1,3,3-trimethyl-N-(2-methylpropylidene)-5-[(2-methylpropylidene)amino]cyclohexanemethylamine

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

No developmental toxicity/teratogenicity studies are available for 1,3,3-trimethyl-N-(-2-methylpropylidene)-5-[(2-methylpropylidene)amino]cyclohexanemethylamine. Due to the likely hydrolysis of the substance 1,3,3 -trimethyl-N-(2 -methylpropylidene)-5 -[(2 -methylpropylidene)amino]cyclohexanemethylamine during an in vivo study, to the hydrolysis products isophoron diamine (IPDA) and isobutyraldehyde, the studies on toxicity to reproduction will be waived and a supporting assessement for the hydrolysis products was chosen:

IPDA was administered orally to rats at dose levels of 30, 100 or 300 mg/kg b.w./day. Due to mortality, the high dose level was decreased to 240 mg/kg b.w./day, starting on test day 28.
The NOAEL for general toxicity is considered to be 100 mg/ kg bw/day. The NOAEL for reproductive toxicity based on adverse effects on the reproductive parameters of the parental females is considered to be above 300/ 240 mg/ kg bw/day. Based on adverse effects on prenatal development (conceptus to birth) the NOAEL for reproductive toxicity is above 300/240 mg/ kg bw/ day and based on adverse effects on postnatal development (pup) above 300/240 mg /kg b.w./day.

Based on the ECHA decision the registrant conducted the OECD 443 study with the basic study design with a 10-week pre-mating (Cohorts 1A, and 1B) without extension to mate the Cohort 1 B animals to produce the F2 generation.

In this OECD 443 study (Provivo, 2022) no adverse effect on mating performance, fertility or gestation length was detected.

The oral exposure to 25, 80, and 240/160 mg test item/kg bw (male and female Sprague-Dawley rats) led to no detectable changes in fertility. In detail, no test item-related influence was noted on the reproductive performance of the parental animals (number and length of estrous cycles, fertility and gestation index, pre-coital time and gestation length).

The prenatal development of the pups (number of resorptions, stillbirths and live born pups) and the postnatal development of the pups (pup body weight, viability index, ano-genital distance, nipple retention, thyroid hormone levels, pup organ weights) was also not affected by the test item. No malformations or variations were noted during the macroscopic external and internal examinations of the pups at necropsy.

No test item-related influence was noted on the development of the reproductive system (time points of sexual maturations, number and length of oestrous cycles, sperm parameter, detailed histopathological examination of testis and epididymides, number of primordial and growing follicles and number of corpora lutea in the ovaries).

Therefore, the No Observed Adverse Effect Level (NOAEL) for reproductive was considered to be above 240 mg test item/kg/day.

The No Observed Adverse Effect Level (NOAEL) for systemic toxicity in the F0 and in the adult animals of Cohort 1A and 1B was determined to be 80 mg/kg/day.

Link to relevant study records

Reference

Reference 1

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

Study started on 24th of July 2019 and the end of in-life period was on10 October 2019, final report received on 18th of April 2022.

Reliability:

1 (reliable without restriction)

Justification for type of information:

This OECD 422 study is used as dose range finding study for the OECD 443 study (Provivo, 2021).

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

adopted July 29, 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Justification for study design:

The study was performed as dose range finding study for the EOGRTS (OECD 443).

Species:

rat

Strain:

Crj: CD(SD)

Details on species / strain selection:

Rat / CD® / Crl:CD(SD)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST 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): Males: 411.8 g - 479.2 g, Females: 246.4 g - 278.9 g
- Age (at start of dosing): 70 days (young adults; sexually mature)
- Selection of species: The rat is a commonly used rodent species for such studies.
- Number of animals: Pre-exposure period: 60 female animals will be evaluated pre-exposure for oestrus cyclicity to yield 40 females with a regular oestrus cycle for the study.
Main study: 80 animals (40 males and 40 females) A sufficient number in order to grant at least 8 pregnant females per group for evaluation of the F0 generation.
- Adaption period: 7 days
- Diet (ad libitum): ssniff® R/Z V1324, ssniff Spezialdiäten GmbH, 59494 Soest, Germany
-Drinking water ad libitum
- Housing: With the exception of the mating period, the males and females (F0-Generation) were 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. Except during the mating period the animals were placed in the animal room as follows:
Male animals: On one side of the room with each dose group separated by an empty row.
Female animals: On the other side of the room with each dose group separated by an empty row.
Granulated textured wood released for animal bedding (Granulat A2, J. Brandenburg, 49424 Goldenstedt/Arkeburg, Germany) was used as bedding material in the cages. The cages were changed and cleaned once a week.

ENVIRONMENTAL CONDITIONS
- Temperature: 22°C ± 3°C
- Humidity: 55% ± 10%
- Air changes per hour: 15 to 20
- Photoperiod: 12 hours dark/12 hours light

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

Route of administration: Oral, via gavage
Frequency of administration: Once daily
Vehicle: sterile water (Batch no. 184928001, B. Braun Melsungen AG, 34212 Melsungen)
Application volume: 10 mL/kg b.w./day
Dosages: 0, 30, 100, 300 (240) mg/kg bw/d; High dose reduction as of test day 28, due to mortality and poor general condition of the animals.
Selection of administration route: According to OECD guideline 422
The test item formulations were freshly prepared every day and volumes administered were adjusted to the animal's actual body weight daily.
The test item was suspended in the vehicle and was administered orally at a constant volume once daily.
The control animals received the vehicle at the same administration volume daily in the same way.

Details on mating procedure:

Sexually mature male and female rats are paired monogamously, i.e. 1 male and 1 female animal are placed in one cage during the dark period. The female is placed with the same male until evidence of mating is observed or two weeks have elapsed. The females are examined each morning for the presence of sperm. The day of conception (day 0 of gestation) is considered to be the day on which sperm is found. In case pairing is unsuccessful, re-mating of females with proven males of the same group can be considered after approximately 2 weeks of unsuccessful mating.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

For each test item that is mixed with a vehicle, tests by appropriate analytical methods shall be conducted to determine the concentration, stability and homogeneity of the test item in the formulations.
For the analysis of the test item-vehicle mixtures, samples of 2 x 5 mL are taken as scheduled (and stored at -20°C±10% until analysis:
- At start of the treatment period (first dosing day; test day 15), Analysis of concentration, Immediately after preparation of the administration formulation (1 sample / dose level group; groups 2 - 4). Number of samples: 3
- At dose change (group 4), test day 28, Analysis of concentration, Immediately after preparation of the administration formulation (1 sample of group 4). Number of samples: 1
- Towards the end of the treatment period (when the majority of animals was dosed, test day 48), 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: 7

Results of test item formulation analysis:
Sampling /Percent of nominal concentration [%]
- immediately after preparation at the start of dosing on test day 15: 100.1 % - 102.6 %
- at dose change on test day 28: 101.7 %
- before administration to the last animal on test day 48: 100.2 % - 103.4 %

These results indicated correctly prepared test item vehicle mixtures.

Duration of treatment / exposure:

The study animals will be treated during the following periods:
- Males+ Females: Pre-mating (test days 15 to 29 (pairing was in the evening of test day 29)), Mating (test days 30 to 43 (first mating day, confirmed by positive sperm detection, was in the morning of test day 30))
- Males: Post-mating (until test day 48 (one day before necropsy on test day 49))
- Females: Gestation and lactation period (until test days 64 to 78 (corresponding to lactation days 13 to 15). The last dosing was always one day before necropsy (necropsy was between test days 65 to 79).)

Frequency of treatment:

daily

Details on study schedule:

study schedule according to OECD 422 guideline, s. also "duration of treatment/ exposure"

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

control group

Dose / conc.:

30 mg/kg bw/day (nominal)

Remarks:

low dose group

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

intermediate dose group

Dose / conc.:

300 mg/kg bw/day (nominal)

Remarks:

high dose group; 240 mg/ kg bw/ day as of test day 28, due to mortality and poor general condition of the animals.

No. of animals per sex per dose:

10 males and 10 females

Control animals:

yes, concurrent vehicle

Details on study design:

Justification for dose selection
The dose levels were selected in agreement with the Sponsor based on the results of a 14-day dose range finding study.
In this 14-day dose range finding study the test item was administered orally to rats by gavage at dose levels of 250 or 500 mg/kg b.w./day for 2 weeks. Due to mortality and a reduced body weight, the high dose level was reduced to 350 mg/kg b.w./day on test day 6.
One male animal of the high dose group died at a dose level of 500 mg/kg b.w./day and another male animal after the reduction of the high dose level to 350 mg/kg b.w./day.
Changes in behaviour in the form of salivation (in many cases consistently during the whole day) and breathing sounds were noted for some male and female animals of the low dose group and / or the high dose group (250 or 500/350 mg/kg b.w./day).
Statistically significant reductions in body weight at the end of the first test week (test day 8) were noted for the female animals of the low dose group (250 mg/kg b.w./day) and for the male and female animals of the high dose group (500/350 mg/kg b.w./day). During the second test week a slight recovery of the body weight was noted for all affected sexes and dose levels. However, the values of body weight still remained below the control values.
Necropsy revealed test item related gastrointestinal changes for some male and female animals of the high dose group (500/350 mg/kg b.w./day).
No changes of toxicological relevance were noted for food consumption and no test item-related changes were noted for the haematological or biochemical parameters and the organ weights. Based on these results, the rats of the OECD 422 study were treated orally with 30, 100 or 300 mg IPDA/kg b.w./day.

