Registration Dossier

Toxicological information

Toxicity to reproduction

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Administrative data

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.

Data source

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

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 443 (Extended One-Generation Reproductive Toxicity Study)
Version / remarks:
adopted June 25, 2018
Deviations:
yes
Remarks:
see section "Details on Study Design"
GLP compliance:
yes (incl. certificate)
Limit test:
no
Justification for study design:
According to the ECHA final decision on a compliance check from 2018-10-29 (Decision number: CCH-D-2114447795-35-01/F and submission number: MJ448798-11) the study design of the EOGRTS according to OECD 443 is as follows:

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

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






Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Details on test material:
2,4,6-(dimethylaminomethyl)phenol from Evonik, Batch: NC18A05920

Test animals

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

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

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

ENVIRONMENTAL CONDITIONS
Diet: Commercial diet ssniff® R/M-Z V1324 (ssniff Spezialdiäten GmbH, 59494 Soest, Composition of the diet will be stated in the report)
Drinking Water: Tap water is offered daily ad libitum.
Housing: Animals are kept singly in MAKROLON cages (type III plus) with a basal surface of approx. 39 cm x 23 cm and a height of approx. 18 cm
at a room temperature of 22°C ± 3 °C (maximum range) and a relative humidity of 55% ± 15% (maximum range).
The rooms are alternately lit (about 150 lux at approx. 1.50 m room height) and darkened in a 12 hours dark/12 hours light cycle.

Administration / exposure

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.
Doses / concentrationsopen allclose all
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

Examinations

Parental animals: Observations and examinations:
CLINICAL SIGNS
All animals
All animals
Throughout the test period, each animal was observed for clinical signs at least once daily.
Individual animals were observed before and after dosing at each time of dosing for any signs of behavioural changes, signs of difficult or prolonged parturition, reaction to treatment or illness. Any signs of illness or reaction to treatment were recorded for each individual animal.
Cage side observations included skin/fur, eyes, mucous membranes, respiratory and circulatory systems, somatomotor activity and behaviour patterns. The onset, intensity and duration of any signs observed were recorded.
In addition, animals were checked regularly throughout the working day from 7:00 a.m. to 3:45 p.m. On Saturdays and Sundays animals were checked regularly from 7:00 a.m. to 11:00 a.m. with a final check performed at approximately 3:30 p.m.
Dated and signed records of appearance, change and disappearance of clinical signs were maintained on clinical history sheets for individual animals.

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



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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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


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

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


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


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


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


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


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


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

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

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

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

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

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

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

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

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

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

HISTOPATHOLOGY

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

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


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

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

Evaluation was performed by LPT.


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

Dissection

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

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

HISTOPATHOLOGY

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

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



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

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

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

Significantly different data are indicated in the summary tables of the result sections (Sections 7 to 9) and the result tables in the tables section (Section 12) of this report.
The mean values and standard deviations were calculated to the highest possible degree of accuracy and then rounded to the reported number of decimal places. Hence, deviations to the last decimal place of up to ± 1 may occur caused by rounding.
Reproductive indices:
Gestation Index, Female Fertility Index, Birth index, Live birth index
Offspring viability indices:
Birth Index, Live Birth Index, Viability Birth Index, Post Implantation loss

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
no effects observed
Description (incidence and severity):
Males and females
No test item-related changes in behaviour, external appearance or the faeces were noted for the male and female animals of the control group and the treatment groups (15, 50 or 150 mg test item/kg b.w./day).
The male animal no. 107 dosed with 50 mg test item/kg b.w./day was noted with piloerection on TD 67 to TD 71 and with breathing sounds on TD 67 and TD 68.
In the high dose group (150 mg test item/kg b.w./day), the female animal no. 183 displayed piloerection from GD 8 until LD 18. Furthermore, the female animal no. 177 was noted with breathing sounds on LD 4 to LD 8. However, as only one animal displayed piloerection or breathing sounds, piloerection and breathing sounds were considered to be spontaneous and not test item-related.

