Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

In an OECD 422 reproduction screening study the test item was tested in rats for reproductive (fertility) effects (SafePharm, 2006). No adverse effect on mating performance, fertility, or gestation length was detected. Treatment at 50 and 150 mg/kg/day appeared to be associated with a higher (not statistically significant) level of preimplantation loss. The NOEL for repeated dose oral systemic and reproductive toxicity was 15 mg/kg/day. These findings on fertility are further investigated in the currently ongoing OECD 443 study.

Based on ECHA decision (CCH-D-2114447795-35-01/F) the extended one-generation reproductive toxicity study (OECD 443) started in March 2019 and is expected to be finished in October 2020.

Based on the ECHA decision the registrant conducted the OECD 443 study with the basic study design (Cohorts 1A, and 1B) with extension to mate the Cohort 1 B animals to produce the F2 generation.

Link to relevant study records

Referenceopen allclose all

Endpoint:
extended one-generation reproductive toxicity - with F2 generation (Cohorts 1A, and 1B with extension)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2019-03-13 to 2019-10-11
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
According to the ECHA final decision on a compliance check from 2018-10-29 (Decision number: CCH-D-2114447795-35-01/F and submission number: MJ448798-11) the study design of the EOGRTS according to OECD 443 is as follows:

Based on Article 41 of Regulation (EC) No 1907/2006 (the REACH Regulation), ECHA requests you to submit information on:

Extended one-generation reproductive toxicity study (Annex X, Section 8.7.3.; test method: OECD TG 443) in rats, oral route with the registered substance specified as follows:
- At least two weeks premating exposure duration for the parental (P0) generation;
- Dose level setting shall aim to induce systemic toxicity at the highest dose level;
- Cohort 1A (Reproductive toxicity);
- Cohort 1B (Reproductive toxicity) with extension to mate the Cohort 1B animals to produce the F2 generation.
Qualifier:
according to
Guideline:
OECD Guideline 443 (Extended One-Generation Reproductive Toxicity Study)
Version / remarks:
adopted June 25, 2018
Deviations:
yes
Remarks:
see section "Details on Study Design"
GLP compliance:
yes (incl. certificate)
Limit test:
no
Justification for study design:
According to the ECHA final decision on a compliance check from 2018-10-29 (Decision number: CCH-D-2114447795-35-01/F and submission number: MJ448798-11) the study design of the EOGRTS according to OECD 443 is as follows:

Based on Article 41 of Regulation (EC) No 1907/2006 (the REACH Regulation), ECHA requests you to submit information on:

Extended one-generation reproductive toxicity study (Annex X, Section 8.7.3.; test method: OECD TG 443) in rats, oral route with the registered substance specified as follows:
- At least two weeks premating exposure duration for the parental (P0) generation;
- Dose level setting shall aim to induce systemic toxicity at the highest dose level;
- Cohort 1A (Reproductive toxicity);
- Cohort 1B (Reproductive toxicity) with extension to mate the Cohort 1B animals to produce the F2 generation.






Species:
rat
Strain:
other: CD® / Crl:CD(SD)
Details on species / strain selection:
Based on the extent of background data and the comparability to general toxicity tests, the rat is the preferred species, and criteria and recommendations given in the TG refer to this species.
Sex:
male/female
Details on test animals and environmental conditions:
DETAILS ON ANIMALS

Species / Strain / Stock: Rat / CD® / Crl:CD(SD)
Breeder: Charles River Laboratories Germany GmbH, Sandhofer Weg 7,97633 Sulzfeld, Germany
Body weight (at start of dosing): Male: 386.8 g – 472.0 g
Female: 214.4 g – 291.4 g
Age at start of dosing: 78 days

Age (at start of mating): Males: 91 days, females: 91 days (young adults; sexually mature)
Selection of species: The rat is a commonly used rodent species for such studies and required by the guideline.
Number of parental animals: Pre-exposure period:
At least 120 female animals were evaluated pre-exposure for oestrus cyclicity to yield 96 females with a regular oestrus cycle for the study.
Main study:
192 animals (96 males and 96 females)
A sufficient number in order to grant at least 20 pregnant females per group for evaluation of the F0 generation.
Adaption period: 7 days

ENVIRONMENTAL CONDITIONS
Diet: Commercial diet ssniff® R/M-Z V1324 (ssniff Spezialdiäten GmbH, 59494 Soest, Composition of the diet will be stated in the report)
Drinking Water: Tap water is offered daily ad libitum.
Housing: Animals are kept singly in MAKROLON cages (type III plus) with a basal surface of approx. 39 cm x 23 cm and a height of approx. 18 cm
at a room temperature of 22°C ± 3 °C (maximum range) and a relative humidity of 55% ± 15% (maximum range).
The rooms are alternately lit (about 150 lux at approx. 1.50 m room height) and darkened in a 12 hours dark/12 hours light cycle.
Route of administration:
oral: gavage
Vehicle:
polyethylene glycol
Remarks:
PEG400
Details on exposure:
Route of administration: Oral, via gavage
Frequency of administration: Once daily
Vehicle: PEG400
Administration volume: 4 mL/kg b.w.
Dosages: 0, 15, 50, 150 mg/kg bw/d
Selection of route of administration: According to international guidelines.
Details on mating procedure:
Sexually mature male and female rats of the same dose group were paired randomly monogamously, i.e. 1 male and 1 female animal were placed in one cage during the dark period. The female was placed with the same male until evidence of mating was observed or two weeks had elapsed.
The females were examined each morning for the presence of sperm. The day of conception (day 0 of gestation) was considered to be the day on which sperm was found.
Females without a positive mating sign were separated from its male partner after 2 weeks without further opportunity for mating. After a pseudo gestation period of approx. 24 test days these females were laparotomized and their non-pregnancy status was confirmed by Salewski Staining. F0 Females without a positive mating sign after 2 weeks of mating were noted in group 2 (no. 93) and in group 4 (no. 189).
If there would have been insufficient males, for example due to male death before pairing, then males which had already mated would have been paired with a second female such that all females would have been paired. However, as no male died prematurely, sufficient male animals were always available in this study.
For the establishment of the F2 Generation, males and females of the same dose group were paired, sibling mating was avoided.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The test item formulations were freshly prepared every day.
The test item was diluted in the vehicle to the appropriate concentrations. The dosing formulations were administered orally at a constant volume/kg b.w..
The amount of the test item was adjusted daily to the animal’s current body weight.
The control animals received the vehicle at the same administration volume daily in the same way.
The stability, homogeneity and the concentration of the administration formulations were monitored (see section 3.7 'Test item-formulation analysis').
The F1 animals of Cohort 1A and Cohort 1B received the same concentration of the test item in the test item formulations, only the in-life periods of the different cohorts were of different lengths.
For the test item that was mixed with a vehicle, tests by appropriate analytical methods were conducted to determine the concentration, stability and homogeneity of the test item in the test item-vehicle formulations.
For the analysis of the test item-vehicle formulations, two (2) samples of approximately 3 mL each were taken according to the following schedule and stored at -20°C ±10% until analysis at LPT.

At start of the treatment period of the F0 animals
(first dosing day)

Analysis of stability and concentration
Immediately after preparation of the test item-vehicle formulation as well as after 8 and 24 hours storage at room temperature.
(3 samples / dose level group; groups 2 - 4).
Number of samples: 3 x 3 = 9

Analysis of homogeneity
At the start of administration, during (middle) administration and before administration to the last animal of the dose level group.
(3 samples / dose level group; groups 2 - 4).
Number of samples: 3 x 3 = 9

At the time when most F0 females had littered

Analysis of concentration
During treatment always before administration to the last animal/dose level group
(1 sample / dose level group; groups 2 - 4)
Number of samples: 3

At termination of the F0 dosing period at a time when the majority of animals was dosed

Analysis of concentration
During treatment always before administration to the last animal/dose level group
(1 sample / dose level group; groups 2 - 4)
Samples were not taken (see section 2.13)

Sum of all samples: 21

Sampling for the F1 Generation

At start of the treatment period of the F1 animals
(first dosing day)

Analysis of stability and concentration
Immediately after preparation of the test item-vehicle formulation as well as after 8 and 24 hours storage at room temperature.
(3 samples / dose level group; groups 2 - 4).
Number of samples: 3 x 3 = 9

Analysis of homogeneity
At the start of administration, during (middle) administration and before administration to the last animal of the dose level group.
(3 samples / dose level group; groups 2 - 4).
Number of samples: 3 x 3 = 9

At termination of Cohort 1A at a time when the majority of animals was dosed

Analysis of concentration
During treatment always before administration to the last animal/dose level group
(1 sample / dose level group; groups 2 - 4)
Number of samples: 3

At termination of Cohort 1B at
a time when the majority of animals was dosed

Analysis of concentration
During treatment always before administration to the last animal/dose level group
(1 sample / dose level group; groups 2 - 4)
Number of samples: 3

Sum of all samples: 24


Duration of treatment / exposure:
The study animals were treated during the following periods:

F0 animals
Males: 2 weeks prior to mating, during the mating period, and at least until weaning of the F1 generation (up to and including the day before sacrifice).
Females: 2 weeks prior to mating, during the mating, gestation and lactation period and until termination after weaning of their litters (up to and including the day before sacrifice).

F1 animals
F1 Pups Until weaning: F1 pups were indirectly exposed to the test item through the breast milk.
After weaning: the selected F1 pups for the F1 cohorts were individually dosed via gavage.
Cohort 1A The male and female animals of cohort 1A were dosed for 10 weeks (up to and including the day before sacrifice (sacrifice of males on TD 71 and 72 (PNDs 91 to 96); sacrifice of females on TD 67 and 68 (PNDs 88 to 92).
Cohort 1B The male and female animals of cohort 1B were dosed until sacrifice of their F2 pups (up to and including the day before sacrifice (males and females on TD 99 to TD 131 (PNDs 120 to 152)).
F2 animals Until sacrifice on PBD 21: The male and female F2 pups were indirectly exposed to the test item through the breast milk.


Frequency of treatment:
daily
Details on study schedule:
The study animals were treated during the following periods:
F0 animals
Males 2 weeks prior to mating, during the mating period, and at least until weaning of the F1 Generation (up to and including the day before sacrifice).
Females 2 weeks prior to mating, during the mating and lactation period and until termination after weaning of their litters (up to and including the day before sacrifice).

F1 animals
F1 Pups Until weaning: F1 pups were indirectly exposed to the test item through the breast milk.
After weaning: the selected F1 pups for the F1 cohorts were individually dosed via gavage.
Cohort 1A The male and female animals of cohort 1A were dosed for 10 weeks (up to and including the day before sacrifice (sacrifice of males on TD 71 and 72 (PNDs 91 to 96); sacrifice of females on TD 67 and 68 (PNDs 88 to 92).
Cohort 1B The male and female animals of cohort 1B were dosed until sacrifice of their F2 pups (up to and including the day before sacrifice (males and females on TD 99 to TD 131 (PNDs 120 to 152)).
F2 animals Until sacrifice on PBD 21: The male and female F2 pups were indirectly exposed to the test item through the breast milk.
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Control group
Dose / conc.:
15 mg/kg bw/day (nominal)
Remarks:
Low dose group
Dose / conc.:
50 mg/kg bw/day (nominal)
Remarks:
Intermediate dose group
Dose / conc.:
150 mg/kg bw/day (nominal)
Remarks:
High dose group
No. of animals per sex per dose:
20 males and females in P generation and 20 males and females in Cohort 1A and Cohort 1B
Control animals:
yes, concurrent vehicle
Details on study design:
The dose levels had been selected in agreement with the Sponsor based on available toxicological data and the results of an OECD 408 study in rats (LPT Study No. 34962), an OECD 414 study in female rats (LPT Study No. 34964) as well as an OECD 422 study in rats (SafePharm laboratories, project no. 936/068). In all studies, dose levels of 15, 50 and 150 mg/kg b.w. per day were employed.
In the OECD 408 study, animals treated with 150 mg/kg showed multiple adverse test item-related effects such as decreased serum levels of albumin, globulin and total protein, increased serum levels of urea, reduced food consumption and body weight, increased organ weights for liver and spleen, decreased organ weights of epididymis, the prostate and the seminal vesicles of the male animals and of the ovaries and uterus of the female animals and very frequent histopathological changes (vacuolation). After further investigation by Dr. Weber from AnaPath, it was concluded that the alterations were not associated with further inflammatory and/or degenerative lesions, and showed partial or complete recovery. Thus, by morphology and distribution, the findings are indicative for phospholipidosis. Female animals treated with 150 mg/kg also displayed pilo-erection, while male animals showed reduced water consumption. At 50 mg/kg, histopathological changes (vacuolation) were only seen in some organs/tissues, and female animals showed reduced water consumption. No effects occurred at the 15 mg/kg b.w. dose level.
In the OECD 414 study, adult animals treated with 150 mg/kg showed slightly reduced food consumption and body weight gain as well as a high incidence of salivation. Furthermore, one animal died. No test item-related changes were noted in the macroscopic examination during laparotomy; reproductive parameters and the offspring were unaffected by the test item. No effects occurred at the 50 mg/kg and 15 mg/kg dose level, therefore the NOAEL was considered to be 50 mg/kg b.w./day for the dams. However, the NOAEL for the fetal organism was above 150 mg/kg b.w./day.
In the OECD 422 study, adult animals treated with 150 mg/kg b.w. displayed a non-significant increase in pre-implantation loss and a lower corpora lutea count. No further adverse effects were noted on the adult animals or their offspring. For animals treated with 50 mg/kg, a non-significant increase in pre-implantation loss was also observed, however adult animals and their offspring showed no further adverse affects. No effects occurred at the 15 mg/kg b.w. dose level.
Hence, 150 mg/kg b.w./day was considered the maximum tolerated dose for the F0-generation of the OECD 443 study, with no systemic effects to be expected in the F1 Generation. 

Deviations:
-No samples were taken at the termination of the F0 dosing period since already samples for the start of the treatment period of the F1 animals (1stt dosing day) were taken
-Food consumption for the males of the F0 Generation and the males of the F1 Cohort 1B was only regularly determined until start of mating. The following weekly determination of food intake during mating was not carried out as this examination as specified in the Study Plan had been overlooked.
-No sampling of urine was carried out for the male and female animals at the end of the F0 dosing period as this examination as specified in the Study Plan had been overlooked
-Male animals were euthanized under ether narcosis to guarantee best possible results for sperm analysis.
-Pup no. 79 7 (F1 Pup) was scheduled for culling on PND 4 (Provantis randomisation program: reduction of litter size to 10 pups per litter). However, pup no. 79 7 was not culled as scheduled due to the fact that another pup of the same litter (no. 79 13) had died on PND 4.
-The relative food consumption of the F1 Cohort 1B females on PND 21 was not determined due to the fact that the Provantis food intake is based on data input on PND 21. However, the respective body weight had already been measured on PND 20 instead of PN 21 as stated in the Study Plan.
These minor deviations do not invalidate the results of the study.
Positive control:
no
Parental animals: Observations and examinations:
CLINICAL SIGNS
All animals
All animals
Throughout the test period, each animal was observed for clinical signs at least once daily.
Individual animals were observed before and after dosing at each time of dosing for any signs of behavioural changes, signs of difficult or prolonged parturition, reaction to treatment or illness. Any signs of illness or reaction to treatment were recorded for each individual animal.
Cage side observations included skin/fur, eyes, mucous membranes, respiratory and circulatory systems, somatomotor activity and behaviour patterns. The onset, intensity and duration of any signs observed were recorded.
In addition, animals were checked regularly throughout the working day from 7:00 a.m. to 3:45 p.m. On Saturdays and Sundays animals were checked regularly from 7:00 a.m. to 11:00 a.m. with a final check performed at approximately 3:30 p.m.
Dated and signed records of appearance, change and disappearance of clinical signs were maintained on clinical history sheets for individual animals.

Clinical signs - Detailed clinical observations
F0 animals and F1 animals after weaning
Additionally, a more detailed examination of the F0 animals and the F1 animals of Cohort 1B was performed on a weekly basis.
This more detailed examination started for the F0 main study animals on test day 14 (one day before the start of treatment) to allow for within-subject comparisons. Thereafter the examination was performed weekly 2 hours post administration until termination.
The F1 animals of Cohort 1B were examined weekly after weaning until termination.
These observations were made outside the home cage in a standard arena and at the same time, each time preferably by observers unaware of the treatment. Signs observed included changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions and autonomic activity (e.g. lacrimation, piloerection, pupil size, and unusual respiratory pattern), as well as changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypy (e.g. excessive grooming, repetitive circling), difficult or prolonged parturition and bizarre behaviour (e.g. self-mutilation, walking backwards).
Dated and signed records of appearance, change, and disappearance of clinical signs would have been maintained on clinical history sheets for individual animals.



MORTALITY
Further checks were made early in the morning and again in the afternoon of each working day to look for dead or moribund animals. This allowed post mortem examination to be carried out during the working period of that day. On Saturdays and Sundays, a similar procedure was followed with a final check at approximately 3.30 p.m..
In the case of prematurely sacrificed animals, laboratory examinations are performed, if possible. However, no animal of the F0 Generation and no animal of the F1 Generation (after weaning) was sacrificed prematurely.
For the prematurely deceased pups during the lactation period an external macroscopic examination for gross abnormalities was performed.

BODY WEIGHT
F0 animals
The male and female animals were weighed on the first day of dosing (test day 15) and daily thereafter for dose adjustment, and at sacrifice. The individual body weights were recorded.
The report includes weekly values as well as those of the female animals determined on gestation days 0, 7, 14 and 21 and on post-natal days (PND) 1, 4, 7, 14 and 21.
F1 animals
Live pups were weighed during the lactation period on their post-natal days (PND) 1, 4, 7, 14 and 21. Starting on PND 22, the animals were weighed daily for dose adjustment, and at sacrifice.
The report includes the values determined on post-natal days (PND) 1, 4, 7, 14 and 21. After weaning they were weighed weekly. The female animals of Cohort 1B were additionally weighed on their gestation days 0, 7, 14 and 21.

F2 animals
The F2 Pups were weighed on their post-natal days (PND) 1, 4, 7, 14 and 21 (at sacrifice).

FOOD CONSUMPTION
Food consumption is recorded weekly during pre-mating period in males and females and during gestation period on GD 7, 14, 21 and during lactation period on PND 1, 7, 14, 21 in females.
In males food consumption ois further recorded during post mating period on a weekly basis.

Food consumption
Food intake per rat (g/rat/week) was calculated using the total amount of food given to and left by each rat in each group
The days on which food start (or total food given) and food residue (or total food left) were weighed for the calculation of the relative food consumption are listed in Table below.

Food consumption of parental animals.
Study period F0 Males F0 Females
Pre-mating period Weekly Weekly
Mating period None#1 None#1
Gestation period Not applicable GD 7, 14, 21
Lactation period Not applicable PND 1, 7, 14, 21
Post-mating period Weekly#2 See gestation and lactation period

#1: No food consumption was measured during the mating period, as the animals were housed together.
#2: Starting on a suitable day after the mating period to consolidate all male animals.

Food consumption of offspring animals started after weaning and was performed weekls for 1A and 1B.
Water Water consumption is monitored by visual appraisal daily throughout the study.

HAEMATOLOGY
10 males and 10 females randomly selected from each F0 group.

Differential blood count (relative)
Differential blood count (absolute)
Erythrocytes (RBC)
Leucocytes (WBC)
Haematocrit value (HCT)
Haemoglobin content (HGB)
Platelets (PLT)
Reticulocytes (RET)
Mean corpuscular volume (MCV)
Mean corpuscular haemoglobin (MCH)
Mean corpuscular haemoglobin concentration (MCHC)

CLINICAL CHEMISTRY
Sodium
Potassium
Calcium
Chloride
Albumin
Total bilirubin
Total cholesterol
Glucose
Total protein
Blood urea (BUN)
Creatinine
Alanine amino-transferase (ALAT/GPT)
Alkaline phosphatase (aP)
Aspartate aminotransferase (ASAT/GOT)
Bile acids
Lactate dehydrogenase (LDH)
Sodium/Potassium ratio
Globulin
Albumin/globulin ratio
BUN/creatinine ratio

T4 and TSH Determination
From 10 males and females of F0 at sacrifice

Urinanalysis
At the end of the F0 dosing period and at the end of the F1 cohort 1 A dosing

Parameters: Volume, pH and specific gravity
Protein, Glucose, Bilirubin, Urobilinogen, Ketones, Haemoglobin, Nitrite
The deposit will be examined for the presence of the following parameters:
- Epithelial cells
- Leucocytes
- Erythrocytes
- Organisms
- Further constituents (i.e. sperm, casts)
- Crystalluria

Reproductive performance for F0 and 1B
Reproductive parameters
- Number of pregnant females
- Pre-coital time
- Gestation length calculated from day 0 of pregnancy
Implantation sites
- number per dam
- distribution in the uterine horns
- absolute number per group
- mean per group
Number of pups per group and per dam
- at birth (live and dead)
- on post-natal day 4
- on post-natal days 7, 14 and 21
Number of male and female pups per group and per dam
- at birth (alive and dead)
- on post-natal day 4
- on post-natal days 7, 14 and 21
Number of stillbirths
- per group
- per dam
Number of pups with malformations (see Appendix 4)
- per group
- per dam

Reproductive indices
For each group of the F0 females and the females of Cohort 1B the fertility index and the gestation index was determined:
Female Fertility Index [%] = (Number of pregnant rats / Number of rats paired with a male) x 100


The female fertility index reflects the total number of dams that had achieved pregnancy, including dams which delivered at term, aborted or had fully resorbed litters.

Gestation Index [%] = (Number of dams with live pups) / Number of pregnant rats) x 100


For each litter and group the following indices were determined for the F0 females and the females of Cohort 1B:
Birth Index [%] = (Total number of pups born (alive + dead) / Number of implantation scars) x 100


Live Birth Index [%] = (Number of pups alive on PND 0/1 / Total number of pups (alive + dead)) x 100


Viability Index [%] pre-cull = (Number of pups alive on PND 4 (pre cull) / Number of pups alive on PND 0/1) x 100


Post-implantation loss [%] = (Implantations - number of pups born alive / Implantations) x 100


Viability Index [%] post cull =(Number of pups alive on PND 21 / Number of pups alive on PND 4 (post cull)) x 100


Oestrous cyclicity (parental animals):
Vaginal smears were taken and the stages of the estrous cycle were determined on the following time points.
F0 animals 14-day pre-exposure period to select 96 animals with regular oestrus cycles (4-5 days)
During 2 weeks of premating until evidence of mating.
F1 animals, cohort 1A Start after onset of vaginal patency until first appearance of cornified cells and two weeks starting around PND 75.
F1 animals, cohort 1 B starting from the time of paining until mating evidence is detected.
F0 and F1 animals On the day of sacrifice, Shortly before necropsy.
Sperm parameters (parental animals):
All F0 males and all F1 Cohort 1A males:
One epididymis and one testicle were used for the sperm count. The sperm viability was determined and the sperm morphology was examined according
to the method described by I. Chahoud and R. Franz (1993) as well as by S. Plassmann and H. Urwyler (2001).
Additionally, from each F0 male animal one drop of sperm was collected and preserved in an Eppendorf tube with 500 μL of 2.5% glutaraldehyde11.
Litter observations:
Examinations of F1 Pups
As soon as possible after delivery, each litter was examined to establish the number and sex of pups, stillbirths, live births, runts (pups were considered as runts if their weight was less than 70% of the mean litter weight) and the presence of gross abnormalities. Any abnormal behaviour of the offspring would have been recorded. However, no abnormal behaviour was noted for the pups.
The following examinations/observations were done for the offspring.

