2,4,6-tris(dimethylaminomethyl)phenol

Endpoint summary

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 OECD 443 study.

The OECD 422 (SafePharm 2006) and OECD 408 study (LPT, 2017) were used as dose range finding study for the sub-chronic OECD 443 study (LPT, 2020).

In an OECD 443 extended one-generation reproductive toxicity study the test item was tested in rats for reproductive effects (LPT, 2020). No adverse effect on mating performance, fertility or gestation length was detected. 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.

The oral exposure to 15, 50, and 150 mg test item/kg bw (male and female Sprague-Dawley rats; OECD 443; LPT, 2020) in the extended one generation reproductive toxicity study led to histopathological changes, at 150 mg /kg b.w./day and assumingly 50 mg/kg b.w./day, and reduced body weight at 150 mg /kg b.w./day.

Additionally, secondary effects have been observed in form of a decreased food consumption, decreased organ weights and implantation loss compared to the control group at 150 mg/kg b.w./day. Histopathology revealed vacuolation of the smooth muscle fibers and the smooth muscle fibers in the arterial media in multiple organs. This is indicative for phospholipidosis, which were confirmed in additional examinations. These findings were verified as equivalent to the ones in the OECD 408.

Phospholipidosis was proven by electron microscopy within the 90-day LPT study 34962 (Weber, 2018).The electron microscopy evaluation revealed in all tissues examined the presence of membrane bound vacuoles indicative for lysosome containing myelin figures. They were noted in the brain plexus epithelia, in smooth muscle fibers of the heart, in liver macrophages, in renal tubular cells and in the smooth muscle fibers of lungs. The finding of lysosomes containing myelin figures is proving the hypothesis of phospholipidosis (Chatman et al., 2009; Nolte et al., 2016).

 

As in the OECD 443, the alterations were not associated with further inflammatory and/or degenerative lesions and showed partial or complete recovery.

The reduced reproductive organ weights (epididymides, prostate glands, seminal vesicles, and ovaries and uterus) in the OECD 443 were not related with specific changes in oocytes or sperm cells but with vacuolation of arteries and smooth muscle cells. Hence a primary effect on reproductive organs is not considered. Changes in reproductive parameters, however, are deemed to be related to the morphologically observed indicators of phospholipidosis in the uterine blood vessels, myometrium and endometrial glands and blood vessels in ovaries.

The same was considered for the effects in endocrine organs, i.e., the pituitary gland and adrenal glands. A primary affection of the endocrine system was excluded. The findings were consistent with phospholipidosis. Regarding the phospholipidosis, the slight changes in T4 or TSH cannot be considered due to a primary effect by the test item inducing hormonal dyshomeostasis but are secondary to morphological changes, i.e., degeneration of thyroid cells. Overt thyroid disfunction related to phospholipidosis was previously reported from other test items (Nonoyama and Fukuda, 2008).

The No Observed Adverse Effect Level (NOAEL) for systemic toxicity was determined to be 15 mg/kg/day.

At 150 mg/kg/day, treatment was associated with a higher level of preimplantation loss. However, this observation was a secondary effect in consequence of the vacuolisation in the reproductive organs which is a mechanism of phospholipidosis. The vacuolisation lead to an insufficient blood supply of the uterus. Hence, insufficient nourishment of the blastocyst.

The No Observed Adverse Effect Level (NOAEL) for reproductive toxicity and pre- and post -natal development was therefore considered to be above 150 mg/kg/day.

 

Link to relevant study records

Reference

Reference 1

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

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. QA statement)

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 or test system 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
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:

mortality observed, non-treatment-related

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 and was considered as a sign of general toxicity.

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 and a sign of general toxicityd.

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.

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.

Clinical biochemistry findings:

effects observed, non-treatment-related

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). As a dose-dependence relationship is present and also a decrease for the T4 levels was noted for the male animals of Cohort 1A the decreased T4 levels were considered to be a secondary effect due to observed vacuolisation in almost all organs

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Males and females
Histopathologic examination was conducted for the organs listed in Table 4-5 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.
The observed a vacuolation was already observed and examined in more depths in the LPT Study 34962 (OECD 408/ Repeated Dose 90-Day Oral Toxicity Study of 2,4,6-tris[(dimethylamino)methyl]phenol in Rats). In the latter study, electron microscopy was performed on selected organs and tissues. The cause of vacuolation at the doses 50 and 150 mg/kg/day was proven to be related to phospholipidosis. However, for the dose 15 mg/kg/day no vacuolation was observed. Due fact that the same concentrations were tested in the OECD 408 as well as in the OECD 443, the similar histopathology results in the present study at 150 mg/kg/day are deemed to represent phospholipidosis. The results of the dose 50 mg/kg/day in the OECD 443 are comparable to the ones for the treatment with 50 mg/kg/day in the OECD 408, which indicates that the observed effects are also related to phospholipidosis in this case. The doses 15 and 50 mg/kg/day have not been investigated histopathological in the present OECD 443. Since the doses 15 and 50 mg/kg/day were already examined in more depth in the OECD 408 and the examination of the doses 15 and 50 mg/kg/day were omitted for the OECD 443.

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:

no effects observed

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. Furthermore, this observation did not recurrence in the Cohort 1A .

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).

General toxicity
Parental male and female animals (F0 Generation)
No test item-related deaths of the animals were noted for any of the treatment groups.
No test item-related changes were noted for the behaviour, the external appearance or the faeces.
Reductions were noted for the body weight and body weight gain of the male high dose animals from TD 22 until sacrifice and for the female high dose animals from GD 7 until LD 14.
A decreased food consumption was noted for the male animals of the high dose group for all evaluated time points and for the female high dose animals from test week 3 until lactation week 1.
No test item-related influence was noted for the male and female animals drinking water consumption, the haematological and biochemical parameters, the levels of the thyroid hormones and the sperm parameter.
No test item-related changes were noted during the macroscopic examination, the examination of bone marrow and for the relative and absolute organ weights.

Reproductive toxicity
Parental females
No test item-related influence was noted on the length and numbers of the estrous cycles during the pre-mating period, the fertility index, the gestation index, the duration of the pre-coital time interval and the gestation period

Key result

Dose descriptor:

NOAEL

Effect level:

15 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

other: Vacuolisation of smooth muscle fibers and smooth muscle fibers in the arterial media in multiple organs.

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

50 mg/kg bw/day (nominal)

System:

other: multiple organs

Organ:

other: smooth muscle fibers and in smooth muscle fibers in the arterial media in multiple organd

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

no

Clinical signs:

no effects observed

Description (incidence and severity):

Cohort 1A:
MALES 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.

Cohort 1B:
MALES and FEMALES
No changes in behaviour, the external appearance 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 female high dose animal no. 502 was noted with diarrhea on TD 59 and TD 60. However, the observation of diarrhea for two days for one animal was considered to be spontaneous.

Dermal irritation (if dermal study):

no effects observed

Mortality:

mortality observed, non-treatment-related

Description (incidence):

Cohort 1A:
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.

Cohort 1B:
MALES and FEMALES
In the low dose group (15 mg test item/kg b.w./day), the male animal no. 404 was found dead in the morning of TD 101. Necropsy revealed a dark-red discoloured and thickened liver and a reddened brain.
Furthermore, the male animal no. 457 dosed with 50 mg test item/kg b.w./day was found dead on TD 112. The post mortem examination revealed reddened cervical and mesenterial lymph nodes, reddened lungs and the thorax filled with clear liquid.
In the high dose group (150 mg test item/kg b.w./day), the female animal no. 510 was found dead on its lactation day 21. No pathologic changes were noted during macroscopic examination. Furthermore, the female animal no. 516 was found dead during its lactation day 16. During the external examination opacity of both eyes was noted. Also, necropsy revealed reddened lungs, thymus and cervical lymph node as well as a brownish discoloured uterus and ovaries and diffuse haemorrhagic foci in the stomach.
However, as no dose-response relationship was noted for the mortality of the male animals of Cohort 1B and necropsy revealed no common pathologic changes, the premature deaths were considered to be not test item-related.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Cohort 1A:
MALES
Body weight:
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
Body weight
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.

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.

Cohort 1B
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 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), a reduced body weight was noted from TD 22 until the end of body weight recording TD 115 (at maximum 18.3% below the value of the control group on TD 99, statistically significant at p ≤ 0.01 for all time points between TD 22 and TD 113).
The body weight of the high dose group was already slightly decreased on TD 1 (5.8% below the value of the control group, statistically not significant). However, the body weight further decreased and did not recover throughout the whole examination period. Therefore, the reduced body weight was considered to be test item-related.
No differences in body weight gain were 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), in accordance with the reduced body weight, also a reduction was noted for the body weight gain. Although also the percentages of the low and intermediate dose group are decreased, the absolute body weight gain shows the decrease only in the high dose group. Therefore, the decreased body weight gain in the high dose group was considered to be test item-related.

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 dose groups (15, 50 or 150 mg test item/kg b.w./day) during the pre-mating period. The weight of the high dose group on LD 1 was already decreased by 6.2% (statistically not significant) in comparison to the control group. During the pre-mating period, no further decrease was noted and the transiently statistically significant differences on TD 15 and TD 64 (6.9% or 6.6% below the value of the control group, statistically significant at p ≤ 0.05) were considered to be spontaneous.
During the gestation and lactation period, a more pronounced decrease in comparison to the control group was noted for the female animals of the high dose group from GD 0 until LD 14. In detail, between GD 0 and LD 14 the difference of the body weight between the high dose group and the control group ranged between -7.2% and -10.4% (statistically significant at p ≤ 0.05 or 0.01). Therefore, the decreased body weight was considered to be test item-related.
No influence on the body weight gain was noted for any of the dose groups.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Cohort 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.

Cohort 1B
MALES
No test item-related difference in food consumption was noted between the control group and the treatment groups (15 or 50 mg test item/kg b.w./day). The transiently statistically significantly decreased food consumption between TD 8 and TD 22 for the intermediate dose group (5.3% or 4.3% below the value of the control group, for both p ≤ 0.05) were considered to be spontaneous.
In the high dose group (150 mg test item/kg b.w./day), a decreased food consumption was noted between TD 64 and TD 71 (6.4% below the value of the control group, statistically significant at p ≤ 0.01). As a decreased food consumption was already noted between TD 50 and TD 64 (4.0% or 3.3% below the value of the control group, statistically not significant), the decreased food consumption between TD 64 and TD 71 was considered to be test item-related.


FEMALES
No test item-related difference in food consumption was noted between the control group and the dose groups (15, 50 or 150 mg test item/kg b.w./day). No food intake of female animals was recorded during the mating period as both sexes were housed together. Statistically significant differences in food consumption which were considered to be not test item-related but spontaneous.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

Cohort 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.
Finally, the individual values of all test groups were in the range 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 (see Appendix 5). 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, non-treatment-related

Description (incidence and severity):

Cohort 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 in T4 and TSH levels of the male animals were considered to be a secondary effect. The slight (T4 and TSH) and dose independent (T4) changes in T4 or TSH cannot be considered due to a primary effect by the test item inducing hormonal dyshomeostasis but are secondary to morphological changes, i.e., degeneration of thyroid cells due to the phospholipidosis.