Positive control:

not needed

Parental animals: Observations and examinations:

CLINICAL SIGNS
Throughout the test period, each animal (parental animals and pups) was observed for clinical signs at least once daily. Behavioural changes, signs of difficult or prolonged parturition, and all signs of toxicity were recorded. Mortality was recorded twice daily. Animals which died prematurely were necropsied as soon as possible after exitus.
Individual animals were observed before and after dosing at each time of dosing for any signs of behavioural changes, 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.

Detailed clinical observations (parental animals):
Additionally, once before the first exposure (to allow for within-subject comparisons) and once a week thereafter, detailed clinical observations were made in all animals of the parental generation. These detailed clinical observations were performed at least 2 hours after dosing.
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). 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), bizarre behaviour (e.g. self-mutilation, walking backwards) were also recorded.
Dated and signed records of appearance, change, and disappearance of clinical signs were maintained on clinical history sheets for individual animals.

VAGINAL SMEARS
Daily monitoring of vaginal smears will continue throughout the premating period until evidence of mating. A vaginal smear will also be taken in the morning of the day of scheduled necropsy.

NEUROLOGICAL SCREENING
Screening of sensory reactivity to stimuli of different types (e.g. auditory, visual and proprioceptive stimuli) (based on Gad ), as well as the assessment of grip strength (Meyer ) and motor activity assessment were conducted as described on the following pages in five males and five females randomly selected from each study group.
The screening was conducted between approx. 2.0 hours and 4.0 hours after dosing and before any blood sampling for laboratory examinations:
5 male F0 animals per group (randomly selected) On test days 44 and 45.
5 female F0 animals per group (randomly selected) Between lactation days 10 to 13.

OBSERVATIONAL SCREENING
Righting reflex
The animal was grasped by its tail and flipped in the air approximately 60 cm above the cart surface so that it turned head over heels. The normal animal should land squarely on its feet; that means zero (0) points were scored. If it landed on its side, 1 point was scored; if it landed on its back, 2 points were scored. This test was repeated five times and the total scores were recorded.

Body temperature
An electronic probe thermometer with a blunt probe was used to take the rectal temperature, being allowed to equilibrate for 30 seconds before the reading was recorded.

Salivation
Discharge of clear fluid from the mouth is most frequently seen as beads of moisture on lips in rats. The normal state is to see none, in which case a zero (0) was recorded in the blank space of the scoring sheet. If present, a plus sign (+) was recorded in the blank.

Startle response
With the animal on the cart, the metal cage was struck with the blunt probe. The normal animal should exhibit a marked but short-lasting response, whereby a zero (0) was recorded in the blank space of the scoring sheet. If there was no response, a plus sign (+) was recorded.

Respiration
While at rest on the cart, the animal's respiration cycle was observed and evaluated in terms of a scale from 1 (reduced) to 5 (increased), with 3 being normal.

Mouth breathing
Rats are normally obligatory nose-breathers. Each animal was observed whether or not it was breathing through its mouth. If the rat was breathing through its nose, a zero (0) was recorded; mouth breathing was documented by a plus sign (+).

Urination
When an animal was removed from its cage, the pan beneath the animal's cage was examined while returning the animal to its cage. The signs of urination were evaluated on a scale of 0 (lacking) to 5 (polyuria) with 3 being normal.

Convulsions
If clinic or tonic convulsions were observed, their intensity was graded on a scale of 1 (minor) to 5 (marked) and the type was recorded. In the normal animal no convulsions should be observed, in which case a score of zero (0) was recorded.

Piloerection
The fur of the animal's back was observed whether it was raised or elevated. In the normal animal no piloerection should be observed and a score of zero (0) was recorded. If piloerection was present, a plus sign (+) was recorded.

Diarrhoea
In examining the pan beneath an animal's cage, it was noted if there were any signs of loose or liquid stools. The normal state is for there to be none (0); in case of diarrhoea the intensity was recorded on a scale of 1 (slight) to 5 (much increased).

Pupil size
It was determined if the pupils were constricted or dilated and the observations were evaluated in terms of a scale from 1 (constricted) to 5 (dilated), with 3 being normal.
 
Pupil response
The beam of light from the pen light was played across the eyes of the animal and the changes in pupil size were noted. In the normal animal, the pupil is constricted when the beam is on it and then dilates back to normal when the light is removed, whereby a score of zero (0) was recorded. If there was no pupil response, a minus sign (-) was recorded in the blank space.

Lacrimation
The animal was observed for the secretion and discharge of tears. The tears of rats contain a reddish pigment. No discharge is normal, whereby a score of zero (0) was recorded in the blank space of the scoring sheet. If a discharge was present, a plus sign (+) was recorded.

Impaired gait
The occurrence of abnormal gait was evaluated. The most frequent impairments are waddling (W), hunched gait (H), or ataxia (A, the inability of all the muscles to act in unison). The extent of any impairment was recorded on a scale of 1 (slight) to 5 (marked). A normal gait was documented by a score of zero (0).

Stereotypy
Each animal was evaluated for stereotypic behaviour (isolated motor acts or partial sequences of more complex behavioural patterns occurring out of context and with an abnormally high frequency). These were graded on a scale of 1 (slight) to 5 (marked). Normal behaviour was documented by a score of zero (0).

Toe pinch
The blunt probe was used to bring pressure to bear on one of the digits of the hind limb. This should evoke a response from the normal animal. The response or lack thereof was graded on a scale from 1 (absent) to 5 (exaggerated) with 3 being the normal response.

Tail pinch
The toe pinch procedure was utilized with the animal's tail instead of its hind limb and was graded on the same scale from 1 (absent) to 5 (exaggerated), with 3 being the normal response.

Wire maneuver
The animal was placed on the metal rod suspended parallel to the cart approximately 60 cm above the cart's surface. The animal's ability to move along the rod was evaluated. If impaired, a score from 1 (slightly impaired) to 5 (unable to stay on wire) was recorded. Normal movement was documented by a score of zero (0).
 
Hind leg splay
The hind paws were marked with ink using an ink pad. The rat was then held 30 cm above a sheet of blotting paper on the cart. The animal was dropped and the distance between the prints of the two hind paws was measured (in cm).

Positional passivity
The animal was observed after being placed in an awkward position, such as on the edge of the top of the wire-bottomed cage on the cart surface. If the animal immediately moved into a normal position, a score of zero (0) was recorded. If not, a score was recorded on a scale of 1 (slightly impaired) to 5 (cataleptic).

Tremors
Periods of continued fine movements, usually starting in the limbs and perhaps limited to them. The normal case is to have none, in which case a score of zero (0) was recorded. If tremors were present, they were graded on a scale of 1 (slight and infrequent) to 5 (continuous and marked).

Positive geotropism
The animal was placed on the inclined (approximately 30°) top surface of the wire cage with its head facing downward. It should turn 180° and face "uphill", in which case a score of zero (0) was recorded in the blank space of the scoring sheet. If this did not occur, a negative sign was recorded in the blank.

Limb rotation
One of the animal's hind limbs was taken and moved through its normal plane of rotation. In the normal state, it should rotate readily but there should be some resistance. The variations from normal were from no resistance (1) to markedly increased resistance or rigidity (5), with 3 being normal.

Auditory function
Each animal was placed in a container and observed for Preyer's reflex (twitching of the pinna) in response to a high frequency sound stimulus. The stimulus was repeated, if necessary, up to 3 times. A normal response was recorded with a plus sign (+); if there was no response a zero (0) was recorded.

FUNCTIONAL TESTS
Grip strength
Prior to testing, the gauge (Chatillon, Modell DPP - 1.0 kg) was calibrated with a set of known weights and the apparatus adjusted for the size of the animal (about 1 cm clearance on both sides of the animal). After the strain gauge was zeroed and set in the record mode, the animal was placed into the trough with the forepaws inside the triangular grasping ring. Using one hand, the animal was grasped about 2.5 cm of the way up toward the base of the tail and steadily pulled (approx. 2.5 cm/sec) away from the ring until the grip was broken. It was continued to pull the animal along the trough until the hind limbs grasp the T-bar. The trial was completed when grip of the hind limbs was broken. Three successive readings were taken for each animal with an intertrial interval long enough to record the data and zero both meters for the next trial.

Locomotor activity
The motility was measured using the TSE InfraMot system . The infrared sensor was placed on the cage and any movements were measured for a duration of 12 min by sensing the body heat image, i.e. the infrared radiation, and its spatial displacement over time.
Any movements within the cage, even brief movement events of only a few milliseconds duration, were detected and included in the activity data.

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..

BODY WEIGHT
The adult animals will be weighed on each day of dosing for dose adjustment and at sacrifice; The report included weekly values for the male animals (starting on test day 15) and the body weight on the day of sacrifice.
Days on which the female body weights are reported:
Pre-mating period: Test days 15, 22, 29
Gestation period: Gestation days 0, 7, 14, 20
Lactation period: Lactation days 1, 4, 8, 13

FOOD AND DRINKING WATER CONSUMPTION
Food intake per rat (g) was calculated using the total amount of food given to and left by each rat in each group on those days:
Study period/ Males /Females
Pre-mating period/ TD15(#1), TD22 and TD29 (#2)/ TD15 (#1), TD22 and TD29 (#2)
Mating period/ None/ None
Gestation period/ Not applicable/ GD0 (#1), GD7, GD14 and GD20 (#2)
Lactation period/ Not applicable/ LD1 (#1), LD8 and LD13 (#2)
#1: Days on which only the amount of food at food start was weighed.
#2: Days on which only the amount of food at food residue was weighed.
On the other days, the amount of food at food residue, followed by food start was weighed.
From these data the relative food consumption (in g/kg b.w./day) was determined using the following formula:
Relative food consumption (g/kg b.w./day)= ((Total food given (g) - Total food left (g))/ (Number of animal days# x Body weight (kg))
# The term 'animal days' counts one animal day for each animal alive for a whole
day; it is assumed that on the day of death an animal does not eat.

