Registration Dossier

Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
extended one-generation reproductive toxicity - with both developmental neuro- and immunotoxicity (Cohorts 1A, 1B without extension, 2A, 2B, and 3)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
September 2018 - September 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2020

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 443 (Extended One-Generation Reproductive Toxicity Study)
Version / remarks:
June 2018
Deviations:
no
Principles of method if other than guideline:
OECD guidance document supporting OECD test guideline 443 on the extended one-generation reproductive toxicity test, No. 151, July 2013.
GLP compliance:
yes (incl. certificate)
Remarks:
d.d. 22 January 2018
Limit test:
no
Justification for study design:
The design of this study was based on the final decision on a compliance check of DGEBADA by ECHA (Decision no. CCH-D-2114375349-37-01/F, date 24 Oct 2017).
The European authorities required an “ Extended one-generation reproductive toxicity study (Annex X, Section 8.7.3.; test method: EU B.56./OECD TG 443) in Wistar rats, oral route with the registered substance specified as follows: Ten weeks premating exposure duration for the parental (P0) generation; Dose level setting shall aim to induce some toxicity at the highest dose level; Cohort 1A (Reproductive toxicity); Cohort 1B (Reproductive toxicity) without extension to mate the Cohort 1B animals to produce the F2 generation; Cohorts 2A and 2B (Developmental neurotoxicity); and Cohort 3 (Developmental immunotoxicity).
The oral gavage route of administration was selected as this allows for a more accurate dosing than dietary administration, and since previous toxicity studies conducted with this test item were also conducted using this route of administration.

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid: viscous

Test animals

Species:
rat
Strain:
Wistar
Remarks:
(Wistar Han rats)
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 toxicity testing by regulatory agencies. Charles River Den Bosch has general and reproduction/developmental/neurological/immunological internal data in this species from the same strain and source. This animal model has been proven to be susceptible to the effects of reproductive, neurological and immunological toxicants.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany.
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: (P) 6-7 wks
- Weight at study initiation: (P) Males: 133-173 g; Females: 112-147 g

- Housing:
On arrival, 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 and cohort together) in polycarbonate cages (Macrolon type IV; height 18 cm).
During the mating phase, males and females were cohabitated on a 1:1 basis in Macrolon plastic cages (type III; height 18 cm).
During the post-mating phase, males were housed in their home cage (Macrolon plastic cages, type IV; height 18 cm) with a maximum of 5 males/cage. Females were individually housed in Macrolon plastic cages (type III, height 18 cm).
During the lactation phase, females were housed in Macrolon plastic cages (type III, height 18 cm). Pups were housed with the dam until termination (unscheduled deaths, spares, and pups of Cohort 2B) or until weaning on PND 21 (Cohorts 1A, 1B, 2A and 3).
During locomotor activity monitoring, F1- Cohort 2A animals were housed individually in a Hi-temp polycarbonate cage (Ancare corp., USA; dimensions: 48.3 x 26.7 x 20.3 cm) without cage-enrichment, bedding material, food and water for a maximum of 2 hours.
The cages contained appropriate bedding (Lignocel S 8-15, JRS - J.Rettenmaier & Söhne GmbH + CO. KG, Rosenberg, Germany) and were equipped with water bottles. For psychological/environmental enrichment and nesting material, animals were provided with paper (Enviro-dri, Wm. Lilico & Son (Wonham Mill Ltd), Surrey, United Kingdom), except when interrupted by study procedures/activities.
Animals were separated during designated procedures/activities.

- Diet: ad libitum
Pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany) was provided ad libitum throughout the study, except during designated procedures. During motor activity measurements, F1- Cohort 2A animals had no access to food for a maximum of 2 hours.
The feed was analyzed by the supplier for nutritional components and environmental contaminants. It is considered that there were no known contaminants in the feed that would interfere with the objectives of the study.

- Water: ad libitum
Municipal tap water was freely available to each animal via water bottles. During motor activity measurements, F1- Cohort 2A animals had no access to water for a maximum of 2 hours.
Periodic analysis of the water was performed. It is considered that there were no known contaminants in the water that would interfere with the objectives of the study.

- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-22
- Humidity (%): 43-67
- Air changes (per hr): Ten or greater
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 25 September 2018 To: 16 April 2019

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
polyethylene glycol
Remarks:
PEG400
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Test item dosing formulations (w/w) were homogenized to visually acceptable levels at appropriate concentrations to meet dose level requirements. Formulations were heated to a maximum temperature of 44°C for at least 30 minutes to a maximum of approximately 8 hours to obtain visual homogeneity. The dosing formulations were prepared in daily portions, maximally 7 days prior to dosing. If these were dosed on the day of formulating, they were dosed within 6 hours after adding the vehicle to the test item. Formulations were released for dosing when they had obtained a temperature of 40°C or lower. If not dosed on the day of formulating, the formulations were stored in the refrigerator and removed from the refrigerator and stirred at room temperature for at least 30 minutes before dosing. From 16 Jan 2019 onwards, formulations that had solidified in the refrigerator were heated to maximally 30°C for maximally 2 hours in a water bath in order to accelerate liquefying the formulations.
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 vehicle and test item. No correction was made for the purity/composition of the test item.
An additional formulation was prepared for the Group 2 F1 concentration in Week 21 and 25 of the study for intended stability measurements (these formulations were used for sampling only).

VEHICLE
- Justification for use and choice of vehicle: Trial preparations were performed at the Test Facility to select the suitable vehicle and to establish a suitable formulation procedure. In these trial preparations, water (Elix), 1% aqueous carboxymethyl cellulose, propylene glycol and polyethylene glycol 400 were tested. From these trial preparations, polyethylene glycol 400 was selected as a suitable vehicle. Polyethylene glycol 400 is a commonly used vehicle for repeated toxicity studies in rats, and is regarded not harmful for rats. Moreover, polyethylene glycol (300) was also used in a 90-day toxicity study with DGEBADA in Wistar Han rats according to OECD TG 408 animals.
- Concentration in vehicle: F0 animals 8, 20 and 40 mg/mL; F1 animals 2, 5 and 10 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg bw

A positive control item was used for the TDAR assay in cohort 3
- vehicle: Sterile saline solution (0.9% NaCl)
Each day of dosing, a dose formulation of 1.5 mg/mL Cyclophosphamide was freshly prepared by dissolving cyclophosphamide monohydrate in sterile saline solution. No adjustment was made for specific gravity of the vehicle and cyclophosphamide monohydrate. No correction was made for the purity/composition of cyclophosphamide monohydrate.
Cyclophosphamide formulations were homogenized to visually acceptable levels at appropriate concentrations to meet dose level requirements. Formulations were dosed within 6 hours after adding the vehicle to the test item.
Dosing formulations were kept at room temperature until dosing. The dosing formulations were gently inverted until and during dosing.
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: A maximum of 14 days was allowed for mating, after which females who have not shown evidence of mating were separated from their males.
- Proof of pregnancy: sperm in vaginal smear or vaginal plug referred to as day 0 of pregnancy
- Further matings after unsuccessful attempt: no
- After successful mating each pregnant female was caged: Individually
- Any other deviations from standard protocol:
Detection of mating was not confirmed in first instance for one Group 2 and one Group 3 female. Evidence of mating was obtained indirectly by delivery of a litter. Apparently, mating was overlooked in the assessment of the vaginal lavage, which explains the continuation of di-estrus during the mating in these females. The mating date of these animals was estimated at 21 days prior to the actual delivery date This day was designated Day 0 post-coitum.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Stability analyses performed previously in conjunction with the method development and validation study (Test Facility Study No. 20153909) demonstrated that the test item is stable in the vehicle for at least 6 hours at room temperature, at least 8 days in the refrigerator and for at least 3 weeks in the freezer (= -15°C) when prepared and stored under the same conditions at concentrations bracketing those used in the present study. In order to cover the time period required and concentrations used for dosing all F0 and F1 animals in the study, an additional stability measurement was required over 24 hours at room temperature under normal laboratory light conditions.

Concentration, homogeneity and stability analysis
Duplicate sets of samples (approximately 500 mg) for each sampling time point were sent to the analytical laboratory. Analyses were performed using a validated analytical procedure.

The following dose formulation samples were collected:
Week 1, 2, 3, 5, 9, 13 and 15: concentration analyses (all groups), homogeneity analysis (groups 2 and 4)
Week 21: concentration analyses (all groups + group 2 (F1)), homogeneity analysis (groups 2 and 4 + group 2 (F1)), stability assessment (group 4 (F0) + group 2 (F1))
Week 25: concentration analyses (all groups + group 2 (F1)), homogeneity analysis (groups 2 and 4 + group 2 (F1)), stability assessment (group 2 (F1))

It was noted that the concentration of the Group 2 formulations prepared for the Week 21 analyses was well outside the acceptance criterion due to a weighing error during formulation preparation. It was therefore decided not to analyze stability of this formulation. Instead, new formulations were prepared as part of the Week 25 analyses, including stability analyses. Accuracy and homogeneity was also determined from the Group 2 formulation with a concentration that was used for dosing the F1 animals between PND 7 and 20. The homogeneity data served as t=0 for the stability measurement.

The homogeneity results obtained from the top, middle and bottom for the Group 2 and 4 preparations were averaged and utilized as the concentration results.

