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

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Link to relevant study records

Reference

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:

guideline study

Reason / purpose for cross-reference:

reference to same study

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

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Karl THOMAE, Biberach an der Riss, FRG
- Age at study initiation: 35 (± 1) days
- Weight at study initiation: mean male 140 g; mean female 114.5 g
- Housing: housed individually in type DK III stainless steel wire mesh cages
- Diet (e.g. ad libitum): ad libitum; Kliba maintenance diet rat/
mouse/harnster, 343 meal, supplied by KLINGENTALMÜHLIE AG
- Water (e.g. ad libitum): ad libitum from waterbottles
- Acclimation period: 10 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24°C
- Humidity (%): 30-70 %
- Air changes (per hr): fully air-conditioned
- Photoperiod (hrs dark / hrs light): 12 h/12 h

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).

Details on mating procedure:

- M/F ratio per cage: 1/1
- Length of cohabitation: max. 3 weeks during the nights (4.00 p.m. until 7.00 - 9.00 a.m. the following morning)
- After 3 weeks of unsuccessful pairing replacement of first male by another male of the control group with proven fertility.
- Further matings after two unsuccessful attempts: no
- Proof of pregnancy: [sperm in vaginal smear] referred to as [day 0] of pregnancy
- After successful mating each pregnant female was caged (how): individually; after birth together with the litter

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

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. 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).

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:

DOSE SELECTION RATIONALE:
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:

-

Parental animals: Observations and examinations:

Food consumption: Food consumption of the F0 rats was determined once a week (each time for a period of 7 days). During the lactation period (animals with litter) food consumption was determined twice within the first week. 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. 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. 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. The littering behaviour of the dams was also inspected on weekdays (except holidays) in the afternoons in addition to the evaluations in the mornings. 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.

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.

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 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 within 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):

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: anesthetized animals, liver, kidneys, adrenal glands, testes, epididymides, ovaries, brain,

Histopathology: The following organs or tissues were fixed in 4% formaldehyde solution: all gross lesions, brain, pituitary gland, thyroid glands with parathyroid glands, thymus, trachea, lungs, heart, aorta, salivary glands (mandibular and sublingual glands), liver, spleen, kidneys, adrenal glands, pancreas, testes/ovaries, uterus/vagina/oviducts, epididymides, prostate, seminal vesicle, skin, esophagus, stomach (glandular and non-glandular), duodenum, jejunum, ileum, cecum, colon, rectum, urinary bladder, mandibular and mesenteric lymph nodes, female mammary gland, skeletal muscle, sciatic nerve, sternum with sternal bone marrow, bone marrow (femur), eyes, femur with knee joint, spinal cord (cervical, thoracic and lumbar cord), extraorbital lacrimal glands

Postmortem examinations (offspring):

Pup necropsy observations: All pups with scheduled sacrifice were killed by means of CO2. All pups (including the stillborn ones) 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 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

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.

Clinical observations for females during gestation
There were no particular substance-related clinical findings in F0 females during the gestation period for F1 litter.

Clinical observations for females during lactation
No substance-related clinical findings were recorded for the F0 dams during the lactation period.

Male reproduction data
The male mating index in all test groups was 100% . The male fertility index varied between 92% and 100%, but did not show a clear relation to dosing. 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 a new 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% but did not show a dose relationship.
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%). 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.
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
In total 8 animals distributed through all dose groups had to be reevaluated for fertility. Fertility was confirmed for all of the above mentioned animals with exception of 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.

Dose descriptor:

NOAEL

Remarks:

reproduction

Effect level:

>= 1 000 mg/kg bw/day

Sex:

male/female

Basis for effect level:

other: 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.

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 number 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 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.

Pup clinical observations
The F1 generation pups did not show any clinical signs up to weaning which could be attributed to treatment.

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

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

> 1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: 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.

Reproductive effects observed:

not specified

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

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Species:

rat

Additional information

Oral

In a 1-generation study conforming to GLP requirements and to OECD test guideline 415, KEROFLUX ES 3241 was administered as an oily emulsion/solution to groups of 25 male and 25 female sexually immature Wistar rats (F0 parental generation) by stomach tube at dosages of 100, 300 or 1000 mg/kg bw/day continuously throughout the whole study period. A standard dose volume of 5 ml/kg bw was used. The control group, consisting of 25 males/25 females was dosed with the vehicle only (olive oil DAB 10). At least 70 days after the beginning of treatment, F0 animals were mated to produce one litter (F1). Mating pairs were from the same dose group. The study was terminated with the terminal sacrifice of the F1 weanlings and F0 adult animals.

Food consumption of the F0 parents was determined regularly during premating (once weekly), and additionally during gestation and lactation periods. In general, body weights of F0 parents were determined once weekly. However, during gestation and lactation F0 females were weighed on days 0, 7, 14 and 20 of gestation, on the day of parturition, and on days 1, 4, 7, 14 and 21 after birth.

The F1 pups were weighed on the day after birth and on days 4, 7, 14 and 21 post partum. The parents' and the pups' state of health was checked each day, and parental animals were examined for their mating and reproductive performances. Pups were sexed and monitored with respect to their development stages and their behavior was assessed in certain tests. Their viability was recorded. All pups were exainined macroscopically at necropsy; if necessary, certain pups were additionally inspected for any organ/skeletal findings.