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 were considered to be a secondary effect based on the observed vacuolisation of smooth muscle fibers in the arterial media in nearly all organs .test item-related. The increase in TSH can not be considered due to a primary effect by the test item inducing hormonal dyshomeostasis but as a secondary to morphological changes, i.e., degeneration of thyroid cells due to the phospholipidosis.

Urinalysis findings:

no effects observed

Description (incidence and severity):

Cohort 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.

Behaviour (functional findings):

not examined

Immunological findings:

no effects observed

Description (incidence and severity):

Cohort 1A
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).

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

Cohort 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.
The nourishment of the organs is not given any longer, which results in an organ weight decrease. The histopathological observation did not show changes in sperm cells. Therefore, a primary effect on male reproductive organs was not considered. The same was considered for the effects in endocrine organs, i.e., the pituitary gland and adrenal glands. A primary affection of the endocrine system was excluded.

Cohort 1B:
MALES
Males
No test item-related differences for the examined absolute and relative organ weights were noted between the control group and the dose groups (15, 50 or 150 mg test item/kg b.w./day).
Statistically significant differences between the control group and the high dose group were considered to be not test item-related. The observed significant organ weight changes in the highest dose are considered to the decreased body weight at autopsy (-17,3%).
Histopathologic vacuolation of smooth muscle fibers and smooth muscle fibers in the arterial media in multiple organs e.g. male reproductive organs (epididymides, prostate glands, seminal vesicles) have been observed at 150 mg/kg/day. This accessory symptom of the phospholipidosis contributed also to the observed reduction of the absolute organ weights e.g. male reproductive organs. Due to the vacuolation of smooth muscle fibers in the arterial media the nourishment of the organs is not given any longer, which results in an organ weight decrease. The histopathological observation did not show changes in sperm cells. Therefore, a primary effect on male reproductive organs was not considered. The same was considered for the effects in endocrine organs, i.e., the pituitary gland and adrenal glands. A primary affection of the endocrine system was excluded.

FEMALES
No test item-related differences for the examined absolute and relative organ weights were noted between the control group and the dose groups (15, 50 or 150 mg test item/kg b.w./day).
In the high dose group, a statistically significant increase was noted for the relative liver weight (8.6% above the value of the control group, p ≤ 0.05). However, as no statistically significant difference was noted for the absolute liver weight the slightly higher relative weight was considered to be spontaneous.

Gross pathological findings:

no effects observed

Description (incidence and severity):

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.


Cohort 1B
MALES
The macroscopic findings that were noted during necropsy are listed below.
Macroscopic post mortem findings noted during necropsy.
Macroscopic post mortem findings
Group Animal no. Observation
Group 1
Control 365 Spleen: - enlarged
Kidney (l.): - enlarged (approx. 60 x 40 x 40 mm in diameter, 49.7 g)
- dark-red, beige discoloured
- soft consistency

368 Testis (l.): - reduced in size
Group 2
15 mg/kg 404 Liver: - dark-red discoloured
Brain: - reddened

441 Testis (l. + r.): - reduced in size
Epididymis (l. + r.): - reduced in size

Group 3
50 mg/kg 441 Testis (r.): - absent
457 Lymph node (cerv.): - reddened
Lymph node (mes.): - reddened
Lungs: - reddened
Thorax: - filled with clear and colourless liquid (3 mL)
Group 4
150 mg/kg - no pathological findings were noted

However, as also pathologic changes were noted for the control group and the findings were only single occurrences, the findings were considered to be not test item-related.

FEMALES
Females
No test item-related observations were noted for the female animals of the dose groups (15, 50 or 150 mg test item/kg b.w./day).
The following isolated observations were considered to be spontaneous:
Group 2: Kidney (l. + r.) enlarged (no. 434)
Group 4: Uterus: dilatation (no. 503)
In addition the prematurely deceased high dose animal no. 516 was noted the cervical lymph node, the lungs and the thymus being reddened, a brownish discoloured uterus, the ovaries partly brownish discoloured and diffusely distributed hemorrhagic foci in the stomach

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Cohort 1A
Histopathologic examination was conducted for the organs listed in Table 4-5 of the male and female animals of the control group and the high dose (150 mg test item/kg b.w./day).
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 (see Appendix 7) leading to inflammatory cell infiltration in the liver. The observed vacuolation was already seen and examined in more depths in the LPT Study 34962 (OECD 408/ Repeated Dose 90-Day Oral Toxicity Study of 2,4,6-tris[(dimethylamino)methyl]phenol in Rats). In the latter study, electron microscopy was performed on selected organs and tissues. The cause of vacuolation at the doses 50 and 150 mg/kg/day was proven to be related to phospholipidosis. Therefore, the similar histopathology results in the present study at 150 mg/kg/day are deemed to represent phospholipidosis. However, for the dose 15 mg/kg/day no vacuolation was observed. Due fact that the same concentrations were tested in the OECD 408 as well as in the OECD 443, the similar histopathology results in the present study at 150 mg/kg/day are deemed to represent phospholipidosis. The results of the dose 50 mg/kg/day in the OECD 443 are comparable to the ones for the treatment with 50 mg/kg/day in the OECD 408, which indicates that the observed effects are also related to phospholipidosis in this case. The doses 15 and 50 mg/kg/day have not been investigated histopathological in the present OECD 443. Since the doses 15 and 50 mg/kg/day were already examined in more depth in the OECD 408 and the examination of the doses 15 and 50 mg/kg/day were omitted for the OECD 443.

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Description (incidence and severity):

Cohort 1A
Bone marrow:
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) in males and females.

Details on 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. The slight and dose independent changes in T4 or TSH cannot be considered due to a primary effect by the test item inducing hormonal dyshomeostasis but are secondary to morphological changes, i.e., degeneration of thyroid cells due to the phospholipidosis.
No test item-related changes were noted during the macroscopic examination, the examination of bone marrow and for the relative and absolute organ weights.

Cohort 1B
This part of LPT Study 37260 reports the effects of the test item on the reproductive performance of the F1 male and female animals of Cohort 1B, including the postnatal development of the F2 generation pups until postnatal day 21.
General toxicity
Parental male and female animals (F1 Generation)
No test item-related premature deaths were noted in the dose groups.
No changes were noted in behaviour, the external appearance and the consistency of the faeces.
A test item-related decrease was noted for the body weight of the male and female animals of the high dose group.
No test item-related changes were noted for the food consumption.
The body weight at autopsy was decreased for the male animals of the high dose group.
No test item-related changes were noted during the macroscopic examination at necropsy and for the relative and absolute organ weights.

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

Cohort 1A
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).
Text Table 8 11: Mean length and mean number of estrous cycles.
Parameter Group 1 Group 2 Group 3 Group 4
Control 15 mg/kg 50 mg/kg 150 mg/kg
Evaluation period: test days 50 to 63
Mean cycle length (days) 4.25 4.42 4.42 4.49
Number of cycles 1.8 1.8 1.9 1.8


Cohort 1B
The stage of the estrous cycle was determined on every day of the mating period until the day before sperm detection.
No signs of impaired mating behaviour was noted for any animal of the dose groups (15, 50 or 150 mg test item/kg b.w./day). The stage of diestrus (D) was commonly noted for all female animals and only a few stages of metestrus (M), estrus (E) or proestrus (P). The stages of estrus and proestrus were followed by a positive mating sign on the next day.

Estrous cycle data - Mating Period
The stage of the estrous cycle was determined on every day of the mating period until the day before sperm detection.
No signs of impaired mating behaviour was noted for any animal of the dose groups (15, 50 or 150 mg test item/kg b.w./day). The stage of diestrus (D) was commonly noted for all female animals and only a few stages of metestrus (M), estrus (E) or proestrus (P). The stages of estrus and proestrus were followed by a positive mating sign on the next day.

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

Cohort 1A
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.

Reproductive performance:

no effects observed

Description (incidence and severity):

Cohort 1B
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).
The fertility indices of the test groups (ratio of pregnant females and females with positive mating sign) ranged between 90% and 100%.

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). All pregnant female animals delivered live pups.

Pre-coital time
No test item-related differences were noted in the length of the pre-coital time between the control group and the dose groups (15, 50 or 150 mg test item/kg b.w./day).
One female with an increased pre-coital time of 14 test days was noted in the control group, three females with a pre-coital time of 11 to 14 days in the low dose group and two females with an increased pre-coital time of 11 or 14 test days were noted in the intermediate dose group. In consequence due to the increased pre-coital time of the control group, a decreased pre-coital time (30.9% below the value of the control group, statistically not significant) was noted for the high dose group (low dose group: 17.6% above the value of the control group, intermediate dose group: 7.4% below the value of the control group). However, the observation of 3 females with an elongated pre-coital time in the low dose group was considered to be spontaneous, as also one female with an elongated pre-coital time was noted in the control group and none in the high dose group. Hence, also the decreased pre-coital time noted in the high dose group was considered to be not test item-related but spontaneous.

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).

Parental females
No influence was noted on the fertility index of the females, the gestation index, the duration of the pre-coital time interval and the gestation period.

Key result

Dose descriptor:

NOAEL

Effect level:

15 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

other: Vacuolisation of smooth muscle fibers and smooth muscle fibers in the arterial media in multiple organs.

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

50 mg/kg bw/day (nominal)

System:

other: multiple organs

Organ:

other: Vacuolation of smooth muscle fibers and smooth muscle fibers in the arterial media in multiple organs.

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

no

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

Number of live 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 treatment groups (15 or 50 mg test item/kg b.w./day) on LD 4 before culling.
In the high dose group (150 mg test item/kg b.w./day), a reduced number of live pups was noted on LD 4 (17.8% below the value of the control group (statistically significant at p ≤ 0.01). This was due to the reduced number of live born pups and was considered to be a secondary effect due to the vacuolisation of the smooth muscle cells in the arterial media of the uterus, which lead to insufficient nourishment.
However, as no influence was noted on the viability of the pups in the dose groups, after culling, no difference was noted for the number of live pups at the end of the lactation period on LD 21.

Viability index
Pre- and post-cull period
No test item-related differences between the control group and the treatment groups (15, 50 or 150 mg test item/kg b.w./day) were noted for the viability indices of the pre- and the post-cull period.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

No test item-related influence on the body weight of pups was noted for the dose groups (15, 50 or 150 mg test item/kg b.w./day).
In the high dose group, decreased body weights were noted for the male and female pups on LD 7 and LD 14 (at maximum 12.8% below the value of the control group for the male pups and the male and female pups combined on LD 14, statistically significant at p ≤ 0.01). However, on LD 21, the body weight recovered and was only between 5.0% and 5.3% below the values of the control group. Therefore, the temporary decreased pup body weight in the high dose group was considered to be not test item-related.

