Ethyldimethylamine

Toxicity to reproduction

Administrative data

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:

From 25 Nov 2020 to 14 Apr 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

The design of this study was based on the final decision on a compliance check of Dimethylethylamine by ECHA (Decision no. CCH-D-2114369268-37-01/F, date 23 Aug 2017).

The objective of this study is to provide an evaluation of the pre- and postnatal effects of Dimethylethylamine on development as well as a thorough evaluation of systemic toxicity in pregnant and lactating females and young and adult offspring of Wistar Han rats. Detailed examination of key developmental endpoints, such as offspring viability, neonatal health, developmental status at birth, and physical and functional development until adulthood, is expected to identify specific target organs in the offspring. In addition, the study will provide and/or confirm information about the effects of Dimethylethylamine on the integrity and performance of the adult male and female reproductive systems. Specifically, but not exclusively, the following parameters are considered: gonadal function, the estrous cycle, epididymal sperm maturation, mating behavior, conception, pregnancy, parturition, and lactation.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2022

Report date:

2022

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 443 (Extended One-Generation Reproductive Toxicity Study)

Version / remarks:

June 2018

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.56 (Extended One-Generation Reproductive Toxicity Study)

Version / remarks:

15 July 2014

Principles of method if other than guideline:

The study was also conducted according to OECD guidance document supporting OECD test guideline 443 on the extended one-generation reproductive toxicity test, No. 151, version July 2013.

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Justification for study design:

The design of this study was based on the final decision on a compliance check of Dimethylethylamine by ECHA (Decision no. CCH-D-2114369268-37-01/F, date 23 Aug 2017).

SPECIFICATION OF STUDY DESIGN FOR EXTENDED ONE-GENERATION REPRODUCTION TOXICITY STUDY WITH JUSTIFICATIONS:

- Premating exposure duration for parental (P0) animals: F0-animals were treated for 10 weeks prior to mating. Ten weeks premating exposure duration is required because there is no substance specific information in the dossier supporting shorter premating exposure duration as advised in the ECHA Guidance on information requirements and chemical safety assessment R.7a, chapter R.7.6 (version 5.0, July 2016).

- Basis for dose level selection: The dose levels were selected based on the results of a reproduction/developmental toxicity screening test with oral exposure of Dimethylethylamine in rats (Charles River Study No. 20240951, OECD 421), and in an attempt to produce graded responses to the test item. In this study, Wistar Han rats were dosed at 0, 80, 150 or 225 mg/kg bw/day for at least 28 days. The following major findings were noted:
1) At 225 mg/kg bw/day, severe toxicity was observed. Three females were euthanized on study day 4 based on marked body weight loss and observed clinical signs (i.e. piloerection, salivation and breathing difficulties). For remaining high dose group animals, clinical signs were limited to salivation, but mild to marked body weight loss was observed for the majority of the animals on study day 6. To prevent further suffering, the remainder of Group 4 males and females were sacrificed for ethical reasons on study day 6. Test item-related macroscopic findings were noted in the stomach for the majority of the animals and consisted of red foci (glandular mucosa and/or forestomach), a thickened limiting ridge, irregular surface and/or discoloration of the stomach. These alterations in the stomach were considered to be the main cause of the poor condition of these animals.
2) At 150 mg/kg bw/day, four males and two females were euthanized between day 6 and day 33 of the study based on marked body weight loss and observed clinical signs (i.e. breathing difficulties, piloerection, hunched posture, lethargic appearance and pale appearance). The majority of remaining animals showed salivation after dosing. Body weight gain and food consumption (males only) were decreased. Necropsy findings of the stomach included red foci (5 males, 2 females), thickened limiting ridge (2 males) and irregular surface (1 male, 1 female). Stomach alterations included minimal-slight inflammation (correlating to an irregular surface in 1 male), mild to marked erosions, minimal-moderate hemorrhage (correlating to reddish foci in 5 males and 1 female) and/or slight edema of the glandular mucosa and slight-moderate inflammation and/or slight hyperplasia of the forestomach.
3) At 80 mg/kg bw/day, one male was euthanized on Day 6 based on marked body weight loss. No other mortality was observed and no test-item related changes were observed on clinical signs, body weight, food consumption or organ weight. At necropsy, red foci were observed in the stomach of 2 males (of which one was the preterm decedent). Microscopic changes in the glandular stomach of males were observed at low degrees. No clear test item-related microscopic changes were noted for females and there was no indication of test item-related effects on observed reproduction and developmental parameters.
Based on the mortality and stomach findings observed during the previous reproduction/ developmental toxicity screening test, dose levels higher than 80 mg/kg/day were considered inappropriate as the high dose for the current OECD 443 study. Except for one male euthanized in extremis and minimal stomach findings, no toxicity was observed at 80 mg/kg/day. As such, dose levels of 0, 10, 30 and 80 mg/kg/day were selected. The high-dose level should produce some toxic effects, but not death nor obvious suffering. The mid-dose level is expected to produce minimal to moderate toxic effects. The low-dose level should produce no observable indications of toxicity.

- Exclusion of extension of Cohort 1B: There is no evidence in the available data that is relevant for triggers for extension of cohort1B.

- Exclusion of developmental neurotoxicity Cohorts 2A and 2B: There is no evidence in the available data of specific mechanism/modes of action and/or neurotoxicity which may meet the particular concern criteria for the developmental neurotoxicity cohorts.

- Exclusion of developmental immunotoxicity Cohort 3: There is no evidence in the available data of specific mechanism/modes of action and/or immunotoxicity which may meet the particular concern criteria for the developmental immuntoxicity cohort.

- Route of administration: oral gavage

- Other considerations, e.g. number of animals: The total number of animals used in this study was considered to be the minimum required to properly characterize the effects of the test item. This study was designed such that it does not require an unnecessary number of animals to accomplish its objectives. At this time, studies in laboratory animals provide the best available basis for extrapolation to humans and are required to support regulatory submissions. Acceptable models which do not use live animals currently do not exist.
As indicated in the OECD guidance document 151 supporting OECD TG 443, in case the developmental neurotoxicity (DNT) (cohort 2A; consisting of 10 animals/group/sex) and/or developmental immunotoxicity (DIT) (cohort 3; consisting of 10 animals/group/sex) cohorts are omitted, a sufficient number of pups (3/sex/litter/dose) for reproductive assessment as detailed in TG 443 should still be maintained until sexual maturation. In this way, the probability to detect rare or low incidence malformations which would appear postnatally is increased. Thus, as in this specific study, cohort 2A and 3 were omitted, in order to ensure that the required number of animals are available for evaluation of critical endpoints, an extra cohort named "cohort C" consisting of 20 animals/group/sex) was maintained and monitored to sexual maturation (vaginal patency or preputial separation).

Test material

Test material information

Constituent 1

Reference substance name:

Ethyldimethylamine

EC Number:

209-940-8

EC Name:

Ethyldimethylamine

Cas Number:

598-56-1

Molecular formula:

C4H11N

IUPAC Name:

ethyldimethylamine

Test material form:

liquid: volatile

Details on test material:

Name of test material (as cited in study report): Dimethylethylamine
- Source (i.e. manufacturer or supplier):Arkema
- Lot/batch number of test material: J093973621
- Expiry date: 03 April 2021
- Purity :99.63 %
- Volatile: Yes, vapour pressure: 527 hPa at 20¿
- Specific gravity / density: 660 Kg/ m3 at 20¿

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: In refrigerator (2-8°C)
- Stability and homogeneity of the test material in the vehicle/solvent under test conditions (e.g. in the exposure medium) and during storage: Stability for at least 4 hours at room temperature under normal laboratory light conditions and stability for at least 10 days in the refrigerator confirmed over the concentration range 1 to 125 mg/mL (solutions), Project 20240947

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: the test substance was kept cold in a refrigerator or in an ice bath during the preparation of formulations

Test animals

Species:

rat

Strain:

Wistar

Remarks:

Crl: WI(Han)

Details on species / strain selection:

The Wistar Han rat was chosen as the animal model for this study as it is an accepted rodent species for reproduction and developmental toxicity testing by regulatory agencies. The testing facility has general and reproduction/developmental historical data in this species from the same strain and source. This animal model has been proven to be susceptible to the effects of reproductive toxicants.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Females nulliparous and non-pregnant: yes

- Age at study initiation: (F0) 6 weeks old

- Weight at study initiation: (F0) Males: 152 – 196 g; Females: 110 – 145 g

- Fasting period before study: No.

- Housing: Prior to mating and during the post-weaning period, animals were group housed (up to 5 animals of the same sex and same dosing group together) in polycarbonate cages (Macrolon, MIV type, height 18 cm). During the mating phase, males and females were cohabitated on a 1:1 basis in Macrolon plastic cages (MIII type, height 18 cm). During the post-mating phase, males were housed in their home cage (Macrolon plastic cages, MIV type) with a maximum of 5 males/cage. Females were individually housed in Macrolon plastic cages (MIII type) during pregnancy and lactation phase. Pups were housed with the dam until termination (unscheduled deaths, spares, and pups of Cohorts Surplus) or until weaning (Cohorts 1A, 1B, 1C).

- Diet: pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany), ad libitum. The feed is analyzed by the supplier for nutritional components and environmental contaminants.

- Water: municipal tap water, ad libitum. Periodic analysis of the water is performed.

No known contaminants were present in the feed or water that would interfere with the objectives of the study.

- Acclimation period: 12 days (F0-generation)

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21-23 (actual daily mean)
- Humidity (%): 33-57 (actual daily mean)
- Air changes (per hr): Ten or greater with 100% fresh air (no air recirculation)
- Photoperiod (hrs dark / hrs light): A 12-hour light/12-hour dark cycle was maintained

IN-LIFE DATES: From: 07 Dec 2020 To: 28 Jun 2021

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

Elix

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Dosing formulations (w/w) were prepared at appropriate concentrations to meet dose level requirements using the following method:
Water (Elix) was chilled in an ice bath or in the refrigerator before formulating. A stir bar was added to an empty container suitable to hold the final volume with the minimum amount of head space. The scale was tared with the container (plus the seal). The required volume of chilled water (Elix) was transferred to the container. The filled container (plus the seal) was weighed. The test substance was kept cold in a refrigerator or ice bath. A syringe or pipette was used to slowly add the required volume of cold test substance into the chilled water (Elix) under stirring. Part of the required amount of test item (about 70-80%) was added to the vehicle under stirring while the container was in a bath filled with ice water. The container was closed, taken out of the ice water, dried off and placed on the scale to add the remaining amount of test item up to the correct weight. The container was sealed and weighed after this addition. The formulation was stirred magnetically to mix.

The dosing formulations were prepared at least weekly, filled out in daily portions with the use of a syringe or pipette and stored in the refrigerator. The dosing formulations were removed from the refrigerator, stirred at room temperature for at least 30 minutes, and kept at room temperature until dosing. Test substance formulations were dosed within 4 hours after removal from the refrigerator.

On the day of dosing, the regular seal was replaced by a seal with a septum/ small hole. Test item dosing formulations were kept at room temperature until dosing. If practically possible, the dosing formulations and vehicle were continuously stirred until and during dosing. Adjustment was made for specific gravity of the test item. No correction was made for the purity/ composition of the test item. Any residual volumes were discarded.