Drinking water consumption was monitored daily by visual appraisal throughout the study.

REPRODUCTIVE PERFORMANCE
The following parameters and indices were evaluated:
Reproductive parameters
- stages of the estrous cycle
- pre-coital time
- gestation length calculated from day 0 of pregnancy
Implantation sites
- number per dam
- distribution in the uterine horns (left or right)
- absolute number per group
- mean per group
Number of pups per group and per dam
- at birth (live and dead)
- on postnatal days 1, 4 and 13
Number of male and female pups per group and per dam
- at birth (alive and dead)
- on postnatal days 1, 4 and 13
Number of stillbirths
- per group
- per dam
Number of pups with malformations (see Appendix 4)
- per group
- per dam

LABORATORY EXAMINATIONS
Blood samples were taken from the retrobulbar venous plexus under isoflurane anaesthesia from animals fasted overnight at the following times:
At study termination (before necropsy) 5 male and 5 female F0 animals randomly selected from each group.
The blood samples collected were divided into tubes as follows:
- EDTA anticoagulant (whole blood) -> for haematological investigations
- Citrate anticoagulant (plasma) -> for coagulation tests
- Li-Heparin anticoagulant (plasma) -> for clinical chemistry tests

HAEMATOLOGY
The parameters listed below were determined (Instrument: ADVIATM 120, Siemens Diagnostics GmbH, 35463 Fernwald, Germany):
Parameter/ Units
Differential blood count (relative)/ %
Differential blood count (absolute)11/ 10^3/µL
Erythrocytes (RBC)/ 10^6/µL
Leucocytes (WBC)/10^3/µL
Haematocrit value (PCV or HCT)/ %
Haemoglobin content (HGB)/ mmol/L
Platelets (PLT)/ 10^3/µL
Reticulocytes (Reti)/ % of erythrocytes
Mean corpuscular volume (MCV)/ fL
Mean corpuscular haemoglobin (MCH)/ fmol/L
Mean corpuscular haemoglobin concentration (MCHC)/ mmol/L
Following the haematological examinations using the ADVIA system, blood smears were prepared from all samples, dried and stained for possible histopathological examinations in case of pathological findings (see section 4.2 'Histopathology').
 
COAGULATION
The parameters listed below were determined (Instrument: Amax Destiny Plus™, Tcoag Deutschland GmbH, 32657 Lemgo, Germany):
Parameter/ Units
Prothrombin time (PT)/ sec
Activated partial thromboplastin time (aPTT)/ sec

BIOCHEMISTRY
The parameters listed below were determined (Instrument: KONELAB 30i, Thermo Fisher Scientific, 63303 Dreieich, Germany):
Parameter/ Units
Sodium/ mmol/L
Potassium/ mmol/L
Calcium/ mmol/L
Chloride/ mmol/L
Albumin/ g/L
Total bilirubin/ µmol/L
Total cholesterol/ mmol/L
Glucose/ mmol/L
Total protein/ g/L
Blood urea (BUN)/ mmol/L
Creatinine/ µmol/L
Alanine aminotransferase (ALAT/GPT)/ U/L
Alkaline phosphatase (aP)/ U/L
Aspartate aminotransferase (ASAT/GOT)/ U/L
Bile acids/ µmol/L
Lactate dehydrogenase (LDH)/ U/L
Globulin/ g/L -> by subtraction
Albumin / Globulin ratio/ on-dimensional-> by calculation

THYROID HORMONE (T4) DETERMINATION
Blood samples were taken under isoflurane anaesthesia from animals fasted overnight always at the same time of day (in the morning between 7.30 a.m. and 9.30 a.m. for the adult animals) as scheduled below.
Blood withdrawal was performed by randomization of the parental male and female animals. The male animals of all test groups were completely randomized in a one- step process, the female animals in different staggers according to their litter day.

Animals/ Time of sampling/ Number of samples/ Feeding status/ Analysis T4/ Sample Volume
All evaluated dams/ At scheduled sacrifice/ 32/ Fasted/ Not yet/ 6x 100 µl
All adult males/ At scheduled sacrifice/ 38/ Fasted/ Yes/ 6x 100 µl

Blood samples were processed for serum, divided into aliquots and stored at - 20°C ± 10 % at the Test Facility.
The T4 ELISA (Total Thyroxine (T4) ELISA, IBL INTERNATIONAL cat. no. RE55261; batch no. 304K079; Instrument: Tecan Sunrise) was conducted at the test institute.
Additional serum samples will be analysed for different hormones (T3 and / or TSH) based on findings and only upon agreement with the Sponsor.

Oestrous cyclicity (parental animals):

During a 14-day pre-exposure period, the oestrus cycle of the female animals will be monitored to select 40 animals with regular oestrus cycles. Animals that fail to exhibit typical 4-5 day cycles will not be included in the study.
Daily monitoring of vaginal smears will continue throughout the premating period until evidence of mating.
A vaginal smear will also be taken in the morning of the day of scheduled necropsy.

Sperm parameters (parental animals):

Detailed histopathologic examination was performed on one testicle and one epididymis (with special emphasis on the qualitative stages of spermatogenesis and histopathology or interstitial testicular structure) of the selected males of groups 1 and 4 following H-E and PAS staining.

Litter observations:

CLINICAL SIGNS
Daily observations (parental animals and pups)
Throughout the test period, each animal (parental animals and pups) was observed for clinical signs at least once daily. Behavioural changes, signs of difficult or prolonged parturition, and all signs of toxicity were recorded. Mortality was recorded twice daily. Animals which died prematurely were necropsied as soon as possible after exitus. Individual animals were observed before and after dosing at each time of dosing for any signs of behavioural changes, 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.

EXAMINATION OF THE 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:

COUTING, SEXING AND WEIGHING
Live pups were counted, sexed and weighed on post-natal days (lactation days) 1, 4 and 13.

ANO-GENITAL DISTANCE
On post-natal day 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 by eliminating (culling) surplus pups using a randomization scheme generated by Provantis®.

BLOOD SAMPLING FOR THYROID HORMONE (T4) DETERMINATION
On PND 4 and on PND 13 blood samples for T4 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.
Determination of the pups used for blood withdrawal:
On lactation day 4 (PND4) and lactation day 13 (PND13) the litter sequence of pup blood withdrawal was determined by randomization of the dams. The collection of the pups for blood withdrawal from the individual litters occurred in an ascending order (the male and female pups per dam with the lowest number were used, if possible).
Animals/ Time of sampling/ Number of samples/ Feeding status/ Analysis T4 /Sample Volume
Pups (at least 2 surplus pups per litter, all litters)/ PND 4/ 57/ Non-fasted/ Not yet/ 1x 75 µl
Pups (at least 2 per litter, all litters)/ PND 13/ 64/ Non-fasted/ Yes/ 1x 75 µl (for T4), 1x 70 µl, 1x 100 µl
#1: 57 culled pups from altogether 32 litters (dam no. 17 with only 1 culled pup and dam nos. 60, 74 and 78 with zero culled pups).

Blood samples were processed for serum, divided into aliquots and stored at - 20°C ± 10 %.
The T4 ELISA (Total Thyroxine (T4) ELISA, IBL INTERNATIONAL cat. no. RE55261; batch no. 304K079; Instrument: Tecan Sunrise) was conducted at the test institute.
Additional serum samples will be analysed for different hormones (T3 and / or TSH) based on findings and only upon agreement with the Sponsor.

MALE NIPPLES COUNTING
Nipples were counted in all male pups on PND 13 (shortly before scheduled sacrifice).

Postmortem examinations (parental animals):

GROSS NECROPSY
Vaginal smears were examined on the day of necropsy to determine the stage of the oestrus cycle and allow correlation with the histopathology of the female reproductive organs.
The animals were euthanized by carbon dioxide (CO2) inhalation, exsanguinated by cutting the aorta abdominalis, weighed, dissected and inspected macroscopically (gross necropsy) at the following times:
Males: On test day 49
Dams: On test days 65 to 79 (corresponding to lactation days 14 to 16)

DISSECTION OF ADULT ANIMALS
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.

ORGANS WEIGHED
The weight of the following organs of all adult male and female animals was determined. Paired organs were weighed individually and identified as left or right.
Adrenal gland (left and right)
Spleen
Brain
Testicle (left and right)
Epididymis (left and right)
Thyroid (left)
Heart
Thymus
Kidney (left and right)
As a whole: prostate, seminal vesicles
Liver with coagulating glands
Ovary (left and right)
Uterus including cervix
The weights of the organs were determined before fixation. Only the weight of the thyroid glands was determined after fixation.

ORGANS FIXED FOR PRESERVATION
The following organ(s) or parts thereof of all adult male and female animals were fixed in modified Davidson's solution or 7% buffered formalin:
Fixative: modified Davidson's solution
Epididymis (left and right)
Testicle (left and right)
Fixative: 7 % buffered formalin
Gross lesions observed
Thyroid (including parathyroids)
Ovary and oviduct (left and right)
Uterus (including cervix)
Prostate Vagina
Seminal vesicles with coagulating glands
Any other organs displaying macroscopic changes were also preserved.

