Registration Dossier

Diss Factsheets

Administrative data

Endpoint:
repeated dose toxicity: oral, other
Remarks:
extended one-generation reproductive toxicity study
Type of information:
experimental study
Adequacy of study:
key study
Study period:
july 2019 - november 2020
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-reference
Reason / purpose for cross-reference:
reference to same study
Reference
Endpoint:
extended one-generation reproductive toxicity - basic test design (Cohorts 1A, and 1B without extension)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
july 2019 - november 2020
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 443 (Extended One-Generation Reproductive Toxicity Study)
Version / remarks:
25 Jun 2018
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
Landesamt für Umwelt, Kaiser-Friedrich-Straße 7, 55116 Mainz
Limit test:
no
Justification for study design:
SPECIFICATION OF STUDY DESIGN FOR EXTENDED ONE-GENERATION REPRODUCTION TOXICITY STUDY WITH JUSTIFICATIONS [please address all points below]:

- Test design: was administered to groups of 25 male and 25 female healthy young Wistar rats as a homogeneous addition to the food in different concentrations (0, 1250, 3750 and 12500 ppm). F0 animals were treated at least for 10 weeks prior to mating to produce a litter (F1 generation). Mating pairs were from the same dose group. Pups of the F1 litter were selected (F1 rearing animals) and assigned to 2 different cohorts (1A and 1B) which were subjected to specific postweaning examinations. The study was terminated with the terminal sacrifice of the F1 rearing animals of cohort 1B. Test diets containing 2-(2-aminoethoxy)ethanol were offered continuously throughout the study.
- Premating exposure duration for parental (P0) animals: at least .10 weeks.
- Basis for dose level selection: OECD Guideline 443.
- Inclusion/exclusion of extension of Cohort 1B: Yes.
- Inclusion/exclusion of developmental neurotoxicity Cohorts 2A and 2B: No.
- Inclusion/exclusion of developmental immunotoxicity Cohort 3: No.
- Route of administration: oral, feed.
Specific details on test material used for the study:
TEST MATERIAL
- CAS No.: 929-06-6
- Batch identification: 22057368E0
- Purity: 99.4 area % corrected with the water content
- Homogeneity: Given (visually)
- Storage stability: Expiry date: 06 Apr 2021. The stability of the test substance under storage conditions over the test period was guaranteed by the sponsor, and the sponsor holds this responsibility.
- Physical state/Appearance: Liquid / colorless clear
- Storage conditions: Room temperature; under N2

Species:
rat
Strain:
Wistar
Details on species / strain selection:
Crl:WI(Han)
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories
- Females nulliparous and non-pregnant: yes
- Age at study initiation: (P) 35 (±1) days
- Weight at study initiation: (P) Males: means 121.9 - 122.3 g; Females: means 97.9 - 99.5 g
- Fasting period before study: no.
- Fasting period before blood sampling: 16 hours.
- Housing: During the study period, the rats were housed together in Polysulfonate cages supplied by TECHNIPLAST, Hohenpeißenberg, Germany and Becker & Co., Castrop-Rauxel, Germany, with the following exceptions: During overnight matings, male and female mating partners were housed together in Polycarbonate cages type III (supplied by TECHNIPLAST, Hohenpeißenberg, Germany and Becker & Co., Castrop-Rauxel, Germany). Dams and their litters were housed together until PND 21 in Polycarbonate cages type III. Females after weaning were housed individually in Polycarbonate cages type III.
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 8 days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24°C
- Humidity (%): 45 - 65%
- Air changes (per hr): 15 times per hour.
- Photoperiod (hrs dark / hrs light): 12 hours (12 hours light from 6.00 h to 18.00 h and 12 hours darkness from 18.00 h to 06.00 h).
Route of administration:
oral: feed
Details on exposure:
DIET PREPARATION
The required quantity of test substance was weighed in a beaker depending on the dose group and thoroughly mixed with a small amount of food. Then further amounts of food were added to this premix and thoroughly mixed for 3 minutes. Afterwards, further amounts of food, depending on the dose group, were added to this premix in order to obtain the desired concentrations. Mixing of this final mix was carried out for about 10 minutes in a laboratory mixer.
Analytical verification of doses or concentrations:
yes
Remarks:
The observed concentrations of the test substance correspond with the expected concentrations by recoveries between 90 and 96%, and demonstrated the correctness of the diet preparations
Duration of treatment / exposure:
P: 132 days,
F1 A: 66 days,
F1 B: 60 days
Frequency of treatment:
daily
Dose / conc.:
1 250 ppm
Remarks:
113 mg/kg bw/d
Dose / conc.:
3 750 ppm
Remarks:
340 mg/kg bw/d
Dose / conc.:
12 500 ppm
Remarks:
1129 mg/kg bw/d
No. of animals per sex per dose:
25 per sex and dose
Control animals:
yes, concurrent vehicle
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes.
- Time schedule: at least once daily.
- Mortality: A check for moribund or dead animals was made twice daily on working days or once daily (Saturday, Sunday or on public holidays). If animals were in a moribund state, they were sacrificed and necropsied.
- Clinical observations: A cageside examination was conducted at least once daily for any signs of morbidity, pertinent behavioral changes and signs of overt toxicity. Abnormalities and changes were documented daily for each animal and reported. The parturition and lactation behavior of the dams was generally evaluated in the mornings in combination with the daily clinical inspection of the dams. Only particular findings (e.g. disability to deliver) were documented on an individual dam basis. On weekdays (except Saturday, Sunday and public holidays) the parturition behavior of the dams was inspected in the afternoons in addition to the evaluations in the mornings. The day of parturition was considered the 24-hour period from about 15.00 h of one day until about 15.00 h of the following day.


DETAILED CLINICAL OBSERVATIONS: Yes.
- Detailed clinical observations (DCO) were performed in all F0 parental animals once before the administration and supsequently once per week and in cohorts 1A and 1B at weekly intervals during the administration period. The examinations started in the morning. The findings were ranked according to the degree of severity, if applicable.
- Parameters assessed: Abnormal behavior in handling, Fur, Skin, Posture, Salivation, Respiration, Activity/arousal level, Tremors, Convulsions, Abnormal movements, Gait abnormalities, Lacrimation, Palpebral closure, Exophthalmos (Protruding eyeball), Assessment of the feces excreted during the examination (appearance/consistency), Assessment of the urine excreted during the examination, Pupil size.


BODY WEIGHT: Yes.
- In general, the body weight of the male and female F0 parental animals and F1 rearing animals was determined once a week at the same time of the day (in the morning), with the following exceptions. During pregnancy, body weight of the F0 females with evidence of sperm was determined weekly for GD 0, 7, 14 and 20. During lactation, body weight of the F0 and F1 females, which gave birth to a litter was determined for PND 1, 4, 7, 10, 14, 18 and 21. The body weight change of the animals was calculated from these results. Body weight was not determined in the F0 females without positive evidence of sperm during mating and gestation periods, in the females without litter during lactation period and in the females after weaning.


FOOD CONSUMPTION AND COMPOUND INTAKE: Yes.
- Food consumption: Generally, food consumption was determined once a week for male and female F0 parental animals and F1 rearing animals, with the following exceptions. Food consumption was not determined after the 10th premating week (male F0 animals) and during the mating period (male and female F0 parental animals). During pregnancy, food consumption of the F0 females with evidence of sperm was determined weekly for GD 0-7, 7-14 and 14-20. During lactation, food consumption of the F0 females, which gave birth to a litter was determined for PND 1-4, 4-7, 7-10, 10-14, 14-18 and 18-21. Food consumption was not determined in the F0 females without positive evidence of sperm during mating and gestation periods, in the females without litter during lactation period and in females after weaning.
- Intake of test substance: The intake of test substance was calculated from the amount of food consumed and expressed in mg/kg body weight per day (mg/kg bw/d). The calculation of the group values/day was carried out according to the following formula:
ITx = FCx x C/ BWy
with
ITx = Intake of test substance on day x in mg/kg bw/d
FCx = Daily food consumption on day x in grams
C = Concentration in ppm
BWy = Body weight on day y in grams (last weighing before day x)
- The values are group means determined from daily intakes of test substance by the individual animals. The means represent interpolated values from the beginning and end of each respective test week.
- Additionally, a weighted mean of mean over all mean substance intakes throughout all study phases, across all cohorts, and both sexes are calculated considering the different time period of phases.


MALE REPRODUCTION DATA
- The pairing partners, the number of mating days until vaginal sperm was detected in the female animals, and the gestational status of the females were recorded for F0 breeding pairs.
- For the males, mating and fertility indices were calculated for F1 litters according to the following formulas:

Male mating index (%) = number of males with confirmed mating* x 100 / number of males placed with females

* defined by a female with vaginal sperm or with implants in utero

Male fertility index (%) = number of males proving their fertility* x 100 / number of males placed with females

* defined by a female with implants in utero


FEMALE REPRODUCTION AND DELIVERY DATA
- The pairing partners, the number of mating days until vaginal sperm were detected, and gestational status were recorded for F0 females.
- For the females, mating, fertility and gestation indices were calculated for F1 litters according to the following formulas:

Female mating index (%) = number of females mated* x 100 / number of females placed with males

* defined as the number of females with vaginal sperm or with implants in utero

Female fertility index (%) = number of females pregnant* x 100 / number of females mated**

* defined as the number of females with implants in utero
** defined as the number of females with vaginal sperm or with implants in utero

Gestation index (%) = number of females with live pubs on the day of birth* x 100 / number of females pregnant*

* defined as the number of females with implants in utero

The total number of pups delivered and the number of liveborn and stillborn pups were noted, and the live birth index was calculated for F1 litters according to the following formula:

Live birth index (%) = number of liveborn pubs at birth* x 100 / total number of pubs born

The implantations were counted2 and the postimplantation loss (in %) was calculated according the following formula:

Postimplantation loss (%) =(number of implantations – number of pups delivered)* x 100 / number of implantations


CLINICAL PATHOLOGY IN F0 PARENTAL ANIMALS
- Samples were withdrawn from 10 F0 parental males and females per group at
termination.
- Blood samples were taken from animals by puncturing the retrobulbar venous plexus following isoflurane anesthesia.
- Blood sampling and blood examinations were carried out in a randomized sequence. The list of randomization instructions was compiled with a computer.
- In the afternoon preceding the day of urinalysis, the animals were individually transferred into metabolism cages (no food or drinking water provided); on the following morning, the individual urine specimens were examined in a randomized sequence (the list of randomization instructions was compiled with a computer).
- The assays of blood and serum parameters were performed under internal laboratory quality control conditions with reference controls to assure reliable test results.
- The results of clinical pathology examinations were expressed in International System (SI) units. The following parameters will be examined:


HEMATOLOGY: Yes
- Parameters listed in "Any other information on materials and methods incl. tables"


CLINICAL CHEMISTRY: Yes
- Parameters listed in "Any other information on materials and methods incl. tables"


HORMONES: Yes
- Parameters listed in "Any other information on materials and methods incl. tables"


URINANALYSIS: Yes
- Parameters listed in "Any other information on materials and methods incl. tables"


SPERM PARAMETERS: Yes
- After the organ weight determination, the following parameters were determined in the right testis or right epididymis of all male F0 parental animals and all cohort 1A males sacrificed on schedule
- Sperm parameter investigations were carried out in a randomized sequence.
- Parameters listed in "Any other information on materials and methods incl. tables"
Oestrous cyclicity (parental animals):
Estrous cycle length was evaluated by daily analysis of vaginal smear for all F0 female parental rats for a minimum of 3 weeks prior to mating. Determination was continued throughout the pairing period until the female exhibited evidence of copulation.

In all cohort 1A females, vaginal smears were collected after vaginal opening until the first cornified smear (estrous) was recorded. The estrous cycle also was evaluated in cohort 1A and 1B females for 2 weeks around PND 75.

At necropsy, an additional vaginal smear was examined to determine the stage of estrous cycle for each F0 female and cohort 1A and 1B female with scheduled sacrifice.
Sperm parameters (parental animals):
Various sperm parameters (motility, sperm head count, morphology) were assessed in the F0 generation males at scheduled sacrifice or after appropriate staining
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes.


PUB NUMBER AND STATUS OF DELIVERY
- All pups delivered from the F0 parents (F1 litter) were examined as soon as possible on the day of birth to determine the total number of pups, the sex and the number of liveborn and stillborn pups in each litter.
- At the same time, the pups were also being examined for macroscopically evident changes.
- Pups, which died before this initial examination, were defined 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.
- Dead pups were evaluated via necropsy.
- The number and percentage of dead pups on the day of birth (PND 0) and of pups dying between PND 1-4, 5-7, 8-14 and 15-21 (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 after birth, and on lactation days 4, 7, 14, and 21.
- Furthermore, viability and lactation indices were calculated according to the following formulas:

Viability index (%) = number of live pubs on day 4* after birth x 100 / number of live pubs on day of birth

* before standardization of litters (i.e. before culling)

Lactation index (%) = number of live pups on day 21 after birth x 100 / nuSmber of live pups on day 4* after birth

* after standardization of litters (i.e. after culling)


SEX RATIO
- On the day of birth (PND 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.
- Later, during the course of lactation, this initial sex determination was followed up by surveying the external appearance of the anogenital region and the mammary line. The sex of the pups was finally confirmed at necropsy.
- The sex ratio was calculated at PND 0 and PND 21 according to the following formula:

Sex ratio = number of live male or female pups on PND 0 and 21 * 100 / number of live male and female pups on PND 0 and 21


PUP CLINICAL OBSERVATION
- The live pups were examined daily for clinical symptoms (including gross-morphological findings) during the clinical inspection of the dams. If pups showed particular findings, these were documented with the dam concerned.


NIPPLE/AEROLA ANLAGEN
- All surviving male pups were examined for the presence of nipple/areola anlagen on PND 13 and were re-examined on PND 20.
- The number of nipple/areola anlagen were counted.


PUP BODY WEIGHT DATA
- The pups were weighed on the day after birth (PND 1) and on PND 4 (before standardization), 7, 14 and 21.
- 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 PND 4 immediately before standardization of the litters.


ANOGENITAL DISTANCE
- Anogenital distance (AGD) is defined as the distance from the center of the anal opening to the base of the genital tubercle. AGD was determined in all live male and female pups on PND 1.
- These measurements were performed in randomized order, using a measuring ocular.
- They were conducted by technicians unaware of treatment group in order to minimize bias.


ANOGENITAL INDEX
- The anogenital index was calculated according to the following formula:

Anogenital index = anogenital distance [mm] / cubic root of pub weight [g]


PUP NECROPSY OBSERVATIONS
- On PND 4, as a result of standardization, all surplus F1 pups were sacrificed by decapitation under isoflurane anesthesia and blood was sampled for determination of serum thyroid hormone concentrations.
- After sacrifice, these pups were examined externally, eviscerated and their organs were assessed macroscopically.
- On PND 22, the surplus F1 generation pups that were not used for the formation of the cohorts or any investigations were sacrificed under isoflurane anesthesia with CO2 and were examined in the pathology lab.
- The selected pups for hormone analyses were sacrificed by decapitation under isoflurane anesthesia in the pathology lab and blood was sampled for thyroid
hormone analyses.
- Pups showing clinical symptoms or gross-morphological findings were examined using appropriate methods. Organs/tissues with gross morphological findings were preserved in a suitable manner for potential histopathological examination.
- All F1 pups which were not used for other purposes without any notable findings were discarded after their macroscopic evaluation.


PREMATURELY DEAD OR SACRIFICED PUPS
- Pups that died or were sacrificed in a moribund state were eviscerated and examined for possible defects and/or the cause of death using appropriate methods. These animals were preserved for this purpose, if necessary.


SEXUAL MATURATION: VAGINAL OPENING
- All female F1 pups selected to become the F1 rearing animals (cohort 1A and 1B) were evaluated daily for vaginal patency beginning on PND 27.
- On the day of vaginal opening the body weights of the respective animals were determined.


SEXUAL MATURATION: PREPUTIAL SEPARATION
- All male F1 pups selected to become the F1 rearing animals (cohort 1A and 1B) were evaluated daily for preputial separation beginning on PND 38.
- On the day of preputial separation the body weights of the respective animals were determined.


CLINICAL PATHOLOGY IN F0 PARENTAL ANIMALS
- Samples were withdrawn from 10 cohort 1A males and females per group at
termination.
- Blood samples were taken from animals by puncturing the retrobulbar venous plexus following isoflurane anesthesia.
- Blood sampling and blood examinations were carried out in a randomized sequence. The list of randomization instructions was compiled with a computer.
- In the afternoon preceding the day of urinalysis, the animals were individually transferred into metabolism cages (no food or drinking water provided); on the following morning, the individual urine specimens were examined in a randomized sequence (the list of randomization instructions was compiled with a computer).
- The assays of blood and serum parameters were performed under internal laboratory quality control conditions with reference controls to assure reliable test results.
- The results of clinical pathology examinations were expressed in International System (SI) units. The following parameters will be examined:


HEMATOLOGY: Yes
- Parameters listed in "Any other information on materials and methods incl. tables"


CLINICAL CHEMISTRY: Yes
- Parameters listed in "Any other information on materials and methods incl. tables"


HORMONES: Yes
- Parameters listed in "Any other information on materials and methods incl. tables"


URINANALYSIS: Yes
- Parameters listed in "Any other information on materials and methods incl. tables"


SPERM PARAMETERS: Yes
- After the organ weight determination, the following parameters were determined in the right testis or right epididymis of all male F0 parental animals and all cohort 1A males sacrificed on schedule
- Sperm parameter investigations were carried out in a randomized sequence.
- Parameters listed in "Any other information on materials and methods incl. tables"

HORMONES IN PND 4 AND 22 F1-OFFSPRING: BLOOD SAMPLING
- Blood samples were withdrawn from 10 surplus (culled) PND 4 offspring (as far as possible of different litters) per sex and group.
- PND 4 samples were pooled per sex and litter if the available amount is not sufficient for a hormone analysis.
- Blood samples were withdrawn from 10 surplus PND 22 offspring (as far as possible of different litters) per sex and group.
- The blood samples were collected after decapitation (following isoflurane anesthesia) from the Vena cava cranialis.


