A mixture of: 2,2',2'',2'''-(ethylenedinitrilotetrakis-N,N-di(C16)alkylacetamide; 2,2',2'',2'''-(ethylenedinitrilotetrakis-N,N-di(C18)alkylacetamide

Toxicity to reproduction

Administrative data

Endpoint:

one-generation reproductive toxicity

Remarks:

based on test type (migrated information)

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP and guideline study

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1996

Report date:

1996

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 415 [One-Generation Reproduction Toxicity Study (before 9 October 2017)]

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

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 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.

Details on mating procedure:

At least 70 days after the beginning of treatment, males and females from the same dose group were mated at a ratio of 1:1 for a maximum of 3 weeks. Generally, throughout the mating period, each male animal was mated with a predetermined female animal from the same dose group. Mating occurred by placing the female in the cage of a male from 4.00 p.m. until 7.00 - 9.00 a.m. the following morning. Deviations from the specified times were possible on weekends and public holidays and were reported in the raw data.
A vaginal smear was prepared after each mating and examined for sperm. If sperm was detected, pairing of the animals was discontinued. The day on which sperm were detected was denoted "day 0" and the following day "day 1" P.C. (post coitum).

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:

The study was terminated with the terminal sacrifice of the F1 weanlings and F0 adult animals.

Frequency of treatment:

once daily

Details on study schedule:

At least 70 days after the beginning of treatment, F0 animals were mated to produce one litter (F1).

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

Parental animals: Observations and examinations:

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.
Male reproduction data: The mating partners, the number of mating days until vaginal sperm could be detected in the female, and the gestational status of the female were noted for F0 breeding pairs.
Female reproduction and delivery data: The mating partners, the number of mating days until vaginal sperm could be detected, and gestational status were recorded for F0 females.

Oestrous cyclicity (parental animals):

see above

Sperm parameters (parental animals):

see above

Litter observations:

Pup number and status at delivery: All pups derived from the F0 parents (F1 litter) were examined as soon as possible on the day of birth to determine the total number of pups and the number of liveborn and stillborn members of each litter. Pups which died before the first determination of their status on the day of birth were designated as stillborn pups.
Pup viability/mortality: In general, a check was made for any dead or moribund pups twice daily on workdays (once in the morning and once in the afternoon) or as a rule, only in the morning on Saturdays, Sundays or public holidays. The number and percentage of dead pups on the day of birth (day 0) and of pups dying between days 1-4, 5-7, 8-14 and 15-21 of the lactation period were determined; however, pups which died accidentally or had to be sacrificed due to maternal death were not included in these calculations. The number of live pups/litter was calculated on the day of birth, and on lactation days 4, 7, 14 and 21.
Sex ratio: On the day of birth (day 0) the sex of the pups was determined by observing the distance between the anus and the base of the genital tubercle; normally, the anogenital distance is considerably greater in male than in female pups. Subsequently the sex of the pups was assessed by the external appearance of the anogenital region and/or the mammary line of the animals and was finally confirmed at necropsy.
Pup body weight data: The pups were weighed on the day after birth (day 1 p.p.) and on days 4 (before standardization), 7, 14 and 21 after birth. Pups' body weight change was calculated from these results. The individual weights were always determined at about the same time of the day (in the morning) and on day 4 p.p. immediately before standardization of the litters.
Pup clinical observations: All live pups were examined each day for clinical symptoms (including gross-morphological findings).

Postmortem examinations (parental animals):

The uteri of the females re-evaluated for fertility were examined at the reproduction toxicology laboratory for live and dead implantations. In the case of an apparently non-pregnant animal or of an empty uterus horn in the case of single-horn pregnancy, the uterus was stained with sodium sulfide and assessed for early implantations according to the method of SALEWSKI. Then the uteri were rinsed carefully under running water. After these examinations were completed, the uteri were transferred to the pathology lab for further fixation and evaluation.