SELECTION OF ANIMALS AND ORGANS FOR HISTOPATHOLOGY
The 5 male and female animals from each group were randomly selected for histopathology examination.
Telected parental animals for histopathological examination.
Group Randomly selected animals
and prematurely deceased or sacrificed animals
Group Males no. Females no.
1 2, 3, 5, 6, 9 12, 13, 15, 17, 19
2 23, 24, 27, 28, 30 34, 35, 36, 37 , 38
3 41, 42, 43, 44, 45 52, 54, 56, 58, 60
4 61, 64#, 65, 66, 67#, 68, 69 71, 72, 73, 74, 75#, 77#, 79#, 80
#: Prematurely deceased or sacrificed animals (no histopathological examination could be performed for animal no. 75, as no organs were preserved at necropsy, see section 2.13 ‘Study Plan deviations‘).
The organs or parts thereof from the animals listed above that were fixed for histopathology examination are listed below:
Fixative: Davidson's solution
Eye with optic nerve (2)
Fixative: modified Davidson's solution
Epididymis (2)
Testicle (2)
Fixative: 7 % buffered formalin
Adrenal gland (2)
Nerve (sciatic)
Bone
Oesophagus
Bone marrow (os femoris)
Ovary and oviduct (2)
Brain (cerebrum, cerebellum, brain stem (pons))
Pituitary
Gross lesions observed
Prostate
Heart (3 levels: right and left ventricle, septum)
Seminal vesicles with coagulating glands
Intestine, small (duodenum, jejunum, ileum, incl. Peyer's patches, Swiss roll method)
Spinal cord (3 sections)
Intestine, large (colon, rectum)
Spleen
Kidney and ureter (2)
Stomach
Liver
Thyroid (including parathyroids)
Lungs (with mainstem bronchi and bronchioles, preserved by inflation with fixative and then immersion)
Thymus
Lymph node (1, cervical)
Trachea (including larynx)
Lymph node (1, mesenteric)
Urinary bladder
Mammary gland
Uterus (including cervix)
Muscle (skeletal)
Vagina

HISTOPATHOLOGY
ANIMALS TO BE EXAMINED AND PREPARATION OF SLIDES
Full histopathology was performed on the preserved organs of the selected parental animals of groups 1 and 4, and the thyroids of the selected pups.
Due to test item-related findings, stomachs of the selected male and female animals and the kidneys of the selected male animals of groups 2 and 3 were additionally examined histopathologically.
The organs listed above were examined histologically after preparation of paraffin sections and haematoxylin-eosin staining. Parathyroids cannot always be identified macroscopically. They were examined microscopically if in the plane of section and in all cases where they are noted were grossly enlarged.
In addition, frozen sections of the heart, liver and one kidney were prepared, stained with Oil Red O and examined histologically.
Detailed histopathologic examination was performed on one testicle and one epididymis (with special emphasis on the qualitative stages of spermatogenesis and histopathology or interstitial testicular structure) of the selected males of groups 1 and 4 following H-E and PAS staining.
The blood smears prepared from all animals during the haematological examination were available for possible examination of pathological changes but examined and evaluated only depending on necropsy findings and upon agreement with the Sponsor. So far, no examination was performed.

HISTOPATHOLOGICAL EVALUATION
The histotechnique was performed by the test institute. The slides (labelled with study number, species, animal number, block number) were dispatched to AnaPath Services GmbH on November 26, 2019 and on February 27, 2020 (additional examinations). The transport of the slides for the histopathology work to AnaPath Services GmbH was arranged by the Test Facility, whereas the return transport to the Test Facility will be arranged by AnaPath Services GmbH.

Postmortem examinations (offspring):

The animals were euthanized by carbon dioxide (CO2) inhalation, exsanguinated by cutting the aorta abdominalis, weighed, dissected and inspected macroscopically (gross necropsy) at the following times: Pups: On PND 13

Dead pups and pups sacrificed at day 13 post-partum were carefully examined externally for gross abnormalities. The external reproductive genitals were examined for signs of altered development.
On lactation day 13, the thyroid of 1 male and 1 female pup from each litter was fixed in 7% formalin. In summary, 64 thyroids were selected from 32 litters. The same pups were used for T4 ELISA sampling.

Statistics:

DATA ACQUISITION
The following data were captured or calculated by the departmental computerized system (Provantis® integrated preclinical software, version 10.2.1, Instem LSS Ltd):
Parental clinical signs, body weight, body weight gain, food consumption, haematological and biochemical parameters.
Raw data not fully compatible with the computerized system (e.g. data from the neurological screening or pup data) were maintained on paper according to the appropriate SOPs.
Data maintained on paper (e.g. data from the neurological screening or pup data) was entered in Provantis in a retrospective manner using the laboratory records according to the appropriate SOPs.

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).

NON-PARAMETRICAL DATA
No statistical evaluation of non-parametrical values (reproductive group indices or macroscopic and microscopic findings) was performed.
Significantly different data are indicated in the summary tables of the result sections of the report.

Reproductive indices:

The following indices were calculated for each group:
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:
Birth Index [%] #1 = (Total number of pups born (alive + dead)/Number of implantation sites) x 100
Live Birth Index [%] = (Number of pups alive on day 0/1 of lactation/ Total number of pups (alive + dead)) x 100
Viability Index [%] pre-cull = (Number of pups alive on day 4 (pre cull)/ Number of pups alive on day 0/1) x 100
Viability Index [%] post cull = (Number of pups alive on day 13/ Number of pups alive on day 4 (post cull)) x 100
Post-implantation loss [%]#1 = ((Implantations - number of pups born alive)/ Implantation sites) x 100
#1: Twins (shared placentae) are considered as additional implantation sites in the calculation of the birth index and the post-implantation loss, to avoid a birth index above 100% or a negative post-implantation loss.

Offspring viability indices:

Birth and live birth index, post-impantation loss and viability indices of the pups

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Males: No observations were noted for the surviving male animals. Females: At 300/240 mg/kg b.w./day salivation, haemorrhagic nose/snout, a reduced motility, breathing sounds and / or piloerection were noted for 3 of 7 surviving females during the gestation and / or lactation period on one or several test days. Start and duration: Salivation started immediately to 5 min after administration and disappeared again between 20 and 60 min after administration. The reduced motility which was noted for female no. 71 during the gestation period started in the first 5 min after administration and disappeared between 20 and 60 min after administration.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, treatment-related

Description (incidence):

Males: None of the males treated with 30 or 100 mg/kg b.w./day died prematurely. At 300/240 mg/kg b.w./day two premature deaths were noted. Male no. 67 was found dead on test day 41 and male no. 64 was found dead on test day 44 (12 or 15 days after dose reduction). Both premature deaths were considered to be test item-related. A loss of body weight, breathing sounds, a reduced motility and / or reduced faeces were noted on several days before death. Histopathology revealed test item-related changes in the stomach (e.g. erosion/ulcer) and the kidneys (e.g. tubular cell degeneration, tubular basophilia) for male no. 67 (but not for male no. 64).
Females: None of the females treated with 30 or 100 mg/kg b.w./day died prematurely.
At 300/240 mg/kg b.w./day two females (no.75 and no. 77) were prematurely sacrificed on test days 27 or 29 (one day before or one day after dose reduction on test day 28) due to a poor general condition. A loss of body weight, breathing sounds, reduced motility, piloerection, laboured breathing and / or gasping were noted several days before death. Histopathology revealed test item-related changes in the stomach (e.g. erosion/ulcer) and the kidneys (e.g. tubular cell degeneration, tubular basophilia) for female no. 77. No organs were examined for no. 75. A further high dosed female (no. 79) was prematurely sacrificed on test day 48 after misgavage.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Males: At 300/240 mg/kg b.w./day, a slightly, statistically not significantly, reduced body weight was noted during the course of the study (at maximum 6.4 % below the control group on test day 36). This was due to the 2 prematurely deceased high dosed males (premature death on test days 41 and 44). On test day 48 (last live body weight before necropsy), when only the surviving high dosed males were left, no differences between the high dose group and the control group were noted anymore. Body weight gain: A reduced body weight gain was noted at the high dose level for the period between test days 15 and 36, when both prematurely deceased animals were still in the study. For the period between test days 15 and 48, when both prematurely deceased animals were no longer present, no differences in body weight gain were noted between the animals of the high dose group and the control group.

Females:
At 300/240 mg/kg b.w./day, a minimal reduction in body weight was noted during the pre-mating period on test day 22 (3.9 % below the control; statistically not significant), due to the 2 females that were prematurely sacrificed at the end of the pre-mating period on test days 27 and 29. The surviving high dosed females revealed reduced body weights on gestation day 7 and lactation days 8 and 13 (5.9 %, 11.7 % and 5.7 % respectively below the control; statistically not significant). Body weight gain: Accordingly, slightly reduced body weights were noted at the high dose level during the gestation and the lactation period.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Males: No adverse effects on food consumption were noted. Slight, but statistically significant reductions in food consumption that were noted at the high dose level during the first treatment week were considered to be test item-related but not adverse. Females: No test item-related effect on food consumption was noted.

Food efficiency:

no effects observed

Description (incidence and severity):

s. "food consumption"

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 was noted for the male and female rats treated with 30, 100 or 300/240 mg/kg b.w./day by visual appraisal.

