: roomtemperature
- Today handle under EC 909-125-3 Reaction mass of N,N-Dimethyldecan-1-amide and N,N-Dimethyloctan-1-amide.
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Lot/batch No.of test material: 002949

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature
- Stability of the test substance in the solvent/vehicle: Before the start of the study it was assured for a period of at least 8 days that the test substance is chemically stable within the concentration range used in the study.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The suspensions were prepared weekly.
- Preliminary purification step (if any):
- Final dilution of a dissolved solid, stock liquid or gel:
- Final preparation of a solid:

FORM AS APPLIED IN THE TEST (if different from that of starting material): suspended in 0.5% aqueous tylose

Test animals

Species:
dog
Strain:
Beagle
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Winkelmann, Borchen, Germany
- Age at study initiation: 19-22 weeks old (at week -1)
- Weight at study initiation: 6.4-9.0 kg (at week -1)
- Housing: individual cages
- Diet: daily, about 2 hours after administering the test substance, ssniff HH complete food for dogs, 12 mm pellets from ssniff Versuchstierdiäten GmbH, Soest, Germany
- Water: ad libitum, common drinking water quality
- Acclimation period: 17 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.0 - 23.5°C
- Humidity (%): 25-50%
- Photoperiod (hrs dark / hrs light): Premises illuminated by diffuse daylight, but mainly fluorescents lamps with regulated the day/night cycle (12h each)
- Cleaning: spray cleaned each afternoon (hot water)

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: 0.5% aqueous tylose
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
- suspensions were prepared weekly
- stability ensured for 8 days in range of concetrations used
- Analytical content check of the formulation performed during the course of the study

ADMINISTRATION:
Administration via gavage: Administration volume: 10ml
Application once daily 2 hours before feeding in the morning
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analytical content checks (via GC) of the formulations performed during the course of the study verified that the suspension actually contained the amount of test substance requested for the different groups (reported recovery >97%).
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
once daily 2 hours before feeding in the morning
Doses / concentrationsopen allclose all
Dose / conc.:
40 mg/kg bw/day (nominal)
Dose / conc.:
200 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Remarks:
reduced to 800 (from the 2nd day of the study onwards) and further to 500 mg/kg bw (from the 4th day of the study onwards)
No. of animals per sex per dose:
4
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The doses were based upon the results of a subacute oral toxicity study in dogs (6-week study by oral administration via gavage)
Positive control:
not necessary

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: repeatedly every day (during feeding, during maintenance and cleaning of the stall, and during the exercise period)
- Cage side observations: appearance and behavior

DETAILED CLINICAL OBSERVATIONS: Yes
testing of each animal's reflexes (pupillary, corneal, patellar, extensor-, postural-, and flexor reflex), body weight temperature, pulse rate at the femoral artery
- Time schedule: before the start of the study (week-2) and in weeks 7 and 13 of the study
Electrocardiograms (ECG) and blood pressure measurements
- Time schedule: once before the start of the study (week -2) and before and 2h after administration in week 7 and 13 of the study

BODY WEIGHT: Yes
- Time schedule for examinations: weekly, generally at intervals of 7 days

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

WATER CONSUMPTION: Yes (differences in the frequencies with which the water bowls had to be filled up)
- Time schedule for examinations: repeatedly every day (during feeding, during maintenance and cleaning of the stall, and during the exercise period)

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: before the start of the study (week -2) and in week 7 and 12 of the study
- Dose groups that were examined: all

HAEMATOLOGY: Yes
- Time schedule for collection of blood: before the start of the study (week -3/-2) and in weeks 4, 7, and 13 of the study
- Anaesthetic used for blood collection: No
- Animals fasted: Not specified
- How many animals: all
- Parameters checked in table no. 1 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: before the start of the study (week -3/-2) and in weeks 4, 7, and 13 of the study
- Animals fasted: Not specified
- How many animals: all
- Parameters checked in table no. 2 were examined.

URINALYSIS: Yes
- Time schedule for collection of urine: before the start of the study (week -2) and in weeks 4, 7, and 13 of the study
- Metabolism cages used for collection of urine: Yes, for a period of approx. 6h
- Animals fasted: Feed and water were not available during the sampling period. Before being transferred to the metabolism cages, the animals were given a large dose of water (approximately 250 ml of tap water) by oesophageal tube.
- How many animals: all
- Parameters checked in table no. 3 were examined.

OTHER:
At necropsy samples of the liver were taken for enzymatic measurements and for the determination of the triglyceride content in the liver.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
- The brain, heart, liver, lungs, spleen, adrenals, kidneys, pancreas, thyroid, pituitary, testes, prostate gland, uterus, thymus, and ovaries were weighed.