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:

effects observed, non-treatment-related

Description (incidence and severity):

Thyroid hormone
No test item-related differences between the control group and the treatment groups (15, 50 or 150 mg test item/kg b.w./day) were noted for the T4 level on LD 4 and no statistically significant differences were noted for the TSH levels on LD 22.
For the T4 levels on LD 22 however, a statistically significant increase was noted for the intermediate and high dose group (13.1% or 12.4% above the value of the control group for the male and female pups combined, statistically significant at p ≤ 0.01) (see figure 10 on the following page). However, as no dose response-relationship was present the increased T4 levels on LD 22 were considered to be not test item-related.

Urinalysis findings:

not examined

Sexual maturation:

no effects observed

Description (incidence and severity):

for more infromation please refere to P1.

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).

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

Cohort 1A
No test item-related differences for the examined absolute organ weights were noted between the control group and the treatment groups (15, 50 or 150 mg test item/kg b.w./day).

Gross pathological findings:

no effects observed

Description (incidence and severity):

External examination (pups terminally sacrificed on LD 4 or LD 22 or found dead):
No gross abnormalities (e.g. malformations or variations) were noted during the macroscopic external examination of the control pups and the pups from the dams treated with 15, 50 or 150 mg test item/kg b.w./day after terminal sacrifice on lactation day 4 (culled pups), on lactation day 22 or for the pups that died (stillborn or found dead) during the lactation period.

Internal examination (pups terminally sacrificed on lactation day 22):
No malformations were noted during the macroscopic examination of the inner organs and tissues of the control pups and the pups of the dose groups (15, 50 or 150 mg test item/kg b.w./day).
Variations in form of enlarged spleens were noted for the pups of the control group (5 pups of 3 litters), for pups of the low dose group (6 pups of 4 litters), the intermediate dose group (5 pups of 2 litters) and the high dose group (2 pups of 1 litter).

Macroscopic Examination of the Inner Organs
Group Dam no. Pup no. Observation
Group 1
(Control) 29 2m Spleen: -enlarged
3m Spleen: -mildly enlarged
9f Spleen: -enlarged
32 4m Spleen: -mildly enlarged
33 3m Spleen: -mildly enlarged
Group 2
(15 mg/kg) 79 8m Spleen: -enlarged
17f Spleen: -mildly enlarged
86 11f Spleen: -enlarged
89 14f Spleen: -mildly enlarged
94 1m Spleen: -mildly enlarged
9f Spleen: -mildly enlarged
Group 3
(50 mg/kg) 127 2m Spleen: -mildly enlarged
3m Spleen: -enlarged
6m Spleen: -enlarged
9f Spleen: -enlarged
129 6m Spleen: -mildly enlarged
Group 4
(150 mg/kg) 178 5m Spleen: -mildly enlarged
9f Spleen: -mildly enlarged
(m: male; f: female)
However, as also the pups of the control dams displayed an enlarged spleen, the observations of the spleen being enlarged was considered to be not test item-related. Also, no difference was noted for the weight of the respective spleens.

Histopathological findings:

not examined

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

Birth indices and post-implantation loss
No test item-related differences were noted for the mean number of implantation sites per dam, the mean number of pups born (alive and dead) per dam and the mean number of live born pups per dam between the control group and the 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), a decrease was noted in the number of implantation sites and consequently also decreased numbers of pups born alive and dead and also in the number of live born pups (15.9% or 17.1% below the value of the control group, statistically significant at p ≤ 0.01). Nevertheless, the reduced numbers of implantation sites and of pups born per dam were within the range of the LPT background data. The reduced numbers of implantation sites and live born pups were considered to be a secondary effect based on the observed vacuolisation in the smooth muscle fibers in the arterial media in the reproductive organs (ovaries and uterus). The vacuolisation lead to an insufficient blood supply of the uterus . Hence, insufficient nourishment of the blastocyst.
The reproductive indices as the birth index, the live birth index and the percentage of post implantation loss revealed no test item-related differences between the control group and the treatment groups (15, 50 or 150 mg test item/kg b.w./day).

Male to female ratio of the pups
No test item-related influence on the male to female ratio was noted for all treatment groups (15, 50 or 150 mg test item/kg b.w./day).
ale to female ratios in the test groups on lactation day 1 and lactation day 4.
Male / Female ratio of the pups:
Group 1 Group 2 Group 3 Group 4
Time point (Control) 15 mg/kg 50 mg/kg 150 mg/kg
Lactation day 1 # 0.98 1.20 1.06 0.86
Lactation day 4 # 0.97 1.22 1.04 0.88

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

For the pre-natal development of the pups, a reduction was noted for the number of implantation sites and consequently for the number of live born pups in the high dose group and the litter weight. Due to the accessory symptom of the phospholipidosis, vacuolisation of the smooth muscle cells in the arterial media of the uterus, these observations are considered to be secondary effects.
No test item-related effect was noted on the birth and live birth index and the percentage of post implantation loss.
For the postnatal development, decreased number of live pups on LD 4 and consequently decreased litter weights between LD 1 and LD 14 were noted for the high dose group. Due to the accessory symptom of the phospholipidosis, vacuolisation of the smooth muscle cells in the arterial media of the uterus, these observations are considered to be secondary effects.
No test item-related differences were noted for the viability indices (pre- and post-cull), the ano-genital distance and the nipple retention.
The examination of the thyroid hormone levels on lactation days 4 and 22 revealed no test item related differences.
The gross inspection (external and / or internal) of the pups at necropsy on lactation days 4 (culled pups) and 22 revealed no test item-related changes.
No test item-related influence was noted on the organ weights from pups sacrificed on lactation day 22.

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

> 150 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: pre- and post-natal development

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Critical effects observed:

no

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, non-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 a secondary effect due to the vacuolisation of the smooth muscle cells in the arterial media of the uterus, which lead to insufficient nourishment of the uterus.

Viability index - F2 Pups
No test item-related differences between the control group and the treatment groups (15, 50 or 150 mg test item/kg b.w./day) were noted for the viability index (difference between the number of live born pups (pups alive on lactation day 0/1) and the number of live pups on lactation day 4).

Body weight and weight changes:

effects observed, non-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 lower number of born pups in combination with the slight reduction of the pup body weights from LD 7 to LD 21. Tthe reduced litter weight was considered to be a secondary effect due to the vacuolisation of the smooth muscle cells in the arterial media of the uterus, which lead to insufficient nourishment of the uterus.

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

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% elevated above 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).

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Description (incidence and severity):

External examination (pups terminally sacrificed on lactation day 4 or found dead):
No gross abnormalities (e.g. malformations or variations) were noted during the macroscopic external examination of the F2 pups from the control group and the F2 pups from the dose groups (15, 50 or 150 mg test item/kg b.w./day) after culling on lactation day 4 or for the pups that died (stillborn or found dead) during the lactation period.
Internal examination (pups terminally sacrificed on lactation day 21):
No gross abnormalities (e.g. malformations or variations) were noted during the macroscopic examination of the inner organs or tissues of the F2 pups from the control group and the F2 pups from the dose groups (15, 50 or 150 mg test item/kg b.w./day) after terminal sacrifice on lactation day 21.

Histopathological findings:

not examined

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

Birth indices and post-implantation loss - F2 Pups
No test item-related differences were noted for the mean number of implantation sites per dam, the mean number of pups born (alive and dead) per dam and the mean number of live born pups per dam between the control group and the low and intermediate dose group (15 or 530 mg test item/kg b.w./day).
In the high dose group (150 mg test item/kg b.w./day), in parallel to the high dose group of the F0 generation, a reduced number was noted for the implantation sites (14.7% below the number of the control group, statistically significant at p ≤ 0.01). Which was, like in the F1 generation, a secondary effect due to the observed vacuolisation of the smooth muscle fibers in the arterial media in the reproductive organs . Accordingly, also the number of pups born alive and dead and also the number of pups born alive were reduced (for both parameters 18.0% below the number of the control group, statistically significant at p ≤ 0.01). Furthermore, as in the high dose group litters with less than 10 pups were obtained, also the number of pups per dam after litter adjustment was decreased. Regardless, of the slightly lowerthe number of implantation sites and the number of live born pups, those numbers were still within the range of the LPT background data.
The reproductive indices as the birth index, the live birth index and the percentage of post implantation loss revealed no test item-related differences between the control group and the treatment groups (15, 50 or 150 mg test item/kg b.w./day).

Male to female ratio of the pups - F2 Pups
No test item-related influence on the male to female ratio was noted for any treatment group (15, 50 or 150 mg test item/kg b.w./day).

Male / Female ratio of the pups
Group 1 Group 2 Group 3 Group 4
Time point (Control) 15 mg/kg 50 mg/kg 150 mg/kg
Lactation day 1 # 1.16 1.09 1.03 0.86
Lactation day 4 # 1.16 1.11 1.03 0.88

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

No test item-related influence was noted on the pre-natal development, regarding the post-implantation loss, the birth index, or the live birth index. However, reductions were noted for the number of implantation sites and consequently for the number of born pups of the high dose group. Both observations were considered to be a secondary effect due to the vacuolisation of the smooth muscle cells in the arterial media of the uterus, which lead to insufficient nourishment of the uterus.

No test item-related effect was noted on the postnatal development of the pups regarding the viability index (surviving live born pups), the pup body weight, and the ano-genital distance.
Reductions were noted for the litter weights of the high dose group. Due to the vacuolisation of the smooth muscle cells in the arterial media of the uterus and the following decreased nourishment of the uterus the reduced litter weights was considered a secondary effect.
The gross inspection of the pups at necropsy revealed no test item-related changes.

Key result

Dose descriptor:

NOAEL

Generation:

F2 (cohort 1B)

Effect level:

> 150 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: pre- and post-natal development

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Critical effects observed:

no

Key result

Reproductive effects observed:

no

Treatment related:

no

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 [x10^6/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.