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

Postmortem examinations (offspring):

Pup necropsy observations: All pups with scheduled sacrifice (i.e. pups which were culled on day 4 p.p. and pups which were sacrificed on day 21 after birth or subsequent days) were killed by means of CO2. These pups were examined externally and eviscerated, their organs were assessed macroscopically. If there were notable findings or if abnormalities were found in the daily clinical observation of the animals after their delivery, the affected animals were, if it was deemed necessary, examined additionally using appropriate methods (e.g., skeletal staining according to modified DAWSON's method (Dawson, A.B., 1926)) and/or further processing of head according to WILSON's method. All stillborn pups and all pups that died up to weaning were examined externally, eviscerated, and their organs assessed macroscopically. If there were notable findings or if abnormalities were found in the daily clinical observation of the animals after their delivery, the affected animals were, if it was deemed necessary, examined additionally using appropriate methods (e.g., skeletal staining according to modified DAWSON's method and/or further processing of head according to WILSON's method. The stained skeletons were evaluated under a stereomicroscope or a magnifying glass. All pups without any notable findings or abnormalities were discarded after their macroscopic evaluation.

Statistics:

Statistics of the clinical examinations, statistics of clinical pathology, statistics of pathology

Reproductive indices:

Male mating index (%), female mating index (%), male fertility index (%), female fertility index (%), gestation index (%), live birth index (%)

Offspring viability indices:

viability index (%), lactation index (%), sex ratio

Results and discussion

Results: P0 (first parental generation)

Details on results (P0)

Test group 3 (1,000 mg/kg body weight/day)
F0 parental animals:
no substance-related adverse effects

Test group 2 (300 mg/kg body weight/day)
F0 parental animals:
no substance-related adverse effects

Test group 1 (100 mg/kg body weight/day)
F0 parental animals:
no substance-related adverse effects

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.

Clinical observations for females during gestation
There were no particular substance-related clinical findings in F0 females during the gestation period for F1 litter. Insufficient nesting activity was
observed for several dams of in the control group and the low and mid dose test group, but not in any of the high dose dams; thus, this finding
occurred without any relation to dosing. Single females of the 100, 300 and 1,000 mg/kg bw/d groups did not become pregnant and consequently did not deliver; one high dose female rat, however, which did not deliver, proved to be pregnant by stain (resorptions in utero) later.

Clinical observations for females during lactation
No substance-related clinical findings were recorded for the F0 dams during the lactation period. After parturition one control dam and one dam
of the 300 mg/kg bw/d group did not cut the umbilical cords and did not consume the placentae.

Male reproduction data
For all F0 males which were placed with females to generate F1 pups mating was confirmed, thus the male mating index in all test groups was 100% (see table below). For nearly all F0 males fertility could be confirmed within the scheduled mating interval; two low dose, one mid dose and one high
dose male/males did not prove their fertility within the scheduled mating period. Thus, the male fertility index varied between 92% and 100%,
but did not show a clear relation to dosing (see table below). All F0 males for which fertility could not be proven within the scheduled mating phase, however, proved to be fertile within the reevaluation of fertility when mated anew with a control female with confirmed fertility .
Thus the fertility of the F0 parental males was not influenced by the oral administration of Keroflux ES 3241.

Female reproduction and delivery data
The female mating index calculated after the mating period for F1 litter was 100% for all groups. The mean duration until sperm was detected (day 0
p.c.) varied between 2.7 and 3.1 days without a clear relation to dosing. Several females of the treatment groups did not become pregnant within the scheduled mating interval. Therefore the fertility index varied between 92% and 100%.
The low dose females in question proved to be fertile after being mated again with control males (with confirmed fertility). According to the results of pathology no relevant gross lesions or microscopic findings were obtained that may explain the infertility of the impregnant high dose female. A reevaluation of the fertility of the impregnant mid dose female could not be performed due to the fact that this rat developed a marked vaginal prolapse. The occurrence of one infertile high dose female is regarded as an incidental finding.
The mean duration of gestation was similar in all groups and the gestation index did not show any biologically relevant differences between the groups (it varied between 96% and 100%; see table below). The mean number of pups delivered/dam was uninfluenced by the test substance administered. The number of liveborn and stillborn pups was comparable between the groups (see table below).
Thus, the administration of Keroflux ES 3241 by intubation did not adversely affect reproduction and delivery data of the F0 generation parental females.

