Registration Dossier

Toxicological information

Repeated dose toxicity: oral

Currently viewing:

Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP Guideline study with acceptable restrictions
Cross-reference
Reason / purpose for cross-reference:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1996

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
other: OECD Guideline 415 (One-Generation Reproduction Toxicity Study)
Deviations:
no
Principles of method if other than guideline:
The principles of a subchronic oral study according to OECD 408 was followed especially with respect to number of animals per dose group and examination parameters. Based on the guideline requirements of OECD 415 the administration period exceeded 13 weeks for both sexes. Further deviations were obvious with regard to the female test group since tested animals were not nulliparous and non-pregnant at the end of the study period. Since there has been no differences between male and females in previous studies as well as this study, this deviation is considered acceptable. Although histopathology was performed in all organs requested by OECD 408, no organ weights were recorded for uterus, thymus, spleen and heart, which is also considered acceptable.
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
A mixture of: 2,2',2'',2'''-(ethylenedinitrilotetrakis-N,N-di(C16)alkylacetamide; 2,2',2'',2'''-(ethylenedinitrilotetrakis-N,N-di(C18)alkylacetamide
EC Number:
406-640-0
EC Name:
A mixture of: 2,2',2'',2'''-(ethylenedinitrilotetrakis-N,N-di(C16)alkylacetamide; 2,2',2'',2'''-(ethylenedinitrilotetrakis-N,N-di(C18)alkylacetamide
Cas Number:
136920-07-5
Molecular formula:
C58H114N4O6 - C154H308N6O4
IUPAC Name:
Reaction mass of 2,2',2'',2'''-(ethylenedinitrilotetrakis-N,N-di(C12-C18)alkylacetamide and {[2-(Carboxymethyl-di(C12-C18)alkylcarbamoylmethyl-amino)-ethyl]-di(C12-C18)alkylcarbamoylmethyl-amino}-acetic acid
Constituent 2
Reference substance name:
Keroflux ES 3241
IUPAC Name:
Keroflux ES 3241
Details on test material:
Batch No.: (T 75492/ST 1414/90) Partie 1
Manufacturing/Sampling Date: 10.10.1990
Physical state: pale yellow wax at room temperature
Storage conditions: refrigerator

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
Male and female Wistar rats supplied by Karl THOMAE, Biberach an der Riss, D, which were free from any clinical signs of disease, were used for the investigations. The 112 male and 112 female rats were 25 (± 1) days old when they arrived from the breeding facilities. During an acclimatization period of 10 days, animals with lowest and highest body weights were eliminated, used for other purposes and finally sacrificed. The 100 male and 100 female animals required for the study were 35 (± 1) days old at the beginning of treatment, and their mean weights and weight ranges were: male animals: 140.0 (129.9 - 152.0) g and female animals: 114.5 (104.0 - 124.5) g.
The females were nulliparous and non-pregnant at the beginning of the study. According to a written statement from the breeder, male and female animals were derived from different litters. This was necessary to rule out the possibility of sibling mating. These animals were taken to form the F0 parental generation. All other animals used in this study (F1 pups) were derived from these animals.
The rats of the parental generation (F0 generation) were identified uniquely by ear tattoo. The unit digit of the animal number was tattooed on the outside of a rat's left ear, the ten digit on the inside of the left ear and the hundred digit was tattooed on the inside of the right ear. All live pups were identified by skin tattoo on day 1 post partum (p.p.) and with picric acid between days 10 and 15 after birth.
During the study period, the rats were housed individually in type DK III stainless steel wire mesh cages supplied by BECKER & CO., Castrop-Rauxel, FRG (floor area of about 800 cm2), with the following exceptions: during mating periods, the males designated for mating were kept individually in Makrolon cages, type M III (floor area of about 800 cm2); for the overnight mating the females were put into the cages of the males. From day 18 of gestation until day 14 after birth, the pregnant animals and their litters were also housed in Makrolon type M III cages. The M III cages were again supplied by BECKER & CO. Pregnant females were provided with nesting material (cellulose wadding) toward the end of gestation. The cages with the test animals were arranged on the racks in such a way that uniform experimental conditions (ventilation and light) were ensured.
The animals were accommodated in fully air-conditioned rooms (floor area about 22 m2) in which central air conditioning guaranteed a range of temperature of 20 - 24°C and a range of relative humidity of 30 - 70%. There were no or only minimal deviations from these limits.
The day/night rhythm was 12 hours (12 hours light from 6.00 a.m. to 6.00 p.m. and 12 hours darkness from 6.00 p.m. to 6.00 a.m.) in general.
Before use each room was completely disinfected using a disinfector. Usually, each week the walls and the floor were cleaned with water containing about 0.5% Mikro-Quat.
The food used was ground Kliba maintenance diet rat/mouse/hamster, 343 meal, supplied by KLINGENTALMUEHLE AG, Kaiseraugst, CH, which was available to the animals ad libitum throughout the study (from the day of supply to the day of or the day before necropsy). Drinking water was supplied from water bottles. The bedding used throughout the study was Ssniff (type 3/4) supplied by SSNIFF SPEZIALDIAETEN GmbH, Soest, D).

