2-(2-aminoethoxy)ethanol

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

oral

Under the conditions of an OECD 422 combined repeated dose toxicity study with the reproductive/developmental screening test in Wistar rats, the no observed adverse effect level (NOAEL) for general systemic toxicity, fertility and reproductive performance and for developmental toxicity in the offspring was 15000 ppm for male (about 793 mg/kg bw/d) and female (about 1175 mg/kg bw/d) Wistar rats, the highest concentration tested. The study was performed as a dose-range-finding-study before an OECD 443. In the subsequent extended one-generation reproduction toxicity study the NOAEL for general, systemic toxicity is 340 mg/kg bw/d, 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 1129 mg/kg bw/d, the highest dose tested. The NOAEL for reproductive performance for the female parental rats is 340 mg/kg bw/d. The NOAEL for developmental toxicity in the F1 progeny is 340 mg/kg bw/d.

 

inhalation

In a combined repeated dose toxicity study with the reproduction/developmental toxicity screening test the no observed adverse effect concentration (NOAEC) for reproductive performance and fertility in male and female Wistar rats was 40 mg/m³ (aerosol). The NOAEC for local signs of toxicity in male and female Wistar rats was 4 mg/m³.

Link to relevant study records

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).

 

Effect on fertility: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

340 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Effect on fertility: via inhalation route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEC

40 mg/m³

Species:

rat

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Study duration:

subchronic

Additional information

In an OECD 422 study (BASF SE, 2022), the test 2-(2-aminoethoxy)ethanol was administered daily as a constant homogeneous addition to the food in different concentrations to groups of 10 male and 10 female Wistar rats to screen for potential systemic, reproductive and developmental toxicity. After a two-week premating period, these parental animals were paired and the females were allowed to give birth and bring up the offspring until sacrifice on PND 4 or PND 13. In males the overall mean dose of 2-(2-aminoethoxy)ethanol throughout the study was approx. 78 mg/kg body weight/day (mg/kg bw/d) in the 1500 ppm group, approx. 262 mg/kg bw/d in the 5000 ppm group and approx. 793 mg/kg bw/d in the 15000 ppm group; in females it was approx. 115 mg/kg body weight/day (mg/kg bw/d) in the 1500 ppm group, approx. 397 mg/kg bw/d in the 5000 ppm group and approx. 1175 mg/kg bw/d in the 15000 ppm group. The no observed adverse effect level (NOAEL) for general systemic toxicity, fertility and reproductive performance and for developmental toxicity in the offspring was 15000 ppm for male (about 793 mg/kg bw/d) and female (about 1175 mg/kg bw/d) Wistar rats, the highest concentration tested. This study was used as a dose-range-finder before a subsequent OECD 443.

 

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) in a study conducted accordning to OECD 443 (BASF SE, 2021). 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).

 

 

In a combined repeated dose toxicity study with the reproduction/developmental toxicity screening test according to OECD 422 (BASF SE, 2010) 2-(2-Aminoethoxy)ethanol was administered via inhalation (aerosol) to groups of 10 male and 10 female Wistar rats (F0 animals) at concentrations of 4, 16, and 40 mg/m³. For all F0 parental males, which were placed with females to generate F1 pups, copulation was confirmed. Thus, the male mating index was 100% in all test groups (0, 4, 16 and 40 mg/m³). Fertility was proven for most of the F0 parental males within the scheduled mating interval for F1 litter. One male of the 4 mg/m³ test group did not generate F1 pups. The apparently infertile male rat did not show histopathological findings that could explain infertility. Thus, the male fertility index was 90% in the 4 mg/m³ test group and 100% in all remaining groups including the control. This reflected the normal range of biological variation inherent in the strain of rats used for this study. All respective values were within the range of the historical control data of the test facility. The female mating index calculated after the mating period for F1 litter was 100% in all test groups (0, 4, 16 and 40 mg/m³). The mean duration until sperm was detected (GD 0) amounted to 3.1, 3.1, 2.7 and 3.3 days (0, 4, 16 and 40 mg/m³). All sperm positive rats delivered pups or had implants in utero with the exception of one low-dose female, that did not become pregnant. The female fertility index varied between 90% (4 mg/m³) and 100% (all other test groups). These values reflect the normal range of biological variation inherent in the strain of rats used for this study. All respective values were within the range of the historical control data of the test facility and did not show any relation to dosing. There were no corroborative histopathological findings in the sexual organs of the nonpregnant female rat. The mean duration of gestation values varied between 22.0 (test groups 4 and 16 mg/m³) and 22.2 days (test groups 0 and 40 mg/m³) without any relation to dosing. The gestation index was 100% in all test groups including the control. Implantation was not affected by the treatment since the mean number of implantation sites was comparable between all test substance-treated groups and the controls, taking normal biological variation into account (10.6, 11.1, 12.3 and 11.4 implants per dam at 0, 4, 16 and 40 mg/m³, respectively). Furthermore, there were no indications for test substance-induced intrauterine embryo-/fetolethality since the postimplantation loss did not show any significant differences between the test groups, and the mean number of F1 pups delivered per dam remained unaffected (9.3, 10.1, 11.6 and 10.7 pups per dam at 0, 4, 16 and 40 mg/m³, respectively). The rate of liveborn pups was not affected by the test substance, as indicated by live birth indices of 97% (test group 16 mg/m³) and 100% (all other test groups). Moreover, the number of stillborn pups was comparable between the test groups.Thus, the NOAEC for reproductive performance and fertility in male and female Wistar rats was 40 mg/m³.

 

 

In a subchronic dermal study following OECD test guideline 411 (Huntsman, 2002) no effects on the male and female reproductive organs were observed up to the highest dose tested (175 mg/kg/day). Application of the test substance to an intact cutaneous site for approximately 6 hours, once daily for 90 consecutive days resulted in ulceration, epidermal hyperplasia, fibrosis and inflammation at doses of 87 and 175 mg/kg bw/d. These changes represent local irritation following topical administration. Based on the results of the study, the NOAEL for local effects was at least 17 mg/kg bw/d while the systemic NOAEL was at least 175 mg/kg bw/d.

Effects on developmental toxicity

Description of key information

In a prenatal developmental toxicity study (OECD 414, GLP) on rats, the oral administration of the test substance caused maternal toxicity, such as a nonregenerative, normocytic, normochromic anemia, a disturbance of platelet homeostasis and a decrease in ALP activities, at the highest dose level of 1000 mg/kg bw/d. The NOAEL for maternal toxicity is the mid dose level of 300 mg/kg bw/d. There was no evidence for toxicologically relevant adverse effects of the test substance on fetal morphology at any dose. In conclusion, the NOAEL for prenatal developmental toxicity is 1000 mg/kg bw/d.

 

Under the conditions of a prenatal developmental toxicity study (OECD 414, GLP), the oral administration of the test substance to pregnant New Zealand White rabbits from implantation to one day prior to the expected day of parturition (GD 6-28) at doses as high as 200 mg/kg bw/d caused neither evidence of maternal nor developmental toxicity. In conclusion, the no observed adverse effect level (NOAEL) for maternal and prenatal developmental toxicity is 200 mg/kg bw/d.

 

In an inhalatory (aerosol) combined repeated dose toxicity study with the reproduction/developmental toxicity screening test (OECD422, GLP) the no observed adverse effect concentration (NOAEC) for developmental toxicity in male and female Wistar rats was 40 mg/m³. The NOAEC for local signs of toxicity in male and female Wistar rats was 4 mg/m³.

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Oct 2020 - May 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

25 Jun 2018

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

Version / remarks:

Aug 1998

Qualifier:

according to guideline

Guideline:

EU Method B.31 (Prenatal Developmental Toxicity Study)

Version / remarks:

Commission Regulation (EC) No 440/2008, 30 May 2008

GLP compliance:

yes (incl. QA statement)

Remarks:

Landesamt für Umwelt, Kaiser-Friedrich-Straße 7, 55116 Mainz

Limit test:

no

Specific details on test material used for the study:

- CAS No.: 929-06-6
- Batch No.: 22057368E0
- Purity: 99.4 area % corrected with the water content
- Identity: confirmed
- Homogeneity: given
- Storage stability: The stability of the test substance under storage conditions over the test period was guaranteed by the sponsor.
- Expiry date: 06 Apr 2021
- appearance: liquid / colorless, clear
- Storage conditions: Room temperature/under N2


Test substance preparations:
- Stability analysis: The stability of the test substance in deionized water at room temperature over a period of 7 days was demonstrated prior to the start of the study.
- Homogeneity analyses of the test substance preparations: Given that the test substance is completely miscible with deionized water, solutions are considered to be homogenous without further analysis.

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH.
- Age at study supply: 14-17 weeks of age.
- Age at study initiation: 15 - 18 weeks of age.
- Weight at study initiation: 3757 - 3814 g.
- Housing: During the acclimatization and study period, the rabbits were housed singly in Type 4X03B700CP cages supplied by TECNIPLAST Deutschland GmbH, Hohenpeißenberg, Germany (floor space 4264 mm², internal height 450 mm). For enrichment, wooden gnawing blocks were added (Typ SAFE® gnawing block), supplied by Söhne GmbH + Co KG, Rosenberg, Germany).
- Diet: ad libitum.
- Water: ad libitum.
- Acclimation period: 7 days
- Only animals free from clinical signs of disease were used for the investigations.
- Identification: ear tattoo.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17-21°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 6.00 h).