Concentration results were considered acceptable if mean sample concentration results were within or equal to ± 10% for solutions of target concentration.
Homogeneity results were considered acceptable if the coefficient of variation (CV) of concentrations was =10%.
Stability results were considered acceptable if the sample analysis results were within or equal to ±10% of the concentration determined by the initial analysis of each formulation.
Duration of treatment / exposure:
(P) Males: 12 weeks (including 10 weeks pre-mating)
(P) Females: 13-16 weeks; 10 weeks before mating, up to 2 weeks during mating, approximately 3 weeks during resulting pregnancies, one week through weaning of their F1 offspring.
(F1) Cohort 1A: 12-13 weeks; direct dosing of pups on PND 7-20 and post-weaning for 10-11 weeks
(F1) Cohort 1B: 13-15 weeks; direct dosing of pups on PND 7-20 and post-weaning for 11-13 weeks
(F1) Cohort 2A: 10-12 weeks; direct dosing of pups on PND 7-20 and post-weaning for 8-10 weeks
(F1) Cohort 2B: 2 weeks; direct dosing of pups on PND 7-20, except for animals that were necropsied on PND 22 which were also dosed on PND 21 using dose levels for PND 7-20
(F1) Cohort 3: 6-8 weeks; direct dosing of pups on PND 7-20 and post-weaning for 4-6 weeks
F1 animals allocated to the positive control group (group 5) were not directly dosed with the test item.
Frequency of treatment:
once daily, 7 days a week
Details on study schedule:
Cohort 1B was not extended to produce a F2 generation
Doses / concentrationsopen allclose all
Dose / conc.:
40 mg/kg bw/day (actual dose received)
Remarks:
Group 2.
F1 animals directly dosed between PND 7 and 20 received 10 mg/kg bw/day (actual dose received)
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Remarks:
Group 3.
F1 animals directly dosed between PND 7 and 20 received 25 mg/kg bw/day (actual dose received)
Dose / conc.:
200 mg/kg bw/day (actual dose received)
Remarks:
Group 4.
F1 animals directly dosed between PND 7 and 20 received 50 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
F0: 25
F1 cohort 1A: 20
F1 cohort 1B: 20
F1 cohort 2A: 10
F1 cohort 2B: 10
F1 cohort 3: 10 males and 9 females
F1 positive control: 10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
The dose levels for F0 animals and for F1 animals post-weaning were selected based on the results of previously conducted (repeated and reproduction) toxicity studies with oral exposure of DGEBADA in rats as detailed below.
In a reproductive/developmental toxicity screening study in Sprague-Dawley rats according to OECD TG 422, animals were dosed at 100, 300 and 900 mg/kg/day by oral gavage (vehicle: propylene glycol). A NOAEL of 900 mg/kg/day was established for parental and reproductive/developmental effects, in absence of adverse effect observed at this maximal dose.
In a 90-day toxicity study in Wistar Han rats according to OECD TG 408 animals were dosed at 100, 300 and 1000 mg/kg/day by oral gavage (vehicle PEG 300). Males at 1000 mg/kg/day had lower body weights (22% lower weight gain than controls) and locomotor activity of males was statistically significantly lower at 300 and 1000 mg/kg/day without concurrent changes in clinical observations or other functional observation parameters. Various clinical pathology changes were noted at 300 and 1000 mg/kg/day including higher cholesterol and phospholipid values. Lower prostate weights were recorded at all dose levels, but occurred in the absence of supportive histopathological lesions and organ weights of other sex organs were normal. Kidney weights were higher at 300 and 1000 mg/kg/day in males. Reduced sperm motility was noted at all dose levels. However, there were no effects on sperm counts and more than 50% of the sperm cells showed progressive motility (mean progressive motility showed no clear dose-related trend). Histopathological changes were noted at 1000 mg/kg/day, consisting of hepatocellular hypertrophy and necrosis, decreased lymphocyte counts in mesenteric lymph nodes and multifocal hypertrophy in the pituitary gland in individual animals. Based on the results of this 90-day repeated toxicity study, a NOAEL for systemic toxicity was below the lowest dose level of 100 mg/kg bw/day of DGEBADA.
In a developmental toxicity study in Wistar Han rats according to OECD TG 414, animals were dosed at 100, 300 and 1000 mg/kg/day by daily oral gavage (vehicle PEG 300). A NOAEL of 1000 mg/kg/day was derived for maternal toxicity and developmental toxicity, in absence of adverse effect observed at this maximal dose.
Based on adverse effects observed in the 90-day repeated toxicity in which no NOAEL was established (LOAEL = 100 mg/kg/day), dose levels were selected to be 40, 100 and 200 mg/kg/day in an attempt to produce graded responses to the test item. The high-dose level should produce some toxic effects, but not death nor obvious suffering. The mid-dose level was expected to produce minimal to moderate toxic effects. The low-dose level should produce no observable indications of toxicity.
The dose levels for F1 animals between PND 7 and 20 were selected based on the dose levels used for F0 animals. Available F0 in-life data indicated that treatment up to 200 mg/kg/day was well tolerated. However, pups may be more vulnerable to treatment than F0 animals given that physiological processes and systems (such as the blood-brain barrier and metabolic processes) of these pups are still developing. Therefore, in order to avoid potential overdosing, the highest dose selected for F1 animals (50 mg/kg/day) was 25% of the high dose administered to F0 animals (200 mg/kg bw/day). The low and mid dose levels for F1 animals (10 and 25 mg/kg bw/day, respectively) were selected using the same relative dose level spacing as for F0 animals.


- Rationale for animal assignment:
F0 animals were assigned to groups by a computer-generated random algorithm according to body weights, with all animals within ± 20% of the sex mean. Males and females were randomized separately.
On PND 4, eight pups from each litter of equal sex distribution (if possible) were selected to reduce variability among the litters. The non-selected pups were culled on PND 4. To reduce variability among the litters, on PND 4 eight pups from each litter of equal sex distribution (if possible) were selected. Selective elimination of pups, e.g. based upon body weight or AGD, was not done. Whenever the number of male or female pups prevented having four of each sex per litter, partial adjustment (for example, five males and three females) was acceptable.
On PND 21, pups from available litters per group were selected and assigned into cohorts.
Positive control:
F1 positive control animals were treated with cyclophosphamide 2 to 4 hours prior to immunization with KLH (Group 5). Cyclophosphamide was administered via intraperitoneal injection using a disposable needle and syringe once daily on five consecutive days prior to necropsy (i.e. starting between PND 48-54), approximately the same time each day with a maximum of 6 hours difference between the earliest and latest dose. Formulations were gently inverted prior to dosing. The dose volume for each animal was based on body weight measurement on Day 1 of treatment.

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily
- General health/mortality and moribundity

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Clinical 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. 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, 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
- Time schedule for examinations: Water consumption was monitored on regular basis throughout the study by visual inspection of the water bottles.

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)
- 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: according to OECD TG 443

CLINICAL CHEMISTRY: 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
- Parameters checked: according to OECD TG 443

URINALYSIS: Yes
- Time schedule for collection of urine: Overnight (15-20 hrs)
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters checked: according to OECD TG 443

THYROID HORMONE
- 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
Blood samples at a target volume of 1.0 mL were collected into tubes without anticoagulant. Blood samples were processed for serum and used for measurement of both T4 and TSH. Any remaining sample was discarded.
Oestrous cyclicity (parental animals):
Estrous stages were determined by examining the cytology of vaginal lavage samples.
Daily vaginal lavage was performed for all F0-females beginning 14 days prior to mating and during mating until evidence of copulation was observed. Vaginal lavage was continued for those females with no evidence of copulation until termination of the mating period. On the day of scheduled necropsy, a vaginal lavage was also taken.
Sperm parameters (parental animals):
For all surviving F0-males and Cohort 1A F1-males, the following assessments were performed:
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.
One epididymis (left) was removed, placed in labeled bags, and kept in the freezer at =-15°C. After thawing the left epididymis was weighed, homogenized and evaluated for sperm numbers. Evaluation was performed for all samples.
As for one Group 4 F0-male, abnormalities were noted in the left epididymis, the left side organ(s) was fixed in modified Davidson's solution, and the right side organ was used for evaluation of sperm numbers.
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
The following parameters were examined in F1 offspring until weaning:
- Mortality/morbundity: twice daily until weaning. The number of live and dead pups was determined on PND 1 and daily thereafter.
- Clinical observations: at least once daily
- Body weights: Live pups were weighed individually on PND 1, 4, 6, 7, 8, 9, 11, 13, 15, 17, 19 and 21. Several litters were also weighed on PND 8.
- Sex: externally determined for all pups on PND 1, 4 and 13.
- Anogenital distance (AGD): measured for all live pups on PND 1. The AGD was normalized to the cube root of body weight.
- Areola/Nipple Retention: all male pups on PND 13.

The following parameters were examined in F1 offspring from weaning onwards (Cohorts 1A, 1B, 2A and 3):
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily
- General health/mortality, simultaneously with the mortality/moribundity check of the dam

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Clinical observations were conducted in a standard arena 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 2A, 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
- Time schedule for examinations: Water consumption was monitored on regular basis throughout the study by visual inspection of the water bottles.