HORMONES IN PND 4 AND 22 F1-OFFSPRING: HORMONES
- The concentrations of TSH were determined by radioimmunoassay (RIA), using commercially available RIA test kits and a Gamma-Counter (LB 2111, Berthold, Germany).
- T4 ELISA was measured with a Sunrise MTP-reader, Tecan AG, Maennedorf, Switzerland, and evaluated with the Magellan-Software of the instrument producer.
- Parameters listed in "Any other information on materials and methods incl. tables"
Postmortem examinations (parental animals):
NECROPSY
- All F0 generation parental animals were sacrificed by decapitation under isoflurane anesthesia.
- The exsanguinated animals were necropsied and assessed by gross pathology; special attention being given to the reproductive organs.
- Moribund animals were sacrificed, necropsied and assessed by gross pathology as soon as possible after their death.


ORGAN WEIGHTS
- The following weights were determined in all animals sacrificed on schedule: Anesthetized animals (final body weight), Adrenal glands (fixed), Brain, Cauda epididymis, Epididymides, Heart, Kidneys, Liver, Ovaries, Pituitary gland (fixed), Prostate (ventral and dorsolateral part together, fixed), Testes, Seminal vesicles including coagulating glands (fixed), Spleen, Thymus (fixed), Thyroid glands (with parathyroid glands) (fixed), Uterus with cervix.
- All paired organs were weighed together (left and right).


ORGAN TISSUE FIXATION
- The following organs or tissues were fixed in 4% neutral-buffered formaldehyde solution or in modified Davidson’s solution: All gross lesions, Adrenal glands, Bone marrow (femur), Brain, Cecum, Cervix, Coagulating glands, Colon, Duodenum, Epididymis, left (fixed in modified Davidson´s solution), Esophagus, Eyes with optic nerve (fixed in modified Davidson’s solution), Heart, Ileum, Jejunum (with Peyer’s patches), Kidneys, Liver, Lungs, Lymph nodes/axillary, Lymph nodes/mesenteric, Mammary gland (male and female), Ovaries (fixed in modified Davidson´s solution), Oviducts, Pancreas, Pituitary gland, Prostate, Rectum, Sciatic nerve, Seminal vesicles, Skeletal muscle, Spinal cord (cervical, thoracic and lumbar cord), Spleen, Stomach (forestomach and glandular stomach), Testis, left (fixed in modified Davidson ´s solution), Thymus, Thyroid glands (with parathyroid glands), Trachea, Urinary bladder, Uterus, Vagina, Vas deferens.
- The ovaries and eyes with optic nerve of animals that were sacrificed intercurrently were fixed in 4% neutral buffered formaldehyde solution.
- The left testis and left epididymis of all male F0 parental animals sacrificed at scheduled dates were fixed in modified Davidson’s solution, whereas the right testis and epididymis were used for sperm parameters analysis.
- For technical reasons, after about 24 hours fixation the ovaries of all F0 females of all test groups were transferred to 70% ethanol.
- The uteri of all cohabited female F0 generation parental animals were examined for the presence and number of implantation sites. The uteri of apparently nonpregnant animals or empty uterus horns were placed in 1% ammonium sulfide solutions for about 5 minutes in order to be able to identify early resorptions or implantations. Then the uteri were rinsed carefully in physiologic salt solution (0.9% NaCl). When the examinations were completed, the uteri were transferred to the Pathology Laboratory for further processing.


HISTOPATHOLOGY
- Organs listed in "Any other information on materials and methods incl. tables"
- The organs were trimmed according to the “Revised guides for organ sampling and trimming in rats and mice” (Ruehl-Fehlert et al., 2003; Kittel et al., 2004; Morawietz et al., 2004).
- The kidneys of males No. 10, 80 and 87 were exemplarily stained immunohistochemically with an antibody against alpha-2u microglobulin.
- For technical reasons, the left testis, left epididymis, and the eyes with optic nerves of all animals of the F0 parental control and high dose groups sacrificed at scheduled dates were embedded in paraplast after fixation. For the same reason, the ovaries of all F0 females in all test groups were embedded in paraplast.
- A correlation between gross lesions and histopathological findings was attempted. Special attention was given to stages of spermatogenesis in the male gonads.
- Special attention was also given to the synchrony of the morphology in ovaries, uterus, cervix, and vagina to the estrous cycle status. Whenever in the ovary the diagnosis: ”no abnormalities detected” was used, that implies that all different, stages of functional bodies (especially corpora lutea) were present and normal.
- Animals No. 119 and 194 that were sacrificed in a moribund state were processed histotechnically and assessed like control animals.
- Reproductive organs of all F0 animals suspected of reduced fertility (maited pairs Nos. 148/48 and 159/59) were subjected to histopathological investigation.
Postmortem examinations (offspring):
PATHOLOGICAL EXAMINATIONS OF F1 GENERATION, REARING ANIMALS, COHORT 1A

NECROPSY
- All F1 generation, rearing animals, cohort 1A animals were sacrificed by decapitation under isoflurane anesthesia.
- The exsanguinated animals were necropsied and assessed by gross pathology; special attention being given to the reproductive organs.
- Moribund animals were sacrificed, necropsied and assessed by gross pathology as soon as possible after their death.

ORGAN WEIGHTS
- The following weights were determined in all animals sacrificed on schedule: Anesthetized animals (final body weight), Adrenal glands (fixed), Brain, Cauda epididymis, Epididymides, Heart, Kidneys, Liver, Lymph nodes, axillary (10 animals per sex per group, cohort 1A animals only), Lymph nodes, mesenteric (10 animals per sex per group, cohort 1A animals only), Ovaries, Pituitary gland (fixed), Prostate (ventral and dorsolateral part together, fixed), Testes, Seminal vesicles including coagulating glands (fixed), Spleen, Thymus (fixed), Thyroid glands (with parathyroid glands) (fixed), Uterus with cervix
- All paired organs were weighed together (left and right).

ORGAN TISSUE FIXATION
- The following organs or tissues were fixed in 4% neutral-buffered formaldehyde solution or in modified Davidson’s solution: All gross lesions, Adrenal glands, Bone marrow (femur), Brain, Cecum, Cervix, Coagulating glands, Colon, Duodenum, Epididymis, left (fixed in modified Davidson´s solution), Esophagus, Eyes with optic nerve (fixed in modified Davidson’s solution), Heart, Ileum, Jejunum (with Peyer’s patches), Kidneys, Liver, Lungs, Lymph nodes/axillary, Lymph nodes/mesenteric, Mammary gland (male and female), Ovaries (fixed in modified Davidson´s solution), Oviducts, Pancreas, Pituitary gland, Prostate, Rectum, Sciatic nerve, Seminal vesicles, Skeletal muscle, Spinal cord (cervical, thoracic and lumbar cord), Spleen, Stomach (forestomach and glandular stomach), Testis, left (fixed in modified Davidson ´s solution), Thymus, Thyroid glands (with parathyroid glands), Trachea, Urinary bladder, Uterus, Vagina, Vas deferens.
- The ovaries and eyes with optic nerve of animals that were sacrificed intercurrently were fixed in 4% neutral buffered formaldehyde solution.
- The left testis and left epididymis of all male cohort 1A animals sacrificed at scheduled dates were fixed in modified Davidson’s solution, whereas the right testis and epididymis were used for sperm parameters analysis.
- For technical reasons, after about 24 hours fixation the ovaries of all cohort 1A females of all test groups were transferred to 70% ethanol.

HISTOPATHOLOGY
- Organs listed in "Any other information on materials and methods incl. tables"
- The organs were trimmed according to the “Revised guides for organ sampling and trimming in rats and mice” (Ruehl-Fehlert et al., 2003; Kittel et al., 2004; Morawietz et al., 2004).
- The kidneys of males No. 10, 80 and 87 were exemplarily stained immunohistochemically with an antibody against alpha-2u microglobulin.
- For technical reasons, the ovaries of all animals of the cohort 1A females and high dose groups sacrificed at scheduled dates were embedded in paraplast after fixation.
- A correlation between gross lesions and histopathological findings was attempted. Special attention was given to stages of spermatogenesis in the male gonads.
- Special attention was also given to the synchrony of the morphology in ovaries, uterus, cervix, and vagina to the estrous cycle status. Whenever in the ovary the diagnosis: ”no abnormalities detected” was used, that implies that all different, stages of functional bodies (especially corpora lutea) were present and normal.
- Animals No. 119 and 194 that were sacrificed in a moribund state were processed histotechnically and assessed like control animals.

DIFFERENTIAL OVARIAN FOLLICLE COUNT IN COHORT 1A FEMALES
- A differential ovarian follicle count (DOFC) was conducted in test groups 10 and 13 (cohort 1A females) according to Plowchalk et.al. (1993).
- In general, sections were prepared with 2 - 3 μm thickness and serial sections were taken every 100 μm to complete up to 15 – 20 cut levels across the whole ovarian tissue. For the counting of primordial and growing follicles, H&E
stained slides were prepared from all cut levels. Counting was performed on slides digitalized with a Hamamatsu NanoZoomer 2.0 slide scanner using the Hamamatsu viewing software (NDP.view).


PATHOLOGICAL EXAMINATIONS OF F1 GENERATION, REARING ANIMALS, COHORT 1B

NECROPSY
- All cohort 1B animals were sacrificed by decapitation under isoflurane anesthesia.
The exsanguinated animals were necropsied and assessed by gross pathology; special attention was given to the reproductive organs.

ORGAN WEIGHTS
- The following weights were determined in all animals sacrificed on schedule: Anesthetized animals (final body weight), Adrenal glands (fixed), Cauda epididymis, Epididymides, Liver, Ovaries, Pituitary gland (fixed), Prostate (ventral and dorsolateral part together, fixed), Testes, Seminal vesicles including coagulating gland (fixed), Uterus (with cervix)
- All paired organs were weighed together (left and right)

ORGAN/TISSUE FIXATION
- The following organs or tissues were fixed in 4% neutral-buffered formaldehyde solution or in modified Davidson’s solution: All gross lesions, Adrenal glands, Cervix uteri, Coagulating glands, Epididymis, left (fixed in modified Davidson ´s solution), Liver, Ovaries (fixed in modified Davidson´s solution), Pituitary gland, Prostate, Seminal vesicles including coagulating glands, Testis (fixed in modified Davidson ´s solution), Uterus, Vagina

HISTOPATHOLOGY
- Histotechnical processing and examination by light microscopy was not performed.
- For technical reasons, the ovaries of all cohort 1B females of all test groups were embedded in paraplast.

PATHOLOGICAL EXAMINATIONSOF SURPLUS F1 GENERATION PUPS ON PND 22 (F1 weanlings not selected for cohorts)

NECROPSY
- All surplus F1 generation pups that were not used for the following organ weight determinations were sacrificed under isoflurane anesthesia with CO2.
- The selected pups for organ weight determination were sacrificed by decapitation under isoflurane anesthesia.
- All animals were necropsied and assessed by gross pathology with special emphasis on the reproductive organs.

ORGAN WEIGHTS
- The following weights were determined in up to 10 animals per sex per group sacrificed on schedule: Anesthetized animals (final body weight), Brain, Spleen, Thymus (fixed)

ORGAN/TISSUE FIXATION
- The following organs or tissues of up to 10 animals per sex per group were fixed in 4% neutral- buffered formaldehyde solution: All gross lesions, Brain, Mammary gland (male and female), Spleen, Thymus, Thyroid glands
- Liver samples of the control surplus F1 generation pups on PND 22 were taken and deep frozen. The livers of these animals were not previewed for any further examination in this study. Liver sampling has no impact on the study results.

HISTOPATHOLOGY
- Histotechnical processing and examination was not performed
Statistics:
Means, medians and standard deviations of each test group were calculated for several parameters (see "Any other information on materials and methods incl. tables").
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
CLINICAL OBSERVATIONS

Clinical observations for males and females (except gestation and lactation period)

- No clinical signs or changes of general behavior, which may be attributed to the test substance, were detected in any of the male and female F0 parental animals in any of the groups.
- One mid-dose female animal (No. 156 - 3750 ppm) showed opacity on the right eye during premating days 56 - 74 and one high-dose female animal (No. 200 - 12500 ppm) showed protruding eyeball during premating days 49 - 74. This observation was not considered to be associated with the test compound.


Clinical observations for females during gestation of F1 litters

- There were no test substance-related clinical findings in any females of all dose groups during the gestation period for F1 litter.
- One female (No. 119 - control) of test group 00 showed palpable mass through the skin at axillary region during GD 9 to 21.
- One female (No. 156 - 3750 ppm) showed opacity on the right eye and one female (No. 200 - 12500 ppm) showed protruding eyeball during the whole gestation period.
- One sperm negative female of the low-dose group (No. 148 - 1250 ppm) and one sperm positive female of the mid-dose group (No. 159 - 3750 ppm) did not deliver F1 pups. This observation was not considered to be associated with the test compound.

Clinical observations for females and offspring during lactation of F1 litters
- One female (No. 119 - control) of test group 00 showed palpable mass through the skin at axillary region and severe poor general condition, therefore the animal was sacrificed in a moribund state on PND 22.
- One female (No. 137 - 1250 ppm) of test group 01 showed pups not properly nursed on PND 0 and had complete litter loss on PND 1.
- One female (No. 154 - 3750 ppm) of test group 02 showed an injury on the right side of inguinal region on PND 23, therefore the animal was scheduled sacrificed slightly earlier on PND 24.
- One female (No. 156 - 3750 ppm) showed opacity on the right eye during the whole lactation period.
- One female (No. 171 - 3750 ppm) of test group 02 showed a diffuse injury, diffuse skin lesion and moderate poor general condition between PND 6 to 25, therefore the animal was sacrificed in a moribund state on PND 25.
- One female (No. 176 - 12500 ppm) of test group 03 showed pups not properly nursed on PND 0 to PND 1 and had complete litter loss on PND 2.
- One female (No. 192 - 12500 ppm) of test group 03 showed pups not properly nursed on PND 0.
- One female (No. 194 - 12500 ppm) of test group 03 showed respiration sounds, moderate/severe poor general condition, pups not properly nursed, moderate reduced nutritional condition, piloerection, high stepping gate, hypothermia, complete litter loss between PND 12 to 19, therefore the animal was sacrificed in a moribund state on PND 19.
- One female (No. 196 - 12500 ppm) of test group 03 showed moderate poor general condition, pale skin, pups palpable in abdomen after delivery and complete litter loss on PND 0, therefore the animal was scheduled sacrificed slightly earlier on PND 28.
- One female (No. 200 - 12500 ppm) showed protruding eyeball during the whole lactation period.


DETAILED CLINICAL OBSERVATIONS
- No additional clinical signs or changes of general behavior, which were attributed to the test substance, were detected in any of the male and female animals in any of the groups which were not observed during the general clinical examinations before.
- Male animals of all dose groups (1250, 3750 and 12500 ppm) did not show any abnormalities.
- One female animal (No. 156) of test group 02 (3750 ppm) showed opacity on the right eye from premating day 56 until sacrificed.
- One female animal (No. 200) of test group 03 (12500 ppm) showed protruding eyeball on the right eye from premating day 49 until sacrificed.
- One control female (No. 119) showed palpable mass through the skin on the left axillary region during DCO days 84 – 112 and severe poor general condition on DCO day 119.
- One mid-dose female (No. 171) showed diffuse injury on animal body during DCO days 112 - 119 and diffuse skin lesions on DCO day 119.
- One female animal (No. 194) of test group 03 (12500 ppm) showed moderate poor general condition and reduced nutritional condition, respiration sound and pups not properly nursed on study day 112.
- One female animal (No. 196) of test group 03 (12500 ppm) showed moderate poor general condition, pale skin and pups palpable in abdomen after delivery on study day 105.
- These observations were not considered to be associated with the test compound.
Mortality:
mortality observed, treatment-related
Description (incidence):
Female animal No. 119 of test group 0 (control; 0 ppm) was sacrificed moribund on PND 22, female animal No. 171 of test group 02 (3750 ppm) was sacrificed moribund on PND 25 and female animal No. 194 of test group 03 (12500 ppm) was sacrificed moribund on PND 19.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
- Body weights and body weight change of all test substance treated male and female F0 rats were essentially comparable to the concurrent control values throughout the entire study.
- The statistically significantly decreased body weight change in the high-dose males during premating days 14 - 21 and increased during premating days 21 - 28, as well as increased body weight change in the mid- dose males during premating days 49 - 56, respectively, were considered to be spontaneous in nature and not treatment-related.
- The statistically significantly decreased body weight change in the high-dose females during premating days 49 - 56, as well as decreased body weight change in the high- dose females during gestation days 7 - 14 respectively, were considered to be spontaneous in nature and not treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
FOOD CONSUMPTION
- During lactation the food consumption was only decreased in female animals of test group 03 (12500 ppm) between PND 10 to 14 (-10.8%) and but over the whole lactation period only slightly and non-statistically significantly reduced (-8.2%).
- Food consumption of the low- and mid-dose animal rats was comparable to the concurrent control values throughout the entire study.