VEHICLE
- Concentration in vehicle: 0, 2, 6, 16 mg/mL
- Amount of vehicle: 5 mL/kg

Details on mating procedure:

- M/F ratio per cage: 1:1 mating
- Length of cohabitation: A maximum of 14 days was allowed for mating.
- Proof of pregnancy: Appearance of intravaginal copulatory plug or by evidence of sperm in vaginal
smear, referred to as day 0 of pregnancy.
- After a maximum of 14 days of unsuccessful pairing females without evidence of mating were separated from their males without further opportunity of mating.
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged: individually

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Accuracy and homogeneity were determined for formulations prepared for use in weeks 1, 4, 7, 10, 14, 18, 21, 24, 27. Samples were stored in normal glassware causing the samples stored at room temperature to be exposed to normal laboratory light conditions.
Additional formulations were prepared at a larger bulk volume during Study Week 10, which were only used for trial analysis and not for dosing. Formulations were destroyed after all measurements were performed and results were evaluated.

Duplicate samples (approximately 500 mg) taken from the formulations using a pipette, were accurately weighed. For determination of accuracy, samples were taken at middle position (50% height) or at top, middle and bottom position (90%, 50% and 10% height). The samples taken at 90%, 50% and 10% height were also used for the determination of the homogeneity of the formulations.

Concentration results were considered acceptable if mean sample concentration results were within or equal to 85-115% of target concentration. Homogeneity results were considered acceptable if the coefficient of variation is = 10%.

Stability analyses performed previously in conjunction with the method development and validation study (Test Facility Study No. 20240947) demonstrated that the test item is stable in the vehicle when prepared and stored under the same conditions at concentrations bracketing those used in the present study. Stability data have been retained in the study records of Test Facility Study No. 20240947.

Duration of treatment / exposure:

F0-males were treated for a minimum of 11 weeks, including 10 weeks prior to mating and during the mating and post-mating period, up to and including the day before scheduled necropsy. F0-females were treated for a minimum of 16 weeks, including 10 weeks prior to mating, the mating period, the duration of pregnancy and at least 21 days after delivery, up to and including the day before scheduled necropsy.

Prior to weaning, pups were not treated directly but could potentially be exposed to the test item in utero, via maternal milk, or from exposure to maternal urine/ feces. From weaning onwards (PND 21), F1-animals of Cohorts 1A, 1B and 1C were dosed up to and including the day before scheduled necropsy. The F1-animals of Cohort Surplus and spare animals were not dosed from weaning onwards.

Frequency of treatment:

once daily, 7 days a week

Details on study schedule:

F1 animals were not mated.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Remarks:

group 2 - low dose

Doses / concentrations 2

Dose / conc.:

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

Remarks:

group 3 - mid dose

Doses / concentrations 3

Dose / conc.:

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

Remarks:

group 4 - high dose

No. of animals per sex per dose:

F0: 25 animals/sex/dose
F1 cohort 1A: 20 animals/sex/dose
F1 cohort 1B: 20 animals/sex/dose
F1 cohort 1C: 20 animals/sex/dose
F1 cohort Surplus: 10 animals/sex/dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Fasting period before blood sampling for clinical biochemistry: Selected F0-animals and Cohort 1A animals (10 animals/sex/group) were fasted overnight with a maximum of 24 hours before blood sampling, but water was available.

- Dose selection rationale: see "Justification for study design" above

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: During treatment, animals were observed at least twice daily, up to the day prior to necropsy. These clinical observations were conducted before dosing and between 0 and 30 min after dosing.
- Additional observations were conducted in a standard arena once before the first administration of the test item and at weekly intervals during the treatment period.

BODY WEIGHT: Yes
- Time schedule for examinations: Animals were weighed individually on the first day of treatment (prior to dosing), and weekly thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on PND 1, 4, 7, 14 and 21. In order to monitor the health status, animals may be weighed more often.

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
Food consumption was quantitatively measured weekly, except for males and females which were housed together for mating and for females without evidence of mating. Food consumption of mated females was measured on Days 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on PND 1, 4, 7, 14 and 21.

WATER CONSUMPTION: Yes
Subjective appraisal was maintained during the study.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Between 7.00 and 10.30 a.m. on the day of scheduled necropsy.
- Anaesthetic used for blood collection: Yes (isoflurane), from the retro-orbital sinus.
- Animals fasted: Yes, overnight with a maximum of 24 hours before blood sampling, but water was available.
- How many animals: 10 selected animals/sex/group F0-animals
- Parameters checked: White Blood Cell Count (WBC), Reticulocyte (absolute), Neutrophils (absolute), Red Blood Cell Distribution Width Gated (RDWG), Lymphocytes (absolute), Hemoglobin, Monocytes (absolute), Hematocrit, Eosinophils (absolute), Mean corpuscular volume (MCV), Basophils (absolute), Mean corpuscular hemoglobin (MCH), Large unstained cells (LUC) (absolute), Mean corpuscular hemoglobin concentration (MCHC), Red Blood Cell Count (RBC), Platelets

COAGULATION: Yes
- Time schedule for collection of blood: see HAEMATOLOGY.
- Anaesthetic used for blood collection: see HAEMATOLOGY.
- Animals fasted: see HAEMATOLOGY.
- How many animals: 10 selected animals/sex/group F0-animals
- Parameters checked: Prothrombin Time (PT), Activated Partial Thromboplastin Time (APTT)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: see HAEMATOLOGY.
- Anaesthetic used for blood collection: see HAEMATOLOGY.
- Animals fasted: see HAEMATOLOGY.
- How many animals: 10 selected animals/sex/group F0-animals
- Parameters checked: Alanine aminotransferase (ALT), Creatinine, Aspartate aminotransferase (AST), Glucose, Alkaline Phosphatase (ALP), Cholesterol, Total protein, Sodium, Albumin, Potassium, Total Bilirubin, Chloride, Bile Acids, Calcium, Urea, Inorganic Phosphate (Inorg. Phos)

URINALYSIS: Yes
- Time schedule for collection of urine: Overnight (15-20 hrs)
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- How many animals: 10 selected animals/sex/group F0-animals
- Parameters checked: Volume, Specific gravity, Clarity, Colour, pH, Blood, Leukocyte esterase, Bilirubin, Protein, Ketones, Glucose, Sediment: White blood cells (WBC-sed.), Red blood cells (RBC-sed.), Casts, Epithelial cells, Crystals, Bacteria, Other

THYROID HORMONE ANALYSIS: Yes
- Time schedule for collection of blood: Between 7.00 and 10.30 a.m. on the day of scheduled necropsy.
- Animals fasted: Yes, overnight with a maximum of 24 hours before blood sampling, but water was available.
- How many animals: 10 selected animals/sex/group F0-animals
Blood samples were processed for serum and used for measurement of both T4 and TSH.

OTHER:
Females were allowed to litter normally. Postnatal day (PND) 1 was defined as the day when a litter is found completed (i.e. membranes and placentas cleaned up, nest built and/or feeding of pups started). The day prior to PND 1 was considered to be the day when the female started to deliver and was defined as PND 0 and used for recording of delivery. Females that were littering were left undisturbed. Cage debris of pregnant females was examined for evidence of premature delivery and pregnant females were examined to detect signs of difficult or prolonged parturition or deficiencies in maternal care.

Oestrous cyclicity (parental animals):

Estrous cycles was evaluated by examining the vaginal cytology of samples obtained by vaginal lavage. Daily vaginal lavage was performed for all females beginning 14 days prior to mating and during mating until evidence of copulation was observed. Vaginal lavage continued for those females with no evidence of copulation until termination of the mating period. On the day of necropsy, a vaginal lavage was also taken to determine the stage of estrous. This was done for all females, except for females that died spontaneously.

Sperm parameters (parental animals):

For all surviving F0-males the following assessments were performed:

1) Sperm samples was taken from the proximal part of the vas deferens (right) at necropsy. Sperm motility and progressive motility was assessed from all samples. Sperm smears for morphological evaluation was fixed from all samples and stained with hematoxylin and eosin. Abnormal forms of sperm from a differential count of at least 200 spermatozoa (if possible) per animal was recorded. Evaluation was performed for all samples.

2) One epididymis (right) was removed and kept in the freezer at =-15°C. After thawing, the right epididymis was weighed, homogenized and evaluated for sperm numbers. Evaluation was performed for all samples.

In the case of any abnormalities in the right epididymis, the right side organ(s) was fixed in modified Davidson's solution, and the left side organ was used for evaluation of sperm numbers. If abnormalities were found in both epididymis, both these organs were fixed in modified Davidson's solution and no evaluation of sperm numbers was performed.

Parameters examined in all surviving F0-males: testis weight, epididymis weight.
For the testes of all control and high dose group F0-males, and all males that failed to sire or died before mating (except for males in the high dose group), a detailed qualitative examination was made, taking into account the tubular stages of the spermatogenic cycle.

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 8 pups/litter (4/sex/litter as nearly as possible); excess pups were killed and discarded.

PARAMETERS EXAMINED F1 UNTIL WEANING
The following parameters were examined in F1 offspring until weaning:
Number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities, anogenital distance (AGD), pup weight on the day of AGD, presence of nipples/areolae in male pups. Particular attention was paid to the external reproductive genitals which was examined for signs of altered development (gross evaluation of external genitalia).
Pups were checked for mortality/morbundity twice daily until weaning and detailed clinical observations were performed at last once daily. The number of live and dead pups was determined on PND 1 and daily thereafter. Live pups were weighed individually on PND 1, 4, 7 ,13 and 21. Anogenital distance (AGD) was measured for all live pups on PND 1. The AGD was normalized to the cube root of body weight. All male pups in each litter were examined for the number of areola/nipples on PND 13. Sex was externally determined for all pups on PND 1, 4 and 13.

Thyroid hormone analysis:
Blood of F1-animals was collected on PND 4, at culling, from two surplus pups per litter by decapitation between 7.00 and 10.30 a.m. and samples were pooled to one sample per litter. Blood samples were collected into tubes without anticoagulant, processed for serum and analyzed for total Thyroxine (T4) only.
For F1-cohort surplus animals (10/sex/group) on PND 22-24 blood was collected between 8.00 and 11.30 a.m. Blood samples at a target volume of 1.0 mL were collected into tubes without anticoagulant, processed for serum and used for measurement of both T4 and TSH.

GROSS EXAMINATION OF DEAD PUPS UNTIL WEANING:
Yes, for external and internal abnormalities; cause of death was determined for pups born or found dead between birth and PND 13, if possible: Pups that died before scheduled termination were examined externally with emphasis on developmental morphology and sexed (both externally and internally). For pups found dead or sacrificed in extremis from PND 14 onwards a limited necropsy was performed including sex determination (both externally and internally, if possible). The stomach of pups not surviving to the scheduled necropsy date was examined for the presence of milk, if possible.

The following parameters were examined in F1 offspring from weaning onwards (Cohorts 1A, 1B and 1C):

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: At least twice daily
- General health/mortality

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: During treatment, animals were observed at least twice daily, up to the day prior to necropsy. These clinical observations were conducted before dosing and between 0 and 30 min after dosing.
- Additional observations were conducted in a standard arena once when the first animals have reached day 8 of weaning and at weekly intervals during the treatment period.

BODY WEIGHT: Yes
- Time schedule for examinations: Animals were weighed weekly from weaning onwards. In addition, the body weight was recorded of each female on the day of acquisition of vaginal patency and of each male on the day of acquisition of balanopreputial separation. For animals of Cohorts 1A, 1B and Surplus, a terminal weight was recorded on the day of scheduled necropsy.