Counting, sexing, weighing
Live pups were counted, sexed and weighed on post-natal days (PND) 1, 4, 7, 14 and 21.

Ano-genital distance
On PND 4 before litter adjustment the ano-genital distance (AGD) of all pups was determined using a scale.

Litter adjustment on PND 4
After counting on PND 4, the litters were adjusted to 10 pups per litter (5 pups per sex and litter) by eliminating (culling) surplus pups using a randomization scheme generated by Provantis®. Selective elimination of pups e.g. based upon body weight is not appropriate and was not performed. In case of unequal gender distribution, a partial litter size adjustment was performed (e.g. 6 male and 4 female pups).

Blood sampling for thyroid hormone determination
On PND 4 (determination of T4) and on PND 22 (determination of T4 and TSH) blood samples for thyroid hormone level determination were taken from 2 selected pups per litter, if possible from one male and one female pup.
On PND 4 the culled surplus pups were used for blood collection and on PND 22 those pups were used which were not selected for the cohorts of the F1 Generation.

Nipples/areolae counting
Nipples/areolae were counted in all male pups on PND 13.

Sexual maturation
All F1 Pups that were selected for the Cohorts of the F1 Generation were evaluated daily for balano-preputial separation or vaginal opening before expected achievement of these endpoints to detect if sexual maturation occurred early. Any abnormalities of the genitals were recorded.
Sexual maturity of the F1 animals was compared to physical development. The body weight of the animals at the time point of balano-preputial separation or vaginal opening was recorded.

Postmortem examinations (parental animals):
Gross necropsy (see table Necropsy Schedule in "Any other information on methods")
For adult F0 and F1 animals a vaginal smear taken on the day of sacrifice, shortly before necropsy, was examined to determine the stage of the estrous cycle and allow correlation with the histopathology of the female reproductive organs.
The female animals were euthanized by carbon dioxide (CO2) inhalation, the male animals were euthanized under ether narcosis to guarantee best possible results for sperm analysis. Immediately thereafter, the animals were exsanguinated by cutting the aorta addominalis and weighed as scheduled.

Dissection
At the time of sacrifice or premature death during the study, the adult animals were examined macroscopically for any abnormalities or pathological changes. Special attention was paid to the organs of the reproductive system.
During necropsy the number of implantation sites was recorded in the female animals.
Apparently non-pregnant uteri were placed in a 10% aqueous solution of ammonium sulfide for about 10 minutes to stain possible implantation sites in the endometrium according to SALEWSKI
All superficial tissues were examined visually and by palpation and the cranial roof removed to allow observation of the brain, pituitary gland and cranial nerves. After ventral midline incision and skin reflection all subcutaneous tissues were examined. The condition to the thoracic viscera was noted with due attention to the thymus, lymph nodes and heart.
The abdominal viscera were examined before and after removal; the urinary bladder was examined externally and by palpation. The gastro-intestinal tract was examined as a whole and the stomach and the caecum were incised and examined. The lungs were removed and all pleural surfaces were examined under suitable illumination.
The liver and the kidneys were examined. Any abnormalities in the appearance and size of the gonads, adrenals, uterus, intra-abdominal lymph nodes and accessory reproductive organs were recorded.
The weights of the organs listed below were weighed from all male and female animals of the F0 Generation and the Cohort 1A. With the exception of the thyroid weight (determined after fixation), all organs were weighed before fixation. Paired organs were weighed individually and identified as left or right.

Organ weights
Adrenal gland (2)
Ovary (2)
Brain
Spleen
Epididymis (2)
Testicle (2)
Heart
Thyroid (fixed)
Kidney (2)
Thymus
Liver
As a whole: prostate, seminal vesicles with coagulating glands
Lymph node (1, cervical)#
Pituitary
Lymph node (1, mesenteric)#
Uterus including cervix

HISTOPATHOLOGY

Full histopathology was performed on the preserved organs of:
• F0 animals: groups 1 and 4
• F1 animals: groups 1 and 4 of Cohort 1A
• all deceased or prematurely sacrificed animals
Histopathology of the following organs:
Fixative: Davidson’s solution
Eye with optic nerve (2)
Fixative: modified Davidson’s solution
Epididymis (1)#
Testicle (1)#
Fixative: 7% buffered formalin
Adrenal gland (2)
Oesophagus
Bone
Ovary and oviduct (2)
Bone marrow (os femoris)
Pituitary
Brain (cerebrum, cerebellum, pons)
Prostate
Gross lesions observed
Seminal vesicles with coagulating glands
Heart (3 levels: right and left ventricle,septum)
Spinal cord (3 sections)
Intestine, small (duodenum, jejunum, ileum, including Peyer’s patches, Swiss roll method)
Spleen##
Intestine, large (colon, rectum)
Stomach
Kidney and ureter (2)
Thyroid (including parathyroids)
Liver
Thymus
Lungs (with mainstem bronchi and bronchioles), preserved by inflation with fixative and then immersion
Trachea (including larynx)
Lymph node (1, cervical)###
Urinary bladder
Lymph node (1, mesenteric)###
Uterus (including cervix)
Mammary gland
Vagina
Muscle (skeletal)
Vas deferens
Nerve (sciatic)

Histopathological evaluation
The preserved organs/tissues, labelled with study number, species, and animal number, were shipped on 29 October 2019 to the Test Site for histopathology (see section 2.6) following advance notice.
The histotechnique and the histopathological examination is performed at the Test Site under the responsibility of the PIs according to all relevant AnaPath SOPs.
All observations upon final assessment will be reported per animal and the findings considered relevant for the treatment in an incidence and occurrence table. All microscopic findings are recorded, reported and archived with the PathData system version 6.2e2.
The report of this phase of the study comprises a description of objective, materials and methods, results (including results of macroscopic and microscopic changes) and conclusions. The unaudited Draft Phase Report will be presented electronically to the Study Director for comments/review. Comments will be addressed and the Draft Final Phase Report of the histopathology phase of the study will be presented to the TSQAU for auditing.
The audited Draft Final Phase Report of the histopathology phase of the study will be sent electronically to the Study Director who will provide documented approval on the contents of the Phase Report by e-mail to the Principal Investigator. An original of the final, signed Phase Report will be sent (on paper and electronically) to the Study Director; a copy will be archived by AnaPath Services GmbH.
The AnaPath Phase Report will be given in Appendix 7 'Histopathological Phase Report' of the final LPT Study Report No. 37260.


Bone marrow
During dissection fresh bone marrow was obtained from the os femoris (3 airdried smears / animal) form 10 male and 10 female animals from all groups of the F0 Generation and the F1 Generation Cohort 1A animals and stained according to PAPPENHEIM. The same animals were used as those that were selected for the laboratory examinations and the hormone level determinations.
The myeloid:erythroid ratio was determined from animals of groups 1 and 4 by cell differentiation (counting of 200 nuclei-containing cells).
Following a randomization scheme, the animals were euthanized by carbon dioxide (CO2) inhalation and exsanguinated by cutting the aorta abdominalis.

Phenotypic analysis of spleen cells
After weighing at necropsy, the spleen of the selected Cohort 1A animals was split in 2 parts (see Text table 4-5. The part of the spleen not preserved for histopathololgy (histopathology of the spleen was only performed for Cohort 1A) was minced using a mechanic dissociator to prepare single cell suspensions.
The prepared spleen samples were used for the determination of the following lymphocyte subpopulations via flow cytometry using the MACSQuant®Analyzer 10 :
- CD4+ T-Lymphocytes helper T cells
- CD8+ T-Lymphocytes suppressor / cytotoxic T cells
- Pan T-Lymphocytes (CD3+) T cells
- B-Lymphocytes (CD45RA+) B cells
- Natural killer cells (CD161+) NK cells

Evaluation was performed by LPT.


Postmortem examinations (offspring):
Gross necropsy (see table Necropsy Schedule in attachment)

Dissection

The weights of the following organs of all adult F0 and F1 cohort 1A animals were determined before fixation, where applicable. Thyroid weight was determined after fixation. Paired organs were weighed individually and identified as left or right.

Organ weights
Adrenal gland (2)
Ovary (2)
Brain
Spleen
Epididymis (2)
Testicle (2)
Heart
Thyroid (fixed)
Kidney (2)
Thymus
Liver
As a whole: prostate, seminal vesicles with coagulating glands
Lymph node (1, cervical)#
Pituitary
Lymph node (1, mesenteric)#
Uterus including cervix

HISTOPATHOLOGY

Full histopathology was performed on the preserved organs of:
• F0 animals: groups 1 and 4
• F1 animals: groups 1 and 4 of Cohort 1A
• all deceased or prematurely sacrificed animals
Histopathology of the following organs:
Fixative: Davidson’s solution
Eye with optic nerve (2)
Fixative: modified Davidson’s solution
Epididymis (1)#
Testicle (1)#
Fixative: 7% buffered formalin
Adrenal gland (2)
Oesophagus
Bone
Ovary and oviduct (2)
Bone marrow (os femoris)
Pituitary
Brain (cerebrum, cerebellum, pons)
Prostate
Gross lesions observed
Seminal vesicles with coagulating glands
Heart (3 levels: right and left ventricle,septum)
Spinal cord (3 sections)
Intestine, small (duodenum, jejunum, ileum, including Peyer’s patches, Swiss roll method)
Spleen##
Intestine, large (colon, rectum)
Stomach
Kidney and ureter (2)
Thyroid (including parathyroids)
Liver
Thymus
Lungs (with mainstem bronchi and bronchioles), preserved by inflation with fixative and then immersion
Trachea (including larynx)
Lymph node (1, cervical)###
Urinary bladder
Lymph node (1, mesenteric)###
Uterus (including cervix)
Mammary gland
Vagina
Muscle (skeletal)
Vas deferens
Nerve (sciatic)

F1 Cohort 1B animals
Determination of organ weight and preservation of the F1 Cohort 1B animals
Endocrine system:
Pituitary
Reproductive system:
Epididymis (2)
Testicle (2)
Ovary and oviduct (2)
Uterus (including cervix)
Prostate
Vagina#
Seminal vesicles with coagulating glands
Target organs:
Liver
Spleen
In case of test item-related changes in group 4, the Sponsor was given sufficient notice before the corresponding organs of the F0 and F1 Cohort 1A of the intermediate and the low dose level groups are sectioned and examined histopathologically.



Statistics:
Parametrical data
The statistical evaluation of the parametrical values was done by Provantis (Provantis® integrated preclinical software, version 10.2.1, Instem LSS Ltd) using the following settings:
Homogeneity of variances and normality of distribution were tested using the BARTLETT’s and SHAPIRO-WILK’s test. In case of heterogeneity and/or non-normality of distribution, stepwise transformation of the values into logarithmic or rank values was performed prior to ANOVA. If the ANOVA yielded a significant effect (p ≤ 0.05), intergroup comparisons with the control group were made by the DUNNETT’s test (p ≤ 0.01 and p ≤ 0.05) (see the decision tree on the following page).

Non-parametrical data
The statistical evaluation of non-parametrical values was done with the following software:
Bone marrow: statistical evaluation of the myeloid / erythroid ratio using the Chi2 test with StatXact 4.0.1 software.

Histopathology data
The statistical methods that were used in the Histopathology Report (e.g. for the quantitative evaluation of ovary follicles and corpora lutea) are described in the Histopathology Report.

Significantly different data are indicated in the summary tables of the result sections (Sections 7 to 9) and the result tables in the tables section (Section 12) of this report.
The mean values and standard deviations were calculated to the highest possible degree of accuracy and then rounded to the reported number of decimal places. Hence, deviations to the last decimal place of up to ± 1 may occur caused by rounding.
Reproductive indices:
Gestation Index, Female Fertility Index, Birth index, Live birth index
Offspring viability indices:
Birth Index, Live Birth Index, Viability Birth Index, Post Implantation loss
Clinical signs:
no effects observed
Description (incidence and severity):
Males and females
No test item-related changes in behaviour, external appearance or the faeces were noted for the male and female animals of the control group and the treatment groups (15, 50 or 150 mg test item/kg b.w./day).
The male animal no. 107 dosed with 50 mg test item/kg b.w./day was noted with piloerection on TD 67 to TD 71 and with breathing sounds on TD 67 and TD 68.
In the high dose group (150 mg test item/kg b.w./day), the female animal no. 183 displayed piloerection from GD 8 until LD 18. Furthermore, the female animal no. 177 was noted with breathing sounds on LD 4 to LD 8. However, as only one animal displayed piloerection or breathing sounds, piloerection and breathing sounds were considered to be spontaneous and not test item-related.

Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Description (incidence):
No premature death was noted in the control group and in the intermediate and high dose groups (50 or 150 mg test item/kg b.w./day) for the male and female animals.
In the low dose group (15 mg test item/kg b.w./day), the female animal no. 78 was found dead in the morning of LD 13. Necropsy revealed dark red discoloured lungs and the thorax being filled with clear liquid. The single occurrence of one prematurely deceased low dose animal was considered to be due to a misgavage and not test item-related.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Males:
Pre-mating-, mating- and post-mating period
No test item-related changes in body weight and body weight gain were noted for the male animals between the control group and the low and intermediate dose groups (15 or 50 mg test item/kg b.w./day).
In the high dose group (150 mg test item/kg b.w./day), the body weight and also the body weight gain were decreased from TD 22 until termination of the male F0 animals on TD 85 or TD 86 (at maximum 9.1% below the value of the control group on TD 71, statistically significant at p ≤ 0.01).
The distinctly and constantly decreased body weight for the male animals of the high dose group was considered to be test item-related.

Females:
Pre-mating, gestation and lactation period
No differences in body weight and body weight gain were noted between the female animals of the control group and the female animals of the treatment groups (15, 50 or 150 mg test item/kg b.w./day) during the pre-mating period.
However, a decreased body weight in comparison to the control group was noted for the high dose group (150 mg test item/kg b.w./day) between GD 7 and LD 14 (at maximum 11.5% below the value of the control group on LD 4, statistically significant at p ≤ 0.01 for all time points evaluated between GD 7 and LD 14). This constantly decreased body weight that was also noted for the male animals was considered to be test item-related.

Body weight gain
In accordance with the development of the body weight, the body weight gain was decreased during the gestation period for the female high dose animals. As the body weight of the high dose females recovered during the lactation period, the body weight gain of the high dose group was above the values of the control group.

Body weight at autopsy
Males and females
No test item-related differences between the male and female animals of the control group and the low and intermediate dose group (15 or 50 mg test item/kg b.w./day) and the female animals of the high dose group (150 mg test item/kg b.w./day) were noted for the body weight at autopsy.
In the male animals of the high dose group (150 mg test item/kg b.w./day), a decreased body weight at autopsy was noted for the male animals (8.8% below the value of the control group, statistically significant at p ≤ 0.01). As this was due to the test item-related decrease in body weight until termination of the male F0 animals, also the decreased body weight at autopsy was considered to be test item-related.


Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Males
No test item-related difference in food consumption was noted between the control group and the low and intermediate dose group (15 or 50 mg test item/kg b.w./day).
In the high dose group (150 mg test item/kg b.w./day), a reduced food consumption was noted in the week between TD 15 and TD 22 and between TD 22 and TD 29 (7.3% and 6.7% below the value of the control group, statistically for both at p ≤ 0.01). This reduction of the food consumption was considered to be test item-related. No food intake of male animals was recorded during the mating period as both sexes were housed together.

Females:
Pre-mating, gestation and lactation period
No test item-related difference in food consumption was noted between the control group and the low and intermediate dose group (15 or 50 mg test item/kg b.w./day).
In the high dose group (150 mg test item/kg b.w./day), a decreased food consumption in comparison to the control group was noted starting in test week 3 (TD 15 to TD 22) until the first week of the lactation period (LD 1 to LD 7). The decrease was at maximum 13.5% below the value of the control group in the week of GD 7 to GD 14 (statistically significant at p ≤ 0.01 for all evaluated time points between TD 15 and LD 7). The constantly decreased food consumption that was also noted for the male animals was considered to be test item-related. No food intake of female animals was recorded during the mating period as both sexes were housed together.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
Males and females
The water consumption for the male and female animals was monitored by visual appraisal. No influence on the water consumption was noted for any animal of the F0 generation.
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Males and females
No test item-related differences for the examined haematological parameters were noted between the control group and the treatment groups (15, 50 or 150 mg test item/kg b.w./day).
Statistically significant changes in comparison to the control group were noted at the intermediate dose males and for the male and female animals of the high dose group.
However, as the differences were only observed for one sex and/or no dose response-relationship was noted, the differences were considered to be spontaneous.
Description (incidence and severity):
General biochemical parameters
Males and females
No test item-related differences for the examined biochemical parameters were noted between the control group and the treatment groups (15, 50 or 150 mg test item/kg b.w./day).
Statistically significant changes in comparison to the control group were noted for the male animals of the intermediate dose group and for the male and female animals of the high dose group. However, these differences were considered to be spontaneous.

Thyroid hormone levels
Males and females
No test item-related differences for the examined thyroid hormone levels (T4 and TSH) were noted between the females of the control group and the treatment groups (15, 50 or 150 mg test item/kg b.w./day) and for the male animals of the low and intermediate dose group.
A statistically significantly decreased T4 level in comparison to the control group was noted for the high dose males (34.6% below the value of the control group, statistically significant at p ≤ 0.01) (see figure 9 below). As a dose-dependence relationship is present and also a decrease for the T4 levels was noted for the male animals of Cohort 1A (see section 8.9) the decreased T4 levels were considered to be test item-related.
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
Detailed clinical observations
The detailed clinical observations were performed once weekly.
Males and females
No further observations in addition to those made during the daily cage side observations were noted for the animals of the control group and the animals of the treatment groups (15, 50 or 150 mg test item/kg b.w./day).

Immunological findings:
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
Males and females
Histopathologic examination was conducted of the male and female animals of the control group and the high dose (150 mg test item/kg b.w./day). The examination revealed test item-related changes in form of vacuolation or vacuolar changes in smooth muscle cells/fibers of several organs. However, in a detailed examination of these effects it was concluded that these findings are form of phospholipidosis and therefore not relevant for human health.
Detailed examination of testis and one epididymis:
The histopathological examination performed on one testicle and one epididymis of the examined males of groups 1 and 4 (with special emphasis on the qualitative stages of spermatogenesis (proliferative, meiotic and spermiogenic phases) and histopathology of the interstitial testicular structure) revealed no toxicologically relevant lesions.
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Description (incidence and severity):
Bone marrow
Males and females
No test item-related differences for the myeloid/erythroid ratio of the bone marrow were noted between the control group and the high dose group (150 mg test item/kg b.w./day).
Reproductive function: oestrous cycle:
effects observed, non-treatment-related
Description (incidence and severity):
Stage of estrous cycle at necropsy
During necropsy, a vaginal smear was taken for determination of the estrous stage from the female animals of the control group and the treatment groups (15, 50 or 150 mg test item/kg b.w./day). The stage of diestrus was the predominantly noted in groups 1 to 3. However, the numbers of female animals in the diestrus stage decreased and the number of animals in the estrus stage increased gradually from group 1 to 4. Thus, in group 4, the number of animals in the diestrous stage was equal to the number of animals in the estrous stage.
However, the numbers of female animals in the different estrous cycles differ from the numbers given in the histopathological phase report. This was due to different methods of evaluation (evaluation of a vaginal smear versus histopathological examination of the vagina).
The histopathologic evaluation of the vagina revealed similar numbers for the control group (4 animals in diestrus and 9 animals in lactational diestrus) and the high dose group (1 animal in diestrus and 10 animals in lactational diestrus). Therefore, the different numbers of animals in diestrus determined by evaluation of vaginal smears was considered to be spontaneous.



Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
Sperm number
No test item-related difference was noted between the rats of the control group and the rats treated with 15, 50 or 150 mg test item/kg b.w./day for the number of ultrasound-resistant sperm heads (sperm count) per gram testicular tissue.

Sperm motility
No test item-related differences were noted between the rats of the control group and the treatment groups (15, 50 or 150 mg test item/kg b.w./day) for the percentage of motile spermatozoa in the epididymal cauda on the total number of motile and non-motile spermatozoa.
A decrease was noted for the sperm motility of the high dose group (150 mg test item/kg b.w./day) (24.2% below the value of the control group, statistically significant at p ≤ 0.01). However, as no influence on the fertility and the reproductive performance was noted the decreased sperm motility was considered to be spontaneous and not test item-related.

Sperm morphology
The examination of spermatozoa from the epididymal cauda revealed no increased number of spermatozoa with a malformation in the treatment groups (15, 50 or 150 mg test item/kg b.w./day) in comparison to the control group.
In detail, the examination of 200 spermatozoa per animal from each test group revealed 51 spermatozoa with a malformation in the control group, 32 in the low dose group, 31 in the intermediate dose group and 6 in the high dose group. In all cases, the observed malformation was in the form of a banana-like sperm head.
Reproductive performance:
no effects observed
Description (incidence and severity):
Female fertility
No test item-related influence on the fertility index of the female rats was noted for any of the treatment groups (15, 50 or 150 mg test item/kg b.w./day).
One female of the low dose group (no. 93) and one female of the high dose group (no. 189) did not show a positive mating sign. Furthermore, one female animal of the low dose group (no. 82) and one female of the high dose group (no. 176) were not pregnant although a positive mating sign was noted. However, the occurrence of single not mated/non-pregnant females in the low and high dose group was considered to be spontaneous. A fertility index of 92 % is in the range of normal variability.

Gestation index
No test item-related influence on the gestation index was noted for the female rats of the treatment groups (15, 50 or 150 mg test item/kg b.w./day).

Pre-coital time
No test item-related differences were noted in the length of the pre-coital time between the control group and the treatment groups (15, 50 or 150 mg test item/kg b.w./day).
A statistically significantly decreased pre-coital time was noted for the animals of the intermediate dose group (39.0% below the value of the control group, statistically significant at p ≤ 0.05). However, a decreased pre-coital interval was considered to be not test item-related.

Gestation length
No test item-related differences were noted for the length of the gestation period between the rats of the control group and the treatment groups (15, 50 or 150 mg test item/kg b.w./day).



Key result
Dose descriptor:
NOAEL
Effect level:
50 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
food consumption and compound intake
clinical biochemistry
Critical effects observed:
not specified
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
1A
Male and females
No test item-related changes in behaviour, the external appearance and the consistency of the faeces were noted for the male and female animals of the control group and the treatment groups (15, 50 or 150 mg test item/kg b.w./day).
Breathing sounds were noted for one male animal (no. 321) of the high dose group on 3 consecutive test days and salivation was noted for one female animal (no. 360) of the high dose group on 6 consecutive test days. As both observations were only noted for one animal each, these observations were considered to be spontaneous.