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

TEST SUBSTANCE PREPARATION AND PREPARATION FREQUENCY:
- The test substance preparations were prepared at the beginning of the administration period and thereafter at intervals, which took into account the period of established stability. The preparations were kept at room temperature.
- For the test substance preparation, the specific amount of the test substance was weighed, topped up with deionized water in a calibrated beaker and intensely mixed with a magnetic stirrer until it was completely dissolved.
- Before and during administration, the preparations were kept homogeneous with a magnetic stirrer.

EXPERIMENTAL EXPOSURE:
- During the acclimatization period the animals were assigned to the different test groups according to a randomization plan (NIJENHUIS and WILF) and on the basis of their body weights.
- The day of insemination was designated as GD 0 (beginning of the study) and the following day as GD 1.
- Based on the pregnant animals the body weight on GD 0 varied between 3342 – 4300 g.
- The test substance was administered to the animal orally by gavage from implantation to one day prior to the expected day of parturition (GD 6-28) always at approximately the same time of day. The animals of the control group were treated in the same way with the vehicle (deionized water). The volume administered each day was 10 mL/kg body weight. The calculation of the administration volume was based on the most recent individual body weight.
- On GD 29, the females were euthanized in randomized order and examined. The fetuses were removed from the uterus and investigated.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The results of the analyses of the test substance preparations in deionized water confirmed the correctness of the prepared concentrations. The measured concentrations of the samples of treatment groups corresponded to the expected values within the limits of the analytical method, i.e. were above 90% and below 110% of the nominal concentrations.

Details on mating procedure:

After an acclimatization period of at least 5 days, the does were fertilized by means of artificial insemination. A synthetic hormone (0.2 mL), which stimulates release of LH and FSH from the anterior pituitary lobe (Receptal®) was injected intramuscularly to the female rabbits about 1 hour before insemination. The ejaculate samples used for the artificial insemination were obtained from male New Zealand White rabbits of the same breed as the females. Each female was inseminated with the sperm of a defined male donor as documented in the raw data. The male donors were kept under conditions (air conditioning, diet, water) comparable to those of the females participating in this study.

Duration of treatment / exposure:

23 days

Frequency of treatment:

daily

Duration of test:

Complete duration of the test (all cohorts): 43 days

Dose / conc.:

20 mg/kg bw/day (actual dose received)

Dose / conc.:

70 mg/kg bw/day (actual dose received)

Dose / conc.:

200 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

25

Details on study design:

- Dose selection rationale: In a previous test study , the test substance was tested orally by gavage at dose levels of 1000, 600, 300 and 0 mg/kg bw/d in each three non- pregnant female New Zealand White rabbits. At 1000 mg/kg bw/d, one female was found dead on study day 4 and clinical findings (reduced/no feces) were observed. Mean food consumption was statistically significantly reduced. High-dose females consumed remarkably less food than controls (95% below control during study days 3-4). For animal welfare reasons, this dose group was terminated. At 600 mg/kg bw/d, one female was found dead on study day 12. Females showed also reduced/no defecation and a reduction in mean food consumption (around about half of the controls, range of 61-116 g) throughout the study. Body weight was decreased in the beginning of the study but recovered in the remaining two females from study day 12 onwards. At 300 mg/kg bw/d, no clinical signs were observed. Mean food consumption was reduced in the beginning of the study (first week: up to 43% below control during study days 0-1) but recovered towards the end of the study to almost control level. Body weight was almost comparable to controls. At necropsy, erosions and ulcerations were observed in the stomach of several treated females. Ulcerations were seen in three high-dose and two mid-dose females. At 300 mg/kg bw/d, one female showed an erosion. Based on the above-mentioned findings, dose levels of 300 (as half-lethal dose) and 100 mg/kg bw/d were used in the following maternal toxicity study in pregnant rabbits. At 300 mg/kg bw/d, food consumption was reduced during GD6-28 (11%) with a maximum of 27% below control during GD15-16. Correspondingly, body weight change was decreased during GD 14-16 (17g versus 68g in controls). At 100 mg/kg bw/d, food consumption and body weight were not affected. At necropsy, foci in the glandular stomach
were seen in three out of five high-dose and one low-dose female. Based on the above-mentioned findings at 300 mg/kg bw/d and at request of the sponsor, the following doses were chosen for the present prenatal developmental toxicity study in New Zealand White rabbits: 20, 70, 200 mg/kg bw/day.
- Exposure roue rationale: The oral route was selected since this has proven to be suitable for the detection of a toxicological hazard.
- Reason for species selection: The strain was selected since historical control data is available from the test facility for New Zealand White rabbits. This specific strain has been proven to be sensitive to substances with a teratogenic potential.

Maternal examinations:

CLINICAL EXAMINATIONS OF THE DOES
- Mortality: A check was made twice a day on working days or once a day on Saturdays, Sundays or on public holidays (GD 0-29).
- Clinical symptoms: A cage-side examination was conducted at least once daily for any signs of morbidity, pertinent behavioral changes and signs of overt toxicity. During the administration period (GD 6-28) all animals were checked daily for any abnormal clinical signs before the administration as well as within 5 hours after the administration.
- Food consumption: The consumption of food was recorded daily during GD 0-29.
- Body weight data: All animals were weighed on GD 0, 2, 4, 6, 9, 11, 14, 16, 19, 21, 23, 25, 28 and 29. The body weight change of the animals was calculated based on the obtained results.
- Corrected (net) body weight gain: Furthermore, the corrected body weight gain was calculated after terminal sacrifice (terminal body weight on GD 29 minus weight of the unopened uterus minus body weight on GD 6).
- Blood samples for non-GLP research purposes: Blood samples were collected for external research projects beyond the scope of this study and without GLP status. The results of the study were not influenced by this procedure because blood sampling occurred just prior to sacrifice/at necropsy. The sampling procedures did not affect the outcome and compliance of this GLP study. The data from these research projects did not affect the outcome, assessment and compliance of this GLP study.


TERMINAL EXAMINATIONS OF THE DOES
- Cesarean section: On GD 29, the surviving does were euthanized in randomized order by intravenous injection of pentobarbital (Narcoren®; dose 2 mL/animal). After exsanguination, the animals were necropsied and assessed by gross pathology. The uteri and the ovaries were removed and the following data were recorded:
- Weight of the unopened uterus
- Number of corpora lutea
- Number and distribution of implantation sites classified as:
• Live fetuses
• Dead implantations:
a) Early resorptions (only decidual or placental tissues visible or according to SALEWSKI from uteri from apparently non-pregnant animals and the empty uterus horn in the case of single horn pregnancy)
b) Late resorptions (embryonic or fetal tissue in addition to placental tissue visible)
c) Dead fetuses (hypoxemic fetuses which did not breathe spontaneously after the uterus had been opened)
- After the weight of the uterus had been determined, all subsequent evaluations of the does (except of gross pathology including organ sampling and weights) and the gestational parameters were conducted by technicians unaware of treatment group in order to minimize bias. For this purpose animal numbers were encoded.
These data were used to calculate conception rate and pre- and postimplantation losses for each individual pregnant animal which underwent scheduled sacrifice according to the following formulas:

Conception rate (%) = number of pregnant animals x 100 / number of fertilized animals

Preimplantation loss (%) = (number of corpora lutea – number of implantations) x 100 / number of corpora lutea

Postimplantation loss (%) = (number of implantations – number of live fetuses) x 100 / number of implantations


PATHOLOGY
- Necropsy: On GD29 all surviving animals were sacrificed by an intravenous injection of pentobarbital (e.g. Narcoren®; dose: 2mL/animal) in a randomized sequence. The exsanguinated animals were necropsied and assessed by gross pathology, special attention being given to the reproductive organs.
- Organ/tissue fixation: The following organs or tissues were fixed in 4% neutral buffered formaldehyde solution:
1. All gross lesions
2. Stomach
No further examinations or procedures were performed in the study.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes.data
Examinations included:
- Gravid uterus weight: Yes.
- Number of corpora lutea: Yes.
- Number of implantations: Yes.
- Number of early resorptions: Yes.
- Number of late resorptions: Yes.