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

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

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

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


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

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

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

Immunological findings:
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
Males and females
Histopathologic examination was conducted of the male and female animals of the control group and the high dose (150 mg test item/kg b.w./day). The examination revealed test item-related changes in form of vacuolation or vacuolar changes in smooth muscle cells/fibers of several organs. However, in a detailed examination of these effects it was concluded that these findings are form of phospholipidosis and therefore not relevant for human health.
Detailed examination of testis and one epididymis:
The histopathological examination performed on one testicle and one epididymis of the examined males of groups 1 and 4 (with special emphasis on the qualitative stages of spermatogenesis (proliferative, meiotic and spermiogenic phases) and histopathology of the interstitial testicular structure) revealed no toxicologically relevant lesions.
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Description (incidence and severity):
Bone marrow
Males and females
No test item-related differences for the myeloid/erythroid ratio of the bone marrow were noted between the control group and the high dose group (150 mg test item/kg b.w./day).

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
effects observed, non-treatment-related
Description (incidence and severity):
Stage of estrous cycle at necropsy
During necropsy, a vaginal smear was taken for determination of the estrous stage from the female animals of the control group and the treatment groups (15, 50 or 150 mg test item/kg b.w./day). The stage of diestrus was the predominantly noted in groups 1 to 3. However, the numbers of female animals in the diestrus stage decreased and the number of animals in the estrus stage increased gradually from group 1 to 4. Thus, in group 4, the number of animals in the diestrous stage was equal to the number of animals in the estrous stage.
However, the numbers of female animals in the different estrous cycles differ from the numbers given in the histopathological phase report. This was due to different methods of evaluation (evaluation of a vaginal smear versus histopathological examination of the vagina).
The histopathologic evaluation of the vagina revealed similar numbers for the control group (4 animals in diestrus and 9 animals in lactational diestrus) and the high dose group (1 animal in diestrus and 10 animals in lactational diestrus). Therefore, the different numbers of animals in diestrus determined by evaluation of vaginal smears was considered to be spontaneous.



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

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

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

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

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

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



Effect levels (P0)

Key result
Dose descriptor:
NOAEL
Effect level:
50 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
food consumption and compound intake
clinical biochemistry

Target system / organ toxicity (P0)

Critical effects observed:
not specified

Results: F1 generation

General toxicity (F1)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
1A
Male and females
No test item-related changes in behaviour, the external appearance and the consistency of the faeces were noted for the male and female animals of the control group and the treatment groups (15, 50 or 150 mg test item/kg b.w./day).
Breathing sounds were noted for one male animal (no. 321) of the high dose group on 3 consecutive test days and salivation was noted for one female animal (no. 360) of the high dose group on 6 consecutive test days. As both observations were only noted for one animal each, these observations were considered to be spontaneous.



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

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

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




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

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

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


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

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




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

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


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

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

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


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

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

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

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Details on results (F1)

Summarized results - Cohort 1A
This part of LPT Study 37260 reports the effects of the test item on the development of the F1 male and female animals of cohort 1A after weaning on lactation day 21 until necropsy. The F1 Generation animals were dosed with the same levels as the animals of the F0 Generation. (15, 50 or 150 mg test item/kg b.w./day). Dosing started immediately after weaning and lasted until one day before necropsy.
None of the animals of cohort 1A died prematurely.
No test item-related changes were noted in behaviour, the external appearance and the consistency of the faeces.
A slightly reduced body weight was noted for the male animals at 150 mg test item/kg b.w./day, whereas no influence was noted on the body weight of the females.
No influence was noted on food consumption, the haematological and biochemical parameters, lymphocyte typing in the spleen, urinalysis, the sperm parameter and sexual maturation.
Increased levels of TSH were noted for the male and female high dose animals and a decrease was noted for the T4 levels of the male animals of the high dose group.
No test item-related changes were noted during the macroscopic examination, the examination of bone marrow and for the relative and absolute organ weights.

Effect levels (F1)

Dose descriptor:
other: No dose descriptor derived yet
Generation:
F1
Remarks on result:
other: study not yet completed

Target system / organ toxicity (F1)

Critical effects observed:
not specified

Results: F2 generation

General toxicity (F2)