1B
Dermal irritation (if dermal study):
not examined
Mortality / viability:
no mortality observed
Description (incidence and severity):
1A
Males and females
No premature death was noted in the control group and in the treatment groups (15, 50 or 150 mg test item/kg b.w./day) for the male and female animals.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Litter weight of F1 pups:

1A
Males
No test item-related changes in body weight and body weight gain were noted for the male rats between the control group and the low and the intermediate dose group (15 or 50 mg test item/kg b.w./day).
A marginally to slightly reduced body weight (statistically not significant) was noted at the low and the intermediate dose level during the later course of the study until test day 70 (last reported live weight). At the low dose level the maximum difference to the control was noted on test day 70 (3.9% below the control) and at the intermediate dose level the maximum difference to control was noted on test day 64 (6.0% below the control). These marginal to slight differences were considered to be spontaneous.
At the high dose level (150 mg test item/kg b.w./day) a statistically significantly (p ≤ 0.05 or 0.01) reduced body weight was noted from test day 22 (8.9% below the value of the control group) onwards. The maximum difference was noted on test day 70 (last reported live weight) (13.8% below the control, statistically significant at p ≤ 0.01).
However, it has to be considered, that a slightly reduced body weight was already noted for the high dosed male animals at the start of the F1 Generation study on test day 1 (7.3% below the value of the control group, statistically not significant). Nevertheless, the increasing difference between the body weight of the high dosed animals and the control animals from test day 22 onwards was considered to be test item-related.
Body weight gain
A slightly reduced body weight gain in comparison to the control group was noted for all treatment groups.
Though there was a more pronounced reduction in body weight at the high dose level on test day 70 as at the low and the intermediate dose level (see above), body weight gain at the high dose level was nearly the same as at the low and the intermediate dose level. This was due to the slightly reduced body weight that was already noted at the high dose level on test day 1 (7.3% below the control) in comparison to the low and the intermediate dose levels (3.4% or 4.7% above the control on test day 1).
Females
No differences in body weight and body weight gain were noted between the female animals of the control group and the female animals of the treatment groups (15, 50 or 150 mg/kg b.w./day).
However, at the high dose level a constantly reduced body weight was noted from test day 1 (6.6% below the value of the control group, statistically not significant) until test day 66 (last reported live weight) (6.7% below the value of the control group, statistically significant at p ≤ 0.05). The maximal difference was noted on test day 8 (7.9% below the value of the control group, statistically significant at p ≤ 0.05).
As the reduced body weight of the high dosed animals was already noted on test day 1 and the difference remained at the same level during the course of the study, a test item-related influence on the body weight of the growing females could not be detected (see also body weight gain below).
Body weight gain
No test item-related differences in body weight gain were noted. Body weight gain in the control group was the same as in the high dose group.

Body weight at autopsy
Males and females
No test item-related differences were noted between the control group and the low and intermediate dose group (15 or 50 mg test item/kg b.w./day).
Slight but statistically significant reductions in the body weight at autopsy were noted for the male and female animals of the high does group (150 mg test item/kg b.w./day) (males: 12.9% below the control; p ≤ 0.01; females: 7.2% below the control; p ≤ 0.05). These differences correspond well with the differences between the control group and the high dose group that were noted for the last live body weight (males: 13.8% below the control; p ≤ 0.01; females: 6.7% below the control; p ≤ 0.05)




Description (incidence and severity):
1A
Males
No test item-related difference in food consumption was noted between the control group and the treatment groups (15, 50 or 150 mg test item/kg b.w./day).
A slightly but statistically significantly increased food consumption was noted at the intermediate dose level between test days 1 to 8 (4.8% below the value of the control group, statistically significant at p ≤ 0.05) and at the high dose level between test days 1 to 8 and test days 36 to 43 (8.2% or 5.2% below the value of the control group, statistically significant at p ≤ 0.01 or 0.05) (see figure 3 Co1A below). These slight and transient differences were considered to be spontaneous.
Females
No test item-related difference in food consumption was noted between the control group and the treatment groups (15, 50 or 150 mg test item/kg b.w./day).
A slightly but statistically significantly increased food consumption was noted at the high dose level between test days 1 to 8 (8.4% above the value of the control group, statistically significant at p ≤ 0.01). These slight and transient difference was considered to be spontaneous.

Food efficiency:
not examined
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):
1A
Males
No test item-related differences for the examined haematological parameters were noted between the control group and the treatment groups (15, 50 or 150 mg test item/kg b.w./day).
Statistically significantly increased numbers of white blood cells, lymphocytes and basophilic granulocytes were noted at the intermediate dose level. However, no dose response-relationship was noted.
Furthermore, the mean values of the white blood cells and the lymphocytes at the high dose level were nearly identical with the mean values of the LPT background data.
Values above the LPT background range were only noted for the number of basophilic granulocytes (see Appendix 5). However, the population of basophilic granulocytes is very small and the values from the individual animals showed a high variability (for example between 0.1 and 0.6 x 10³ cells/µL at the high dose level).
Taken together, the observed changes that were noted for the number of white blood cells, lymphocytes and basophilic granulocytes were considered to be spontaneous.

Females
No test item-related differences for the examined haematological parameters were noted between the control group and the treatment groups (15, 50 or 150 mg test item/kg b.w./day).
Statistically significantly increased values were noted at the high dose level and / or the intermediate dose level for the number of white blood cells, lymphocytes, neutrophilic granulocytes, monocytes, large unstained cells and basophilic granulocytes.
However, the increased values were considered to be spontaneous, due to the following reasons:
All individual values of the white blood cells and the lymphocytes from all test groups were in the range of the LPT background data.
The mean values at the high dose level for the number of white blood cells and lymphocytes were nearly identical with the mean values of the LPT background data, leading to the assumption, that the increased values at the high dose level were due to low values in the control group.
Mean values that were above the mean values of the LPT background data were only noted for the number of neutrophilic granulocytes, monocytes, large unstained cells and basophilic granulocytes. However, these populations are small and the individual values showed a high variability, which made a comparison with the LPT background data difficult. Due to these reasons the observed changes for these cell populations were considered to be spontaneous.


Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
1A
Males and females
No test item-related differences for the examined biochemical parameters were noted between the control group and the treatment groups (15, 50 or 150 mg test item/kg b.w./day).
However, slight but statistically significant differences in comparison to the control group were noted for the males and / or females at the low or the high dose group for the following parameters: albumin (males and females), protein (females), calcium (males and females) and potassium (females) concentrations, the sodium / potassium ratio (females) and the ALAT activity (females).
The observed differences were considered to be spontaneous due to the following reasons:
The differences were only slight and only noted for one sex (differences in the albumin concentration were noted for both sexes, but with different plus / minus signs).
Additionally, the statistically significant differences for the potassium concentration and the sodium / potassium ratio were noted at the low dose level and no dose response relationship was noted.
Finally, an increased ALAT activity is a sign of organ damage and a decreased ALAT activity, as noted in this study, cannot be explained by a toxicological mechanism and has to be considered as spontaneous.

Thyroid hormone levels
Males
No test item-related differences for the examined thyroid hormone levels (T4 and TSH) were noted between the control group and the low and the intermediate dose group (15 or 50 mg test item/kg b.w./day).
A statistically significantly decreased T4 level in comparison to the control group was noted at the high dose level (150 mg test item/kg b.w./day) and statistically significantly increased TSH values were noted at the intermediate and the high dose level. As also the males of the F0 generation were noted with decreased T4 levels and also the females of Cohort 1A displayed increased levels for TSH, the differences for the levels of T4 and TSH for the male animals were considered to be test item-related. Females
No test item-related differences for the examined thyroid hormone levels were noted between the control group and the dose groups (15, 50 or 150 mg test item/kg b.w./day).
An increased TSH level was noted at the high dose level (150 mg test item/kg b.w./day). The distinctly increased TSH levels in the high dose group (see figure 7-Co1A below) were considered to be test item-related.




Urinalysis findings:
effects observed, non-treatment-related
Description (incidence and severity):
1A
Males and females
No test item-related differences for the examined urinalysis parameters were noted between the control group and the treatment groups (15, 50 or 150 mg test item/kg b.w./day) for the male and female animals.
A statistically significantly increased specific gravity, a decreased pH value and a decreased relative urine volume (statistically significant or not) in comparison to the control group were noted for the male animals of all treatment groups. However, no dose response-relationship was noted (the difference in comparison to the control was nearly equal for all treatment groups) and no statistically significant changes were noted for the female animals.
Hence, the changes that were observed for the male animals were most probably due to unusually high values at the control group and considered to be spontaneous
Description (incidence and severity):
1A
Males
No test item-related differences between the control group and the test item-treated groups (15, 50 or 150 mg test item/kg b.w./day) were noted for the time point of balanoprepuital gland cleavage.
A slight but statistically significantly reduced body weight at the time point of balanopreputial gland cleavage (6.8% below the value of the control group, statistically significant at p ≤ 0.05) was noted at the high dose level which corresponds to the slightly reduced body weight that was noted on lactation day 21 (5.0% below the value of the control group). However, though the body weight was slightly reduced at the high dose level, no delay in sexual maturation was noted for the male animals of the high dose group.
Females
No test item-related differences between the control group and the test item-treated groups (15, 50 or 150 mg test item/kg b.w./day) were noted for the time point of vaginal opening and the body weight at the time point of vaginal opening.
A slight delay was noted at the high dose level for the day of vaginal opening (postnatal day 36.2 in comparison to postnatal day 34.3 in the control). However, a similar delay was noted for the female animals of cohort 1B without an influence on the reproductive performance of the female animals of cohort 1B. Hence, the delay maybe test item-related, but is regarded to be not adverse.
Appearance of cornified cells in the vaginal smear
A slight delay was noted at the intermediate dose level for the day of the appearance of cornified cells (postnatal day 40.8 in comparison to postnatal day 38.5 for the control), leading to an increased period between the day of vaginal opening and the day of the appearance of cornified cells (6.3 test days in comparison to 4.4 test days in the control). However, as this period was again in the range of the control group at the high dose level (4.3 test days at the high dose level in comparison to 4.4 test days in the control, no dose response relationship), the delay in the appearance of cornified cells that was noted at the intermediate dose level was considered to be spontaneous.

Examination of the sperm number, viability and morphology
Sperm number
No test item-related difference was noted between the rats of the control group and the rats treated with 15, 50 or 150 mg test item/kg b.w./day for the number of ultrasound-resistant sperm heads (sperm count) per gram testicular tissue.
Sperm motility
No test item-related differences were noted between the rats of the control group and the treatment groups (15, 50 or 150 mg test item/kg b.w./day) for the percentage of motile spermatozoa in the epididymal cauda on the total number of motile and non-motile spermatozoa.
A slightly increased number of motile spermatozoa was noted at the intermediate dose level (76.9% motile spermatozoa in comparison to 64.0% in the control group, statistically significant at p ≤ 0.05). However, an increased number of motile spermatozoa is not an adverse effect. Hence, this observation was considered to be spontaneous.
Sperm morphology
The examination of spermatozoa from the epididymal cauda revealed no increased number of spermatozoa with a malformation in the treatment groups (15, 50 or 150 mg test item/kg b.w./day) in comparison to the control group.
In detail, the examination of 4000 spermatozoa (200 per animal) from each test group (only 3800 spermatozoa were evaluated at the high dose level, as no evaluable sperms were available from male no. 323) revealed one malformation at the control group, the low and the intermediate dose group. No malformed spermatozoa was noted at the high dose level.


Anogenital distance (AGD):
no effects observed
Description (incidence and severity):
No test item-related difference in the absolute and the relative ano-genital distance (value normalized to cube root of pup body weight) of the male and the female pups was noted between the control group and the treatment groups (15, 50 or 150 mg test item/kg b.w./day).
Nipple retention in male pups:
no effects observed
Description (incidence and severity):
No test item-related difference in the number of nipples was noted between the male pups of the control group and in the male pups of the treatment groups (15, 50 or 150 mg test item/kg b.w./day).

Description (incidence and severity):
1A
Males and females
No primary toxic effect of the test item on the absolute and relative organ weights of the examined organs was noted for all treatment groups (15, 50 or 150 mg test item/kg b.w./day).
Statistically significant differences that were noted for different organs at the intermediate and the high dose level were considered to be secondary effects that were due to a decreased body weight at autopsy.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
1A
Males
No observations were noted in the control group and no test item-related observations were noted in the treatment groups (15, 50 or 150 mg test item/kg b.w./day).
The following observations were considered to be spontaneous:
Group 2:
The right testis of male no. 259 was absent.
Group 3:
The right testis of male no. 281 was absent.
The left and the right kidney of male no. 299 were enlarged and pale.
Group 4:
A yellowish discoloured left and right kidney and multiple foci at the stomach mucosa (fundus region) were noted for male no. 329.
The right seminal vesicle of male no. 340 was reduced in size.

Females
No test item-related observations were noted in the treatment groups (15, 50 or 150 mg test item/kg b.w./day).
The following observations were considered to be spontaneous:
Group 1:
The uterus of female no. 226 was dilated and filled with clear liquid.
Group 2:
The uterus of female no. 266 was dilated and filled with clear liquid.
The uterus of female no. 269 was dilated and filled with clear liquid.
The left and the right kidney of female no. 271 were yellowish discoloured.
A solid tissue enlargement (approx.. 2 x 2 5 mm) in the right uterus horn was noted for female no. 273.
The uterus of female no. 276 was dilated and filled with clear liquid.
Group 3:
The mandibular lymph node and the right and the left adrenal gland of female no. 305 were enlarged.
The uterus of female no. 313 was dilated.
The left and the right kidney of female no. 319 were enlarged and yellowish discoloured.
Group 4:
A left thyroid that was reduced in size and an enlarged right thyroid were noted for female no. 343.
An eschar formation was noted at neck of animal no. 345.
An enlarged and yellowish discoloured left and right kidney was noted for animal no. 353.


Description (incidence and severity):
1A
The histopathological examination of the organs of male and female organs of the control group and the high dose group (150 mg test item/kg b.w./day) revealed vacuolation or vacuolic changes in the smooth muscle cells of almost all organs leading to inflammatory cell infiltration in the liver.
Other effects:
no effects observed
Description (incidence and severity):
1A
Lymphocyte typing in spleen
Males and females
No test item-related influence was noted in the proportion of the examined lymphocyte subtypes to each other between the male and female animals of the control group and the treatment groups (15, 50 or 150 mg test item/kg b.w./day).

Monitoring of estrous cycles - 2 week period
The stages of the estrous cycle of the cohort 1A animals were monitored on 14 test days between test days 50 and 63.
No test item-related differences were noted for the mean length and the mean number of estrous cycles per female animal between the females of the control group and the treatment groups (15, 50 or 150 mg test item/kg b.w./day)

Bone marrow
Males and females
No test item-related differences for the myeloid/erythroid ratio of the bone marrow were noted between the control group and the high dose group (150 mg test item/kg b.w./day).

Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
Summarized results - Cohort 1A
This part of LPT Study 37260 reports the effects of the test item on the development of the F1 male and female animals of cohort 1A after weaning on lactation day 21 until necropsy. The F1 Generation animals were dosed with the same levels as the animals of the F0 Generation. (15, 50 or 150 mg test item/kg b.w./day). Dosing started immediately after weaning and lasted until one day before necropsy.
None of the animals of cohort 1A died prematurely.
No test item-related changes were noted in behaviour, the external appearance and the consistency of the faeces.
A slightly reduced body weight was noted for the male animals at 150 mg test item/kg b.w./day, whereas no influence was noted on the body weight of the females.
No influence was noted on food consumption, the haematological and biochemical parameters, lymphocyte typing in the spleen, urinalysis, the sperm parameter and sexual maturation.
Increased levels of TSH were noted for the male and female high dose animals and a decrease was noted for the T4 levels of the male animals of the high dose group.
No test item-related changes were noted during the macroscopic examination, the examination of bone marrow and for the relative and absolute organ weights.
Dose descriptor:
other: No dose descriptor derived yet
Generation:
F1
Remarks on result:
other: study not yet completed
Critical effects observed:
not specified
Mortality / viability:
mortality observed, treatment-related
Description (incidence and severity):
Number of live pups – F2 Pups
No test item-related or statistically significant differences were noted for the mean number of live pups per dam between the control group and the low and intermediate dose group (15 or 50 mg test item/kg b.w./day).
In the high dose group (150 mg test item/kg b.w./day), a reduced number was noted for the number of live born pups leading to reductions in the numbers of pups per dam on LD 1 and LD 4 (17.4% or 17.0% below the value of the control group for male and female pups combined, statistically significant at p ≤ 0.05). As 5 of the 20 dams were noted with 9 pups and one dam with only 3 pups on LD 4, the reduced number of pups per dam was also noted after litter adjustment to 10 pups per dam (6.0%, 6.0% and 6.3% for the male and female pups combined on LD 7, LD 14 and LD 20, statistically significant at p ≤ 0.01).
The reduced number of live pups was considered to be test item-related.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Body weight of pups - F2 Pups
No test item-related influence on the body weight of pups was noted for any dose group (15, 50 or 150 mg test item/kg b.w./day).
In the high dose group, a statistically significant decrease was noted for the male pup body weights and for the combined body weights of the male and female pups on LD 14 (7.9% or 8.0% below the value of the control group, statistically significant at p ≤ 0.05). However, as no difference was noted on LD 1 and as the pup body weights of the high dose group recovered on LD 21, the decreased pup body weights on LD 14 was considered to be not test item-related.

Litter weight of F2 pups
No test item-related influence on the litter weight was noted for the low and intermediate dose group (15 or 50 mg test item/kg b.w./day).
In the high dose group (150 mg test item/kg b.w./day), a decrease was noted for the weight of the litters during the whole lactation period (between 10.2% and 18.9% below the value of the control group for the male and female litters combined, with the exception of LD 21 statistically significant at p ≤ 0.05 or 0.01).
As this was due to the test item-related lower number of born pups in combination with the slight reduction of the pup body weights from LD 7 to LD 21 the reduced litter weight was considered to be test item-related.
Anogenital distance (AGD):
no effects observed
Description (incidence and severity):
No test item-related difference in the absolute and the relative ano-genital distance (value normalized to cube root of pup body weight) of the male and the female pups was noted between the control group and the dose groups (15, 50 or 150 mg test item/kg b.w./day).
In the intermediate and high dose group, a statistically significant increase was noted for the absolute and relative ano-genital distance of the female pups (14.1%/12.3% or 14.8%/13.6% below the value of the control group, p ≤ 0.01). However, as no difference was noted for the male pups, the increased ano-genital distance was considered to be spontaneous.
Nipple retention in male pups:
no effects observed
Description (incidence and severity):
No test item-related difference for the number of nipples/areolae of the male pups between the control group and those of the dose groups (15, 50 or 150 mg test item/kg b.w./day).
Dose descriptor:
other: no dose descriptor derived yet
Generation:
F2 (cohort 1B)
Sex:
male/female
Remarks on result:
other: study not yet completed
Reproductive effects observed:
not specified

Text Table7‑1:  Outcome of the female animals (P0) per group.

Test item

Group 1

Control

Group 2

15 mg/kg

Group 3

50 mg/kg

Group 4

150 mg/kg

Females examined for general toxicity

24

24

24

24

Females used for pairing

24

24

24

24

Females with positive mating sign

24

23

24

23

Females without a positive mating sign

0

1 (93)

0

1 (189)

Females not pregnant

0

1 (82)

0

1 (176)

Females pregnant

24

22

24

22

Pregnancy rate (Fertility index)

100%

92%

100%

92%

Females with resorption of all implants

0

0

0

0

Females with litter

24

22

24

22

 

Text Table7‑3: Body weight gain of the female animals during the pre-mating period, the gestation period and the lactation period.

Females

Group 1

Control

Group 2

15 mg/kg

Group 3

50 mg/kg

Group 4

150 mg/kg

Body weight gain [%] #1

(pre-mating period)

(test days 15 to 29)

+4.3%

+5.8%

+5.4%

-0.2%

Body weight gain [g]

(pre-mating period)

(test days 15 to 29)

+10.7

+14.3

+13.5

-0.7

Body weight gain [%] #2

(gestation period)

+63.5%

+56.1%

+61.4%

+52.8%

Body weight gain [g]

(gestation period)

+169.6

+149.1

+162.5

+133.9

Body weight gain [%] #3

(lactation period)

+0.1%

+2.8%

+2.8%

+14.5%

Body weight gain [g]

(lactation period)

+0.0

+6.2

+7.8

+40.2

Text Table7‑4: Statistically significant changes of the haematological parameters that werenotconsidered to be test-item-related.

 

 

Changes in comparison to control

[%]

Reason

Parameter #1

Sex

Group 2

Group 3

Group 4

 

 

HGB

[mmol/L]

Males

+0.4

-1.3

-6.0**

B

RBC

[x106/µL]

Males

-0.9

-2.4

-8.5**

B

Reticulocytes

[%]

Males

+5.6

+24.9*

+11.3

A, B

HCT

[%]

Males

+0.7

-0.6

-5.7**

A, B

Neutrophils

[x10³/µL]

Females

-2.3

-1.2

+78.8**

A, B

Monocytes

[x10³/µL]

Females

-2.6

+33.3

+109.4**

A, B

LUC

[x10³/µL]

Females

-13.9

0.0

+111.1*

A, B

aPTT

[seconds]

Males

+0.9

-2.1

-5.9*

A, B

*/**:

Statistically significant from control at p ≤ 0.05/0.01 (DUNNETT test)

#1:

Values taken from table8-1'Haematological Parameters - Summary - Males'or table8-2'Haematological Parameters - Summary - Females'.

A:

No dose response-relationship was noted.

B:

No difference was noted for the opposite sex.

Text Table7‑5:  Statistically significant changes of the biochemical parameters that werenotconsidered to be test item-related.

 

 

Changes in comparison to control

[%]

Reason

Parameter #1

Sex

Group 2

Group 3

Group 4

 

 

Albumin

[g/L]

Males

-3.1

-1.0

-6.4**

A

Females

+2.9

+0.5

-4.0*

A

Bile acids

[µmol/L]

Males

+4.9

-4.4

+67.7**

A, B

Cholesterol (total)

[mmol/L]

Males

+14.7

+20.6

+29.9*

B

BUN/Creatinine

[ratio]

Males

+2.4

-6.6

+23.4**

A

Females

-3.6

-1.4

+18.6**

A

Glucose

[mmol/L]

Males

+7.9

+15.7*

+16.8

B, C

Protein (total)

[g/L]

Males

-3.9

+0.6

-6.5*

A, B

Urea (in blood)

[mmol/L]

Males

+5.0

-7.1

+22.6**

A, B

Calcium

[mmol/L]

Males

-1.2

-0.4

-4.8**

A, B

*/**:

Statistically significant from control at p ≤ 0.05/0.01 (DUNNETT test)

#1:

Values taken from table9-1'Biochemical Parameters - Summary - Males' or from table9-2'Biochemical Parameters - Summary - Females'.

A:

No dose response-relationship was noted.

B:

No difference was noted for the opposite sex.

C:

No statistically significant difference was noted for group 4.

Text Table7‑6:    Macroscopic post mortem findings during necropsy.

Macroscopicpost mortemfindings

Group

Animal no.

Observation

Group 1

(Control)

45

Kidney (l. + r.):          -pale

Group 2

(15 mg/kg)

75

78

Uterus:           -dilated

Thorax:          -filled with clear liquid

Lungs:            -dark-red discoloured

Group 3

(50 mg/kg)

142

Uterus:           -dilated

                       -filled with clear liquid

Group 4

(150 mg/kg)

171

182

 

189

Spleen:          -enlarged

Uterus:           -dilated

                       -filled with liquid

Ovary (l. + r.):-cystic

                       -no positive mating sign

Text Table7‑7:    Stage of the estrous cycle at necropsy determined by evaluation of vaginal smears.