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
Food consumption was quantitatively measured weekly, from weaning onwards up to the day prior to scheduled necropsy.

WATER CONSUMPTION: Yes
Subjective appraisal was maintained during the study.

VAGINAL PATENCY: Yes
- Time schedule for examinations: monitored daily for all females from PND 25 onwards until vaginal patency was present, by visual inspection of the vaginal area.

BALANOPREPUTIAL SEPARATION
- Time schedule for examinations: monitored daily for all males from PND 35 onwards until balanopreputial separation was present, by visual inspection of the genital area.


The following parameters were examined in cohort 1A animals only:

STAGE OF ESTROUS CYCLE
Estrous stages was determined by examining the cytology of a vaginal lavage sample, taken on the day of scheduled necropsy.

ESTOUS CYCLE DETERMINATION
Estrous stages were determined by examining the cytology of vaginal lavage samples, taken during two periods:
During the first period, daily vaginal lavage was performed for all Cohort 1A females starting on the day of onset of vaginal patency and was minimally continued until the first estrus is determined, in order to determine the time interval between these two events. During the second period, daily vaginal lavage was performed from PND 75 to 88.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Between 7.00 and 10.30 a.m. on the day of scheduled necropsy.
- Anaesthetic used for blood collection: Yes (isoflurane), from the retro-orbital sinus.
- Animals fasted: Yes, overnight with a maximum of 24 hours before blood sampling, but water was available.
- How many animals: 10 selected animals/sex/group
- Parameters checked: White Blood Cell Count (WBC), Reticulocyte (absolute), Neutrophils (absolute), Red Blood Cell Distribution Width Gated (RDWG), Lymphocytes (absolute), Hemoglobin, Monocytes (absolute), Hematocrit, Eosinophils (absolute), Mean corpuscular volume (MCV), Basophils (absolute), Mean corpuscular hemoglobin (MCH), Large unstained cells (LUC) (absolute), Mean corpuscular hemoglobin concentration (MCHC), Red Blood Cell Count (RBC), Platelets

COAGULATION: Yes
- Time schedule for collection of blood: see HAEMATOLOGY.
- Anaesthetic used for blood collection: see HAEMATOLOGY.
- Animals fasted: see HAEMATOLOGY.
- How many animals: 10 selected animals/sex/group
- Parameters checked: Prothrombin Time (PT), Activated Partial Thromboplastin Time (APTT)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: see HAEMATOLOGY.
- Anaesthetic used for blood collection: see HAEMATOLOGY.
- Animals fasted: see HAEMATOLOGY.
- How many animals: 10 selected animals/sex/group
- Parameters checked: Alanine aminotransferase (ALT), Creatinine, Aspartate aminotransferase (AST), Glucose, Alkaline Phosphatase (ALP), Cholesterol, Total protein, Sodium, Albumin, Potassium, Total Bilirubin, Chloride, Bile Acids, Calcium, Urea, Inorganic Phosphate (Inorg. Phos)

URINALYSIS: Yes
- Time schedule for collection of urine: Overnight (15-20 hrs)
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- How many animals: 10 selected animals/sex/group
- Parameters checked: Volume, Specific gravity, Clarity, Colour, pH, Blood, Leukocyte esterase, Bilirubin, Protein, Ketones, Glucose, Sediment: White blood cells (WBC-sed.), Red blood cells (RBC-sed.), Casts, Epithelial cells, Crystals, Bacteria, Other

THYROID HORMONE ANALYSIS: Yes
- Time schedule for collection of blood: Between 7.00 and 10.30 a.m. on the day of scheduled necropsy on PND 89-95.
- Animals fasted: Yes, overnight with a maximum of 24 hours before blood sampling, but water was available.
- How many animals: 10 selected animals/sex/group.
Blood samples were processed for serum and used for measurement of both T4 and TSH.

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving males after successful mating and a minimum of 10 weeks of treatment.
- Maternal animals: All surviving females which delivered on LD 23-25. Females which failed to deliver were euthanized at post-coitum days 25-27 (with evidence of mating), or approximately 24-26 days after the last day of the mating period (without evidence of mating).

Unscheduled deaths - F0-Generation

If an animal dies on study, a necropsy was conducted and specified tissues were saved, but not weighed. If necessary, the animal was refrigerated to minimize autolysis. Animals were euthanized for humane reasons as per Test Facility SOPs. These animals were deeply anesthetized using isoflurane and subsequently exsanguinated. They underwent necropsy, and specified tissues were retained, but not weighed.

GROSS NECROPSY
- All animals surviving until scheduled euthanasia were subjected to a full post mortem examination, with special attention being paid to the reproductive organs.
The numbers of former implantation sites were recorded for all paired females. In case no macroscopically visible implantation sites were present, non-gravid uteri were stained using the Salewski technique in order to detect any former implantation sites and the number of corpora lutea was also recorded.

HISTOPATHOLOGY / ORGAN WEIGHTS
Organs were weighed at necropsy for all scheduled euthanasia animals according to the guideline.
Paired organs were weighed together. In the event of gross abnormalities, in addition to the combined weight, the weight of the aberrant organ was taken and recorded in the raw data. Organ to body weight ratios (using the terminal body weight) were calculated.
Representative samples of the tissues were collected from all animals, preserved, processed and evaluated according to the guideline and as described in Table 1 below.

Postmortem examinations (offspring):

SACRIFICE
- The F1 offspring were sacrificed at:
Cohort 1A: PND 89-95
Cohort 1B: PND = 97
Cohort 1C: after positive determination of vaginal patency or balanopreputial separation
Cohort Surplus: PND 22-24
Spare animals: PND 22-24

Unscheduled Deaths– F1 -Generation
Recognizable fetuses of females that died spontaneously or were euthanized in extremis were examined externally and sexed (both externally and internally, if possible). Live fetuses were euthanized by decapitation.

Pups sacrificed in extremis on or after PND 7 were euthanized by an intraperitoneal injection of sodium pentobarbital.
Stillborn pups and pups found dead between birth and PND 13 were sexed (both externally and internally) and externally examined with emphasis on developmental morphology. For pups found
dead or sacrificed in extremis from PND 14 onwards a limited necropsy was performed including sex determination (both externally and internally).
Descriptions of all external abnormalities were recorded. The stomach of pups not surviving to the scheduled necropsy date was examined for the presence of milk, if possible, to evaluate any possible defects or cause of death.

Culled Pups (PND 4) – F1-Generation
On PND 4, the pups scheduled for culling (> 8 pups per litter) were euthanized by decapitation. Sex was determined both externally and internally. Pups were externally examined, with particular attention to the external reproductive genitals to examine signs of altered development. Descriptions of all external abnormalities were recorded.

Cohort surplus (10/sex/group)
Scheduled necropsy of Cohort Surplus was conducted on PND 22-24. Cohort Surplus animals were not deprived of food overnight before necropsy and a terminal body weight was recorded. All animals were subjected to a limited examination, with special attention being paid to the reproductive organs. Descriptions of all external abnormalities were recorded. Representative samples of the tissues were weighed and collected.

Spare F1-animals which were not assigned to one of the Cohorts were sacrificed between PND 22-24 by intraperitoneal injection of sodium pentobarbital. Animals were externally examined, with particular attention to the external reproductive genitals to examine signs of altered development, and sex was determined (both externally and internally). Descriptions of all external abnormalities were recorded.

Cohort 1A
Cohort 1A animals surviving to scheduled necropsy were deprived of food overnight (with a maximum of 24 hours) before necropsy, weighed and deeply anaesthetized using isoflurane and subsequently exsanguinated. All animals were subjected to a full post mortem examination, with special attention being paid to the reproductive organs. Descriptions of all external abnormalities were recorded.
HE stained step sections of ovaries and corpora lutea at a thickness of 5 micrometers (5 step sections in total, including the routine section) were prepared for the Cohort 1A females of Group 1 and 4 for quantitative evaluation of follicles (one ovary; primordial and small growing follicles counted together), as well as corpora lutea.

Sperm Analysis – Cohort 1A
For all males of Cohort 1A, the following assessments were performed:
1) Sperm samples were taken from the proximal part of the vas deferens (right) at necropsy. Sperm motility and progressive motility were assessed from all samples. Sperm smears for morphological evaluation were fixed from all samples and stained with haematoxylin and eosin. Abnormal forms of sperm from a differential count of at least 200 spermatozoa (if possible) per animal was recorded. Evaluation was performed for all samples.

2) One epididymis (right) was removed, placed in labeled bags, and kept in the freezer at =-15°C. After thawing the right epididymis was weighed, homogenized and evaluated for sperm numbers. Evaluation was performed for all samples.

In the case of any abnormalities in the right epididymis, the right side organ(s) were fixed in modified Davidson's solution, and the left side organ was used for evaluation of sperm numbers. If abnormalities were found in both epididymis, both these organs were fixed in modified Davidson's solution and no evaluation of sperm numbers were performed.

Parameters examined in all surviving Cohort 1A F1-males: testis weight, epididymis weight.
For the testes of all control and high dose group males, a detailed qualitative examination was made, taking into account the tubular stages of the spermatogenic cycle.

Splenic Lymphocyte Subpopulation Analysis – Cohort 1A
From 10 selected animals/sex/group of Cohort 1A, splenic lymphocyte subpopulation analysis was performed at termination. If possible, one pup (male or female) was selected per litter (20 litters in total). One half of the spleen was kept on ice until splenic lymphocytes were isolated using 70 µm cell strainers. The other half of the spleen was preserved for histopathological evaluation. Splenocytes were counted with the Coulter Counter Z1. The following subpopulations were determined in isolated splenic lymphocytes using the BD FACSCanto™ flow cytometer system on the day of necropsy:
T-cells (CD3+/CD45RAT-), T-helper cells (CD3+/CD4+/CD8-), T-cytotoxic cells (CD3+/CD4-/CD8+), B-cells (CD3-/CD45RA+), NK-cells (CD3-/CD161a+), Ratio T-helper cells/ T-cytotoxic cells (Th/Tc)

Cohort 1B & Cohort 1C
Cohort 1B and 1C animals were not deprived of food overnight before necropsy. These animals were weighed and deeply anaesthetized using isoflurane and subsequently exsanguinated. All animals were subjected to a limited examination, with special attention being paid to the reproductive organs. Descriptions of all macroscopic abnormalities were recorded.

HISTOPATHOLOGY / ORGAN WEIGTHS
Organs were weighed at necropsy for all scheduled euthanasia animals according to the guideline.
Paired organs were weighed together. In the event of gross abnormalities, in addition to the combined weight, the weight of the aberrant organ was taken and recorded in the raw data. Organ to body weight ratios (using the terminal body weight) were calculated.
Representative samples of the tissues were collected from all cohort 1A and 1B animals, preserved, processed and evaluated according to the guideline and as indicated in Table 2 and 3 below.
From Cohort 1C animals only animal identification and any gross lesions observed during necropsy were collected.
From all animals of the cohort surplus, the brain, thymus and spleen were weighed. The following tissues were collected: brain, mammary gland (of both sexes), spleen, thymus and any gross lesions observed during necropsy.