Mortality / viability:
mortality observed, treatment-related
Description (incidence and severity):
Number of live pups – F2 Pups
No test item-related or statistically significant differences were noted for the mean number of live pups per dam between the control group and the low and intermediate dose group (15 or 50 mg test item/kg b.w./day).
In the high dose group (150 mg test item/kg b.w./day), a reduced number was noted for the number of live born pups leading to reductions in the numbers of pups per dam on LD 1 and LD 4 (17.4% or 17.0% below the value of the control group for male and female pups combined, statistically significant at p ≤ 0.05). As 5 of the 20 dams were noted with 9 pups and one dam with only 3 pups on LD 4, the reduced number of pups per dam was also noted after litter adjustment to 10 pups per dam (6.0%, 6.0% and 6.3% for the male and female pups combined on LD 7, LD 14 and LD 20, statistically significant at p ≤ 0.01).
The reduced number of live pups was considered to be test item-related.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Body weight of pups - F2 Pups
No test item-related influence on the body weight of pups was noted for any dose group (15, 50 or 150 mg test item/kg b.w./day).
In the high dose group, a statistically significant decrease was noted for the male pup body weights and for the combined body weights of the male and female pups on LD 14 (7.9% or 8.0% below the value of the control group, statistically significant at p ≤ 0.05). However, as no difference was noted on LD 1 and as the pup body weights of the high dose group recovered on LD 21, the decreased pup body weights on LD 14 was considered to be not test item-related.

Litter weight of F2 pups
No test item-related influence on the litter weight was noted for the low and intermediate dose group (15 or 50 mg test item/kg b.w./day).
In the high dose group (150 mg test item/kg b.w./day), a decrease was noted for the weight of the litters during the whole lactation period (between 10.2% and 18.9% below the value of the control group for the male and female litters combined, with the exception of LD 21 statistically significant at p ≤ 0.05 or 0.01).
As this was due to the test item-related lower number of born pups in combination with the slight reduction of the pup body weights from LD 7 to LD 21 the reduced litter weight was considered to be test item-related.
Anogenital distance (AGD):
no effects observed
Description (incidence and severity):
No test item-related difference in the absolute and the relative ano-genital distance (value normalized to cube root of pup body weight) of the male and the female pups was noted between the control group and the dose groups (15, 50 or 150 mg test item/kg b.w./day).
In the intermediate and high dose group, a statistically significant increase was noted for the absolute and relative ano-genital distance of the female pups (14.1%/12.3% or 14.8%/13.6% below the value of the control group, p ≤ 0.01). However, as no difference was noted for the male pups, the increased ano-genital distance was considered to be spontaneous.
Nipple retention in male pups:
no effects observed
Description (incidence and severity):
No test item-related difference for the number of nipples/areolae of the male pups between the control group and those of the dose groups (15, 50 or 150 mg test item/kg b.w./day).

Effect levels (F2)

Dose descriptor:
other: no dose descriptor derived yet
Generation:
F2 (cohort 1B)
Sex:
male/female
Remarks on result:
other: study not yet completed

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

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

Test item

Group 1

Control

Group 2

15 mg/kg

Group 3

50 mg/kg

Group 4

150 mg/kg

Females examined for general toxicity

24

24

24

24

Females used for pairing

24

24

24

24

Females with positive mating sign

24

23

24

23

Females without a positive mating sign

0

1 (93)

0

1 (189)

Females not pregnant

0

1 (82)

0

1 (176)

Females pregnant

24

22

24

22

Pregnancy rate (Fertility index)

100%

92%

100%

92%

Females with resorption of all implants

0

0

0

0

Females with litter

24

22

24

22

 

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

Females

Group 1

Control

Group 2

15 mg/kg

Group 3

50 mg/kg

Group 4

150 mg/kg

Body weight gain [%] #1

(pre-mating period)

(test days 15 to 29)

+4.3%

+5.8%

+5.4%

-0.2%

Body weight gain [g]

(pre-mating period)

(test days 15 to 29)

+10.7

+14.3

+13.5

-0.7

Body weight gain [%] #2

(gestation period)

+63.5%

+56.1%

+61.4%

+52.8%

Body weight gain [g]

(gestation period)

+169.6

+149.1

+162.5

+133.9

Body weight gain [%] #3

(lactation period)

+0.1%

+2.8%

+2.8%

+14.5%

Body weight gain [g]

(lactation period)

+0.0

+6.2

+7.8

+40.2

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

 

 

Changes in comparison to control

[%]

Reason

Parameter #1

Sex

Group 2

Group 3

Group 4

 

 

HGB

[mmol/L]

Males

+0.4

-1.3

-6.0**

B

RBC

[x106/µL]

Males

-0.9

-2.4

-8.5**

B

Reticulocytes

[%]

Males

+5.6

+24.9*

+11.3

A, B

HCT

[%]

Males

+0.7

-0.6

-5.7**

A, B

Neutrophils

[x10³/µL]

Females

-2.3

-1.2

+78.8**

A, B

Monocytes

[x10³/µL]

Females

-2.6

+33.3

+109.4**

A, B

LUC

[x10³/µL]

Females

-13.9

0.0

+111.1*

A, B

aPTT

[seconds]

Males

+0.9

-2.1

-5.9*

A, B

*/**:

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

#1:

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

A:

No dose response-relationship was noted.