VAGINAL PATENCY: Yes
- Time schedule for examinations: monitored daily for alle 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.

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

The following parameters were examined in cohort 1A animals:
ESTOUS CYCLE ETERMINATION
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 was 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
- 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)
- 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: according to OECD TG 443

CLINICAL CHEMISTRY
- 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
- Parameters checked: according to OECD TG 443

URINALYSIS
- Time schedule for collection of urine: Overnight (15-20 hrs)
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters checked: according to OECD TG 443

THYROID HORMONE
- Time schedule for collection of blood: between 7.00 and 10.30 a.m. on the day of scheduled necropsy (cohort 1A and 2B), at culling
- Animals fasted: Yes, overnight with a maximum of 24 hours before blood sampling, but water was available (cohort 1A only).
- How many animals: 10 selected animals/sex/group (cohort 1A and 2B), on PND 4 from two surplus pups per litter
Blood samples at a target volume of 1.0 mL (Cohort 1A), 0.5 mL (pooled PND 4 pups) and 0.4 mL (Cohort 2B PND 21-22 pups) were collected into tubes without anticoagulant. Blood samples were processed for serum.
Serum of Cohort 1A animals and Cohort 2B animals (i.e. PND 21-22 pups) was used for measurement of both T4 and TSH. Pooled serum of culled PND 4 pups was used for measurement of T4. Any remaining sample was discarded.


ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY (cohort 2A):
- Acoustic startle response:
Acoustic startle response (habituation) was assessed using the StartleMonitor System (Kinder Scientific, Poway, USA). This was performed once between PND 23-25 in a sound-attenuated room.
To the extent possible, treatment groups were balanced across devices and the time of testing was counterbalanced across dose group and sex. The animals were tested in sets of up to 3. The test sessions consisted of a five-minute acclimation period with a 65 ± 5-dB broadband background white noise. The startle stimulus for each trial was a 115 ± 5-dB mixed frequency noise burst stimulus (approximately 20 milliseconds in duration). Responses were recorded during the first 250 milliseconds following onset of the startle stimulus for each trial. The test session consisted of 50 trials with an eight-second intertrial interval. Average response amplitude (AveN), average maximum response amplitude (MaxN) and average latency to achieving the maximum response amplitude (Tmax) was analyzed in five blocks of 10 trials each.

- Functional observation battery: tests were conducted once between PND 63-75 in the order of sequence indicated below and were divided between several days.
1. Detailed clinical observations, consisting of a number of tests conducted in- and out-side the home cage
2. Rectal temperature, measured immediately after the detailed clinical observations.
3. Locomotor activity, tested using the Kinder Scientific Motor Monitor System. Recording period was one hour under normal laboratory light conditions.
To the extent possible, treatment groups were balanced across devices and the time of testing was counterbalanced across dose groups. Total movements and ambulations were reported. Ambulations represent movements characterized by a relocation of the entire body position like walking, whereas total movements represent all movements made by the animals, including ambulations but also smaller or finer movements like grooming, weaving or movements of the head.
4. Hearing ability.
5. Fore- and hindlimb grip strength, recorded per animal as the mean of three measurements, using a grip strength meter (Series M4-10, Mark-10 Corporation).
6. Pupillary reflex .
7. Landing (hind) foot splay, recorded per animal as the mean of three measurements.

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY (cohort 3 and positive control animals):
All F1-animals of Cohort 3 and all positive control animals were immunized once, 5 days before scheduled necropsy (i.e. once between PND 48-54) via intravenous injection into the tail vein (approximately 1 mL/min) with 1.0 mL of the 300 µg/ml of Keyhole Limpet Hemocyanin KLH solution.
For the positive control animals, this immunization was performed 2 to 4 hours after treatment with cyclophosphamide.
For Cohort 3 animals, this immunization was performed at least 2 hours after treatment with the test item formulations/vehicle.
The animals were restrained during the injection procedure and injected using a disposable needle and butterfly needle. After injection, the injection site was marked with indelible ink.
On the day of KLH injection, clinical sign observations was performed after this injection for animals of Cohort 3 and the positive control animals.

Inject site evaluation: at least daiy, starting one day after KLH injection up to and including the day before scheduled necropsy
The irritation scores and a description of all other (local) effects were recorded as part of the “1-hour post-dose” observation. Adjacent areas of skin of each animal served as controls.
The irritation was assessed according to the following numerical scoring system. At each observation, the highest scores given were recorded:

Erythema and eschar formation:
Very slight erythema (barely perceptible) 1
Well-defined erythema 2
Moderate to severe erythema 3
Severe erythema (beet redness) 4
Where signs of necrosis or corrosion (injuries in depth) prevent erythema scoring, the maximum grade for erythema (= 4) is given.

Oedema formation:
Minimal oedema 1
Slight oedema 2
Moderate oedema 3
Severe oedema 4

In-life procedures positive control animals:
- Mortality/moribundity checks: twice daily
- Clinical observations: at least once daily, up to the day prior to necropsy.
- Body weight: Animals were individually weighed once, on the first day of cyclophosphamide treatment.
- Food and water consumption was not determined for positive control animals.

T-Cell Dependent Antibody Response (TDAR) Assay (cohort 3 and positive control animals)
- Time schedule for collection of blood: between 7.00 and 10.30 a.m. pre-immunisation and 5 day after immunization (PND 53-59)
- Animals fasted: No
- How many animals: 10 selected animals/sex/group (cohort 3) and 10 selected animals/sex (positive control animals)
Blood samples at a target volume of 0.3 mL were collected into tubes without anticoagulant. Blood samples were allowed to clot for at least 30 minutes and centrifuged within 2 hours after collection. Within 1 hour after centrifugation, serum of these samples were divided into 2 aliquots and subsequently stored in labeled polypropylene tubes at =-75°C until analysis.
The antibody response to the immunization with KLH was determined by measuring the anti-KLH IgM levels in serum using Rat Anti-KLH IgM ELISA Kits (Life Diagnostics, Inc., West Chester, PA, USA) and an EL808™ Absorbance Microplate reader including Gen5 Secure software version 1.11.5 (BioTek Instruments, Inc., Winooski Vermont, USA) according to a validated method.
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals after successful mating or at the end of the mating period
- Maternal animals: All surviving animals on LD 23-25, or after total litter loss, or failure to deliver

GROSS NECROPSY
- All animals 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 recorded in addition.

Organ Weights and Tissue Collection/Preservation – F0-Generation
Organs were weighed at necropsy for all scheduled euthanasia animals according to the guideline. Organ weights were not recorded for animals found dead or euthanized in poor condition or in extremis. 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.
For females which failed to deliver a complete litter, uterine contents (i.e. any fetuses, placenta and implantation sites) was fixed (if applicable), but was not be examined histopathologically in first instance.
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring were sacrificed at:
Cohort 1A: PND 87-97
Cohort 1B: PND 97-110
Cohort 2A: PBD 76-90
Cohort 2B: PND 21-22
Cohort 3: PND 53-59
Positive control animals: PND 53-59

Unscheduled Deaths– F1 -Generation
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 were examined for the presence of milk, if possible. If possible, defects or cause of death were evaluated.

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.

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. Organs were weighed and tissues prepared according to the guideline.

Sperm Analysis – Cohort 1A
For all males of Cohort 1A, the following assessments were performed: 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.
One epididymis (left) was removed, placed in labeled bags, and kept in the freezer at =-15°C. After thawing the left epididymis was weighed, homogenized and evaluated for sperm numbers. Evaluation was performed for all samples.

Splenic Lymphocyte Subpopulation Analysis – Cohort 1A
From 10 selected animals/sex/group of Cohort 1A, splenic lymphocyte subpopulation analysis was performed at termination. 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:

Parameter (Abbreviation) Unit Markers for identification
T-cells % Lymphoid cells CD3+/CD45RA-
T-helper cells % Lymphoid cells CD3+/CD4+/CD8-
T-cytotoxic cells % Lymphoid cells CD3+/CD4-/CD8+
B-cells % Lymphoid cells CD3-/CD45RA+
NK-cells % Lymphoid cells CD3-/CD161a+
Ratio T-helper cells/ T-cytotoxic cells (Th/Tc) - -

Cohort 1B
Cohort 1B animals were not deprived of food overnight before necropsy. These animals were weighed 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.

Cohort 2A and 2B
The animals were not deprived of food overnight before necropsy and a terminal body weight was recorded. The animals were first anaesthetized using isoflurane and subsequently sacrificed by whole body (in situ) perfusion using heparinized saline (0.9% NaCl) followed by a 4% paraformaldehyde solution (adjusted to pH 7.4; HCl, KCl, NaH2PO4 x H2O, Na2HPO4 x 2H2O, paraformaldehyde and NaOH, aqua dest.).
All animals were subjected to a limited examination, with special attention being paid to the reproductive organs.
After perfusion, the cranium was removed, exposing the brain. The skull including the brain was placed in 10% buffered formalin and allowed to fix for at least 7 days prior to removal from the skull.
The fixed brains were removed and weighed, and the length and maximum width of the brain was measured for all animals selected for neuropathology. Subsequently, the brain was fixed in 10% buffered formalin together with selected PNS tissues.
Morphometric (quantitative) analyses of CNS tissues was performed for Cohort 2A and 2B animals of Groups 1 and 4.