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. Table7‑10:          Mean length and mean number of estrous cycles. Parameter Group 1 Control Group 2 15 mg/kg Group 3 50 mg/kg Group 4 150 mg/kg Pre-mating: Test day 15 (start of treatment) until pairing (test day 29) Number of females evaluated 24 24 24 24 Number of females with ≥ 1 cycle 21 19 18 21 Mean cycle length (days) 4.85 4.55 5.28 5.50 Number of cycles 2.0 2.2 2.1 1.9 # : Values taken from table18-1'Estrous Cycle Data – Start of Dosing until Mating - Summary' Text Table7‑11:  Fertility indices per group. Group / Dose level Fertility index [%] Pregnant rats / Rats paired with a male partner on test day 85 #1 #2 Group 1 (Control) 100 24 / 24 Group 2 (15 mg/kg) 92 22 / 24 Group 3 (50 mg/kg) 100 24 / 24 Group 4 (150 mg/kg) 92 22 / 24 */**: p ≤ 0.05 / p ≤ 0.01, Fisher test #1: Values taken from table20-1'Reproductive Outcomes and Indices per Group - Females'. #2: The females which were excluded from calculation of reproductive data were not considered for the calculation of the fertility index. Text Table7‑12:  Gestation indices per group. Group / Dose level Gestation index % Dams with live pups / Pregnant rats #1#2 Group 1 (Control) 100 24 / 24 Group 2 (15 mg/kg) 100 22 / 22 Group 3 (50 mg/kg) 100 24 / 24 Group 4 (150 mg/kg) 100 22 / 22 */**: p ≤ 0.05 / p ≤ 0.01, Fisher test #1: Values taken from table20-1'Reproductive Outcomes and Indices per Group - Females'. #2: The females which were excluded from calculation of reproductive data were not considered for the calculation of the fertility index.Text Table7‑13:  Overview of the reproductive data.   Reproductive data   Group 1 (Control) Group 2 15 mg/kg Group 3 50 mg/kg Group 4 150 mg/kg Parametrical values (mean number per dam)#1 Implantation sites #3 16.6 15.4 15.0 13.8** Pups #3 (born alive and dead) 15.1 14.5 14.4 12.5** Pups born alive #3 15.1 14.5 14.4 12.5** Indices [%]#2 Birth Index mean per dam #3 group            #4 91.57 90.73 94.16 93.84 95.59 95.59 91.93 92.08 Live birth Index mean per dam #3 group            #4 100.00 100.00 99.68 99.69 99.75 99.71 98.60 98.57 Post-implantation loss   mean per dam #3 group            #4   8.43 9.27   6.12 6.45   4.66 4.68   9.42 9.24 Resorptions and stillbirths Difference between no. of implantation sites and no. of pups born alive mean per dam #5     sum per group #5 1.5     37 1.0     22 0.7     17 1.3     28 Number of stillbirths #5 0 1 1 4 #1: Statistical calculation was performed by ANOVA / DUNNETT (*/**: p ≤ 0.05 / p ≤ 0.01). #2: Statistical calculation was performed byANOVA / DUNNETT (mean values) (*/**: p ≤ 0.05 / p ≤ 0.01). #3: Mean values per group were taken from table22-1'Birth Indices and Post-implantation loss - Values per Dam - Summary'. #4: Group values were taken from table21'Number of Pups and Indices - Overview per Group'. #5: Values taken from table22-2'Birth Indices and Post-implantation loss - Values per Dam - Individual Data'; column'No. Resorptions + Stillborns'.Text Table7‑14:LPTbackground data on number of implantation sites and pups born alive. Parameter Values from this study Group mean values ± SD #2 LPTBackground Data #1 obtained from the control groups of 27 OECD 422/421 studies performed atLPTfrom 2016 to 2019 Number of implantation sites Group 1 16.6 ± 2.7 Mean of the group mean values of the control groups 14.70 ± 0.87   Range of the group mean values 13.6 - 16.1 Group 2 15.4 ± 3.2 Group 3 15.0 ± 2.8 Group 4 13.8 ± 1.8 ** Number of pups born (alive) Group 1 15.1 ± 2.2 Mean of the group mean values of the control groups 13.9 ± 0.88   Range of the group mean values 12.0 - 15.1 Group 2 14.5 ± 2.9 Group 3 14.4 ± 2.8 Group 4 12.5 ± 1.9 ** **:      Statistically significant at p ≤ 0.01 (DUNNETT test). #1:      Data not audited by QAU. #2:      Values taken from table22-1'Birth Indices and Post-implantation Loss'.   Text Table7‑15:  Viability indices and prematurely deceased pups during the pre-cull period. Pre-cull period Parameter Group 1 (Control) Group 2 15 mg/kg Group 3 50 mg/kg Group 4 150 mg/kg Prematurely deceased pups between lactation days 0/1 to 4  / Total number of live born pups# 3 / 362 5 / 319 4 / 346 3 / 275 Viability index (group values)# 99.17 98.75 98.84 98.91   (*/**: p ≤ 0.05 / p ≤ 0.01) Chi^2test #: Taken from table21'Number of Pups and Indices - Overview per Group'. Table7‑16:          Dams with prematurely deceased pups during the pre-cull period. Number of prematurely deceased pups per dam (pre-cull period) # Group 1 (Control) Group 2 15 mg/kg Group 3 50 mg/kg Group 4 150 mg/kg dam no. deceased pups dam no. deceased pups dam no. deceased pups dam no. deceased pups 40 1 79 1 122 1 180 1 42 1 87 1 123 1 186 1 48 1 92 1 136 1 192 1     94 1 139 1     #: Taken from table23-2'Dead Pups per Dam and Viability Index - Individual Data'. Text Table7‑17:  Viability indices and prematurely deceased pups during the post-cull period. Post-cull period Parameter Group 1 (Control) Group 2 15 mg/kg Group 3 50 mg/kg Group 4 150 mg/kg Prematurely deceased pups between lactation days 5 and 21 / Number of pups alive on lactation day 4 after culling # 1 / 240 2 / 213 5 / 235 1 / 218 Viability index (group values) # 99.58 93.90 97.87 99.54   (*/**: p ≤ 0.05 / p ≤ 0.01) Chi^2test #: Taken from table21'Number of Pups and Indices - Overview per Group'. Text Table7‑18:  Dams with prematurely deceased pups during the post-cull period. Number of prematurely deceased pups per dam (post-cull period) # Group 1 (Control) Group 2 15 mg/kg Group 3 50 mg/kg Group 4 150 mg/kg dam no. deceased pups dam no. deceased pups dam no. deceased pups dam no. deceased pups 34 1 73 1 126 1 192 1     74 1 127 1         86 1 129 1             139 2   #: Taken from table23-2'Dead Pups per Dam and Viability Index - Individual Data'.Text Table7‑19:  Male to female ratios in the test groups on lactation day 1 and lactation day 4. Male / Female ratio of the pups Time point Group 1 (Control) Group 2 15 mg/kg Group 3 50 mg/kg Group 4 150 mg/kg Lactation day 1 # 0.98 1.20 1.06 0.86 Lactation day 4 # 0.97 1.22 1.04 0.88 #: Taken from table21'Number of Pups and Indices - Overview per Group'. Text Table7‑20:  Mean number of live pups per dam during the lactation period. Parameter (males + females combined) # Mean number of live pups per dam Group 1 (control) Group 2 (15 mg/kg) Group 3 (50 mg/kg) Group 4 (150 mg/kg)   Lactation day 1 - 15.1 14.5 14.4 12.5** Lactation day 4 before culling 15.0 14.3 14.3 12.4** after culling 10.0 9.7 9.8 9.9 Lactation day 7 - 10.0 9.5 9.8 9.9 Lactation day 14 - 10.0 9.5 9.6 9.9 Lactation day 21 - 10.0 9.5 9.6 9.9 */**: Statistically significant from control at p ≤ 0.05/0.01 (DUNNETT test) #: Values are taken fromtable 24-1'Number of Live Pups per Dam - Summary'. Text Table8‑1:    Body weight gain of the male animals from test day 1 to test day 70. Males Group 1 Control Group 2 15 mg/kg Group 3 50 mg/kg Group 4 150 mg/kg Body weight gain [%] # (test day 1 to test day 70) +747% +691% +668% +691% Body weight gain [g] (test day 1 to test day 70) +447.5 +425.4 +415.8 +382.0 #: Values taken from table4-1-Co1A'Body Weight Gain - Summary - Males'. Text Table8‑2:    Body weight gain of the female animals from test day 1 to test day 66. Females Group 1 Control Group 2 15 mg/kg Group 3 50 mg/kg Group 4 150 mg/kg Body weight gain [%] # (test day 1 to test day 66) +400.0% +371.6% +351.1% +400.4% Body weight gain [g] (test day 1 to test day 66) +224.6 +221.8 +224.1 +207.5 Text Table8‑3:   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 15 mg/kg Group 3 50 mg/kg Group 4 150 mg/kg     White blood cells [x10^3/µl] Males +2.4 +38.7* +18.7 A Lymphocytes [x10^3/µl] Males +3.2 +42.6* +26.2 A Basophilic granulocytes [x10^3/µl] Males +31.8 +104.5** +59.1 B */**: Statistically significant from control at p ≤ 0.05/0.01 (DUNNETT test) #1: Values taken from table6-1-Co1A'Haematological Parameters - Summary – Males’. A: The increase was considered to be spontaneous (see discussion above) B: The increase was considered to be spontaneous (see discussion above). Text Table8‑4:    Comparison with theLPTbackground data. Parameter Values from this study Group mean values ± SD Range of individual animals #2 LPT Background Data #1 obtained from the control groups of OECD 422 / 421studies performed atLPTfrom 2016 - 2017 WBC (x10^3/µL) (males)   Group 1 8.626 ± 1.805 6.65 - 12.56 Mean value from 90 control animals from 18 studies: 10.290 ± 2.987   Range of 90 control animals from 18 studies: 5.83 – 18.49 Group 2 8.830 ± 2.941 5.87 - 15.45 Group 3 11.962 ± 2.612** 7.94 - 16.18 Group 4 10.236 ± 2.045 6.31 - 13.55 Lympho-cytes (x10^3/µL) (males)   Group 1 6.718 ± 1.708 4.41 - 9.80 Mean value from 90 control animals from 18 studies: 8.315 ± 2.486   Range of 90 control animals from 18 studies: 4.44 – 14.98 Group 2 6.935 ± 2.568 4.24 - 12.42 Group 3 9.582 ± 2.307* 6.23 - 13.60 Group 4 8.475 ± 1.813 5.41 - 11.28 */**: Statistically significant from control at p ≤ 0.05/0.01 (DUNNETT test) #1: Data not audited by QAU #2: Values are taken fromtable6-1-Co1A'Haematological Parameters - Summary - Males' and table6-3-Co1A'Haematological Parameters - Individual Data - Males'. Text Table8‑5:    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 15 mg/kg Group 3 50 mg/kg Group 4 150 mg/kg     White blood cells [x10^3/µl] Females +18.7 +38.0 +62.2** A Lymphocytes [x10^3/µl] Females +21.8 +45.9* +63.8** A Neutrophilic granulocytes [x10^3/µl] Females +9.1 -0.9 +46.2* B Monocytes [x10^3/µl] Females +17.2 +49.1 +112.2** B Large unstained cells [x10^3/µl] Females +35.7 +77.8 +160.0** B Basophilic granulocytes [x10^3/µl] Females +33.3 +122.2 +188.9** B */**: Statistically significant from control at p ≤ 0.05/0.01 (DUNNETT test) #1: Values taken from table6-2-Co1A'Haematological Parameters - Summary - Females'. A: The increase was considered to be spontaneous (see above). B: The increase was considered to be spontaneous (see above).  Text Table8‑6:    Comparison with theLPTbackground data. Parameter Values from this study Group mean values ± SD Range of individual animals #2 LPT Background Data #1 obtained from the control groups of OECD 422 / 421studies performed atLPTfrom 2016 - 2017 WBC (x10^3/µL) (females)   Group 1 5.417 ± 1.156 4.10 - 7.92 Mean value from 90 control animals from 18 studies 8.224 ± 2.441   Range of 90 control animals from 18 studies: 3.52 – 15.08 Group 2 6.431 ± 1.853 4.18 - 9.34 Group 3 7.478 ± 2.558 4.13 - 12.35 Group 4 8.785 ± 1.682** 5.62 - 11.55 Lympho-cytes (x10^3/µL) (females)   Group 1 4.210 ± 1.143 2.80 - 6.57 Mean value from 90 control animals from 18 studies 6.997 ± 2.290   Range of 90 control animals from 18 studies: 2.95 – 14.76 Group 2 5.126 ± 1.420 3.13 - 7.45 Group 3 6.141 ± 2.211* 3.33 - 10.15 Group 4 6.894± 1.784** 4.00 - 9.90 */**: Statistically significant from control at p ≤ 0.05/0.01 (DUNNETT test) #1: Data not audited by QAU #2: Values are takenfromtable6-2-Co1A'Haematological Parameters - Summary - Females' and table6-4-Co1A'Haematological Parameters - Individual Data - Females'. Text Table8‑7:    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 15 mg/kg Group 3 50 mg/kg Group 4 150 mg/kg     Albumin (g/L) Males +0.1 - 2.1 +5.9* A Albumin (g/L) Females - 1.7 - 2.5 - 7.8** A Protein (total) (g/L) Females - 1.9 0.0 - 6.8* A Calcium (mmol/L) Males - 0.8 - 0.9 - 3.7* A Calcium (mmol/L) Females - 2.9 - 0.4 - 3.8*   Potassium (mmol/L) Females - 8.1* +1.3 +3.0 B Sodium / Potassium ratio Females +9.3* -1.7 -2.9 B ALAT (U/L) Females +8.5 +0.0 - 24.4** C */**: Statistically significant from control at p ≤ 0.05/0.01 (DUNNETT test) #1: Values taken fromtable7-1-Co1A'Biochemical Parameters - Summary - Males'and from table7-2-Co1A'Biochemical Parameters - Summary - Females' A: Considered to be spontaneous (see above). B: Considered to be spontaneous (see above). C: Considered to be spontaneous (see above).  Text Table8‑8:    Statistically significant changes of the urinalysis that were noted for the male animals andnotconsidered to be test item-related.     Mean values per group Changes in comparison to control [%] Reason Parameter # Sex Group 1 control Group 2 15 mg/kg Group 3 50 mg/kg Group 4 150 mg/kg     Specific gravity (g/mL) Males 1.0614 - 1.0918** 2.9% 1.0829* 2.0% 1.0907** 2.8% A pH Males 6.80 - 6.39** - 6.0% 6.52 - 4.1% 6.29** -7.5% A Urine volume (relative) (mL/kg b.w./24h) Males 30.83 - 22.51* - 27.0% 24.60 - 20.2% 21.32* -30.8% A */**: Statistically significant from control at p ≤ 0.05/0.01 (DUNNETT test) #: Values takenfromtable9-1-Co1A'Urinalysis - Parameters - Summary - Males'. A: The changes were considered to be spontaneous, as no dose-response relationship was noted and no changes were noted for the female animals. Text Table8‑9:    Sexual maturation of male animals of Cohort 1A. Parameters # Parameter of balanopreputial gland cleavage Mean values per group (differences in comparison to control %) Group 1 (Control) Group 2 15 mg/kg Group 3 50 mg/kg Group 4 150 mg/kg - day of life(PND[HC1] ) 22.2 22.6 22.4 22.2 - body weight (g) 55.6 (-) 57.1 (+2.8%) 55.1 (- 0.9%) 51.8* (- 6.8%) *: (p ≤ 0.05), ANOVA / DUNNETT #: Values taken fromtable11-1-Co1A'Balanopreputial Gland Cleavage - Summary'.  