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
olive oil
Details on oral exposure:
Before the beginning of the study the test substance was melted at temperatures up to about 80°C, thoroughly mixed and bottled in small portions determined for separate use for each day of dosing. Each day before dosing the test substance was heated to about 80°C, intensively shaken and thereafter, the amounts necessary for each dose group were weighed and topped up with olive oil DAB 10 (heated to about 80°C). These test substance preparations were then stirred continuously in a water bath of about 60°C until the preparations turned into clear solutions. Finally, the test substance preparations were cooled down in a water bath of about 33°C (under continuous stirring). The stability of the test substance preparations for a period of 4 hours at room temperature was proven in a previous study (Project No. 34S0763/90086) as was the homogeneous distribution within the test substance preparations. Samples of each concentration were drawn for concentration control analyses at the start of the administration period, thereafter at intervals of about 4 weeks and at study termination.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The content of KEROFLUX ES 3241 in the test substance preparations was determined by titration of functionalized nitrogen by trifluoromethane sulfonic acid.
Duration of treatment / exposure:
19 weeks
Frequency of treatment:
continuously throughout the whole study period
Doses / concentrations
Remarks:
Doses / Concentrations:
100; 300 or 1,000 mg/kg body weight/day
Basis:
actual ingested
No. of animals per sex per dose:
25
Control animals:
yes, concurrent vehicle
Details on study design:
The doses were chosen on the basis of a previous oral toxicity study, in which Wistar rats received the test substance by gavage over 4 weeks (21 administrations). In this study KEROFLUX ES 3241 was administered to each 5 male and 5 female Wistar rats per dose at doses of 15, 150 and 1000 mg/kg body weight/day. There were no substance-related effects on food consumption, body weight, body weight change, clinical observations, hematological and clinicochemical examinations and concerning pathology (absolute and relative organ weights, gross and histopathological findings) in any of the test groups. Thus, the "no observed adverse effect level" (NOAEL) was at least 1.000 mg/kg body weight/day.
Positive control:
-

Examinations

Observations and examinations performed and frequency:
Food consumption: During the first 10 test weeks, food consumption of the F0 rats was determined once a week (each time for a period of 7 days). After the 10th test week, food consumption of the females during gestation (animals with evidence of sperm) was determined for days 0 - 7, 7 - 14 and 14 - 20 p.c. During the lactation period (animals with litter) food consumption was determined for days 1 - 4, 4 - 7 and 7 - 14 p.p. Food consumption was not determined between days 14 and 21 after parturition as required in the test guidelines, since during this time pups begin to consume considerable amounts of solid food offered, and therefore there was no point in such a measurement. Food consumption of the F0 males was not determined after the 10th test week through sacrifice. Furthermore, there was no determination of food consumption in the F0 females during the mating periods, in the F0 females without positive evidence of sperm during the programmed gestation phase, or in the F0 females without litters during the lactation phase. The food consumption of those animals whose fertility had to be re-evaluated and those controls which were chosen as partners for these re-evaluations was not determined, neither during the additional matings nor until sacrifice.
Body weight data: In general, the body weight of the male and female parental animals was determined once a week at the same time of the day (in the morning); if possible, the weighings were carried out until the end of the study. The body weight change of the animals was calculated from these results.
Clinical observations: All parental animals were checked for clinically evident signs of toxicity shortly before and after the daily intubation; in case of findings, these were documented. For technical reasons, however, the clinical observations recorded during the premating periods were printed out on a weekly basis. The nesting, littering, and lactation behaviour of the dams was generally evaluated in the mornings in connection with the daily clinical inspection of the dams. Only special findings (e.g., animal could not litter, umbilical cord not cut), were documented on an individual dam basis. The littering behaviour of the dams was also inspected on weekdays (except holidays) in the afternoons in addition to the evaluations in the mornings. The day of littering was considered the 24 hour period from about 3.00 p.m. of one day until about 3.00 p.m. of the following day. Deviations from this procedure were possible on Saturdays, Sundays and on public holidays, when the weighings of the dams took place as early as about 7.00 a.m.. Animals in a moribund state were sacrificed and examined in the laboratory of pathology.
Sacrifice and pathology:
Hematology and clinical chemistry: Blood was taken from the retroorbital venous plexus in the morning from 10 non-fasted, unanesthetized animals per sex per dose. The blood sampling procedure and the subsequent analysis of the blood and serum samples were carried out in a randomized sequence. The following parameters were determined in blood: leukocytes, erythrocytes, hemoglobin, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, platelets, prothrombin time (Hepato Quick's test), alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, serum-gamma-glutamyltransferase, sodium, potassium, chloride, inorganic phosphate, calcium, urea, creatinine, glucose, total bilirubin, total protein, albumin, globulins, triglycerides, cholesterol, magnesium

Urinalysis: For urinalysis the individual animas were transferred to metabolism cages (withdrawal of food and water) and urine was collected overnight. The urine samples were evaluated in a randomized sequence. The following examinations were carried out: volume, colour, turbidity, nitrite, pH, protein, glucose, ketones, urobilinogen, bilirubin, blood, specific gravity, sediment.