Fetal examinations:

EXAMINATIONS OF THE FETUSES
- All fetal analyses were conducted by technicians unaware of the treatment group, in order to minimize bias.
- Examinations of the fetuses after dissection from the uterus: At necropsy each fetus was weighed and examined macroscopically for external findings. Furthermore, the viability of the fetuses and the condition of placentas, umbilical cords, fetal membranes, and fluids were examined. Individual placental weights were recorded. Thereafter, the fetuses were sacrificed by an intraperitoneal injection of pentobarbital (Narcoren®; dose: 0.2 mL/fetus; one part Narcoren® diluted with one part physiological saline).
- Soft tissue examination of the fetuses: After the fetuses had been sacrificed, the abdomen and thorax were opened in order to examine the organs in situ before they were removed. The heart and the kidneys were sectioned in order to evaluate the internal structure. The sex of the fetuses was determined by examination of the gonads in situ. After these examinations, the heads of approximately one half of the fetuses per doe (and the heads of any fetus which revealed severe findings during the external examination, e.g. anophthalmia, microphthalmia or hydrocephalus) were severed from the trunk. These heads were fixed in BOUIN’s solution and were, after fixation, processed and evaluated according to WILSON’s method (WILSON and WARKANY). About 10 transverse sections were prepared per head. After the examination the heads were discarded. All fetuses (including those without heads) were skinned and fixed in ethyl alcohol. After fixation for approx. 1-5 days, the intact fetuses were removed from the fixative and a transversal incision was made into the frontal/parietal head bones. The two halves of the calvarium were cautiously bent outward and the brain was thoroughly examined. Subsequently, these fetuses were placed back into the fixative for further fixation.
- Skeletal examination of the fetuses: After fixation in ethyl alcohol, the skeletons were stained according to a modified method of KIMMEL and TRAMMELL. The stained skeletons were placed on an illuminated plate, investigated and archived individually.
- Evaluation criteria for assessing the fetuses: Classification and assessment of fetal findings is a matter of ongoing discussion (see e.g. BELTRAME and GIAVINI, CHAHOUD, SOLECKI). Despite considerable efforts to harmonize the
nomenclature used to describe observations of fetal morphology, the terms still vary considerably between laboratories, investigators and textbooks in the fields of teratology and developmental toxicity.
- In the present study the internationally harmonized glossary of WISE et al. and the updated version MAKRIS et al. was essentially used to describe findings in fetal morphology.Classification of these findings was based on the terms and definitions proposed by CHAHOUD and SOLECKI:
- Malformation: A permanent structural change that is likely to adversely affect survival or health.
- Variation: A change that also occurs in the fetuses of control animals and/or is unlikely to adversely affect survival or health. This includes delays in growth or morphogenesis that have otherwise followed a normal pattern of development.
- The term "unclassified observation" was used for those fetal findings, which could not be classified as malformations or variations.

Statistics:

See "Any other information on materials and methods incl. tables"

Historical control data:

Yes.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

One high-dose doe (No. 78 - 200 mg/kg bw/d) was sacrificed moribund before treatment on GD 21 after showing several clinical findings on the respective day of
sacrifice or the days before: reduced defecation (GD 17-18), no defecation (GD 20-21), blood in bedding (GD 20-21), hypothermia (GD 21) and a poor general state (GD 21). Furthermore, one control doe (No. 5) had blood in bedding before/after treatment on GD 24.
In total, reduced defecation was observed in seven control, nine low-dose, five mid dose and twelve high-dose females (0, 20, 70 and 200 mg/kg bw/d). No defecation was observed in two control females and in one female, each, of the low-dose and high-dose groups. Incidence and distribution of these findings do not indicate a relationship to the test substance.
There were no further clinical findings in the other does in this study.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

One high-dose female (No. 78 - 200 mg/kg bw/d) was sacrificed moribund on GD 21 (before treatment) for animal welfare reasons. The animal showed numerous clinical findings such as no/reduced defecation, blood in bedding, hypothermia and poor general state. Since only one single animal in the high-dose group was affected and the above-mentioned findings can be also seen in control animals, this was assessed as not related to treatment and as incidental.
There were no further substance-related or spontaneous mortalities in any female of all test groups (0, 20, 70 or 200 mg/kg bw/d).

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

The mean body weights and the average body weight gain of the low-, mid- and high-dose groups (20, 70 and 200 mg/kg bw/d) were generally comparable to the concurrent control group throughout the entire study period.
The statistically significantly increased body weight gain value in test group 1 on GD 21-23 is assessed as incidental.

Mean carcass weights and the corrected body weight gain (terminal body weight on GD 29 minus weight of the unopened uterus minus body weight on GD 6) were comparable among all test groups.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

The food consumption of the low-, mid- and high-dose rabbits (20, 70 and 200 mg/kg bw/d) was comparable to the concurrent control (0 mg/kg bw/d) throughout the entire study period.
The statistically significantly reduced food consumption value in test group 1 on GD 7-8 is assessed as incidental since there was no relation to dose.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Endocrine findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

Uterus weights
- The mean gravid uterus weight of the rabbits of test groups 1-3 (20, 70 or 200 mg/kg bw/d) was not influenced by the test substance. The differences between these groups and the control group showed no dose-dependency and were assessed to be without biological relevance.

Weight of the placentae
- The mean placental weights in test groups 1, 2 and 3 were not influenced by the test substance and were comparable to the control value.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Macroscopically, in 2/25 high-dose animals, a focus in the stomach was observed. There was a minimally increased incidence compared to the control group, where none of the animals showed a focus. Therefore, a relation to treatment can not be excluded.
A histopathologic examination of the stomach was not performed.
All other findings occurred only individually. They were considered to be incidental or spontaneous in origin and without any relation to treatment.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

There were no test substance-related and/or biologically relevant differences between the different test groups in the values calculated for pre- and post implantation losses.

Total litter losses by resorption:

no effects observed

Description (incidence and severity):

There were no test substance-related and/or biologically relevant differences between the different test groups in the number of resorptions.

Early or late resorptions:

no effects observed

Dead fetuses:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test substance-related and/or biologically relevant differences between the different test groups in the number of viable fetuses.

One dead fetus was found at cesarean section of high-dose doe No. 80 (200 mg/kg bw/d) which is a rare finding but occurs also spontaneously in this rabbit strain. Therefore, this is considered to be an incidental finding.

Changes in pregnancy duration:

not examined

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

Female rabbits were placed into the study in five cohorts. Each dose group was represented in each cohort. The conception rate was 76% in the control, 84% in the mid-dose group (70 mg/kg bw/d) and 92% in the low- and high-dose groups (20 and 200 mg/kg bw/d). A sufficient number (approximately 20, but not fewer than 16 females with implantation sites) of pregnant females was available for the purpose of the study.

There were no test substance-related and/or biologically relevant differences between the different test groups in conception rate, in the mean number of corpora lutea and implantation sites.

Dose descriptor:

NOAEL

Remarks:

maternal toxicity

Effect level:

>= 200 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

other: No treatment-related adverse effects observed up to the highest tested dose.

Fetal body weight changes:

no effects observed

Description (incidence and severity):

The mean fetal weights of test groups 1, 2 and 3 were not influenced by the test substance and did not show any biologically relevant differences in comparison to the control group.

Reduction in number of live offspring:

not examined

Changes in sex ratio:

no effects observed

Description (incidence and severity):

The sex distribution of the fetuses in test groups 1-3 (20, 70 and 200 mg/kg bw/d) was comparable to the control fetuses. Any observable differences were without biological relevance.

Changes in litter size and weights:

no effects observed

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Two external malformations were recorded (see "Any other informations on results incl. tables"): an umbilical hernia occurred in one fetus of test group 1, which had additionally a malrotated limb, and in one fetus of test group 3. However, these findings were isolated events in single fetuses and can be found in the historical control data at comparable incidences (Supplement, HCD, fetal external malformations, umbilical hernia: affected fetuses per litter: range of 0.0 – 1.3%). Thus, they are considered to be incidental. No statistically significant differences of overall incidences were noted between the groups.

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Skeletal malformations were detected in single fetuses of all test groups including the control (0, 20, 70 and 200 mg/kg bw/d), as shown in "Any other informations on results incl. tables". One fetus (No. 96-01) had associated soft tissue malformations. No statistically significant differences between the groups were noted (see "Any other informations on results incl. tables"). The overall incidences were well within the historical control range of the test facility.
Two of the high-dose findings, such as absent cervical center, absent lumbar vertebra, were within the range of the historical control data. The other two were isolated events in single fetuses. Therefore, they were not assessed as treatment related. Findings in test group 2 were without relation to dose and, therefore, assessed as not treatment-related.

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Soft tissue malformations occurred in test groups 0, 2 and 3 (0, 70 and 200 mg/kg bw/d), as listed in "Any other informations on results incl. tables". One female fetus in test group 3 (No. 96-01) had additional skeletal malformations. Furthermore, for one male fetus of test group 3 (No. 99-11) multiple external malformations, such as diaphragmatic hernia, small spleen, small pancreas, absent subclavian, malpositioned carotid branch and absent lung lobes, were recorded.
Most of the findings, such as aortic arch atresia, absent subclavian, heart: membranous ventricular septum defect, small spleen, can be found in the historical control data at comparable incidences. All other findings in the high dose group were isolated events in single fetuses. Therefore, they were assessed to be not related to treatment.
The distribution of the findings about the test groups does not indicate an association to the treatment and no statistically significant differences between the groups were noted. The total incidence of soft tissue malformations in treated animals did not differ significantly from the control group.

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

Fetal external variations:
- No external variations were recorded.

Fetal external unclassified observations:
- One unclassified external observation (placentae necrobiotic) was recorded in one control fetus. This finding is considered to be incidental (see "Any other informations on results incl. tables").