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

Males: No test item-related differences for the examined haematological parameters were noted between the control group and the treatment groups (30, 100 or 300/240 mg/kg b.w./day).
Statistically significantly increased values were noted for the percentage of reticulocytes at the low and at the intermediate dose level (62.2% or 51.5% above the control; p ≤ 0.05). However, with one exception from the low dose group (no. 24 with 4.6% reticulocytes), all individual values for the reticulocytes of the low dose group (2.8 to 4.6%) and the intermediate dose group (2.6 to 3.6%) were within the range of the background data (1.6% to 3.9%). Therefore, and because no dose response relationship was noted (the mean value of the high dose group was only 7.3% above the control, statistically not significant) the changes were considered to be spontaneous.
Females: No test item-related differences for the examined haematological parameters were noted between the control group and the low and the intermediate dose group (30 or 100 mg/kg b.w./day).
Decreased values were noted for the number of lymphocytes in the low, the intermediate and the high dose group (35.3%, 39.9% or 21.4% below the control, statistically significant at the low and the intermediate dose group at p ≤ 0.05). However, with one exception at the low dose level, all individual values were within the range of the background data. Furthermore, as no dose response-relationship was noted, the decreased numbers of lymphocytes were considered to be spontaneous.
At the high dose level (300/240 mg /kg b.w./day), a statistically significantly increased number was noted for neutrophilic granulocytes (186.1% above the control, p ≤ 0.01). The numbers of neutrophilic granulocytes from 4 (2.74 to 5.28 x10E3 cells/µL) of 5 high dosed females were above the background range (0.41 to 1.68 x10E3 cells/µL). In contrast, with one exception at the low dose level, all individual values of the control group, the low and the intermediate dose group were within the range of the background data.
Hence, the increased number of neutrophilic granulocytes that was noted for the females of the high dose group was considered to be test item-related. This was considered a consequence of the severe stomach erosion/ulcer due to the corrosivity of the test item. Hence, the increased number in neutrophilic granulocytes was considered as a secondary effect to the test item treatment.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

Males: No test item-related differences for the examined biochemical parameters were noted between the control group and the treatment groups (30, 100 or 300/240 mg/kg b.w./day).
Increased cholesterol concentrations were noted at the low, the intermediate and the high dose level (53.1%, 29.2% or 16.5% above the control; statistically significant at p ≤ 0.01 at the low dose level). However, only one individual value each of the low and the high dose level was above the background data. All other individual values were within the background range. Furthermore, no dose response-relationship was noted. Hence, the statistically significantly increased cholesterol concentration at the low dose level was considered to be spontaneous.
Females:
No test item-related differences for the examined biochemical parameters were noted between the control group and the low and the intermediate dose group (30 or 100 mg/kg b.w./day).
A statistically significantly increased LDH activity was noted at the high dose level (300/240 mg /kg b.w./day). In detail, 2 of 5 individual values (no. 73 with 211 U/L and no. 80 with 267 U/L) were above the background range (32 – 159 U/L). For both animals (nos. 73 and 80) the histopathological finding in the stomach with necrotic cellular/tissue debris (no. 73) or intestinal metaplasia (no. 80) can be regarded as the cause for the LDH increase for these animals. Hence, the increased LDH activity was considered a secondary effect to the test item treatment.

Endocrine findings:

no effects observed

Description (incidence and severity):

T4 serum level (test day 49), Males: No test item-related changes were noted between the control group and the treatment groups (30, 100 or 300/240 mg/kg b.w./day) for the T4 levels of the parental males.
A statistically significantly decreased T4 concentration was noted at the intermediate and the high dose level (18.4% or 17.9% below the control; p ≤ 0.01 or 0.05).
One individual value of the low (no. 27 with 37.684 nmol T4/L), one of the intermediate (no. 43 with 38.123 nmol T4/L) and 2 values of the high dose group (nos. 65, 70 with 42.529 or 41.446 nmol/L) were slightly below the background data range (42.661 to 90.506 nmol T4/L).
However, as from the high dose group only 2 values were slightly below the background range and no changes were observed for the thyroids during the histopathological examination, the statistically significantly decreased T4 concentrations that were noted at the intermediate and the high dose levels were considered to be spontaneous.
Furthermore, no influence was noted on the thyroid weight and no changes were noted during the histopathological examination of the thyroid glands from the high dosed animals. It is generally known that histopathological examination of the thyroid is usually more sensitive than thyroid weight and hormone levels . The validation report of OECD 407 states that “thyroid histopathology was consistently the most reliable and most sensitive endpoint for the detection of thyroid modulation. Thyroid weigth was reliable, but was somewhat less sensitive when compared to thyroid histopathology. Circulating thyroid hormone levels (T3, T4, and TSH) were not always reliable and sensitive, but the standard operating procedures for blood sampling and for thyroid hormone analyses were not standardized to reduce stress induced variability and to reduce analytical variability, respectively. Circulating T4 levels were the most promising of the three thyroid hormonal values.

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

Males and females: No test item-related observations of abnormal behaviour, no adverse effects on motoric skills, changes in the external appearance and the appearance of the faces were noted for the male and female animals of all treatment groups (30, 100 or 300/240 mg/kg b.w./day), approx. 2 hours after treatment.
Furthermore, no test item-related differences were noted in body temperature or the hind-leg splay in comparison to the control group.
Grip strength, Males and females:
No test item-related influence on the fore- and hindlimb grip strength was noted for the male and female animals of all treatment groups (30, 100 or 300/240 mg/kg b.w./day), approx. 2 hours after treatment.
spontaneous motility, Males and females: No test item-related influence on the spontaneous motility was noted for the male and female animals of all treatment groups (30, 100 or 300/240 mg/kg b.w./day), approx. 2 hours after treatment.

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):

Males and females: At the high dose level (300/240 mg/kg b.w./day), microscopic changes that could be attributed to treatment with the test item were observed in the stomach (male and female animals) and in the kidneys (male animals only).
Stomach (male and female animals):
In both sexes of group 4, forestomach and/or glandular stomach lesions were observed, which included forestomach erosion/ulcer, glandular stomach erosion, and inflammation in the forestomach or the glandular stomach. Intestinal metaplasia of glandular stomach, glandular dilatation and squamous cell hyperplasia were also observed as the secondary changes to the ulcerative and inflammatory lesions.
The stomach lesions recorded in both sexes of the high dose group were most likely due to local effect associated with the corrosive property of the test item, and were considered not to be due to systemic effects of the test item.
No stomach lesions were observed for the additionally examined stomachs from the male and female animals of the low and the intermediate dose group (30 or 100 mg/kg b.w./day).
Kidneys (male animals only):
Tubular basophilia and mononuclear cell foci with a minimal severity grade were noted for a few or several male and female animals of the control group and the high dose group, as well as the additionally examined kidneys from the male animals of the low and the intermediate dose groups. However, an increased severity grade was noted for the male animals from the high dose group. Due to the increased severity grade (slight instead of minimal) the kidney observations were considered as test item-related for the male animals of the high dose group.
As described above, only a minimal severity grade was noted for the above mentioned kidney findings for the surviving female animals. Only for the prematurely deceased high dosed female no. 77 tubular basophilia of a moderate grade was noted and considered to be test item-related
In addition, pyelonephritis was observed in 2 males of group 4. Pyelonephritis could arise occasionally in rats as spontaneous lesions. However, in the present study, this lesion was found in 2 of 5 males (40%) in the high dose group. Due to higher incidence of it, the possible relationship of pyelonephritis with the treatment could not be denied.
No test item-related kidney changes were noted for the additionally examined kidneys from the male animals of the low and the intermediate dose group (30 or 100 mg/kg b.w./day).
Reproductive organs (males and females):
There was no histological evidence of toxicity in the reproductive organs including testes, epididymides, prostate gland, seminal vesicles, coagulating glands, ovaries, uterus, cervix and vagina.
Detailed examination for testes:
Testes were also checked on completeness of cell populations and stages, while taking account into the interstitial cell structure and presence/absence of any degenerative changes. As a result, no treatment-related effects on the testicular histomorphology were observed.

Histopathological findings: neoplastic:

no effects observed

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

No test item-related influence was noted during the examination from test day 15 until a positive mating sign was noted. Estrous stage at necropsy: A cycle stage of diestrus was mostly noted for the females of the control group and the treatment groups

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

Testes were checked on completeness of cell populations and stages, while taking account into the interstitial cell structure and presence/absence of any degenerative changes. As a result, no treatment-related effects on the testicular histomorphology were observed.

Reproductive performance:

no effects observed

Description (incidence and severity):

FERTILITY: No test item-related influence on the fertility index of the female rats was noted for any of the treatment groups (30, 100 or 300/240 mg /kg b.w./day).
One non-pregnant female each was noted at the low (no. 32) and the high dose level (no. 76), though a positive mating sign (sperm detection) was noted for both animals. A non-pregnant animal despite a positive sperm detection per group is in the range of normal variability and considered to be spontaneous.
At the intermediate dose level two non-pregnant animals were noted. For one of these non-pregnant animals a positive mating sign was noted (no. 51), whereas for the other non-pregnant animal (no. 55) no positive mating sign was noted after a mating period of 14 days. A non-pregnant animal without a positive mating sign (non-mated animal) is rare in comparison to non-pregnant animals with a positive mating sign. This could be related to the male animal or the behaviour of the female animal. However, as only one such case was noted and without a dose response-relationship, the occurrence of this non-mated animal was considered to be spontaneous. The number of 7 pregnant females is below the recommendation in the OECD guideline 422 of at least 8 pregnant females. However, the OECD guideline 422 states, that the number of pregnant females may be lower 'in case of marked toxic effects'. As two female animals were noted in a moribund condition that led to a decrease of the high dose level, this exception was met. Therefore, the number of 7 pregnant females out of the remaining 8 females does not affect the validity of the study.
GESTATION INDEX:
No test item-related influence on the gestation index was noted for the female rats of the treatment groups (30, 100 or 300/240 mg/kg b.w./day).
All pregnant females of the treatment groups delivered live pups (the pregnant high dose female no. 79 which deceased on gestation day 17 due to a misgavage was excluded), leading to a gestation index of 100% for all treatment groups.
Only in the control group one female was noted with a total resorption of all implantation sites, leading to a gestation index of 90 % in the control group.
PRE-COITAL TIME: No test item-related influence was noted.
GESTATION LENGTH: No test item-related influence was noted.