INTAKE OF TEST SUBSTANCE
- See "Any other information on results incl. tables"


Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
No treatment-related changes among hematological parameters were observed. At the end of the administration period in F0 females of test groups 02 and 03 (3750 and 12500 ppm) hemoglobin and hematocrit values as well as mean corpuscular volume (MCV) and mean corpuscular hemoglobin content (MCH) were significantly decreased. In females of test group 01(1250 ppm) hematocrit values were already significantly lower compared to controls. However, all values were within historical control ranges (F0 females, hematocrit 0.421-0.470 L/L, hemoglobin 8.9-10.0 mmol/L, MCV 52.7-54.5 fL; MCH 1.11-1.18 fmol). In females of test group 02, prothrombin time (Hepatoquick’s test, HQT) was significantly reduced, relative neutrophil counts were significantly increased, and relative lymphocyte counts were significantly decreased. However, the changes were not dose dependent. Therefore, the mentioned alterations in this paragraph were regarded as incidental and not treatment related.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
No treatment-related changes among clinical chemistry parameters were observed.
At the end of the administration period, in F0 females of test groups 02 and 03 (3750 and 12500 ppm), total bilirubin values were significantly decreased, but the change was not dose dependent. Therefore, this change was regarded as incidental and not treatment related.

In parental males and females (test groups 01, 02 and 03; 1250 3750 and 12500 ppm) no treatment-related alterations of T4 and TSH levels were observed.

Urinalysis findings:
no effects observed
Description (incidence and severity):
No treatment-related changes among urinalysis parameters were observed.
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
- Treatment-related findings were observed in the kidneys of male animals with incidences and grading given in the table in "Any other information on results incl. tables"
- Eosinophilic droplets, representing proteinaceous material, were found in the cytoplasm of proximal tubular cells in all test groups including control animals. Compared to the control animals, a slight dose-dependent increase in incidence and grading was noted starting in test group 02 up to test group 03, which was considered treatment-related. This change was characterized by an increase of droplet number, size and distribution. An immunhistochemical stain, performed exemplarily in animals 10, 80 and 87, revealed that the staining pattern was different to alpha 2u staining pattern.
This finding was not associated with tubular cell injury and was therefore considered treatment- related but not adverse.
- All other findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.
- Fertility: The female animal No. 148 and 159, which were not pregnant as well as the male mating partners No. 48 and 59 did not show relevant histopathological findings consistent with impaired fertility.
- Decedents: The female animal Nos. 119, 171 and 194 were sacrificed moribund. The female No. 119 showed macroscopically a mass (diameter 75 mm) in the skin of the axillary region which correlated with a spontaneous malignant basal cell tumor and was the cause of the moribund state. The female No. 171 showed in the skin of the head and in the abdominal and back region many lesions with incrusted surface, which correlated with multifocal ulcers and crusts. The axillary lymph nodes displayed a neutrophilic inflammation, sinus histiocytosis, increased plasma cells and sinus dilation. These findings were consistent with macroscopically enlarged axillary lymph nodes and represented a reactive response to the skin lesions. The skin lesions most likely contributed to the moribund state of this animal. Additionally, a slight hyperplasia of the mammary gland was noted. The female No. 194 showed in the thymus a massive decreased cellularity of the cortex and medulla and a moderate atrophy of the genital organs (uterus, vagina and interstitial glands in the ovary). These signs reflected the moribund state in this female but were not considered the cause of morbidity. Animal Nos. 119 and 194 also displayed also displayed a decreased cellularity in the mesenteric lymph node (cortical and paracortical lymphocytes).
Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
- Estrous cycle data, generated during the last 3 weeks prior to mating to produce the F1 litter, revealed regular cycles in the females of all test groups including the control.
- The mean estrous cycle number was similar: 4.6 / 4.6 / 5.5 and 4.4 in test groups 00 - 03, respectively.
- The mean estrous cycle duration was similar: 4.0 / 3.9 / 4.0 and 4.2 days in test groups 00 - 03, respectively.
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
Concerning motility of the sperms and the incidence of abnormal sperms in the cauda epididymidis as well as sperm head counts in the testis and in the cauda epididymidis of parental males no treatment-related effects were observed.
Reproductive performance:
effects observed, treatment-related
Description (incidence and severity):
MALE REPRODUCTION DATA
- For all F0 parental males, which were placed with females to generate F1 pups, copulation was confirmed.
- Thus, the male mating index was 96% in test group 01 (1250 ppm) and 100% in the
remaining test groups (00, 02 - 03).
- Fertility was proven for most of the F0 parental males within the scheduled mating interval for F1 litter.
- One low dose male (No. 48 - 1250 ppm) did not generate pregnancy.
- Thus, the male fertility index ranged between 96% and 100%, reflecting the normal range of biological variation inherent in the strain of rats used for this study.
- The apparently infertile male rat did not show histopathological findings that could explain infertility.


FEMALE REPRODUCTION AND DELIVERY DATA
- The female mating index calculated after the mating period for F1 litter ranged between 96% and 100%.
- The mean duration until sperm was detected (GD 0) varied between 2.5 and 3.1 days.
- All female rats delivered pups or had implants in utero with the following exception: Test group 02, female No. 159 (mated with male No. 59) did not become pregnant.
- The apparently infertile female rats did not show histopathological findings that could explain infertility.
- The fertility index was 100% in in all test groups 00 - 03. The mean duration of gestation values varied between 22.0 and 22.1 days without any relation to dosing. The gestation index varied between 96% and 100%.
- Implantation was not affected by the treatment since the mean number of implantation sites was comparable between all test substance-treated groups and the control, taking normal biological variation into account (13.5 / 12.8 / 12.9 and 12.3 implants/dam in test groups 00 - 03, respectively). Furthermore, there were no indications for test substance-induced intrauterine embryo-/fetolethality since the postimplantation loss did not show any statistically significant differences between the groups (5.1% / 5.8% / 11.7% and 10.3% in test groups 00 – 03, respectively), and the mean number of F1 pups delivered per dam remained unaffected (12.8 / 12.1 / 12.5 and 11.2 pups/dam, respectively in test groups 00 - 03).
- The rate of liveborn pups was also not affected by the test substance, as indicated by live birth indices of 99% / 98% / 98% and 93% in test groups 00 - 03. - The incidence of the high dose group is slightly below the historical control range.
- The numbers of stillborn pups with 1.2% / 2.4% / 2.0% and 7.1% in test groups 00-03 were not statistically significantly different between the test groups and but the high dose group was outside of the historical control range of 0.0% to 5.5%.
- The higher number of stillborn pups in test group 03 were caused by one dam (No. 192) with 9 stillborn pups and one dam (No. 196) with 7 stillborn pups.
Dose descriptor:
NOAEL
Remarks:
general systemic toxicity
Effect level:
340 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects observed at this dose.
Remarks on result:
other: 3750 ppm
Dose descriptor:
LOAEL
Remarks:
general systemic toxicity
Effect level:
1 129 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
mortality
food consumption and compound intake
Remarks on result:
other: 11250 ppm
Dose descriptor:
NOAEL
Remarks:
fertility
Effect level:
>= 1 129 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effect observed up to the highest tested dose.
Remarks on result:
other: 11250 ppm
Dose descriptor:
NOAEL
Remarks:
reproductive performance
Effect level:
340 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No observed adverse effects at this dose.
Remarks on result:
other: 3750 ppm
Dose descriptor:
LOAEL
Remarks:
reproductive performance
Effect level:
1 129 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reproductive performance
Remarks on result:
other: 11250 ppm
Critical effects observed:
no
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
PUPS
- There was no test substance-related adverse clinical sign observed in any of the surviving F1 generation pups of the different test groups.

F1 REARING ANIMALS, COHORT 1A
- No clinical signs or changes of general behavior, which may be attributed to the test substance, were detected in any of the male and female animals in any of the groups.

F1 REARING ANIMALS, COHORT 1B
- No clinical signs or changes of general behavior, which may be attributed to the test substance, were detected in any of the male and female animals in any of the groups.
Mortality / viability:
mortality observed, treatment-related
Description (incidence and severity):
F1 PUBS
- The viability index indicating pup survival during early lactation (PND 0 - 4) varied between 99% / 96% / 99% and 90% in test groups 00 - 03. The value of the high dose was slightly outside of the historical control range 94-100.0% (see Part III, Supplement).
- The survival index indicating pup survival on PND 4 - 21 (lactation index) was 100% / 100% / 100% and 95% in test groups 00 - 03. The value of the high dose was slightly outside of the historical control range 95.7-100.0%. On postnatal day 21 significant lower number of mean pups were observed 8.7 pups per litter vs 9.9 pups per litter in control.

F1 REARING ANIMALS, COHORT 1A
- There were no test substance-related or spontaneous mortalities in any of the groups.

F1 REARING ANIMALS, COHORT 1B
- There were no test substance-related or spontaneous mortalities in any of the groups.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
F1 PUPS
- The mean body weights and body weight change of all male and female pups in all test substance-treated groups were comparable to the concurrent control values throughout the entire study.

F1 REARING ANIMALS, COHORT 1A
- The mean body weights/body weight change of all test substance-treated F1 male and female animals were comparable to the concurrent control values throughout the entire study.

F1 REARING ANIMALS, COHORT 1B
- The body weights of the mid-dose females were statistically significantly increased to the control values between study day 21 and 28 of the inlife period (up to 5%). This finding without dose dependency was considered as incidental and not related to treatment.
- The body weight gain of all test substance treated male and female F1 rats was comparable to the concurrent control values throughout the entire study.


Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
F1 REARING ANIMALS, COHORT 1A
- Food consumption of the all test substance-treated male and female rats (1250, 3750 and 12500 ppm) was comparable to the concurrent control values throughout the entire study of F1.

F1 REARING ANIMALS, COHORT 1B
- Food consumption of the all test substance-treated male and female rats (1250, 3750 and 12500 ppm) was comparable to the concurrent control values throughout the entire study of F1.

COMPOUND INTAKE
- Tables see "Any other information on results incl. tables"
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
F1 GENERATION
- No treatment-related changes among hematological parameters were observed.
- At study day 90, in males of test group 13 (12500 ppm) prothrombin time (Hepatoquick’s test, HQT) was significantly prolonged, but the mean was within the historical control range (F1 males, HQT 32.6-38.3 sec). In F1 females of test group 11 (1250 ppm) relative eosinophil counts were significantly increased, but the alteration was not dose dependent. Therefore, the mentioned changes were regarded as incidental and not treatment related.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
F1 GENERATION
- No treatment-related, adverse changes among clinical chemistry parameters were observed.
- In females of test group 13 (12500 ppm) sodium levels were significantly decreased. The values were marginally below the historical control range (F1 females, sodium 141.7-144.0 mmol/L). However, this was the only changed clinical chemistry parameter among these individuals and therefore, it was regarded as maybe treatment-related, but non-adverse (ECETOC Technical Report No. 85, 2002).
- In males of test group 12 (3750 ppm) chloride levels were significantly lower compared to study controls, and in females of test group 11 (1250 ppm) total bilirubin levels were significantly decreased. However, the alterations were not dose dependent. Therefore, the mentioned changes were regarded as incidental and not treatment related.
- In F1 male PND4 as well as male and female PND22 pups (test groups 01, 02 and 03; 1250, 3750 and 12500 ppm) as well as in F1A adult males and females (test groups 11, 12 and 13; 1250, 3750 and 12500 ppm) no treatment-related alterations of T4 and TSH levels were observed.
- In female PND4 pups of test group 03 (12500 ppm) T4 values were significantly increased. However, T4 as well as TSH mean in this group was within historical control ranges (PND4 females, T4 17.88-53.89 nmol/L, TSH 3.05-7.58). Therefore, the T alteration in female PND4 pups was regarded as incidental and not treatment related.

Urinalysis findings:
no effects observed
Description (incidence and severity):
F1 GENERATION
- No treatment-related changes among urinalysis parameters were observed.
Sexual maturation:
no effects observed
Description (incidence and severity):
SEX RATIO
- The sex distribution and sex ratios of live F1 pups on the day of birth and on PND 21 did not show substantial differences between the control and the test substance-treated groups; slight differences were regarded to be spontaneous in nature.

VAGINAL OPENING
- Each female F1 pup, which was selected to become a rearing female, was evaluated for commencement of sexual maturity. The first day when vaginal opening was observed was PND 27, the last was PND 38. The mean number of days to reach the criterion in the control and 1250/625, 3750/1875 and 12500/6250 ppm test groups was 31.6; 30.9; 31.4 and 31.7 days, respectively. The mean body weight on the day, when vaginal opening was recorded, amounted to 95.3 g, 92.5 g, 97.0 g and 96.4 g in test groups 00-03. Neither a statistically significant nor a toxicologically relevant effect was noted in any of the treatment groups.

PREPUTIAL SEPARATION
- Each male F1 pup, which was selected to become a rearing male, was evaluated for commencement of sexual maturity. The first day when preputial separation was observed was PND 38, the last was PND 48. The mean number of days to reach the criterion in the control and 1250/625, 3750/1875 and 12500/6250 ppm test groups was 40.6, 40.9, 41.9** (** = p::0.01) and 41.1 days, respectively. The mean body weight on the day, when preputial separation was recorded, amounted to 171.1 g, 173.2 g, 178.3* g (* = p::0.05) and 172.6 g in test groups 00-03. All values for days and weights are well within the historical control range (40.1-45.2 days, 158.2-221.1 gram ), thus any observed statistical change is considered
incidental and not treatment-related.

Anogenital distance (AGD):
no effects observed
Description (incidence and severity):
Anogenital distance and anogen tal index of all test substance treated pups were comparable to the concurrent control values.
Nipple retention in male pups:
no effects observed
Description (incidence and severity):
- The percentage of male pups having nipples/areolae was not influenced by the test substance when examined on PND 13.
- During the re-examination on PND 20 no nipples/areolae were detected in any male pup of all test groups.
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
F1 REARING ANIMALS, COHORT 1A
- When compared with control group 10 (=100%), the kidney mean absolute weights were significantly changed (see "Any other information on results incl. tables")
- The significant absolute weight increase of the kidneys in females of test groups 12 and 13 (1.589 and 1.585 g, respectively) was marginally above the historical control range (1.305 –1.542 g). Since neither relative weight deviations nor histopatological changes occurred in the kidneys, this finding was assessed as not treatment-related.


F1 REARING ANIMALS, COHORT 1B
- see "Any other information on results incl. tables"
- The significantly increased terminal body weight occurred without dose dependency. The significantly increased absolute weights of the adrenal glands, although minimally above the historical control values, occurred without statistical deviation in the relative weight, and the liver weight increases were within the historical control ranges (see Part III, supplement). The significant decreases of the absolute and relative weights of the pituitary gland occurred without dose-dependency and were still within the historical control ranges (absolute: 10.840 – 11.400 mg; relative: 0.005 – 0.006%). Therefore, all these changes were not regarded as treatment-related.

SURPLUS F1 GENERATION PUPS ON PND 22 (F1 weanlings not selected for cohorts)
- see "Any other information on results incl. tables"
- The significant absolute and relative spleen weight increase of females in test group 02 occurred without a dose-response relationship and was considered incidental and not treatment-related.

Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
PUP NECROPSY OBSERVATION
- A few F1 pups showed spontaneous findings at gross necropsy, such as partly cannibalized, post-mortem autolysis, discolored testis, discolored liver lobe, discolored lung, skin lesion (biting wound from the mother) and empty stomach.
- Male pup no. 05 from animal No. 149 (test group 01 - 625 ppm) showed bilateral slightly malrotated limb towards the center. Male pup no. 07 from animal No. 182 (test group 03 - 6250 ppm) showed small tongue and small mandible in addition of the finding to the stained skeleton. Male pups nos. 04 - 09 and female pup no. 12 from animal No. 192 (test group 03 - 6250 ppm) showed right or bilateral slightly malrotated limb towards the center. Male pup no. 02 and female pup nos. 04 and 05 from animal No. 196 (test group 03 - 6250 ppm) showed left or bilateral slightly malrotated limb towards the center.
- The malrotated limbs observed in 10 pups of two litters in the high dose group were considered as treatment-related. The single finding of malrotated limbs in the low dose group with no corresponding finding in the mid dose group was considered as incidental and not related to treatment. All other findings occurred without any relation to dosing and/or can be found in the historical control data at comparable or even higher incidences and were not considered to be associated with the test substance.

F1 REARING ANIMALS, COHORT 1A
- All findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.
- Decedents: No decedent observed.

F1 REARING ANIMALS, COHORT 1B
- All findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.
- Decedents: No decedent observed.

SURPLUS F1 GENERATION PUPS ON PND 22 (F1 weanlings not selected for cohorts)
- No gross changes were observed at necropsy.
Histopathological findings:
effects observed, treatment-related
Description (incidence and severity):
F1 REARING ANIMALS, COHORT 1A
- See "Any other information on results incl. tables"
- Eosinophilic droplets, with the same characteristics as in the kidneys of the F0 generation males, were seen in all test groups including control animals. Compared to the control animals, a dose-dependent increase in the incidence and grading was noted in test groups 12 and 13, which was considered treatment-related. As observed in the F0 generation males, this finding was not associated with tubular cell injury and was therefore considered treatment-related but not adverse.
- All other findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.
- Differential ovarian follicle count (see "Any other information on results incl. tables*): The results of the differential ovarian follicle count (DOFC) – comprising the numbers of primordial and growing follicles, as well as the combined incidence of primordial plus growing follicles – did not reveal significant differences between the control group 10 and animals of test group 13 (12500 ppm).

F1 REARING ANIMALS, COHORT 1B
- Histotechnical processing and examination by light microscopy was not performed.