Stage of estrous cycle

at necropsy

Group 1

Control

Group 2

15 mg/kg

Group 3

50 mg/kg

Group 4

150 mg/kg

Proestrus

1 of 24

2 of 24

2 of 24

4 of 24

Estrus

3 of 24

6 of 24

7 of 24

9 of 24

Metestrus

4 of 24

4 of 24

5 of 24

2 of 24

Diestrus

16 of 24

12 of 24

10 of 24

9 of 24

Text Table7‑8:    Statistically significant but considered to benottest item-related differences in the relative and absolute organ weights of the male animals.

Organ #1

Parameter

(absolute: g)

(relative: g/kg b.w.)

Changes in comparison to control

[%]

Reason

Group 2

Group 3

Group 4

 

Brain

g/kg b.w.

-1.2

-1.0

+6.3*

A, C

Heart

g/kg b.w.

-2.7

-4.0

+4.8**

A, C

Liver

g/kg b.w.

-1.1

-0.8

+10.6*

A, C

Spleen

g/kg b.w.

+0.6

+5.8

+15.8**

A

Testis (left)

g/kg b.w.

-0.6

-0.9

+11.8**

A

Testis (right)

g/kg b.w.

-1.4

-0.7

+11.9**

A, C

Epididymis (left)

g

-0.1

+2.3

-10.5*

B, C

Epididymis (right)

g

-1.9

+2.5

-8.5**

B, C

Prostate and Seminal Vesicle

g

-1.3

-5.6

-15.3**

B

*/**:

Statistically significant from control at p ≤ 0.05/0.01 (DUNNETT test).

#1:

Values taken from tables16-1'Relative Organ Weights - Summary - Males' and17-1'Absolute Organ Weights - Summary - Males'.

A:

Increased relative organ weights were considered to be secondary due to the decreased body weight at autopsy for the male group 4 animals.

B:

Decreased absolute organ weights were considered to be secondary due to the decreased body weight at autopsy for the male group 4 animals.

C:

No dose dependence-relationship present.

  Text Table7‑9:   Statistically significant but considered to benottest item-related differences in the relative and absolute organ weights of the female animals.

Organ #1

Parameter

(absolute: g)

(relative: g/kg b.w.)

Changes in comparison to control

[%]

Reason

Group 2

Group 3

Group 4

 

Brain

g

-0.9

-1.3

-3.6**

A

Liver

g/kg b.w.

-1.1

-1.9

+6.5*

C

Ovary (left)

g/kg b.w.

-4.3

-4.2

-20.0**

B

Ovary (left)

g

-7.5

-4.8

-22.0**

B, C

Ovary (right)

g

+2.7

+0.7

-13.1*

B, C

Thymus

g/kg b.w.

+10.7

+1.5

-16.2**

A, C

*/**:

Statistically significant from control at p ≤ 0.05/0.01 (DUNNETT test).

#1:

Values taken fromtables16-2'Relative Organ Weights - Summary - Females'and17-2'Absolute Organ Weights - Summary - Females'.

A:

No difference was noted for the respective organ weights of the male animals.

B:

No difference was noted for the respective organ weights of the F1 animals of Cohort 1A and Cohort 1B.

C:

No dose dependence-relationship present.

Conclusions:
Up to now, there are no results of the study available.
Executive summary:

The OECD 443 study started in March 2019 and the first draft report is expected to be available in August 2020.

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2005-05-06 to 2005-12-09
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source:Charles River (UK) Limited, Margate, Kent
- Strain:Sprague-Dawley Cr1:CD (SD) IGS BR strain
- Sex. male and female
- Age: Young adult rats, approx 8 weeks old at the start of the treatment
- Weight at study initiation: Males:193 - 251 g, females:150 - 195 g
- Housing:group-housed, with up to 5 animals of the same sex and dose group/cage, with the exception of the mating and gestation/delivery period
- Diet (e.g. ad libitum): Rodent PMI 5002 (Certified) diet, BCM IPS Limited, London, UK
- Water (e.g. ad libitum): tap water
- Acclimation period: 8 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 +/- 2 °C
- Humidity (%): 55 +/- 15 %
- Air changes (per hr): was at least fifteen air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours daily
Route of administration:
oral: gavage
Vehicle:
polyethylene glycol
Remarks:
PEG400
Details on exposure:
Vehicle: Polyethylene glycol 400
The test material was administered daily by gavage. Control animals were treated in an identical manner with 4 ml/kg/day of polyethylene glycol 400.
The volume of test and control material administered to each animal was based on the most recent bodyweight and was adjusted at regular intervals.
Details on mating procedure:
Animals were paired on a one male: one female basis within each dose group, for a period of up to fourteen days. Cage tray-liners were checked each morning for the presence of ejected
copulation plugs and each female was examined for the presence of a copulation plug in the vagina. A vaginal smear was prepared for each female and the stage of the oestrous cycle or the
presence of sperm was recorded. The presence of sperm within the vaginal smear andfor vaginal plug in situ was taken as positive evidence of mating and the males were subsequently returned to
their original holding cages. Mated females were housed individually during the period of gestation and lactation.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The stability and homogeneity of the test material formulations were determined by Safepharm Analytical Laboratory. The concentration of test item in the test material formulations was determined spectrophotometrically.
The analytical method has been satisfactorily validated in terms of linearity, specificity and accuracy for the purposes of the study.
Duration of treatment / exposure:
The test material was administered daily by gavage until termination (up to fifty-four consecutive days). Males were terminated on Day 43, followed by the termination of all surviving females and offspring on Day 5 post partum.
Frequency of treatment:
The test material was administered once daily.
Details on study schedule:
- Groups of ten male and ten female animals were treated daily at the appropriate dose level throughout the study (except for females during parturition where applicable).
- Prior to the start of treatment and once weekly, all animals were observed for signs of functional/behavioural toxicity.
- One day prior to pairing (Day 14), blood samples were taken from five males and five females, randomly selected from each assessment.
- On Day 15, all animals were paired on a 1 male:1 female basis within each dose group for a maximum of fourteen days.
- Following evidence of mating, the males were returned to their original cages and females were transferred to individual cages.
- On completion of mating (during Week 6), five selected males per dose group were evaluated for functional/sensory responses to various stimuli.
- Pregnant females were allowed to give birth and maintain their offspring until Day 4 post partum. Evaluation of each litter size, litter weight, mean pup weight, clinical observations and
landmark developmental signs were also performed during this period.
- At Day 4 post partum, five selected females per - Following completion of the female gestation and lactation phases, the male dose groups were killed and examined macroscopically on Day 43.
- At Day 5 post partum, all females and offspring were killed and examined macroscopically.
Dose / conc.:
0 mg/kg bw/day
Remarks:
Control Group
Dose / conc.:
15 mg/kg bw/day (nominal)
Remarks:
Low Group
Dose / conc.:
50 mg/kg bw/day (nominal)
Remarks:
Intermediate Group
Dose / conc.:
150 mg/kg bw/day (nominal)
Remarks:
High Group
No. of animals per sex per dose:
Ten
Control animals:
yes, concurrent vehicle
Details on study design:
The rat was selected for this study as it is a readily available rodent species historically used in safety evaluation studies and is acceptable to appropriate regulatory authorities.
The dose levels were chosen based on the results of a preliminary range-finder presented in Part 2 of this report. The oral route was selected as the most appropriate route of exposure, based on the physical properties of the test material, and the results of the study are believed to be of value in predicting the likely toxicity of the test material to man and to screen for potential adverse effects on reproduction.
Positive control:
not required
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS:
All animals were examined for overt signs of toxicity, ill-health and behavioural change immediately before and after dosing, and one and five hours after dosing, during the working week. Animals were observed immediately before and after dosing, and one hour after dosing at weekends and bank holidays (except for females during parturition where applicable).

DETAILED CLINICAL OBSERVATIONS:
All animals were examined for overt signs of toxicity, ill-health and behavioural change immediately before and after dosing, and one and five hours after dosing, during the working week.
Prior to the start of treatment and at weekly intervals thereafter, all animals were observed for signs of functionalhehavioural toxicity. Functional performance tests were also performed on
five selected males and females per dose level, prior to termination, together with an assessment of sensory reactivity to various stimuli.

Detailed individual clinical observations were performed for each animal using a purpose-built arena. The following parameters were observed:
Gait
Tremors
Twitches
Convulsions
Bizarre/Abnormal/Stereotypie behaviour
Salivation
Pilo-erection
Exophthalmia
Lachrymation
Hyper/Hypothermia
Skin colour
Respiration
Palpebral closure
Urination
Defecation
Transfer arousal
Tail elevation

Functional Performance Tests
Motor Activify. Purpose-built 44 infra-red beam automated activity monitors were used to assess
motor activity. Animals were randomly allocated to the activity monitors. The tests were
performed at approximately the same time each day, under similar laboratory conditions. The
evaluation period was thirty minutes for each animal. The percentage of time each animal was
active and mobile was recorded for the overall thirty minute period and also during the final 20%
of the period (considered to be the asymptotic period).

Forelimb/Hindlimb Grips Strength.
An automated grip strength meter was used. Each animal was allowed to grip the proximal metal bar of the meter with its forepaws. The animal was pulled by the base of the tail until its grip was broken. The animal was drawn along the trough of the meter by the tail until its hind paws gripped the distal metal bar. The animal was pulled by the base of the tail until its grip was broken. A record of the force required to break the grip for each animal was made. Three consecutive trials were performed for each animal.

Sensory Reactivity
Each animal was individually assessed for sensory reactivity to auditory, visual and proprioceptive stimuli. The following parameters were observed:
Grasp response
Vocalisation
Toe pinch
Tail pinch
Finger approach
Touch escape
Pupil reflex
Startle reflex
Blink reflex


BODY WEIGHT:
Individual bodyweights were recorded on Day 0 (the day before the start of treatment) and then weekly for males until termination. Females were weighed weekly until mating was evident.
Bodyweights were then recorded on Days 0, 7, 14 and 20 post coitum, and on Days 1 and 4 post partum.

FOOD CONSUMPTION:
During the maturation period, weekly food consumption was recorded for each cage of adults. This was continued for males after the mating phase. For females showing evidence of mating,
food consumption was recorded for the periods covering Days 0-7, 7-14 and 14-20. For females with live litters, food consumption was recorded on Days 1 and 4 postpartum.

WATER CONSUMPTION:
Water intake was observed daily by visual inspection of water bottles for any overt change.

OTHER:
Pregnancy and Parturiton
Each pregnant female was observed at approximately 08.30, 12.30 and 16.30 hours and around the period of expected parturition. Observations were carried out at approximately 08.30 and
12.30 hours at weekends and public holidays. The following was recorded for each female:
- Date of mating
- Date and time of observed start of parturition
- Date and time of observed completion of parturition
- Duration of gestation.



Oestrous cyclicity (parental animals):
A vaginal smear was prepared for each female and the stage of the oestrous cycle or the presence of sperm was recorded
Sperm parameters (parental animals):
The presence of sperm within the vaginal smear andfor vaginal plug in situ was taken as positive evidence of mating and the males were subsequently returned to their original holding cages.
Organ weight and Histopathology see below
Litter observations:
On completion of parturition, the number of live and dead offspring was recorded.
For each litter the following was recorded:
- Number of pups born
- Number and sex of pups alive recorded daily and reported on Day 1 and 4 postpartum
- Clinical condition of pups from birth to Day 4postpartum
- Individual pup and litter weights on Day 1 and 4 postpartum

Physical Development
All live offspring were observed for the detachment of pinna and assessed for reflexological response to stimuli by assessing surface righting reflex on Day 1 postpartum.
Postmortem examinations (parental animals):
Adult males were killed by intravenous overdose of sodium pentobarbitone followed by exsanguination on Day 43. Adult females were killed by intravenous overdose of sodium pentobarbitone followed by exsanguination on Day 5 post partum. Surviving offspring were terminated via intracardiac overdose of sodium pentobarbitone.
In addition, the corpora lutea of all ovaries from pregnant females were counted at necropsy. The uterine implantation sites were counted. The procedure was enhanced by staining the uteri with a
1 % ammonium polysulphide solution.
All adult animals and offspring, including those dying during the study, were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.

Organ Weights
The following organs, removed from adult animals that were killed at the end of the study, were dissected free from fat and weighed before fixation:
Adrenals
Brain
Epididymides
Heart
Kidneys
Liver
Ovaries
Spleen
Testes
Thymus

Histopathology
Samples of the following tissues were preserved from five males and five females from each dose group, in buffered 10% formalin. The tissues shown in bold were also removed from the remaining animals:
Adrenals
Aorta (thoracic)
Bone & bone marrow (femur including stifle joint)
Bone & bone marrow (sternum)
Brain (including cerebrum, cerebellum and pons)
Caecum
Coagulating gland *
Colon
Duodenum
Epididymides *
Eyes
Gross lesions
Heart
Ileum
Jejunum
Kidneys
Liver
Lungs (with bronchi)
Lymph nodes (cervical and mesenteric)
Mammary gland
Muscle (skeletal)
Ovaries *
Pancreas
Pituitary *
Prostate *
Oesophagus
Rectum
Salivary glands (submaxillary)
Sciatic nerve
Seminal vesicles *
Skin (hind limb)
Spinal cord (cervical)
Spleen
Stomach
Thyroidlparathyroid
Trachea
Testes *
Thymus
Urinary bladder
UterusICewix
Vagina

All tissues were despatched to Propath (Principal Investigator: T Hilling). The tissues from five selected control and 150 mg/kg/day dose group animals were prepared as paraffin blocks, sectioned at nominal thickness of 5 pm and stained with haematoxylin and eosin for subsequent microscopic examination. The tissues markes with * from the remaining control and 150 mg/kg/day were also processed.
Since there were indications of treatment-related changes in the brain, liver and kidneys, examination was subsequently extended to include similarly prepared sections of these tissues from five selected males and females from the 50 and 15 mg/kg/day dose groups.
Microscopic examination was conducted by the Study Pathologist. All findings were entered into the ROELEE Pathology computerisation system for tabulation and report production.









Postmortem examinations (offspring):
Surviving offspring were terminated via intracardiac overdose of sodium pentobarbitone.
Offspring were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.
Statistics:
Haematological, blood chemical, organ weight (absolute and relative to terminal bodyweight), weekly bodyweight gain, litter weights, offspring bodyweights and quantitative functional performance data were assessed for dose response relationships by linear regression analysis, followed by one way analysis of variance (ANOVA) incorporating Levene's test for homogeneity of variance. Where variances were shown to be homogenous, painvise comparisons were conducted using Dunnett's test. Where Levene's test showed unequal variances the data were analysed using non-parametric methods: Kruskal-Wallis ANOVA and Mann-Whitney 'U' test.
The non-parametric methods were also used to analyse implantation loss, offspring sex ratio and landmark developmental markers.
The haematology variable basophils was not analysed since consistently greater than 30% of the data were recorded as the same value.
Probability values (p) are presented as follows:
p < 0.001 ***
p < 0.01 **
p < 0.05 *
p 0.05 (not significant)
Reproductive indices:
Mating Performance and Fertility
Pre-coital Interval: Calculated as the time elapsing between initial pairing and the observation of positive evidence of mating.
Fertility Indices: Mating Index (%), Pregnancy Index (%)

Gestation and Parturition Data
Gestation Length: Calculated as the number of days of gestation including the day for observation of mating and the start of parturition.
Parturition Index (%)

Litter Responses
Implantation Losses (%): % pre- implantation loss and % post - implantation loss
Offspring viability indices:
Live Birth Index (%)
Viability Index (%)
Sex Ratio (% males)
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Two males treated with 150 or 50 mg/kg/day displayed an isolated incident of noisy respiration on Day 38 and Day 42 respectively, additionally incidents of increased salivation were detected immediately after dosing in animals of either sex from the 150 mg/kg/day treatment group (from Day 33 in males and from Day 26 in females). Observations of this nature are commonly reported following oral administration of an unpalatable or slightly irritant test material formulation and, in the absence of correlating histopathological evidence and, in isolation, are considered not to be toxicologically significant. Incidents of generalised redbrown staining of the fur and mouth/snout was observed in control animals of either sex and animals of either sex from all treatment groups from Day 15 onwards. As the staining is not confined to test group animals it was considered incidental and therefore of no toxicological significance.

Generalised fur loss was detected in a single female treated with 150 mgkglday from Day 3 onwards. One other female in this treatment group developed fix loss on Day 21 but then
recovered by Day 23. This observation is sometimes observed when females are approaching their littering period so in isolation is considered unrelated to toxicity.

One female treated with 50 mgikglday developed a damaged tail tip on Day 9 of the study, this is a physical injury and is unrelated to treatment.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Description (incidence):
There were no unscheduled deaths during the study.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
There were no adverse effects on body weights for males throughout the study.
Body weight gains for treated females throughout the maturation, gestation and lactation phases of the study were considered to have been unaffected by treatment. Lower body weight gain was evident at both 50 and 150 mgkglday, compared with the controls, during the last week of gestation, but this was considered to reflect the lower litter size at these dosages rather than any effect on the parental females.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
For females receiving 150 mg/kg/day, food consumption and food utilisation were unaffected by treatment during the maturation phase of the study. Subsequent food intake during the first two weeks of gestation was lower than controls, with differences attaining statistical significance (p<0.05), although food conversion efficiency for this period was unaffected. Differences in food intake for the last week of gestation and during early lactation were considered to reflect the lower litter size at this dosage.
There was no adverse effect of treatment on food consumption of females during the maturation, gestation or lactation phases of the study at 15 or 50 mg/kg/day. Food conversion efficiency during maturation and the first two weeks of gestation at these dosages were also unaffected by treatment.
Food efficiency:
no effects observed
Description (incidence and severity):
see "Food consumption"
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
Daily visual inspection of water bottles revealed no intergroup differences.
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Description (incidence and severity):
No treatment-related changes were detected in the haematological parameters examined.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
No treatment-related changes were detected in the blood chemical parameters examined.
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
Behavioural Assessment: There were no treatment-related behavioural changes. All inter and intra group differences in urination, defecation and transfer arousal scores were considered to be a result of normal variation for rats of the strain and age used and were of no toxicological importance.
Functional Performance Tests: There were no treatment-related changes in the functional performance parameters measured. Statistical analysis of the data revealed no significant intergroup differences.
Sensory Reactivity Assessments: There were no treatment-related changes in sensory reactivity. All inter and intra group differences in sensory reactivity scores were considered to be a result of normal variation for rats of the strain and age used and were of no toxicological importance.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
There were no treatment-related changes observed in the reproductive or accessory reproductive organs for rats of either sex.
Liver: Periportal lipid vacuolation of hepatocytes was observed in animals of either sex treated with 150 mg/kg/day, but not at any other dose level. The nature of the vacuolation was not
established but it was considered not to result from lipid accumulation.
Spleen: Vacuolated cells were observed scattered throughout the red pulp of three male and of one female rat treated with 150 mg/kg/day. Animals from the remaining treatment levels were not similarly affected.
Brain: Vacuolation of the ventricular choroid plexus was seen for animals of either sex treated with 150 mg/kg/day, and for three males treated with 50 mg/kg/day
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
No adverse effects were detected on mating performance, fertility or gestation length.

One control female showed positive evidence of mating after three days of co-habitation with its male partner but subsequently was not observed to litter. The female was found to have two implantation sites at necropsy and therefore had achieved pregnancy. Examination of the bodyweight profile during gestation and around the time of lactation indicated that this total litter loss had most likely occurred in utero and this female has, therefore, been classified as a total litter resorption. It is however, possible that the dam gave birth and consumed its small litter before parturition had been detected.
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
No adverse effects were detected on mating performance, fertility or gestation length.
Reproductive performance:
effects observed, treatment-related
Description (incidence and severity):
At 150 mglkglday corpora lutea count was slightly lower than the control, although differences failed to attain statistical significance. Subsequent pre-implantation loss was higher than control, resulting in an even lower number of implantations; however, differences again were not statistically significant. Post-implantation and post natal losses were then similar to the control, with litter size remaining lower than the control to termination on Day 4 of lactation/age.
At 50 mg/kg/day, corpora lutea count was essentially similar to the control; however, higher preimplantation losses were again evident leading to a lower number of implantations, although no statistical significance was achieved. Subsequent post-implantation and post natal losses were similar to the control, with litter size remaining lower than the control to termination.
At 15 mg/kg/day, the numbers of corpora lutea and implantations, litter size on Day 1 and Day 4 of age and pre and post-natal losses at 15 mg/kg/day, were similar to control and were unaffected by treatment.
Sex ratio at birth, on Day 1 and on Day 4 were similar in all groups and did not indicate any obvious selective effect on survival for either sex.

One control female showed positive evidence of mating after three days of co-habitation with its male partner but subsequently was not observed to litter. The female was found to have two implantation sites
at necropsy and therefore had achieved pregnancy. Examination of the bodyweight profile during gestation and around the time of lactation indicated that this total litter
loss had most likely occurred in utero and this female has, therefore, been classified as a total litter resorption. It is however, possible that the dam gave birth and consumed its small litter
before parturition had been detected.
There were no unscheduled deaths during the study and no clinical signs of toxicity were observed. A functional observational battery did not detect any treatment-related behavioral effects, changes in sensory reactivity, or changes in the functional performance parameters measured. Bodyweight gains were considered to be unaffected by treatment. No toxicologically significant effects on dietary intake or food efficiency were detected and no overt intergroup differences in water consumption were detected. No significant hematological or serum chemistry effects were detected prior to mating. Organ weight analysis and macroscopic necropsy did not indicate any adverse effects of treatment, however microscopic examination revealed histopathological changes, involving minimal to moderate vacuolation of cells, for the liver, spleen and brain (ventricular choroids plexus) in 5/10 males and 6/10 females at 150 mg/kg/day. No similar histopathological changes in the liver and spleen were apparent at 50 mg/kg/day, however, 3/5 males did show minimal vacuolation of the ventricular choroid plexus of the brain. While the aetiology of this finding is unknown, such vacuolation is rare and within the context of this study is considered to be toxicologically significant, precluding 50 mg/kg/day as a NOAEL for systemic toxicity.