General toxicity
Parental male and female animals
At 300 mg/kg b.w./day 2 females were prematurely sacrificed due to a poor general condition, one female one day before and the other female one day after dose reduction.
At 300/240 mg/kg b.w./day 2 male animals were found dead 12 or 15 test days after dose reduction.
At 300/240 mg/kg b.w./day a few of the surviving females showed salivation, a haemorrhagic nose/snout, piloerection, breathing sounds and / or reduced motility during the gestation and or lactation period. No changes in behaviour, the external appearance or the faeces were noted for the surviving male animals of the high dose group.
A marked body weight loss was noted at 300/240 mg/kg b.w./day for the prematurely deceased or sacrificed male and female animals.
The surviving female animals showed a slightly reduced body weight in comparison to control during the gestation and the lactation period. This and the observed general bad condition of the females are signs of maternal toxicity. No differences in body weight were noted for the surviving high dosed male animals.
At 300/240 mg/kg b.w./day an increased number of neutrophilic granulocytes and an increased LDH activity were noted for the female animals. Both were the secondary effects due to the corrosivity of the test item, which is manifesting in the very severe histopathological observations of erosions/ulcer in the female animals. No test item-related differences for the haematological and biochemical parameters were noted for the male animals.
The macroscopic examination at necropsy revealed kidney changes for 2 of the 8 surviving male animals of the high dose group (300/240 mg/kg b.w./day) that could possibly be test item-related.

Kidney changes that were considered to be test item-related were noted during the microscopic examination for the surviving male animals of the high dose group (300/240 mg/kg b.w./day), as well as for one of the prematurely deceased male animals and one of the prematurely sacrificed female animals, but not for the surviving female animals.
Test item-related changes for the surviving animals from both sexes were noted during the microscopic examinations of the stomachs from the male and female animals of the high dose group (300/240 mg/kg b.w./day) in form of an erosion of the stomach wall and an inflammatory response. These stomach changes were considered as local and secondary effects that were caused by the corrosive properties of the test item.
The additional microscopic examinations of the kidneys from the male animals and the stomachs from the male and female animals of the low and the intermediate dose groups revealed no test item-related changes.
No test item-related influence or adverse effects were noted for the male and female animals on food consumption, on the parameters of the neurological screening, for the organ weights and on the T4 serum levels of the male animals.

Reproductive toxicity
Parental females
No influence was noted on the number and length of the estrous cycles, 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:

> 240 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

reproductive performance

Remarks on result:

other: 300/ 240 mg/ kg bw/ day

Key result

Dose descriptor:

NOAEL

Effect level:

100 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

mortality

gross pathology

histopathology: non-neoplastic

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

240 mg/kg bw/day (nominal)

Organ:

kidney

stomach

Treatment related:

yes

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

Mortality / viability:

no mortality observed

Description (incidence and severity):

VIABILITY INDEX: Pre- and post-cull period: No test item-related differences between the control group and the treatment groups (30, 100 or 300/240 mg/kg b.w./day) were noted for the viability indices between lactation days 0/1 and 4 (pre-cull) and lactation days 5 and 13 (post-cull).
Overall, 4 prematurely deceased pups were noted during the pre-cull period (3 prematurely deceased pups in the control group and 1 in the high dose group. No prematurely deceased pups were noted during the post-cull period.
NUMBER OF LIVE PUPS: A slightly (statistically not significantly) reduced mean number of live pups per dam was noted at the high dose level (300/240 mg/kg b.w./day) on lactation days 1 and 4.
This was due to the high number of stillbirths at the high dose level (altogether 9 stillbirths from dam nos. 78 and 80 in comparison to 1 stillbirth in the control group). This led to a reduction from 14.3 pups born alive and dead per dam to 12.8 live born pups per dam in the high dose group. In comparison, in the control group 14.6 live born pups per dam were noted from 14.7 pups that were born alive and dead per dam.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

BODY WEIGHT: No test item-related difference was noted between the mean body weight of the pups from the dams of the control group and the mean body weight of the pups from the treatment groups (30 or 100 mg/kg b.w./day) on lactation days 1, 4 and 13.
However, on PND13 a decreased pup body weight was noted at the high dose level (300/240 mg/kg b.w./day). In detail, the mean pup body weight (mean value from the mean pup body weights from each dam) of the male and female pups combined was 7.2% below the control group (statistically not significant). This was due to the decreased pup body weights of the high dose female nos. 74 and 80 with 8 pups each on PND 13. The mean pup body weights of these 2 females (22.93 g for no. 74 and 19.83 g for no. 80) were clearly below the mean pup body weights of the remaining 4 high dose dams (between 28.10 g and 34.30 g). Furthermore, the mean pup body weights of these 2 dams (nos, 74, 80) were also clearly below the range of the control group (27.48 g to 32.00 g) and the background range (24.34 to 32.75 g).
The observation, that 2 of 6 high dosed females showed a clearly reduced mean pup body weight was considered to be not test item-related. The decreased pup body weight of the dams nos. 74 and 80 is a secondary effect, due to the observed maternal toxicity. Both dams showed an overall lower body weight between the end of the gestation and the lactation period compared to the other four dams of the high dose group. Furthermore, the mean pup body weights of the other 4 females of the high dose group (28.10 to 34.40 g; each dam with 10 pups on PND13) were clearly in the range of the background data on PND13 or even above (24.34 to 32.75 g). RUNTS: One runt was noted in the control group (no. 12-17).

LITTER WEIGHT: No test item-related differences in litter weight were noted between the control group and the low and the intermediate dose groups (30 or 100 mg/kg b.w./day).
At the high dose level (300/240 mg/kg b.w./day) a reduced litter weight (male and female pups combined) was noted on lactation days 1, 4 and 13 (20.5%, 19.2% and 13.7%, respectively below the control value, statistically not significant).
In detail, on lactation day 1 the litter weights from 2 of 6 high dosed dams (nos. 74 with 63.7 g and no. 78 with 66.9 g) were below the lower range of the background data on lactation day 1 (70.3 g – 122.6 g). This was due to a slightly reduced number of live pups on lactation day 1 for dam nos. 74 and 78 (8 or 9 pups in comparison to 12 to 19 pups for the remaining high dosed dams).
After the adjustment of the pup number per dam due to the culling process, the remaining difference in litter weight between the high dose group and the control group was due to the reduced pup body weight that was noted for the pups of the high dose group on lactation day 13. As discussed above, this was due to the reduced pup body weights of dam nos. 74 and 80.
Due to the increased male to female ratio, the difference in litter weight between the control group and the high dose group was mostly pronounced for the female pups.

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:

no effects observed

Description (incidence and severity):

No test item-related differences between the control group and the treatment groups (30, 100 or 300/240 mg/kg b.w./day) were noted for the T4 levels of the male and female pups.
However, at the intermediate and the high dose level statistically significantly increased T4 levels were noted for the female pups (16.7% or 20.5% above the control, p ≤ 0.05), whereas no statistically significant changes were noted for the male pups. For the male and female pups combined a statistically significantly increased T4 level was only noted at the high dose level (15.7% above the control, p ≤ 0.05).
As all individual values were in the range of the background data, the observed increased T4 concentrations at the intermediate and the high dose level were considered to be spontaneous. Furthermore, no changes were noted during the microscopic examination of the pup thyroids.

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 (ano-genital distance normalized to the cube root of pup body weight) of the male and the female pups was noted between the control group and the treatment groups (30, 100 or 300/240 mg/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 (30, 100 or 300/240 mg/kg b.w./day).
However, a spontaneously increased number of nipples was noted in the control group in comparison to the high dose group. In detail, 7 pups with nipples were noted in the control group, 5 in the low dose group and 2 in the intermediate dose group. No pup with nipples was noted at the high dose level.
However, male pups are supposed to have no nipples and the number of pups with nipples in the control group is at the upper limit of the background data. Therefore, the absence of male pups with nipples in the high dose group was considered to be of no toxicological relevance but due to spontaneously high number of male nipples in the control group.
Mammary gland development begins similarly in male and female rats, but only female rats have nipples, whereas male rats possess only rudimentary mammary glands but no nipples, as in male pups locally produced testosterone causes the regression or apoptosis of the nipple anlagen. Therefore, the retention of nipples in male pups is an indicator of impaired androgen action during the development.

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Description (incidence and severity):

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 30, 100 or 300/240 mg/kg b.w./day after terminal sacrifice on lactation day 13.
Also no gross abnormalities were noted for the pups that prematurely deceased during the lactation period (stillbirth or found dead).

Histopathological findings:

no effects observed

Description (incidence and severity):

No test item-related changes were noted for the thyroid glands from the pups of the treatment groups (30, 100 or 300/240 mg/kg b.w./day).