Cohort 3 and Positive Control Animals
Scheduled necropsy of Cohort 3 was conducted on PND 53-59 (except for one animal which was necropsied on PND 49). Positive control animals were euthanized on the same date(s). The animals were deeply anaesthetized using isoflurane and subsequently exsanguinatedand were subjected to a limited examination, with special attention being paid to the reproductive organs.
Statistics:
All statistical tests were conducted at the 5% significance level. All pairwise comparisons were 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 according to sex and occasion. Descriptive statistics number, mean and standard deviation were reported whenever 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

Parametric
Datasets with at least 3 groups (the designated control group and 2 other groups) were compared using Dunnett-test (many-to-one-t-test).
For the motor activity data set (at least 3 groups) parametric (ANOVA) tests on group means was applied with Bonferroni correction for multiple testing. Mixed modelling techniques, comparing six different covariance structures, were used in order to select the best fitting statistical model.

Non-Parametric
Datasets with at least 3 groups were compared using a Steel-test (many-to-one rank test).
The startle data set and the follicle count data set (at least 3 groups) was compared using an overall Kruskal-Wallis. Whenever, the overall test is significant, the Wilcoxon Rank-Sum test was applied to compare the treated groups to the control group.

Incidence
An overall Fisher’s exact test was used to compare all groups. The above pairwise comparisons were conducted using Fisher’s exact test whenever the overall test is significant.
Reproductive indices:
Mating index males (%): Number of males mated/Number of males paired x 100
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 males(%): Number of pregnant females/Number of males mated x 100
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:
no effects observed
Description (incidence and severity):
No clinical signs were noted during daily detailed clinical observations that were considered to be signs of toxicity. No findings were noted during the weekly arena observations in this study.
Salivation seen after dosing among all test item treated groups in a dose-related manner was considered to be a physiological response rather than a sign of systemic toxicity considering the nature and minor severity of the effect and its time of occurrence (i.e. shortly after dosing). This sign may be related to taste of the test substance.
Other findings 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 signs of no toxicological relevance.
Mortality:
no mortality observed
Description (incidence):
No mortality related to treatment with the test item occurred during the study period.
One female of the 200 mg/kg/day group was euthanized on Lactation Day 3, as she had a total litter loss.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
At 200 mg/kg bw/day, mean body weight gain of females was statistically significantly lower than controls on Days 14 and 21 of the lactation phase (0.7x and 0.5x, respectively), which was considered to be related to treatment with the test item. Mean body weight gain at the end of the lactation phase was 0.5x of the control mean. Two females at 200 mg/kg/day showed weight loss of 8-11% and 1-5% of body weight on PND 1 throughout the lactation phase, respectively.
Other variations in mean absolute body weights noted during the premating and post-coitum phase were considered not to be related to treatment. During the premating period these variations consisted of slightly higher mean absolute body weights in females at 40, 100 and 200 mg/kg/day. A level of statistical significance was achieved on most occasions during the premating phase at 100 and 200 mg/kg/day, and at 40 mg/kg/day on Day 22 only. Mean body weight gain at the end of the premating period was similar between the groups.
During the post-coitum phase, these variations consisted of statistically significantly higher mean absolute body weights at 200 mg/kg/day on Day 7. Mean body weight gain during the post-coitum phase was similar between the groups. Since a clear dose-related trend was absent and since these variations were of a slight degree and/or not consistently noted as treatment progressed, they were considered not to be related to treatment.
Body weights and body weight gain of males remained in the same range as controls over the treatment period.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
Food consumption before or after correction for body weight was considered not affected by treatment with the test item in males and females.
A statistically significant higher mean absolute food consumption was recorded for females at 200 mg/kg/day over Days 1-15 of the premating phase. However, since this apparent difference was very minor and did not become more pronounced as treatment progressed, this was considered not to be related to treatment.
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Haematological parameters of treated rats were considered not to have been affected by treatment with the test item.
Any statistically significant changes in haematology parameters achieving a level of statistical significance when compared to controls, occurred in the absence of a dose-related response. As such, these slight differences were considered not related to treatment.

Coagulation
At 200 mg/kg bw/day, a statistically significantly elongated prothrombin time (PT) was recorded for males (1.08x; mean remained within the internal control range (mean = 17.6; P5 – P95 = 15.50-19.40 (n=79)).
At 40 and 100 mg/kg bw/day in males and females, and at 200 mg/kg bw/day in females, coagulation parameters were considered not to have been affected by treatment.

Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Higher mean cholesterol was recorded in males at 200 mg/kg bw/day (1.26x; mean remained within the internal control range (mean = 1.81; P5 – P95 = 1.315-2.465 (n=80)), which was considered to be related to treatment.
Slightly lower total protein in males at 100 and 200 mg/kg bw/day and slightly lower albumin in males at 40, 100 and 200 mg/kg/day were considered not to be related to treatment with the test item, since these variations occurred in the absence of a dose-related trend and since means remained within the internal control range.
Clinical chemistry parameters in F0-females were considered not affected by treatment.
Any other statistically significant changes in clinical chemistry parameters achieving a level of statistical significance when compared to controls, occurred in the absence of a dose-related response. As such, these slight differences were considered not related to treatment.
Urinalysis findings:
no effects observed
Description (incidence and severity):
Urinalysis revealed no changes that were considered to be related to treatment with the test item.
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
There were no test item-related microscopic observations.
All 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.
Histopathological findings: neoplastic:
not examined
Other effects:
not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
Length and regularity of the estrous cycle were considered not to have been affected by treatment with the test item.
Most females had regular cycles of 4 days (one female at 100 mg/kg bw/day had a cycle length of 5 days). For two control females, regularity of the estrous cycle could not be determined. An irregular cycle was noted for one female at 40 mg/kg/day (with normal litter). Given their incidental nature, absence of a dose-related incidence and absence of an apparent correlation to pregnancy status, these findings did not indicate a relation with treatment.
Reproductive function: sperm measures:
effects observed, non-treatment-related
Description (incidence and severity):
Sperm motility, concentration and morphology were considered not affected by treatment with the test item.
A statistically significantly higher or lower mean epididymal sperm count was recorded at 40 and 100 mg/kg bw/day, respectively. At 40 mg/kg bw/day, the standard deviation from the mean was relatively high, and 6/25 individual values at this dose level exceeded the historical data range. However, these variations in mean epididymal sperm count occurred in the absence of a dose-related trend, and in absence of macroscopic changes on male reproductive organs and means remained within the internal control range. Therefore, these variations were considered not to be related to treatment.
Reproductive performance:
no effects observed
Description (incidence and severity):
Mating index was considered not affected by treatment with the test item.
The mating indices were 100% for the control group, and 40 and 100 mg/kg bw/day groups. Two females at 200 mg/kg bw/day did not show evidence of mating, resulting in a mating index of 92% in this group which was considered to be within normal ranges (mean = 99 (n=7 studies; no confidence interval due to the limited number of data); Historical control data for OECD422 studies (mean: 99; P5 – P95 = 90-100 (n=120 studies)). As such, the incidence of two non-mated females in this group was considered not to be related to treatment, also given the absence of treatment-related changes in other reproductive parameters.

Precoital time was considered not affected by treatment with the test item. Mean precoital time was 2.8 days in the control group, 3.8 days at 40 and 100 mg/kg bw/day and 3.3 days at 200 mg/kg bw/day.
Most females showed evidence of mating within 5 days, except for three females at 40 mg/kg bw/day, two females at 100 mg/kg bw/day and two females at 200 mg/kg bw/day for which mating took 13 or 14 days. As this variation in precoital time remained within the normal range of biological variation, occurred at low incidence and in absence of a dose-related trend, this was considered to be unrelated to treatment.

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

Fertility index was considered not affected by treatment. The fertility indices were 100% for the control and 200 mg/kg/day group, and 88% for the 40 and 100 mg/kg/day groups.
A total of three females at 40 mg/kg/day and three females at 100 mg/kg/day were not pregnant. In the absence of a dose-related incidence of non-pregnancy and since mean fertility indices remained within the internal control data range , this was considered not to be related to treatment.

Gestation index and duration of gestation were considered not affected by treatment with the test item; the gestation indices were 100% for all dose groups. All pregnant females had live offspring. Mean duration of gestation ranged from 21.3 to 21.5 days across the dose groups.

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.
For two females at 100 mg/kg bw/day and one female at 200 mg/kg bw/day, the number of pups was higher than the number of implantations. This phenomenon is observed from time to time and is caused by normal resorption of these areas during lactation. No toxicological relevance was attached to this finding in the current study.

The total number of offspring born compared to the total number of uterine implantations was considered not affected by treatment with the test item.
Post-implantation survival index was 94% for the control group and 95, 100 and 97% for the 40, 100 and 200 mg/kg bw/day groups, respectively. These means remained within the range considered normal for rats of this age and strain (due to the limited number of data, internal control data for OECD 421/422 studies were used (period 2015-2018)): mean: 92%, P5-95: 83-99 (n=98 studies)).
Litter size was considered not affected by treatment with the test item. Live litter sizes were 11.2, 11.2, 11.5 and 11.3 living fetuses/litter for the control, 40, 100 and 200 mg/kg bw/day groups, respectively.

