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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:
November - December 1985
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: GLP-study equivalent to OECD Guideline 407 with acceptable restrictions (i.e. fewer organs weighed and miroscopically examined as specified in OECD Guideline 407, adopted 2008)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1986
Report date:
1986

Materials and methods

Test guideline
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)
Version / remarks:
adopted 1981
Deviations:
not applicable
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
1-(N,N-bis(2-ethylhexyl)aminomethyl)-1,2,4-triazole
EC Number:
401-280-0
EC Name:
1-(N,N-bis(2-ethylhexyl)aminomethyl)-1,2,4-triazole
Cas Number:
91273-04-0
Molecular formula:
C19 H38 N4
IUPAC Name:
1-(N,N-bis(2-ethylhexyl)aminomethyl)-1,2,4-triazole
Details on test material:
- Physical state: liquid
- Storage condition of test material: room temperature protected from light

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River (76410 St Aubin les Elbeuf, France)
- Age at study initiation: 5 weeks
- Weight at study initiation: 137 g for males and 86 g for females.
- Housing: five per cage from the same sex and the same group in labelled polycarbonate cages
- Diet: standard laboratory animal diet ref. A 04C, ad libitum
- Water: filtered tap water, ad libitum


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 2°
- Humidity (%): 50 ± 20%
- Air changes (per hr): at least 15 cycles/hour of filtered non-recycled air
- Photoperiod (hrs dark / hrs light): 12 h/12 h

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: Distilled water containing 0.5 % carboxymethylcellulose and 0.1 % polysorbate 80.
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The test substance was suspended in the vehicle by means of a magnetic stirrer.

VEHICLE
- Justification for use and choice of vehicle (if other than water): homogeneity and stability of test item in vehicle
- Amount of vehicle (if gavage): 5 mL/kg bw per day
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
- Homogeneity of the test suspensions was checked before the beginning of the study.
- Stability of the test suspensions kept at 4°C was checked before the beginning of the study at days: 0 - 1 - 3 - 5.
- Concentrations of all suspensions (vehicle included) were verified on the first and the last week of the study.
The homogeneity of the suspension of 20, 60 and 200 mg/kg/day groups was found to be satisfactory.
The stability of the test item in aqueous suspensions at the concentrations of 4 - 12 - 40 mg/mL stored at + 4°C during five days was also satisfactory.
Concentrations checked for all the suspensions on weeks 1 and 4 revealed a good correlation between obtained and theoretical values (the difference was always lower than 10 %).
Duration of treatment / exposure:
28 days
Frequency of treatment:
Daily
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 20, 60, 200 mg/kg bodyweight per day
Basis:
actual ingested
No. of animals per sex per dose:
5 males and 5 females
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The LD 50 has been determined by the sponsor and noted to be about 2500 mg/kg bodyweight : due to the observation of the symptoms, such as dyspnea (from 1000 mg/kg) or sedation (from 2000 mg/kg), the highest dose for this 28 day study is chosen equal to 200 mg/kg/day.

Examinations

Observations and examinations performed and frequency:
MORTALITY:
All animals were checked twice a day during the treatment period (in the morning and in the afternoon) to look for dead or moribund animals, except during week-ends and Public Holidays (once only in the morning).

CLINICAL OBSERVATIONS: Yes
All signs of ill health, together with behavioural changes or reaction to treatment, were recorded for each animal. These examinations of were carried out daily, at approximately the same time each day.

BODY WEIGHT: Yes
- Time schedule for examinations: The weight of each animal was recorded at the time of allocation of animals into groups, on the day of commencement of treatment and at weekly intervals during the treatment period.

FOOD CONSUMPTION:
The quantity of food consumed by the animals of each cage was recorded on a weekly basis during treatment. Food intake per animal was calculated using the amount of food given and left in each cage per group and the number of rats surviving in each cage for the majority of days during the week.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Before commencement of treatment and at the end of treatment
- Dose groups that were examined: 0 mg/kg/d, 200 mg/kg/d

HAEMATOLOGY: Yes
- Time schedule for collection of blood: on Week 5 in all surviving males and females
- Anaesthetic used for blood collection: Yes (light ether anesthesia)
- Animals fasted: overnight
- How many animals: all surviving animals
- Parameters examined: Red Cell Count, Haemoglobin, Mean Cell Volume, Packed Cell Volume, Mean Cell Haemoglobin Concentration, White Cell Count, Differential White Cell Count (Neutrophils, Eosinophils, Basophils, Lymphocytes, Monocytes), Platelet Count, Quick time, Activated partial Thromboplastin Time, Fibrinogen