Other effects:

no effects observed

Description (incidence and severity):

BIRTH INDICES AND POST-IMPLANTATION LOSS:
No test item-related influence on prenatal development (number of implantation sites per dam, number of resorptions, number of stillbirths) was noted at the low and the intermediate dose level (30 or 100 mg/kg b.w./day). The increased number of resorptions that was noted at the low dose level (17 resorptions in comparison to 12 in the control group and 5 in the intermediate dose group) was considered to be spontaneous, as no dose response-relationship was noted.

A test item-related effect on prenatal development was noted at the high dose level (300/240 mg/kg b.w./day).
In detail, 9 stillbirths were noted at the high dose level in comparison to 1 stillbirths each in the control group and the intermediate dose group. This led to a markedly decreased live birth index in the high dose group (89.5 % in comparison to 99.3 % in the control group, 100.0 % in the low dose group and 99.2 % in the intermediate dose group; group values).
The 9 stillbirths of the high dose group were noted from two (no. 78 with 5 stillbirths and no. 80 with 4 stillbirths) of 6 high dosed females that had littered. Furthermore, 5 resorptions were noted for dam no. 78 and 2 resorptions for dam no. 80. No resorptions and no stillbirths were noted for the remaining 4 females of the high dose group which littered live pups.
Due to the high number of stillbirths from 2 of 6 females with live pups, an adverse effect of the test item on prenatal development has to be considered. However, compared to the OECD 443 study of the test item the increased number of stillbirths in the high dose group can be regarded to be within the range of biological variability as also 9 stillbirths were noted in the low dose group of the OECD 443 study. No dose response-relationship was observed.

MALE TO FEMALE RATIO OF THE PUPS
No test item-related influence on the male to female ratio was noted for any treatment group (30, 100 or 300/240 mg/kg b.w./day).
However, a male to female ratio that markedly differed from the expected value (around 1.0) was noted at the intermediate dose group and the high dose group (0.70 or 1.41). However, as no dose-response relationship was noted (the values at the intermediate and the high dose group have opposite directions), the reduced male to female ratio that was noted at the intermediate dose group, was not considered to be test item-related but spontaneous.

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

no abnormal behaviour was noted for the pups.

Developmental immunotoxicity:

not examined

Reproductive toxicity
Pups
At 300/240 mg/kg b.w./day an effect was noted on the prenatal development in the form of an increased number of stillbirths. However, compared to the OECD 443 study of the test item the increased number of stillbirths in the high dose group can be regarded to be within the range of biological variability as also 9 stillbirths were noted in the low dose group of the OECD 443 study. No dose response-relationship was observed.
During the postnatal development a reduced pup body weight was noted at 300/240 mg/kg b.w./day, which was a secondary effect due to maternal toxicity.
No influence was noted on the viability of the pups, the ano-genital distance, the number of nipples per male pup and on the T4 concentration. No changes were noted for the thyroid glands from the pups during the microscopic examination.
No external gross abnormalities (malformations or variations) were noted for any of the pups.

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

> 240 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other:

Remarks on result:

other: > 300/ 240 mg/ kg bw/day

Key result

Critical effects observed:

no

Key result

Reproductive effects observed:

no

Conclusions:

The aim of the study was to obtain information on possible effects of the test item on general toxicity, reproduction and/or development according to OECD guideline 422. The test item was administered orally to rats at dose levels of 30, 100 or 300 mg/kg b.w./day. Due to mortality, the high dose level was decreased to 240 mg/kg b.w./day, starting on test day 28.
Additionally, this OECD 422 serves as dose range finding study for the OECD 443 study. The NOAEL for general toxicity is considered to be 100 mg/ kg bw/day. The NOAEL for reproductive toxicity based on adverse effects on the reproductive parameters of the parental females is considered to be above 300/ 240 mg/ kg bw/day. Based on adverse effects on prenatal development (conceptus to birth) the NOAEL for reproductive toxicity is above 300/240 mg/ kg bw/ day and based on adverse effects on postnatal development (pup) above 300/240 mg /kg b.w./day.

Executive summary:

Conclusion

The aim of the study was to obtain information on possible effects of the test item on general toxicity, reproduction and/or development according to OECD guideline 422. The test item was administered orally to rats at dose levels of 30, 100 or 300 mg/kg b.w./day. Due to mortality, the high dose level was decreased to 240 mg/kg b.w./day, starting on test day 28.

Additionally, this OECD 422 serves as dose range finding study for the OECD 443 study.

 

General toxicity

Parental male and female animals

At 300 mg/kg b.w./day 2 females were prematurely sacrificed due to a poor general condition, one female one day before and the other female one day after dose reduction.

At 300/240 mg/kg b.w./day 2 male animals were found dead 12 or 15 test days after dose reduction.

At 300/240 mg/kg b.w./day a few of the surviving females showed salivation, a haemorrhagic nose/snout, piloerection, breathing sounds and / or reduced motility during the gestation and or lactation period. No changes in behaviour, the external appearance or the faeces were noted for the surviving male animals of the high dose group.

A marked body weight loss was noted at 300/240 mg/kg b.w./day for the prematurely deceased or sacrificed male and female animals.

The surviving female animals showed a slightly reduced body weight in comparison to control during the gestation and the lactation period. This and the observed general bad condition of the females are signs of maternal toxicity. No differences in body weight were noted for the surviving high dosed male animals.

At 300/240 mg/kg b.w./day an increased number of neutrophilic granulocytes and an increased LDH activity were noted for the female animals. Both were secondary effects due to the corrosivity of the test item, which is manifesting in the very severe histopathological observations of erosions/ulcer in the female animals. No test item-related differences for the haematological and biochemical parameters were noted for the male animals.

The macroscopic examination at necropsy revealed kidney changes for 2 of the 8 surviving male animals of the high dose group (300/240 mg/kg b.w./day) that could possibly be test item-related.

Kidney changes that were considered to be test item-related were noted during the microscopic examination for the surviving male animals of the high dose group (300/240 mg/kg b.w./day), as well as for one of the prematurely deceased male animals and one of the prematurely sacrificed female animals, but not for the surviving female animals.

Test item-related changes for the surviving animals from both sexes were noted during the microscopic examinations of the stomachs from the male and female animals of the high dose group (300/240 mg/kg b.w./day) in form of an erosion of the stomach wall and an inflammatory response. These stomach changes were considered as local and secondary effects that were caused by the corrosive properties of the test item.

The additional microscopic examinations of the kidneys from the male animals and the stomachs from the male and female animals of the low and the intermediate dose groups revealed no test item-related changes.

No test item-related influence or adverse effects were noted for the male and female animals on food consumption, on the parameters of the neurological screening, for the organ weights and on the T4 serum levels of the male animals.

 

Reproductive toxicity

Parental females

No influence was noted on the number and length of the estrous cycles, the fertility index, the gestation index, the duration of the pre-coital time interval and the gestation period.

Pups

At 300/240 mg/kg b.w./day an effect was noted on the prenatal development in the form of an increased number of stillbirths. However, compared to the OECD 443 study of the test item the increased number of stillbirths in the high dose group can be regarded to be within the range of biological variability as also 9 stillbirths were noted in the low dose group of the OECD 443 study. No dose response-relationship was observed.

During the postnatal development a reduced pup body weight was noted at 300/240 mg/kg b.w./day, which was a secondary effect due to maternal toxicity.

No influence was noted on the viability of the pups, the ano-genital distance, the number of nipples per male pup and on the T4 concentration. No changes were noted for the thyroid glands from the pups during the microscopic examination.

No external gross abnormalities (malformations or variations) were noted for any of the pups.

 

The following no-observed-adverse-effect levels (NOAEL) were established:

General toxicity
NOAEL (for systemic toxicity)
100 mg/kg b.w./day, p.o.
Reproductive toxicity
a) adverse effects on the reproductive parameters of the parental females
	NOAEL                                                                    above 300/240 mg/kg b.w./day, p.o.
b) adverse effects on pre- and postnatal development
- b1) adverse effects on prenatal development (conceptus to birth)
		NOAEL                                                                                     above 300/240 mg/kg b.w./day, p.o.
- b2) adverse effects on postnatal development (pup)
		NOAEL                                                                    above 300/240 mg/kg b.w./day, p.o.

Reference 2

Endpoint:

reproductive toxicity, other

Remarks:

14 days dose range finding study for OECD 422

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

2019-04-30 until 2020-07-08

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

other: dose range finding study for OECD 422 Selection of route of administration: According to OECD guidelines 421 and 422.

Principles of method if other than guideline:

- Principle of test:
14-day dose-range-finding study to select the dose levels for a reproduction/developmental toxicity study of the test item administered by oral
administration to rats daily over 14 days.