Reevaluation of fertility
4 animals of the 100 mg/kg bw/d group (two males, two females), 2 animals of 300 mg/kg bw/d group (one male and one female) and 2 animals of the high dose group (one male and one female) had to be reevaluated for fertility. Fertility was confirmed for all of the above mentioned animals with exception of the 1,000 mg/kg female and the mid dose female. A reevaluation of fertility could not be performed for the latter female, because it developed a vaginal prolapse, which made further matings impossible.
Moreover, another high dose pair was supposed to be infertile and therefore remated with proven fertile animals. However, the female proved signs of implantations from the scheduled first mating period were seen in the uterus of female (i.e. pregnant by stain). Thus reevaluation of fertility for this dam and her first male mating partner were superfluous.

Effect levels (P0)

Dose descriptor:

NOAEL

Remarks:

reproduction

Effect level:

>= 1 000 mg/kg bw/day

Sex:

male/female

Results: F1 generation

Details on results (F1)

Test group 3 (1,000 mg/kg body weight/day):
F1 pups:
CLINICAL EXAMINATIONS: no substance-related adverse effects

Test group 2 (300 mg/kg body weight/day):
F1 pups:
CLINICAL EXAMINATIONS: no substance-related adverse effects.

Test group 1 (100 mg/kg body weight/day):
F1 pups:
CLINICAL EXAMINATIONS: no substance-related adverse effects.


Pup number and status of delivery
The mean numnber of delivered Fl pups/dam and the rate of liveborn and stillborn F1 pups were not influenced by the administration of the test substance. The observable differences are without any biological relevance; all values are fully within the historical control range.

Pup viability/ mortality
The number of F1 pups which died during the entire lactation period was similar in all groups and did not show any dose-response relationship. Consequently, viability and lactation indices were substantially similar between the treatment groups and the control group; the corresponding values are fully within the historical control range for rats of this strain. Thus, pup viability/mortality was not influenced by the oral administration of Keroflux ES 3241 to the lactating F0 dams.

Pup body weight data
Mean body weights and body weight gains of F1 pups were not adversely affected. Sporadically, statistically significantly increased pup body vieights/body weight gains occurred in the 300 and 1,000 mg/kg body weight/day treatment groups; however, as mean pup body weights on day 21 p.p. and pup weight gains (days 4 - 21 p.p.> were quite similar between the control and the pups of the substance treated groups (max. deviation 6%>, these differences are considered to be spontaneous in nature.

Pup clinical observations
The F1 generation pups did not show any clinical signs up to weaning which could be attributed to treatment.
Kinky tail was recorded for one control pup and two pups of low dose test group. Moreover, one pup of the
100 mg/kg group showed bilateral anophthalmia and hydrocephaly. All of these findings, for which no dose-response relationship
exists occur also occasionally in control pups of the rat strain used.

Behavioral tests
No remarkable differences between the groups were observed in the different behavioral tests which the pups underwent up to weaning.

Overall reproductive toxicity

Reproductive effects observed:

not specified

Any other information on results incl. tables

Reproductive indices
Treatment (mg/kg bw/d)	0 (control)	100	300	1000
Male mating index (%)	100	100	100	100
Male fertility index (%)	100	92	96	96
Treatment (mg/kg bw/d)	0 (control)	100	300	1000
Female mating index (%)	100	100	100	100
Female fertility index (%)	100	92	96	96
Gestation index (%)	100	100	100	96
Live Birth index (%)	92	98	94	95
Viability indices
Treatment (mg/kg bw/d)	0 (control)	100	300	1000
Viability index (%)	97	95	95	96
Lactation index (%)	98	99	99	99
Sex ratio: males (%; d0)	52	53	52	51

---

Conclusion

Due to the absence of respective effects in all treatments, the NOAEL (no observed adverse effect level) for reproductive function of the F0 parental rats and for reproductive toxicity of the F1 pups is at least 1000 mg/kg body weight/day in this study.