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: on Week 5 in all surviving males and females
- Animals fasted: overnight
- How many animals: all surviving animals
- Parameters examined: Sodium, Potassium, Chloride, Calcium, Inorganic Phosphorus, Glucose, Urea, Creatinine, total bilirubin, total proteins, Cholesterol, Triglycerides, Alkaline phosphatase, Aspartate aminotransferase, Alanine aminotransferase, Gamma glutamyl transpeptidase, Albumin, alpha-1-globulins, alpha-2-globulins, beta globulins, gamma globulins, albumin/globulins

URINALYSIS: Yes
- Time schedule for collection of urine: on Week 5 in all surviving males and females
- Metabolism cages used for collection of urine: Yes
- Animals fasted: overnight
- Parameters examined: Volume, pH, specific gravity, urobilinogen, proteins, glucose, ketones, bilirubin, blood, nitrites, sediment (leukocytes, erythrocytes, hyaline and granular casts, ammonium magnesium phosphate, calcium phosphate and calcium oxalate crystals, cells (epithelial, bladder and kidney).

NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes. All superficial tissues were examined before and after dissection. All abnormalities were recorded. All the rats killed in extremis, or found dead in the cages, were subjected to detailed macroscopic examination. When possible, a full set of tissues were preserved.

-Organ weights: The following organs from all animals killed at termination were dissected free of fat and weighed: adrenals - brain - heart -kidneys - liver - ovaries - spleen - testes - thymus.

HISTOPATHOLOGY: Yes
Samples of the following tissues from all animals, together with any macroscopically abnormal tissues, were preserved in appropriate fixative:
aorta - caecum - brain (bulb, cerebellum, medulla) - heart - colon - duodenum - stomach (glandular, non glandular) - liver (all lobes) - mammary gland - salivary glands (parotid, submandibular) - pituitary - ileum - jejunum - spinal cord - skeletal muscle - sciatic nerve - Lymph nodes (mesenteric , thoracic ) - femur with joint - oesophagus - ovaries - pancreas - lungs (all lobes and mainstem bronchi) - prostate - spleen - rectum - kidneys - sternum - testes with epididymides - adrenals - thymus - thyroids and parathyroids - trachea - ureters - urethra - uterus (corpus, cervix) - vagina - seminal vesicles - urinary bladder.
The samples were embedded in paraffin and sections cut at 4 to 5 microns were then stained with hemalum and eosin. HistomorphologicaI examinations were performed on all animals from the control and 200 mg groups.
Statistics:
Analysis was done according to the following evaluations:
The normality of the distribution of values through the Kolmogorov-Smirnov test, then the homogeneity of variances through the Bartlett test (more than two samples).
The comparison between treated and control groups was made through Dunnett test in case of homogeneity of variances and through Mann Whitney in case of no homogeneity of variances.

Results and discussion

Results of examinations

Details on results:
CLINICAL SIGNS AND MORTALITY
No clinical signs were noted in male and female animals.
Male animals: there was no death in the 0, 60 and 200 mg/kg/day groups. In the 20 mg/kg/day group, one animal was found dead on Day 18 after treatment without clinical signs preceding death.
Female animals: there was no death in the 0, 20 and 60 mg/kg/day groups. In the 200 mg/kg/day group, three animals were found dead on days 4, 6 and 7, without clinical signs preceding death.

BODY WEIGHT AND WEIGHT GAIN
There were no statistically significant mean-bodyweight variations in male or female animals. Only a loss of bodyweight was noted in one female from the 20 mg/kg/day group (- 10 g) between weeks 4 and 5.

FOOD CONSUMPTION AND COMPOUND INTAKE
There was no change in food consumption of the male animals.
On week 1, a decrease in food consumption was observed in the females from the 200 mg/kg/day group. This change was no more seen on week 2 and later.
The food consumption was normal in all the other groups.

OPHTHALMOSCOPIC EXAMINATION
The only abnormalities were observed before treatment start and were commonly seen in the laboratory rat. No abnormality was recorded at the end of the treatment period. No abnormality was recorded at the end of the treatment period.

HAEMATOLOGY
No test item-related changes were observed in the animals from the 20 mg/kg/day groups.
A slight decrease of platelet count was noted in males from the 60 and 200 mg/kg/day groups. Nevertheless, the individual values were within normal limits, and this change has no clear toxicological significance.
The other values were normal.