Statistics:

All statistical tests were conducted at the 5% significance level. All pairwise comparisons was conducted using two sided tests and were reported at the 1% or 5% levels.
Numerical data collected on scheduled occasions for the listed variables were analyzed as indicated according to sex and occasion. Descriptive statistics number, mean and standard deviation were reported when possible. Inferential statistics were performed according to the matrix below when possible, but excluded semi-quantitative data, and any group with less than 3 observations.
The following pairwise comparisons were made:
Group 2 vs. Group 1
Group 3 vs. Group 1
Group 4 vs. Group 1
Datasets with at least 3 groups (control group and 2 other groups) were compared using Dunnett-test (many-to-one-t-test). Datasets with at least 3 groups was compared using a Steel-test (many-to-one rank test). An overall Fisher’s exact test was used to compare all groups at the 5% significance level. The above pairwise comparisons were conducted using Fisher’s exact test whenever the overall test is significant.

For hematology, coagulation and clinical chemistry parameters, Levene’s test was used to assess the homogeneity of group variances. The groups were compared using an overall one-way ANOVA F-test if Levene’s test was not significant or the Kruskal-Wallis test if it was significant. If the overall F-test or Kruskal-Wallis test was found to be significant, then pairwise comparisons were conducted using Dunnett’s or Dunn’s test, respectively. A Fisher’s exact test was used to conduct pairwise group comparisons of interest.

Reproductive indices:

Mating index females (%): (Number of females mated/Number of females paired) x 100

Precoital time: Number of days between initiation of cohabitation and confirmation of mating

Fertility index females (%): (Number of pregnant females/Number of females mated) x 100

Gestation index (%): (Number of females with living pups on Day 1/Number of pregnant females) x 100

Duration of gestation: Number of days between confirmation of mating and the beginning of parturition

Offspring viability indices:

Post-implantation survival index (%): (Total number of offspring born/Total number of uterine implantation sites) x 100

Live birth index (%): (Number of live offspring on Day 1 after littering/Total number of offspring born) x 100

Percentage live males at First Litter Check (%): (Number of live male pups at First Litter Check/ Number of live pups at First Litter Check) x 100

Percentage live females at First Litter Check (%): (Number of live female pups at First Litter Check/ Number of live pups at First Litter Check) x 100

Viability index (%): (Number of live offspring on Day 4 before culling/Number live offspring on Day 1 after littering) x 100

Weaning index (%): (Number of live offspring on Day 21 after littering/Number live offspring on Day 4 (after culling)) x 100

Percentage live males at weaning (%): (Number of live male pups on Day 21 after littering / Number of live pups on Day 21 after littering) x 100

Percentage live females at weaning (%): (Number of live female pups on Day 21 after littering / Number of live pups on Day 21 after littering) x 100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

No findings were noted during the weekly arena observations in this study.

Test item-related clinical signs were observed in males at 30 and 80 mg/kg bw/day and in females at all dose levels and consisted of the following observations:

Rales: (mostly) directly after dosing in 11 males and 19 females at 80 mg/kg bw/day. This clinical sign was noted mostly on incidental occasions, with a maximum of 7 consecutive days.

Piloerection (females only): in two, four, five and nine females at 0, 10, 30 and 80 mg/kg bw/day, respectively. For most females, this clinical sign was noted for a single period of maximally 8 consecutive days during the Dosing Period.

Salivation: (mostly) directly after dosing in one female at 10 mg/kg bw/day, two males and seven females at 30 mg/kg/day and all males and females at 80 mg/kg bw/day. This clinical sign was noted at low severity and mostly on incidental occasions, with a maximum of 5 consecutive days.

Any other clinical signs noted during the treatment period occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study and did not show any apparent dose-related trend. At the incidence observed, these were considered to be unrelated to treatment with the test item.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

There were two unscheduled deaths in total, one male and one female at 80 mg/kg bw/day.

The male at 80 mg/kg bw/day was euthanized in extremis before dosing on Day 71. This animal had a labored respiration, flat posture, blue discoloration of extremities (cyanosis), and was lethargic. Except for salivation on incidental days, no clinical signs and no changes in body weight were observed for this male up to the morning of early sacrifice. At necropsy, the lungs did not collapse, stomach, jejunum and ileum were distended with gas and many reddish foci were noted on the glandular mucosa of the stomach. These mucosal foci were related to the microscopic findings of moderate hemorrhages; additional microscopic findings in the stomach were moderate erosion, slight inflammatory infiltrates, and edema. These abnormalities in the stomach were likely the cause of moribundity and therefore considered to be test item-related.

The female at 80 mg/kg bw/day was found dead on Day 2 of lactation prior to dosing. No relevant clinical signs or changes in body weight were noted during the days prior to its death. At necropsy, the trachea was found to be perforated which was microscopically confirmed by the presence of a massive ulceration and acute inflammation. Further necropsy findings consisted of lungs that failed to collapse, intestines distended with gas, an enlarged clitoral gland (left side) and reddish foci on the thymus (left side). Given the macroscopic and microscopic findings in the trachea, this mortality was considered to be related to the gavage procedure and not the treatment with the test item.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Body weights and body weight gain were considered to have been unaffected by treatment with the test item up to and including 80 mg/kg bw/day.
Any statistically significant changes in body weights were considered to be unrelated to treatment with the test item, as the difference was noted on Day 1 (prior to start dosing) only or occurred in the absence of a dose-related response.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

No toxicologically relevant changes in food consumption before or after correction for body weight were noted.
Females at 80 mg/kg bw/day had a slightly lower relative food consumption during the premating and post-coitum periods (not always statistically significant), mean of mean food consumption was 4 and 6% lower than control, respectively. Based on the magnitude and absence of relevant differences in food consumption during the lactation period, this change was considered not to be toxicologically relevant.
Any other statistically significant changes in food consumption before or after correction for body weight were considered to be unrelated to treatment since no trend was apparent regarding dose and duration of treatment.

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Hematological parameters of treated rats were considered not to have been affected by treatment with the test item up to and including80 mg/kg bw/day.
The higher mean values noted in males at 80 mg/kg bw/day (not statistically significant) for white blood cell (WBC), neutrophil (NEUT), monocyte (MONO), and eosinophil (EOS) counts were mainly attributed to a single male in each case with relatively high values.
As individual values of other animals at the same dose level generally remained within concurrent control range, these changes were considered to be unrelated to treatment with the test item.

The statistically significantly lower mean neutrophil count of females at 10 mg/kg bw/day was considered to be unrelated to administration of the test item due to absence of a dose response.

Coagulation parameters of treated rats were considered not to have been affected by treatment with the test item up to 80 mg/kg bw/day.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

No toxicologically relevant changes were noted in clinical biochemistry parameters after treatment with the test item up to and including 80 mg/kg bw/day.
Mean bile acid (BILEAC) concentration was increased in females at 30 and 80 mg/kg bw/day (1.62 and 1.94x of control, respectively; not statistically significant). At the individual level, 3/10 mid-dose and 4/10 high-dose females had values that were above the range of controls (8.7-53.0 µmol/L), with a maximum of 150.1 µmol/L in a single mid-dose female. In absence of any corroborative histopathological findings, these changes were considered to be non-adverse.
The statistically significantly lower mean aspartate aminotransferase (AST) noted for females at 10 mg/kg bw/day was considered to be unrelated to administration of the test item due to the absence of a dose response.

Endocrine findings:

effects observed, non-treatment-related

Description (incidence and severity):

Serum levels of total T4 and TSH in males and females were considered to be unaffected by treatment with the test item up to and including 80 mg/kg bw/day.
Mean TSH concentrations of treated males were 1.34x, 1.32x and 1.48x of control at 10, 30 and 80 mg/kg bw/day, respectively (not statistically significant). In the absence of a clear dose response and as individual values generally remained within historical control range, this increase was considered not to be test item-related.

[Mean values were 0.1219, 0.1633, 0.1612 and 0.1799 mU/L for the control, 10, 30 and 80 mg/kg bw/day groups, respectively. Historical control data of TSH (mU/L) in male Wistar Han rats (2017-2021): Mean = 0.174, P5 – P95 = 0.030 – 0.514 (n=120)].

Urinalysis findings:

no effects observed

Description (incidence and severity):

Urinalysis parameters of treated rats were considered not to be affected by treatment with the test item up to and including 80 mg/kg bw/day.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Test item-related microscopic findings were noted in the stomach of the 80 mg/kg bw/day group males and are summarized in Table 5 below.

Test item-related microscopic findings in the glandular stomach of males at 80 mg/kg bw/day
consisted of:
- Degeneration/Regeneration of the glandular mucosa was observed (up to slight degree). In these animals, hypertrophy of mucous cells was additionally present.
- Erosion of the glandular stomach was observed (up to moderate degree).
- An increased incidence and severity in hemorrhage of the glandular mucosa (up to moderate degree) was observed.
- An increased incidence and severity in submucosal glandular infiltrate was observed (up to slight degree).

The remainder of the recorded microscopic findings were considered to be incidental or within the range of background pathology encountered in rats of this age and strain. There was no test item-related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

Length and regularity of the estrous cycle were not affected by treatment with the test item, all females had regular cycles of 4 to 5 days.

Reproductive function: sperm measures:

effects observed, non-treatment-related

Description (incidence and severity):

No toxicologically relevant changes were noted in sperm analysis parameters.
The mean sperm count in males at 80 mg/kg bw/day was increased (1.20x of control). Due to the direction of change, this difference was considered not to be toxicologically relevant.
Mean percentage of progressive sperm was lower in males at 10 and 30 mg/kg bw/day (0.80 and 0.77x of control, respectively). In absence of a dose-related response, this change was considered to be unrelated to treatment with the test item.

Reproductive performance:

effects observed, non-treatment-related

Description (incidence and severity):

Mating index was considered not to be affected by treatment with the test item. Except for one control female and two females of the 10 and 80 mg/kg vw/day group each, all females showed evidence of mating.
The mating indices were 96, 92, 100 and 92% for the control, 10, 30 and 80 mg/kg bw/day groups, respectively.

Precoital time was considered not to be affected by treatment with the test item. All females showed evidence of mating within 5 days, with exception of one control female, four females at 10 mg/kg bw/day and one female at 80 mg/kg bw/day, for which mating was confirmed during the second week of the mating period.

Number of implantation sites was considered not to be affected by treatment with the test item.

Fertility index was considered not to be affected by treatment with the test item. The fertility indices were 96, 96, 100 and 91% for the control, 10, 30 and 80 mg/kg/day groups, respectively.
In total, one control female, one female at 10 mg/kg bw/day and two females at 80 mg/kg bw/day were not pregnant. At the incidence observed and given the absence of any reproductive/ developmental toxicity, these cases of non-pregnancy were considered not to be related to treatment with the test item.

In the control group, there was 1/25 couple that did not mate and one female that was not pregnant after mating. In the 10 mg/kg/day group, there were 2/25 couples that did not mate, one female that was not pregnant after mating and one female with implantation sites only. In the 30 mg/kg bw/day group, there was one female with implantation sites only. In the 80 mg/kg bw/day group, there were two 2/25 couples that did not mate and two females that were not pregnant after mating. There were no histopathologic changes which could account for their lack of healthy offspring.
There were no morphological findings in the reproductive organs of either sex which could be attributed to the test item and stage aware evaluation of the testes did not show any indication for abnormal spermatogenesis (see table 6 below). The testes revealed normal progression of the spermatogenic cycle, and the expected cell associations and proportions in the various stages of spermatogenesis were present.