SURPLUS F1 GENERATION PUPS ON PND 22 (F1 weanlings not selected for cohorts)
- Histotechnical processing and examination by light microscopy was not performed.
Other effects:
effects observed, treatment-related
F1 PUB NUMBER AND STATUS OF DELIVERY
- The mean number of delivered F1 pups per dam and the rates of liveborn, stillborn and dead F1 pups were evenly distributed about the groups. The respective values reflect the normal range of biological variation inherent in the strain used in this study.
- The higher number of stillborn pups in test group 03 were caused by one dam (No. 192) with 9 stillborn pups, one dam (No. 196) with 7 stillborn pups. In test group 03 the higher number of pups they were found dead on PND 19 were caused of one dam (No. 194) with 10 pups.

F1 REARING ANIMALS, COHORT 1A - ESTROUS CYCLE
- Estrous cycle data, generated during 2 weeks, revealed regular cycles in the females of all test groups including the control. The mean estrous cycle duration in the different test groups was similar: 4.0 days in control, 3.9 days in the low dose group, 3.9 days in the mid-dose group and 4.0 days in the high-dose group.

F1 REARING ANIMALS, COHORT 1B - ESTROUS CYCLE
- Estrous cycle data, generated during 2 weeks, revealed regular cycles in the females of all test groups including the control. The mean estrous cycle duration in the different test groups was similar: 4.0 days in control, 4.0 days in the low-dose group, 3.9 days in the mid-dose group and 4.0 days in the high-dose group.

F1 GENERATION - SPERM ANALYSIS
- Concerning motility of the sperms and the incidence of abnormal sperms in the cauda epididymidis as well as sperm head counts in the testis and in the cauda epididymidis of F1A males no treatment-related effects were observed.



Dose descriptor:
LOAEL
Remarks:
developmental toxicity
Generation:
F1
Effect level:
1 129 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: decreased lactation index in F1 pubs, decreaesed viability index, decreased number of surviving pubs per litter, increased number of F1 pups with malrotated limbs
Remarks on result:
other: 12500 ppm
Dose descriptor:
NOAEL
Remarks:
developmental toxicity
Generation:
F1
Effect level:
340 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects observed at this dose.
Remarks on result:
other: 3750 ppm
Critical effects observed:
no
Reproductive effects observed:
yes
Lowest effective dose / conc.:
1 129 mg/kg bw/day (actual dose received)
Treatment related:
yes
Relation to other toxic effects:
reproductive effects as a secondary non-specific consequence of other toxic effects

ANALYSES


- Stability analyses: The stability of test substance in rat diet was demonstrated for a period of 35 days at room temperature.


- Homogeneity analyses: The homogeneity of the mixtures was verified.


- Concentration control analyses: The observed concentrations of the test substance correspond with the expected concentrations by recoveries between 90 and 96, and demonstrated the correctness of the diet preparations.


- Food analyses: With regard to the analytical findings of chemical and microbiological contaminants and the duration of application, the diet was found to be suitable. Fed. Reg. Vol. 44, No. 91 of 09 May 1979, p. 27354 (EPA), served as a guideline for maximum tolerable chemical contaminants. The concentration of microorganisms did not exceed 1*10^5/g feed.


- Drinking water analyses: On the basis of the analytical findings, the drinking water was found to be suitable. German Drinking Water Regulation (Trinkwasserverordnung) served as a guideline for maximum  tolerable contaminants.


- Bedding and enrichment analyses: On the basis of the analytical findings, bedding and cage enrichment were found to be suitable. Levels given in Lab Animal (Nov-Dec 1979, pp. 24-34) served as a guideline for maximum tolerable contaminants.


 


 


Tab. 10: Mean test substance intake of test substance (mg/kg bw/d; minimum value/maximum value) in F0 animals


 






























 



Test group 01 (1250/625 ppm)



Test group 02 (3750/1875 ppm)



Test group 03 (12500/6250 ppm)



F0 males



98.5 (72.6 / 153.6)



299.4 (217.4 / 460.7)



997.4 (736.6 / 1541.7)



F0 females (premating)



106.2 (90.6 / 140.5)



320.2 (270.4 / 438.4)



1088.0 (930.6 / 1431.3)



F0 females


*  gestation period


*  lactation period



93.4 (88.6 / 98.2)


127.3 (84.3 / 182.8)



280.2 (262.7 / 293.0)


374.9 (247.7 / 509.2)



923.3 (881.6 / 949.4)


1129.4 (741.7 / 1548.2)



 


 


Tab. 11: Mean test substance intake of test substance (mg/kg bw/d; minimum value/maximum value) in F1 rearing animals, Cohort 1A


 
























 



Test group 11


(1250 ppm)



Test group 12


(3750 ppm)



Test group 13


(12500 ppm)



Males



118.6 (81.5 / 174.0)



359.2 (241.3 / 529.2)



1192.1 (818.8 / 1758.2)



Females



121.0 (97.5 / 167.2)



364.8 (291.8 / 498.7)



1192.8 (951.2 / 1660.0)



 


 


Tab. 12: Mean test substance intake of test substance (mg/kg bw/d; minimum value/maximum value) in F1 rearing animals, Cohort 1B


 
























 



Test group 11


(1250 ppm)



Test group 12


(3750 ppm)



Test group 13


(12500 ppm)



Males



121.5 (84.9 / 171.1)



365.6 (257.7 / 512.5)



1231.7 (876.7 / 1723.1)



Females



120.6 (99.7 / 161.6)



360.7 (293.1 / 490.1)



1214.7 (994.6 / 1641.4)



 


 


Tab. 13: Mean test substance intake of test substance


 


























































































 



 



Test group 01/11 (1250 ppm;


lactation 625 ppm)



Test group 02/12 (3750 ppm;


lactation 1875 ppm)



Test group 03/13 (12500 ppm;


lactation 6250 ppm)



 



Duration of phase (weeks)



Mean (mg/kg b/d)



Mean (mg/kg b/d)



Mean (mg/kg b/d)



F0 males



 


10



 


98.5



 


299.4



 


997.4



F1 males rearing 1A



8



118.6



359.2



1192.1



F1 males rearing 1B



 


7



 


121.5



 


365.6



 


1231.7



Mean / Weighted mean of mean



 



 


112.9 / 111.4



 


341.4 / 337.1



 


1140.4 / 1125.3



F0 females premating



10



 


106.2



 


320.2



 


1088.0



F0 females gestation



3



 


93.4



 


280.2



 


923.3



F0 females lactation



3



127.3



374.9



1129.4



F1 females rearing 1A



8



 


121.0



 


364.8



 


1192.8



F1 females rearing 1B



7



 


120.6



 


360.7



 


1214.7



Mean / Weighted mean of mean



 



 


112.0 / 112.7



 


341.1 / 339.7



 


1131.9 / 1128.5



 


 


Tab. 14: F0 generation parental animals, Absolute organ weights


 






















F0



Female animals



Test group (ppm)



01


(1250)



02


(3750)



03


(12500)



Kidneys



100%



101%



107%**



* for p ≤0.05, ** for p ≤0.01


 


 


Tab. 15: F0 generation parental animals, Relative organ weights


 




























F0



Male animals



Test group (ppm)



01


(1250)



02


(3750)



03


(12500)



Kidneys



102%



100%



106%**



Liver



101%



102%



107%**



* for p ≤0.05, ** for p ≤0.01


 


 


Tab. 16: F0 generation parental animals, Histopathology findings


 













































Kidneys



Male animals (F0)



Test group (ppm)



00


(0)



01


(1250)



02


(3750)



03


(12500)



No. of animals



20



20



20



20



Eosinophilic droplets



8



10



14



18



Grade 1



8



8



8



11



Grade 2



 



2



6



7



 


 


Tab. 17: F1 generation, rearing animals, cohort 1A, Absolute organ weights


 






















Cohort 1A



Female animals



Test group (ppm)



11


(1250)



12


(3750)



13


(12500)



Kidneys



100%



105%*



105%*



for p ≤0.05, ** for p ≤0.01


 


 


 


Tab. 18: F1 generation, rearing animals, cohort 1A, Histopathology findings


 













































Kidneys



Male animals (Cohort 1A)



Test group (ppm)



10


(0)



11


(1250)



12


(3750)



13


(12500)



No. of animals



20



20



20



20



Eosinophilic droplets



9



10



19



20



Grade 1



4



6



16



8



Grade 2



5



4



3



12



 


 


Tab. 19: F1 generation, rearing animals, cohort 1A, Differential ovarian follicle count, Absolute values


 






























Number of animals



Absolute values



Group



Primordial



Growing



Primordial + growing



20



10



4069



464



4533



20



13



4116



531



4647



for p ≤0.05, ** for p ≤0.01


 


 


Tab. 20: F1 generation, rearing animals, cohort 1A, Differential ovarian follicle count, Mean values


 






























Number of animals



Mean values



Group



Primordial



Growing



Primordial + growing



20



10



203



23



227



20



13



206



27



232



for p ≤0.05, ** for p ≤0.01


 


 


Tab. 21: F1 generation, rearing animals, cohort 1B, Absolute organ weights


 








































Cohort 1B



Female animals



Test group (ppm)



11


(1250)



12


(3750)



13


(12500)



Terminal body weight



100%



105%**



104%



Adrenal glands



99%



107%



107%*



Liver



103%



107%*



107%**



Pituitary gland



90%*



90%**



90%*



for p ≤0.05, ** for p ≤0.01


 


 


Tab. 22: F1 generation, rearing animals, cohort 1B, Relative organ weights


 






















Cohort1B



Female animals



Test group (ppm)



11


(1250)



12


(3750)



13


(12500)



Pituitary gland



90%*



85%**



87%**



for p ≤0.05, ** for p ≤0.01


 


 


Tab. 23: Surplus F1 generation pups on PND 22 (F1 weanlings not selected for cohorts), Absolute organ weights


 






















F1 Pups PND 22



Female animals



Test group (ppm)



01


(1250)



02


(3750)



03


(12500)



Spleen



113%



111%*



94%



for p ≤0.05, ** for p ≤0.01


 


 


Tab. 24: Surplus F1 generation pups on PND 22 (F1 weanlings not selected for cohorts), Relative organ weights


 






















F1 Pups PND 22



Female animals



Test group (ppm)



01


(1250)



02


(3750)



03


(12500)



Spleen



112%



114%**



98%



for p ≤0.05, ** for p ≤0.01


 


 


 


 


 

Conclusions:
Under the conditions of the present extended one-generation reproduction toxicity study the NOAEL (no observed adverse effect level) for general, systemic toxicity is 12500 ppm (about 1129 mg/kg bw/d) for males and 3750 ppm (340 mg/kg bw/d) for females, based on clinical signs of toxicity and mortality during lactation, in the F0 parental animals as well as adolescent and adult F1 offspring. The NOAEL for fertility for the parental rats is 12500 ppm (about 1129 mg/kg bw/d), the highest dose tested. The NOAEL for reproductive performance for the female parental rats is 3750 ppm (about 340 mg/kg bw/d). The NOAEL for developmental toxicity in the F1 progeny is 3750 ppm (about 340 mg/kg bw/d).
Executive summary:

The test substance was administered to groups of 25 male and 25 female healthy young Wistar rats as a homogeneous addition to the food in different concentrations (0, 1250, 3750 and 12500 ppm). F0 animals were treated at least for 10 weeks prior to mating to produce a litter (F1 generation). Mating pairs were from the same dose group. Pups of the F1 litter were selected (F1 rearing animals) and assigned to 2 different cohorts (1A and 1B) which were subjected to specific postweaning examinations. The study was terminated with the terminal sacrifice of the F1 rearing animals of cohort 1B. Test diets containing test substance were offered continuously throughout the study.


 


Observations: The parents' and the pups' state of health was checked each day, and parental animals were examined for their mating and reproductive performances. Food consumption of the F0 parents and F1 rearing animals was determined regularly once weekly and weekly during gestation (days 0 - 7, 7 - 14, 14 - 20) and lactation periods (days 1 - 4, 4 - 7, 7 - 10, 10 - 14, 14 - 18 and 18 - 21). In general, body weights of F0 parents and F1 rearing animals were determined once weekly. However, during gestation and lactation F0 females were weighed on gestation days (GD) 0, 7, 14, 20 and on postnatal days (PND) 1, 4, 7, 10, 14, 18 and 21. A detailed clinical observation (DCO) was performed in all F0 parents and F1 animals in cohorts 1A and 1B at weekly intervals. Estrous cycle data were evaluated for F0 females over a three week period prior to mating until evidence of mating occurred. In all cohort 1A females, vaginal smears were collected after vaginal opening until the first cornified smear (estrous) was recorded. The estrous cycle also was evaluated in cohort 1A and 1B females for 2 weeks around PND 75. Moreover, the estrous stage of each female was determined on the day of scheduled sacrifice. The F1 pups were sexed on the day of birth (PND 0) and were weighed on the first day after birth (PND 1) as well as on PND 4, 7, 14 and 21. Their viability was recorded. At necropsy, all pups were examined macroscopically. Date of sexual maturation, i.e. day of vaginal opening (females) or balanopreputial separation (males), of all F1 pups selected to become F1 rearing animals was recorded. All surviving pups were examined for the presence or absence of nipple/areola anlagen on PND 13 and were re-examined on PND 20. If nipple/areola anlagen were recorded, all surviving male pups were carefully re-examined one day prior to necropsy. The number of nipple/areola anlagen were counted. Anogenital distance (defined as the distance from the anus [center of the anal opening] to the base of the genital tubercle) measurements were conducted in a blind randomized fashion, using a measuring ocular on all live male and female pups on PND 1. Urine samples for clinical pathological investigations were withdrawn from 10 selected F0 and cohort 1A animals per sex and group. Blood samples for clinical pathological investigations were withdrawn from 10 selected F0 and cohort 1A animals per sex and group. Further blood samples were taken from a maximun of 10 surplus (culled) PND 4 pups per sex and group as well as from 10 surplus PND 22 pups per sex and group. Various sperm parameters (motility, sperm head count, morphology) were assessed in the F0 generation males and cohort 1A males at scheduled sacrifice or after appropriate staining. 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. A quantitative assessment of primordial and growing follicles in the ovaries was performed for all control and high-dose F1 rearing females cohort 1A. All F1 rearing animals were assessed by different pathological and histopathological examinations.


 


Analyses: The various analyses: 1.) Demonstrated the stability of the test substance preparations over a period of 35 days at room temperature, 2.) Confirmed the homogeneous distribution of the test substance in the diet, 3.) Verified correct concentrations of the test substance in the diet preparations.


Intake of test substance:  The weighted mean of mean dose of test substance throughout all study phase and across all cohorts was approx. 113 mg/kg body weight/day (mg/kg bw/d) in the 1250 ppm group, approx. 340 mg/kg bw/d in the 3750 ppm group and approx. 1129 mg/kg bw/d in the 12500 ppm group.


 


Effects: The following test substance-related and relevant effects/findings were noted:


 


12500 ppm (Substance intake overall mean of mean 1129 mg/kg bw/d)


F0 PARENTAL ANIMAL


CLINICAL EXAMINATIONS/ REPRODUCTIVE PERFORMANCE/ CLINICAL PATHOLOGY/ PATHOLOGY



  • Decresased number of live born pups in comparison to control (93% vs. 99% in control)

  • Increased number of stillborn in comparison to control (7.1% vs. 1.2% in control)

  • Pups not properly nursed in 3 females during lactation

  • Complete litter loss in 3 females during lactation

  • Pups palpable in abdomen after delivery in 1 female during lactation

  • General conditions poor in 2 females during lactation

  • Hypothermia and respiratory sounds in 1 female during lactation

  • Two females sacrificed moribund on postnatal day 19 and 28

  • Reduced food consumption in females between lactation days 10 and 14 (-10.8%)


 


F1 PUPS


CLINICAL EXAMINATIONS/ SEXUAL MATURATION/ GROSS FINDINGS



  • Decreased viability index with 90% (not statistically significant) in comparison to control (99%)

  • Decreased lactation index with 95% (not statistically significant) in comparison to control (100%)

  • Decreased mean number of surviving pups on post natal day 21 8.7 pups per litter in comparison to control (9.9 pups per litter)

  • Increased number of pups with malrotated limbs 10 out of 92


 


F1 REARING ANIMALS, COHORT 1A


CLINICAL EXAMINATIONS/ CLINICAL PATHOLOGY



  • No treatment-related relevant findings


 


F1 REARING ANIMALS, COHORT 1B


CLINICAL EXAMINATIONS/ CLINICAL PATHOLOGY/ PATHOLOGY



  • No treatment-related relevant findings


 


3750 ppm (Substance intake overall mean of mean 340 mg/kg bw/d)


F0 PARENTAL ANIMALS


CLINICAL EXAMINATIONS/ REPRODUCTIVE PERFORMANCE/ CLINICAL PATHOLOGY/ PATHOLOGY



  • No test substance-related adverse findings


 


F1 PUPS


CLINICAL EXAMINATIONS/ SEXUAL MATURATION/ GROSS FINDINGS



  • No test substance-related relevant findings


 


F1 REARING ANIMALS, COHORT 1A


CLINICAL EXAMINATIONS/ CLINICAL PATHOLOGY/ PATHOLOGY



  • No test substance-related relevant findings


 


F1 REARING ANIMALS, COHORT 1B


CLINICAL EXAMINATIONS/ CLINICAL PATHOLOGY/ PATHOLOGY



  • No test substance-related relevant findings


 


1250 ppm (Substance intake overall mean of mean 113 mg/kg bw/d)


F0 PARENTAL ANIMALS


CLINICAL EXAMINATIONS/ REPRODUCTIVE PERFORMANCE/ CLINICAL PATHOLOGY/ PATHOLOGY



  • No test substance-related relevant findings


 


F1 PUPS


CLINICAL EXAMINATIONS/ SEXUAL MATURATION/ GROSS FINDINGS



  • No test substance-related relevant findings


 


F1 REARING ANIMALS, COHORT 1A


CLINICAL EXAMINATIONS/ CLINICAL PATHOLOGY/ PATHOLOGY



  • No test substance-related relevant findings


 


F1 REARING ANIMALS, COHORT 1B


CLINICAL EXAMINATIONS/ CLINICAL PATHOLOGY/ PATHOLOGY



  • No test substance-related relevant findings


 


Conclusion: Thus, under the conditions of the present extended one-generation reproduction toxicity study the NOAEL (no observed adverse effect level) for general, systemic toxicity is 12500 ppm (about 1129 mg/kg bw/d) for males and 3750 ppm (340 mg/kg bw/d) for females, based on clinical signs of toxicity and mortality during lactation, in the F0 parental animals as well as adolescent and adult F1 offspring. The NOAEL for fertility for the parental rats is 12500 ppm (about 1129 mg/kg bw/d), the highest dose tested. The NOAEL for reproductive performance for the female parental rats is 3750 ppm (about 340 mg/kg bw/d). The NOAEL for developmental toxicity in the F1 progeny is 3750 ppm (about 340 mg/kg bw/d).