Effect levels (P0)

open allclose all
Key result
Dose descriptor:
NOAEL
Remarks:
General toxicity F0
Effect level:
>= 200 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: absence of adverse effects up to and including the highest dose level.
Remarks on result:
other: Possible adversity of the higher TSH in males at 40, 100 and 200 mg/kg/day and higher total T4 in males at 100 and 200 mg/kg/day could not be assessed within this type of study and was therefore not taken into account when determining the NOAEL.
Key result
Dose descriptor:
NOAEL
Remarks:
Reproductive toxicity F0
Effect level:
>= 200 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Absence of adverse effcets on reproduction parameters up to and including the highest dose level.

Target system / organ toxicity (P0)

Key result
Critical effects observed:
no

Results: F1 generation

General toxicity (F1)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
No clinical signs occurred among pups that were considered to represent signs of toxicity.
Primary clinical signs among pups that were found dead or were sacrificed in extremis before weaning consisted of abdominal swelling and pallor (PND 10) for one control pup, lean appearance (PND 12 and 13) and lateral recumbency (PND 13) for one pup at 40 mg/kg bw/day, and dehydrated and lean appearance (PND 12) for one pup at 100 mg/kg bw/day. Given that the incidence of these findings showed no relationship to the dose and since their unscheduled sacrifice was considered to be gavage-related, these clinical signs were considered not to represent signs of toxicity.
The nature and incidence of other clinical signs remained within the range considered normal for pups of this age, and were therefore not considered to be toxicologically relevant.

Breathing rales were recorded for 3/60 males at 40 mg/kg bw/day, 12/60 males and 12/60 females at 100 mg/kg bw/day, and 17/60 males and 8/60 females at 200 mg/kg bw/day, primarily during the first two weeks of treatment post-weaning. Rales were also recorded for 2/60 control females.
No other treatment-related clinical signs were noted among surviving animals during daily detailed clinical observations or during weekly arena observations.
Salivation was recorded after dosing among several males and females at 100 mg/kg bw/day and most males and females at 200 mg/kg w/day during the treatment period with a dose-related incidence. This clinical sign was considered to be a physiological response related to gavage administration of the test item rather than a sign of systemic toxicity, taking into account the nature and minor severity of the effect and its time of occurrence (i.e. after dosing).
No clinical signs were noted during treatment of Positive Control animals.
Clinical signs of animal that did not survive until scheduled necropsy are specified under Mortality - F1 generation.
Any other clinical signs noted during the treatment period among surviving animals 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.
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
The number of live offspring on Day 1 after littering compared to the total number of offspring born was not affected by treatment with the test item. One pup at 100 mg/kg bw/day and one pup at 200 mg/kg/day were found dead at first litter check. No toxicological relevance was attributed to these dead/missing pups since the mortality incidence did not show a dose-related trend and remained within the range considered 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 was considered not affected by treatment with the test item. Viability indices were 100% for the control group and 40 and 100 mg/kg bw/day groups, and 98% for the 200 mg/kg bw/day group. One pup at 40 mg/kg bw/day, one pup at 100 mg/kg bw/day and five pups at 200 mg/kg bwday were found missing between PND 2 and 4. These pups were most likely cannibalised. No toxicological relevance was attributed to these missing pups since the mortality incidence did not show a dose-related trend and remained within the range considered 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 not affected by treatment with the test item.
The weaning indices were 97% for the control group, and 90, 93 and 86% for the 40, 100 and 200 mg/kg bw/day groups, respectively. Note that the weaning index did not include dead animals that were replaced by another animal.
Five pups of the control group, seventeen pups at 40 mg/kg bw/day, twelve pups at 100 mg/kg bw/day, and twenty-nine pups at 200 mg/kg bw/day did not survive the scheduled treatment period. This included one male at 40 mg/kg bw/day and four males at 200 mg/kg bw/day that were sacrificed in extremis or found dead between PND 21 and 25, i.e. after weaning. Most of these pups were sacrificed in extremis or found dead between PND 10 and 20. Three pups at 40 mg/kg bw/day and one pup at 200 mg/kg bw/day were found missing between PND 14 and 22 and were most likely cannibalised.
Most of these mortality cases occurred on a single day, when a total number of 1, 3, 9 and 21 pups were found dead or missing at 0, 40, 100 and 200 mg/kg bw/day, respectively. The number of pups that died outside this date in the period between culling and weaning was 4, 14, 3 and 8 at 0, 40, 100 and 200 mg/kg bw/day, respectively. This incidence did not show a correlation to the dose administered. The most relevant macroscopic observations for these pups that did not survive until scheduled necropsy were essentially confined to lungs that were not collapsed, and fluid was found in the thoracic and/or abdominal cavity. None of these macroscopic findings were recorded for F1 animals that survived until scheduled necropsy. The nature of these findings is suggestive of a gavage-related occurrence.
Overall, it was considered that this pup mortality was not related to toxicity of the test item but had occurred principally as a result of the dosing procedure. Any clear relationship between the dose and mortality incidence was absent, most cases of mortality occurred on a single day, and the nature of the macroscopic observations was specific for the pups that were found dead/sacrificed and were suggestive of a gavage-related occurrence.

No post-weaning mortality occurred that was considered to be related to treatment with the test item.
A total of five animals were sacrificed in extremis or found dead after weaning between PND 21 and 25. Except for one female, all these animals could be replaced by another animal in order to supplement the cohorts. At 40 mg/kg bw/day, one Cohort 1B male was found dead on PND 23. This animal showed rales and gasping prior to death and a necropsy finding (lungs not collapsed) similar to that seen for several pups that were sacrificed/found dead before weaning. At 200 mg/kg bw/day, four males were sacrificed in extremis or found dead after weaning between PND 21 and 25, i.e. two Cohort 1B animals, one Cohort 2B animal and one Cohort 3 animal. Three of these males at 200 mg/kg bw/day showed hunched posture, uncoordinated movements, piloerection, deep respiration, rales, brown staining of the genital region, and/or lethargy prior to sacrifice.

No further mortality occurred on subsequent treatment days among any group. It was therefore conceivable that the death/sacrifice of these animals after weaning had a similar cause as those pups that died or were found dead prior to weaning. In the absence of an overall dose-related trend in mortality incidence (pre- and post-weaning deaths combined), these deaths were considered not related to treatment with the test item.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Body weights of pups were considered not affected by treatment with the test item. Means of test item treated groups were similar to the control group means on all occasions.
No changes in body weight or body weight gain of F1 animals post-weaning were noted that were considered to be related to treatment with the test item.
In females, statistically significant increases in body weight gain in Week 29 and 36 (less than 10% different to control means) were considered not related to treatment as a dose-related trend was absent and these increases were transient (final body weights were similar between groups).
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Food consumption (before and/or after correction for body weight) was considered not affected by treatment with the test item.
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
The following statistically significant changes were noted in haematology parameters that were considered to be related to treatment with the test item.
- Lower platelet counts in males at 200 mg/kg bw/day (0.87x; mean remained within the internal control range (mean = 726; P5 – P95 = 601-851 (n=75)))
- Higher red cell distribution width (RDW) in females at 200 mg/kg bw/day (1.06x; mean remained within the internal control range (mean = 11.0; P5 – P95 = 10.10-12.40 (n=75)))
Any other statistically significant changes in haematology parameters achieving a level of statistical significance when compared to controls, occurred in the absence of a dose-related response. As such, these slight differences were considered not related to treatment.

Coagulation parameters were considered not affected by treatment with the test item. The statistically significantly lower activated partial thromboplastin time (APTT) in males at 100 mg/kg bw/day occurred in the absence of a dose-related trend. Also, the opposite effect (i.e. a delayed APTT) is expected in case of target organ toxicity. Therefore, this variation was considered not to be related to treatment.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
The following statistically significant changes were noted in clinical chemistry parameters that were considered to be related to treatment with the test item.
- Higher urea in males at 200 mg/kg bw/day (1.15x; mean remained within the internal control range (mean = 4.9; P5 – P95 = 3.75-6.40 (n=80)))
- Higher cholesterol in males and females at 100 and 200 mg/kg bw/day (1.29x and 1.25x, respectively for males, and 1.39x and 1.42x, respectively for females; means remained within the internal control range (males: mean = 1.90; P5 – P95 = 1.295-2.530 (n=80);females: mean = 1.40; P5 – P95 = 0.825-2.175 (n=80)))
Any other statistically significant changes in clinical chemistry parameters achieving a level of statistical significance when compared to controls, occurred in the absence of a dose-related response (including higher urea and chloride in females at 40 and 200 mg/kg bw/day, and higher alkaline phosphatase activity (ALP) in females at 200 mg/kg bw/day), or were considered not toxicologically relevant as the opposite effect would be expected in case of target organ toxicity (lower alanine aminotransferase activity (ALAT) in males at 200 mg/kg bw/day). As such, these differences were considered not related to treatment.