Gestation index (females with living pups on Day 1 compared to the number of pregnant females) and duration of gestation were considered not to be affected by treatment with the test item.
Except for one female of the 10 and 30 mg/kg bw/day groups each with implantation sites only, all pregnant females had live offspring. The gestation indices were 100, 95, 96 and 100% for the control, 10, 30 and 80 mg/kg bw/day groups, respectively.

No signs of difficult or prolonged parturition were noted among the pregnant females. Examination of cage debris of pregnant females revealed no signs of abortion or premature birth. No deficiencies in maternal care were observed.

The total number of offspring born compared to the total number of uterine implantations was considered not to be affected by treatment with the test item.

Post-implantation survival index (total number of offspring born as percentage of total number of uterine implantation sites) was 94, 84, 96 and 94% for the control, 10, 30 and 80 mg/kg bw/day groups, respectively. The lower post-implantation survival index at 10 mg/kg bw/day was partially caused by two females with a relatively small litter when compared to the number of implantation sites (i.e. 4 pups vs. 12 or 14 implantation sites). However, no relationship with the test item was indicated given the absence of a dose-related response.

Litter size was considered not to be affected by treatment with the test item. Live litter sizes were 11.1, 11.0, 11.3 and 11.1 living pups/litter for the control, 10, 30 and 80 mg/kg bw/day groups, respectively.

Sex ratio was considered not to be affected by treatment with the test item.

The number of live offspring on Day 1 after littering compared to the total number of offspring born was considered not to be affected by treatment with the test item. Live birth index (number of live offspring on PND 1 as percentage of total number of offspring born) was considered not to be affected by treatment with the test item. The live birth indices were 99, 99, 98 and 100% for the control, 10, 30 and 80 mg/kg b w/day groups, respectively. Two pups of the control group (both from the same litter), two pups at 10 mg/kg bw/day (both from the same litter) and five pups at 30 mg/kg bw/day (all from the same litter) were found dead at first litter check. No toxicological relevance was attributed to these dead pups since the mortality incidence did not show a dose-related trend and remained within the range considered to be normal for pups of this age.

The number of live offspring on Day 4 before culling compared to the number of offspring on Day 1 (viability index) was considered to be unaffected by treatment with the test item. Viability indices (number of live offspring on PND 1 as a percentage of total number of offspring born) were 98, 100, 100 and 93% for the control, 10, 30 and 80 mg/kg bw/day groups, respectively. The slightly lower viability index at 80 mg/kg bw/day was mostly attributed to the 15 pups of a single dam that were sacrificed on Day 2 of lactation as this dam died shortly after the morning check on that day. As such, this pup mortality was considered to be unrelated to treatment with the test item. In addition, four pups of the control group (from two different litters), one pup at 10 mg/kg bw/day, one pup at 30 mg/kg bw/day and two pups at 80 mg/kg bw/day (from the same litter) were found dead or missing on PND 2-4. Pups missing were most likely cannibalized. No toxicological relevance was attributed to these dead/missing pups since the mortality incidence did not show a doserelated trend and remained within the range considered to be normal for pups of this age.

The number of live offspring at weaning (PND 21) compared to the number of live offspring on Day 4 (after culling) was considered to be unaffected by treatment with the test item. The weaning indices were 100, 100, 99 and 100% for the control, 10, 30 and 80 mg/kg bw/day groups, respectively. One pup of the 30 mg/kg bw/day group was found dead on PND 15. No toxicological relevance was attributed to this single dead pup since the mortality incidence did not show a dose-related trend and remained within the range considered to be normal for pups of this age.

Effect levels (P0)

1

Key result

Dose descriptor:

NOAEL

Remarks:

Local toxicity

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

gross pathology

histopathology: non-neoplastic

2

Key result

Dose descriptor:

NOAEL

Remarks:

Systemic toxicity

Effect level:

>= 80 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Absence of additional adverse effects other than the local effects observed above up to and including the highest dose level tested

3

Key result

Dose descriptor:

NOAEL

Remarks:

Reproduction

Effect level:

>= 80 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Absence of adverse effects observed up to and including the highest dose level tested.

Target system / organ toxicity (P0)

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

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

System:

gastrointestinal tract

Organ:

stomach

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

Results: F1 generation

General toxicity (F1)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

F1 - pre weaning:
No clinical signs occurred among pups that were considered to be related to treatment with the test item.
For the pup of a single dam at 30 mg/kg bw/day that was missing on PND 2, absence of milk in the stomach was noted at first litter check on PND 1. The nature and incidence of this and other clinical signs remained within the range considered to be normal for pups of this age, and were therefore considered not to be toxicologically relevant.

F1 - post weaning:
No findings were noted during the weekly arena observations in this study.
Test item-related clinical signs were observed in males and females at 80 mg/kg bw/day and consisted of the following observations:
• Rales: pre- and/or post-dose in 38/60 males and 34/60 females. This clinical sign was noted at slight to moderate severity, mostly on incidental occasions, with a maximum of seven consecutive days, except for one female at 80 mg/kg bw/day that presented with rales over a period of 17 days.
• Gasping (males only): pre- and/or post-dose in 5/60 males, with a maximum of two consecutive days. In all males that showed gasping, also rales were noted in (almost) the same period.
• Piloerection (males only): pre- and/or post-dose in 6/60 males. This clinical sign was noted for a single period of maximally four consecutive days.
• Slight lethargy (post-dose), slightly labored respiration (pre-dose), hunched posture and/or a pale appearance (both pre- and post-dose): in 3/60 males on 1-3 days; observed together with other signs of respiratory difficulties (rales, gasping) and/or poor condition (piloerection).
• Salivation: (mostly) directly after dosing in 36/60 males and 37/60 females. This clinical sign was noted at slight severity and mostly on isolated occasions, with a maximum of three consecutive days.
Any other clinical signs noted during the treatment period (including rales or squeaking in a single male at 30 and 80 mg/kg bw/day, respectively) occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study. At the incidence observed, these were considered to be unrelated to treatment with the test item.

Mortality / viability:

mortality observed, treatment-related

Description (incidence and severity):

F1 – pre weaning:
see reproductive performance P0


F1 – post weaning:
There were six unscheduled deaths at 80 mg/kg bw/day, three males and three females.
A summary of the data available is given in Table 7 below.

Three males (one f cohort 1A and 2 of cohort 1B) died during the first 6-12 days of treatment. One female was found dead on the morning of treatment Day 90 (pre-dose). Available data from observations during in-life and/or necropsy indicated respiratory difficulties for these animals. As similar findings were noted for surviving animals in this high dose group, a relation of these premature deaths to treatment with the test item is very likely.
The death of a second female on Day 3 of treatment was mainly considered to be related to the challenges of the gavage procedure in relatively young and small animals, based on the necropsy finding (trachea perforation).
The third female was sacrificed for humane reasons on Day 9 of treatment as it was observed with paralyzed hindlegs. It should be noted that despite this paralysis, the withdrawal reflex was still present (taken from study daybook). At the single incidence, this unscheduled death was considered to be unrelated to treatment with the test item.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

F1 - pre weaning:
Body weights of pups were considered not to be affected by treatment with the test item.

F1- post weaning:
Body weights and body weight gain were considered to be unaffected by treatment with the test item up to and including 80 mg/kg bw/day. Any statistically significant changes in body weights were considered to be unrelated to treatment with the test item, as the difference was noted on Day 1 (prior to start dosing) or occurred in absence of a relation to duration of treatment and/or dose.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

F1 – post weaning:
No toxicologically relevant changes in food consumption before or after allowance for body weight were noted. The slightly lower absolute food consumption noted for males at all dose levels up to Week 8 of treatment (not always statistically significant at 30 mg/kg bw/day) was attributed to minimal differences in body weights, as relative food consumption remained similar to concurrent control. The statistically significantly lower absolute food consumption observed in females at 80 mg/kg bw/day in Week 5 was considered to be unrelated to administration of the test item due to the minimal magnitude of the change and absence of a relation to duration of treatment.

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

F1 – post weaning:
Test item-related changes in hematology parameters were observed in females at 80 mg/kg bw/day only. Mean counts of neutrophils (NEUT), lymphocytes (LYMPH) and basophils (BASO) were increased when compared with concurrent control, resulting in an increased mean total white blood cell (WBC) count. The fold changes for these parameters when compared with concurrent control are presented in Table 8 below.
The higher mean counts of monocytes (MONO), eosinophils (EOS) and large unstained cells (LUC) in females at 80 mg/kg bw/day (1.46, 1.75 and 1.80x of control, respectively; not statistically significant) were attributed to relatively low control means. The lower mean corpuscular volume (MCV) in females at 30 mg/kg bw/day was considered to be unrelated to treatment with the test item in absence of a dose-related response. In absence of any corroborative histopathological findings, these changes in hematology parameters were considered to be non-adverse. Hematology parameters in males were considered to be unaffected by treatment with the test item.

Clotting parameters in males and females were considered to be unaffected by treatment with the test item. The longer mean activated partial thromboplastin time (APTT) in males at 80 mg/kg bw/day (1.19x of control; not statistically significant) was attributed to a single male with an extremely high value (58.9 sec), which was confirmed after re-analysis of the sample (60.6 sec). After exclusion of this unexpected high value, a mean value of 20.74 sec is reached which is in the same range as for controls.

[Mean values for Cohort 1A females were: Monocytes (109/L): 0.063, 0.084, 0.086, 0.092 for the control, 10, 30 and 80 mg/kg bw/day groups, respectively, Eosinophils (109/L): 0.059, 0.088, 0.077, 0.103 for the control, 10, 30 and 80 mg/kg bw/day groups, respectively, Large unstained cells (109/L): 0.010, 0.017, 0.013, 0.018 for the control, 10, 30 and 80 mg/kg bw/day groups, respectively. Historical control data for selected hematology parameters in Cohort 1A females (period 2017-2021): Monocytes (109/L): Mean = 0.1, P5 - P95 = 0.04 - 0.17 (n=114), Eosinophils (109/L): Mean = 0.1, P5 - P95 = 0.03 - 0.11 (n=114), Large unstained cells (109/L): Mean = 0.0, P5 - P95 = 0.01 - 0.07 (n=38)]

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

F1 – pre weaning:

1) PND 4 (T4 only)
Serum T4 levels in male and female pups, culled on PND 4, were considered not to be affected by treatment with the test item.
For several animals within each group, values of 5.0 ng/mL were reported (i.e. ½ LOQ) as the original value was below the LOQ, which is causing relatively low mean values. If the 5.0 ng/mL values were not taken into account, mean values would be 13.4, 12.5, 13.6 and 12.4 ng/mL for control, 10, 30 and 80 mg/kg/day groups, respectively. These corrected mean serum T4 levels in male and female pups, were 0.93x, 1.01x and 0.93x of control at 10, 30 and 80 mg/kg bw/day, respectively. Based on the low magnitude of change and in absence of a dose-related response, these changes were considered not to be related to treatment with the test item.

2) PND 22 (TSH and T4)
At 80 mg/kg bw/day, mean TSH concentration in males of Cohort Surplus was increased (1.29x of control; not statistically significant). All individual values remained within the historical control range.
Serum levels of TSH (females) and total T4 (both sexes) in animals of Cohort Surplus were considered to be unaffected by treatment with the test item up to 80 mg/kg/day. The slightly increased concentration of total T4 in males at 10 mg/kg/day (1.24x of control; not statistically significant) was not test item-related in the absence of a dose-related response.