 

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2021

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
other: OECD Guideline 443 (Extended One-Generation Reproduction Toxicity Study)
Version / remarks:
25 Jun 2018
GLP compliance:
yes (incl. QA statement)
Remarks:
Landesamt für Umwelt, Kaiser-Friedrich-Straße 7, 55116 Mainz
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
2-(2-aminoethoxy)ethanol
EC Number:
213-195-4
EC Name:
2-(2-aminoethoxy)ethanol
Cas Number:
929-06-6
Molecular formula:
C4H11NO2
IUPAC Name:
2-(2-aminoethoxy)ethan-1-ol
Test material form:
liquid
Specific details on test material used for the study:
TEST MATERIAL
- CAS No.: 929-06-6
- Batch identification: 22057368E0
- Purity: 99.4 area % corrected with the water content
- Homogeneity: Given (visually)
- Storage stability: Expiry date: 06 Apr 2021. The stability of the test substance under storage conditions over the test period was guaranteed by the sponsor, and the sponsor holds this responsibility.
- Physical state/Appearance: Liquid / colorless clear
- Storage conditions: Room temperature; under N2

Test animals

Species:
rat
Strain:
Wistar
Details on species / strain selection:
Crl:WI(Han)
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories
- Females nulliparous and non-pregnant: yes
- Age at study initiation: (P) 35 (±1) days
- Weight at study initiation: (P) Males: means 121.9 - 122.3 g; Females: means 97.9 - 99.5 g
- Fasting period before study: no.
- Fasting period before blood sampling: 16 hours.
- Housing: During the study period, the rats were housed together in Polysulfonate cages supplied by TECHNIPLAST, Hohenpeißenberg, Germany and Becker & Co., Castrop-Rauxel, Germany, with the following exceptions: During overnight matings, male and female mating partners were housed together in Polycarbonate cages type III (supplied by TECHNIPLAST, Hohenpeißenberg, Germany and Becker & Co., Castrop-Rauxel, Germany). Dams and their litters were housed together until PND 21 in Polycarbonate cages type III. Females after weaning were housed individually in Polycarbonate cages type III.
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 8 days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24°C
- Humidity (%): 45 - 65%
- Air changes (per hr): 15 times per hour.
- Photoperiod (hrs dark / hrs light): 12 hours (12 hours light from 6.00 h to 18.00 h and 12 hours darkness from 18.00 h to 06.00 h).

Administration / exposure

Route of administration:
oral: feed
Vehicle:
water
Details on oral exposure:
DIET PREPARATION
The required quantity of test substance was weighed in a beaker depending on the dose group and thoroughly mixed with a small amount of food. Then further amounts of food were added to this premix and thoroughly mixed for 3 minutes. Afterwards, further amounts of food, depending on the dose group, were added to this premix in order to obtain the desired concentrations. Mixing of this final mix was carried out for about 10 minutes in a laboratory mixer.
Analytical verification of doses or concentrations:
yes
Remarks:
The observed concentrations of the test substance correspond with the expected concentrations by recoveries between 90 and 96%, and demonstrated the correctness of the diet preparations
Duration of treatment / exposure:
P: 132 days,
F1 A: 66 days,
F1 B: 60 days
Frequency of treatment:
daily
Doses / concentrationsopen allclose all
Dose / conc.:
113 mg/kg bw/day (actual dose received)
Remarks:
1250 ppm
Dose / conc.:
340 mg/kg bw/day (actual dose received)
Remarks:
3750 ppm
Dose / conc.:
1 129 mg/kg bw/day (actual dose received)
Remarks:
12500 ppm
No. of animals per sex per dose:
25 per sex and dose
Control animals:
yes, concurrent vehicle
Positive control:
no

Examinations

Observations and examinations performed and frequency:
PARENTAL ANIMALS:

CAGE SIDE OBSERVATIONS: Yes.
- Time schedule: at least once daily.
- Mortality: A check for moribund or dead animals was made twice daily on working days or once daily (Saturday, Sunday or on public holidays). If animals were in a moribund state, they were sacrificed and necropsied.
- Clinical observations: A cageside examination was conducted at least once daily for any signs of morbidity, pertinent behavioral changes and signs of overt toxicity. Abnormalities and changes were documented daily for each animal and reported. The parturition and lactation behavior of the dams was generally evaluated in the mornings in combination with the daily clinical inspection of the dams. Only particular findings (e.g. disability to deliver) were documented on an individual dam basis. On weekdays (except Saturday, Sunday and public holidays) the parturition behavior of the dams was inspected in the afternoons in addition to the evaluations in the mornings. The day of parturition was considered the 24-hour period from about 15.00 h of one day until about 15.00 h of the following day.


DETAILED CLINICAL OBSERVATIONS: Yes.
- Detailed clinical observations (DCO) were performed in all F0 parental animals once before the administration and supsequently once per week and in cohorts 1A and 1B at weekly intervals during the administration period. The examinations started in the morning. The findings were ranked according to the degree of severity, if applicable.
- Parameters assessed: Abnormal behavior in handling, Fur, Skin, Posture, Salivation, Respiration, Activity/arousal level, Tremors, Convulsions, Abnormal movements, Gait abnormalities, Lacrimation, Palpebral closure, Exophthalmos (Protruding eyeball), Assessment of the feces excreted during the examination (appearance/consistency), Assessment of the urine excreted during the examination, Pupil size.


BODY WEIGHT: Yes.
- In general, the body weight of the male and female F0 parental animals and F1 rearing animals was determined once a week at the same time of the day (in the morning), with the following exceptions. During pregnancy, body weight of the F0 females with evidence of sperm was determined weekly for GD 0, 7, 14 and 20. During lactation, body weight of the F0 and F1 females, which gave birth to a litter was determined for PND 1, 4, 7, 10, 14, 18 and 21. The body weight change of the animals was calculated from these results. Body weight was not determined in the F0 females without positive evidence of sperm during mating and gestation periods, in the females without litter during lactation period and in the females after weaning.


FOOD CONSUMPTION AND COMPOUND INTAKE: Yes.
- Food consumption: Generally, food consumption was determined once a week for male and female F0 parental animals and F1 rearing animals, with the following exceptions. Food consumption was not determined after the 10th premating week (male F0 animals) and during the mating period (male and female F0 parental animals). During pregnancy, food consumption of the F0 females with evidence of sperm was determined weekly for GD 0-7, 7-14 and 14-20. During lactation, food consumption of the F0 females, which gave birth to a litter was determined for PND 1-4, 4-7, 7-10, 10-14, 14-18 and 18-21. Food consumption was not determined in the F0 females without positive evidence of sperm during mating and gestation periods, in the females without litter during lactation period and in females after weaning.
- Intake of test substance: The intake of test substance was calculated from the amount of food consumed and expressed in mg/kg body weight per day (mg/kg bw/d). The calculation of the group values/day was carried out according to the following formula:
ITx = FCx x C/ BWy
with
ITx = Intake of test substance on day x in mg/kg bw/d
FCx = Daily food consumption on day x in grams
C = Concentration in ppm
BWy = Body weight on day y in grams (last weighing before day x)
- The values are group means determined from daily intakes of test substance by the individual animals. The means represent interpolated values from the beginning and end of each respective test week.
- Additionally, a weighted mean of mean over all mean substance intakes throughout all study phases, across all cohorts, and both sexes are calculated considering the different time period of phases.


MALE REPRODUCTION DATA
- The pairing partners, the number of mating days until vaginal sperm was detected in the female animals, and the gestational status of the females were recorded for F0 breeding pairs.
- For the males, mating and fertility indices were calculated for F1 litters according to the following formulas:

Male mating index (%) = number of males with confirmed mating* x 100 / number of males placed with females

* defined by a female with vaginal sperm or with implants in utero

Male fertility index (%) = number of males proving their fertility* x 100 / number of males placed with females

* defined by a female with implants in utero


FEMALE REPRODUCTION AND DELIVERY DATA
- The pairing partners, the number of mating days until vaginal sperm were detected, and gestational status were recorded for F0 females.
- For the females, mating, fertility and gestation indices were calculated for F1 litters according to the following formulas:

Female mating index (%) = number of females mated* x 100 / number of females placed with males

* defined as the number of females with vaginal sperm or with implants in utero

Female fertility index (%) = number of females pregnant* x 100 / number of females mated**

* defined as the number of females with implants in utero
** defined as the number of females with vaginal sperm or with implants in utero

Gestation index (%) = number of females with live pubs on the day of birth* x 100 / number of females pregnant*

* defined as the number of females with implants in utero

The total number of pups delivered and the number of liveborn and stillborn pups were noted, and the live birth index was calculated for F1 litters according to the following formula:

Live birth index (%) = number of liveborn pubs at birth* x 100 / total number of pubs born

The implantations were counted2 and the postimplantation loss (in %) was calculated according the following formula:

Postimplantation loss (%) =(number of implantations – number of pups delivered)* x 100 / number of implantations


CLINICAL PATHOLOGY IN F0 PARENTAL ANIMALS
- Samples were withdrawn from 10 F0 parental males and females per group at
termination.
- Blood samples were taken from animals by puncturing the retrobulbar venous plexus following isoflurane anesthesia.
- Blood sampling and blood examinations were carried out in a randomized sequence. The list of randomization instructions was compiled with a computer.
- In the afternoon preceding the day of urinalysis, the animals were individually transferred into metabolism cages (no food or drinking water provided); on the following morning, the individual urine specimens were examined in a randomized sequence (the list of randomization instructions was compiled with a computer).
- The assays of blood and serum parameters were performed under internal laboratory quality control conditions with reference controls to assure reliable test results.
- The results of clinical pathology examinations were expressed in International System (SI) units. The following parameters will be examined:


HEMATOLOGY: Yes
- Parameters listed in "Any other information on materials and methods incl. tables"


CLINICAL CHEMISTRY: Yes
- Parameters listed in "Any other information on materials and methods incl. tables"


HORMONES: Yes
- Parameters listed in "Any other information on materials and methods incl. tables"


URINANALYSIS: Yes
- Parameters listed in "Any other information on materials and methods incl. tables"


SPERM PARAMETERS: Yes
- Various sperm parameters (motility, sperm head count, morphology) were assessed in the F0 generation males at scheduled sacrifice or after appropriate staining
- After the organ weight determination, the following parameters were determined in the right testis or right epididymis of all male F0 parental animals and all cohort 1A males sacrificed on schedule
- Sperm parameter investigations were carried out in a randomized sequence.
- Parameters listed in "Any other information on materials and methods incl. tables"

OESTROUS CYCLICITY
- Estrous cycle length was evaluated by daily analysis of vaginal smear for all F0 female parental rats for a minimum of 3 weeks prior to mating. Determination was continued throughout the pairing period until the female exhibited evidence of copulation.



LITTER OBSERVATIONS:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes.


PUB NUMBER AND STATUS OF DELIVERY
- All pups delivered from the F0 parents (F1 litter) were examined as soon as possible on the day of birth to determine the total number of pups, the sex and the number of liveborn and stillborn pups in each litter.
- At the same time, the pups were also being examined for macroscopically evident changes.
- Pups, which died before this initial examination, were defined 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.
- Dead pups were evaluated via necropsy.
- The number and percentage of dead pups on the day of birth (PND 0) and of pups dying between PND 1-4, 5-7, 8-14 and 15-21 (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 after birth, and on lactation days 4, 7, 14, and 21.
- Furthermore, viability and lactation indices were calculated according to the following formulas:

Viability index (%) = number of live pubs on day 4* after birth x 100 / number of live pubs on day of birth

* before standardization of litters (i.e. before culling)

Lactation index (%) = number of live pups on day 21 after birth x 100 / nuSmber of live pups on day 4* after birth

* after standardization of litters (i.e. after culling)


SEX RATIO
- On the day of birth (PND 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.
- Later, during the course of lactation, this initial sex determination was followed up by surveying the external appearance of the anogenital region and the mammary line. The sex of the pups was finally confirmed at necropsy.
- The sex ratio was calculated at PND 0 and PND 21 according to the following formula:

Sex ratio = number of live male or female pups on PND 0 and 21 * 100 / number of live male and female pups on PND 0 and 21


PUP CLINICAL OBSERVATION
- The live pups were examined daily for clinical symptoms (including gross-morphological findings) during the clinical inspection of the dams. If pups showed particular findings, these were documented with the dam concerned.


NIPPLE/AEROLA ANLAGEN
- All surviving male pups were examined for the presence of nipple/areola anlagen on PND 13 and were re-examined on PND 20.
- The number of nipple/areola anlagen were counted.


PUP BODY WEIGHT DATA
- The pups were weighed on the day after birth (PND 1) and on PND 4 (before standardization), 7, 14 and 21.
- 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 PND 4 immediately before standardization of the litters.


ANOGENITAL DISTANCE
- Anogenital distance (AGD) is defined as the distance from the center of the anal opening to the base of the genital tubercle. AGD was determined in all live male and female pups on PND 1.
- These measurements were performed in randomized order, using a measuring ocular.
- They were conducted by technicians unaware of treatment group in order to minimize bias.


ANOGENITAL INDEX
- The anogenital index was calculated according to the following formula:

Anogenital index = anogenital distance [mm] / cubic root of pub weight [g]


PUP NECROPSY OBSERVATIONS
- On PND 4, as a result of standardization, all surplus F1 pups were sacrificed by decapitation under isoflurane anesthesia and blood was sampled for determination of serum thyroid hormone concentrations.
- After sacrifice, these pups were examined externally, eviscerated and their organs were assessed macroscopically.
- On PND 22, the surplus F1 generation pups that were not used for the formation of the cohorts or any investigations were sacrificed under isoflurane anesthesia with CO2 and were examined in the pathology lab.
- The selected pups for hormone analyses were sacrificed by decapitation under isoflurane anesthesia in the pathology lab and blood was sampled for thyroid
hormone analyses.
- Pups showing clinical symptoms or gross-morphological findings were examined using appropriate methods. Organs/tissues with gross morphological findings were preserved in a suitable manner for potential histopathological examination.
- All F1 pups which were not used for other purposes without any notable findings were discarded after their macroscopic evaluation.


PREMATURELY DEAD OR SACRIFICED PUPS
- Pups that died or were sacrificed in a moribund state were eviscerated and examined for possible defects and/or the cause of death using appropriate methods. These animals were preserved for this purpose, if necessary.


SEXUAL MATURATION: VAGINAL OPENING
- All female F1 pups selected to become the F1 rearing animals (cohort 1A and 1B) were evaluated daily for vaginal patency beginning on PND 27.
- On the day of vaginal opening the body weights of the respective animals were determined.


SEXUAL MATURATION: PREPUTIAL SEPARATION
- All male F1 pups selected to become the F1 rearing animals (cohort 1A and 1B) were evaluated daily for preputial separation beginning on PND 38.
- On the day of preputial separation the body weights of the respective animals were determined.


CLINICAL PATHOLOGY IN F0 PARENTAL ANIMALS
- Samples were withdrawn from 10 cohort 1A males and females per group at
termination.
- Blood samples were taken from animals by puncturing the retrobulbar venous plexus following isoflurane anesthesia.
- Blood sampling and blood examinations were carried out in a randomized sequence. The list of randomization instructions was compiled with a computer.
- In the afternoon preceding the day of urinalysis, the animals were individually transferred into metabolism cages (no food or drinking water provided); on the following morning, the individual urine specimens were examined in a randomized sequence (the list of randomization instructions was compiled with a computer).
- The assays of blood and serum parameters were performed under internal laboratory quality control conditions with reference controls to assure reliable test results.
- The results of clinical pathology examinations were expressed in International System (SI) units. The following parameters will be examined:


HEMATOLOGY: Yes
- Parameters listed in "Any other information on materials and methods incl. tables"


CLINICAL CHEMISTRY: Yes
- Parameters listed in "Any other information on materials and methods incl. tables"


HORMONES: Yes
- Parameters listed in "Any other information on materials and methods incl. tables"


URINANALYSIS: Yes
- Parameters listed in "Any other information on materials and methods incl. tables"


SPERM PARAMETERS: Yes
- After the organ weight determination, the following parameters were determined in the right testis or right epididymis of all male F0 parental animals and all cohort 1A males sacrificed on schedule
- Sperm parameter investigations were carried out in a randomized sequence.
- Parameters listed in "Any other information on materials and methods incl. tables"

HORMONES IN PND 4 AND 22 F1-OFFSPRING: BLOOD SAMPLING
- Blood samples were withdrawn from 10 surplus (culled) PND 4 offspring (as far as possible of different litters) per sex and group.
- PND 4 samples were pooled per sex and litter if the available amount is not sufficient for a hormone analysis.
- Blood samples were withdrawn from 10 surplus PND 22 offspring (as far as possible of different litters) per sex and group.
- The blood samples were collected after decapitation (following isoflurane anesthesia) from the Vena cava cranialis.