Text Table8‑10:  Sexual maturation of female animals of Cohort 1A Parameters Parameters of sexual maturation Mean values per group Group 1 (Control) Group 2 15 mg/kg Group 3 50 mg/kg Group 4 150 mg/kg - day of vaginal opening (PND) 34.3 33.4 34.5 36.2 - body weight on the day of vaginal opening (g) 133.0 131.2 133.2 131.5 - day of appearance of cornified cells (PND) 38.5 38.3 40.8** 40.4 - period between day of vaginal opening and day of appearance of cornified cells (test days) 4.4 4.9 6.3 4.3 */**: (p ≤ 0.05 / 0.01), ANOVA / DUNNETT #: Values taken from table11-3-Co1A'Vaginal Opening - Summary'. PND: Post-natal day Text Table8‑11:  Mean length and mean number of estrous cycles. Parameter Group 1 Control Group 2 15 mg/kg Group 3 50 mg/kg Group 4 150 mg/kg Evaluation period: test days 50 to 63 Mean cycle length (days) 4.25 4.42 4.42 4.49 Number of cycles 1.8 1.8 1.9 1.8 # : Values taken from table12-1-Co1A'Estrous Cycle Data - Test Days 50 to 63 - Summary' Text Table8‑12:  Stage of the estrous cycle at necropsy. The stage of the estrous cycle was evaluated from vaginal smears that were taken at necropsy. Stage of estrus at necropsy (Cohort 1A females) Group 1 Control Group 2 15 mg/kg Group 3 50 mg/kg Group 4 150 mg/kg Proestrus - 3 of 20 1 of 20 2 of 20 Estrus 13 of 20 8 of 20 6 of 20 10 of 20 Metestrus 1 of 20 2 of 20 - 2 of 20 Diestrus 6 of 20 7 of 20 12 of 20 6 of 20 - : not detected Text Table8‑13:  Statistically significant changes in male organ weights that were considered to be secondary to a reduced body weight at autopsy. Organs #1 Parameter (absolute: g) (relative: g/kg b.w.) Changes in comparison to control [%] Reason Group 2 15 mg/kg Group 3 50 mg/kg Group 4 150 mg/kg   Heart g +0.2 - 9.5* - 13.8** A g/kg b.w. +3.6 - 4.2 - 0.8 Spleen g +0.8 +3.6 - 2.5 B g/kg b.w. +4.6 +10.4 +12.1* Brain g - 1.4 - 2.6 - 4.3** B, C g/kg b.w. +1.3 +3.1 +9.8** Prostate and seminal vesicle g +4.8 - 13.2** - 14.4** A g/kg b.w. +7.4 - 8.8 - 1.6 Testis (left) g - 1.9 - 0.6 - 3.1 B g/kg b.w. +0.8 +5.1 +11.1** Testis (right) g - 3.4 - 1.2 - 4.0 g/kg b.w. - 0.5 +4.1 +10.3* Epididymis (left) g +2.8 - 3.0 - 9.9** A g/kg b.w. +5.8 +3.0 +3.8 Epididymis (right) g +4.0 - 4.2 - 6.8 g/kg b.w. +7.0 +1.1 +6.8 */**: Statistically significant from control at p ≤ 0.05/0.01 (DUNNETT test) #1: Values taken fromtable17-1-Co1A'Relative Organ Weights - Summary - Males' and table18-1-Co1A'Absolute Organ Weights - Summary - Females'. A: The slightly decreased absolute organ weights were considered to be a secondary effect of the reduced body weight at autopsy (adaptation of the organ weight to the reduced body weight). No differences for the relative organ weights were noted. B: The slightly increased relative organ weights were considered to be a secondary effect to the decreased body weight at autopsy. No influence on the absolute organ weights were noted (no adaptation of the organ weight to the decreased body weight at autopsy). C: The decrease in the absolute brain weight that was noted in group 4 was considered to be spontaneous, as the difference was only marginally.Text Table8‑14:  Statistically significant changes in females organ weights that were considered to be secondary to a reduced body weight at autopsy. Organs #1 Parameter (absolute: g) (relative: g/kg b.w.) Changes in comparison to control [%] Reason Group 2 15 mg/kg Group 3 50 mg/kg Group 4 150 mg/kg     Spleen g +6.2 +0.7 +5.2 A g/kg b.w. +6.3 +3.4 +13.3** Kidney (left) g +2.0 3.2 2.6 A g/kg b.w. +1.8 5.8 +10.4** Kidney (right) g +0.6 +2.6 +2.1 g/kg b.w. +0.6 +5.4 +9.9** */**: Statistically significant from control at p ≤ 0.05/0.01 (DUNNETT test) #1: Values taken fromtable17-2-Co1A‘Relative Organ Weights - Summary - Females’ and table18-2-Co1A'Absolute Organ Weights - Summary - Females'. A: The slightly increased relative organ weights in group 4 were considered to be a secondary effect of the decreased body weight at autopsy in group 4. No differences were noted for the absolute organ weights. Text table9‑1:     Reproductive outcome of the female animals per group. test item Group 1 Control Group 2 15 mg/kg Group 3 50 mg/kg Group 4 150 mg/kg Females used for pairing 20 20 20 20 Females with positive mating sign 20 20 20 20 Females pregnant 19 18 20 20 Females not pregnant 1 2 0 0 Pregnancy rate (Fertility index) 95% 90% 100% 100% Females with live born pups 19 18 20 20 Text Table9‑2:    Body weight gain of the male animals from test day 1 to test day 115. Males Group 1 Control Group 2 15 mg/kg Group 3 50 mg/kg Group 4 150 mg/kg Body weight gain [%] # (test day 1 to test day 115) +880% +797% +763% +760% Body weight gain [g] (test day 1 to test day 115) +513.9 +493.6 +491.3 +417.6 #: Values taken from table6-1-Co1B'Body Weight Gain - Summary - Males'. Text Table9‑3:    Body weight gain of the female animals during the pre-mating period and the gestation 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 1 to 71) +395% +391% +375% +399% Body weight gain [g] (pre-mating period) (test days 1 to 71) +223.6 +223.7 +226.6 +208.7 Body weight gain [%] #2 (gestation period) +54% +50% +52% +50% Body weight gain [g] (gestation period) +157.6 +156.6 +149.2 +128.2 Body weight gain [%] #3 (lactation period) +7% +3% +7% +11% Body weight gain [g] (lactation period) +0.8 +11.8 +17.0 +30.1 #1: Values taken from table6-2-Co1B'Body Weight Gain - Summary - Females - Pre-mating Period' #2: Values taken from table6-3-Co1B'Body Weight Gain - Summary - Females - Gestation Period' #3: Values taken from table6-4-Co1B'Body Weight Gain - Summary - Females - Lactation Period' Text Table9‑4:    Statistically significant differences in food consumption of the female animals that were considered to be spontaneous. Parameter Ref. Table no. Increase ­Decrease¯ Group / sex Test days Statistical significance Reason Relative food consumption 7-2-Co1B ­ 4 f 1 - 8 p£0.05 A, B Relative food consumption 7-2-Co1B ¯ 3 f 22 - 29 p£0.01 B Relative food consumption 7-2-Co1B ¯ 4 f 22 - 29 p£0.01 B Relative food consumption 7-2-Co1B ¯ 4 f 43 - 50 p£0.01 B A:        An increased food consumption was considered to be not test item-related. B:          The reduction was only temporary Text Table9‑6:    Examination of stage of estrous cycle at necropsy from vaginal smears taken at necropsy. Stage of estrus at necropsy (Cohort 1B females) Group 1 Control # Group 2 15 mg/kg Group 3 50 mg/kg # Group 4 150 mg/kg # Proestrus - 2 of 18 - 1 of 18 Estrus 2 of 19 5 of 18 3 of 20 3 of 18 Metestrus 4 of 19 4 of 18 4 of 20 2 of 18 Diestrus 12 of 19 7 of 18 12 of 20 9 of 18 -: not detected #: For one female of group 1, one female of group 3 and three females of group 4 the estrous stage was not determined as no evaluable cells could be obtained. Table9‑7:            Statistically significant changes in organ weights unrelated to the test item. Organ (Relative: g/kg b.w.)/ (Absolute: g) Changes in comparison to control [%] Reason Group 2 Group 3 Group 4   Liver #1 g/kg b.w. -0.1 -2.5 +10.9** A, C g -2.6 -4.5 -9.0 Spleen #1 g/kg b.w. -4.9 -3.7 +20.9** A, C g -7.0 -4.9 +0.4 Pituitary #1 g/kg b.w. -3.1 +3.3 +23.2** A, C g -4.5 +2.0 +1.7 Testis (left) #1 g/kg b.w. +0.9 +3.6 +18.6** A g -1.4 +1.3 -1.9 Testis (right) #1 g/kg b.w. -2.6 +2.0 +13.8** A, C g -4.8 -0.3 -5.8 Prostate and seminal vesicle #2 g/kg b.w. +6.3 +1.0 -2.7 B, C g +3.3 -1.1 -19.9** Epididymis (left) #2 g/kg b.w. +2.0 -2.6 -2.8 B g -0.3 -4.9 -19.5** Epididymis (right) #2 g/kg b.w. +1.3 +2.0 +4.0 B g -0.9 -0.1 -14.0** */**: Statistically significant from control at p ≤ 0.05/0.01 (DUNNETT test) #1: Values taken fromtable12-1-Co1B'Relative Organ Weights - Summary - Males' #2: Values taken from table13-1-Co1B'Absolute Organ Weights - Summary - Males' A: The increased relative organ weight was considered to be due to the decreased body weight at autopsy. B: The decreased absolute organ weight was considered to be due to the decreased body weight at autopsy. C: No dose response-relationship was present. Text Table9‑8:    Fertility indices per group. Group / Dose level Fertility index [%] Pregnant rats / Rats paired with a male partner #1 Group 1 (Control) 95 19 / 20 Group 2 (15 mg/kg) 90 18 / 20 Group 3 (50 mg/kg) 100 20 / 20 Group 4 (150 mg/kg) 100 20 / 20 #1: Values taken from table16-1-Co1B'Reproductive Outcomes and Indices per Group'. Text Table9‑9:    Gestation indices per group. Group / Dose level Gestation index % Dams with live pups / Pregnant rats # Group 1 (Control) 100 19 / 19 Group 2 (15 mg/kg) 100 18 / 18 Group 3 (50 mg/kg) 100 20 / 20 Group 4 (150 mg/kg) 100 20 / 20 */**: p ≤ 0.05 / p ≤ 0.01, Fisher test #: Values taken from table16-1-Co1B'Reproductive Outcomes and Indices per Group' Text Table9‑10:  Females with an elongated mating period. Females with an elongated mating period between 11 and 14 test days Group Female no. Pre-coital time [days] Male partner no. Control 397 14 378 Group 2 421 11 402 426 13 405 433 # 14 414 Group 3 468 14 447 478 11 457 #: The female animal was noted to be not pregnant. Text Table9‑11:  Overview on the reproduction data.   Reproduction data Group 1 (Control) Group 2 15 mg/kg Group 3 50 mg/kg Group 4 150 mg/kg Parametrical values (mean number per dam)#1 Implantation sites #3 15.9 15.3 15.8 13.6** Pups #3 (born alive and dead) 15.1 15.0 14.8 12.4* Pups born alive #3 15.0 14.9 14.8 12.3* Indices [%]#2 Birth Index mean per dam  #3 group              #4 94.00 93.77 93.24 93.10 91.98 92.21 89.34 90.48 Live birth Index mean per dam  #3 group              #4 99.62 99.65 99.67 99.63 99.72 99.66 99.62 99.60 Post-implantation loss   mean per dam  #3 group              #4   6.27 6.56   7.08 7.24   8.30 8.10   11.02 9.89 Resorptions and stillbirths Prenatal loss (sum of resorptions and stillbirths) mean per group#5   sum per group#5 1.1   19 1.2   21 1.3   26 1.4   27 Number of stillbirths #5 1 1 1 1 #1: Statistical calculation was performed by ANOVA / DUNNETT (*/**: p ≤ 0.05 / p ≤ 0.01). #2: Statistical calculation was performed by ANOVA / DUNNETT (mean values) (*/**: p ≤ 0.05 / p ≤ 0.01). #3: The group mean values were taken from table18-1-Co1B'Birth Indices and Post-implantation loss - Values per Dam - Summary'. #4: The group values were taken fromtable17-Co1B'Number of Pups and Indices - Overview per Group'. #5: Values taken from table18-2-Co1B'Birth Indices and Post-implantation loss - Values per Dam - Individual Data'; column'No. Resorptions + Stillborns'. Text Table9‑12:  LPTbackground data on number of implantation sites and live born pups. Parameter Values from this study Group mean values ± SD #2 LPTBackground Data #1 obtained from the control groups of 27 OECD 422/421 studies performed atLPTfrom 2016 to 2019 Number of implantation sites Group 1 15.9 ± 1.4 Mean of the group mean values of the control groups 14.70 ± 0.87   Range of the group mean values 13.6 - 16.1 Group 2 15.3 ± 1.7 Group 3 15.8 ± 2.0 Group 4 13.6 ± 2.7** Number of pups born (alive) Group 1 15.0 ± 1.3 Mean of the group mean values of the control groups 13.9 ± 0.88   Range of the group mean values 12.0 - 15.1 Group 2 14.9 ± 2.1 Group 3 14.8 ± 2.8 Group 4 12.3 ± 3.3* */**:   Statistically significant at p ≤ 0.05/0.01 (DUNNETT test). #1:      Data not audited by QAU. #2:         Values taken from table18-1-Co1B'Birth Indices and Post-implantation Loss' Text Table9‑13:  Viability index and prematurely deceased pups. Viability index Parameter Group 1 (Control) Group 2 15 mg/kg Group 3 50 mg/kg Group 4 150 mg/kg Prematurely deceased pups between lactation days 0/1 to 4  / Total number of live born pups# 7 / 285 5 / 269 5 / 295 3 / 246 Viability index (group values)# 97.54 98.14 98.31 98.78 #: Taken from table17-Co1B'Number of Pups and Indices - Overview per Group'.   Text Table9‑14:  Dams with prematurely deceased pups. Number of prematurely deceased pups between LD 0/1 (live born pups) and LD 4 # Group 1 (Control) Group 2 15 mg/kg Group 3 50 mg/kg Group 4 150 mg/kg dam no. deceased pups dam no. deceased pups dam no. deceased pups dam no. deceased pups 382 1 430 2 468 1 505 3 395 1 437 3 472 3   396 2   473 1   400 3       #: Taken from table19-2-Co1B'Dead Pups per Dam and Viability Index - Individual Data'. Text Table9‑15:  Male to female ratio in the test groups on LD 1 and LD 4. Male / Female ratio of the pups Time point Group 1 (Control) Group 2 15 mg/kg Group 3 50 mg/kg Group 4 150 mg/kg Lactation day 1 # 1.16 1.09 1.03 0.86 Lactation day 4 # 1.16 1.11 1.03 0.88 #: Taken from table17-Co1B'Number of Pups and Indices - Overview per Group'.Text Table10‑1:  Results of the test item-formulation analysis. Parameter Sampling Range of % nominal concentration Start of dosing of F0 Generation Concentration and Stability immediately after preparation of test item formulation, 8 h after preparation and 24 h after preparation 99.0 - 106.3 Homogeneity before administration to the first animal, during (middle) administration and before administration to the last animal 99.9 - 104.8 After littering of the F0 females Concentration before administration to the last animal of the group at the end of the study 98.6 - 102.5 End of dosing of F0 Generation Concentration before administration to the last animal of the group at the end of the study 97.6 - 99.9 Start of dosing of F1 Generation Concentration and Stability immediately after preparation of test item formulation, 8 h after preparation and 24 h after preparation 97.0 - 109.1 Homogeneity before administration to the first animal, during (middle) administration and before administration to the last animal 95.4 - 103.0 End of dosing of Cohort 1A Concentration before administration to the last animal of the group at the end of the study 96.8 - 102.9 End of dosing of Cohort 1B Concentration before administration to the last animal of the group at the end of the study 100.3 - 102.3