[Mean values for TSH (mU/L) in Cohort Surplus males were 0.0896, 0.0847, 0.0938, 0.1158 mU/L for the control, 10, 30 and 80 mg/kg bw/day groups, respectively. Historical control data for TSH (mU/L) in Cohort Surplus males (period 2017-2021): mean = 0.081, P5 - P95 = 0.0245 - 0.2065 (n=100)]

F1 – post weaning:

Test item-related changes in clinical chemistry parameters were observed starting at 30 mg/kg bw/day. In males at 80 mg/kg bw/day, a slight decrease in mean concentration of albumin (ALB) and consequently in total protein (TPROT) was recorded (0.97 and 0.96x of control, respectively). In females at 30 and 80 mg/kg bw/day, an increase in mean concentration of bile acids (BILEAC) was noted (1.68 and 2.64x of control, respectively; not statistically significant). At the individual level, 5/10 mid-dose and 6/10 high-dose females had values that were above the range of controls (5.5-20.5 µmol/L), with a maximum of 95.1 µmol/L in a single high-dose female. In absence of any corroborative histopathological findings, these changes in clinical biochemistry parameters were considered to be non-adverse. Any other changes in clinical biochemistry parameters were considered to be unrelated to administration of the test item due to the minimal magnitude, variation in direction of change and/or absence of a dose-related response.

Thyroid hormone analyses:
Serum levels of total T4 and TSH in Cohort 1A males and females were considered to be unaffected by treatment with the test item up to 80 mg/kg bw/day.
Mean concentrations of total T4 in treated males were 0.83, 0.84 and 0.82x of control at 10, 30 and 80 mg/kg bw/day, respectively (not statistically significant at the mid-dose). This finding was regarded unrelated to treatment with the test item, as it lacked a dose relationship and individual values showed great overlap.
Any differences in mean levels of total T4 and TSH in treated females compared to controls were considered to be unrelated to treatment with the test item, as no dose relationship was evident.
Note: The relatively high mean TSH concentration in females at 30 mg/kg bw/day (2.65x of control; not statistically significant) was attributed to a single animal with an unusual high value (2.360 mU/L). When excluding this female, a mean value of 0.0822 mU/L was obtained which is comparable to that of the low-dose group and 0.70x of control.

Urinalysis findings:

effects observed, non-treatment-related

Description (incidence and severity):

Urinalysis parameters of treated rats were considered to be unaffected by treatment with the test item up to 80 mg/kg bw/day.
At the individual level, occult blood (BLD) was noted in the urine samples of 2/10 males at 80 mg/kg bw/day, whereas all remaining samples (including controls) were negative for blood. This finding was considered not to be test item-related, as it occurred at a relative low incidence and severity (maximum score of 2 on a scale of 1-3) and no red blood cells (RBC) were found in these urine samples.

Sexual maturation:

effects observed, non-treatment-related

Description (incidence and severity):

Balanopreputial separation (prepuce opening; BPS) in males and vaginal patency (vaginal opening; VO), occurrence of first estrus and time between vaginal opening and first estrus in females was considered not to be affected by treatment with the test item.
At 80 mg/kg bw/day, it was noted that three males did not reach BPS before PND 51 or 53, while in all remaining high dose males BPS was attained between PND 38-47 (i.e. same range as for controls). It should be noted that all three high dose males came from the same litter and also one male in the low dose group did not attain BPS before PND 50. Therefore, the incidentally higher individual age at attainment of BPS in the high dose group was considered to be unrelated to treatment with the test item.
At 30 mg/kg bw/day, mean age at attainment of both BPS and VO was slightly reduced in males (40.8 days vs. 41.6 in controls; not achieving statistical significance) and females (31.4 days vs. 32.1 in controls), respectively. Also mean body weights at attainment of BPS and VO were slightly lower (males: 160 gram vs. 169 in controls; females: 92 gram vs. 97 in controls; not statistically significant in females). In the absence of a dose-related trend, this observation was considered to be unrelated to treatment with the test item.

Length and regularity of the estrous cycle were not affected by treatment with the test item; all females had regular cycles of 4 to 5 days. At both 10 and 30 mg/kg bw/day, a single female was noted with an irregular cycle. Based on the single incidence and in the absence of a dose-related trend, this finding was considered not to be test item-related.

Anogenital distance (AGD):

no effects observed

Description (incidence and severity):

Anogenital distance (absolute and normalized for body weight) in male and female pups was considered not to be affected by treatment with the test item.

Nipple retention in male pups:

no effects observed

Description (incidence and severity):

Treatment up to and including 80 mg/kg/day had no effect on areola/nipple retention. For none of the examined male pups nipples were observed on PND 13.

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

F1- pre weaning:
Cohort surplus (PND 22): Brain, thymus and spleen weight of Cohort Surplus were considered to be unaffected by treatment with the test item up to 80 mg/kg bw/day.

F1- post weaning:
Cohort 1A (PND 89-95)
There were no test item-related organ weight changes. All organ weight differences observed, regardless of reaching statistical significance when compared to controls, were considered not to be test item-related due to the lack of a dose-related pattern, lack of a test item-related microscopic correlate and/or general overlap and variability in individual values. The lower terminal body weight recorded for males at 10 mg/kg bw/day when compared to control males was considered to be unrelated to treatment with the test item, as it lacked a dose relationship.

Cohort 1B (= PND 90)
Organ weights and organ:body weight ratios of treated Cohort 1B animals were considered to be similar to those of control animals. The slightly lower mean organ weights and mean organ:body weight ratios noted for testes, epididymides, prostate and seminal vesicles in males at 80 mg/kg bw/day were attributed to a single male which had unusual small reproductive organs. At the isolated incidence and in absence of similar findings in Cohort 1A males, this was regarded as a chance finding.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

F1 - pre weaning: No macroscopic findings were noted among pups sacrificed at the end of the lactation period that were considered to be related to treatment with the test item.

Cohort surplus (PND 22-24): There were no gross observations in animals of Cohort Surplus.

F1 - post weaning:

Cohort 1A (PND 89-95)
There was a test item-related increased incidence in macroscopic findings in the glandular
stomach of 4/20 males treated at 80 mg/kg bw/day consisting of reddish/dark red foci (the
microscopic correlate was hemorrhage). Watery fluid in the uterus, found in 5/20, 5/20 and 3/20 and 6/20 females of respectively the control, 10, 30 and 80 mg/kg bw/day group, is related to the stage in the estrous cycle and is a normal finding. All other recorded macroscopic findings were within the range of background gross observations encountered in rats of this age and strain.

Cohort 1B (= PND 97)
There were no test item-related macroscopic observations. All of the recorded macroscopic findings were within the range of background gross observations encountered in rats of this age and strain.
Note: A single male at 80 mg/kg bw/day was observed with all its reproductive organs (testes, epididymides, prostate and seminal vesicles) reduced in size (see also section Organ weight findings above). At the isolated incidence and in absence of similar findings in Cohort 1A males, this was regarded as a chance finding. Watery fluid in the uterus, found in 3/20, 4/20 and 3/20 and 4/20 females of respectively the control, 10, 30 and 80 mg/kg bw/day group, is related to the stage in the estrous cycle and is a normal finding.

Cohort 1C (males: = PND 35; females: = PND 25)
There were no test item-related macroscopic observations. All of the recorded macroscopic findings in Cohort 1C animals were within the range of background gross observations encountered in rats of this age and strain.

Histopathological findings:

effects observed, treatment-related

Description (incidence and severity):

F1 - post weaning
Test item-related microscopic findings were noted in the stomach of the 30 and 80 mg/kg bw/day group males of Cohort 1A and are summarized in Table 9 below.

Test item-related microscopic findings in the glandular stomach of males at 30 and 80 mg/kg bw/day consisted of:
• Hemorrhage of the glandular mucosa (up to slight degree).
• Minimal erosion observed in a single male at 30 and 80 mg/kg bw/day each.
• An increased incidence and severity in submucosal glandular infiltrate (up to moderate degree).
The remainder of the recorded microscopic findings were within the range of background pathology encountered in rats of this age and strain. There was no test item-related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.

Testis Staging (Cohort 1A)
Stage dependent qualitative evaluation of spermatogenesis in the testis was performed. The testes revealed normal progression of the spermatogenic cycle and the expected cell associations and proportions in the various stages of spermatogenesis were present.

Ovarian Follicle Counts (Cohort 1A)
There were no test item-related effects on the ovarian follicle and corpora lutea counts in F1-females (Cohort 1A) at 80 mg/kg bw/day when compared to control group females. Any variation between group mean counts represented biological variability and was not statistically significant.

Additional Histopathology Early Sacrifices
Additional histopathological examination of the gastro-intestinal tract (stomach, duodenum, jejunum, ileum, cecum, colon, rectum) was performed on the (former) F1-animals that were sacrificed in extremis or found dead and of which mortality was possibly related to treatment with the test item. All animals (except for one female at 80 mg/kg bw/day) were replaced by other animals and therefore not included in the Main study results. Animals included in this part are one male of Cohort 1A at 80 mg/kg bw/day, two males of Cohort 1B at 80 mg/kg bw/day and one female of Cohort 1B at 80 mg/kg bw/day.

Test item-related microscopic findings were observed in former male of Cohort 1B at 80 mg/kg bw/day, which consisted of a few small ulcerations (slight degree) in the glandular mucosa of the stomach together with granulocytic inflammatory infiltrates (moderate) in glandular mucosa/submucosa and edema (slight) in the glandular submucosa. These findings could explain the poor health condition of this animal. There were no findings in the other gastrointestinal organs.
Additionally, a small erosion in the stomach was observed in the glandular mucosa of a female of Cohort 1B at 80 mg/kg bw/day, which was not considered to be the cause of death for this animal. There were no findings in the other gastro-intestinal organs. No test item-related microscopic findings in the glandular stomach were noted for the male of Cohort 1A at 80 mg/kg bw/day and the second male of Cohort 1B at 80 mg/kg bw/day.

Other effects:

no effects observed

Description (incidence and severity):

There were no test item-related effects on splenic lymphocyte subpopulations observed.

Sperm motility, concentration and morphology were considered not to be affected by treatment up to 80 mg/kg bw/day. The mean sperm count in males at 80 mg/kg bw/day was increased (1.26x of control), but without achieving statistical significance. Due to the direction of change, this difference was considered not to be toxicologically relevant.
At the individual level, two males were noted with a remarkably low percentage of motile and/or progressive sperm: One male at 10 mg/kg bw/day, had only 6% motile and 1% progressive sperm; no macroscopic abnormalities were found in the reproductive organs of this low dose male. Another male at 30 mg/kg bw/day, had 90% motile sperm, but 0% progressive sperm. Both, testes and epididymides were reduced in size (bilateral).
Furthermore, there was one male at 30 mg/kg bw/day with a relatively low epididymal sperm count (107.6x10^6 cells/gram). No macroscopic abnormalities were observed in the reproductive organs of this male which could explain this low sperm concentration.
In absence of a dose-related trend, these incidental findings were regarded as unrelated to treatment with the test item.

Effect levels (F1)

1

Key result

Dose descriptor:

NOAEL

Remarks:

Local toxicity

Generation:

F1

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

gross pathology

histopathology: non-neoplastic

2

Key result

Dose descriptor:

NOAEL

Remarks:

Systemic toxocity

Generation:

F1

Effect level:

>= 80 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Absence of adverse effects other than the local effects observed above up to and including the highest dose level tested

3

Key result

Dose descriptor:

NOAEL

Remarks:

Developmental toxicity (F1 until weaning at PND 21)

Generation:

F1

Effect level:

>= 80 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Absence of adverse effects observed up to and including the highest dose level tested.