Fetal soft tissue variations:
- The examinations of the soft tissues revealed malpositioned carotid branches and an absent lung lobe (Lobus inferior medialis) in all test groups including the control (0, 20, 70 and 200 mg/kg bw/d). Other variations, such as cystic dilatation of the brain and narrowed aortic arch (test group 2, respectively), dilated aorta (test groups 1 and 3), dilated pulmonary trunk (test groups 1 and 2), narrowed pulmonary trunk (test group 3) and dilated aortic arch (test group 0) occurred in individual fetuses of the different test groups.
- The overall incidences in test groups 2 and 3 (see "Any other informations on results incl. tables", affected fetuses per litter: mean% 5.9 and 9.1, respectively) were outside the historical control range (Supplement, total soft tissue variations HCD: mean% 3.1 (0.4-5.6)). However, for the overall and also for the individual variations, no statistically significant differences between the groups were noted. No ontogenetic pattern is recognizable. Therefore, this was assessed as not relevant and not related to treatment.

Fetal soft tissue unclassified observations:
- Two unclassified soft tissue observations were recorded. A blood coagulum around urinary bladder was seen in eight control, five low-dose, 14 mid-dose and seven high-dose fetuses. This finding can be found in the historical control data at a comparable incidence. Therefore, it is neither assessed as treatment-related nor as adverse. Furthermore, a fluid-filled abdomen was seen in one control fetus.

Fetal skeletal variations:
- For all test groups, skeletal variations of different bone structures were observed, with or without effects on corresponding cartilages. The observed skeletal variations were related to several parts of fetal skeletons and appeared without a relation to dosing (see "Any other informations on results incl. tables"). The overall incidences of skeletal variations were comparable to the historical control data.
- All skeletal variations with statistically significant differences between the control and any treated groups were compiled in the table below (see "Any other informations on results incl. tables"). All incidences were expressed on a fetus per litter basis.
- Concerning the statistically significant finding, no dose dependency was observed and all values were clearly inside the historical control range. Thus, an association to the test substance and a toxicological relevance is not assumed.

Fetal skeletal unclassified cartilage observations:
- Some isolated cartilage findings without impact on the respective bone structures, which were designated as unclassified cartilage observations, occurred in all test groups (see "Any other informations on results incl. tables"). The observed unclassified cartilage findings were related to the sternum and the ribs and did not show any relation to the dose.
- The incidence of ‘cartilaginous part of ribs not connected with sternum’ was statistically significantly increased in the mid-dose group (test groups 1-3: mean% 11.8 / 14.0* / 10.0 affected fetuses per litter versus 4.8% in control [* = p ≤ 0.05]). However, since this was not related to dose, it was assessed as incidental.
- The overall incidence of unclassified cartilage observations was statistically significantly increased in test group 1 and 2 (20 and 70 mg/kg bw/d). However, both values were well within the historical control range (HCD: mean% 11.3 [3.1-30.5]) and there was no relation to dose. Therefore, it is assessed as not treatment-related.

Details on embryotoxic / teratogenic effects:

Assessment of all fetal external, soft tissue and skeletal observations:
- There were noted external , soft tissue and skeletal malformations in all test groups (0, 20, 70 or 200 mg/kg bw/d). The distribution of total malformations about the groups was not related to dose.
- Six fetuses had more than one malformation. Male low-dose fetus No. 42-08 (20 mg/kg bw/d) had an umbilical hernia and a malrotated limb, while female mid-dose fetus No. 70-01 (70 mg/kg bw/d) showed a severely malformed vertebral column and/or ribs (i.e. thoracic arches fused with ribs, fused thoracic arch cartilages, unilateral, dumbbell, incomplete or bipartite ossification of thoracic centrum). Furthermore, for female mid-dose fetus No. 72-06 a small pancreas, an absent subclavian and a small spleen were recorded. Male high-dose fetus No. 81-09 (200 mg/kg bw/d) had an absent cervical center and a small thoracic arch. Female high dose fetus No. 96-01 showed a membranous ventricular septum defect at the heart combined with severely fused sternebrae (bony plate). Lastly, male high-dose fetus No. 99-11 had multiple visceral malformations, such as diaphragmatic hernia, small spleen, small pancreas, absent subclavian, malpositioned carotid branch and absent lung lobes. No ontogenetic pattern is recognizable for the individual malformations nor was there any cluster of any of these individual malformations seen in the other offspring of these test groups.
- The findings ‘umbilical hernia’, ‘absent subclavian’ and ‘small spleen’ which were seen in the multiple malformed fetuses, occurred also in further individual fetuses of test group 3. Other malformations, such as enlarged lens, cardiomegaly, small interparietal and malpositioned and bipartite sternebra (test group 0, respectively), right-sided aortic arch, asplenia and absent lumbar vertebra (test group 3, respectively), aortic arch atresia (test groups 0 and 3) and absent gallbladder (test groups 0 and 2), cleft sternum (test group 1) and fused rib (test group 2) were scattered observations in individual fetuses. They all were not dose-related and most of them can be found in the historical control data at comparable frequency. An association of these findings to the treatment is not assumed.
- The total incidences of malformations are summarized in "Any other informations on results incl. tables".
- External variations did not occur in any fetus in this study. A spontaneous origin is assumed for the soft tissue variations and the broad range of skeletal variations which were noted in fetuses of all test groups including controls.
- If all different types of variations are summarized, none of the incidences showed a relation to dosing (see "Any other informations on results incl. tables") and can be found in the historical control data at comparable frequency.
- A spontaneous origin is assumed for the unclassified external, unclassified soft tissue and unclassified skeletal cartilage observations which were observed in several fetuses of all test groups (0, 20, 70 and 200 mg/kg bw/d). The distribution and type of these findings do not suggest any relation to treatment. Finally, fetal examinations revealed that there is no adverse effect of the compound on the respective morphological structures up to the highest dose tested (200 mg/kg bw/d).

Dose descriptor:

NOAEL

Effect level:

>= 200 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No treatment-related adverse effects observed up to the highest tested dose.

Abnormalities:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related adverse effects observed up to the highest tested dose.

Developmental effects observed:

no

Clinical examinations
Only pregnant does were used for the calculations of mean maternal food consumption, body weight and body weight change. Only pregnant does with scheduled sacrifice (GD 29) were used for the calculation of mean gravid uterine weights, mean organ weights, corrected (net) body weight gain and summary of reproduction data. The following females were excluded from the above-mentioned calculations:
• Test group 0 (0 mg/kg bw/d): females Nos. 3, 13, 15, 20, 22, 24 - not pregnant
• Test group 1 (20 mg/kg bw/d): females Nos. 37, 38 - not pregnant
• Test group 2 (70 mg/kg bw/d): females Nos. 62, 71, 74, 75 - not pregnant
• Test group 3 (200 mg/kg bw/d): female No. 97- not pregnant, female No. 78 - sacrificed moribund
Thus, according to the requirements of the corresponding test guidelines, each test group including the controls contained a sufficient number of females with implantation sites at necropsy (approximately 20, but not fewer than 16 females with implantation sites).

 

Tab. 3: Individual fetal external malformations

Test group	Doe No.-Fetus No., Sex	Finding
0 (0 mg/kg bw/d)	none
1 (20 mg/kg bw/d)	42-08 M	umbilical hernia, malrotated limb
2 (70 mg/kg bw/d)	none
3 (200 mg/kg bw/d)	89-06 F	umbilical hernia

mg/kg bw/d = milligram per kilogram body weight per day; No. = number; M = male; F = female

 

Tab. 4: Total external malformations

		Test group 0 0 mg/kg bw/d	Test group 1 20 mg/kg bw/d	Test group 2 70 mg/kg bw/d	Test group 3 200 mg/kg bw/d
Litter Fetuses	N N	19 192	23 217	21 205	23 211
Fetal incidence	N (%)	0.0	1 (0.5)	0.0	1 (0.5)
Litter incidence	N (%)	0.0	1 (4.3)	0.0	1 (4.3)
Affected fetuses/litter	Mean%	0.0	0.4	0.0	0.4

mg/kg bw/d = milligram per kilogram body weight per day; N = number; % = per cent

 

Tab. 5: Total external unclassified observations

		Test group 0 0 mg/kg bw/d	Test group 1 20 mg/kg bw/d	Test group 2 70 mg/kg bw/d	Test group 3 200 mg/kg bw/d
Litter Fetuses	N N	19 192	23 217	21 205	23 212
Fetal incidence	N (%)	1 (0.5)	0.0	0.0	0.0
Litter incidence	N (%)	1 (5.3)	0.0	0.0	0.0
Affected fetuses/litter	Mean%	0.5	0.0	0.0	0.0

mg/kg bw/d = milligram per kilogram body weight per day; N = number; % = per cent

 

Tab. 6: Individual fetal soft tissue malformations

Test group	Doe No.-Fetus No., Sex	Finding
0 (0 mg/kg bw/d)	5-07 M	aortic arch atresia
	9-03 F, 9-05 F, 9-08 M	absent gallbladder
	16-11 M	cardiomegaly
	23-02 F	enlarged lens
1 (20 mg/kg bw/d)	none
2 (70 mg/kg bw/d)	72-04 F	absent gallbladder
	72-06 F	small pancreas, absent subclavian, small spleen
	72-12 F	small spleen
3 (200 mg/kg bw/d)	77-03 M	right-sided aortic arch
	80-13 D	aortic arch atresia
	81-10 F	asplenia
	93-02 F	small spleen
	96-01 F a)	heart: membranous ventricular septum defect
	99-07 F	absent subclavian
	99-11 M	multiple visceral malformations

mg/kg bw/d = milligram per kilogram body weight per day; No. = number; M = male; F = female; D = dead. a) fetus with additional skeletal malformations.