B:

No difference was noted for the opposite sex.

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

 

 

Changes in comparison to control

[%]

Reason

Parameter #1

Sex

Group 2

Group 3

Group 4

 

 

Albumin

[g/L]

Males

-3.1

-1.0

-6.4**

A

Females

+2.9

+0.5

-4.0*

A

Bile acids

[µmol/L]

Males

+4.9

-4.4

+67.7**

A, B

Cholesterol (total)

[mmol/L]

Males

+14.7

+20.6

+29.9*

B

BUN/Creatinine

[ratio]

Males

+2.4

-6.6

+23.4**

A

Females

-3.6

-1.4

+18.6**

A

Glucose

[mmol/L]

Males

+7.9

+15.7*

+16.8

B, C

Protein (total)

[g/L]

Males

-3.9

+0.6

-6.5*

A, B

Urea (in blood)

[mmol/L]

Males

+5.0

-7.1

+22.6**

A, B

Calcium

[mmol/L]

Males

-1.2

-0.4

-4.8**

A, B

*/**:

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

#1:

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

A:

No dose response-relationship was noted.

B:

No difference was noted for the opposite sex.

C:

No statistically significant difference was noted for group 4.

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

Macroscopicpost mortemfindings

Group

Animal no.

Observation

Group 1

(Control)

45

Kidney (l. + r.):          -pale

Group 2

(15 mg/kg)

75

78

Uterus:           -dilated

Thorax:          -filled with clear liquid

Lungs:            -dark-red discoloured

Group 3

(50 mg/kg)

142

Uterus:           -dilated

                       -filled with clear liquid

Group 4

(150 mg/kg)

171

182

 

189

Spleen:          -enlarged

Uterus:           -dilated

                       -filled with liquid

Ovary (l. + r.):-cystic

                       -no positive mating sign

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

Stage of estrous cycle

at necropsy

Group 1

Control

Group 2

15 mg/kg

Group 3

50 mg/kg

Group 4

150 mg/kg

Proestrus

1 of 24

2 of 24

2 of 24

4 of 24

Estrus

3 of 24

6 of 24

7 of 24

9 of 24

Metestrus

4 of 24

4 of 24

5 of 24

2 of 24

Diestrus

16 of 24

12 of 24

10 of 24

9 of 24

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

Organ #1

Parameter

(absolute: g)

(relative: g/kg b.w.)

Changes in comparison to control

[%]

Reason

Group 2

Group 3

Group 4

 

Brain

g/kg b.w.

-1.2

-1.0

+6.3*

A, C

Heart

g/kg b.w.

-2.7

-4.0

+4.8**

A, C

Liver

g/kg b.w.

-1.1

-0.8

+10.6*

A, C

Spleen

g/kg b.w.

+0.6

+5.8

+15.8**

A

Testis (left)

g/kg b.w.

-0.6

-0.9

+11.8**

A

Testis (right)

g/kg b.w.

-1.4

-0.7

+11.9**

A, C

Epididymis (left)

g

-0.1

+2.3

-10.5*

B, C

Epididymis (right)

g

-1.9

+2.5

-8.5**

B, C

Prostate and Seminal Vesicle

g

-1.3

-5.6

-15.3**

B

*/**:

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

#1:

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

A:

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

B:

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

C:

No dose dependence-relationship present.

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

Organ #1

Parameter

(absolute: g)

(relative: g/kg b.w.)

Changes in comparison to control

[%]

Reason

Group 2

Group 3

Group 4

 

Brain

g

-0.9

-1.3

-3.6**

A

Liver

g/kg b.w.

-1.1

-1.9

+6.5*

C

Ovary (left)

g/kg b.w.

-4.3

-4.2

-20.0**

B

Ovary (left)

g

-7.5

-4.8

-22.0**

B, C

Ovary (right)

g

+2.7

+0.7

-13.1*

B, C

Thymus

g/kg b.w.

+10.7

+1.5

-16.2**

A, C

*/**:

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

#1:

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

A:

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

B:

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

C:

No dose dependence-relationship present.

Applicant's summary and conclusion

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

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