CLINICAL CHEMISTRY
No changes were observed in animals from the 20 mg/kg/day group.
Slight increase of urea and creatinine level was noted as statistically significant in males from the 60 and 200 mg/kg/day groups, in comparison with control values. This significance was due to low values noted in three control males out of five. All the individual values were within normal limits.
Slight changes in protein electrophoresis were statistically significant:
- slight increase of albumin level in males from the 60 mg/kg/day groups,
- slight decrease of alpha-1-globulin level in males from the 60 and 200 mg/kg/day groups,
- slight increase of alpha-2-globulin level in females from the 60 mg/kg/day group,
- slight increase of beta-globulin in males from the 60 and 200 mg/kg/day groups.
Due to their marginal character, the changes observed in dose levels 60 and 200 mg/kg/day have no toxicological significance.
In conclusion, no test item-related changes were observed in animals from all the three treated groups.

URINALYSIS
The results of urinalysis were normal.

ORGAN WEIGHTS
In the treated groups, no difference in the organ weights when compared to the control groups, presented any dose response relationship.

GROSS PATHOLOGY
At terminal necropsy, signs of gastric irritation consisting of wall thickening and/or focal hemorrhages of the forestomach were observed in 5 out of 7 surviving rats from the 200 mg group. No such changes were seen in the other treatment groups.
The abnormalities seen at necropsy in the respiratory tract of two rats (200 mg group) found dead during the study were attributed to a gavage error.
Due to cannibalism, the cause of the death of one rat was not established.
One animal from the 20 mg group (found dead on day 18) showed a dilatation of the forestomach.

HISTOPATHOLOGY: NON-NEOPLASTIC
Test item-related changes were observed in the stomach of rats from the 200 mg group (7 animals). In 5 animals (4 males and 1 female) moderate or marked signs of irritation were observed; the two other rats had a slight submucosal edema. One animal from the 60 mg group presented a gastric submucosal edema; no such changes were seen in controls.
Other minor changes were found in different organs of control or treated rats without any dose response relationship.

Effect levels

Dose descriptor:
NOEL
Effect level:
60 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: mortality and signs of gastric irritation (observed during gross pathology and histopathology) at the next higher dose level

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
Under the experimental conditions of this study, the test substance given to rats at the dose levels of 0, 20, 60 and 200 mg/kg/day did not induce any clinical change. The mortality in the 20 mg/kg/day group (one animal out of ten) cannot be certainly related to the treatment. In laboratory investigations, the slight changes observed at the two higher dose levels have no toxicological significance. At necropsy, drug-related signs were seen in the higher dose group. The NOEL was estimated to be 60 mg/kg/day.
Executive summary:

In a GLP-compliant 28-day repeated dose toxicity study, four groups of 5 male and 5 female Sprague Dawley rats each were given the test substance by oral gavage at dose levels of 0, 20, 60 and 200 mg/kg body weight per day. Clinical signs and mortality were checked each day. Body weight and food consumption were recorded weekly. Ophthalmological examinations were performed during the predosing period and on drug week 4. Hematology, blood biochemistry and urinalysis were performed at the end of the treatment period in all surviving animals. On completion of the study, the surviving animals were sacrificed; all the superficial tissues were examined before and after dissection; the main organ weights were recorded and the samples of main tissues were subjected to a microscopic examination.

No treatment-related clinical signs and no marked changes in bodyweight and food consumption were noted in all treated animals. The very slight changes recorded in bodyweight or food consumption were without toxicological significance. No mortality occurred in male animals from the 0, 60 and 200 mg/kg/day groups. In the 20 mg/kg/day group, one animal was found dead, without demonstration of any clinical sign during the period preceding death. No mortality occurred in female animals from the 0, 20 and 60 mg/kg/day groups. Three animals were found dead in the 200 mg/kg/day group, without clinical signs preceding death. No hematological changes were seen in female animals from all treated groups and in males from the 20 mg/kg/day group. A very slight decrease in platelet count was noted in males from the 60 and 200 mg/kg/day groups. This change has no toxicological significance. In females from the 60 mg/kg/day group and in the males from the 60 and 200 mg/kg/day groups, there were slight changes in protein electrophoresis. This change has no toxicological significance. There were no changes in urinalysis in males and females from all treated groups. At necropsy, signs of gastric irritation consisting in wall thickening and/or focal hemorrhages of the forestomach were observed in 5 out of 7 surviving rats from the 200 mg/kg/day group. Microscopic examinations revealed changes in the stomach of the 7 surviving rats from the 200 mg/kg/day group: moderate or marked signs of irritation in 5 animals (4 males and 1 female); a slight submucosal edema in the 2 other rats. One female from the 60 mg/kg/day group presented a gastric submucosal oedema.

Based on these results and under the described experimental conditions, the No observed effect level was estimated to be 60 mg/kg/day for both sexes.