4

Key result

Dose descriptor:

NOAEL

Remarks:

Developmental toxicity (F1 post-weaning)

Generation:

F1

Effect level:

>= 80 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Absence of adverse effects observed up to and including the highest dose level tested.

Target system / organ toxicity (F1)

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

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

System:

gastrointestinal tract

Organ:

stomach

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

Overall reproductive toxicity

Key result

Reproductive effects observed:

no

Any other information on results incl. tables

Analysis of dose preparations:

Accuracy
A small response at the retention time of the test item was observed in the chromatograms of the Group 1 formulations prepared for use in Weeks 4, 7 and 24. It was considered to derive from carry-over in the analytical system since similar responses were found in the analytical blanks. The maximum contribution to the Group 2 samples analyzed in the same series based on peak area and taking the dilution factors into account was 0.22%, 0.22% and 0.33%, respectively. This was considered negligible and regarded to have no impact on the outcome of the study.
In all other formulations for Group 1, no test item was detected.
The concentrations analyzed in the formulations of Groups 2, 3 and 4 were in agreement with target concentrations (i.e., mean sample concentration within or equal to 85-115% of target concentration) except for:
- Group 3 prepared for use in Week 7 (mean concentration 120% of target);
- Group 2 prepared for use in Week 10 (mean concentration 118% of target);
- Group 2 prepared for use in Week 14 (mean concentration 127% of target);
- Group 2 prepared for use in Week 21 (mean concentration 84% of target);
- Group 2 prepared for use in Week 24 (mean concentration 83% of target);
- Groups 2 and 4 prepared for use in Week 27 (mean concentrations 79 and 83% of target, respectively).
Out of Specification investigations were performed in order to determine the cause of the relatively low or high recovery. During these investigations, both the dose preparation procedure and analytical procedure were scrutinized. No explanation could be found for the observed high recoveries. The low recoveries may have been due to evaporation of test item during preparation of the formulations and/or analysis even though all possible precautions to keep evaporation as limited as possible had been taken.

Homogeneity
All Group 2 and 4 formulations were homogeneous (i.e., coefficient of variation ≤ 10% (actual min-max was 0.25-6%)).

 

Table 4: Mean Percent THYROID GLAND Weight Differences from Control Groups: F₀-Females

	Females
Dose level (mg/kg bw/day):	10	30	80
THYROID GLAND
Absolute	8	10	13*
Relative to body weight	5	9	13*
*: P<0.05

 

Table 5: Summary Test Item-Related Microscopic Findings: Males F₀-Generation

	Males
Dose level (mg/kg bw/day):	0	10	30	80
STOMACH ^a	25	25	25	25
Degeneration/Regeneration, glandular mucosa
Minimal	-	-	-	1
Slight	-	-	-	1
Erosion, glandular mucosa
Minimal	-	-	-	2
Slight	-	-	-	1
Moderate	-	-	-	1
Hemorrhage, glandular mucosa
Minimal	1	-	-	2
Slight	-	-	-	2
Moderate	-	-	-	1
Infiltrate, glandular submucosa
Minimal	3	5	6	8
Slight	-	-	-	3
^a  =Number of tissues examined from each group. Bold used for test item-related findings.

 

Table 6: Summary F₀-Generation Males that Failed to Sire and Females that Failed to Deliver Healthy Pups

Group	Dose Level (mg/kg bw/day)	Female/Male Nos.	In-Life Reason	Histopathology Related to Reproductive Failure
1	0	105/5	Not mated	no/no
		114/14	Not pregnant	no/no
2	10	126/26	Not pregnant	no/no
		127/27	Not mated
		140/40	Not mated	no/no
		142/42	Implantation sites only
3	30	155/55	Implantation sites only	no/no
4	80	183/83	Not pregnant	no/no
		191/91	Not mated	no/no
		192/92	Not pregnant	no/no
		196/96	Not mated	no/no

 

Table 7 Mortality Incidence F₁-Animals

Dose Level		Animal No.	Cohort	Sex	Day of Death	Day of Treatment^a	Death Status	Relevant Observations prior to Death	Relevant Necropsy Findings ^b	Histopathological Findings Gastro-Intestinal Tract^c
80 mg/kg bw/day		426A^$	1A	M	13 Apr 2021 (PND 32)	12 Note: This male was not dosed on Days 11 and 12 due to breathing difficulties.	Euthanized in extremis	Piloerection, rales (slight to moderate), gasping, abdomen distended with gas. In addition, this male barely gained weight between Days 8 and 15 of treatment (from 78 to 79 grams).	Emaciated, intestines distended with gas and lungs failed to collapse.	No abnormalities
		433A*	1B	M	05 Apr 2021 (PND 26)	6 (pre-dose)	Euthanized in extremis	Piloerection, pale appearance, dull and gasping for air with a gurgling sound. In addition, body weight loss between Days 1 and 6 after dosing (from 56 to 41 grams; taken from study daybook).	Stomach and intestines distended with gas; lungs failed to collapse.	No abnormalities
		441A^#	1B	M	09 Apr 2021 (PND 29)	9 (pre-dose)	Euthanized in extremis	Piloerection; gasping for air with a gurgling sound and an extended abdomen.	Stomach and intestines distended with gas, several reddish foci on the forestomach, and lungs failed to collapse.	Small ulcerations (slight degree) in the glandular mucosa of the stomach, granulocytic inflammatory infiltrates (moderate) in glandular mucosa/submucosa and edema (slight) in the glandular submucosa.
		692A^#	1A	F	08 Apr 2021 (PND 29)	9 (pre-dose)	Euthanized in extremis	Paralyzed hindlegs. Note: Withdrawal reflex was still present (taken from study daybook).	Many reddish foci on the thymus.	-
		714A*	1B	F	02 Apr 2021 (PND 23)	3 (pre-dose)	Euthanized in extremis	Piloerection; animal was squeaking and gasping for air with a gurgling sound.	Perforation of the trachea.	-
		726	1B	F	09 Jun 2021 (PND 89)	90 (pre-dose)	Spontaneous death	Slight rales.	Beginning autolysis; lungs failed to collapse and many dark-red foci on the thymus.	Small erosion in the stomach
	^aTime point of death compared to dosing (i.e. pre-dose or post-dose) was taken from the study daybook/raw data. Dosing commenced on the day that the respective animals were weaned. ^b      No full histopathological examination was conducted on animals that died preterm. Except for Cohort 1B-Female No. 726, all preterm decedents were replaced by either a Cohort 1C animal or pups that were not allocated yet. Therefore, sufficient Cohort 1A animals were available for a thorough evaluation of possible test item-related effects. ^c       Histopathological examination of the gastro-intestinal tract was conducted on Animal Nos. 426, 433, 441 and 726 that died preterm. Note: ‘A’ denotes animals that were replaced after their preterm death. Information regarding replacement and data recording is given below: ^*      Male No. 433A and Female No. 714A (both Cohort 1B) were replaced by spare animals. ^$      Male No. 426A (Cohort 1A) was replaced by Male No. 469 (Cohort 1C). Data from Male No. 469 were transferred to Male No. 426 (and vice versa). Note: Before the replacement, all in-life measurements were identical for both males. Therefore, no data are missing. ^#Male No. 441A (Cohort 1B) and Female No. 692A (Cohort 1A) were replaced with the pups that were intended to be weaned as Nos. 470 (Cohort 1C) and 760 (Cohort Surplus), respectively. For Animal Nos. 470 and 760, the necropsy date was recorded as being the first date of weaning. These animal numbers were planned in the computer system but were not used in the study as no animal was available at weaning.

 

Table 8: Test Item-Related Hematology Changes – F₁-Cohort 1A Females

	Females
Dose Level (mg/kg bw/day)	10	30	80
WBC (10^9/L)	1.08x	1.10x	1.39x**
NEUT (10^9/L)	0.95x	1.02x	1.29x
LYMPH (10^9/L)	1.10x	1.10x	1.40x**
BASO	1.50x	1.50x	2.25x

** = p ≤ 0.01.

 

Table 9: Summary Test Item-Related Microscopic Findings: Males F₁-Generation – Cohort 1A

	Males
Dose level (mg/kg bw/day):	0	10	30	80
STOMACH ^a	20	20	20	20
Hemorrhage, glandular mucosa
Minimal	-	-	2	2
Slight	-	-	-	3
Erosion, glandular mucosa
Minimal	-	-	1	1
Infiltrate, glandular submucosa
Minimal	1	3	8	4
Slight	-	1	1	5
Moderate	-	-	-	1
^a  =Number of tissues examined from each group. Bold used for test item-related findings.

 

Applicant's summary and conclusion

Conclusions:

In an extended one generation reproductive toxicity study according to OECD TG 443 (including Cohort 1) without extension to F2, exposure of parental F0-Wistar (Han) rats and their F1 offspring in utero, through nursing during lactation and by oral gavage with Dimethylethylamine at doses of 10, 30 and 80 mg/kg bw/day, resulted in a combination of clinical signs and histopathological findings (both macroscopically and microscopically) in the F0- and F1 generation (both unscheduled deaths and animals surviving until scheduled necropsy) that might be interpreted as local rather than systemic effects.
Clinical signs indicative of local effects were respiratory difficulties (i.e., rales, gasping and/or labored respiration) and salivation, observed in both F0 and F1 animals. In addition, general signs of discomfort consisted of flat posture, cyanosis, lethargy, hunched posture, piloerection and/or a pale appearance (mostly observed for F1-animals only). Adverse test item-related morphologic alterations were observed in the (sub)mucosa of the glandular stomach of the F0- and F1-males at 80 mg/kg bw/day, which were likely the cause of moribundity in one early sacrificed F0-male. The findings consisted of a combination of degeneration/regeneration, hemorrhages, erosions and/or inflammatory infiltrates up to moderate degree. In addition, four early sacrificed/found dead F1-animals at 80 mg/kg/day, showed similar signs of local toxicity as observed for surviving animals and as such a relation to treatment with the test item was suspected.

In conclusion, based on the results the following no-observed-adverse-effect levels (NOAELs) of Dimethylethylamine were established:

Local toxicity (F0 and F1): 30 mg/kg bw/day.
Systemic toxicity (F0): at least 80 mg/kg bw/day.
Reproduction toxicity (F0): at least 80 mg/kg bw/day.
Developmental toxicity (F1 until weaning at PND 21): at least 80 mg/kg bw/day.
Developmental toxicity (F1 post-weaning): at least 80 mg/kg bw/day.
Higher doses were not tolerated, based on the results from the previous reproduction/developmental toxicity screening test (Test Facility Study No. 20240951; OECD 421). In this latter study, high mortality and severe stomach findings were observed at 150 mg/kg bw/day (mid dose) and 225 mg/kg bw/day (high dose). At the low dose of 80 mg/kg bw/day, however, only a single male had to be euthanized in extremis and only minimal stomach findings were noted.

Executive summary:

The dose levels in this study were selected to be 0, 10, 30 and 80 mg/kg/day, based on the results of a preliminary reproductive toxicity study (reproduction/developmental toxicity screening test) with oral exposure of Dimethylethylamine in rats (Test Facility Study No. 20240951, OECD 421). In this latter study, high mortality and severe stomach findings were observed at 150 mg/kg/day (mid dose) and 225 mg/kg/day (high dose). At the low dose of 80 mg/kg/day, however, only a single male had to be euthanized in extremis and only minimal stomach findings were noted.