HORMONES IN PND 4 AND 22 F1-OFFSPRING: HORMONES
- The concentrations of TSH were determined by radioimmunoassay (RIA), using commercially available RIA test kits and a Gamma-Counter (LB 2111, Berthold, Germany).
- T4 ELISA was measured with a Sunrise MTP-reader, Tecan AG, Maennedorf, Switzerland, and evaluated with the Magellan-Software of the instrument producer.
- Parameters listed in "Any other information on materials and methods incl. tables"
Sacrifice and pathology:
PARENTAL ANIMALS:

NECROPSY
- All F0 generation parental animals were sacrificed by decapitation under isoflurane anesthesia.
- The exsanguinated animals were necropsied and assessed by gross pathology; special attention being given to the reproductive organs.
- Moribund animals were sacrificed, necropsied and assessed by gross pathology as soon as possible after their death.


ORGAN WEIGHTS
- The following weights were determined in all animals sacrificed on schedule: Anesthetized animals (final body weight), Adrenal glands (fixed), Brain, Cauda epididymis, Epididymides, Heart, Kidneys, Liver, Ovaries, Pituitary gland (fixed), Prostate (ventral and dorsolateral part together, fixed), Testes, Seminal vesicles including coagulating glands (fixed), Spleen, Thymus (fixed), Thyroid glands (with parathyroid glands) (fixed), Uterus with cervix.
- All paired organs were weighed together (left and right).


ORGAN TISSUE FIXATION
- The following organs or tissues were fixed in 4% neutral-buffered formaldehyde solution or in modified Davidson’s solution: All gross lesions, Adrenal glands, Bone marrow (femur), Brain, Cecum, Cervix, Coagulating glands, Colon, Duodenum, Epididymis, left (fixed in modified Davidson´s solution), Esophagus, Eyes with optic nerve (fixed in modified Davidson’s solution), Heart, Ileum, Jejunum (with Peyer’s patches), Kidneys, Liver, Lungs, Lymph nodes/axillary, Lymph nodes/mesenteric, Mammary gland (male and female), Ovaries (fixed in modified Davidson´s solution), Oviducts, Pancreas, Pituitary gland, Prostate, Rectum, Sciatic nerve, Seminal vesicles, Skeletal muscle, Spinal cord (cervical, thoracic and lumbar cord), Spleen, Stomach (forestomach and glandular stomach), Testis, left (fixed in modified Davidson ´s solution), Thymus, Thyroid glands (with parathyroid glands), Trachea, Urinary bladder, Uterus, Vagina, Vas deferens.
- The ovaries and eyes with optic nerve of animals that were sacrificed intercurrently were fixed in 4% neutral buffered formaldehyde solution.
- The left testis and left epididymis of all male F0 parental animals sacrificed at scheduled dates were fixed in modified Davidson’s solution, whereas the right testis and epididymis were used for sperm parameters analysis.
- For technical reasons, after about 24 hours fixation the ovaries of all F0 females of all test groups were transferred to 70% ethanol.
- The uteri of all cohabited female F0 generation parental animals were examined for the presence and number of implantation sites. The uteri of apparently nonpregnant animals or empty uterus horns were placed in 1% ammonium sulfide solutions for about 5 minutes in order to be able to identify early resorptions or implantations. Then the uteri were rinsed carefully in physiologic salt solution (0.9% NaCl). When the examinations were completed, the uteri were transferred to the Pathology Laboratory for further processing.


HISTOPATHOLOGY
- Organs listed in "Any other information on materials and methods incl. tables"
- The organs were trimmed according to the “Revised guides for organ sampling and trimming in rats and mice” (Ruehl-Fehlert et al., 2003; Kittel et al., 2004; Morawietz et al., 2004).
- The kidneys of males No. 10, 80 and 87 were exemplarily stained immunohistochemically with an antibody against alpha-2u microglobulin.
- For technical reasons, the left testis, left epididymis, and the eyes with optic nerves of all animals of the F0 parental control and high dose groups sacrificed at scheduled dates were embedded in paraplast after fixation. For the same reason, the ovaries of all F0 females in all test groups were embedded in paraplast.
- A correlation between gross lesions and histopathological findings was attempted. Special attention was given to stages of spermatogenesis in the male gonads.
- Special attention was also given to the synchrony of the morphology in ovaries, uterus, cervix, and vagina to the estrous cycle status. Whenever in the ovary the diagnosis: ”no abnormalities detected” was used, that implies that all different, stages of functional bodies (especially corpora lutea) were present and normal.
- Animals No. 119 and 194 that were sacrificed in a moribund state were processed histotechnically and assessed like control animals.
- Reproductive organs of all F0 animals suspected of reduced fertility (maited pairs Nos. 148/48 and 159/59) were subjected to histopathological investigation.




PATHOLOGICAL EXAMINATIONS OF F1 GENERATION, REARING ANIMALS, COHORT 1A:

NECROPSY
- All F1 generation, rearing animals, cohort 1A animals were sacrificed by decapitation under isoflurane anesthesia.
- The exsanguinated animals were necropsied and assessed by gross pathology; special attention being given to the reproductive organs.
- Moribund animals were sacrificed, necropsied and assessed by gross pathology as soon as possible after their death.

ORGAN WEIGHTS
- The following weights were determined in all animals sacrificed on schedule: Anesthetized animals (final body weight), Adrenal glands (fixed), Brain, Cauda epididymis, Epididymides, Heart, Kidneys, Liver, Lymph nodes, axillary (10 animals per sex per group, cohort 1A animals only), Lymph nodes, mesenteric (10 animals per sex per group, cohort 1A animals only), Ovaries, Pituitary gland (fixed), Prostate (ventral and dorsolateral part together, fixed), Testes, Seminal vesicles including coagulating glands (fixed), Spleen, Thymus (fixed), Thyroid glands (with parathyroid glands) (fixed), Uterus with cervix
- All paired organs were weighed together (left and right).

ORGAN TISSUE FIXATION
- The following organs or tissues were fixed in 4% neutral-buffered formaldehyde solution or in modified Davidson’s solution: All gross lesions, Adrenal glands, Bone marrow (femur), Brain, Cecum, Cervix, Coagulating glands, Colon, Duodenum, Epididymis, left (fixed in modified Davidson´s solution), Esophagus, Eyes with optic nerve (fixed in modified Davidson’s solution), Heart, Ileum, Jejunum (with Peyer’s patches), Kidneys, Liver, Lungs, Lymph nodes/axillary, Lymph nodes/mesenteric, Mammary gland (male and female), Ovaries (fixed in modified Davidson´s solution), Oviducts, Pancreas, Pituitary gland, Prostate, Rectum, Sciatic nerve, Seminal vesicles, Skeletal muscle, Spinal cord (cervical, thoracic and lumbar cord), Spleen, Stomach (forestomach and glandular stomach), Testis, left (fixed in modified Davidson ´s solution), Thymus, Thyroid glands (with parathyroid glands), Trachea, Urinary bladder, Uterus, Vagina, Vas deferens.
- The ovaries and eyes with optic nerve of animals that were sacrificed intercurrently were fixed in 4% neutral buffered formaldehyde solution.
- The left testis and left epididymis of all male cohort 1A animals sacrificed at scheduled dates were fixed in modified Davidson’s solution, whereas the right testis and epididymis were used for sperm parameters analysis.
- For technical reasons, after about 24 hours fixation the ovaries of all cohort 1A females of all test groups were transferred to 70% ethanol.

HISTOPATHOLOGY
- Organs listed in "Any other information on materials and methods incl. tables"
- The organs were trimmed according to the “Revised guides for organ sampling and trimming in rats and mice” (Ruehl-Fehlert et al., 2003; Kittel et al., 2004; Morawietz et al., 2004).
- The kidneys of males No. 10, 80 and 87 were exemplarily stained immunohistochemically with an antibody against alpha-2u microglobulin.
- For technical reasons, the ovaries of all animals of the cohort 1A females and high dose groups sacrificed at scheduled dates were embedded in paraplast after fixation.
- A correlation between gross lesions and histopathological findings was attempted. Special attention was given to stages of spermatogenesis in the male gonads.
- Special attention was also given to the synchrony of the morphology in ovaries, uterus, cervix, and vagina to the estrous cycle status. Whenever in the ovary the diagnosis: ”no abnormalities detected” was used, that implies that all different, stages of functional bodies (especially corpora lutea) were present and normal.
- Animals No. 119 and 194 that were sacrificed in a moribund state were processed histotechnically and assessed like control animals.

DIFFERENTIAL OVARIAN FOLLICLE COUNT IN COHORT 1A FEMALES
- A differential ovarian follicle count (DOFC) was conducted in test groups 10 and 13 (cohort 1A females) according to Plowchalk et.al. (1993).
- In general, sections were prepared with 2 - 3 μm thickness and serial sections were taken every 100 μm to complete up to 15 – 20 cut levels across the whole ovarian tissue. For the counting of primordial and growing follicles, H&E
stained slides were prepared from all cut levels. Counting was performed on slides digitalized with a Hamamatsu NanoZoomer 2.0 slide scanner using the Hamamatsu viewing software (NDP.view).



PATHOLOGICAL EXAMINATIONS OF F1 GENERATION, REARING ANIMALS, COHORT 1B:

NECROPSY
- All cohort 1B animals were sacrificed by decapitation under isoflurane anesthesia.
The exsanguinated animals were necropsied and assessed by gross pathology; special attention was given to the reproductive organs.

ORGAN WEIGHTS
- The following weights were determined in all animals sacrificed on schedule: Anesthetized animals (final body weight), Adrenal glands (fixed), Cauda epididymis, Epididymides, Liver, Ovaries, Pituitary gland (fixed), Prostate (ventral and dorsolateral part together, fixed), Testes, Seminal vesicles including coagulating gland (fixed), Uterus (with cervix)
- All paired organs were weighed together (left and right)

ORGAN/TISSUE FIXATION
- The following organs or tissues were fixed in 4% neutral-buffered formaldehyde solution or in modified Davidson’s solution: All gross lesions, Adrenal glands, Cervix uteri, Coagulating glands, Epididymis, left (fixed in modified Davidson ´s solution), Liver, Ovaries (fixed in modified Davidson´s solution), Pituitary gland, Prostate, Seminal vesicles including coagulating glands, Testis (fixed in modified Davidson ´s solution), Uterus, Vagina

HISTOPATHOLOGY
- Histotechnical processing and examination by light microscopy was not performed.
- For technical reasons, the ovaries of all cohort 1B females of all test groups were embedded in paraplast.




PATHOLOGICAL EXAMINATIONS OF SURPLUS F1 GENERATION PUPS ON PND 22 (F1 weanlings not selected for cohorts):

NECROPSY
- All surplus F1 generation pups that were not used for the following organ weight determinations were sacrificed under isoflurane anesthesia with CO2.
- The selected pups for organ weight determination were sacrificed by decapitation under isoflurane anesthesia.
- All animals were necropsied and assessed by gross pathology with special emphasis on the reproductive organs.

ORGAN WEIGHTS
- The following weights were determined in up to 10 animals per sex per group sacrificed on schedule: Anesthetized animals (final body weight), Brain, Spleen, Thymus (fixed)

ORGAN/TISSUE FIXATION
- The following organs or tissues of up to 10 animals per sex per group were fixed in 4% neutral- buffered formaldehyde solution: All gross lesions, Brain, Mammary gland (male and female), Spleen, Thymus, Thyroid glands
- Liver samples of the control surplus F1 generation pups on PND 22 were taken and deep frozen. The livers of these animals were not previewed for any further examination in this study. Liver sampling has no impact on the study results.

HISTOPATHOLOGY
- Histotechnical processing and examination was not performed

Statistics:
Means, medians and standard deviations of each test group were calculated for several parameters (see "Any other information on materials and methods incl. tables").

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
PARENTAL ANIMALS:

CLINICAL OBSERVATIONS

Clinical observations for males and females (except gestation and lactation period)

- No clinical signs or changes of general behavior, which may be attributed to the test substance, were detected in any of the male and female F0 parental animals in any of the groups.
- One mid-dose female animal (No. 156 - 3750 ppm) showed opacity on the right eye during premating days 56 - 74 and one high-dose female animal (No. 200 - 12500 ppm) showed protruding eyeball during premating days 49 - 74. This observation was not considered to be associated with the test compound.


Clinical observations for females during gestation of F1 litters

- There were no test substance-related clinical findings in any females of all dose groups during the gestation period for F1 litter.
- One female (No. 119 - control) of test group 00 showed palpable mass through the skin at axillary region during GD 9 to 21.
- One female (No. 156 - 3750 ppm) showed opacity on the right eye and one female (No. 200 - 12500 ppm) showed protruding eyeball during the whole gestation period.
- One sperm negative female of the low-dose group (No. 148 - 1250 ppm) and one sperm positive female of the mid-dose group (No. 159 - 3750 ppm) did not deliver F1 pups. This observation was not considered to be associated with the test compound.

Clinical observations for females and offspring during lactation of F1 litters
- One female (No. 119 - control) of test group 00 showed palpable mass through the skin at axillary region and severe poor general condition, therefore the animal was sacrificed in a moribund state on PND 22.
- One female (No. 137 - 1250 ppm) of test group 01 showed pups not properly nursed on PND 0 and had complete litter loss on PND 1.
- One female (No. 154 - 3750 ppm) of test group 02 showed an injury on the right side of inguinal region on PND 23, therefore the animal was scheduled sacrificed slightly earlier on PND 24.
- One female (No. 156 - 3750 ppm) showed opacity on the right eye during the whole lactation period.
- One female (No. 171 - 3750 ppm) of test group 02 showed a diffuse injury, diffuse skin lesion and moderate poor general condition between PND 6 to 25, therefore the animal was sacrificed in a moribund state on PND 25.
- One female (No. 176 - 12500 ppm) of test group 03 showed pups not properly nursed on PND 0 to PND 1 and had complete litter loss on PND 2.
- One female (No. 192 - 12500 ppm) of test group 03 showed pups not properly nursed on PND 0.
- One female (No. 194 - 12500 ppm) of test group 03 showed respiration sounds, moderate/severe poor general condition, pups not properly nursed, moderate reduced nutritional condition, piloerection, high stepping gate, hypothermia, complete litter loss between PND 12 to 19, therefore the animal was sacrificed in a moribund state on PND 19.
- One female (No. 196 - 12500 ppm) of test group 03 showed moderate poor general condition, pale skin, pups palpable in abdomen after delivery and complete litter loss on PND 0, therefore the animal was scheduled sacrificed slightly earlier on PND 28.
- One female (No. 200 - 12500 ppm) showed protruding eyeball during the whole lactation period.


DETAILED CLINICAL OBSERVATIONS
- No additional clinical signs or changes of general behavior, which were attributed to the test substance, were detected in any of the male and female animals in any of the groups which were not observed during the general clinical examinations before.
- Male animals of all dose groups (1250, 3750 and 12500 ppm) did not show any abnormalities.
- One female animal (No. 156) of test group 02 (3750 ppm) showed opacity on the right eye from premating day 56 until sacrificed.
- One female animal (No. 200) of test group 03 (12500 ppm) showed protruding eyeball on the right eye from premating day 49 until sacrificed.
- One control female (No. 119) showed palpable mass through the skin on the left axillary region during DCO days 84 – 112 and severe poor general condition on DCO day 119.
- One mid-dose female (No. 171) showed diffuse injury on animal body during DCO days 112 - 119 and diffuse skin lesions on DCO day 119.
- One female animal (No. 194) of test group 03 (12500 ppm) showed moderate poor general condition and reduced nutritional condition, respiration sound and pups not properly nursed on study day 112.
- One female animal (No. 196) of test group 03 (12500 ppm) showed moderate poor general condition, pale skin and pups palpable in abdomen after delivery on study day 105.
- These observations were not considered to be associated with the test compound.

PUPS:
- There was no test substance-related adverse clinical sign observed in any of the surviving F1 generation pups of the different test groups.

F1 REARING ANIMALS, COHORT 1A:
- No clinical signs or changes of general behavior, which may be attributed to the test substance, were detected in any of the male and female animals in any of the groups.

F1 REARING ANIMALS, COHORT 1B:
- No clinical signs or changes of general behavior, which may be attributed to the test substance, were detected in any of the male and female animals in any of the groups.
Mortality:
mortality observed, treatment-related
Description (incidence):
PARENTAL ANIMALS
Female animal No. 119 of test group 0 (control; 0 ppm) was sacrificed moribund on PND 22, female animal No. 171 of test group 02 (3750 ppm) was sacrificed moribund on PND 25 and female animal No. 194 of test group 03 (12500 ppm) was sacrificed moribund on PND 19.

F1 PUBS
- The viability index indicating pup survival during early lactation (PND 0 - 4) varied between 99% / 96% / 99% and 90% in test groups 00 - 03. The value of the high dose was slightly outside of the historical control range 94-100.0% (see Part III, Supplement).
- The survival index indicating pup survival on PND 4 - 21 (lactation index) was 100% / 100% / 100% and 95% in test groups 00 - 03. The value of the high dose was slightly outside of the historical control range 95.7-100.0%. On postnatal day 21 significant lower number of mean pups were observed 8.7 pups per litter vs 9.9 pups per litter in control.

F1 REARING ANIMALS, COHORT 1A
- There were no test substance-related or spontaneous mortalities in any of the groups.