Offspring Litter Size and Viability:
The most important reproductive findings were the lower corpora lutea count at 150 mg/kg/day and the higher pre-implantation loss at 50 and 150 mgikglday. The number of corpora lutea at
150 mg/kg/day was not significantly lower than the concurrent control, only marginally outside the recent control range and could possibly represent normal biological variation. The higher preimplantation
losses observed at 50 and 150 mg/kg/day, however, are probably more biologically significant. While values are similar to the control, when the control animal with total litter resorption is included, excluding this atypical
animal demonstrates a higher incidence of preimplantation loss at these dosages. Although differences in pre-implantation loss do not attain statistical significance, at 150 mg/kg/day these higher losses occur despite the already reduced
number of corpora lutea. Within the confines of this study it may be argued that a reIationship between these findings and treatment is equivocal and has not been proven; however an association with treatment cannot
be discounted and it is considered that this precludes assigning 50 or 150 mg/kg/day as a reproductive No Observable Adverse Effect level (NOAEL).
Key result
Dose descriptor:
NOEL
Effect level:
15 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Histopathology
Key result
Critical effects observed:
not specified
Clinical signs:
no effects observed
Description (incidence and severity):
No toxicologically significant clinical findings were observed. The clinical observations recorded for offspring throughout the dose groups were consistent with normally expected incidence findings in the offspring of the age examined and therefore considered of no toxicological importance.
Dermal irritation (if dermal study):
not examined
Mortality / viability:
no mortality observed
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Offspring body weight on Day 1 of age and subsequent bodyweight gain to Day 4 of age was unaffected by treatment at 15, 50 or 150 mg/kg/day. The lower litter weight observed for litters at 50 and 150 mg/kg/day were a consequence of the smaller litter size at these dosages.
Assessment of pinna unfolding and surface righting reflex did not indicate any adverse effect on offspring development at dosages up to 150 mg/kg/day.
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Description (incidence and severity):
No treatment related macroscopic abnormalities were detected for offspring at terminal kill. The macroscopic findings observed for interim and terminal kill offspring throughout the treatment groups, were consistent with normally expected low incidence findings in pups of the age examined and were of no toxicological importance.
Histopathological findings:
not examined
Other effects:
not examined
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
At 50 and 150 mg/kg/day treatment appeared to be associated with a higher level of preimplantation loss although there was no subsequent effect on post-natal survival, growth or development.

see table overall remarks "TABULAR SUMMARY REPORT OF EFFECTS ON REPRODUCTION/DEVELOPMENT"
Key result
Dose descriptor:
NOEL
Generation:
F1
Effect level:
15 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: higher level of preimplantation loss
Remarks on result:
other: At 50 and 150 mg/kg/day treatment appeared to be associated with a higher level of preimplantation loss although there was no subsequent effect on post-natal survival, growth or development.
Key result
Critical effects observed:
not specified
Reproductive effects observed:
not specified

Mortality - There were no unscheduled deaths during this study.

Clinical Observations – No clinical observable signs of toxicity were observed.

Behavioral Assessment – No treatment-related effects were detected.

Function Performance Tests – There were no treatment-related changes in the functional performance parameters measured.

Sensory Reactivity Assessments – There were no treatment-related changes in sensory reactivity.

Bodyweight – Bodyweight gains for males throughout the study and for females during the maturation, gestation or lactation phases of the study were considered to be unaffected by treatment.

Food Consumption – No toxicologically significant effects on dietary intake or food efficiency were detected for males throughout the treatment period or for females during maturation, gestation or lactation phases of the study.

Water Consumption – No overt intergroup differences were detected.

Hematology – No significant hematological effects were detected prior to mating.

Blood Chemistry – No significant biochemical effects were detected prior to mating.

Mating – No adverse effect on mating performance. Fertility or gestation length was detected.

Offspring Litter Size and Viability – At 150 mg/kg/day there was a lower corpora lutea count and a higher pre-implantation loss compared with the controls. At 50 mg/kg/day, pre-implantation loss was again higher than controls. Resultant litter size at this dosage was lower than the control but potential offspring survival was unaffected by treatment. Litter data at 15 mg/kg/day were unaffected by treatment.

Offspring Growth and Development – There were no adverse affects of maternal treatment on offspring growth or development.

Offspring Observations – there were no adverse effects of maternal treatment in pinna unfolding or surface righting reflex.

Necropsy:

Offspring – No treatment related macroscopic abnormalities were detected for the interim death or terminal death offspring of all treatment groups.

Adults – No treatment-related effects were detected at terminal kill.

Organ Weights – No adverse effects of treatment were detected.

Histopathology:

Liver – Periportal lipid vacuolation of hepatocytes was observed in animals of either sex treated with 150 mg/kg/day, but not at any other dose level.

Spleen – Vacuolated cells were observed scattered throughout the red pulp of three male and of one female rat treated with 150 mg/kg/day. Animals from the remaining treatment levels were not similarly affected.

Brain – Vacuolation of the ventricular choroid plexus was seen for animals of either sex treated with 150 mg/kg/day, and for three males treated with 50 mg/kg/day.

There were no treatment-related changes observed in the reproductive or accessory reproductive organs for rats of either sex.

Conclusions:
The oral administration of test item to rats by gavage at a maximum dose level of 150 mg/kg/day resulted in toxicologically significant histopathological findings in the liver, spleen and brain. Similar brain histopathology was observed for a few animals at 50 mg/kg/day.
The No Observed Effect Level (NOEL) for systemic toxicity was therefore considered to be 15 mgkglday.

At 50 and 150 mg/kg/day, treatment appeared to be associated with a higher level of preimplantation loss (not statistical significant) although there was no subsequent effect on post-natal survival, growth or development.
The NOEL for reproductive toxicity was therefore, also considered to be 15 mg/kg/day.
Executive summary:

Introduction. The study was designed to investigate the systemic toxicity and potential adverse effects on reproduction (including offspring development) of the test material and complies with the recommendations of the OECD Guidelines for Testing of Chemicals No. 422 "Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test" (adopted 22 March 1996).

Methods. The test material was administered by gavage to three groups each of ten male and ten female Sprague-Dawley Crl.:CD (SD) IGS BR strain rats, for up to fifty-four consecutive days, at dose levels of 15, 50 and 150 mg/kg/day. A control group of ten males and ten females was dosed with vehicle alone (polyethylene glycol 400).

Clinical signs, behavioural assessments, bodyweight development, food and water consumption were monitored during the study. Haematology and blood chemistry were evaluated prior to mating on five selected males and females from each dose group.

Pairing of animals within each dose group was undertaken on a one male to one female basis on Day 15 of the study, to produce litters.

During the lactation phase, daily clinical observations were performed on all surviving offspring, together with litter size and offspring weights and assessment of developmental landmarks.

Functional observations were performed on five selected parental males from each dose group after the completion of the mating phase, and for five selected parental females from each dose group on Day 4 postpartum.

Males were terminated on Day 43, followed by the termination of all surviving females and offspring on Day 5 postpartum. All animals were subjected to a gross necropsy examination and histopathological evaluation of selected tissues was performed.

 

Results.

Mortality. There were no unscheduled deaths during the study.

Clinical Observations. No clinical observable signs of toxicity were observed.

Behavioural Assessment. No treatment-related effects were detected.

Functional Performance Tests. There were no treatment-related changes in the functional performance parameters measured.

Sensory Reactivity Assessments. There were no treatment-related changes in sensory reactivity.

Bodyweight, Bodyweight gains for males throughout the study and for females during the maturation, gestation or lactation were considered to be unaffected by treatment.

Food Consumption. No toxicologically significant effects on dietary intake or food efficiency were detected for males throughout the treatment period or for females during maturation, gestation or lactation phases of the study.

Water Consumption. No overt intergroup differences were detected.

Haematology. No significant haematological effects were detected prior to mating.

Blood Chemistry. No significant biochemical effects were detected prior to mating.

Reproductive Screening:

Mating. No adverse effect on mating performance, fertility or gestation length was detected.

Offspring Litter Size and Viability. At 150 mg/kg/day there was a lower corpora lutea count and a higher pre-implantation loss compared with the controls. At 50 mg/kg/day, pre-implantation loss was again higher than controls. Resultant litter size at this dosage were lower than the control but potential offspring survival were unaffected by treatment. Litter data at 15 mg/kg/day were unaffected by treatment.

Offspring Growth and Development, There were no adverse affects of maternal treatment on offspring growth or development.

Offspring Observations. There were no adverse effects of maternal treatment in pinna unfolding or surface righting reflex.

 

Pathology:

Necropsy.

Offspring: No treatment related macroscopic abnormalities were detected for the interim death or terminal death offspring of all treatment groups.

Adults: No treatment-related effects were detected at terminal kill.

Organ Weights. No adverse effects of treatment were detected.

Histopathology.

Liver: Periportal lipid vacuolation of hepatocytes was observed in animals of either sex treated with 150 mg/kg/day, but not at any other dose level.

Spleen: Vacuolated cells were observed scattered throughout the red pulp of three male and of one female rat treated with 150 mg/kg/day. Animals from the remaining treatment levels were not similarly affected.

Brain: Vacuolation of the ventricular choroid plexus was seen for animals of either sex treated with 150 mg/kg/day, and for three males treated with 50 mg/kg/day.

There were no treatment-related changes observed in the reproductive or accessory reproductive organs for rats of either sex.

Conclusion:

As part of an OECD 422 study, rats were exposed via oral gavage to test item at dose levels of 0, 15, 50, and 150 mg/kg/day for two weeks prior to mating, during gestation and for 4 days post partum. 

There were no unscheduled deaths during the study and no clinical signs of toxicity were observed. No adverse effect on mating performance, fertility, or gestation length was detected. While not statistically significant, at 150 mg/kg/day there was a lower corpora lutea count and a higher pre-implantation loss compared with the controls and at 50 mg/kg/day, pre-implantation loss was again higher than controls. Resultant litter size at this dosage was lower than the control but offspring survival was unaffected by treatment. Litter data at 15 mg/kg/day were unaffected by treatment. There were no adverse affects of maternal treatment on offspring growth or development including pinna unfolding and surface righting reflex. No treatment-related macroscopic abnormalities were detected in the offspring of all treatment groups. There were no treatment-related changes observed in the reproductive or accessory reproductive organs for parental rats of either sex.

At 50 and 150 mg/kg/day treatment appeared to be associated with a higher level of preimplantation loss although there was no subsequent effect on post-natal survival, growth or development. The NOEL for reproductive (fertility) toxicity was therefore, considered to be 15 mg/kg/day.

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Study duration:
subacute
Species:
rat
Quality of whole database:
Klimisch 1
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

OECD 422 (SafePharm 2006):

The most important reproductive findings were the lower corpora lutea count at 150 mg/kg/day and the higher pre-implantation loss at 50 and 150 mg/kg/day. The number of corpora lutea at 150 mg/kg/day was not significantly lower than the concurrent control, only marginally outside the recent control range and could possibly represent normal biological variation. The higher preimplantation losses observed at 50 and 150 mg/kg/day, however, are probably more biologically significant. While values are similar to the control, when the control animal with total litter resorption is included, excluding this atypical animal demonstrates a higher incidence of preimplantation loss at these dosages. Although differences in pre-implantation loss do not attain statistical significance, at 150 mg/kg/day these higher losses occur despite the already reduced number of corpora lutea. Within the confines of this study it may be argued that a relationship between these findings and treatment is equivocal and has not been proven; however an association with treatment cannot be discounted and it is considered that this precludes assigning 50 or 150 mg/kg/day as a reproductive No Observable Adverse Effect level (NOAEL).

Subsequent post-natal survival, bodyweight, growth and development of the offspring to termination (Day 5 of age) was unaffected by treatment.

Organ weight analysis and macroscopic necropsy of the adults did not indicate any adverse effects of treatment, however microscopic examination revealed histopathological changes, involving vacuolation of cells, for the liver, spleen and brain (ventricular choroid plexus) at 150 mg/kg/day.

No similar histopathological changes in the liver and spleen were apparent at 50 mg/kg/day, however, three males did show vacuolation of the ventricular choroid plexus of the brain. While the aetiology of this finding is unknown, such vacuolation is rare and within the context of this study is considered to be toxicologically significant, precluding 50 mg/kg/day as a NOAEL for systemic toxicity.

Effects on developmental toxicity

Description of key information

The test substance was tested in rats in an OECD 422 screening test for reproductive (developmental) effects (SafePharm, 2006). No effect on post-natal survival, growth or development was observed. The NOEL for repeated dose oral systemic toxicity was 15 mg/kg/day.  The NOEL for repeated dose oral developmental toxicity was 150 mg/kg/day.

In a prenatal developmental toxicity study according to OECD 414, the test item was administered orally to female rats at dose levels of 15, 50 or 150 mg/kg b.w./day from the 6th to 20th day of pregnancy (LPT, 2018).

Under the present test conditions, the no-observed-adverse-effect level (NOAEL) was 50 mg test item/kg b.w./day for the dams.

Dam no. 79 of the high dose group (150 mg test item/kg b.w./day) prematurely deceased during GD 14.

A high incidence of salivation, a slightly reduced body weight gain and a reduced food consumption were further noted at the high dose level.

No test item-related changes were noted in the macroscopic examination during laparotomy.

The no-observed-adverse-effect level (NOAEL) for the fetal organism was above 150 mg test item/kg b.w./day.

The reproductive parameters (number of implantation sites, number of resorptions and number of fetuses) were not influenced by the test item.

No dead fetuses, no malformations and no test item-related variations or retardations were noted.

Under the conditions of the study, test item did not show any teratogenic potential.

In a further prenatal developmental toxicity study according to OECD 414 guideline in a second species (rabbits), the test item 2,4,6tris[(dimethylamino)methyl]phenol was administered orally to 99 inseminated female rabbits at dose levels of 0, 15, 50 or 100 mg/kg b.w./day from the 6th to 28th day of pregnancy.

One group served as control group, in which the animals received the vehicle (tap water) without test substance. The body weight of the 5-month-old animals at day 6 of gestation was 3.48 kg to 4.60 kg. 20 dams per dose group were examined and evaluated at the end of the study.

Under the present test conditions, the no-observed-adverse-effect level (NOAEL) was 15 mg test item/kg b.w./day for the dams.

Three test item-related premature deaths were noted for the high dose group (100 mg test item/kg b.w./day).

A decrease in food consumption was noted at 50 and 100 mg test item/kg b.w./day.

Test item-related pathological findings in form of a pale or pale and brittle liver were noted for the intermediate (50 mg test item/kg b.w./day) and high dose group (100 mg test item/kg b.w./day) and in form of a thickened stomach mucosa for the high dose group.

No changes in behaviour, external appearance or faeces were noted for the treatment groups.

No differences between the dose groups and the control groups were noted for the body weight and the body weight gain.

The no-observed-adverse effect level (NOAEL) for the fetal organism was 15 mg test item/kg b.w./day.

At the maternotoxic dose levels of 50 and 100 mg test item/kg b.w./day the following observations were noted:

At 50 or 100 mg test item/kg b.w./day, dose-related reductions were noted for the placental and fetal weights. Additionally, dose-related increased incidences were observed for a skeletal variation in form of an enlarged fontanelle, for a skeletal retardation in form of unossified sternebrae and for the total number of skeletal retardations.

At 100 mg test item/kg b.w./day, 2 animals were noted with a total loss of implantations. Furthermore, post mortem examinations revealed increased incidences of a soft tissue variation in form of a dilatation of the 4th cerebral ventricle and of an unclassified observation in form of a pericardial cyst.

However, the decreased fetal and placental weights and the increased number of retardations and the increased incidence of unossified sternebrae were considered to be secondary to the reduced food consumption of the intermediate and high dose dams as it is known that a reduced maternal food intake can result in a retarded fetal development (Cappon GD, 2005).

No test item-related malformations were noted in any of the dose groups

Link to relevant study records

Referenceopen allclose all

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
2017-06-19 to 2017-07-13
Reliability:
1 (reliable without restriction)
Justification for type of information:
This developmental toxicity study is used as dose range finding study for the OECD 414 main study with rats (LPT, 2017).
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
adopted January 22, 2001
Qualifier:
according to
Guideline:
EU Method B.31 (Prenatal Developmental Toxicity Study)
Version / remarks:
May 30, 2008
GLP compliance:
no
Remarks:
The study was performed based on 'Good Laboratory Practice' Regulations of the EC enacted in Germany in the 'Chemikaliengesetz' [Chemicals Act], current edition; 'OECD Principles of Good Laboratory Practice' Document Nos. 1, 8 and 13 ENV/MC/CHEM (98) 17,
Limit test:
no
Species:
rat
Strain:
other: CD /Crl:CD(SD)
Details on test animals and environmental conditions:
TEST ORGANISMS: 
- Species: Rat
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH
- Strain: CD / Crl: CD(SD)
- Age: 61 days
- body weight: 206.3 g - 233.1 g
- Diet: ad libitum, Commercial diet ssniff® R/Z V1324 (ssniff Spezialdiäten GmbH, 59494 Soest, Germany)
- Water: ad libitum
- Acclimatisation period: 5 days
-Housing: Except during the mating period, the dams were kept singly in MAKROLON cages
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 °C +/- 3° C
- Humidity (%): 55% +/- 15 %
- Illumination: 12 hours artifical fluorescent light and 12 hours dark
- Ventilation rate: between fifteen to twenty air changes per hour.
Route of administration:
oral: gavage
Vehicle:
polyethylene glycol
Remarks:
PEG400
Details on exposure:
ADMINISTRATION: 
- Frequency: once daily, day 6 to 20 of gestation
- Dose volume: 4 ml/kg b.w.
- Dose: 0, 15, 50, 150 mg/kg/bw
- Animals: Treated animals: Groups 1 - 4: 3 females per group
Evaluated dams: Groups 1 - 4: 2 females per group
Evaluated litters: Groups 1 - 4: 2 litters per group

- DOSAGE PREPARATION:
The test item formulations were freshly prepared every day.
The test item was diluted in the vehicle to the appropriate concentrations using a mag-netic stirrer and was administered orally at a constant volume once daily from the 6th to the 20th day of gestation.
The amount of the test item was daily adjusted to the current body weight of the animal. The control animals received the vehicle at the same administration volume daily in the same way.
The male rats for mating remained untreated.

Analytical verification of doses or concentrations:
no
Remarks:
Tests by appropriate analytical methods will be carried out for the main study
Details on mating procedure:
Sexually mature ('proved') male rats of the same breed served as partners. The female breeding partners were randomly chosen.

Mating was monogamous: 1 male and 1 female animal were placed together in one cage during the dark period. Each morning a vaginal smear was
taken to check for the presence of sperm. If findings were negative, mating was repeated with the same partner. The day on which sperm was found
was considered as the day of conception (day 0 of pregnancy). This procedure was repeated until enough pregnant dams were available for all
groups. Rats which did not become pregnant were excluded from the analysis of the results and replaced by other animals. A post-mortem negative
staining according to SALEWSKI was carried out in the replaced animals in order to confirm the non-pregnancy status.
Duration of treatment / exposure:
From gestation day 6 until gestation day 20
Frequency of treatment:
Once daily
Duration of test:
On gestation day 21, the rats were laparotomised under ether narcosis.
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
vehicle control
Dose / conc.:
15 mg/kg bw/day (nominal)
Remarks:
low dosw
Dose / conc.:
50 mg/kg bw/day (nominal)
Remarks:
intermediate dose
Dose / conc.:
150 mg/kg bw/day (nominal)
Remarks:
high dose
No. of animals per sex per dose:
3 female rats/dose , orally dosed with 0, 15, 50 or 150 mg test item/kg b.w.
Evaluated litters: 2 litters per group
Control animals:
yes, concurrent vehicle
Details on study design:
Dose selection rationale:
The dose levels have been selected by the sponsor based on available toxicological data of a OECD422 study already conducted..

Selection of species:
The rat is a commonly used rodent species for such prenatal developmental studies.
Maternal examinations:
Dated and signed records of all activities relating to the day to day running and maintenance of the study within the animal units, as well as to the
group observations and examinations outlined in the Study Plan, were recorded in the appropriate documentation. In addition, observations relating
to the individual animals made throughout the study were recorded.

The following observations were made during the course of the study:
Clinical signs
Individual animals were observed daily for behavioural changes, reaction to treatment, or illness.
Immediately after administration, any signs of illness or reaction to treatment were recorded. In case of changes, the animals were observed until the symptoms disappeared. In addition, animals were checked regularly throughout the working day from 7.00 a.m. to 3.45 p.m.
On Saturdays and Sundays, the animals were checked regularly starting from 7.00 a.m. to 11.00 a.m. with a final check performed at approximately
3.30 p.m.
Dated and signed records of appearance, change and disappearance of clinical signs were maintained on clinical history sheets for individual animals.


Viability
Further checks were made early in the morning and again in the afternoon of each working day to look for dead or moribund animals. This would have allowed post mortem examinations to be carried out during the working period of that day. On Saturdays and Sundays, a similar procedure was followed except that the final check was carried out at approximately midday.
Animals showing signs of abortion or premature delivery would have been sacrificed on the same day. Fetuses obtained this way were examined for abnormal development, whenever possible. No abortion occurred in the study.


Body weight
The weight of each rat was recorded on day 0 of gestation (the day of detection of a positive mating sign), followed by daily weighing - always at the
same time of the day.
The body weight gain was calculated in intervals (i.e. day 0-3, 3 6, 6-9, 9-12, 12 15, 15-18 and 18-21), for the whole study (gestation day 0 - 21) and
for the period after the start of dosing (gestation day 6 to gestation day 21). Furthermore the carcass weight and the net weight gain from day 6 are
given.
These values are stated in the report.
These measurements were also used for calculating the daily amount of test item to be administered.


Food and drinking water consumption
The quantity of food consumed by each rat was recorded daily. Food intake per rat (g/rat/day) was calculated using the total amount of food given to and left by each rat in each group on completion of a treatment day.
The relative food consumption (g/kg b.w./day) was calculated using the following formula:

Daily food consumption [g/kg b.w./day]= Total food intake in g / Body weight in kg

Daily monitoring by visual appraisal of the drinking water bottles was maintained throughout the study.


EXAMINATIONS (NECROPSY), Examination of the dams
Dissection technique and evaluation of the animals:
On gestation day 21, the rats were laparotomised under ether narcosis. The ovaries and the uteri of the dams were removed; the gravid uteri (in toto) were weighed. In order to check for possible drug effects, a dissection with macroscopic examination of the internal organs and placentae of the dams was carried out on the day of sacrifice or on the day on which the animals were found dead. In case of macroscopical findings, the affected maternal tissues were preserved in 7% buffered formalin for possible future histopathological examinations.