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 multiple organs. Similar as in the OECD 408 vacuolation of the smooth muscle fibers and the smooth muscle fibers in the arterial media have been observed in multiple organs. Similar histopathology were observed for the animals at 50 mg/kg/day also in the OECD 408.
The No Observed Adverse Effect Level (NOAEL) for systemic toxicity was therefore considered to be 15 mg/kg/day.

At 150 mg/kg/day, treatment was associated with a higher level of preimplantation loss. However, this observation was a secondary effect in consequence of the vacuolisation in the reproductive organs which is a mechanism of phospholipidosis. The vacuolisation lead to an insufficient blood supply of the uterus. Hence, insufficient nourishment of the blastocyst.
The No Observed Adverse Effect Level (NOAEL) for reproductive toxicity and pre- and postnnatal development was therefore considered to be above 150 mg/kg/day.

Executive summary:

The aim of the study was to evaluate the effects of the test item on general and reproductive toxicity of the F0 Parents and of the F1 Pups from weaning until adulthood including mating and post-mating (F2 Pups) until lactation day 21 employing dose levels of 15, 50 or 150 mg/kg b.w./day per gavage.

General and reproductive toxicity (F0 Generation and F1 Pups)

Test item-related influence was noted on general toxicity parameters in form of reductions of the body weight and food consumption for the male and female of the high dose group.

No test item-related influence however, was noted on the reproductive performance of the parental animals of the F0 Generation.

In the high dose group changes were noted on the pre-natal development in form of a reduced number of implantation sites and a resulting decreased number of born pups. However, this observation was a secondary effect in consequence of the vacuolisation in the reproductive organs which is a mechanism of phospholipidosis.The vacuolisation lead to an insufficient blood supply of the uterus. Hence, insufficient nourishment of the blastocyst.