 

Tab. 7: Total soft tissue malformations

		Test group 0 0 mg/kg bw/d	Test group 1 20 mg/kg bw/d	Test group 2 70 mg/kg bw/d	Test group 3 200 mg/kg bw/d
Litter Fetuses	N N	19 192	23 217	21 205	23 212
Fetal incidence	N (%)	6 (3.1)	0.0	3 (1.5)	7 (3.3)
Litter incidence	N (%)	4 (21)	0.0	1 (4.8)	6 (26)
Affected fetuses/litter	Mean%	3.2	0.0	1.1	3.1

mg/kg bw/d = milligram per kilogram body weight per day; N = number; % = per cent

 

Tab. 8: Total soft tissue variations

		Test group 0 0 mg/kg bw/d	Test group 1 20 mg/kg bw/d	Test group 2 70 mg/kg bw/d	Test group 3 200 mg/kg bw/d
Litter Fetuses	N N	19 192	23 217	21 205	23 212
Fetal incidence	N (%)	6 (3.1)	7 (3.2)	8 (3.9)	11 (5.2)
Litter incidence	N (%)	6 (32)	5 (22)	6 (29)	10 (43)
Affected fetuses/litter	Mean%	3.1	3.6	5.9	9.1

mg/kg bw/d = milligram per kilogram body weight per day; N = number; % = per cent

 

 

Tab. 9: Total soft tissue unclassified observations

		Test group 0 0 mg/kg bw/d	Test group 1 20 mg/kg bw/d	Test group 2 70 mg/kg bw/d	Test group 3 200 mg/kg bw/d
Litter Fetuses	N N	19 192	23 217	21 205	23 212
Fetal incidence	N (%)	9 (4.7)	5 (2.3)	14 (6.8)	7 (3.3)
Litter incidence	N (%)	4 (21)	2 (8.7)	4 (19)	6 (26)
Affected fetuses/litter	Mean%	4.1	2.0	6.6	3.2

mg/kg bw/d = milligram per kilogram body weight per day; N = number; % = per cent

 

Tab. 10: Individual fetal soft skeletal malformations

Test group	Doe No.-Fetus No., Sex	Finding
0 (0 mg/kg bw/d)	11-04 M 12-01 M	malpositioned and bipartite sternebra small interparietal
1 (20 mg/kg bw/d)	33-06 F	cleft sternum
2 (70 mg/kg bw/d)	60-03 F	fused rib
	70-01 F	severely malformed vertebral column and/or ribs
3 (200 mg/kg bw/d)	81-09 M 81-10 F 96-01 F a)	absent cervical center, small thoracic arch absent lumbar vertebra sternebrae severely fused (bony plate)

mg/kg bw/d = milligram per kilogram body weight per day; No. = number; M = male; F = female; D = dead

1. a) fetus with additional soft tissue malformations.

 

Tab. 11: Total skeletal malformations

		Test group 0 0 mg/kg bw/d	Test group 1 20 mg/kg bw/d	Test group 2 70 mg/kg bw/d	Test group 3 200 mg/kg bw/d
Litter Fetuses	N N	19 192	23 217	21 205	23 212
Fetal incidence	N (%)	2 (1.0)	1 (0.5)	2 (1.0)	3 (1.4)
Litter incidence	N (%)	2 (11)	1 (4.3)	2 (9.5)	2 (8.7)
Affected fetuses/litter	Mean%	0.9	0.5	0.9	1.3

 mg/kg bw/d = milligram per kilogram body weight per day; N = number; % = per cent

 

Tab. 12: Total fetal skeletal malformations

		Test group 0 0 mg/kg bw/d	Test group 1 20 mg/kg bw/d	Test group 2 70 mg/kg bw/d	Test group 3 200 mg/kg bw/d
Litter Fetuses	N N	19 192	23 217	21 205	23 212
Fetal incidence	N (%)	174 (91)	193 (89)	189 (92)	197 (93)
Litter incidence	N (%)	19 (100)	23 (100)	21 (100)	23 (100)
Affected fetuses/litter	Mean%	92.0	89.3	92.3	92.1

mg/kg bw/d = milligram per kilogram body weight per day; N = number; % = per cent

 

Tab. 13: Occurrence of statistically significantly increased fetal skeletal variations (expressed as mean percentage of affected fetuses/litter)

Finding	Test group 0 0 mg/kg bw/d	Test group 1 20 mg/kg bw/d	Test group 2 70 mg/kg bw/d	Test group 3 200 mg/kg bw/d	HCD Mean % (range)
Incomplete ossification of sternebra; unchanged cartilage	34.6	40.6	34.3	48.5*	36.6 (13.3 - 49.5)

mg/kg bw/d = milligram per kilogram body weight per day; HCD = Historical control data; % = per cent

* = p ≤ 0.05 (Wilcoxon-test [one-sided])

 

Tab. 14: Total unclassified cartilage observations

		Test group 0 0 mg/kg bw/d	Test group 1 20 mg/kg bw/d	Test group 2 70 mg/kg bw/d	Test group 3 200 mg/kg bw/d
Litter Fetuses	N N	19 192	23 217	21 205	23 212
Fetal incidence	N (%)	20 (10)	39 (18)	47 (23)	34 (16)
Litter incidence	N (%)	10 (53)	17 (74)	16 (76)	15 (65)
Affected fetuses/litter	Mean%	9.6	17.9*	22.1*	14.8

mg/kg bw/d = milligram per kilogram body weight per day; N = number; % = per cent

* = p ≤ 0.05 (Wilcoxon-test [one-sided])

 

Tab. 15: Total fetal malformations

		Test group 0 0 mg/kg bw/d	Test group 1 20 mg/kg bw/d	Test group 2 70 mg/kg bw/d	Test group 3 200 mg/kg bw/d
Litter Fetuses	N N	19 192	23 217	21 205	23 212
Fetal incidence	N (%)	8 (4.2)	2 (0.9)	5 (2.4)	9 (4.2)
Litter incidence	N (%)	6 (32)	2 (8.7)	3 (14)	7 (30)
Affected fetuses/litter	Mean%	4.1	0.9	2.0	4.0

mg/kg bw/d = milligram per kilogram body weight per day; N = number; % = per cent

 

Tab. 16: Total fetal variations

		Test group 0 0 mg/kg bw/d	Test group 1 20 mg/kg bw/d	Test group 2 70 mg/kg bw/d	Test group 3 200 mg/kg bw/d
Litter Fetuses	N N	19 192	23 217	21 205	23 212
Fetal incidence	N (%)	175 (91)	193 (89)	189 (92)	197 (93)
Litter incidence	N (%)	19 (100)	23 (100)	21 (100)	23 (100)
Affected fetuses/litter	Mean%	92.5	89.3	92.3	92.1

mg/kg bw/d = milligram per kilogram body weight per day; N = number; % = per cent

Conclusions:

Under the conditions of this prenatal developmental toxicity study, the oral administration of the test substance to pregnant New Zealand White rabbits from implantation to one day prior to the expected day of parturition (GD 6-28) at doses as high as 200 mg/kg bw/d caused neither evidence of maternal nor developmental toxicity. In conclusion, the no observed adverse effect level (NOAEL) for maternal and prenatal developmental toxicity is 200 mg/kg bw/d.

Executive summary:

The test substance was tested for its prenatal developmental toxicity in New Zealand White rabbits. The test substance was administered as an aqueous solution to groups of 25 inseminated female New Zealand White rabbits orally by gavage in doses of 20, 70 and 200 mg/kg body weight/day (mg/kg bw/d) on gestation days (GD) 6 through 28. The vehicle control group, consisting of 25 females, was dosed with the vehicle (deionized water) in parallel. A standard dose volume of 10 mL/kg body weight was used for each test group. At terminal sacrifice on GD 29, 19-23 females per group had implantation sites.

Food consumption and body weight of the animals were recorded regularly throughout the study period. The state of health of the animals was checked each day. On GD 29, all surviving females were sacrificed and assessed by gross pathology (including weight determinations of the unopened uterus and placentas). For each doe, corpora lutea were counted and number and distribution of implantation sites (differentiated between resorptions, live and dead fetuses) were determined. The fetuses were removed from the uterus, sexed, weighed and further investigated for any external, soft tissue and skeletal (inclusive cartilage) findings.

Analytics: The stability of the test substance in deionized water over a period of 7 days at room temperature was demonstrated. The correctness of the prepared concentrations was shown.

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

• Test group 3 (200 mg/kg bw/d): No test substance-related adverse effects on does, gestational parameters or fetuses.


• Test group 2 (70 mg/kg bw/d): No test substance-related adverse effects on does, gestational parameters or fetuses.


• Test group 1 (20 mg/kg bw/d): No test substance-related adverse effects on does, gestational parameters or fetuses.