Blood and urine samples were taken from 10 F0 animals per sex of each test group towards the end of the study period for clinical pathology examinations. All F0 parental animals were assessed by gross pathology (including weight determinations of several organs) and subjected to an extensive histopathological examination, special attention being paid to the organs of the reproductive system.

The oral administration of KEROFLUX ES 3241 to male and female rats in this one generation reproduction toxicity study had no adverse effects on reproductive parameters of the F0 parental animals and F1 pups of all groups (100, 300 and 1000 mg/kg bw/day) and did not induce any signs of developmental toxicity in the F1 pups.

Treatment-related systemic toxicity effects, however, were observed in the parental (F0) rats. These were considered as not adverse. For details, see the respective robust study summary and endpoint summary in IUCLID chapter 7.5.1.

The NOAELs (no observed adverse effect levels) for reproductive function of the F0 parental rats and F1 pups and for developmental toxicity of the F1 pups is considered to be >= 1000 mg/kg bw/day.

Short description of key information:
oral
1-generation study rat (GLP, OECD 415, BASF 1996)
NOAEL >1000mg/kg bw/day for reproductive function (F0 and F1) and developmental toxicity (F1)

Effects on developmental toxicity

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 415 (One-Generation Reproduction Toxicity Study)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Karl THOMAE, Biberach an der Riss, FRG
- Age at study initiation: 35 (± 1) days
- Weight at study initiation: mean male 140 g; mean female 114.5 g
- Housing: housed individually in type DK III stainless steel wire mesh cages
- Diet (e.g. ad libitum): ad libitum; Kliba maintenance diet rat/
mouse/harnster, 343 meal, supplied by KLINGENTALMÜHLIE AG
- Water (e.g. ad libitum): ad libitum from waterbottles
- Acclimation period: 10 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24°C
- Humidity (%): 30-70 %
- Air changes (per hr): fully air-conditioned
- Photoperiod (hrs dark / hrs light): 12 h/12 h

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).

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

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. The content of KEROFLUX ES 3241 in the test substance preparations was determined by titration of functionalized nitrogen by trifluoromethane sulfonic acid.

Details on mating procedure:

- M/F ratio per cage: 1/1
- Length of cohabitation: max. 3 weeks during the nights (4.00 p.m. until 7.00 - 9.00 a.m. the following morning)
- After 3 weeks of unsuccessful pairing replacement of first male by another male of the control group with proven fertility.
- Further matings after two unsuccessful attempts: no
- Proof of pregnancy: [sperm in vaginal smear] referred to as [day 0] of pregnancy
- After successful mating each pregnant female was caged (how): individually; after birth together with the litter

Duration of treatment / exposure:

The administration of the test substance started at least 70 days before mating and continued until day 21 after parturition of the F1 pups. The study was terminated with the terminal sacrifice of the F1 weanlings and F0 adult animals.

Frequency of treatment:

continuously throughout the whole study period .

Duration of test:

ca. 5 month (21 weeks)

No. of animals per sex per dose:

25

Control animals:

yes, concurrent vehicle

Details on study design:

DOSE SELECTION RATIONALE:
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.

Maternal examinations:

Food consumption: Food consumption of the F0 rats was determined once a week (each time for a period of 7 days). During the lactation period (animals with litter) food consumption was determined twice within the first week. 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. 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. 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. The littering behaviour of the dams was also inspected on weekdays (except holidays) in the afternoons in addition to the evaluations in the mornings. 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.

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.

Ovaries and uterine content:

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. After these examinations were completed, the uteri were transferred to the pathology lab for further fixation and evaluation.

Fetal examinations:

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 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 within 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).

Pup necropsy observations: All pups with scheduled sacrifice were killed by means of CO2. All pups (including the stillborn ones) 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 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

Indices:

Gestation index (%), live birth index (%), viability index (%), lactation index (%), sex ratio

Historical control data:

Historical control data is included in the study report.

Details on maternal toxic effects:

Maternal toxic effects:no effects

Details on maternal toxic effects:
no adverse effects observed; for details see study results described in IUCLID chapter 7.5.1

Dose descriptor:

NOAEL

Effect level:

>= 1 000 mg/kg bw/day (nominal)

Basis for effect level:

other: developmental toxicity

Dose descriptor:

NOAEL

Effect level:

>= 1 000 mg/kg bw/day (nominal)

Basis for effect level:

other: maternal toxicity

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic 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
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.

Pup clinical observations
The F1 generation pups did not show any clinical signs up to weaning which could be attributed to treatment.

Development stages
There were no biologically relevant differences between the control and the substance-treated F1 pups in the several morphological development stages monitored up to weaning.

Pup necropsy observations
Pup necropsy findings (e.g. incisors sloped, anophthalmia, hernia diaphragmatica, dilated renal pelvis, agenesia of kidney(s), ureter(s), uterine horn (s), hydrocephaly and kinky tail) appeared only in very few of the pups examined. The isolated nature of the pup necropsy observations does not suggest any relationship to treatment.

Key result

Dose descriptor:

NOAEL

Effect level:

> 1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Developmental; No signs of developmental toxicity observed.

Abnormalities:

not specified

Developmental effects observed:

not specified

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

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Species:

rat

Justification for classification or non-classification

No signs of toxicity to reproduction or development were observed in the rat after oral dosage up to the limit concentration of 1000 mg/kg bw/d. Therefore, a classification is not warranted.