Ovaries and uterine content:
Corpora lutea
- number per dam
- absolute number per group
- mean per group

Implantations
- number per dam
- distributions in the uterine horns
- absolute number per group
- mean per group

Resorptions
- number per dam
- distributions in the uterine horns
- absolute number per group
- mean per group
- early resorptions < 2 mm
- Late resorptions > 2 mm

Weight of placentae
- individual data per fetus
- mean per litter
- mean per group
- mean per sex and group

Weight of fetuses
- individual data per fetus (alive and dead)
- mean per litter
- mean per group
- mean per sex and group

Fetuses
- number per dam (alive)
- number per dam (dead)
- number of fetuses (alive and dead) per sex and dam
- distribution in the uterine horns
- absolute number of fetuses alive per group
- mean number of fetuses alive per group
- mean % of fetuses alive per group
- male/female ratio (alive and dead)


Runts
- number per dam
- mean per group


Malformed fetuses
- type of malformation
- individual data per fetus
- number and incidence (%) per group and litter

Total malformation rate [%] = malformed fetuses per group / fetuses per group x 100

Fetuses with variations
- type of variation
- individual data per fetus
- number and incidence (%) per group and litter

Total variation rate [%] = fetuses per group with variations / fetuses per group x 100

Indices of pre-implantation loss and post-implantation loss:

Calculation of group indices
Pre implantation
loss [%] = Corpora lutea (per group) - Implantations (per group) / Corpora lutea (per group) x 100

Post implantation
loss [%] = Implantations (per group) - living fetuses (per group) / Implantations (per group) x 100


Calculation of mean indices per litter
Pre implantation
loss [%] = sum of pre-implantation losses per litter in a group [%] / number of litters in a group


Post implantation
loss [%] = sum of post-implantation losses per litter in a group [%] / number of litters in a group


Fetal examinations:
The fetuses were removed and the following examinations performed:
(a) Macroscopic inspection (gross evaluation) of the placentae for example for focal indurations or abnormal appearance (e.g. size, colour, shape).
(b) The number of fetuses (alive and dead) and placentae (location in the uterus and the assignment of the fetuses) was determined.
(c) Sex and viability of fetuses were determined. Animals are said to be viable when they are found alive (spontaneous breathing, spontaneous movement).
(d) Number and size of resorptions were determined.
(e) Corpora lutea in the ovaries, implantations and location of fetuses in the uterus were determined.
(f) Weight of gravid uterus was determined.
(g) Weights of fetuses and weights of the placentae were determined (fetuses were considered as runts if their weight was less than 70% of the mean litter weight).
(h) All fetuses (dead and alive) were inspected externally for damages, especially for malformations .
(i) The fetuses were sacrificed by an ether atmosphere.
Statistics:
No statistical analysis was conducted as only 2 animals per group were evaluated. The data of the spare animal of each group was not included.
Indices:
DRF, Fertility Index, Viability Index, Resorption Index, Pre-Implantation Loss Index, Post-Implantation Loss Index, Runts Index, Variation Index,
Number of litters having abnormalities, Number of abnormalities per litter
Clinical signs:
no effects observed
Description (incidence and severity):
No test item-related changes in behaviour, the external appearance or the faeces were noted in any of the treatment groups (15, 50 or 150 mg test item/kg b.w./day).
Mortality:
no mortality observed
Description (incidence):
No premature deaths were noted in the control group and in the treatment groups (15, 50 or 150 mg test item/kg b.w./day).
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
No test item-related differences in body weight were noted between the dams of the control group and the low and intermediate dose groups (15 or 50 mg test item/kg b.w./day).
However, slight reductions in body weight were noted at the intermediate dose level from GD 19 onwards (at maximum 5.0% below the value of the control group on GD 21). Considering the 3-day intervals of body weight gain, the reduction in body weight was reflected by a decreased body weight gain between GD 18 and 21 (24.6% below the value of the control group). This is regarded to be still within the normal range of variation as only 2 animals were examined.
At 150 mg test item/kg b.w./day, the start of treatment caused a delay in body weight gain from GD 6 until GD 13, leading to a reduction of body weight with a maximum on GD 13 (12.6% below the value of the control group). Considering the 3-day intervals of body weight gain, the reduction in body weight was reflected by a decreased body weight gain between GD 6 and 9 (48.1% below the value of the control group) and between GD 9 and 12 (55.3% below the value of the control group). Thereafter, body weight gain was again in the range of the control group, but the body weight remained below the value of the control group (still 8.7% below the value of the control group on GD 21). This reduction in body weight was considered to be test item-related.

Body weight gain during the whole study period
As described above, the reductions in body weight gain led to a reduced body weight gain for the whole study period for the dams of the intermediate and the high dose group (see table below "any other information"). The reduction in body weight gain noted for the high dose level was due to a test item-related reduction in body weight.

Body weight gain from gestation day 6
No differences in the absolute and the net body weight gain were noted between the dams of the control group and the dams of the low dose group (15 mg test item/kg b.w./day) during the period after the start of the treatment.
At the intermediate and the high dose level (50 or 150 mg test item/kg b.w./day), the above described reductions in the gravid uterus weight and the carcass weight led to a reduced absolute body weight gain and a reduced net body weight gain (body weight gain without gravid uterus) for the period after the start of treatment from GD 6 to 21 (see table below "any other information"). Only the reductions that were noted at the high dose level were considered to be test item-related and adverse, as they were mainly due to test item-related reduction in the carcass weight.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
No test item-related differences were noted between the control group and the treatment groups (15, 50 or 150 mg test item/kg b.w./day).
At 150 mg test item/kg b.w./day, the food consumption was lower between GD 8 and 9 until between GD 11 and 12. As the difference normalized during the further course of the study this was considered to be not test item-related.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
No test item-related changes in drinking water consumption were noted between the dams of the control group and the dams of the treatment groups by visual appraisal.
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
Gravid uterus weight
No test item-related differences were noted between the gravid uterus weight of the con-trol dams and the dams of the treatment groups (15, 50 or 150 mg test item/kg b.w./day).
However, slight reductions in the gravid uterus weight were noted at the intermediate dose level (9.1% below the value of the control group) and the high dose level (11.2% below the value of the control group). As these slight reductions in the gravid uterus weight did not have an effect on the fetal weights (see section 'Weight of placentae and fetuses') they were considered to be not adverse.

Carcass weight see "other effects"
Gross pathological findings:
no effects observed
Description (incidence and severity):
No observations were noted for the dams of the control group and the dams of the treat-ment groups (15, 50 or 150 mg test item/kg b.w./day) during the macroscopic inspection of the organs and tissues.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
effects observed, treatment-related
Description (incidence and severity):
Carcass weight
No test item-related differences were noted between the carcass weight of the control dams and the dams of the low and intermediate dose group (15 or 50 mg test item/kg b.w./day).
However, a slightly reduced carcass weight was noted at the intermediate dose level (3.6% below the value of the control group). This slight difference was considered to be spontaneous and not test item-related.
At the high dose level (150 mg test item/kg b.w./day), a reduced carcass weight by 7.8% was noted in comparison to the control group. This reduction was considered to be test item-related.
Number of abortions:
no effects observed
Description (incidence and severity):
No test item-related influence on the reproductive parameters (number of implantation sites, fetuses, resorptions and the indices of pre- and post-implantation) were noted between the dams of the control group and the dams of the treatment groups (15, 50 or 150 mg test item/kg b.w./day).
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
No test item-related influence on the reproductive parameters (number of implantation sites, fetuses, resorptions and the indices of pre- and post-implantation) were noted between the dams of the control group and the dams of the treatment groups (15, 50 or 150 mg test item/kg b.w./day).
Total litter losses by resorption:
no effects observed
Description (incidence and severity):
No test item-related influence on the reproductive parameters (number of implantation sites, fetuses, resorptions and the indices of pre- and post-implantation) were noted between the dams of the control group and the dams of the treatment groups (15, 50 or 150 mg test item/kg b.w./day).
Early or late resorptions:
no effects observed
Description (incidence and severity):
No test item-related influence on the reproductive parameters (number of implantation sites, fetuses, resorptions and the indices of pre- and post-implantation) were noted between the dams of the control group and the dams of the treatment groups (15, 50 or 150 mg test item/kg b.w./day).
Dead fetuses:
no effects observed
Description (incidence and severity):
No test item-related influence on the reproductive parameters (number of implantation sites, fetuses, resorptions and the indices of pre- and post-implantation) were noted between the dams of the control group and the dams of the treatment groups (15, 50 or 150 mg test item/kg b.w./day).
Changes in pregnancy duration:
not examined
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): not examined
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
No test item-related influence on the reproductive parameters (number of implantation sites, fetuses, resorptions and the indices of pre- and post-implantation) were noted between the dams of the control group and the dams of the treatment groups (15, 50 or 150 mg test item/kg b.w./day).
Details on maternal toxic effects:
Maternal toxic effects:no effects
see table "overall remarks"
Dose descriptor:
NOAEL
Effect level:
50 mg/kg bw/day
Basis for effect level:
other: maternal toxicity
Fetal body weight changes:
no effects observed
Description (incidence and severity):
The fetal weights showed no test item-related differences between the control group and the treatment groups (15, 50 or 150 mg test item/kg b.w./day).

Weight of placentae
The placental weights showed no test item-related differences between the control group and the treatment groups (15, 50 or 150 mg test item/kg b.w./day).

In the low and high dose group (15 or 150 mg test item/kg b.w./day), slightly higher values for the placental weights compared to the control group were noted for the male and female fetuses (at maximum 18.2% above the value of the control group for the pla-centae of the female fetuses of the low dose group). As no dose-response relationship was present, these differences were considered to be spontaneous and not test item-related.

Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): no effects observed
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.DescriptionIncidenceAndSeverityFetalPupBodyWeightChanges): see above
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
No dead fetus was noted in the control group and in the test item treated groups (15, 50 or 150 mg test item/kg b.w./day).
Changes in sex ratio:
no effects observed
Description (incidence and severity):
No test item-related differences were noted between the ratio of male and female fetuses of the treatment groups (15, 50 or 150 mg test item/kg b.w./day, range: 0.76 - 1.36) and the control group (0.80). The wide range of the sex distribution was due to the low number of the evaluated litters.
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
No test item-related influence on the reproductive parameters (number of implantation sites, fetuses, resorptions and the indices of pre- and post-implantation) were noted between the dams of the control group and the dams of the treatment groups (15, 50 or 150 mg test item/kg b.w./day).
Changes in postnatal survival:
not examined
External malformations:
no effects observed
Description (incidence and severity):
No macroscopically visible external alterations (malformations or variations) were noted for the fetuses of the control group and the treatment groups (15, 50 or 150 mg test item/kg b.w./day) during the macroscopic inspection at laparotomy.
Skeletal malformations:
not examined
Visceral malformations:
not examined
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
Number of runts
No runt was noted in the control group or in the low and intermediate dose group (15 or 50 mg test item/kg b.w./day).
One runt (11-1) was noted in the high dose group (150 mg test item/kg b.w./day). The occurrence of one runt was considered to be within the normal range of spontaneous events and not test item-related.


Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects
Dose descriptor:
NOAEL
Effect level:
> 150 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: developmental toxicity
Key result
Developmental effects observed:
no

Mean body weight gain

Mean body weight gain

Group

Time interval

Gestation day 0 – 21 (whole study period)

 

Gain in g

Gain in %

Difference to control in %

Control

167.2

77.5

n.a

Group 2 (15 mg/kg)

176.8

79.1

+5.7

Group 3 (50 mg/kg)

144.0

63.5

-13.9

Group 4 (150 mg/kg)

139.9

64.6

-16.3

n.a.

not applicable

 

 

The values are taken from Table 3 'Maternal Body Weight - Summary'

 

 

Test item-related changes are marked inbold.

 

Body weight gain from gestation day 6

Mean body weight gain

Group

Time interval

Gestation day 6 - 21 (period after start of treatment)

 

absolute body weight gain

net body weight gain

 

g

difference to control (%)

g

difference to control (%)

Control

38.9

n.a.

135.9

n.a.

Group 2

36.2

-6.8

141.1

3.8

Group 3

26.8

-31.1

115.0

-15.4

Group 4

27.6

-29.0

114.0

-16.3

n.a.

Not applicable

 

Test item-related are marked inbold.

 

Conclusions:
Under the conditions of the study, test item did not show any teratogenic potential.
Based on the data obtained in this dose-range-finding study, the following dose levels are suggested for Main Study (Prenatal developmental toxicity study in rats):
Group 1: Control, Group 2: 15 mg test iteml/kg b.w./day, Group 3: 50 mg test item/kg b.w./day, Group 4: 150 mg test item/kg b.w./day
Executive summary:

The aim of this dose-range-finding study was to determine the dose levels for a prenatal developmental toxicity study of the test item in pregnant rat when administered orally during the critical period of organogenesis and the fetal development (6th to 20th day of gestation).

 

In this prenatal developmental toxicity study, the test item was administered orally to female rats at dose levels of 15, 50 or 150 mg/kg b.w./day from the 6th to 20th day of pregnancy.

Findings

Examination of the dams:

 

Mortality

No premature deaths were noted in the control group and in the treatment groups (15, 50 or 150 mg test item/kg b.w./day).

 

 

Clinical signs

No signs of toxicity were noted.

 

 

Body weight and

body weight gain

 

In the high dose group (150 mg test item/kg b.w./day), a reduced body weight and body weight gain was noted.

 

 

Food and drinking water

consumption

 

No test item-related differences were noted.

 

 

Necropsy findings

No test changes were noted during the macroscopic inspection of the dams at necropsy.

 

 

Uterus and carcass weights

At 150 mg test item/kg b.w./day, a reduction was noted in the carcass weight (7.8% below the value of the control group).

 

 

Reproduction data

No test item-related influence was noted on the reproductive parameter (number of implantation sites, resorptions and fetuses).

 

Examination of the fetus:

 

Mortality

No dead fetuses were noted in any of the test groups.

 

 

Body weight of the fetuses

and the placentae

 

No test item-related differences were noted between the control group and the treatment groups.

 

 

Fetal alterations

 

Malformations

No malformations were noted during the external macroscopic examinations at laparotomy.

 

 

Variations

The external macroscopic examinations at laparotomy revealed no test item-related variations.

Under the present test conditions, the no-observed-adverse-effect level (NOAEL) was 50 mg test item/kg b.w./day for the dams.

No prematurely deceased dams were noted during the study.

At 150 mg test item/kg b.w./day, a reduced body weight and body weight gain and a reduced carcass weight were noted.

No changes of behaviour, the external appearance or the faeces and no influence on the food and water consumption were noted.

No changes were noted in the macroscopic examination during laparotomy.

The no-observed-adverse-effect level (NOAEL) for the fetal organism was above 150 mg test item/kg b.w./day.

The reproductive parameters (number of implantation sites, number of resorptions, number of fetuses and pre- and post-implantation loss) were not influenced by the test item.

No dead fetuses, no malformations and no variations were noted.

Under the conditions of the study, test item did not show any teratogenic potential.

Based on the data obtained in this dose-range-finding study, the following dose levels are suggested for the Main Study (Prenatal developmental toxicity study in rats):

Group 1:

Control

Group 2:

 15 mg test item/kg b.w./day, p.o

Group 3:

 50 mg test item/kg b.w./day, p.o

Group 4:

150 mg test item/kg b.w./day, p.o

 

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
2018-08-09 to 2018-09-07
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
This developmental toxicity study is used as dose range finding study for the OECD 414 main study with rabbits (LPT, 2019).
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
adopted January 2001
Deviations:
yes
Remarks:
Dose Range Finding Study with limited scope and only 3 animals per dose group
GLP compliance:
yes
Limit test:
no
Species:
rabbit
Strain:
New Zealand White
Details on test animals and environmental conditions:
TEST ANIMALS:
- Species: Rabbit, pregnant
- Source: Manfred Bauer Kaninchen, Lohe 7/1, 74632 Neuenstein, Germany

- Strain: Rabbit/ New Zealand White
- Age on day 0 of gestation: 4.5 month
- Weight at day 6 of gestation: 3.86 - 4.44 kg
For this experiment, sexually mature, purebred female artificially inseminated rabbits (New Zealand White) were used. Their state of health was checked prior to the start of the study.

- Housing:
- Diet: ad libitum, Commercial ssniff® K-Z V2323
- Water: ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20°C +/- 3°C
- Humidity (%): 55% +/- 10%
- Ventilation: between fifteen to twenty air changes per hour
- Photoperiod (hrs dark / hrs light): 12-hour light/ 12-hour dark cycle
Route of administration:
oral: gavage
Vehicle:
water
Remarks:
tap water
Details on exposure:
ADMINISTRATION:
- Route of administration: Oral, via gavage
- Frequency of administration: Once daily
- Treatment period: Day 6 to 28 of gestation
- Vehicle: Tap water
- Administration volume: 2 mL/kg b.w./day

PREPARATION OF DOSES:
The test item formulations were freshly prepared every day.
The test item was suspended in the vehicle to the appropriate concentration and was administered orally at a constant volume (2 mL/kg b.w.) once daily from the 6th to the 28th day of gestation.
The amount of the test item was daily adjusted to the current body weight of the animal. The control animals received the vehicle at the same administration volume daily in the same way.

Analytical verification of doses or concentrations:
no
Remarks:
Dose Range Finding study
Details on analytical verification of doses or concentrations:
For each test or reference item that is mixed with a vehicle, tests by appropriate analytical methods will be carried out for the main study (LPT study no. 36657) to
determine concentration, homogeneity (if applicable) and, if needed, stability of the test item in the formulations.
Details on mating procedure:
In this study sexually mature, purebred female artificially inseminated rabbits (New Zealand White) were used.
Duration of treatment / exposure:
From gestation day 6 to gestation day 28 (except high dose animals)
Frequency of treatment:
Once daily
Duration of test:
One day before the calculated parturition, i.e. on gestation day 29, all rabbits were sacrificed by lethal intravenous injection of 300 mg Pentobarbital/kg b.w. and exsanguinated.
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Vehicle control group
Dose / conc.:
25 mg/kg bw/day (nominal)
Remarks:
Low Dose group
Dose / conc.:
75 mg/kg bw/day (nominal)
Remarks:
Mid Dose group
Dose / conc.:
220 mg/kg bw/day (nominal)
Remarks:
High Dose group
No. of animals per sex per dose:
3 females per dose group
Maternal examinations:
OBSERVATIONS
Dated and signed records of all activities relating to the day to day running and mainte-nance of the study within the animal units as well as to the group observations and examinations outlined in the Study Plan were recorded in the appropriate documenta-tion. In addition, observations relating to the individual animals made throughout the study were recorded.
The following observations were made during the course of the study:

- Clinical signs
Animals were individually observed at least once daily for any signs of behavioural changes, reaction to treatment or illness.
Immediately after administration any signs of illness or reaction to treatment were recorded. In case of changes the animals were observed until the symptoms disap-peared. In addition, animals were checked regularly throughout the working day from 7.00 a.m. to 3.45 p.m.
On Saturdays and Sundays, animals were checked regularly from 7.00 a.m. to 11.00 a.m. with a final check performed at approximately 3.30 p.m.
Dated and signed records of appearance, change and disappearance of clinical signs were maintained on clinical history sheets for individual animals.

- Viability
Further checks were made early in the morning and again in the afternoon of each working day to look for dead or moribund animals. This would have allowed post mortem examinations to be carried out during the working period of that day. On Saturdays and Sundays, a similar procedure was followed except that the final check was carried out at approximately midday.
Animals showing signs of abortion or premature delivery were sacrificed on the same day. Fetuses obtained this way were examined for abnormal development whenever possible. No abortions or premature delivery occurred in this study.-

- Body weight
The weight of each rabbit was recorded on the day of delivery (used for randomization), followed by daily weighings starting on gestation day 6 - always at the same time of the day. The body weight change was also calculated in intervals (i.e. gestation day 6-9, 9 12, 12-15, 15-18, 18-21, 21-24, 24-27, 27-29). Furthermore, the carcass weight and the net weight change from day 6 are given.

- Food and drinking water consumption
The quantity of food consumed by each rabbit was recorded. Food intake per rabbit (g/rabbit/day) was calculated using the total amount of food given to and left by each rabbit in each group on completion of a treatment day.

EXAMINATIONS (NECROPSY):
One day before the calculated parturition, i.e. on gestation day 29, all rabbits were sacrificed by lethal intravenous injection of 300 mg Pentobarbital/kg b.w. and exsangui-nated.
In order to check for possible drug effects, a dissection with macroscopic examination of the internal organs and placentae of the dams was carried out on the day of sacrifice.
After ventral midline incision and skin reflection, the ovaries and uteri were removed; the gravid uteri (in toto) were weighed. A macroscopic examination of all subcutaneous tissues and internal organs of the dams was carried out. The condition of the thoracic viscera was noted with due attention to the thymus, lymph nodes and heart.
The abdominal viscera were examined before and after removal, the urinary bladder was examined externally and by palpation. The gastro-intestinal tract was examined as a whole and the stomach and caecum were incised and examined. The lungs were removed and all pleural surfaces examined under suitable illumination. The liver and the kidneys were examined. Any abnormalities in the appearance and size of the gonads, adrenal glands, uterus, intraabdominal lymph nodes and accessory reproductive organs were recorded.
In case of macroscopical findings, the affected maternal tissues were preserved in 10% buffered formalin for possible future histopathological examinations.


Indices of pre-implantation loss and post-implantation loss:

Calculation of group indices (see table 7-1)
Pre implantation
loss [%] = Corpora lutea (per group) - implantations (per group) x 100
Corpora lutea (per group)

Post implantation
loss [%] = Implantations (per group) - living fetuses (per group) x 100
Implantations (per group)

Calculation of mean indices per litter (see table 7-2 and A7)
Pre implantation
loss [%] = Sum of pre-implantation losses per litter in a group [%]
Number of litters in a group

Post implantation
loss [%] = Sum of post-implantation losses per litter in a group [%]
Number of litters in a group

Ovaries and uterine content:
Evaluated parameters:
Corpora lutea
- number per dam
- absolute number per group
- mean per group

Implantations
- number per dam
- distributions in the uterine horns
- absolute number per group
- mean per group

Resorptions
- number per dam, % per litter
- distributions in the uterine horns
- absolute number per group
- mean per group
- mean % per group
- number of early resorptions (< 2 g)
- number of late resorptions (> 2 g)
Fetal examinations:
The fetuses were removed and the following examinations performed:
(a) Macroscopic inspection (gross evaluation) of the placentae for example for focal indurations.
(b) The number of fetuses (alive and dead) and placentae (location in the uterus and the assignment of the fetuses) was determined.
(c) Sex and viability of fetuses were determined. Animals are said to be viable when they are found alive (spontaneous breathing, spontaneous movement).
(d) Number and size of resorptions were determined.
(e) Corpora lutea in the ovaries, implantations and location of fetuses in the uterus were determined.
(f) The gravid uterus weight was determined.
(g) Weights of fetuses and weights of the placentae were determined (fetuses were considered as runts if their weight was less than 70% of the mean litter weight).
(h) All fetuses (dead and alive) were inspected externally for damages, especially for malformations .
(i) The fetuses were sacrificed by i.p. injection of 60 mg pentobarbital/fetus (=0.2 mL Release/fetus).

Dissection of fetuses:
The thorax and peritoneal cavity (without damage to ribs and sternum) were opened. Location, size and condition of the internal organs were determined and examined for abnormalities (e.g. liver, discolouration, situs inversus, alterations of urinary bladder, brain, lungs, cleft palate) of soft tissue.
The sex was determined.
The kidneys were removed and incised to check for damages (e.g. dilatation of the renal pelvis).
The abdominal organs were removed.
The diaphragm was carefully removed to check the position of the heart (left - right).
The thoracic organs were removed using surgical forceps; the heart was incised to check for damages.

Evaluated parameters:
Weight of placentae
- individual data per fetus
- mean per litter
- mean per sex and litter
- litter mean per group
- litter mean per sex and group

Weight of fetuses
- individual data per fetus (alive and dead)
- mean per litter
- mean per sex and litter
- litter mean per group
- litter mean per sex and group

Fetuses
- number and % per dam (alive)
- number and % per dam (dead)
- number of fetuses (alive and dead) per sex and dam
- sex ratio per litter
- distribution in the uterine horns
- absolute number of fetuses alive per group
- mean number of fetuses alive per group
- mean % of fetuses alive per group
- mean % per sex and group

Runts
- number per dam
- mean per group

Dead fetuses
- number per dam
- mean per group


Malformed fetuses
- individual data per fetus
- type of malformation: number and incidence (%) per group and litter
- number of affected fetuses per group
Total malformation rate [%] = malformed fetuses per group x 100
fetuses per group



Fetuses with variations
- individual data per fetus
- type of variation: number and incidence (%) per group and litter
- Number of affected fetuses per group13
Total variation rate [%] = fetuses per group with variations x 100
fetuses per group
Statistics:
No statistical analysis was conducted as only 2 or 3 animals/ dose group were evaluated. The data of spare animals were not included.
Indices:
Pre-implantation loss and post-implantation loss indices, total malformation rate, total variation rate
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
No changes in behaviour, the external appearance or faeces were noted for the control group and the low or intermediate dose groups (25 or 75 mg test item/kg b.w./day).
At 220 mg test item/kg b.w./day, changes of behaviour in form of a prone position were noted for 2 of 3 animals between GD 17 and GD 19 before premature death or humane sacrifice.

Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, treatment-related
Description (incidence):
No premature death was noted for the control group and the low and intermediate dose groups (25 or 75 mg test item/kg b.w./day).
However, animal no. 10 of the high dose group (220 mg test item/kg b.w./day) was found dead in the morning of GD 19. Animal no. 10 was noted with signs of toxicity in form of a prone position on GD 17 and GD 18 and necropsy revealed an enlarged, pale and partly marbled liver.
Due to the premature death of animal no. 10 and signs of systemic toxicity for animal no. 11 (prone position on GD 19), the high dose group was terminated on GD 20.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Body weight:
No test item-related changes in body weight or body weight gain were noted in the dams after oral treatment with 25 or 75 mg test item/kg b.w./day.
At 220 mg test item/kg b.w./day, a decreased body weight compared to the control group (at maximum 20.5% below the value of the control group on GD 20) was noted from GD 12 until premature sacrifice on GD 20. This distinctly decreased body weight in comparison to the control group was considered to be test item-related.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
No test item-related changes in food consumption were noted between the dams of the control group and the dams treated with 25 or 75 mg test item/kg b.w./day. However, a decreased food consumption of the low and intermediate dose group was noted for GD 25 to GD 29 (at maximum 34.3% below the value of the control group for group 2 from GD 27 to GD 28). As no dose response-relationship was noted this difference was considered to be not test item-related.
In the high dose group (220 mg test item/kg b.w./day), a distinctly decreased food consumption was noted between GD 10 until termination of the high dose group on GD 20. Between GD 12 and GD 15 and between GD 18 and GD 20 no food intake was noted. This marked difference in food consumption was considered to be test item-related.
Food efficiency:
effects observed, treatment-related
Description (incidence and severity):
Body weight gain:
No test item-related differences were noted between the control group and the low and intermediate dose group (25 or 75 mg test item/kg b.w./day) for the body weight gain (see table below).
In the high dose group (220 mg test item/kg b.w./day), a distinctly decrease was noted for the body weight gain (-10.6% compared to +13.9% for the control group for GD 6 to GD 20). Therefore, the decreased body weight gain was considered to be test item-related.

No test item-related changes between the control group and the low and intermediate dose group (25 or 75 mg test item/kg b.w./day) were noted for the net body weight gain (without gravid uterus) between gestation day 6 and gestation day 29.
As the high dose group was terminated prematurely, no comparison could be done between the uteri and the carcasses of the control group on GD 29 and the uteri and carcasses of group 4 from GD 20 as well as for the net body weight change.

Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
No test item-related changes in the consumption of drinking water were noted for the dams treated with 25, 75 or 220 mg test item/kg b.w./day by visual appraisal.
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
No test item-related changes in the gravid uterus weight and the carcass weight (terminal body weight minus gravid uterine weight) in comparison to the control group were noted for the dams of the low and intermediate dose group (25 or 75 mg test item/kg b.w./day). As the high dose group was terminated prematurely, no comparison could be done between the uteri and the carcasses of the control group on GD 29 and the uteri and carcasses of group 4 from GD 20 as well as for the net body weight change.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
No pathological changes were noted during macroscopic inspection of the dams treated with 25 or 75 mg test item/kg b.w./day.
In the control group, necropsy revealed a pericardium filled with clear liquid for dam no. 1.
At 220 mg test item/kg b.w./day, necropsy revealed pathologic changes in the gastro-intestinal tract and the liver:
As 2 of 3 animals were noted with livers being enlarged and discoloured (pale and/or marbled), the changes in the liver were considered to be test item-related.
In the high dose group (220 mg test item/kg b.w./day), the findings related to the gastro-intestinal tract were considered to be single occurrences and therefore considered to be not test item-related.
The single occurrence of the pericardium filled with liquid for the control dam no. 1 was considered to be spontaneous.

Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not examined
Number of abortions:
no effects observed
Pre- and post-implantation loss:
effects observed, non-treatment-related
Description (incidence and severity):
In the intermediate dose group (75 mg test item/kg b.w./day) a high pre-implantation loss (mean per group: 35%) was noted. However, as implantation is completed before start of treatment on GD 6, the high pre-implantation loss in the intermediate dose group was considered to be spontaneous.
Total litter losses by resorption:
no effects observed
Description (incidence and severity):
No test item-related influence on the number of resorptions and the number of live fetuses were noted for the dams of the evaluated treatment groups (25 or 75 mg test item/kg b.w./day). As group 4 was terminated on GD 20, no reproduction data could be obtained.
Early or late resorptions:
no effects observed
Description (incidence and severity):
No test item-related influence on the number of resorptions and the number of live fetuses were noted for the dams of the evaluated treatment groups (25 or 75 mg test item/kg b.w./day). As group 4 was terminated on GD 20, no reproduction data could be obtained.
Dead fetuses:
no effects observed
Description (incidence and severity):
No test item-related influence on the number of resorptions and the number of live fetuses were noted for the dams of the evaluated treatment groups (25 or 75 mg test item/kg b.w./day). As group 4 was terminated on GD 20, no reproduction data could be obtained.
Changes in pregnancy duration:
no effects observed
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
No test item-related influence on the number of resorptions and the number of live fetuses were noted for the dams of the evaluated treatment groups (25 or 75 mg test item/kg b.w./day). As group 4 was terminated on GD 20, no reproduction data could be obtained.
Other effects:
not examined
Details on maternal toxic effects:
The reproductive parameters (number of corpora lutea, number of resorptions, number of fetuses) were not effected by the test material.
Key result
Dose descriptor:
LOAEL
Effect level:
220 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
clinical signs
mortality
body weight and weight gain
food consumption and compound intake
gross pathology
maternal abnormalities
Key result
Dose descriptor:
NOAEL
Effect level:
75 mg/kg bw/day (nominal)
Based on:
test mat.
Fetal body weight changes:
no effects observed
Description (incidence and severity):
No test item-related influence was noted on the mean fetal weights and no runts were noted after administration of 25 or 75 mg test item/kg b.w./day to the dams.
The high differences between the evaluated dose groups and the control group and the high variability (between 17.8% below the value of the control group for the male fetuses of the low dose group and 18.2% above the value of the control group for the female fetuses of the intermediate dose group) was due to the low number of animals per group.
As the high dose group (220 mg test item/kg b.w./day) was terminated on GD 20, no data on reproduction could be obtained for this group.
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
No dead fetus was noted in the control group and in the evaluated treatment groups (25 or 75 mg test item/kg b.w./day).
As the high dose group (220 mg test item/kg b.w./day) was terminated on GD 20, no data on reproduction could be obtained for this group.
Changes in sex ratio:
no effects observed
Description (incidence and severity):
The sex distribution of the fetuses in the evaluated dose groups (25 or 75 mg test item/kg b.w./day) was within the biological variability. The differences between the groups were due to the small number of animals evaluated.
As the high dose group (220 mg test item/kg b.w./day) was terminated on GD 20, no data on reproduction could be obtained for this group.
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
No test item-related influence was noted on the mean fetal weights and no runts were noted after administration of 25 or 75 mg test item/kg b.w./day to the dams.
The high differences between the evaluated dose groups and the control group and the high variability (between 17.8% below the value of the control group for the male fetuses of the low dose group and 18.2% above the value of the control group for the female fetuses of the intermediate dose group) was due to the low number of animals per group.
As the high dose group (220 mg test item/kg b.w./day) was terminated on GD 20, no data on reproduction could be obtained for this group.
Changes in postnatal survival:
not examined
External malformations:
no effects observed
Description (incidence and severity):
No visible gross alteration (malformation or variation) was noted for fetuses of the evaluated dose groups (25 or 75 mg test item/kg b.w./day).
As the high dose group (220 mg test item/kg b.w./day) was terminated on GD 20, no data on reproduction could be obtained for this group.
Skeletal malformations:
no effects observed
Description (incidence and severity):
A macroscopic internal examination was performed to detect gross alterations of the internal organs. No malformations or variations were noted during the internal examina-tion of the fetuses of the control group and the evaluated treatment groups (25 or 75 mg test item/kg b.w./day).
As the high dose group (220 mg test item/kg b.w./day) was terminated on GD 20, no data on reproduction could be obtained for this group.
Visceral malformations:
not examined
Other effects:
not examined
Details on embryotoxic / teratogenic effects:
No dead fetuses were noted in the litters of the control group and the dose groups (dams treated orally with 25 or 75 mg test item/kg b.w./day). No runts were noted at any tested dose level.
No test item-related malformations or variations were noted during the macroscopic external examination and the macroscopic gross inspection of the internal organs at laparotomy for the treatment groups (25 or 75 mg test item/kg b.w./day).
As the high dose group (220 mg test item/kg b.w./day) was terminated on GD 20, no data on reproduction could be obtained for this group.
Key result
Dose descriptor:
NOAEL
Effect level:
75 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Remarks on result:
other:
Remarks:
All animals in HD were terminated on test day 20. Therefore, no examinations of the fetuses possible in HD.
Key result
Abnormalities:
no effects observed
Key result
Developmental effects observed:
no

Examination of the dams

2,4,6-tris[(dimethylamino)

methyl]phenol

Group 1

Control

Group 2

25 mg/kg

Group 3

75 mg/kg

Group 4

220 mg/kg

Treated females

3

3

3

3

Non-pregnant females

none

none

1

n.a.

Deceased animals

none

none

none

1#1

Prematurely sacrificed animals

none

none

none

2#2

Not examined females

(excluded animals)

none

1

none

n.a.

Evaluated pregnant females

3#3

2

2

none

Dams without viable fetuses

(total post implantation loss)

none

none

none

n.a.

Dams with abortion

none

none

none

n.a.

Evaluated litters with

viable fetuses

3

2

2

n.a.

#1:      Animal no. 10 of group 4 was found dead on GD 19.

#2:      Animal nos. 11 and 12 of group 4 were prematurely sacrificed for animal welfare reasons on GD 20.

#3:      Due to the high post-implantation loss for the control animal no. 2 (10 late resorptions and one living fetus), the spare animal no. 3 was also examined.

n.a.:    not applicable

Conclusions:
In this dose-range-finding for a prenatal developmental toxicity study, the test item 2,4,6-tris[(dimethylamino]methyl)phenol was administered orally (per gavage) to female rabbits at dose levels of 25, 75 or 220 mg/kg b.w./day from the 6th to 28th day of pregnancy.
Based on the data obtained in this dose-range-finding study, the following dose levels are suggested for LPT Study No. 36657 (Prenatal developmental toxicity study in rabbits):
Group 1: Control
Group 2: 15 mg 2,4,6-tris[(dimethylamino)methyl]phenol/kg b.w./day, p.o
Group 3: 50 mg 2,4,6-tris[(dimethylamino)methyl]phenol/kg b.w./day, p.o
Group 4: 150 mg 2,4,6-tris[(dimethylamino)methyl]phenol/kg b.w./day, p.o


Executive summary:

The toxicity of the test substance was analyzed with an OECD 422 DRF study in rabbits using the dose groups 0, 25, 75 and 220 mg/kg bw/d. The goal was to be able to determine optimal dose groups for the main OECD 422 study. Due to animal welfare reasons (low food consumption, decreases body weight) the high dose group was terminated on GD20.

In this dose-range-finding for a prenatal developmental toxicity study, the test item 2,4,6-tris[(dimethylamino]methyl)phenol was administered orally (per gavage) to female rabbits at dose levels of 25, 75 or 220 mg/kg b.w./day from the 6th to 28th day of pregnancy.

Under the present test conditions, the no-observed-adverse-effect level (NOAEL) was 75 mg test item/kg b.w./day for the dams:

In the high dose group (220 mg test item/kg b.w./day), 1 of 3 animals was found dead in the morning of GD 19. On GD 20 the high dose group was terminated due to animal welfare reasons.

At 220 mg test item/kg b.w./day, signs of systemic toxicity was noted in the form of prone position in 2 of 3 animals.

A distinct decrease was noted for the body weight, body weight gain and food consump-tion for the high dose group (220 mg test item/kg b.w./day).

In the high dose group (220 mg test item/kg b.w./day), test item-related pathologic changes were noted in form of an enlarged and discoloured liver.

The no-observed-adverse effect level (NOAEL) for the fetal organism was also 75 mg test item/kg b.w./day:

No test item-related effects were noted for the reproduction parameters (number of implantation sites, number of fetuses, number of resorptions) in the low and intermediate dose group (25 or 75 mg test item/kg b.w./day).

No test item-related influence was noted on the body weight of the fetuses in the low and intermediate dose groups.

No dead fetuses and no test item-related malformations or variations were noted.

Based on the data obtained in this dose-range-finding study, the following dose levels are suggested for LPT Study No. 36657 (Prenatal developmental toxicity study in rabbits):

Group 1:       Control

Group 2:         15 mg 2,4,6-tris[(dimethylamino)methyl]phenol/kg b.w./day, p.o

Group 3:         50 mg 2,4,6-tris[(dimethylamino)methyl]phenol/kg b.w./day, p.o

Group 4:       150 mg 2,4,6-tris[(dimethylamino)methyl]phenol/kg b.w./day, p.o

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017-09-04 to 2017-10-03
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
adopted January 22, 2001
Qualifier:
according to
Guideline:
EU Method B.31 (Prenatal Developmental Toxicity Study)
Version / remarks:
May 30, 2008
GLP compliance:
yes (incl. certificate)
Limit test:
no
Specific details on test material used for the study:
The test item was suspended in the vehicle to the appropriate concentrations and was administered orally at a constant volume once daily from the 6th to the 20th day of gestation
Species:
rat
Strain:
Sprague-Dawley
Details on test animals and environmental conditions:
TEST ORGANISMS: 
- Species: Rat
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH
- Strain: CD / Crl: CD(SD)
- Age: 56 days
- body weight: 200.2 g - 280.3 g
- Diet: ad libitum, Commercial diet ssniff® R/Z V1324 (ssniff Spezialdiäten GmbH, 59494 Soest, Germany)
- Water: ad libitum
- Acclimatisation period: 5 days
-Housing: Except during the mating period, the dams were kept singly in MAKROLON cages
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 °C +/- 3° C
- Humidity (%): 55% +/- 15 %
- Illumination: 12 hours artifical fluorescent light and 12 hours dark
- Ventilation rate: between fifteen to twenty air changes per hour.
ENVIROMENTAL ENRICHMENT
- The animals received one piece of wood (certified for animal use) to gnaw on once weekly at change of the cages.
- Octagon-shaped red-tinted huts (polycarbonate) were placed in the cages to offer the animals a resting and hiding place.
Route of administration:
oral: gavage
Vehicle:
polyethylene glycol
Remarks:
PEG400
Details on exposure:
ADMINISTRATION: 
- Frequency: once daily, day 6 to 20 of gestation
- Dose volume: 4 ml/kg b.w.
- Dose: 0, 15, 50, 150 mg/kg/bw
- Animals: 25 female rats/group
DOSAGE PREPARATION:
The test item formulations were freshly prepared every day.
The test item was suspended in the vehicle to the appropriate concentrations and was administered orally at a constant volume once daily from the 6th to the 20th day of gesta-tion.
The amount of the test item was daily adjusted to the current body weight of the animal. The control animals received the vehicle at the same administration volume daily in the same way.
The male rats for mating remained untreated.
Analytical verification of doses or concentrations:
yes
Remarks:
Concentration of the test item-vehicle mixtures
Details on analytical verification of doses or concentrations:
For the analysis of the test item-vehicle formulations, samples of approximately 4 mL were taken at the following times and stored at -20°C or colder until analysis at LPT:
At start of dosing
Analysis of concentration: During treatment always before administration to the last animal of the group (1 sample/test item group).
Number of samples: 1 x 3 = 3, (Sampling date: September 11, 2017)

At the end of the dosing period (at a time when the majority of animals was dosed):
Analysis of concentration: During treatment always before administration to the last animal of the group (1 sample/test item group).
Number of samples: 3 x 3 = 9, (Sampling date: September 25, 2017)

Sum of all samples: 12
Details on mating procedure:
- Sexually mature ('proved') male rats of the same breed served as partners.
- The female breeding partners were randomly chosen.
- Mating was monogamous: 1 male and 1 female animal were placed together in one cage during the dark period.
- Each morning a vaginal smear was taken to check for the pres-ence of sperm. If findings were negative, mating was repeated with the same partner.
- The day on which sperm was found was considered as the day of conception (day 0 of pregnancy).
- This procedure was repeated until 25 mated dams were available for all groups.
- The non-pregnant animals were excluded from the analysis of the results and replaced by other animals.
- A post-mortem negative staining according to SALEWSKI was carried out in the replaced animals in order to confirm the non-pregnancy status.
Duration of treatment / exposure:
From gestation day 6 until gestation day 20
Frequency of treatment:
Once daily
Duration of test:
On gestation day 21, the rats were laparotomised under CO2 narcosis.
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Vehicle control
Dose / conc.:
15 mg/kg bw/day (nominal)
Remarks:
Low dose
Dose / conc.:
50 mg/kg bw/day (nominal)
Remarks:
Intermediate dose
Dose / conc.:
150 mg/kg bw/day (nominal)
Remarks:
HIgh dose
No. of animals per sex per dose:
25 female rats/dose , orally dosed with 0, 15, 50 or 150 mg test item/kg b.w.
Evaluated litters: 20 litters per group
Control animals:
yes, concurrent vehicle
Details on study design:
The dose levels were selected in agreement with the Sponsor based on the results of a dose-range-finding study for a prenatal developmental toxicity (LPT study no. 34963).
In this dose-range finding study, test item was administered to pregnant female rats at dose levels of 15, 50 or 150 mg/kg b.w./day orally, by gavage, once daily from gestation day 6 to 20.
No premature deaths were noted.
Dams treated with 150 mg test item/kg b.w./day were noted with a reduced body weight and body weight gain.
No embryotoxic properties (no dead fetuses, no malformations and no test item-related variations) were noted at any of the tested dose levels.

Maternal examinations:
Dated and signed records of all activities relating to the day to day running and maintenance of the study within the animal units, as well as to the group observations and examinations outlined in the Study Plan, were recorded in the appropriate documentation. In addition, observations relating to the individual animals made throughout the study were recorded.

The following observations were made during the course of the study:
Clinical signs
Individual animals were observed daily for any signs of behavioural changes, reaction to treatment, or illness.
Immediately after administration, any signs of illness or reaction to treatment were recorded. In case of changes, the animals were observed until the symptoms disappeared. In addition, animals were checked regularly throughout the working day from 7.00 a.m. to 3.45 p.m.
On Saturdays and Sundays, the animals were checked regularly starting from 7.00 a.m. to 11.00 a.m. with a final check performed at approximately 3.30 p.m.
Dated and signed records of appearance, change and disappearance of clinical signs were maintained on clinical history sheets for individual animals.


Viability
Further checks were made early in the morning and again in the afternoon of each working day to look for dead or moribund animals. This allowed post mortem examinations to be carried out during the working period of that day. On Saturdays and Sundays, a similar procedure was followed except that the final check was carried out at approximately midday.
Animals showing signs of abortion or premature delivery would have been sacrificed on the same day. Fetuses obtained this way were examined for abnormal development, whenever possible. No abortion occurred in the study.


Body weight
The weight of each rat was recorded on day 0 of gestation (the day of detection of a positive mating sign), followed by daily weighing - always at the same time of the day.
The body weight gain was calculated in intervals (i.e. day 0-3, 3 6, 6-9, 9-12, 12-15, 15-18 and 18-21), for the whole study (gestation day 0 - 21) and for the period after the start of dosing (gestation day 6 to gestation day 21). Furthermore the carcass weight and the net weight gain from day 6 is given.
These values are stated in the report.
These measurements were also used for calculating the daily amount of test item to be administered.

Food and drinking water consumption
The quantity of food consumed by each rat was recorded daily. Food intake per rat (g/rat/day) was calculated using the total amount of food given to and left by each rat in each group on completion of a treatment day.

The relative food consumption (g/kg b.w./day) was calculated using the following formula:
Daily food consumption [g/kg b.w./day]= Total food intake in g / Body weight in g x 1000

Daily monitoring by visual appraisal of the drinking water bottles was maintained throughout the study. Dehydration of the dams was avoided.

EXAMINATIONS (NECROPSY), Examination of the dams
Dissection technique and evaluation of the animals:
On gestation day 21, the rats were laparotomised under CO2 narcosis. The ovaries and the uteri of the dams were removed; the gravid uteri (in toto) were weighed. In order to check for possible drug effects, a dissection with macroscopic examination of the internal organs and placentae of the dams was carried out on the day of sacrifice or on the day on which the animals were found dead. In case of macroscopical findings, the affected maternal tissues were preserved in 7% buffered formalin for possible future histopatho-logical examinations.
Ovaries and uterine content:
Corpora lutea
- number per dam
- absolute number per group
- mean per group

Implantations
- number per dam
- distributions in the uterine horns
- absolute number per group
- mean per group

Resorptions
- number per dam
- distributions in the uterine horns
- absolute number per group
- mean per group
- early resorptions < 2 mm
- Late resorptions > 2 mm

Weight of placentae
- individual data per fetus
- mean per litter
- mean per group
- mean per sex and group

Weight of fetuses
- individual data per fetus (alive and dead)
- mean per litter
- mean per group
- litter mean per sex and group

Fetuses
- number per dam (alive)
- number per dam (dead)
- number of fetuses (alive and dead) per sex and dam
- distribution in the uterine horns
- absolute number of fetuses alive per group
- mean number of fetuses alive per group
- mean % of fetuses alive per group
- male/female ratio (alive and dead)


Runts
- number per dam
- mean per group


Malformed fetuses
- individual data per fetus
- type of malformation
- number and incidence (%) per group and litter

Total malformation rate [%] = malformed fetuses per group / fetuses per group x 100

Fetuses with variations
- type of variation
- individual data per fetus
- number and incidence (%) per group and litter

Total variation rate [%] = fetuses per group with variations / fetuses per group x 100

Fetuses with retardations
- type of retardation
- individual data per fetus
- number and incidence (%) per group and litter

Total retardation rate [%] = fetuses per group with retardations / fetuses per group x 100


Indices of pre-implantation loss and post-implantation loss:

Calculation of group indices
Pre implantation
loss [%] = [Corpora lutea (per group) - Implantations (per group)] / Corpora lutea (per group) x 100


Post implantation
loss [%] = [Implantations (per group) - living fetuses (per group)] / Implantations (per group) x 100


Calculation of mean indices per litter
Pre implantation
loss [%] = sum of pre-implantation losses per litter in a group [%] / number of litters in a group

Post implantation
loss [%] = sum of post-implantation losses per litter in a group [%] / number of litters in a group



Fetal examinations:
The fetuses were removed and the following examinations performed:
(a) Macroscopic inspection (gross evaluation) of the placentae for example for focal indurations or abnormal appearance (e.g. size, colour, shape).
(b) The number of fetuses (alive and dead) and placentae (location in the uterus and the assignment of the fetuses) was determined.
(c) Sex and viability of fetuses were determined. Animals are said to be viable when they are found alive (spontaneous breathing, spontaneous movement).
(d) Number and size of resorptions were determined.
(e) Corpora lutea in the ovaries, implantations and location of fetuses in the uterus were determined.
(f) Weights of fetuses and weights of the placentae were determined (fetuses were considered as runts if their weight was less than 70% of the mean litter weight).
(g) All fetuses (dead and alive) were inspected externally for damages, especially for malformations .
(h) The fetuses were sacrificed by an ether atmosphere.
(i) Examination of fetuses and determination of number and kind of retardations, variations or malformations:
1) 50% of the number of fetuses in each litter were examined for skeletal anomalies. The thorax and peritoneal cavity (without damage to ribs and sternum) were opened and the location, size and condition of the internal organs were determined.
Then the skeleton was double-stained with Alcian blue for the examination of cartilage and with Alizarin red to reveal ossifications (according to DAWSON). The skeletal system was examined (determination of the number and type of retardations, variations as well as malformations).
2) The remaining 50% of the number of fetuses in each litter were examined for soft tissue anomalies. Body sections were made and examined according to WILSON.
The fetuses were allocated to the evaluation of DAWSON or WILSON on an alternating basis.
Statistics:
Parametrical data:
The statistical evaluation of the parametrical values was done by Provantis using the following settings:
Homogeneity of variances and normality of distribution were tested using the BARTLETT’s and SHAPIRO-WILKS test. In case of heterogeneity and/or non-normality of distribution, stepwise transformation of the values into logarithmic or rank values was performed prior to ANOVA. If the ANOVA yielded a significant effect (p ≤ 0.05), inter-group comparisons with the control group were made by the DUNNETT’s test (p ≤ 0.01 and p ≤ 0.05).