Serum T4 levels in male and female pups culled at PND 4, and serum T4 and TSH levels in male and female pups of Cohort 2B at PND 21-22 were considered not affected by treatment with the test item. Mean total T4 values of Cohort 2B males showed an apparent downward trend over the dose groups, but the variation in means across the dose groups was similar to that recorded for Cohort 2B females where such an apparent trend was not visible.
A higher T4 was recorded at 100 and 200 mg/kg bw/day in Cohort 1A males and females at PND 89-92 (1.21x and 1.33x, respectively for males, and 1.41x and 1.58x, respectively for females (not statistically significant for females at 100 mg/kg bw/day)), which was considered to be related to treatment with the test item. However, means were within the internal control range (males: mean = 6.23; P5 – P95 = 4.180-8.220 (n=70); females: mean = 3.54; P5 – P95 = 1.810-6.060 (n=70)).
TSH was considered not affected by treatment with the test item.
Urinalysis findings:
no effects observed
Description (incidence and severity):
Urinalysis parameters were considered not affected by treatment with the test item. Any statistically significant changes in urinary parameters occurred in the absence of a dose-related response and were therefore considered not related to treatment.
Sexual maturation:
effects observed, non-treatment-related
Description (incidence and severity):
Balanopreputial separation (prepuce opening) in males and vaginal patency (vaginal opening), occurrence of first estrus and time between vaginal opening and first estrus in females was considered not affected by treatment with the test item.
At 200 mg/kg bw/day, a statistically significantly longer period to achieving vaginal opening was recorded (30.5 days vs. 29.1 days in the control); the mean remained well within the internal control data range, as did means of all other groups. Also, a clear dose-related trend was absent, and the control mean was at the lower margin of the internal control data range. The vast majority of individual values remained within the concurrent control and/or internal control data range: 3/60 values (34, 34 and 38) were slightly outside the concurrent control range (control range: 25-33; at 200 mg/kg/day), and 1/60 values (38) were outside the internal control data range. Mean body weight on the day of vaginal opening was considered not affected by treatment. Therefore, overall, these variations in vaginal opening were considered not related to treatment with the test item.
At 200 mg/kg bw/day, a statistically significantly longer period to achieving first estrous was recorded (34.6 days vs. 32.5 days in the control). The control mean was at the lower margin of the internal control data range. Most individual values remained within the concurrent control data range: 2/20 values (38 and 37) were slightly outside the concurrent control range (control range: 30-36; at 200 mg/kg bw/day), and all individual values remained within the internal control data range. The means of all groups remained well within the internal control data range. Therefore, overall, these variations in the period to achieving first estrous were considered not related to treatment with the test item.
The interval between day of vaginal opening and day of first estrous was not significantly different between the groups. All individual values remained within the concurrent control range and internal control data range. The means of all groups remained well within the internal control data range.
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 affected by treatment with the test item.
Mean corrected anogenital distance was 1.46, 1.49, 1.45 and 1.44 mm in males, and 0.57, 0.56, 0.54 and 0.56 mm in females for the control, 40, 100 and 200 mg/kg bw/day groups, respectively.
Nipple retention in male pups:
no effects observed
Description (incidence and severity):
Areola/nipple retention was considered not affected by treatment. For none of the examined male pups nipples were observed at PND 13.
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
Organ weights of Cohort 1A and 1B animals were considered not affected by treatment.
In Cohort 1A males treated at 200 mg/kg bw/day, a statistically significantly higher and possible test item-related increase in thyroid gland weight (absolute (+18%) and relative to body weight (+15%)) and lower adrenal gland weight (absolute (-11%) and relative to body weight (-12%)) was recorded. Similar organ weight variations were not recorded for Cohort 1B males which were necropsied in a similar time interval as Cohort 1A animals. Also, means of these organ weights in Cohort 1A were similar to those recorded across Cohort 1B groups, and remained within the internal control data range (see below). Moreover, these organ weight changes occurred without any macroscopic or microscopic correlate. Therefore, these statistically significant changes in thyroid and adrenal gland weights in Cohort 1A animals were considered not to be of toxicological relevance.
Adrenal and thyroid gland weights were considered not affected in females.
The statistically significantly lower seminal vesicle to body weight ratio recorded for Cohort 1A males at 200 mg/kg bw/day was considered not to be related to treatment, since mean seminal vesicle to body weight ratios of Cohort 1B males which were necropsied in a similar time interval as Cohort 1A animals were similar to the control mean. Also, the difference to the control mean in Cohort 1A was slight, absolute mean seminal vesicle weight in Cohort 1A at 200 mg/kg bw/day was similar to the control mean and remained within the internal control range (see below). Moreover, this variation in seminal vesicle to body weight ratio occurred without any macroscopic or microscopic correlate.
Any other differences, including those that reached statistical significance, were considered to be incidental and not related to the test item.

Internal control data
Thyroid gland weight - absolute (gram): mean = 0.016; P5 – P95 = 0.0105-0.0231 (males, Cohort 1A; n=160).
Thyroid gland weight - relative (% from terminal body weight): mean = 0.005; P5 – P95 = 0.0031-0.0063 (males, Cohort 1A; n=159).
Adrenal gland weight - absolute (gram): mean = 0.060; P5 – P95 = 0.0460-0.0755 (males, Cohort 1A; n=160).
Adrenal gland weight - relative (% from terminal body weight): mean = 0.017; P5 – P95 = 0.0130-0.0221 (males, Cohort 1A; n=159).
Seminal vesicle weight – relative (% from terminal body weight): mean = 0.302; P5 – P95 = 0.2052-0.4189 (males, Cohort 1A; n=159).
Gross pathological findings:
no effects observed
Description (incidence and severity):
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.
There were no test item-related gross observations in Cohort 2B (PND 21-22), Cohort 3 (PND 53-59), Cohort 2A (PND 76-100), Cohort 1A (PND 89-95) and Cohort 1B (= PND 90).
All of the recorded macroscopic findings were within the range of background gross observations encountered in rats of this age and strain.
Histopathological findings:
no effects observed
Description (incidence and severity):
Cohort 1A
There were no test item-related microscopic observations.
All 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.
Spermatogenesis-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 counts in the F1 200 mg/kg bw/day group females (Cohort 1A) when compared to control group females. Any variation between group mean counts represented biological variability and were not statistically significant.

Neuropathology and Morphometry (Cohorts 2A and 2B)
Absolute and relative fixed brain weights at PND 21-22 (Cohort 2B) and PND 76-90 (Cohort 2A) were considered not affected by treatment with the test item.
Brain dimensions (length and width of brain) at PND 21-22 (Cohort 2B) and PND 76-90 (Cohort 2A) were considered not affected by treatment with the test item. The statistically significantly lower brain width of Cohort 2B males at 100 mg/kg/day occurred in the absence of a dose-related trend and was therefore considered not related to treatment.
There were no changes in morphometry of the brain of Cohort 2B animals (PND 21-22) and Cohort 2A animals (PND 76-90) that were considered to be related to treatment. At PND 21-22, Cohort 2B females in the 200 mg/kg bw/day dose group had a significantly lower mean height of the cerebellum compared to controls. This finding was no longer present at PND 76-90 and although not statistically significant, mean cerebellar height of the 200 mg/kg bw/day Cohort 2A females was greater than controls.
The Cohort 2A, 200 mg/kg bw/day males had a statistically significantly greater mean height of the cerebellum compared to controls at PND 76-90. Given the inconsistent direction of change in the cerebellar height in the 200 mg/kg bw/day females at PND 21-22 and PND 76-90, these findings are considered unrelated to DGEBADA administration. Additionally, the increased cerebellar height in the 200 mg/kg bw/day males on PND 76-90 is in a direction opposite of what is expected for a toxicologically relevant effect.
Other effects:
no effects observed
Description (incidence and severity):
Sex ratio F1 pups
Sex ratio was considered not affected by treatment.

Estrous Cycle (Cohort 1A)
Length and regularity of the estrous cycle were not affected by treatment. For most females, regular cycles of 4 to 5 days were recorded between PND 75 and 88. One female at 40 mg/kg bw/day and one female at 200 mg/kg bw/day were acyclic. Given the incidental nature and the absence of a dose-related incidence, these single occurrences of an acyclic estrous cycle were considered not to indicate a relation to treatment with the test item.

Sperm Analysis (Cohort 1A)
Sperm motility, concentration and morphology were considered not affected by treatment with the test item.

Splenic Lymphocyte Subpopulation (Cohort 1A)
Splenic lymphocyte subpopulations (Cohort 1A) were considered not affected by treatment with the test item. No statistically significant changes were observed in splenic lymphocyte subpopulation parameters.

Developmental neurotoxicity (F1)

Behaviour (functional findings):
effects observed, treatment-related
Description (incidence and severity):
Acoustic Startle Response:
At 200 mg/kg/day, males showed a statistically significantly higher average response amplitude (both as overall mean response (1.38x), and during the first four test blocks (1.64x, 1.38x, 1.42x and 1.42x for blocks 1, 2, 3 and 4, respectively)), maximum response amplitude (both as overall mean (1.23x) and during the first test block (1.47x)) and latency to achieving the maximum response amplitude (both as overall mean (1.27x) and during the first test block (1.46x)). These changes were considered to be related to treatment with the test item.
At 40 and 100 mg/kg/day (males and females), and 200 mg/kg/day (females), acoustic startle response (both as overall mean and per block) was considered not affected by treatment. The statistically significantly lower average and maximum response amplitude at 100 mg/kg/day in females occurred in the absence of a dose-related trend, and was therefore considered not to be related to treatment with the test item.