A decrease was noted for the T4 levels of the male high dose animals, which is also based on the vacuolisation in nearly all organs. This symptom of the phospholipidosis resulted also in a decrease in the absolute organs weights in the highest dose groups. This included also the reproductive organs like the prostate and seminal vesicles (-15,3%) in the males and the ovaries (-13,1% or -22%) in the females. Nevertheless, a primary effect on the male and female reproductive organs was not considered. Additionally, a primary affection of the endocrine system was excluded.

No test item-related influence was noted on the postnatal development of the F1 pups.

The results of the histopathological examination indicate test item-related changes in form of vacuolation or vacuolic changes of smooth muscle cells/fibres of almost all organs, which is due to phospholipidosis. This leads to inflammatory cell infiltration in the liver for the high dose animals of the F0 Generation and F1 Generation Cohort 1A.

 

Reproductive and developmental toxicity (F1 Generation and F2 Pups - Cohorts 1A and 1B)

During their post-weaning development the high dose male and female animals of the F1 Generation showed a decrease for the body weight. Additionally, a reduced food consumption was noted for the male animals of Cohort 1B.

A increase in the TSH levels of the male and female animals and decreased T4 levels of the male animals were noted for the high dose group, which were also secondary effects on account of vacuolisation in nearly all organs. Despite that, a primary affection of the endocrine system was excluded. In the male animals this resulted in the decrease of the absolute organ weights of prostate and seminal vesicles (-14,4%) in the highest dose group. Nevertheless, a primary effect on male reproductive organs was not considered.

No test item-related influence was noted on the development of the reproductive system (time points of sexual maturations, number and length of estrous cycles, sperm parameter).

The detailed histopathological examination of testis and epididymides, number of primordial and small growing follicles and number of corpora lutea in the ovaries revealed no toxicologically relevant lesions.

Also, no test item-related influence was noted on the reproductive performance of the F1 females (fertility index, gestation index, pre-coital time and gestation length). A change was noted on the pre-natal development of the high dose group in form of a decreased number of implantation sites and consequently a decreased number of born pups. Also in the F1 generation this observation is based on vacuolisation in nearly all organs. The observed vacuolisation is an accompanying symptom for phospholipidosis.

No test item-related influence was noted on the post-natal development of the F2 pups until sacrifice on lactation day 21 (number of resorptions, stillborns, live born pups and the viability index after birth until lactation day 4 and 21).

The following no-observed-adverse-effect levels (NOAEL) were established for the parental animals of the F0 Generation and the F1 Generation by gavage:

F0 and F1 Generation:

General toxicity

NOAEL                                                   15 mg test item/kg b.w./day

Due to the observed vacuolisation.

Reproductive toxicity

a)   Adverse effects on the reproductive parameters of the parental females

NOAEL                        above 150 mg test item/kg b.w./day

 

b)   Adverse effects on pre- and postnatal development

Pre-natal development

NOAEL                                    above 150 mg test item/kg b.w./day

Post-natal development

NOAEL                                     above 150 mg test item/kg b.w./day

 

F2 Generation:

 

Developmental toxicity

Pre-natal development

NOAEL                                     above 150 mg test item/kg b.w./day

Post-natal development

NOAEL                                     above 150 mg test item/kg b.w./day

 

 

 

 

1.1.1        Findings - F0 Generation and F1 Pups

General toxicity - Parental animals (F0 Generation)
Mortality	Males and females No premature deaths that were considered to be test item-related were noted for any dose group (15,50or150 mg test item/kg b.w./day).
Clinical signs	Males and females No test item-related changes were noted in behaviour, the external appearance and the consistency of the faeces for the male and female animals during the daily cage side observations. Detailed clinical observations: No observations were noted during the weekly detailed clinical observations.
Body weight and body weight gain	Males and females In the high dose group (150 mg test item/kg b.w./day), a reduced body weight compared to the control group was noted for the male animals from TD 22 until termination (at maximum 9.1% below the value of the control group, statistically significant at p ≤ 0.01). Also, a reduced body weight was noted for the high dose females between GD 7 and LD 14 (at maximum 11.5% below the value of the control group, statistically significant at p ≤ 0.01). In accordance with the decreased body weight in the high dose group for the male and female (temporary) animals, also the body weight gain was reduced for the male and female animals.
Food consumption		Males and females A reduced food consumption was noted for the male and female animals dosed with150 mg test item/kg b.w./day. For the males, the reduced food consumption was noted between TD 15 and TD 29 (at maximum 7.3% below the value of the control group in the week from TD 15 to TD 22, statistically significant at p ≤ 0.01) and for the female animals between TD 15 and LD 7 (at maximum 13.5% below the value of the control group in the week from GD 7 to GD 14, statistically significant at p ≤ 0.01).
Drinking water consumption		Males and females No test-item related changes were noted.
Haematology		Males and females No test-item related changes were noted.
Clinical biochemistry		Males and females No test-item related changes were noted.
Thyroid hormone levels		Males and females Decreases were noted for the levels of T4 in the males of the high dose group (150 mg test item/kg b.w./day). This observation is a secondary effect due to the ascertained phospholipidosis which resulted in the vacuolisation of smooth muscle fibers in the arterial media of multiple organs. A primary affection of the endocrine system was excluded.
Sperm parameter		Males No test-item related changes were noted.
Necropsy
Macroscopicpost mortem findings		Males and females No test item-related observations were noted.
Stage of estrous cycle at necropsy		No difference for the stage of the estrous cycle at necropsy was noted between the control group and the dose groups.
Organ weights		Males and females No test item-related differences were noted.
Bone marrow examination		Males and females No test item-related differences were noted.

 

Histopathological examinations (Groups 1 and 4, performed by AnaPath GmbH)	Males and females Histopathological examination of the male and female animals of the control and high dose group revealedtest item-related changes in form of vacuolation or vacuolar changes in smooth muscle cells/fibers of multiple organs.The findings noted by histopathology were consistent with findings noted in a previous study (LPT Study 34962 (Repeated Dose 90-Day Oral Toxicity Study of 2,4,6-tris[(dimethylamino)methyl]phenol in Rats)).   Detailed examination of testis and epididymides: The histopathological examination on one testicle and one epididymis of the examined males (n = 20) 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), did not reveal any toxicologically relevant lesions.
Reproductive toxicity Reproductive parameters of the parental females
Estrous cycle data	No test item-related influence was noted on the mean number and the mean length of the estrous cycles during the pre-mating period and the mating period.
Fertility index	No test item-related influence was noted on the fertility index of the female animals.
Gestation index	No test item-related influence was noted.
Pre-coital time	No test item-related influence was noted.
Gestation length	No test item-related influence was noted.
F1 Pups - Pre- and postnatal development
- Prenatal development (from conceptus to birth)
Reproductive parameters	No test item-related influence was noted on the birth index, the live birth index and the percentage of post-implantation loss. Decreased numbers of implantation sites and consequently of pups born were noted for the high dose group (150 mg test item/kg b.w./day) leading to a decreased litter weight. These observations are secondary as they result from vacuolisation in the reproductive organ, which .lead to an insufficient blood supply of the uterus . Hence, insufficient nourishment of the blastocyst.Already theLPT Study 34962 (Repeated Dose 90-Day Oral Toxicity Study of 2,4,6-tris[(dimethylamino)methyl]phenol in ratsrevealed that phospholipids is the mechanism behind the vacuolisation.

 

 

- Postnatal development (pup)
Mortality (Viability index)	Pre- and post-cull period No test item-related influence was noted.
Pup body weight	No test item-related influence was noted.
Ano-genital distance	No test item-related influence was noted.
Count of male nipples (nipple retention)	No test item-related influence was noted.
F1 Pups - Examination at necropsy (surplus pups; not used for F1 generation)
External and internal examination	No malformations or variations were noted.
T4 determination (LD 4; culled pups)	No test item-related difference was noted.
T4 and TSH determination (LD 22)	No test item-related difference was noted.
Pup organ weights	No test item-related difference was noted.

 

 

1.1.2        Findings - Cohort 1A

Mortality Males and females No animal of the control group and the treatment groups (15,50or150 mg test item/kg b.w./day) died prematurely.     Clinical signs Males and females No test item-related changes were noted in behaviour, the external appearance and the consistency of the faeces for the male and female animals during the daily cage side observations.     Body weight and body weight gain   Males and females Reductions for the body weight were noted for the male animals of the high dose group (150 mg test item/kg b.w./day). For the male animals, the decreased body weight was noted between TD 22 and TD 70 (at maximum 13.8% below the value of the control group, statistically significant at p ≤ 0.01). Also a decrease was noted for the body weight gain of the male animals (691% body weight gain in the high dose group compared to 747% in the control group). For the female animals, no test item-related influence was noted on the body weight and body weight gain.     Food consumption Males and females No test-item related changes were noted.     Haematology Males and females No test-item related changes were noted.     Clinical biochemistry Males and females No test-item related changes were noted.     Lymphocyte typing (spleen) Males and females No test-item related changes were noted.     Urinalysis Males and females No test-item related changes were noted.         Thyroid hormone levels (PND 87 - 96; at necropsy)     Males and females In the high dose group (150 mg test item/kg b.w./day), a decrease was noted for the male T4 levels and an increase for the TSH levels of the male and female animals. These observations are a secondary effect due to the ascertained phospholipidosis which resulted in the vacuolisation of smooth muscle fibers in the arterial media of multiple organs. A primary affection of the endocrine system was excluded.     Sexual Maturation Males No influence was noted on the day of balanopreputial gland cleavage and the body weight on the day of balanopreputial gland cleavage.   Females No influence was noted on the body weight on the day of vaginal opening, the day of the appearance of cornified cells in the vaginal smear and the period between the day of vaginal opening and the appearance of cornified cells in the vaginal smear. A slight delay was noted for the day of vaginal opening in the high dose group (150 mg test item/kg b.w./day), that was considered to be test item-related butnotadverse.     Estrous cycle data Females No test item-related influence on the number and length of the estrous cycles was noted during the examination of a 2 week period between test days 50 and 63.     Sperm parameter Males No test-item related changes were noted.     Necropsy Macroscopicpost mortem findings   Males and females No test item-related observations were noted.     Stage of estrous cycle at necropsy   No difference for the stage of the estrous cycle at necropsy was noted between the control group and the dose groups.       Organ weights Males and females No test item-related differences were noted.     Bone marrow examination Males and females No test item-related differences were noted. Histopathology (groups 1 and 4; performed at AnaPath GmbH)     Males and females The microscopical examinations of the organs of the groups 1 and 4 animals of the F0 Generation and F1 Generation Cohort 1A revealed vacuolation are vacuolic changes in the smooth muscle cells in the arterial media of almost all organs.The findings noted by histopathology were consistent with findings noted in a previous study (LPT Study 34962 (Repeated Dose 90-Day Oral Toxicity Study of 2,4,6-tris[(dimethylamino)methyl]phenol in Rats)). In the latter study, electron microscopy was performed on selected organs and tissues. The cause of vacuolation at the doses 50 and 150 mg/kg/day was proven to be related to phospholipidosis. Beyond he vacuolation the treatment did not lead to any toxicological relevant tissue reactions. Nevertheless, the liver displayed vacuolation and inflammatory cell infiltrate.