F1 REARING ANIMALS, COHORT 1B
- There were no test substance-related or spontaneous mortalities in any of the groups.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
PARENTAL ANIMALS
- Body weights and body weight change of all test substance treated male and female F0 rats were essentially comparable to the concurrent control values throughout the entire study.
- The statistically significantly decreased body weight change in the high-dose males during premating days 14 - 21 and increased during premating days 21 - 28, as well as increased body weight change in the mid- dose males during premating days 49 - 56, respectively, were considered to be spontaneous in nature and not treatment-related.
- The statistically significantly decreased body weight change in the high-dose females during premating days 49 - 56, as well as decreased body weight change in the high- dose females during gestation days 7 - 14 respectively, were considered to be spontaneous in nature and not treatment-related

F1 PUPS
- The mean body weights and body weight change of all male and female pups in all test substance-treated groups were comparable to the concurrent control values throughout the entire study.

F1 REARING ANIMALS, COHORT 1A
- The mean body weights/body weight change of all test substance-treated F1 male and female animals were comparable to the concurrent control values throughout the entire study.

F1 REARING ANIMALS, COHORT 1B
- The body weights of the mid-dose females were statistically significantly increased to the control values between study day 21 and 28 of the inlife period (up to 5%). This finding without dose dependency was considered as incidental and not related to treatment.
- The body weight gain of all test substance treated male and female F1 rats was comparable to the concurrent control values throughout the entire study.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
PARENTAL ANIMALS:
FOOD CONSUMPTION
- During lactation the food consumption was only decreased in female animals of test group 03 (12500 ppm) between PND 10 to 14 (-10.8%) and but over the whole lactation period only slightly and non-statistically significantly reduced (-8.2%).
- Food consumption of the low- and mid-dose animal rats was comparable to the concurrent control values throughout the entire study.

INTAKE OF TEST SUBSTANCE
- See "Any other information on results incl. tables"

F1 REARING ANIMALS, COHORT 1A
- Food consumption of the all test substance-treated male and female rats (1250, 3750 and 12500 ppm) was comparable to the concurrent control values throughout the entire study of F1.

F1 REARING ANIMALS, COHORT 1B
- Food consumption of the all test substance-treated male and female rats (1250, 3750 and 12500 ppm) was comparable to the concurrent control values throughout the entire study of F1.

COMPOUND INTAKE
- Tables see "Any other information on results incl. tables"
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
PARENTAL ANIMALS:
No treatment-related changes among hematological parameters were observed. At the end of the administration period in F0 females of test groups 02 and 03 (3750 and 12500 ppm) hemoglobin and hematocrit values as well as mean corpuscular volume (MCV) and mean corpuscular hemoglobin content (MCH) were significantly decreased. In females of test group 01(1250 ppm) hematocrit values were already significantly lower compared to controls. However, all values were within historical control ranges (F0 females, hematocrit 0.421-0.470 L/L, hemoglobin 8.9-10.0 mmol/L, MCV 52.7-54.5 fL; MCH 1.11-1.18 fmol). In females of test group 02, prothrombin time (Hepatoquick’s test, HQT) was significantly reduced, relative neutrophil counts were significantly increased, and relative lymphocyte counts were significantly decreased. However, the changes were not dose dependent. Therefore, the mentioned alterations in this paragraph were regarded as incidental and not treatment related.

F1 GENERATION
- No treatment-related changes among hematological parameters were observed.
- At study day 90, in males of test group 13 (12500 ppm) prothrombin time (Hepatoquick’s test, HQT) was significantly prolonged, but the mean was within the historical control range (F1 males, HQT 32.6-38.3 sec). In F1 females of test group 11 (1250 ppm) relative eosinophil counts were significantly increased, but the alteration was not dose dependent. Therefore, the mentioned changes were regarded as incidental and not treatment related.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
PARENTAL ANIMALS:
No treatment-related changes among clinical chemistry parameters were observed.
At the end of the administration period, in F0 females of test groups 02 and 03 (3750 and 12500 ppm), total bilirubin values were significantly decreased, but the change was not dose dependent. Therefore, this change was regarded as incidental and not treatment related.

In parental males and females (test groups 01, 02 and 03; 1250 3750 and 12500 ppm) no treatment-related alterations of T4 and TSH levels were observed.

F1 GENERATION
- No treatment-related, adverse changes among clinical chemistry parameters were observed.
- In females of test group 13 (12500 ppm) sodium levels were significantly decreased. The values were marginally below the historical control range (F1 females, sodium 141.7-144.0 mmol/L). However, this was the only changed clinical chemistry parameter among these individuals and therefore, it was regarded as maybe treatment-related, but non-adverse (ECETOC Technical Report No. 85, 2002).
- In males of test group 12 (3750 ppm) chloride levels were significantly lower compared to study controls, and in females of test group 11 (1250 ppm) total bilirubin levels were significantly decreased. However, the alterations were not dose dependent. Therefore, the mentioned changes were regarded as incidental and not treatment related.
- In F1 male PND4 as well as male and female PND22 pups (test groups 01, 02 and 03; 1250, 3750 and 12500 ppm) as well as in F1A adult males and females (test groups 11, 12 and 13; 1250, 3750 and 12500 ppm) no treatment-related alterations of T4 and TSH levels were observed.
- In female PND4 pups of test group 03 (12500 ppm) T4 values were significantly increased. However, T4 as well as TSH mean in this group was within historical control ranges (PND4 females, T4 17.88-53.89 nmol/L, TSH 3.05-7.58). Therefore, the T alteration in female PND4 pups was regarded as incidental and not treatment related.
Urinalysis findings:
no effects observed
Description (incidence and severity):
PARENTAL ANIMALS
- No treatment-related changes among urinalysis parameters were observed.

F1 GENERATION
- No treatment-related changes among urinalysis parameters were observed.
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
PARENTAL ANIMALS
- See "Any other information on results incl. tables"
- The significantly increased absolute weight of the kidneys in females of test group 03 (1.839 g) was within the historical control range (1.550 – 1.959 g) and showed no histopathological correlate. It was regarded therefore not as treatment-related. The minimal but significant relative weight increase of the kidneys in males of test group 03 (0.648%), although still within the historical control range (0.569 – 0.680%), might reflect a minimal increase of eosinophilic droplets noted in the cortical tubular cells. The significant relative weight increase of the liver (2.326%) was within the historical control values (2.13 – 2.49%) and occurred without histopathological changes. Therefore, it was considered not treatment-related.

F1 REARING ANIMALS, COHORT 1A
- When compared with control group 10 (=100%), the kidney mean absolute weights were significantly changed (see "Any other information on results incl. tables")
- The significant absolute weight increase of the kidneys in females of test groups 12 and 13 (1.589 and 1.585 g, respectively) was marginally above the historical control range (1.305 –1.542 g). Since neither relative weight deviations nor histopatological changes occurred in the kidneys, this finding was assessed as not treatment-related.


F1 REARING ANIMALS, COHORT 1B
- see "Any other information on results incl. tables"
- The significantly increased terminal body weight occurred without dose dependency. The significantly increased absolute weights of the adrenal glands, although minimally above the historical control values, occurred without statistical deviation in the relative weight, and the liver weight increases were within the historical control ranges (see Part III, supplement). The significant decreases of the absolute and relative weights of the pituitary gland occurred without dose-dependency and were still within the historical control ranges (absolute: 10.840 – 11.400 mg; relative: 0.005 – 0.006%). Therefore, all these changes were not regarded as treatment-related.

SURPLUS F1 GENERATION PUPS ON PND 22 (F1 weanlings not selected for cohorts)
- see "Any other information on results incl. tables"
- The significant absolute and relative spleen weight increase of females in test group 02 occurred without a dose-response relationship and was considered incidental and not treatment-related.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
PARENTAL ANIMALS:
- All findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.
- Fertility: The female animals No. 148 and 159, which were not pregnant as well as the male mating partners No. 48 and 59 did not show relevant gross lesions.

PUP NECROPSY OBSERVATION
- A few F1 pups showed spontaneous findings at gross necropsy, such as partly cannibalized, post-mortem autolysis, discolored testis, discolored liver lobe, discolored lung, skin lesion (biting wound from the mother) and empty stomach.
- Male pup no. 05 from animal No. 149 (test group 01 - 625 ppm) showed bilateral slightly malrotated limb towards the center. Male pup no. 07 from animal No. 182 (test group 03 - 6250 ppm) showed small tongue and small mandible in addition of the finding to the stained skeleton. Male pups nos. 04 - 09 and female pup no. 12 from animal No. 192 (test group 03 - 6250 ppm) showed right or bilateral slightly malrotated limb towards the center. Male pup no. 02 and female pup nos. 04 and 05 from animal No. 196 (test group 03 - 6250 ppm) showed left or bilateral slightly malrotated limb towards the center.
- The malrotated limbs observed in 10 pups of two litters in the high dose group were considered as treatment-related. The single finding of malrotated limbs in the low dose group with no corresponding finding in the mid dose group was considered as incidental and not related to treatment. All other findings occurred without any relation to dosing and/or can be found in the historical control data at comparable or even higher incidences and were not considered to be associated with the test substance.

F1 REARING ANIMALS, COHORT 1A
- All findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.
- Decedents: No decedent observed.

F1 REARING ANIMALS, COHORT 1B
- All findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.
- Decedents: No decedent observed.

SURPLUS F1 GENERATION PUPS ON PND 22 (F1 weanlings not selected for cohorts)
- No gross changes were observed at necropsy.
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
PARENTAL ANIMALS
- Treatment-related findings were observed in the kidneys of male animals with incidences and grading given in the table in "Any other information on results incl. tables"
- Eosinophilic droplets, representing proteinaceous material, were found in the cytoplasm of proximal tubular cells in all test groups including control animals. Compared to the control animals, a slight dose-dependent increase in incidence and grading was noted starting in test group 02 up to test group 03, which was considered treatment-related. This change was characterized by an increase of droplet number, size and distribution. An immunhistochemical stain, performed exemplarily in animals 10, 80 and 87, revealed that the staining pattern was different to alpha 2u staining pattern.
This finding was not associated with tubular cell injury and was therefore considered treatment- related but not adverse.
- All other findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.
- Fertility: The female animal No. 148 and 159, which were not pregnant as well as the male mating partners No. 48 and 59 did not show relevant histopathological findings consistent with impaired fertility.
- Decedents: The female animal Nos. 119, 171 and 194 were sacrificed moribund. The female No. 119 showed macroscopically a mass (diameter 75 mm) in the skin of the axillary region which correlated with a spontaneous malignant basal cell tumor and was the cause of the moribund state. The female No. 171 showed in the skin of the head and in the abdominal and back region many lesions with incrusted surface, which correlated with multifocal ulcers and crusts. The axillary lymph nodes displayed a neutrophilic inflammation, sinus histiocytosis, increased plasma cells and sinus dilation. These findings were consistent with macroscopically enlarged axillary lymph nodes and represented a reactive response to the skin lesions. The skin lesions most likely contributed to the moribund state of this animal. Additionally, a slight hyperplasia of the mammary gland was noted. The female No. 194 showed in the thymus a massive decreased cellularity of the cortex and medulla and a moderate atrophy of the genital organs (uterus, vagina and interstitial glands in the ovary). These signs reflected the moribund state in this female but were not considered the cause of morbidity. Animal Nos. 119 and 194 also displayed also displayed a decreased cellularity in the mesenteric lymph node (cortical and paracortical lymphocytes).


F1 REARING ANIMALS, COHORT 1A
- See "Any other information on results incl. tables"
- Eosinophilic droplets, with the same characteristics as in the kidneys of the F0 generation males, were seen in all test groups including control animals. Compared to the control animals, a dose-dependent increase in the incidence and grading was noted in test groups 12 and 13, which was considered treatment-related. As observed in the F0 generation males, this finding was not associated with tubular cell injury and was therefore considered treatment-related but not adverse.
- All other findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.
- Differential ovarian follicle count (see "Any other information on results incl. tables*): The results of the differential ovarian follicle count (DOFC) – comprising the numbers of primordial and growing follicles, as well as the combined incidence of primordial plus growing follicles – did not reveal significant differences between the control group 10 and animals of test group 13 (12500 ppm).

F1 REARING ANIMALS, COHORT 1B
- Histotechnical processing and examination by light microscopy was not performed.

SURPLUS F1 GENERATION PUPS ON PND 22 (F1 weanlings not selected for cohorts)
- Histotechnical processing and examination by light microscopy was not performed.
Other effects:
effects observed, treatment-related
Description (incidence and severity):
PARENTAL ANIMALS:

OESTROUS CYCLE
- Estrous cycle data, generated during the last 3 weeks prior to mating to produce the F1 litter, revealed regular cycles in the females of all test groups including the control.
- The mean estrous cycle number was similar: 4.6 / 4.6 / 5.5 and 4.4 in test groups 00 - 03, respectively.
- The mean estrous cycle duration was similar: 4.0 / 3.9 / 4.0 and 4.2 days in test groups 00 - 03, respectively.

SPERM MEASURES
- Concerning motility of the sperms and the incidence of abnormal sperms in the cauda epididymidis as well as sperm head counts in the testis and in the cauda epididymidis of parental males no treatment-related effects were observed.

MALE REPRODUCTION DATA
- For all F0 parental males, which were placed with females to generate F1 pups, copulation was confirmed.
- Thus, the male mating index was 96% in test group 01 (1250 ppm) and 100% in the
remaining test groups (00, 02 - 03).
- Fertility was proven for most of the F0 parental males within the scheduled mating interval for F1 litter.
- One low dose male (No. 48 - 1250 ppm) did not generate pregnancy.
- Thus, the male fertility index ranged between 96% and 100%, reflecting the normal range of biological variation inherent in the strain of rats used for this study.
- The apparently infertile male rat did not show histopathological findings that could explain infertility.


FEMALE REPRODUCTION AND DELIVERY DATA
- The female mating index calculated after the mating period for F1 litter ranged between 96% and 100%.
- The mean duration until sperm was detected (GD 0) varied between 2.5 and 3.1 days.
- All female rats delivered pups or had implants in utero with the following exception: Test group 02, female No. 159 (mated with male No. 59) did not become pregnant.
- The apparently infertile female rats did not show histopathological findings that could explain infertility.
- The fertility index was 100% in in all test groups 00 - 03. The mean duration of gestation values varied between 22.0 and 22.1 days without any relation to dosing. The gestation index varied between 96% and 100%.
- Implantation was not affected by the treatment since the mean number of implantation sites was comparable between all test substance-treated groups and the control, taking normal biological variation into account (13.5 / 12.8 / 12.9 and 12.3 implants/dam in test groups 00 - 03, respectively). Furthermore, there were no indications for test substance-induced intrauterine embryo-/fetolethality since the postimplantation loss did not show any statistically significant differences between the groups (5.1% / 5.8% / 11.7% and 10.3% in test groups 00 – 03, respectively), and the mean number of F1 pups delivered per dam remained unaffected (12.8 / 12.1 / 12.5 and 11.2 pups/dam, respectively in test groups 00 - 03).
- The rate of liveborn pups was also not affected by the test substance, as indicated by live birth indices of 99% / 98% / 98% and 93% in test groups 00 - 03. - The incidence of the high dose group is slightly below the historical control range.
- The numbers of stillborn pups with 1.2% / 2.4% / 2.0% and 7.1% in test groups 00-03 were not statistically significantly different between the test groups and but the high dose group was outside of the historical control range of 0.0% to 5.5%.
- The higher number of stillborn pups in test group 03 were caused by one dam (No. 192) with 9 stillborn pups and one dam (No. 196) with 7 stillborn pups.


F1 ANIMALS
SEX RATIO
- The sex distribution and sex ratios of live F1 pups on the day of birth and on PND 21 did not show substantial differences between the control and the test substance-treated groups; slight differences were regarded to be spontaneous in nature.

VAGINAL OPENING
- Each female F1 pup, which was selected to become a rearing female, was evaluated for commencement of sexual maturity. The first day when vaginal opening was observed was PND 27, the last was PND 38. The mean number of days to reach the criterion in the control and 1250/625, 3750/1875 and 12500/6250 ppm test groups was 31.6; 30.9; 31.4 and 31.7 days, respectively. The mean body weight on the day, when vaginal opening was recorded, amounted to 95.3 g, 92.5 g, 97.0 g and 96.4 g in test groups 00-03. Neither a statistically significant nor a toxicologically relevant effect was noted in any of the treatment groups.

PREPUTIAL SEPARATION
- Each male F1 pup, which was selected to become a rearing male, was evaluated for commencement of sexual maturity. The first day when preputial separation was observed was PND 38, the last was PND 48. The mean number of days to reach the criterion in the control and 1250/625, 3750/1875 and 12500/6250 ppm test groups was 40.6, 40.9, 41.9** (** = p::0.01) and 41.1 days, respectively. The mean body weight on the day, when preputial separation was recorded, amounted to 171.1 g, 173.2 g, 178.3* g (* = p::0.05) and 172.6 g in test groups 00-03. All values for days and weights are well within the historical control range (40.1-45.2 days, 158.2-221.1 gram ), thus any observed statistical change is considered
incidental and not treatment-related.

ANOGENITAL DISTANCE
- Anogenital distance and anogen tal index of all test substance treated pups were comparable to the concurrent control values.

NIPPLE RETENTION IN MALE PUPS
- The percentage of male pups having nipples/areolae was not influenced by the test substance when examined on PND 13.
- During the re-examination on PND 20 no nipples/areolae were detected in any male pup of all test groups.

F1 PUB NUMBER AND STATUS OF DELIVERY
- The mean number of delivered F1 pups per dam and the rates of liveborn, stillborn and dead F1 pups were evenly distributed about the groups. The respective values reflect the normal range of biological variation inherent in the strain used in this study.
- The higher number of stillborn pups in test group 03 were caused by one dam (No. 192) with 9 stillborn pups, one dam (No. 196) with 7 stillborn pups. In test group 03 the higher number of pups they were found dead on PND 19 were caused of one dam (No. 194) with 10 pups.