Detailed clinical observations:
Detailed clinical observations revealed no symptoms that were considered to be related to treatment with the test item.
The clinical symptoms that were observed were considered to be within the normal range of behavioural findings for this type of study, and were generally also observed in control animals. These findings were therefore considered not to be related to treatment.
Decreased locomotor activity was recorded for 2/10 and 1/10 males at 100 and 200 mg/kg/day, respectively. Increased urinating was recorded for 1/10 females at 200 mg/kg/day.
Whilst these symptoms were not recorded among control animals, they were considered not to be related to treatment with the test item given their low incidence, and absence of a clear dose-related response.

Rectal temperature:
Rectal temperature was considered not affected by treatment with the test item.

Motor activity :
Motor activity was considered not affected by treatment with the test item. All groups showed a similar motor activity habituation profile with a decreasing trend in activity over the duration of the test period.
Slightly higher mean activity levels (ambulations and total movements) were recorded for females at 40 and 100 mg/kg/day, but these did not achieve a level of statistical significance and occurred in the absence of a dose-related trend. As such, these variations were considered not to be related to treatment with the test item.

Functional observations :
Functional observation parameters (hearing ability, pupillary reflex, foot splay and grip strength) were considered not affected by treatment with the test item. Hearing ability and pupillary reflex were normal in all examined animals. Mean foot splay values of males at 200 mg/kg/day appeared lower than controls but without statistical significance.
A statistically significantly higher forelimb grip strength was recorded for females at 100 mg/kg/day. In absence of a dose-related response, this variation was considered to be unrelated to treatment with the test item.



Developmental immunotoxicity (F1)

Developmental immunotoxicity:
no effects observed
Description (incidence and severity):
All animals were negative for the presence of anti-KLH IgM antibodies before the administration of KLH.
The incidence of non-responders (< LLOQ) to KLH (IgM) in the control group was higher than expected for this assessment. Indeed, in the vehicle control group, there were 5/10 males and 2/10 females with non-detectable levels of anti-KLH IgM antibodies. It is unclear why a high incidence of vehicle control animals mounted a weak response to KLH in the present study. This trend was also observed in most dosed groups. There were at least 3 animals per sex/group with detectable responses to KLH (IgM), wBased on the data available from the responding animals, there were no DGEBADA related effects on the levels of anti-KLH IgM antibodies detected at any dose levels.
Based on the data available from the responding animals, there were no DGEBADA related effects on the levels of anti-KLH IgM antibodies detected at any dose levels.
It should also be noted that no pathology findings were detected in the lymphoid organs (i.e. histopathology and organ weight) and no treatment related changes in splenic lymphocyte subpopulations were observed in this study (refer to relevant section in the Main Study Report). Therefore, overall, the data indicates that DGEBADA does not appear to induce any immunotoxic effect in young Wistar rats.

Effect levels (F1)

open allclose all
Key result
Dose descriptor:
NOAEL
Remarks:
General toxicity
Generation:
F1
Effect level:
>= 200 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Absence of adverse effects up to and including the highest dose level.
Dose descriptor:
NOEL
Remarks:
General toxicity
Generation:
F1
Effect level:
40 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: The presence of rales and higher cholesterol at 100 and 200 mg/kg bw/day in F1 males and females.

Target system / organ toxicity (F1)

Critical effects observed:
no

Overall reproductive toxicity

Key result
Reproductive effects observed:
no

Any other information on results incl. tables

Dose Formulation Analyses

Accuracy

In the Group 1 formulations (F0-Animals and F1-Animals), no test item was detected.

The concentrations analyzed in the formulations of Groups 2, 3 and 4 for F0-Animals, prepared for use in Weeks 1, 2, 3, 5, 13, 15, 21 and 25 were in agreement with target concentrations (i.e. mean accuracies between 90% and 110%). For the formulations of Group 2, 3 and 4 (F0-Animals), prepared for use in Week 9, the mean accuracy was slightly below the target concentration (i.e. 89%, 84% and 89% of target). No cause for these slightly lower values could be found. Based on overall accuracy results however, it was considered that formulations were prepared accurately for the purpose of this study.

The concentrations analyzed in the formulations of Groups 2, 3 and 4 for F1-Animals, prepared in Week 15 and Group 2 for F1-Animals prepared in Week 25 were in agreement with target concentrations (i.e. mean accuracies between 90% and 110%). The high mean accuracy for the formulation of Group 2 (F1-Animals) prepared in Week 21 (i.e. 976% of target) was due to an incorrect preparation of the formulation (not used for dosing). 

Homogeneity

The formulations of Group 2 (F0-Animals) and Group 4 (F0-Animals) and Group 2 (F1-Animals) were homogeneous (i.e. coefficient of variation= 10%).

Stability

Analysis of Group 4 (F0-Animals) and Group 2 (F1-Animals) formulations after storage yielded a relative difference of= 10%. The formulations were found to be stable during storage at room temperature under normal laboratory light conditions for at least 24 hours.

Mean Total T4 and TSH levels (and Fold Increase compared to Control Mean) in F0and F1animals.

Parameter

Dose level (mg/kg/day)

F0

PND 4

PND 21-22

(Cohort 2B)

PND 89

(Cohort 1A)

M

F

M/F

M

F

M

F

Total T4 (µg/dL)

 

Mean(fold change)

0

6.25

4.98

1.46

4.65

4.32

6.09

3.06

40

6.15

(n.a.)

5.57

(n.a.)

1.56

(n.a.)

4.39

(n.a.)

3.85

(n.a.)

6.39(1.05x)

3.45

(1.13x)

100

7.47

(1.20x)

5.00

(n.a.)

1.59

(n.a.)

4.31

(n.a.)

4.07

(n.a.)

7.37*

(1.21x)

4.30

(1.41x)

200

8.04 **

(1.27x)

5.34

(n.a.)

1.58

(n.a.)

4.09

(n.a.)

4.19

(n.a.)

8.12 **

(1.33x)

4.83**

(1.58x)

TSH (uIU/mL)

 

Mean(fold change)

0

0.111

0.193

-

0.079

0.086

0.153

0.079

40

 

0.125

(1.13x)

0.507

(n.a.)

-

0.088

(n.a.)

0.079

(n.a.)

0.135

(n.a.)

0.072

(n.a.)

100

 

0.153

(1.38x)

0.274

(n.a.)

-

0.093

(n.a.)

0.108

(n.a.)

0.168

(n.a.)

0.072

(n.a.)

200

0.185

(1.67x)

0.246

(n.a.)

-

0.091

(n.a.)

0.111

(n.a.)

0.142

(n.a.)

0.092

(n.a.)

M: male; F: female.

*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level

n.a. Not applicable; no treatment-related effect or mean not in line with increasing or decreasing trend over the dose groups.

Applicant's summary and conclusion

Executive summary:

 The objective of this study was to provide an evaluation of the pre- and postnatal effects of DGEBADA 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, was expected to identify specific target organs in the offspring. 

In addition, the study provided and/or confirmed information about the effects of DGEBADA on the integrity and performance of the adult male and female reproductive systems. Specifically, but not exclusively, the following parameters were considered: gonadal function, the estrous cycle, epididymal sperm maturation, mating behaviour, conception, pregnancy, parturition, and lactation. 

Furthermore, the information obtained from the developmental neurotoxicity and developmental immunotoxicity assessments characterized potential effects in those systems.

The dose levels in this study were selected to be 0, 40, 100 and 200 mg/kg/day, based on the results of previously conducted (reproductive) toxicity studies with oral exposure of DGEBADA in rats.

A Lactational Transfer Study (Test Facility Study No. 20172948) was performed prior to this studyto determine if DGEBADA could transfer to mother’s milk after maternal exposure fromDay 6 post-coitum to Day 13 of lactation, inclusive. All measured milk samples collected from dams on Day 13 of lactation revealed values of DGEBA diacrylate (the main compound present in the test item) that were below the lower limit of quantification (LLOQ). This result indicated that DGEBADA is not excreted in the mother’s milk at detectable levels, when administered up to a dose level of 200 mg/kg/day by once daily oral gavage from Day 6 post-coitum until Day 13 of lactation inclusive.

Chemical analyses of formulations were conducted on nine occasions during the study to assess accuracy, homogeneity and stability over 24 hours at room temperature.

For the F0-generation, the following parameters and end points were evaluated in this study: mortality/moribundity, clinical signs, body weight, food consumption, estrous cycle determination,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 estrous, estrous cycle determination, functional observations including acoustic startle response, immunotoxicity assessments using TDAR assay),clinical pathology including measurement of thyroid hormones and urinalysis,gross necropsy findings, sperm analysis and splenic lymphocyte subpopulation analysis, organ weights and histopathologic examinations, neurohistopathological examinations and morphometric analysis of brain tissue.In addition, the following reproduction/developmental parameters were determined: 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).

Test formulations preparedwere considered homogeneous at the concentrations tested and analysis of accuracy and stability revealed acceptable levels. 

No adverse changes were noted in any of the F0-Generation parameters investigatedup to the highest dose level tested (200 mg/kg/day).

No mortality and no adverse clinical signs related to treatment occurred during the study period.