Conclusion: Under the conditions of this prenatal developmental toxicity study, the oral administration of the test substance to pregnant New Zealand White rabbits from implantation to one day prior to the expected day of parturition (GD 6-28) at doses as high as 200 mg/kg bw/d caused neither evidence of maternal nor developmental toxicity. In conclusion, the no observed adverse effect level (NOAEL) for maternal and prenatal developmental toxicity is 200 mg/kg bw/d.

Reference 2

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Mar 2019 - Feb 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

- Lot/batch No.:18L00117
- Analytical purity: 99.4 g/100g

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH
- Age at study initiation: about 10-12 weeks
- Weight at study initiation: 173.1–229.1 g
- Housing: individually (Polycarbonate cages type III)
- Diet (e.g. ad libitum): ad libitum (ground Kliba maintenance diet mouse/rat “GLP”, meal, supplied by Granovit AG, Kaiseraugst, Switzerland)
- Water (e.g. ad libitum): drinking water (ad libitum)
- Acclimation period: 6 days
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24 °C
- Humidity (%): 45-64 %
- Air changes (per hour): 15 times per hour
- Photoperiod (hours dark / hours light): 12/12

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

deionized

Details on exposure:

The test substance preparations were prepared at the beginning of the administration period and thereafter at intervals, which took into account the period of established stability. The preparations were kept at room temperature. For the test substance preparations, the specific amount of test substance was weighed,topped up with deionized water in a graduated flask and intensely mixed with a magnetic stirrer until it was completely dissolved. Before and during administration, the preparations were kept homogeneous with a magnetic stirrer.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The analytical investigations of the test substance preparations were carried out as a separate study at the test facility Competence Center Analytics. Under the responsibility of the study director of this test facility. The study was carried out in compliance with the Principles of Good Laboratory Practice. Analytical verifications of the stability of the test substance in deionized water over a period of 7 days at room temperature had been verified prior to the start of the study.

Details on mating procedure:

The animals were paired by the breeder (“time-mated”), the day of evidence of mating (= detection of vaginal plug/sperm) was referred to as GD 0. The animals arrived on the same day (GD 0) at the experimental laboratory. The following day was designated as “GD 1”.

Duration of treatment / exposure:

The test substance preparations were administered to the animals once a day orally by gavage, from implantation to one day prior to the expected day of parturition (GD 6 to GD 19), always at approximately the same time in the morning.

Frequency of treatment:

Once daily

Duration of test:

2-(2-aminoethoxy)ethanol was administered to pregnant Wistar rats by gavage, daily as a preparation in deionized water from implantation to one day prior to the expected day of parturition (GD 6-19). On GD 20 all surviving dams were sacrificed and examined.

Dose / conc.:

100 mg/kg bw/day

Dose / conc.:

300 mg/kg bw/day

Dose / conc.:

1 000 mg/kg bw/day

No. of animals per sex per dose:

25

Control animals:

yes, concurrent vehicle

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Mortality: A check was made twice a day on working days or once a day on Saturdays, Sundays or on public holidays (GD 0-20).
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule:
cage-side examination was conducted at least once daily before and after treatment period (GD 0-5 and 20). During treatment period (GD 6-19) all rats were checked daily for any signs of morbidity, pertinent behavioral changes and/or signs of overt toxicity before administration as well as within 2 hours and between 2 and 5 hours after administration.
BODY WEIGHT: Yes
- Time schedule for examinations: All animals were weighed on GD 0, 1, 3, 6, 8, 10, 13, 15, 17, 19 and 20. The body weight change of the animals was calculated based on the obtained results.
FOOD CONSUMPTION: Yes
- The consumption of food was recorded for the intervals GD 0-1, 1-3, 3-6, 6-8, 8-10, 10-13, 13-15, 15-17, 17-19 and 19-20.
POST-MORTEM EXAMINATIONS: Yes
Cesarean section:
On GD 20, the dams were sacrificed under isoflurane anesthesia by decapitation, in randomized order. After the dams had been sacrificed, they were necropsied and assessed for gross pathology.The uteri and the ovaries were removed and the following data were recorded:
- Weight of the unopened uterus
- Number of corpora lutea
- Number and distribution of implantation sites classified as:
• Live fetuses
• Dead implantations:
a) Early resorptions (only decidual or placental tissues visible or according to SALEWSKI from uteri from apparently non-pregnant animals and the empty uterus horn in the case of single horn pregnancy)
b) Late resorptions (embryonic or fetal tissue in addition to placental tissue visible)
c) Dead fetuses (hypoxemic fetuses which did not breathe spontaneously after the uterus had been opened)
After the weight of the uterus had been determined, all subsequent evaluations of the dams and the gestational parameters (except of gross pathology including organ sampling and weights) were conducted by technicians unaware of treatment group in order to minimize bias. For this purpose animal numbers were encoded.
Pathology:
Organ weights:
The following weights were determined in all dams sacrificed on schedule: Liver, Spleen and Thyroid glands (with parathyroid glands) (fixed). All paired organs were weighed together (left and right). The carcass weights (GROSSE-System) were transferred to the ACOPAT-System to calculate the relative organ weights.
Organ / Tissue fixation:
The following organs or tissues were fixed in 4% neutral-buffered formaldehyde solution: all gross lesions, liver, spleen.
Organ / Tissue fixation:
The following organs or tissues were fixed in 4% neutral-buffered formaldehyde solution: all gross lesions, adrenal glands, kidneys, liver, spleen.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Weight of the unopened uterus: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Number of live and dead fetuses: Yes

Blood sampling:

On GD 20, blood samples were obtained from all females by retrobulbar venous puncture following isoflurane anesthesia. The assays of blood and serum parameters were performed under internal laboratory quality control conditions with reference controls to assure reliable test results.

Fetal examinations:

Proportions of fetuses with malformations,variations and/or unclassified observations in each litter. And anogenital distance,anogenital index .
External examinations: YES
- Examinations of the fetuses after dissection from the uterus. At necropsy each fetus was weighed, sexed, and external tissues and all orifices were examined macroscopically. The sex was determined by observing the distance between the anus and the base of the genitalia. Furthermore, the viability of the fetuses and the condition of placentas, umbilical cords, fetal membranes, and fluids were examined. The placentas were weighed and their individual weights were recorded. Thereafter, the fetuses were sacrificed by a subcutaneous injection of pentobarbital (Narcoren®; dose: 0.1 mL/fetus).
The anogenital distance (defined as the distance from the center of the anal opening to the base of the genital tubercle) measurements were conducted, using a measuring ocular, on all liveborn fetuses.
- Soft tissue examination of the fetuses:
The fetuses fixed in Harrison’s fluid were examined for any visceral findings according to the method of BARROW and TAYLOR. After this examination these fetuses were discarded.
- Skeletal examination of the fetuses:
The skeletons of the fetuses fixed in ethanol were stained according to a modified method of KIMMEL and TRAMMELL. Thereafter, the skeletons of these fetuses were examined under a stereomicroscope. After this examination the stained fetal skeletons were retained individually.

Statistics:

KRUSKAL-WALLIS H test (two-sided)/ WILCOXON-test (two-sided) and WILCOXON-test (one-sided) /DUNNETT-test (two-sided)

Indices:

The conception rate (in %) was calculated according to the following formula:
(number of pregnant animals/number of fertilized animals) x100
The preimplantation loss (in %) for each individual pregnant animal which underwent scheduled sacrifice was calculated according to the following formula:
((number of corpora lutea – number of implantations)/number of corpora lutea) x100
The postimplantation loss (in %) for each individual pregnant animal which underwent scheduled sacrifice was calculated according to the following formula:
((number of implantations – number of live fetuses)/number of implantations) x100

Historical control data:

Historical control data were available

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Three females (out of 25) of the high-dose group (1000 mg/kg bw/d) showed salivation during the treatment period. Salivation occurred in the respective animals only shortly, i.e. within 0 - 2 h, after treatment and was observed on GD 10, 11 and 14. No clinical signs or changes of general behavior were detected in any female of all test groups beyond 2 hours after treatment. The occasional salivation was most probably caused by the bad taste or smell of the test substance and was not assessed as sign of systemic toxicity.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

There were no test substance-related or spontaneous mortalities in any females of all test groups.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

The mean body weights and the average body weight gain of test groups 1, 2 and 3 were generally comparable to the concurrent control group throughout the entire study period.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

In test group 3 (1000 mg/kg bw/d), the mean food consumption was statistically significantly reduced on GD 6-8 but recovered afterwards and was comparable to the control values again throughout the remaining study period. If calculated for the entire treatment period (GD 6-19), the mean food consumption of the high-dose dams was comparable to the concurrent control group. Since the marginal decrease was only transient during a short time period, it was not assessed as treatment-related.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

At the end of the gestation period, in dams of test group 3 (1000 mg/kg bw/d) red blood cell (RBC) and absolute reticulocyte counts as well as hemoglobin and hematocrit values were significantly decreased, whereas platelet counts were significantly increased. These changes were regarded as treatment-related and adverse. Hemoglobin values were decreased in dams of test group 2 (300 mg/kg bw/d) and platelet counts were significantly higher compared to controls in this test group. Hemoglobin was the only changed parameter in test group 2. Therefore, this change was regarded as possibly treatment-related but non-adverse (ECETOC Technical Report No. 85, 2002). Platelet counts in test group 2 were within the historical control range (platelets 706-940 Giga/L). Therefore, this alteration was regarded as incidental and not treatment-related. In dams of test groups 1 and 3 (100 and 1000 mg/kg bw/d) relative eosinophil counts were significantly increased, whereas in dams of test group 3 relative basophil counts were significantly decreased. However, all changed values were within historical control ranges (relative eosinophils 0.8-1.7 %; relative basophils 0.1-0.3 %). Therefore, these alterations were regarded as incidental and not treatment-related.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

At the end of the gestation period, in dams of test group 3 (1000 mg/kg bw/d) alkaline phosphatase (ALP) activities were significantly decreased. This change was regarded as treatment-related and adverse. The following significant alterations were within historical control ranges. Therefore, they were regarded as incidental and not treatment-related: in dams of test group 3 (1000 mg/kg bw/d) decreased creatinine, total protein and total bilirubin values and increased inorganic phosphate values; in dams of test group 1 (100 mg/kg bw/d) increased total bilirubin values (creatinine 24.0-33.6 μmol/L; total protein 55.90-63.74 g/L; total bilirubin 0.74-1.30 μmol/L; inorganic phosphate 1.34-1.99 mmol/L).