Non-parametrical data
The statistical evaluation of non-parametrical values was done using the FISHER or Chi2 test:
FISHERs exact test, n < 100; (p ≤ 0.05 and p ≤ 0.01)
or
Chi2 test, n ≤ 0.01 (p ≤ 0.05 and p ≤ 0.01)

The respective calculations for the FISHER and Chi2 test were performed using Provantis (maternal macroscopic findings at necropsy or findings during the external macro-scopic examination of the fetuses). or an internal computer program (e.g. findings during the fetal skeletal or soft tissue examination).
Note:
The statistical evaluation of the pre- and post-implantation index (per group) using the number of corpora lutea, implantation sites and/ or fetuses per group (see table 7-1 Re-production Data - Summary - Values per Group) was done using StatXact 4.0.1 software, as such a calculation is not possible in Provantis.

Significantly different data are indicated in the summary tables of the result sections of the report.
Indices:
Fertility Index, Viability Index, Resorption Index, Pre-Implantation Loss Index, Post-Implantation Loss Index, Runts Index, Variation Index,
Number of litters having abnormalities, Number of abnormalities per litter
Historical control data:
LPT Background Data, see Appendix 4
Summarized results of the 59 last embryotoxicity studies in Sprague-Dawley rats (Charles River Deutschland GmbH performed at LPT in the years 2004 to July 2017 .
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
No test item-related changes in behaviour, the external appearance or the faeces that were considered to be of toxicological relevance were noted in the low and intermediate dose groups (15 or 50 mg test item/kg b.w./day).
The changes in behaviour and external appearance that were noted in the intermediate and high dose group (15 or 50 mg test item/kg b.w./day) are listed in the tables below.
At 15 mg test item/kg b.w./day, slight to moderate salivation was noted for 4 dams.
At 50 mg test item/kg b.w./day, slight to moderate salivation was noted for 6 dams.
Due to the low incidence of at maximum 2 days per dam for the low and intermediate dose group (15 or 50 mg test item/kg b.w./day), salivation was considered to be not adverse, especially as no signs of systemic toxicity were noted at these dose levels.
At 150 mg test item/kg b.w./day, slight to extreme salivation and piloerection were noted for 14 or 2 of 21 animals. The prematurely deceased dam no. 79 was noted with slightly to extremely reduced motility, breathing sounds lateral position and pultaceous faeces.
As reduced motility, breathing sounds, lateral position, pultaceous faeces and piloerec-tion were noted for dam no. 79 shortly before its premature death and therefore, were considered to be pre-mortal symptoms and not test item-related. Piloerection was additionally observed for 3 test days for dam no. 88. This observation was considered to be spontaneous, as none of the other surviving dams showed piloerection.
For salivation, an increase was noted for the severity, number of affected dams and the incidence compared to the low and intermediate dose groups. Therefore, at 150 mg test item/kg b.w./day, salivation was considered to be adverse.

Start and duration
Salivation
In the low and intermediate dose groups (15 or 50 mg test item/kg b.w./day), salivation started immediately to 5 minutes post administration and ended 0 to 20 minutes post administration.
At 150 mg test item/kg b.w./day, salivation started immediately to 5 minutes post administration and disappeared 5 to 20 or 20 to 60 minutes post administration.

Reduced Motility
Reduced Motility was noted for dam no. 79 of the high dose group (150 mg test item/kg b.w./day). It was noted consistently during GD 10 and 11. On GD 12 and 13, reduced motility started immediately to 5 minutes post administration and disappeared 20 to 60 minutes post administration.

Description (incidence and severity):
No test item-related differences in body weight were noted between the dams of the con-trol group and the treatment groups (15, 50 or 150 mg test item/kg b.w./day).
Mortality:
mortality observed, treatment-related
Description (incidence):
No premature deaths were noted in the control group and in the low and intermediate dose groups (15 or 50 mg test item/kg b.w./day).
Dam no. 79 treated with 150 mg test item/kg b.w./day was found dead during GD 14. Dam no. 79 was noted with pre-mortal symptoms in form of piloerection, reduced motility, breathing sounds lateral position and pultaceous faeces. The pre-mortal death was considered to be test item-related. Observations for dam no. 79 that were noted during necropsy
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Body weight
No test item-related differences in body weight were noted between the dams of the control group and the treatment groups (15, 50 or 150 mg test item/kg b.w./day).

Body weight gain from GD 0 to 21
No test item-related difference for the body weight gain from GD 0 to 21 compared to the control group was noted for the animals treated with 15, 50 or 150 mg test item/kg b.w./day.
In the high dose group (150 mg test item/kg b.w./day), decreased values were noted for the body weight gain (33.1% below the value of the control group between GD 6 and 9, p < 0.01 and 12.5% below the value of the control group between GD 10 and 12, not significant) for the first 6 days after the start of the dosing between GD 6 and GD 12. As also a decreased body weight gain (8.3% below the value of the control group, not significant) was noted during the whole study period (see table), the reduced body weight gain was considered to be test item-related.

There were no test item-related influences on the gravid uterus weight. Therefore, the gravid uterus weight had the same influence on the body weight gain for all groups
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Food and drinking water consumption
No test item-related differences were noted between the control group and the low and intermediate dose groups (15 or 50 mg test item/kg b.w./day).
At 150 mg test item/kg b.w./day, statistically significantly lower values were noted for the food consumption of the dams between GD 7 and 12 and between GD 20 and 21 in comparison to the control. This distinct decrease of food consumption was considered to be test item-related.
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
No test item-related changes in drinking water consumption were noted between the dams of the control group and the dams of the treatment groups by visual appraisal.
Ophthalmological findings:
not specified
Haematological findings:
not specified
Clinical biochemistry findings:
not specified
Urinalysis findings:
not specified
Behaviour (functional findings):
not specified
Immunological findings:
not specified
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Net body weight gain from GD 6 to 21
No test item-related differences were noted for the net body weight gain from GD 6 to 21 between the dams of the control group and the dams of the low and intermediate dose groups (15 or 50 mg test item/kg b.w./day).
A distinctly lower net body weight gain was noted for the dams treated with 150 mg test item/kg b.w./day (24.6% below the value of the control group). Although not statistically significant, the reduced net body weight gain was considered to be test item-related.

Gravid uterus weight and Carcass weight
No test item-related differences were noted between the gravid uterus weight and the carcass weight of the control dams and the dams of the treatment groups (15, 50 or 150 mg test item/kg b.w./day).
Slight reductions were noted for the gravid uterus weight for the dams treated with 50 or 150 mg test item/kg b.w./day (6.2% or 6.3% below the value of the control group, not significant). However, as these were only slight and not statistically significant reductions, the reduced gravid uterus weights were considered to be not test item-related.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
No test item-related observations were noted for the dams of the treatment groups (15, 50 or 150 mg test item/kg b.w./day) during the macroscopic inspection of the organs and tissues.
However, macroscopic findings were noted for the kidneys (cystic or dilation of the renal pelvis) for the control and also for the treatment groups or to the uterus (hemorrhage of the left or right uterine horn) for the control and the high dose group. Additionally, the prematurely deceased dam no. 79 was noted with ulcera in the stomach and dark-brown contents in the intestines.
Neuropathological findings:
not specified
Histopathological findings: non-neoplastic:
not specified
Histopathological findings: neoplastic:
not specified
Other effects:
no effects observed
Details on results:
Dam no. 79 of the high dose group (150 mg test item/kg b.w./day) prematurely deceased during GD 14.
A high incidence of salivation, a slightly reduced body weight gain and a reduced food consumption were further noted at the high dose level.
No test item-related changes were noted in the macroscopic examination during laparotomy.
Number of abortions:
no effects observed
Description (incidence and severity):
No test item-related influence on the reproductive parameters (number of implantation sites, fetuses, resorptions and the index of pre- and post-implantation loss) were noted between the dams of the control group and the dams of the treatment groups (15, 50 or 150 mg test item/kg b.w./day). See table below "any other information on results"
Pre- and post-implantation loss:
effects observed, non-treatment-related
Description (incidence and severity):
No test item-related influence on the reproductive parameters (number of implantation sites, fetuses, resorptions and the index of pre- and post-implantation loss) were noted between the dams of the control group and the dams of the treatment groups (15, 50 or 150 mg test item/kg b.w./day). See table below "any other information on results"
At 150 mg test item/kg b.w./day, a statistically significant increase was noted for the pre-implantation loss. As the implantation is completed before start of treatment, this increase was considered to be not test item-related but spontaneous.
Total litter losses by resorption:
no effects observed
Description (incidence and severity):
No test item-related influence on the reproductive parameters (number of implantation sites, fetuses, resorptions and the index of pre- and post-implantation loss) were noted between the dams of the control group and the dams of the treatment groups (15, 50 or 150 mg test item/kg b.w./day). See table below "any other information on results"
Early or late resorptions:
no effects observed
Description (incidence and severity):
No test item-related influence on the reproductive parameters (number of implantation sites, fetuses, resorptions and the index of pre- and post-implantation loss) were noted between the dams of the control group and the dams of the treatment groups (15, 50 or 150 mg test item/kg b.w./day). See table below "any other information on results"
Dead fetuses:
no effects observed
Description (incidence and severity):
No test item-related influence on the reproductive parameters (number of implantation sites, fetuses, resorptions and the index of pre- and post-implantation loss) were noted between the dams of the control group and the dams of the treatment groups (15, 50 or 150 mg test item/kg b.w./day). See table below "any other information on results"
Changes in pregnancy duration:
not examined
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): not examined
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
No test item-related influence on the reproductive parameters (number of implantation sites, fetuses, resorptions and the index of pre- and post-implantation loss) were noted between the dams of the control group and the dams of the treatment groups (15, 50 or 150 mg test item/kg b.w./day). See table below "any other information on results"
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
2 runts were noted for a dam in the control group (nos. 10-08 and 10-10) and one runt (88-11) was noted in the high dose group (150 mg test item/kg b.w./day). The occurrence of one or two runts was within the normal range of variation and therefore not test item-related.
Details on maternal toxic effects:
The reproductive parameters (number of implantation sites, number of resorptions and number of fetuses) were not influenced by the test item.
Key result
Dose descriptor:
NOAEL
Effect level:
50 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Fetal body weight changes:
no effects observed
Description (incidence and severity):
The placental and fetal weights showed no test item-related differences between the control group and the treatment groups (15, 50 or 150 mg test item/kg b.w./day).
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): no effects observed
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.DescriptionIncidenceAndSeverityFetalPupBodyWeightChanges): The placental and fetal weights showed no test item-related differences between the control group and the treatment groups (15, 50 or 150 mg test item/kg b.w./day).
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
No dead fetus was noted in the control group and in the test item treated groups (15, 50 or 150 mg test item/kg b.w./day).
Changes in sex ratio:
no effects observed
Description (incidence and severity):
No test item-related differences between the ratio of male and female fetuses (range: 0.83 - 1.03) were noted between the control group and the treatment groups (15, 50 or 150 mg test item/kg b.w./day).
Changes in litter size and weights:
no effects observed
Changes in postnatal survival:
not examined
External malformations:
no effects observed
Description (incidence and severity):
External inspection at laparotomy
No macroscopically visible external alterations (malformations or variations) were noted for the fetuses of the control and the treatment groups (15, 50 or 150 mg test item/kg b.w./day) during the macroscopic inspection at laparotomy

Gross inspection of the organs and tissues at laparotomy
The macroscopic inspection of the organs and tissues for gross alterations at laparotomy revealed no malformations or variations for the fetuses of the control group and the fetuses of the treatment groups (15, 50 or 150 mg test item/kg b.w./day).
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Skeletal malformations
No skeletal malformations were noted for the fetuses of the control group and the test item-treated groups (15, 50 or 150 mg test item/kg b.w./day) during the skeletal exami-nation according to DAWSON.

Skeletal variations
Skeletal variations were noted for the ribs (less than 13 ribs ossified, wavy or misshapen) and the sternum (bipartite, misshapen or misaligned to a slight degree).
No test item-related difference in the incidence of the observed skeletal variations in comparison to the control group was noted for the fetuses of the treatment groups (15, 50 or 150 mg test item/kg b.w./day).

Skeletal retardations
Retardations (delayed ossifications) were related to the skull (incomplete ossification of nasal, frontal, parietal and/or interparietal areas), the hyoid (unossified), the sternum (sternebra(e) incompletely ossified, reduced in size or unossified), the thoracic vertebral bodies (bipartite or dumbbell-shaped), the caudal vertebral bodies (only one body ossi-fied), the os pubis and the os ischii (incompletely ossified) and the metacarpalia and metatarsalia (absence of ossification in metacarpalia 2 to 5 and metatarsalia 2 to 5).
No test item-related increase in the incidence of skeletal retardations at 15, 50 or 150 mg test item/kg b.w./day was noted during skeletal examination according to DAWSON.
See table below "overall remarks"
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Malformations
No malformations were noted for the fetuses of the control group and the fetuses of the treatment groups (15, 50 or 150 mg test item/kg b.w./day) during the soft tissue examination according to WILSON.

Variations
During the examination of the organs and tissues according to WILSON, variations were noted for the brain (dilatation of the 4th cerebral ventricle or subdural hemorrhage in the cerebellum), the kidneys (uni- or bilateral dilatation of the renal pelvis, formation of a hydro ureter or malpositioned) and the liver (haemorrhagic focus/foci).
No test item-related differences and no statistically significant differences in the incidences of the observed variations were noted between the control group and the low and intermediate dose groups (15 or 50 mg test item/kg b.w./day).
At 150 mg test item/kg b.w./day, a statistically significantly increased incidence was noted for the incidence of the dilatation of the renal pelvis and for the total incidence of variations. As both incidences were within the range of the LPT background data (see table on the following page), these increased incidences were considered to be not test item-related.

Other effects:
no effects observed
Description (incidence and severity):
Unclassified observations
No unclassified observation was noted for any fetus of the control group and the treat-ment groups (15, 50 or 150 mg test item/kg b.w./day).
Details on embryotoxic / teratogenic effects:
No dead fetuses, no malformations and no test item-related variations or retardations were noted.
Key result
Dose descriptor:
NOAEL
Effect level:
150 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: prenatal developmental toxicity; highest dose tested
Key result
Developmental effects observed:
no

Examination of the dams

test item

Group 1

Control

Group 2

15 mg/kg

Group 3

50 mg/kg

Group 4

150 mg/kg

Treated dams

25

25

25

25

Not pregnant dams

1

1

0

0

Dams without viable fetuses

0

0

0

0

Dams with early delivery

0

0

0

0

Prematurely deceased animals

0

0

0

1

Not examined dams

(spare animals)

4

4

5

4

Evaluated litters

20

20

20

20

test item

Group 1

Control

Group 2

15 mg/kg

Group 3

50 mg/kg

Group 4

150 mg/kg

Animal nos. of mated rats

1 - 25

26 - 50

51 - 75

76 - 100

Animal nos. with evaluable litters at laparotomy

1, 3-21

26-28, 30‑46

51‑70

76-78, 80‑96

Dams not pregnant

(animal nos.)

2

29

none

none

Dams with total implantation loss (animal nos.)

none

none

none

none

Prematurely deceased animals (animal nos.)

none

none

none

79

Reserve animals

(animal nos.)

22-25

47-50

71-75

97-100

Reproduction data of the dams



Parameter

Group 1

Control

(n=20)

Group 2

15

mg/kg

(n=20)

Group 3

50

mg/kg

(n=20)

Group 4

150 mg/kg

(n=20)

Corpora lutea

total

mean per dam

304

15.2

319

16.0

291

14.6

311

15.6

Implantation sites

total

mean per dam

300

15.0

315

15.8

283

14.2

298

14.9

Resorptions

total

mean per dam

8

0.4

8

0.4

10

0.5

14

0.7

Early resorptions

total

mean per dam

7

0.4

5

0.3

8

0.4

12

0.6

Late resorptions

total

mean per dam

1

0.1

3

0.2

2

0.1

2

0.1

Live fetuses

total

mean per dam

292

14.6

307

15.4

273

13.7

284

14.2

Dead fetuses

total

0

0

0

0

Pre-implantation loss [%]

per group #1

mean per dam

1.3

1.2

1.3

1.2

2.7

3.3

4.2 *

4.8

Post-implantation loss [%]

per group #2

mean per dam

2.7

2.8

2.5

2.5

3.5

3.5

4.7

5.1

 

 

Statistical analyses were performed for the mean values per dam using an ANOVA/DUNNETT test.

 

 

#1

The statistical comparison of the pre-implantation loss per group was done by comparing the values of implantation sites/corpora lutea of the test group with the ratio of implantation sites/corpora lutea of the control group using the Chi2test (*/**: p ≤ 0.05/p ≤ 0.01).

 

 

#2

The statistical comparison of the post-implantation loss per group was done by comparing the values of live fetuses/implantation sites of the test group with the ratio of fetuses/implantation sites of the control group using the Chi2test (*/**: p ≤ 0.05/p ≤ 0.01).

 

Analysis of test item formulations

The results of the test item-formulation analyses for the investigated parameters are listed in the table below:

Parameter

Sampling / dealing

Range of

% nominal concentration

Concentration

before administration to the last animal of the group on test day 7

95.7% - 100.4%

before administration to the last animal of the group on test day 21

98.7% - 101.2%

The measured actual concentrations of the test item in the test item vehicle-mixtures were between 95.7% and 101.2% of the nominal concentrations, indicating correctly prepared formulations.

Conclusions:
Under the conditions of the study, test item did not show any teratogenic potential.
Executive summary:

The aim of this prenatal developmental toxicity study (OECD 414, oral) was the examination of the influence of the test item administered orally during the critical period of organogenesis and the fetal development (6th to 20th day of gestation) on the pregnant rat and the fetus.

Findings:

Examination of the dams:

 

Mortality

No test item-related premature deaths were noted for the low and intermediate dose groups (15 or 50 mg test item/kg b.w./day).

At 150 mg test item/kg b.w./day, dam no. 79 prematurely deceased during GD 14.

Clinical signs

No test item related influence on the behaviour, external appearance or faeces were noted for the low and intermediate dose groups (15 or 50 mg test item/kg b.w./day).

At 150 mg test item/kg b.w./day, salivation was noted in 14 of 21 evaluated dams.

Body weight and

body weight gain

 

No test item-related difference between the control group and the treatment groups was noted for the body weight. Additionally, no test item-related differences were noted for the body weight gain and the net weight gain at the low and intermediate dose levels (15 or 50 mg test item/kg b.w./day).

At 150 mg test item/kg b.w./day, a reduction was noted for the body weight gain (8.3% below the value of the control group, not significant) and for the net body weight gain (24.6% below the value of the control group, not significant).

 

 

Food consumption

No test item-related difference was noted between the control group and the dams treated with15 or 50 mg test item/kg b.w./day.

In the high dose group (150 mg test item/kg b.w./day), a reduced food consumption (at maximum 17.1% below the value of the control group between GD 11 to 12, p < 0.01) was noted between GD 7 and 12 and between GD 20 and 21.

 

 

Drinking water consumption

No differences were noted.

Necropsy findings

No test item-related changes were noted during the macroscopic inspection of the dams at necropsy.

Uterus and carcass weights

 

No test item-related differences were noted.

Reproduction data

No test item-related influence was noted on the reproductive parameter (number of implantation sites, resorptions and fetuses).

Examination of the fetus:

 

Mortality

No dead fetuses were noted in any of the test groups.

Body weight of the fetuses

and the placentae

 

No test item-related differences were noted between the control group and the treatment groups.

 

 

 

Fetal alterations

 

Malformations

No malformations were noted during the macroscopic examinations at laparotomy (external inspection and inspection of the organs and tissues for gross lesions), the skeletal examination according to DAWSON and the soft tissue examination according to WILSON.

Variations

The macroscopic examinations at laparotomy, the skeletal examination according to DAWSON and the soft tissue examination according to WILSON revealed no test item-related variations.

 

 

Retardations

No test item-related retardations (delays in ossification) were noted.

Analysis of test item

formulations

 

The measured actual concentrations of the test item in the test item vehicle mixtures were between 95.7% and 101.2% of the nominal concentrations, indicating correctly prepared formulations.

Conclusion

In this prenatal developmental toxicity study, the test item was administered orally to female rats at dose levels of 15, 50 or 150 mg/kg b.w./day from the 6th to 20th day of pregnancy.

Under the present test conditions, the no-observed-adverse-effect level (NOAEL) was 50 mg test item/kg b.w./day for the dams.

Dam no. 79 of the high dose group (150 mg test item/kg b.w./day) prematurely deceased during GD 14.

A high incidence of salivation, a slightly reduced body weight gain and a reduced food consumption were further noted at the high dose level.

No test item-related changes were noted in the macroscopic examination during laparotomy.

The no-observed-adverse-effect level (NOAEL) for the fetal organism was above150 mg test item/kg b.w./day.

The reproductive parameters (number of implantation sites, number of resorptions and number of fetuses) were not influenced by the test item.

No dead fetuses, no malformations and no test item-related variations or retardations were noted.

Under the conditions of the study, test item did not show any teratogenic potential.


Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

In an OECD 422 screening study rats were exposed via oral gavage to the test item at dose levels of 0, 15, 50, and 150 mg/kg/day for two weeks prior to mating, during gestation and for 4 days post partum.

There were no adverse affects of maternal treatment on post-natal survival or on offspring growth or development including pinna unfolding and surface righting reflex. No treatment-related macroscopic abnormalities were detected in the offspring of all treatment groups. The NOEL for developmental toxicity was therefore considered to be 150 mg/kg/day.

Justification for classification or non-classification

Based on the available studies (OECD 422 in rats and OECD 414 in rats and rabbits) the test material has not to be classified for reproductive/developmental toxicity according to CLP regulation 1272/2008. However, the marginal effects on the fertility of rats, which might be indicated in the OECD 422 study, are re-evaluated and further assessed in the currently ongoing OECD 443 study.