Non-adverse changes in body weight were recorded among females, however the lower mean body weight gain of females at 200 mg/kg/day was considered to be treatment-related. Statistically significant lower than controls on Days 14 and 21 of the lactation phase (0.7x and 0.5x respectively). The mean body weight gain at the end of the lactation phase was 0.5x of the control mean. The other variations in mean absolute body weights noted during the premating and post-coitum phases were considered not to be related to treatment.

Food consumption before or after correction for body weight was considered not affected by treatment with the test item in males and females.

Haematological parameters of treated rats were considered not to have been affected by treatment with the test item.

Non-adverse changes in coagulation and clinical chemistry parameters consisted of an elongated prothrombin time and higher cholesterol in males at 200 mg/kg/day are considered to be related to treatment. Since means of these parameters remained within the internal control range, and had no corroborative morphological lesions, these were considered non-adverse.

A trend towards higher thyroid stimulating hormone (TSH) was recorded in males at 40, 100 and 200 mg/kg/day, but means remained within the internal control range. Also, higher total T4 was recorded in males at 100 and 200 mg/kg/day; mean was outside the internal control range at 200 mg/kg/day only. Serum levels of TSH and T4 in F0-females were considered not affected by treatment. These changes in males were not accompanied by test item-related changes in thyroid morphology, but were considered as related to treatment. However, possible adversity of this effect could not be assessed within this type of study. No test item-related changes were noted in any of the other F0-Generation parameters investigated in this study (i.e. functional observations, urinalysis, macroscopic examination, organ weights and microscopic examination).

Reproduction results – F0-generation

No test item-related changes were noted in any of the reproductive parameters investigatedup to the highest dose level tested (200 mg/kg/day), i.e.sperm motility, sperm concentration and morphology, mating and fertility indices, precoital time, number of implantations, estrous cycle, and histopathological examination of reproductive organs including stage-dependent qualitative evaluation of spermatogenesis in the testis.

Developmental results – F0 generation / F1 generation (pre-weaning)

No test item-related changes were noted in any of the developmental parameters investigated up to the highest dose level tested (200 mg/kg/day) during the gestation and lactation phases.

Five animals of the control group, seventeen animals at 40 mg/kg/day, twelve animals at 100 mg/kg/day, and twenty-nine animals at 200 mg/kg/day (including one animal at 40 mg/kg/day and four animals at 200 mg/kg/day that were sacrificed or found dead post-weaning) did not survive the scheduled treatment period. It was considered that this mortality was not related to toxicity of the test item but had occurred principally as a result of the dosing procedure. Clear relationship between the dose and mortality incidence was absent, most cases of mortality occurred on a single day, and the nature of the macroscopic observations was specific for the animals that were found dead/sacrificed and were suggestive of a gavage-related occurrence.

No test item-related changes were noted in any of the following developmental parameters investigated in this study: gestation index and duration, live birth, viability and weaning indices, parturition, sex ratio, litter size, maternal care and early postnatal pup development consisting of mortality, pups’ clinical signs, pups’ body weight, anogenital distance, areola/nipple retention, thyroid hormone levels in pups (T4 of PND 4, T4 and TSH of PND 22) and macroscopic examination of pups sacrificed at the end of the lactation period.

 

Developmental results – F1 generation (post-weaning)

A dose-related but non-adverse incidence of breathing rales was recorded at 100 and 200 mg/kg/day, primarily during the first two weeks of treatment. Incidences were 12/60 males and 12/60 females at 100 mg/kg/day, and 17/60 males and 8/60 females at 200 mg/kg/day. Incidences of rales in males at 40 mg/kg/day (3/60 males) were comparable to incidences in control females (2/60 control females), and as such these may not be attributable to the test item. Since these rales essentially resolved with continuing treatment, this was considered not an adverse finding. However, it could not be excluded that occurrence of these rales may be associated with deaths that occurred primarily prior to weaning, as most of these deaths showed lung lesions (lungs that were not collapsed, and fluid in the thoracic and/or abdominal cavity) suggestive of a gavage-related occurrence.

At 200 mg/kg/day, non-adverse treatment-related changes in acoustic startle response parameters of Cohort 2A males were recorded.These changes consisted of a higher average response amplitude (both as overall mean response (1.38x), and during the first four test blocks (1.64x, 1.38x, 1.42x and 1.42x for blocks 1, 2, 3 and 4, respectively)), maximum response amplitude (both as overall mean (1.23x) and during the first test block (1.47x)) and latency to achieving the maximum response amplitude (both as overall mean (1.27x) and during the first test block (1.46x)). Females at this dose showed no such changes in acoustic startle response parameters, and these parameters were considered not affected by treatment with the test item in males at 40 and 100 mg/kg/day. Other developmental neurotoxicity endpoints of Cohort 2A animals (detailed clinical observations, rectal temperature, hearing ability, pupillary reflex, foot splay, grip strength and motor activity) were considered not affected by treatment, and there was no apparent relationship between body weights and the level of acoustic startle response on an individual animal basis. These changes were considered not adverse since the direction of change consisted of an increased response rather than a decreased response, the auditory reflex loop was functioning at least in a comparable manner as in controls, habituation occurred as expected and recorded for control rats,means were only slightly outside historical control data range for maximum response amplitude in Block 1 and since the changes only occurred for males and not for females.

No test item-related changes were recorded for other developmental neurotoxicity endpoints determined in Cohort 2A and 2B animals, consisting of fixed brain weights, brain dimensions (length and width of brain), routine sections of brain or peripheral nervous system and morphometric analysis of the brain.

Non-adverse changes in haematology and clinical chemistry parameters consisted of lower platelet counts and higher urea in males at 200 mg/kg/day, higher red cell distribution width in females at 200 mg/kg/day, and higher cholesterol in males and females at 100 and 200 mg/kg/day. Since means of these parameters remained within the internal control range, and had no corroborative morphological lesions, these were considered non-adverse. Higher cholesterol was also recorded for F0-generation males as outlined above.

Higher T4 was recorded at 100 and 200 mg/kg/day in Cohort 1A males and females at PND 89 (1.21x and 1.33x, respectively for males, and 1.41x and 1.58x, respectively for females). Means remained within the internal control range and were not accompanied by test item-related changes in thyroid morphology or TSH levels. Higher T4 and higher TSH was recorded or F0-generation males as outlined above. Since means remained within the internal control data range, these variations were considered not adverse. T4 and TSH levels at PND 4 and PND 21/22 were considered not affected by treatment with the test item.

No treatment-related effects on F1-animals were recorded for other developmental parameters including mortality, weekly arena observations, body weight and food consumption, balanopreputial separation (prepuce opening), vaginal patency (vaginal opening), occurrence of first estrus, time between vaginal opening and first estrus, length and regularity of the estrous cycle, and sperm motility, concentration and morphology, coagulation parameters, urinalysis, macroscopy and organ weights.

Histopathologically, no test-item related effects were noted including on stage-dependent qualitative evaluation of spermatogenesis, ovarian follicle and corpora lutea counts of females of Cohort 1A and on morphology of reproductive organs.

Due to mortality among pups, no Surplus Cohort was added to the study and priority was given to allocate pups to other cohorts. Hence, no spleen and thymus weights were recorded from “up to 10 pups/sex/group” shortly after weaning as per OECD443 testing guideline. Since there were no test-item related effects noted on weight and morphology of these organs in F0 animals and Cohort 1A animals, this was considered not to have adversely affected interpretation of the study results.

TDAR results were considered not affected by treatment with the test item. The incidence of non-responding animals (i.e. anti-KLH IgM levels <LLOQ) was not affected following administration of DGEBADA. Based on the data available from the responding animals, there were no DGEBADA related effects on the levels of anti-KLH IgM antibodies detected at any dose levels Moreover no pathology findings in the lymphoid organs (i.e. histopathology and organ weight) and no test item-related changes in splenic lymphocyte subpopulations, it is confirmed that the test item did not induce any immunotoxic effect.

 

In conclusion, based on the results of this extended one generation reproductive toxicity study (including Cohorts 1, 2 and 3), the following no-observed-adverse-effect levels (NOAEL) and/or No-Observed-Effect-Levels (NOEL) of DGEBADA were established:

> General toxicity (F0): NOAEL of at least 200 mg/kg/day(possible adversity of the higher TSH in males at 40, 100 and 200 mg/kg/dayandhigher total T4 in males at 100 and 200 mg/kg/day could not be assessed within this type of study and was therefore not taken into account when determining the NOAEL for general toxicity). The No Observed Effect Level (NOEL) could not be determined based on the lower body weight gain observed at 200 mg/kg/day in F0 females during lactation, and the high TSH levels in males from 40 mg/kg/day.

> Reproductive toxicity (F0): NOAEL of at least 200 mg/kg/day in absence of adverse effects at this dose on reproduction parameters.

> General toxicity (F1): NOAELof at least 200 mg/kg/day (possible adversity of the higher T4 in Cohort 1A males and females at 100 and 200 mg/kg/day could not be assessed within this type of study and was therefore not taken into account when determining the NOAEL). A No Observed Effect Level (NOEL) of 40 mg/kg/day was determined based on the presence of rales and higher cholesterol at 100 and 200 mg/kg/day in F1 males and females.