 

 

1.1.3        Findings - Cohort 1B

Mortality	Males and females No test item-related premature death was noted in any of the dose groups (15,50or150 mgtest item/kg b.w./day).
Clinical signs	Males and females No changes were noted in behaviour and the external appearance and no test item-related changes for the consistency of the faeces for the male and female animals during the daily cage side observations. Detailed clinical observations: No observations were noted during the weekly detailed clinical observations.
Body weight and body weight gain	Males and females A reduced body weight was noted for the male and female animals of the high dose group (150 mg test item/kg b.w./day). The reduction was noted for the males from TD 22 until termination on TD 115 (at maximum 18.3% below the value of the control group, statistically significant at p ≤ 0.01) and for the females from GD 0 to LD 14 (at maximum 10.4% below the value of the control group, statistically significant at p ≤ 0.01). Also, the body weight gain was decreased for the high dose group males (760% body weight gain in the high dose group compared to 880% body weight gain in the control group).
Food consumption		Males In the high dose group (150 mg test item/kg b.w./day), a reduced food consumption was noted from TD 64 and TD 71 (6.4% below the control group, statistically significant at p ≤ 0.01). Females No test item-related changes were noted.

 

 

 

Water consumption	Males and females No test-item related changes were noted.
Sexual maturation	Males and females No test item-related differences were noted for the time points of balanopreputial gland cleavage and vaginal opening. Also no test item-related differences were noted for the body weight at the time points of balanopreputial gland cleavage and vaginal opening.
Necropsy
Macroscopicpost mortem findings	Males and females No test item-related observations were noted.
Stage of estrous cycle at necropsy	No difference for the stage of the estrous cycle at necropsy was noted between the control group and the dose groups.
Organ weights	Males and females No test item-related differences were noted.
Reproductive toxicity Reproductive Parameters of the Parental Females
Estrous cycle data	The stages of the estrous cycle during the mating period did not show any sign of an impaired mating behaviour.
Fertility index	No test item-related influence was noted on the fertility index of the female animals.
Gestation index	No test item-related influence was noted.
Pre-coital time	No test item-related influence was noted.
Gestation length	No test item-related influence was noted.

 

 

Pups - Pre- and Postnatal Development (F2 Pups)
- Prenatal development (from conceptus to birth)
Reproductive parameters	No test item-related influence was noted on the birth index, the live birth index and the percentage of post-implantation loss. In the high dose group (150 mg test item/kg b.w./day), a decrease was noted for the number of implantation sites, the number of born pups and for the number live born pups. These resulted from the vacuolisation of arteries and smooth muscle cells observed in the reproductive organs, which is an accessory symptom of phospholipidosis.The vacuolisation lead to an insufficient blood supply of the uterus. Hence, insufficient nourishment of the blastocyst
- Postnatal development (pup)
Mortality (Viability index)	No test item-related influence was noted on the surviving rate of the live born pups (LD 0/1) until LD 4 and LD 21.
Number of live pups on LD 4	Due to the reduced number of born pups, also the number of pups after culling on LD 4 was decreased for the high dose group (150 mg test item/kg b.w./day).
Pup body weight	No test item-related influence was noted.
Ano-genital distance	No test item-related influence was noted.
External and internal examination	No malformations or variations were noted during the macroscopic inspection of the pups at necropsy on LD 4 and LD 21.

 

 

 

1.1.4        Analysis of test item formulation

Concentration, stability and homogeneity

The test item formulation analysis revealed concentrations ranging between 95.4% and 109.1% of the nominal concentration, indicating correctly prepared, stable and homogenous test item formulations.

Reference 2

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

2005-05-06 to 2005-12-09

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

This OECD 422 study is used as dose range finding study for the OECD 443 study (LPT, 2020).

Qualifier:

according to guideline

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 or test system 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

Dose descriptor:

NOAEL

150 mg/kg bw/day

Study duration:

subchronic

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.

OECD 443 (LPT, 2020):

Reduced organ weights and histopathology examination of the adult P0 & F1 generation revealed toxicologically significant histopathological findings in the multiple organs. Similar as in the OECD 408 vacuolation of the smooth muscle fibers and the smooth muscle fibers in the arterial media have been observed in multiple organs.

The No Observed Adverse Effect Level (NOAEL) for systemic toxicity was therefore considered to be 15 mg/kg/day.

At 150 mg/kg/day, treatment was associated with a higher level of preimplantation loss. However, this observation was a secondary effect in consequence of the vacuolisation in the reproductive organs which is a mechanism of phospholipidosis. The vacuolisation lead to an insufficient blood supply of the uterus. Hence, insufficient nourishment of the blastocyst.

Subsequent post-natal survival, bodyweight, growth and development of the offspring to termination (Day 0 & 4 of age) was unaffected by treatment.

The No Observed Adverse Effect Level (NOAEL) for reproductive toxicity and pre- and post -natal development was therefore considered to be above 150 mg/kg/day.

 

Discussion regarding the key observations

In the ECHA decision CCH-D-211447795-35-01/F, ECHA noted that the lowered body weight of the females (-12%) and males (-13%) treated with 150 mg/kg bw /d of the OECD 408 can not singly explain the statistically significant changes in the absolute organ weights of the hormone sensitive organs such as prostate and seminal vesicles (-22%), ovaries (-27%) and uterus incl. cervix (-28%). Similar findings have been observed in the first parental generation (P0) treated with 150 mg/kg bw/d in the OECD 443 at hand. In this study the organ weights for prostate and seminal vesicles decreases concentration dependent at 15,3% at the highest dose (150 mg/kg bw/d). In the male animals the epididymis decreased in the weight between 8.5 and 10.5% in the P0 generation, too. The ovary weight in this high dose group decrease about 22% in the left ovary and 13.1% in the right ovary in the P0 generation. This effect was not reproduced in the female adult animals of the second parental generation (P1). In this generation no changes between the untreated and treated groups in regards of the ovaries and uterus weight were observed. Therefore, it can be concluded that the effect that reduced the ovaries and uterus weight in the P0 is maybe better adapted by the next generation treated with the test substance (CAS 90-72-2).

For the male animals of the second parental generation (P1) on the other hand a reduction in the prostate and seminal vesicles weight were observed in both cohorts 1A and 1B of the P1 generation at 150 mg/kg bw/d. The reductions observed were like the ones in the P1 generation. The prostate and seminal vesicles weight decreased in cohort 1A by 14.4%. Additionally, the weight of the epididymis decreased between 6.6% and 9.9% in cohort 1A. In cohort 1B the weight of the prostate and seminal vesicles decreased even further down by 14% respectively 19.5%. However, also the overall bodyweight of the male animals of cohort 1B decreased more dramatically (18.3%) compared to the control animals. Due to fact that ECHA noted in their last decision (CCH-D-211447795-35-01/F) on CAS 90-72-2 that the observed vacuolisation could not be explain the weight decrease in the hormone sensitive organs, ECHA mentioned that the effects could indicate and relate to an endocrine mode of action. If we lock at substances which have shown similar effects regards the reduction of organs weights in males, like phthalates substances e.g. di-2-ethylexylphthalate (DEHP), we do not see enough similarities. DEHP has been found to reduce the weights of ventral prostate and seminal vesicle (Dalsenter et al., 2006; Ponzo and Carbone, 2013). But additionally, DEHP also altered spermatogenic processes in the adult males (Dalsenter et al., 2006; Ponzo and Carbone, 2013). After treatment with DEHP male pups show a higher incidence of reproductive malformations, a reduced anogenital distance as well as reduction testis weights (Gray et al., 2000; Ponzo and Carbone, 2013). These are all processes which are regulated by androgen. Female animals which have been exposed to androgen during foetus and postnatal development have shown increased clitoral length (Traish & Guay, 2015). Additionally, testosterone propionate treatment results in increased anogenital distance in females (Wolf et al., 2002). Secondly, it was descripted that androgen treatment of immature animals can result in the premature vaginal opening (Traish & Guay, 2015). Thirdly, the vaginal mucosa produces more mucins after androgen treatment (Dorfman and Shipley, 1956). Neither, the changes in the anogenital distances in male pups nor in the female pups have been reported in the OECD 443. Other effects reported for the males treated with DEHP like altered spermatogenic and reduction of the testis have not been observed in the P0 and P1 generation. Parallel also the effects observed for female animals treated with DEHP are not reproduceable in animals treated with the test substance. In the F1 generation we observed a delay in the vaginal opening and not an early vaginal opening like with DEHP. No changes have been reported for the clitoral length or histopathological changes in the vaginal mucosa after treatment with the test substance.

Overall it can be concluded, that all observed changes/adverse effects in the OECD 443 are of secondary nature due to the descripted vacuolisation in the smooth muscle fibers and vacuolisation the smooth muscle fibers of the arteria media in nearly all organs (Weber, 2020) and are therefore not specific for an endocrine mode of action.Phospholipidosis can affect multiple and different organs (van Meer, 2006). This is especially true for the medial affection of arteries in multiple organs (Ishikawa et al., 1988; Shayman and Abe, 2013). Decreased organ weights are considered to be related to the phospholipidosis.

 

The reduced reproductive organ weights (epididymides, prostate glands, seminal vesicles, and ovaries and uterus) were not related with specific changes in oocytes or sperm cells but with vacuolation of arteries and smooth muscle cells. Hence a primary effect on reproductive organs is not considered. Changes in reproductive parameters, however, are deemed to be related to the morphologically observed indicators of phospholipidosis in the uterine blood vessels, myometrium and endometrial glands and blood vessels in ovaries.

 

The same is true for the effected endocrine organs, i.e., the pituitary gland, adrenal glands, and thyroid and parathyroid glands.A primary affection of the endocrine system cannot be concluded. Regarding the phospholipidosis, the slight changes in T4 or TSH cannot be considered due to a primary effect by the test item inducing hormonal dyshomeostasis but are secondary to morphological changes, i.e., degeneration of thyroid cells. Overt thyroid disfunction related to phospholipidosis was previously reported from other test items (Nonoyama and Fukuda, 2008).

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 2,4,6‑tris[(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

Reference

Reference 1

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

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

adopted January 22, 2001

Qualifier:

according to guideline

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 or test system 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

 

Reference 2

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

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 or test system 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:

body weight and weight gain

clinical signs

food consumption and compound intake

gross pathology

maternal abnormalities

mortality

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

Reference 3

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

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

adopted January 22, 2001

Qualifier:

according to guideline

Guideline:

EU Method B.31 (Prenatal Developmental Toxicity Study)

Version / remarks:

May 30, 2008

GLP compliance:

yes (incl. QA statement)

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 or test system 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.

Dermal irritation (if dermal study):

not examined

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 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, 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 examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

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 Chi^2test (*/**: 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 Chi^2test (*/**: 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

Quality of whole database:

Klimisch 1

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, OECD 443 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.