F1 REARING ANIMALS, COHORT 1A - ESTROUS CYCLE
- Estrous cycle data, generated during 2 weeks, revealed regular cycles in the females of all test groups including the control. The mean estrous cycle duration in the different test groups was similar: 4.0 days in control, 3.9 days in the low dose group, 3.9 days in the mid-dose group and 4.0 days in the high-dose group.

F1 REARING ANIMALS, COHORT 1B - ESTROUS CYCLE
- Estrous cycle data, generated during 2 weeks, revealed regular cycles in the females of all test groups including the control. The mean estrous cycle duration in the different test groups was similar: 4.0 days in control, 4.0 days in the low-dose group, 3.9 days in the mid-dose group and 4.0 days in the high-dose group.

F1 GENERATION - SPERM ANALYSIS
- Concerning motility of the sperms and the incidence of abnormal sperms in the cauda epididymidis as well as sperm head counts in the testis and in the cauda epididymidis of F1A males no treatment-related effects were observed.

Effect levels

open allclose all
Dose descriptor:
NOAEL
Remarks:
general systemic toxicity
Effect level:
340 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse effects observed at this dose
Remarks on result:
other: 3750 ppm
Dose descriptor:
LOAEL
Remarks:
general systemic toxicity
Effect level:
1 129 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical biochemistry
food consumption and compound intake
mortality
Remarks on result:
other: 11250 ppm

Target system / organ toxicity

Critical effects observed:
no

Any other information on results incl. tables

ANALYSES


- Stability analyses: The stability of test substance in rat diet was demonstrated for a period of 35 days at room temperature.


- Homogeneity analyses: The homogeneity of the mixtures was verified.


- Concentration control analyses: The observed concentrations of the test substance correspond with the expected concentrations by recoveries between 90 and 96, and demonstrated the correctness of the diet preparations.


- Food analyses: With regard to the analytical findings of chemical and microbiological contaminants and the duration of application, the diet was found to be suitable. Fed. Reg. Vol. 44, No. 91 of 09 May 1979, p. 27354 (EPA), served as a guideline for maximum tolerable chemical contaminants. The concentration of microorganisms did not exceed 1*10^5/g feed.


- Drinking water analyses: On the basis of the analytical findings, the drinking water was found to be suitable. German Drinking Water Regulation (Trinkwasserverordnung) served as a guideline for maximum  tolerable contaminants.


- Bedding and enrichment analyses: On the basis of the analytical findings, bedding and cage enrichment were found to be suitable. Levels given in Lab Animal (Nov-Dec 1979, pp. 24-34) served as a guideline for maximum tolerable contaminants.


 


 


Tab. 10: Mean test substance intake of test substance (mg/kg bw/d; minimum value/maximum value) in F0 animals


 






























 



Test group 01 (1250/625 ppm)



Test group 02 (3750/1875 ppm)



Test group 03 (12500/6250 ppm)



F0 males



98.5 (72.6 / 153.6)



299.4 (217.4 / 460.7)



997.4 (736.6 / 1541.7)



F0 females (premating)



106.2 (90.6 / 140.5)



320.2 (270.4 / 438.4)



1088.0 (930.6 / 1431.3)



F0 females


*  gestation period


*  lactation period



93.4 (88.6 / 98.2)


127.3 (84.3 / 182.8)



280.2 (262.7 / 293.0)


374.9 (247.7 / 509.2)



923.3 (881.6 / 949.4)


1129.4 (741.7 / 1548.2)



 


 


Tab. 11: Mean test substance intake of test substance (mg/kg bw/d; minimum value/maximum value) in F1 rearing animals, Cohort 1A


 
























 



Test group 11


(1250 ppm)



Test group 12


(3750 ppm)



Test group 13


(12500 ppm)



Males



118.6 (81.5 / 174.0)



359.2 (241.3 / 529.2)



1192.1 (818.8 / 1758.2)



Females



121.0 (97.5 / 167.2)



364.8 (291.8 / 498.7)



1192.8 (951.2 / 1660.0)



 


 


Tab. 12: Mean test substance intake of test substance (mg/kg bw/d; minimum value/maximum value) in F1 rearing animals, Cohort 1B


 
























 



Test group 11


(1250 ppm)



Test group 12


(3750 ppm)



Test group 13


(12500 ppm)



Males



121.5 (84.9 / 171.1)



365.6 (257.7 / 512.5)



1231.7 (876.7 / 1723.1)



Females



120.6 (99.7 / 161.6)



360.7 (293.1 / 490.1)



1214.7 (994.6 / 1641.4)



 


 


Tab. 13: Mean test substance intake of test substance


 


























































































 



 



Test group 01/11 (1250 ppm;


lactation 625 ppm)



Test group 02/12 (3750 ppm;


lactation 1875 ppm)



Test group 03/13 (12500 ppm;


lactation 6250 ppm)



 



Duration of phase (weeks)



Mean (mg/kg b/d)



Mean (mg/kg b/d)



Mean (mg/kg b/d)



F0 males



 


10



 


98.5



 


299.4



 


997.4



F1 males rearing 1A



8



118.6



359.2



1192.1



F1 males rearing 1B



 


7



 


121.5



 


365.6



 


1231.7



Mean / Weighted mean of mean



 



 


112.9 / 111.4



 


341.4 / 337.1



 


1140.4 / 1125.3



F0 females premating



10



 


106.2



 


320.2



 


1088.0



F0 females gestation



3



 


93.4



 


280.2



 


923.3



F0 females lactation



3



127.3



374.9



1129.4



F1 females rearing 1A



8



 


121.0



 


364.8



 


1192.8



F1 females rearing 1B



7



 


120.6



 


360.7



 


1214.7



Mean / Weighted mean of mean



 



 


112.0 / 112.7



 


341.1 / 339.7



 


1131.9 / 1128.5



 


 


Tab. 14: F0 generation parental animals, Absolute organ weights


 






















F0



Female animals



Test group (ppm)



01


(1250)



02


(3750)



03


(12500)



Kidneys



100%



101%



107%**



* for p ≤0.05, ** for p ≤0.01


 


 


Tab. 15: F0 generation parental animals, Relative organ weights


 




























F0



Male animals



Test group (ppm)



01


(1250)



02


(3750)



03


(12500)



Kidneys



102%



100%



106%**



Liver



101%



102%



107%**



* for p ≤0.05, ** for p ≤0.01


 


 


Tab. 16: F0 generation parental animals, Histopathology findings


 













































Kidneys



Male animals (F0)



Test group (ppm)



00


(0)



01


(1250)



02


(3750)



03


(12500)



No. of animals



20



20



20



20



Eosinophilic droplets



8



10



14



18



Grade 1



8



8



8



11



Grade 2



 



2



6



7



 


 


Tab. 17: F1 generation, rearing animals, cohort 1A, Absolute organ weights


 






















Cohort 1A



Female animals



Test group (ppm)



11


(1250)



12


(3750)



13


(12500)



Kidneys



100%



105%*



105%*



for p ≤0.05, ** for p ≤0.01


 


 


 


Tab. 18: F1 generation, rearing animals, cohort 1A, Histopathology findings


 













































Kidneys



Male animals (Cohort 1A)



Test group (ppm)



10


(0)



11


(1250)



12


(3750)



13


(12500)



No. of animals



20



20



20



20



Eosinophilic droplets



9



10



19



20



Grade 1



4



6



16



8



Grade 2



5



4



3



12



 


 


Tab. 19: F1 generation, rearing animals, cohort 1A, Differential ovarian follicle count, Absolute values


 






























Number of animals



Absolute values



Group



Primordial



Growing



Primordial + growing



20



10



4069



464



4533



20



13



4116



531



4647



for p ≤0.05, ** for p ≤0.01


 


 


Tab. 20: F1 generation, rearing animals, cohort 1A, Differential ovarian follicle count, Mean values


 






























Number of animals



Mean values



Group



Primordial



Growing



Primordial + growing



20



10



203



23



227



20



13



206



27



232



for p ≤0.05, ** for p ≤0.01


 


 


Tab. 21: F1 generation, rearing animals, cohort 1B, Absolute organ weights


 








































Cohort 1B



Female animals



Test group (ppm)



11


(1250)



12


(3750)



13


(12500)



Terminal body weight



100%



105%**



104%



Adrenal glands



99%



107%



107%*



Liver



103%



107%*



107%**



Pituitary gland



90%*



90%**



90%*



for p ≤0.05, ** for p ≤0.01


 


 


Tab. 22: F1 generation, rearing animals, cohort 1B, Relative organ weights


 






















Cohort1B



Female animals



Test group (ppm)



11


(1250)



12


(3750)



13


(12500)



Pituitary gland



90%*



85%**



87%**



for p ≤0.05, ** for p ≤0.01


 


 


Tab. 23: Surplus F1 generation pups on PND 22 (F1 weanlings not selected for cohorts), Absolute organ weights


 






















F1 Pups PND 22



Female animals



Test group (ppm)



01


(1250)



02


(3750)



03


(12500)



Spleen



113%



111%*



94%



for p ≤0.05, ** for p ≤0.01


 


 


Tab. 24: Surplus F1 generation pups on PND 22 (F1 weanlings not selected for cohorts), Relative organ weights


 






















F1 Pups PND 22



Female animals



Test group (ppm)



01


(1250)



02


(3750)



03


(12500)



Spleen



112%



114%**



98%



for p ≤0.05, ** for p ≤0.01


 


 


 


 


 

Applicant's summary and conclusion

Conclusions:
Under the conditions of the present extended one-generation reproduction toxicity study the NOAEL (no observed adverse effect level) for general, systemic toxicity is 12500 ppm (about 1129 mg/kg bw/d) for males and 3750 ppm (340 mg/kg bw/d) for females, based on clinical signs of toxicity and mortality during lactation, in the F0 parental animals as well as adolescent and adult F1 offspring.
Executive summary:

The test substance was administered to groups of 25 male and 25 female healthy young Wistar rats as a homogeneous addition to the food in different concentrations (0, 1250, 3750 and 12500 ppm). F0 animals were treated at least for 10 weeks prior to mating to produce a litter (F1 generation). Mating pairs were from the same dose group. Pups of the F1 litter were selected (F1 rearing animals) and assigned to 2 different cohorts (1A and 1B) which were subjected to specific postweaning examinations. The study was terminated with the terminal sacrifice of the F1 rearing animals of cohort 1B. Test diets containing test substance were offered continuously throughout the study.


 


Observations: The parents' and the pups' state of health was checked each day, and parental animals were examined for their mating and reproductive performances. Food consumption of the F0 parents and F1 rearing animals was determined regularly once weekly and weekly during gestation (days 0 - 7, 7 - 14, 14 - 20) and lactation periods (days 1 - 4, 4 - 7, 7 - 10, 10 - 14, 14 - 18 and 18 - 21). In general, body weights of F0 parents and F1 rearing animals were determined once weekly. However, during gestation and lactation F0 females were weighed on gestation days (GD) 0, 7, 14, 20 and on postnatal days (PND) 1, 4, 7, 10, 14, 18 and 21. A detailed clinical observation (DCO) was performed in all F0 parents and F1 animals in cohorts 1A and 1B at weekly intervals. Estrous cycle data were evaluated for F0 females over a three week period prior to mating until evidence of mating occurred. In all cohort 1A females, vaginal smears were collected after vaginal opening until the first cornified smear (estrous) was recorded. The estrous cycle also was evaluated in cohort 1A and 1B females for 2 weeks around PND 75. Moreover, the estrous stage of each female was determined on the day of scheduled sacrifice. The F1 pups were sexed on the day of birth (PND 0) and were weighed on the first day after birth (PND 1) as well as on PND 4, 7, 14 and 21. Their viability was recorded. At necropsy, all pups were examined macroscopically. Date of sexual maturation, i.e. day of vaginal opening (females) or balanopreputial separation (males), of all F1 pups selected to become F1 rearing animals was recorded. All surviving pups were examined for the presence or absence of nipple/areola anlagen on PND 13 and were re-examined on PND 20. If nipple/areola anlagen were recorded, all surviving male pups were carefully re-examined one day prior to necropsy. The number of nipple/areola anlagen were counted. Anogenital distance (defined as the distance from the anus [center of the anal opening] to the base of the genital tubercle) measurements were conducted in a blind randomized fashion, using a measuring ocular on all live male and female pups on PND 1. Urine samples for clinical pathological investigations were withdrawn from 10 selected F0 and cohort 1A animals per sex and group. Blood samples for clinical pathological investigations were withdrawn from 10 selected F0 and cohort 1A animals per sex and group. Further blood samples were taken from a maximun of 10 surplus (culled) PND 4 pups per sex and group as well as from 10 surplus PND 22 pups per sex and group. Various sperm parameters (motility, sperm head count, morphology) were assessed in the F0 generation males and cohort 1A males at scheduled sacrifice or after appropriate staining. 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. A quantitative assessment of primordial and growing follicles in the ovaries was performed for all control and high-dose F1 rearing females cohort 1A. All F1 rearing animals were assessed by different pathological and histopathological examinations.


 


Analyses: The various analyses: 1.) Demonstrated the stability of the test substance preparations over a period of 35 days at room temperature, 2.) Confirmed the homogeneous distribution of the test substance in the diet, 3.) Verified correct concentrations of the test substance in the diet preparations.


Intake of test substance:  The weighted mean of mean dose of test substance throughout all study phase and across all cohorts was approx. 113 mg/kg body weight/day (mg/kg bw/d) in the 1250 ppm group, approx. 340 mg/kg bw/d in the 3750 ppm group and approx. 1129 mg/kg bw/d in the 12500 ppm group.


 


Effects: The following test substance-related and relevant effects/findings were noted:


 


12500 ppm (Substance intake overall mean of mean 1129 mg/kg bw/d)


F0 PARENTAL ANIMAL


CLINICAL EXAMINATIONS/ REPRODUCTIVE PERFORMANCE/ CLINICAL PATHOLOGY/ PATHOLOGY



  • Decresased number of live born pups in comparison to control (93% vs. 99% in control)

  • Increased number of stillborn in comparison to control (7.1% vs. 1.2% in control)

  • Pups not properly nursed in 3 females during lactation

  • Complete litter loss in 3 females during lactation

  • Pups palpable in abdomen after delivery in 1 female during lactation

  • General conditions poor in 2 females during lactation

  • Hypothermia and respiratory sounds in 1 female during lactation

  • Two females sacrificed moribund on postnatal day 19 and 28

  • Reduced food consumption in females between lactation days 10 and 14 (-10.8%)


 


F1 PUPS


CLINICAL EXAMINATIONS/ SEXUAL MATURATION/ GROSS FINDINGS



  • Decreased viability index with 90% (not statistically significant) in comparison to control (99%)

  • Decreased lactation index with 95% (not statistically significant) in comparison to control (100%)

  • Decreased mean number of surviving pups on post natal day 21 8.7 pups per litter in comparison to control (9.9 pups per litter)

  • Increased number of pups with malrotated limbs 10 out of 92


 


F1 REARING ANIMALS, COHORT 1A


CLINICAL EXAMINATIONS/ CLINICAL PATHOLOGY



  • No treatment-related relevant findings


 


F1 REARING ANIMALS, COHORT 1B


CLINICAL EXAMINATIONS/ CLINICAL PATHOLOGY/ PATHOLOGY



  • No treatment-related relevant findings


 


3750 ppm (Substance intake overall mean of mean 340 mg/kg bw/d)


F0 PARENTAL ANIMALS


CLINICAL EXAMINATIONS/ REPRODUCTIVE PERFORMANCE/ CLINICAL PATHOLOGY/ PATHOLOGY



  • No test substance-related adverse findings


 


F1 PUPS


CLINICAL EXAMINATIONS/ SEXUAL MATURATION/ GROSS FINDINGS



  • No test substance-related relevant findings


 


F1 REARING ANIMALS, COHORT 1A


CLINICAL EXAMINATIONS/ CLINICAL PATHOLOGY/ PATHOLOGY



  • No test substance-related relevant findings


 


F1 REARING ANIMALS, COHORT 1B


CLINICAL EXAMINATIONS/ CLINICAL PATHOLOGY/ PATHOLOGY



  • No test substance-related relevant findings


 


1250 ppm (Substance intake overall mean of mean 113 mg/kg bw/d)


F0 PARENTAL ANIMALS


CLINICAL EXAMINATIONS/ REPRODUCTIVE PERFORMANCE/ CLINICAL PATHOLOGY/ PATHOLOGY



  • No test substance-related relevant findings


 


F1 PUPS


CLINICAL EXAMINATIONS/ SEXUAL MATURATION/ GROSS FINDINGS



  • No test substance-related relevant findings


 


F1 REARING ANIMALS, COHORT 1A


CLINICAL EXAMINATIONS/ CLINICAL PATHOLOGY/ PATHOLOGY



  • No test substance-related relevant findings


 


F1 REARING ANIMALS, COHORT 1B


CLINICAL EXAMINATIONS/ CLINICAL PATHOLOGY/ PATHOLOGY



  • No test substance-related relevant findings


 


Conclusion: Thus, under the conditions of the present extended one-generation reproduction toxicity study the NOAEL (no observed adverse effect level) for general, systemic toxicity is 12500 ppm (about 1129 mg/kg bw/d) for males and 3750 ppm (340 mg/kg bw/d) for females, based on clinical signs of toxicity and mortality during lactation, in the F0 parental animals as well as adolescent and adult F1 offspring. The NOAEL for fertility for the parental rats is 12500 ppm (about 1129 mg/kg bw/d), the highest dose tested. The NOAEL for reproductive performance for the female parental rats is 3750 ppm (about 340 mg/kg bw/d). The NOAEL for developmental toxicity in the F1 progeny is 3750 ppm (about 340 mg/kg bw/d).