Endocrine findings:

no effects observed

Description (incidence and severity):

Thyroid hormones:In dams of test groups 1, 2 and 3 (100, 300 and 1000 mg/kg bw/d), no treatment-related alterations of T3, T4 and TSH levels were observed.

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

The mean gravid uterus weights of the animals of test groups 1-3 (100, 300 and 1000 mg/kg bw/d) were not influenced by the test substance. The differences between these groups and the control group revealed no dose-dependency and were assessed to be without biological relevance. When compared to control group 0 (=100%), the mean relative weight of the liver was significantly decreased in test group 1 (97%). This change was not dose-dependent and was regarded as incidental. All other mean relative weight parameters did not show significant differences when compared to the control group 0.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

In test group 3, 13 out of 25 dams showed organ-like colored foci with sunken surface (up to 6 mm in diameter) in the forestomach and one out of 25 dams showed foci in the glandular stomach (red, up to 4 mm in diameter). The foci in the forestomach were considered treatment-related. All other findings occurred individually and were considered to be incidental or spontaneous in origin and without any relation to treatment. All other findings occurred individually and were considered to be incidental or spontaneous in origin and without any relation to treatment.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

The conception rate was 100% in all test groups (0, 100, 300 and 1000 mg/kg bw/d). With these rates, a sufficient number of pregnant females were available for the purpose of this study. There were no test substance-related and/or biologically relevant differences between the test groups 0 - 3 in conception rate, in the mean number of corpora lutea and implantation sites or in the values calculated for the pre- and post-implantation losses, the number of resorptionsand viable fetuses. All observed differences are considered to reflect the normal range of fluctuations for animals of this strain and age.
The mean gravid uterus weights of the animals of test groups 1 - 3 (100, 300 and 1000 mg/kg bw/d) were not influenced by the test substance. The differences between these groups and the control group revealed no dose-dependency and were assessed to be without biological relevance.

Total litter losses by resorption:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test substance-related and/or biologically relevant differences between the test groups 0 - 3 in conception rate the number of resorptions and viable fetuses. The total number of resorptions was statistically significantly lower in the low- and mid-dose groups (1.5/ 0.2**[p<=0.01]/ 0.4**[p<=0.01]/ 0.8 in test groups 0 - 3). Consequently, the mean number of live fetuses was also higher in those dams (mean: 88.1% / 98.4**% / 96.8*% / 91.4% in test groups 0 - 3). As specified above, these statistically significantly greater litters came from a comparatively low resorption rate at the low- and mid-dose levels and are neither treatment-related nor adverse.

Early or late resorptions:

no effects observed

Description (incidence and severity):

see " Total litter losses by resorption"

Dead fetuses:

no effects observed

Changes in pregnancy duration:

not examined

Changes in number of pregnant:

not examined

Other effects:

no effects observed

Description (incidence and severity):

The mean placental weights of test groups 1 - 3 were comparable to the concurrent control group.The mean placental weights of the low-, mid- and high-dose groups were comparable to the corresponding control group.

Details on maternal toxic effects:

The conception rate was 100% in all test groups (0, 100, 300 and 1000 mg/kg bw/d). With these rates, a sufficient number of pregnant females were available for the purpose of this study (according to the test guidelines). There were no test substance-related and/or biologically relevant differences between the test groups 0 - 3 in conception rate, in the mean number of corpora lutea and implantation sites or in the values calculated for the pre- and post-implantation losses, the number of resorptions and viable fetuses. All observed differences are considered to reflect the normal range of fluctuations for animals of this strain and age; see also the historical control data.

Dose descriptor:

NOAEL

Effect level:

300 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: No adverse effects observed at this dose.

Dose descriptor:

LOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

clinical biochemistry

haematology

Key result

Abnormalities:

effects observed, treatment-related

Description (incidence and severity):

Haematology (RBC), Clinical biochemistry (ALP)

Fetal body weight changes:

no effects observed

Description (incidence and severity):

The mean fetal weights of test groups 1, 2 and 3 were not influenced by the test substance and did not show any biologically relevant differences in comparison to the control group.

Reduction in number of live offspring:

not examined

Changes in sex ratio:

no effects observed

Description (incidence and severity):

The sex distribution of the fetuses in test groups 1 - 3 (100, 300 and 1000 mg/kg bw/d) was comparable to the control fetuses.

Changes in litter size and weights:

no effects observed

Anogenital distance of all rodent fetuses:

no effects observed

Description (incidence and severity):

The anogenital distance and anogenital index of all male and female fetuses in the test groups 1 - 3 was comparable to the concurrent control values.

Changes in postnatal survival:

not examined

External malformations:

no effects observed

Description (incidence and severity):

External or soft tissue malformations did not occur in any of the fetuses in this study. There were noted skeletal malformations in one fetus, each, of the control, low- and mid-dose groups (0, 100 or 300 mg/kg bw/d). High-dose fetuses showed no malformations at all. One fetus carried more than one malformation: female mid-dose fetus No. 51-12 had malformations concerning the vertebral column and ribs in the area of thoraco-lumbar border, such as an additional vertebral arch and corresponding rib, misshapen thoracic vertebra and a supernumerary thoracic hemivertebra. However, these findings occurred without relation to dose. Further malformations, i.e. fused rib and shortened humerus, were observed in individual fetuses, unrelated to dose and at least ‘shortened humerus’ can be found in the historical control data. All these findings were single cases without relation to treatment, no ontogenetic pattern is recognizable for all these individual malformations nor was there any cluster of any of these individual malformations seen in the other offspring of these test groups. They also do neither form a pattern or syndrome with other minor anomalies which may raise toxicological concern nor do they influence the overall rate of malformations in this study. There is no evidence for any association of these scattered findings with the treatment. Fetal external unclassified observations: No external unclassified observations were recorded

Skeletal malformations:

no effects observed

Description (incidence and severity):

Skeletal malformations were recorded for four control, one mid- and one high-dose fetuses. The mean values of total incidences of skeletal malformations did not differ significantly and were within the historical control data. The finding ‘misshapen basisphenoid’ in the fetuses of test groups 2 and 3 were single events in individual fetuses and the mean values were within the historical control data (affected fetuses per litter, mean: 0.1%, range: 0.0 - 1.0%). Therefore, the observed malformation in substance-treated fetuses was not assessed as treatment-related and adverse.

Visceral malformations:

no effects observed

Other effects:

no effects observed

Details on embryotoxic / teratogenic effects:

No differences of toxicological relevance between the control and the treated groups (100, 300 or 1000 mg/kg bw/d) were determined for any reproductive parameters, such as conception rate, mean number of corpora lutea, mean number of implantations, as well as pre- and postimplantation loss. Similarly, no toxicologically relevant influence of the test substance on sex distribution and anogenital distance/index of the fetuses was noted at any dose.

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects observed up to the highest tested dose.

Abnormalities:

no effects observed

Developmental effects observed:

no

Conclusions:

Under the conditions of this prenatal developmental toxicity study, the oral administration of 2-(2-aminoethoxy)ethanol to pregnant Wistar rats from implantation to one day prior to the expected day of parturition (GD 6-19) provided evidence of maternal toxicity, such as a nonregenerative,normocytic, normochromic anemia, a disturbance of platelet homeostasis and a decrease in ALP activities, at the highest dose level of 1000 mg/kg bw/d.In conclusion, the no observed adverse effect level (NOAEL) for maternal toxicity is the mid dose level of 300 mg/kg bw/d. There was no evidence for toxicologically relevant adverse effects of the test substance on fetal morphology at any dose. In conclusion, the no observed adverse effect level (NOAEL) for prenatal developmental toxicity is 1000 mg/kg bw/d.