Necropsy: The animals were sacrificed by decapitation under CO2 anesthesia. The exsanguinated animals were necropsied and assessed by gross pathology. With the exception of those female FO parental animas that were used for the re-evaluation of fertility, the following weight parameters of all
animals sacrificed at scheduled dates were determined: 1. anesthetized animals, 2. liver, 3. kidneys, 4. adrenal glands, 5. testes, 6. epididymides, 7. ovaries, 8. brain,

Histopathology: The following organs or tissues were fixed in 4% formaldehyde solution: 1. all gross lesions, 2. brain, 3. pituitary gland, 4. thyroid glands with parathyroid glands, 5. thymus, 6. trachea, 7. lungs, 8. heart, 9. aorta, 10. salivary glands (mandibular and sublingual glands), 11. liver, 12. spleen, 13. kidneys, 14. adrenal glands, 15. pancreas, 16. testes/ovaries, 17. uterus/vagina/oviducts, 18. epididymides, prostate, seminal vesicle, 19. skin, 20. esophagus, 21. stomach (glandular and non-glandular), 22. duodenum, jejunum, ileum, 23. cecum, colon, rectum, 24. urinary bladder, 25. mandibular and mesenteric lymph nodes, 26. female mammary gland, 27. skeletal muscle, 28. sciatic nerve, 29. sternum with sternal bone marrow, 30. bone marrow (femur), 31. eyes, 32. femur with knee joint, 33. spinal cord (cervical, thoracic and lumbar cord), 34. extraorbital lacrimal glands
Statistics:
Statistics of the clinical examinations, statistics of clinical pathology, statistics of pathology

Results and discussion

Results of examinations

Details on results:
Test group 3 (1,000 mg/kg body weight/day)
F0 parental animals:
CLINICAL EXAMINATIONS: no substance-related adverse effects.
CLINICAL PATHOLOGY: increase in leukocytes and polymorphonuclear neutrophils in both sexes.
ORGAN WEIGHTS: no substance-related adverse effects.
GROSS AND HISTOPATHOLOGICAL FINDINGS: grossly seen enlargement of mesenteric lymph nodes in five females; granulomatosis in the sinus of the mesenteric lymph nodes of all ten animals of either sex investigated histopathologically; increased number of focal lymphoid cell infiltrates (Kupffer cell granulomas) in the liver of male and female rats.

Test group 2 (300 mg/kg body weight/day)
F0 parental animals:
CLINICAL EXAMINATIONS: no substance-related adverse effects.
CLINICAL PATHOLOGY: increase in leukocytes and polymorphonuclear neutrophils in the females.
ORGAN WEIGHTS: no substance-related adverse effects.
GROSS AND HISTOPATHOLOGICAL FINDINGS: granulomatosis in the sinus of the mesenteric lymph nodes of all ten animals of either sex investigated histopathologically; increased number of focal lymphoid cell infiltrates (Kupffer cell granulomas) in the liver of female rats.

Test group 1 (100 mg/kg body weight/day)
F0 parental animals:
CLINICAL EXAMINATIONS/CLINICAL PATHOLOGY/ORGAN WE IGHTS: no substance-related adverse effects.
GROSS AND HISTOPATHOLOGICAL FINDINGS: granulomatosis in the sinus of the mesenteric lymph nodes of all ten animals of either sex investigated histopathologically.


Mortality
There were no mortalities in any of the F0 generation parental animals in any of the groups.

Clinical observations for males and females
No clinical signs which might be attributed to the test substance were detected in male or female F0 generation parental animais. The 3 doses (100, 300 and 1,000 mg/kg bw/d) administered by gavage did not lead to disturbances of the general behavior in any of the F0 parental animais.
Three males of the control group and one male each of the 100 mg/kgbw/d and 300 mg/kg bw/d) showed transient skin lesions in the region
of the neck on dif ferent study intervas. Another 100 mg/kg male rat had urine smeared fur from study week 17 until scheduled sacrifice, but
showed no unequivocal findings at necropsy or histopathology which could explain this finding.
After weaning of the F1 pups a vaginal prolapse occurred in one dam of the 300 mg/kg bw/d dose which made it impossible to reevaluate the fertility
of this female. One control female developed a cataract (right eye) on the last two weeks of the study period. Due to the isolated and disparate nature of the described clinical observations, these are considered to be spontaneous in nature.

Effect levels

open allclose all
Dose descriptor:
NOAEL
Effect level:
>= 1 000 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
other: no adverse effects observed
Dose descriptor:
NOEL
Effect level:
100 mg/kg bw/day (nominal)
Sex:
male/female

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

"Granulomatosis" of the sinus was refered to as proliferation of histiocytic cell elements in the marginal and intermdiate sinus in the mesenteric lymph node, forming granulomatous clusters and borad stands of phagocytes.

Applicant's summary and conclusion