HISTOPATHOLOGY: Yes (see table 4)
Statistics:
In line with the small number of animals per group, a descriptive statistical evaluation was performed

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
From group II upwards, vomiting and salivation were observed repeatedly.
Additional effects in group III consisted of uncoordinated movements, nasal discharge, lateral/prone position, and trembling.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
One male of group III had to be sacrificed on day 3 (showing lateral position, flat breathing and bloody faeces before sacrificing).
One femal of group III died in week 7 of the study exhibiting lateral position, bloody nasal and oral discharge before death.
Due to the lung findings observed in the histopathological investigations, these two events might be associated with an unintentional intratracheal application.
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
effects observed, treatment-related
Description (incidence and severity):
In group III, a cataractous lens occurred in the right eye of one female in week 12 of the study.
Haematological findings:
no effects observed
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
APh increased in one male and one female of group III;
N-Dem: marginally increased in one male of group II; increased in all females of group II and all animals of group III;
P 450: increased in two females of group III;
Urinalysis findings:
no effects observed
Behaviour (functional findings):
not specified
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
There was an increase in absolute and relative liver weights in males of group III. This is regarded as an indication for a minimal liver enzyme induction but not for an adverse effect.
Gross pathological findings:
no effects observed
Neuropathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
- severe bronchopneumonia in one male of group III (animal had to be sacrificed on day 3)
- acute edema in one female of group III (animal died in week 7 of the study)
An unintentional intratracheal application might have been the cause for these effects in the lungs.
Histopathological findings: neoplastic:
not specified

Effect levels

open allclose all
Dose descriptor:
NOAEL
Remarks:
systemic effects
Effect level:
200 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
clinical biochemistry
clinical signs
mortality
Dose descriptor:
NOEL
Remarks:
local effects
Effect level:
40 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
other: repeated vomiting and salivation, nasal discharge

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
As there were only local effects and slightly increased N-DEM values in the absence of any other finding the NOAEL is established at 200 mg/kg bw/d.
Executive summary:

To assess the subchronic toxicity of the test substance a subchronic test according to OECD Guideline for Testing of Chemicals No. 409 "Subchronic Oral Toxicity - Non rodent", adopted 12th of May 1981 was performed.

Therefore Groups of 4 male and 4 female beagle dogs per dosage were treated once daily by gavage over a period of 13 weeks with following daily oral doses:

Control group: 0 mg/kg b.w.

Group I : 40 mg/kg b.w. test substance

Group II : 200 mg/kg b.w. test substance

Group III : 1000 mg/kg b.w. test substance

From the second day of the study onwards, 1000 mg/kg were reduced to 800 mg/kg, and from the 4th day of the study onwards, 800 mg/kg were reduced to 500 mg/kg due to a markedly reduced health status of some group III-animals.

Tests of the reflexes, body temperatures, pulse rates, blood pressure, and heart rates showed no changes up to and including group III. The same holds true for the electrocardiograms. The nutritional state, feed intake, and body weight gain also yielded no adverse effects up to and including group III. Clinical observations evidenced repeated vomiting and salivation from group II upwards. Additional effects in group III consisted of uncoordinated movements, nasal discharge, lateral/prone position, and trembling. All these effects from group II upwards are considered to be due to treatment with the test compound. One male animal of group III had to be sacrificed on day 3 of the study showing lateral position, flat breathing, and bloody faeces before sacrificing. Another animal (female) of group III died in week 7 of the study exhibiting lateral position, bloody nasal and oral discharge before death. Due to the lung findings observed in the histopathological investigations, these two events might be associated with an unintentional intratracheal application.

Ophthalmoscopy showed a lens cataract in one animal of group III. This effect is seen to be treatment-related. Haematology and differential blood counting showed no changes up to and including group III. Clinical chemistry evidenced a substance-related elevation of APh-values in group III. Increased N-Demethylase values from group II upwards and increased Cytochrome P450-values in group III are seen as treatment-related effects giving evidence for an increased metabolic activity in the liver. In this context the increased liver weights in males of group III are also considered to be treatment-related. Urinalyses showed no changes up to and including group III. Necropsy and histopathological examination did not reveal toxicologically relevant effects up to and including group III. Besides increased liver weights in males of group III (see above), no changes in organ weights were detected up to and including group III.

The author concluded that based on the reported effects it can be concluded that 40 mg/kg test substance administered orally by gavage to beagle dogs daily over a period of 13 weeks were tolerated without adverse effects.

In contrast to this conclusion the applicant assessed to effects observed as follow:

The NOEL at 40 mg/kg bw/d (0.4%) reported is based on local effects caused by oral application of the test substance at irritating concentrations (≥2%). At 200 mg/kg bw/d clinical chemistry revealed only slightly increased N-DEM values in the absence of any other finding. The increase of this parameter is an indication for a minimal liver enzyme induction but not for an adverse effect. Therefore the NOAEL is established at 200 mg/kg bw/d.