Executive summary:

In a prenatal developmental toxicity study, the test substance 2-(2-aminoethoxy)ethanol was administered to pregnant Wistar rats daily by gavage from implantation to one day prior to the expected day of parturition (GD 6-19) to evaluate its potential maternal and prenatal developmental toxicity. Analyses confirmed the correctness of the prepared concentrations and the stability of the test substance in the vehicle. Generally, clinical observations including food consumption and body weight (gain) revealed no toxicologically relevant difference between the animals receiving 100, 300 or 1000 mg/kg bw/d 2-(2-aminoethoxy)ethanol and controls. Three females (out of 25) of the high-dose group (1000 mg/kg bw/d) showed salivation on GD10, 11 and 14. Salivation occurred in the respective animals only shortly, i.e. within 0-2 h, after treatment. No clinical signs or changes of general behavior were detected in any female of all test groups beyond 2 hours after treatment. The occasional salivation was most probably caused by the bad taste or smell of the test substance and was not assessed as sign of systemic toxicity. Regarding clinical pathology, in dams of test group 3 (1000 mg/kg bw/d) a non-regenerative,normocytic, normochromic anemia were observed, due to significantly decreased red bloodcell (RBC) and absolute reticulocyte counts, hemoglobin and hematocrit values. The changed blood flow in the vessels due to the anemia led to a disturbance of the platelet homeostasis with higher platelet counts in dams of this test group. The reason for decreased alkaline phosphatase (ALP) activities in dams of test group 3 cannot be elucidated. For further clarification ALP isoenzyme measurements should be performed. Regarding pathology, no treatment-related organ weight changes were observed. In test group 3, 13 out of 25 dams showed in the forestomach organ-like colored foci with sunken surface (up to 6 mm in diameter). Although no histopathology of these findings was performed, the foci were considered treatment-related and locally adverse. All histopathological findings in the thyroid gland 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. No differences of toxicological relevance between the control and the treated groups (100, 300 or 1000 mg/kg bw/d) were determined for any reproductive parameters, such as conception rate, mean number of corpora lutea, mean number of implantations, as well as pre- and postimplantation loss. Similarly, no toxicologically relevant influence of the test substance on sex distribution and anogenital distance/index of the fetuses was noted at any dose. Overall, there was no evidence for toxicologically relevant adverse effects of the test substance on fetal morphology at any dose.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Species:

rat

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEC

40 mg/m³

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

In a prenatal developmental toxicity study, the test substance 2-(2-aminoethoxy)ethanol was administered to pregnant Wistar rats daily by gavage from implantation to one day prior to the expected day of parturition (GD 6-19) to evaluate its potential maternal and prenatal developmental toxicity. Analyses confirmed the correctness of the prepared concentrations and the stability of the test substance in the vehicle. Generally, clinical observations including food consumption and body weight (gain) revealed no toxicologically relevant difference between the animals receiving 100, 300 or 1000 mg/kg bw/d 2-(2-aminoethoxy)ethanol and controls. Three females (out of 25) of the high-dose group (1000 mg/kg bw/d) showed salivation on GD10, 11 and 14. Salivation occurred in the respective animals only shortly, i.e. within 0-2 h, after treatment. No clinical signs or changes of general behavior were detected in any female of all test groups beyond 2 hours after treatment. The occasional salivation was most probably caused by the bad taste or smell of the test substance and was not assessed as sign of systemic toxicity. Regarding clinical pathology, in dams of test group 3 (1000 mg/kg bw/d) a non-regenerative,normocytic, normochromic anemia were observed, due to significantly decreased red bloodcell (RBC) and absolute reticulocyte counts, hemoglobin and hematocrit values. The changed blood flow in the vessels due to the anemia led to a disturbance of the platelet homeostasis with higher platelet counts in dams of this test group. The reason for decreased alkaline phosphatase (ALP) activities in dams of test group 3 cannot be elucidated. For further clarification ALP isoenzyme measurements should be performed. Regarding pathology, no treatment-related organ weight changes were observed. In test group 3, 13 out of 25 dams showed in the forestomach organ-like colored foci with sunken surface (up to 6 mm in diameter). Although no histopathology of these findings was performed, the foci were considered treatment-related and locally adverse. All histopathological findings in the thyroid gland 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. No differences of toxicological relevance between the control and the treated groups (100, 300 or 1000 mg/kg bw/d) were determined for any reproductive parameters, such as conception rate, mean number of corpora lutea, mean number of implantations, as well as pre- and postimplantation loss. Similarly, no toxicologically relevant influence of the test substance on sex distribution and anogenital distance/index of the fetuses was noted at any dose. Overall, there was no evidence for toxicologically relevant adverse effects of the test substance on fetal morphology at any dose.

Under the conditions of this prenatal developmental toxicity study, the oral administration of 2-(2-aminoethoxy)ethanol to pregnant Wistar rats from implantation to one day prior to the expected day of parturition (GD 6-19) provided evidence of maternal toxicity, such as a nonregenerative,normocytic, normochromic anemia, a disturbance of platelet homeostasis and a decrease in ALP activities, at the highest dose level of 1000 mg/kg bw/d. In conclusion, the no observed adverse effect level (NOAEL) for maternal toxicity is the mid dose level of 300 mg/kg bw/d. There was no evidence for toxicologically relevant adverse effects of the test substance on fetal morphology at any dose. In conclusion, the no observed adverse effect level (NOAEL) for prenatal developmental toxicity is 1000 mg/kg bw/d.

 

In a prenatal developmental toxicity study, the test substance was administered to pregnant New Zealand White rabbits daily by stomach tube from implantation to one day prior to the expected day of parturition (GD 6-28). Analyses confirmed the correctness of the prepared concentrations and the stability of the test substance in the vehicle. Clinical examinations revealed no toxicologically relevant difference between the animals receiving 20, 70 or 200 mg/kg bw/dt of the est substance nd the controls. Neither food consumption nor gross or net body weight gain were adversely influenced by the treatment. One high-dose female (No. 78 - 200 mg/kg bw/d) was sacrificed moribund on GD 21 for animal welfare reasons due to severe clinical findings (no/reduced defecation, blood in bedding, hypothermia and poor general state). Since only one single animal in the high-dose group was affected and the above-mentioned findings can be also seen in control animals, this was assessed as not-related to treatment and as incidental. Regarding pathology, no treatment-related, adverse macroscopic findings were noted. Macroscopically, in 2/25 high-dose animals, a focus in the stomach was observed. There was a minimally increased incidence compared to the control group. Therefore, a relation to treatment can not be excluded. However, a histopathologic examination of the stomach was not performed. No differences of toxicological relevance between the control and the treated groups (20, 70 or 200 mg/kg bw/d) were determined for any reproductive parameters, such as conception rate, mean number of corpora lutea, mean number of implantations, as well as pre- and postimplantation loss. All differences observed are considered to reflect the normal range of fluctuations for animals of this strain and age. Similarly, no influence of the test substance on uterine weight, placental weight, fetal weight and sex distribution of the fetuses was noted at any dose. Fetal examinations revealed no toxicologically relevant adverse effects of the test substance on embryofetal development.

Conclusion: Under the conditions of this prenatal developmental toxicity study, the oral administration of the test substance to pregnant New Zealand White rabbits from implantation to one day prior to the expected day of parturition (GD 6-28) at doses as high as 200 mg/kg bw/d caused neither evidence of maternal nor developmental toxicity. In conclusion, the no observed adverse effect level (NOAEL) for maternal and prenatal developmental toxicity is 200 mg/kg bw/d.

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No. 1272/2008

The available extended one-generation study (OECD 443), reproduction screening study by inhalation (OECD 422) and prenatal toxicity studies (OECD 414 first and second species) are reliable and suitable for classification purposes under Regulation 1272/2008.

In the oral extended one-generation reproduction toxicity study in rats, no treatment-related, adverse effects on fertility were observed up to the highest tested dose level of 1129 mg/kg bw/day administered via diet. Concerning reproductive performance of the F0 females, however, a slightly decreased number of pups were born (93% vs. 99% in control) and a slightly increased number of stillborn were observed (7.1% vs. 1.2% in control) at the highest dose. Three dams did not nurse their pups properly and three females had complete litter losses during lactation. Although assessed as treatment-related and adverse, the findings in the high dose group were observed in the presence of clear signs of systemic toxicity in the female animals. Aside from reduced food consumption in the high dose females during lactation, two females developed poor conditions and had to be sacrificed moribund. These findings indicate that the effects on reproductive performance were secondary to the maternal toxicity of the test substance. In line with this, no effects on the viability of fetuses were observed in a prenatal developmental study in rats at lower but limit dose levels of up to 1000 mg/kg bw/day administered via gavage (vs. 1129 mg/kg bw/day). In this study, no test substance-related and/or biologically relevant differences between the test groups were observed in the mean number of corpora lutea and implantation sites or in the values calculated for the pre- and post-implantation losses, the number of resorptions and viable fetuses. Moreover, no treatment-related adverse effects on reproductive performance were seen in a reproduction screening study in rats following inhalation exposure up to the highest tested dose of 40 mg/m3.

No developmental toxicity was found in the prenatal developmental toxicity studies in rats or in rabbits.

As a result, the substance is not considered to be classified for fertility or developmental toxicity under Regulation (EC) No. 1272/2008, as amended for the fourteenth time in Regulation (EC) No. 2020/217.