For the F₀-generation, the following parameters and end points were evaluated in this study: mortality/ moribundity, clinical signs, body weight, food consumption, clinical pathology including measurement of thyroid hormones and urinalysis, gross necropsy findings, sperm analysis, organ weights and histopathologic examinations.

For the F₁-generation, the following parameters and end points were evaluated in this study: mortality/moribundity, clinical signs, body weight, food consumption, vaginal patency and balanopreputial separation, day of first estrus, clinical pathology including measurement of thyroid hormones and urinalysis, gross necropsy findings, sperm analysis and splenic lymphocyte subpopulation analysis, organ weights and histopathologic examinations.

In addition, the following reproduction/developmental parameters were determined for the F₀-Generation: estrous cycle, mating and fertility indices, precoital time, number of implantation sites, gestation index and duration, parturition, maternal care, sex ratio and early postnatal pup development (mortality, clinical signs, body weights, sex, anogenital distance, areola/nipple retention, macroscopy and measurement of thyroid hormones).

 

 

Parental Results (F₀-Generation)

Test item-related changes were observed in F₀-males at 80 mg/kg/day and F₀-females from all treated groups.

10 mg/kg/day

A small increase in thyroid gland weight (both absolute and relative to body weight) was observed in F₀-females starting at 10 mg/kg/day. Considering the subtle dose-response over the treated groups, this was likely test item-related. However, based on the absence of any macroscopic or microscopic findings it was considered to be non-adverse.

30 mg/kg/day

An increase in mean concentration of bile acids was noted in females at 30 mg/kg/day. In absence of any corroborative histopathological findings, these changes were considered to be non-adverse.

A small increase in thyroid gland weight (both absolute and relative to body weight) was observed in F₀-females at 30 mg/kg/day. Considering the subtle dose-response over all treated groups (starting at 10 mg/kg/day), this was likely test item-related. However, based on the absence of any macroscopic or microscopic findings it was considered to be non-adverse.

80 mg/kg/day

One male in this high dose was euthanized in extremis before dosing on Day 71. This animal had a labored respiration, flat posture, blue discoloration of extremities (cyanosis), and was lethargic. At necropsy, the stomach, jejunum and ileum were distended with gas at necropsy. histopathological examination revealed a combination of microscopical findings in the glandular stomach that were likely the cause of moribundity of this high dose male. These consisted of hemorrhages (macroscopic correlate: many reddish foci on the glandular mucosa), erosion, slight inflammatory infiltrates, and edema. Similar microscopic findings and signs of degeneration/regeneration in the glandular stomach were also observed in individual high dose males that survived until scheduled necropsy. Based on the mortality of the single male, the degenerative nature (degeneration/regeneration and erosion) in combination with severities up to moderate degree, the glandular stomach findings were considered adverse in the 80 mg/kg/day group F₀-males. However, these are signs of local, rather than systemic toxicity of the test item. As such, these are regarded as not relevant for systemic effect level determination and not taken into account for establishing the systemic NOAEL.

In-life findings in the 80 mg/kg/day group included rales (several males and females) and salivation (all animals). Both findings were noted independently from each other. They occurred (mostly) directly after dosing on incidental occasions (with a maximum of 7 consecutive days), without any relation to the duration of treatment. As such they were considered to represent local, rather than systemic effects of the test item. They may have been caused by reflux triggered by the observed test item-related irritation of the glandular stomach. Alternatively, small amounts of test formulation that remained on the outside of the feeding tube after dosing may have come into contact with the epithelial lining of the esophagus/oral cavity upon withdrawal of the tube from the forestomach, thereby causing local effects.

The additionally finding of piloerection in a few females (not males) at 80 mg/kg/day was regarded as a general sign of discomfort. Due to its transient nature and in absence of any other relevant findings in high dose females, it was considered to be non-adverse.

An increase in mean concentration of bile acids was noted in females at 80 mg/kg/day. In absence of any corroborative histopathological findings, these changes were considered to be non-adverse.

The small increase in thyroid gland weight (both absolute and relative to body weight) in F_0-females at 80 mg/kg/day was likely test item-related, as it showed a subtle dose-response over all treated groups. However, this change in thyroid gland weight was considered to be non-adverse, based on the absence of any macroscopic or microscopic findings. Further, serum levels of the thyroid hormones TSH and (total) T4 appeared to be unaffected by treatment with the test item up to 80 mg/kg/day.

No test item-related changes were noted in any of the remaining parameters investigated in this study (i.e. body weight, food consumption, hematology, clotting and clinical biochemistry parameters, except for TSH and total T4 thyroid hormone levels in females).

Reproductive Results

No reproduction toxicity was observed up to the highest dose level tested (80 mg/kg/day).

No test item-related or toxicologically significant changes were noted in any of the reproductive parameters investigated in this study (i.e. mating and fertility indices, precoital time, number of implantations, estrous cycle, sperm analysis, and histopathological examination of reproductive organs, including spermatogenic profiling).

Developmental Results

No developmental toxicity was observed up to the highest dose level tested (80 mg/kg/day).

At 80 mg/kg/day, mean serum TSH level in males of Cohort Surplus (PND 22) were increased (not statistically significant). As all individual values remained within the historical control range, this change was considered to be non-adverse.

No test item-related changes were noted in any of the other developmental parameters investigated in this study (i.e. gestation, post-implantation, live birth, viability and weaning indices, duration of gestation, parturition, sex ratio, maternal care and early postnatal pup development consisting of mortality, clinical signs, body weight, anogenital distance, areola/nipple retention, total T4 thyroid hormone level and macroscopic examination).

F₁-Generation results

Test item-related changes were observed in F₁-males and F₁-females starting at 30 mg/kg/day.

30 mg/kg/day

A dose-related increase in mean concentration of bile acids was noted in F₁-females starting at 30 mg/kg/day. In absence of any corroborative histopathological findings, this was considered to be non-adverse.

Histopathological examination revealed in F₁-males starting at 30 mg/kg/day (mid dose) a combination of microscopic findings in the glandular stomach (minimal erosion and/or minimal to slight hemorrhage and/or inflammatory cell infiltrates) that was comparable to those seen previously in F₀-males at 80 mg/kg/day (high dose). Based on the relatively low severity, this was considered to be non-adverse.

80 mg/kg/day

There were six unscheduled deaths at 80 mg/kg/day, three F₁-males and three F₁-females.

One male in Cohort 1A and two males in Cohort 1B died during the first 6-12 days of treatment. In addition, one female in Cohort 1B was found dead on the morning of treatment Day 90 (pre-dose). Available data from observations during in-life and/or necropsy from these animals indicated respiratory difficulties. As similar findings were also noted for surviving F₁-animals in this high dose group (see below), a relation of these premature deaths to treatment with the test item is very likely.

The deaths of the remaining two females (one in Cohorts 1A and 1B each) on Days 9 and 3 of treatment, respectively were considered unrelated to treatment with the test item. Rather these were caused by incidents during respectively animal handling (clinical observation: paralyzed hindlegs) or the gavage procedure (macroscopic correlate: trachea perforation).

Test item-related clinical signs were observed in F₁-males and F₁-females at 80 mg/kg/day and consisted of signs of respiratory difficulties (i.e., mostly rales, and incidentally gasping, slightly labored respiration), slight lethargy, hunched posture, piloerection, a pale appearance and/or salivation. Similar to the F₀-generation, these findings in F₁-animals were seen on incidental occasions, lasted in general for only a few days, and occurred without any relation to the duration of treatment. Therefore, they were considered to represent local, rather than systemic effects of the test item.

The only observed test item-related change in hematology parameters consisted of increased mean counts of neutrophils, lymphocytes and basophils and consequently an increased mean total white blood cell count in F₁-females at 80 mg/kg/day.
Changes in clinical biochemistry parameters included a slight decrease in mean concentration of albumin and consequently total protein in F₁-males at 80 mg/kg/day, and an increase in mean concentration of bile acids in F₁-females starting at 30 mg/kg/day.
In absence of any corroborative histopathological findings, these changes in hematology and clinical biochemistry parameters were considered to be non-adverse.

Histopathological examination revealed a combination of microscopic findings in the glandular stomach in F₁-males (including one preterm sacrificed male) that was comparable to those seen previously in F₀-males at 80 mg/kg/day. They consisted of erosion, hemorrhage and/or inflammatory cell infiltrates at minimal or slight degree, except for a single F₁-male at 80 mg/kg/day that presented with inflammatory cell infiltrate at a moderate degree. Based on this higher severity, it was considered to be adverse. However, all observed abnormalities in the glandular stomach are signs of local, rather than systemic toxicity of the test item. As such, they are regarded as not relevant for systemic effect level determination and not taken into account for establishing the systemic NOAEL.

No test item-related changes were noted in any of the remaining parameters investigated in this study (i.e. body weights, food consumption, sexual maturation, length and regularity of the estrous cycle, sperm analysis, coagulation parameters, TSH and total T4 thyroid hormone levels, and histopathological examination of reproductive organs, including spermatogenic profiling and ovarian follicle count).

 

 

 

In conclusion, exposure of parental F₀-Wistar (Han) rats and their F₁-offspring in utero, through nursing during lactation and by oral gavage with Dimethylethylamine at doses of 10, 30 and 80 mg/kg/day, resulted in a combination of clinical signs and histopathological findings (both macroscopically and microscopically) in the F₀- and F₁-generation (both unscheduled deaths and animals surviving until scheduled necropsy) that might be interpreted as local rather than systemic effects.
Clinical signs indicative of local effects were respiratory difficulties (i.e., rales, gasping and/or labored respiration) and salivation, observed in both F₀ and F₁-animals. In addition, general signs of discomfort consisted of flat posture, cyanosis, lethargy, hunched posture, piloerection and/or a pale appearance (mostly observed for F₁-animals only). Adverse test item-related morphologic alterations were observed in the (sub)mucosa of the glandular stomach of the F₀- and F₁-males at 80 mg/kg/day, which were likely the cause of moribundity in one early sacrificed F₀-male. The findings consisted of a combination of degeneration/regeneration, hemorrhages, erosions and/or inflammatory infiltrates up to moderate degree. In addition, four early sacrificed/found dead F₁-animals at 80 mg/kg/day, showed similar signs of local toxicity as observed for surviving animals and as such a relation to treatment with the test item was suspected.

Based on the results of this Extended One Generation Reproductive Toxicity Study (including Cohort 1, without extension to the F₂-generation), the following No Observed Adverse Effect Level (NOAEL) of Dimethylethylamine were established:

Local toxicity (F₀ and F₁):           30 mg/kg/day.

Systemic toxicity (F₀):                at least 80 mg/kg/day.

Reproduction toxicity (F₀):         at least 80 mg/kg/day.

Developmental toxicity
(F₁ until weaning at PND 21):     at least 80 mg/kg/day.

Developmental toxicity
(F₁ post-weaning):                       at least 80 mg/kg/day.

Higher doses were not tolerated, based on the results from the previous reproduction/developmental toxicity screening test (Test Facility Study No. 20240951; OECD 421). In this latter study, high mortality and severe stomach findings were observed at 150 mg/kg/day (mid dose) and 225 mg/kg/day (high dose). At the low dose of 80 mg/kg/day, however, only a single male had to be euthanized in extremis and only minimal stomach findings were noted.