- Short description of test conditions:

30 animals (15 males and 15 females)
Oral administration by gavage
10 mL/kg b.w./day
Vehicle Aqua ad injectabilia
single housing in MAKROLON cages
temperature 22°C ± 3°C and the relative humidity 55% ± 10%

Duration of the study:
7 adaptation days
14 days of treatment
Necropsy on test day 15

- Parameters analysed / observed:
Clinical signs
Mortality
Body weight
Food and drinking water consumption
Haematology
Clinical biochemistry
Necropsy

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Crj: CD(SD)

Details on species / strain selection:

The rat is a commonly used rodent species for such studies. CD® rats supplied by Charles River Laboratories Germany GmbH were used.
An initial health check was performed on the day of delivery. Only animals free of signs of illness were selected for the study.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Species / Strain / Stock: Rat / CD® / Crl:CD(SD)
- Source: Charles River Laboratories, Research Models and Services Germany GmbH, Sandhofer Weg 7, 97633 Sulzfeld, Germany
- Age at study initiation: Males and Females: 59 days
- Weight at study initiation: Males: 352.3 g - 388.0 g; Females: 205.9 g - 254.5 g
- Selection of species The rat is a commonly used rodent species for such studies.
- Number and sex of animals 30 animals (15 males and 15 females) In addition, approximately 10% spare animals were available for possible replacement during the adaptation period.
- Acclimation period: 7 days
- Housing: The animals were 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.
- Diet (e.g. ad libitum): A certified commercial diet (ssniff® R/M-H V1534, ssniff Spezialdiäten GmbH, 59494 Soest, Germany) served as food. This food was offered ad libitum. Food residue was removed and weighed. Samples of the food are analysed for contaminants based on EPA/USA3 by LUFAITL4 at least twice a year. Certificates of analysis of the composition and for contaminants are provided by the manufacturer and are included in the raw data. No contaminants above the limitations were noted.
- Water (e.g. ad libitum): Drinking water was offered ad libitum. Samples of the drinking water are taken periodically by the Wasserwerk Wankendorf and periodic analyses are performed by LUFA-ITL according to the 'Deutsche Trinkwasserverordnung, Bundesgesetzblatt 2001' [German Regulations on drinking water, public notice of the law, 20015]. In addition, drinking water samples taken at LPT are analysed by LUFA-ITL once a year for means of bacteriological investigations according to the "Deutsche Trinkwasserverordnung 2001, Anlage 1" [German Regulations on Drinking Water 2001, Addendum 1]. No contaminants above the limitations were noted.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22°C ± 3°C (maximum range)
- Humidity (%): 55% ± 10% (maximum range)
- Air changes (per hr): fifteen to twenty air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours dark/12 hours light cycle

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

Aqua ad injectabilia (Water for injection, batch 184928001, B. Braun, 34212 Melsungen, Germany)

Details on exposure:

Route of administration: Oral administration by gavage
Frequency of administration: Once daily for 14 days
Vehicle: Aqua ad injectabilia1
Administration volume: 10 mL/kg b.w./day
Selection of route of administration: According to OECD guidelines 421 and 422.
The test item formulations were freshly prepared every day. The test item was diluted in the vehicle to the appropriate concentrations and administered orally at a constant volume/kg b.w. once daily. The amount of the test item was daily adjusted to the current body weight of the animals. The control animals received the vehicle at the same administration volume in the same way.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Water for injection, batch 184928001, B. Braun, 34212 Melsungen, Germany
- Concentration in vehicle: 0;250; 500/300 mg/kg b.w.
- Amount of vehicle (if gavage): 10 mL/kg b.w./day

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

Once daily for 14 days

Frequency of treatment:

Once daily for 14 days

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

control

Dose / conc.:

250 mg/kg bw/day (nominal)

Dose / conc.:

300 mg/kg bw/day (nominal)

Remarks:

Dose reduction from 500 to 300 mg/kg bw for all animals of group 3 due to mortality and reduced body weight.

No. of animals per sex per dose:

5 male / 5 female

Control animals:

yes, concurrent no treatment

Details on study design:

- Dose selection rationale:
The dose levels for this study have been selected based on available data.
Study pupose: dose range finding study for OECD 422

- Rationale for animal assignment (if not random):
The animals were allocated to the test groups by using a ProvantisR2-generated randomization based on the body weights of the animals.
Each rat received a continuous number: according to a differentiated number scheme.

- Fasting period before blood sampling for clinical biochemistry:
Blood samples were taken from the retrobulbar venous plexus under isoflurane anaesthesia from animals fasted overnight.


- Section schedule rationale (if not random):
On test day 15 (approximately 24 hours after the last administration) all animals were dissected following a randomisation scheme. Those animals that were found dead in the morning were examined after they had been found dead.

Parental animals: Observations and examinations:

OBSERVATIONS
Dated and signed records of all activities relating to the day by day running and maintenance of the study within the animal unit 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 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 before and after dosing at each time of dosing for any signs of behaviroural changes, reaction to treatment, or illness. In addition, the 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.
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. Dated and signed records of appearance, change, and disappearence of clinical signs of individual animals were maintained on clinical history sheets.

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 examinations to be carried out during the working period of that day. On Saturdays and Sundays, a similar procedure was performed with a final check at approximately 3.30 p.m. Premortal symptoms were recorded in detail; as soon as possible after excitus, a post mortem examination was performed as described in section 3.15 'Necropsy'. In the case of prematurely sacrificed animals, blood sampling for laboratory examinations would have been performed. However, the prematurely deceased animals in this study were found dead in the morning. Hence, no blood sampling was possible.


BODY WEIGHT:
The weight of each rat was determined and recorded at the time of group allocation and daily during the treatment period for dose adjustment. Weekly values are stated in this report.

FOOD AND DRINKING WATER CONSUMPTION:
The quantitiy of food left by each animal was recorded on a wekly basis throughout the experimental period.
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 upon completion of a treatment week.
From these data the relative food consumption (in g/kg b.w./day) was determined using the following formula:

Relative food consumption (g/kg b.w./day)= (Total food given (g) - Total food left (g))/(Number of animal days# x Body weight (kg))
# The term 'animal days' counts one animal day for each animal alive for a whole day; it is assumed that on the day of death an animal does not eat.

Drinking water consumption was monitored daily by visual appraisal throughout the study.

FOOD EFFICIENCY:
- Body weight gain data: Yes

LABORATORY EXAMINATIONS:
Blood samples were taken from the retrobulbar venous plexus under isoflurane anaesthesia from animals fasted overnight. The time points of blood withdrawal are given below.
Blood sampling schedule for laboratory examination: At the end of the dosing period, on the day of necropsy | Taken from all surviving male and female animals of the control group and the treatment groups.
The collected blood samples were divided into tubes as follows:
EDTA anticoagulant (whole blood) ...................... for haematological investigations
Li-Heparin anticoagulant (plasma) ................................ for clinical chemistry tests

HAEMATOLOGY:
The parameters that were determined are listed below (Instrument: ADVIATM 120 Siemens Diagnostics GmbH 35463 Fernwald, Germany.
Parameter/ Units
Haemoglobin content (HGB)/ mmol/L blood
Erythrocytes (RBC)/ 10^6/μL blood
Leucocytes (WBC)/ 10^ 3/μL blood
Differential blood count (relative and absolute)*/ % and 10^3/μL blood
Reticulocytes (RET)/ % of the erythrocytes
Platelets (PLT)/10^3/μL blood
Haematocrit value (HCT)/ %
Mean corpuscular volume (MCV)/ fL
Mean corpuscular haemoglobin (MCH)/ fmoL
Mean corpuscular haemoglobin concentration (MCHC)/ mmol/L

*Neutrophilic, eosinophilic and basophilic granulocytes, lymphocytes and monocytes. Large unstained cells were simultaneously quantified during measurement of the differential blood count.

Following the haematological examinations using the ADVIA system, blood smears were prepared from all samples, dried and stained for possible histopathological examinations in case of pathological findings


CLINICAL BIOCHEMISTRY:
The parameters that were determined are listed in below (Instrument: KONELAB 30i, Thermo Fisher Scientific, 63303 Dreieich, Germany).
Parameter/ Units
Albumin/ g/L
Globulin/ g/L plasma (by subtraction)
Albumin/Globulin ratio/non-dimensional (by calculation)
Bilirubin (total)/ μmol/L
Cholesterol (total)/ mmol/L
Creatinine/ μmol/L
Glucose/ mmol/L
Protein (total)/ g/L
Blood urea (BUN)/ mmol/L
Calcium/ mmol/L
Chloride/ mmol/L
Potassium/ mmol/L
Sodium/ mmol/L
Lactate dehydrogenase (LDH)/ U/L
Alanine aminotransferase (ALAT/GPT)/ U/L
Alkaline phosphatase (aP)/ U/L
Aspartate aminotransferase (ASAT/GOT)/ U/L

Postmortem examinations (parental animals):

NECROPSY
On test day 15 (approximately 24 hours after the last administration) all animals were dissected following a randomisation scheme. Those animals that were found dead in the morning were examined after they had been found dead. The animals were euthanized by carbon dioxide (CO2) inhalation, exsanguinated by cutting the aorta abdominalis, weighed, dissected, and inspected macroscopically under the direction of a pathologist. 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.
Weighed organs of all male and female animals:
Adrenal gland (2)
Liver
Kidney (2)
Spleen
Paired organs were weighed individually and identified as left or right. The organ weights of the animals that were found dead were recorded but not included in the mean value comparison.

The organs or parts thereof that were listed below of all animals (including the prematurely deceased animals) were fixed for preservation in 7%
buffered formalin:
Stomach
Trachea (including larynx)

Histopathological examination was not conducted.

Statistics:

Data acquisition: The following data were captured or calculated by the departmental computerized system (Provantis® integrated preclinical software, version 10.2.1, Instem LSS
Ltd): Clinical signs, body weight, body weight gain, food consumption, haematological and biochemical parameters. Raw data not fully compatible with the computerized system (organ weights, macroscopic post mortem findings) were maintained on paper according to the
appropriate SOPs. Data maintained on paper (organ weights) was entered in Provantis in a retrospective manner using the laboratory records according to the appropriate SOPs.


Statistics: The statistical evaluation of the parametrical values was done by Provantis using the following settings (see also the decision tree on the following page): Homogeneity of variances and normality of distribution were tested using the BARTLETT’s and SHAPIRO-WILK’s test. In case of heterogeneity and/or nonnormality 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). Significantly different data are indicated in the summary tables of the result sections of the report.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Males and females
No changes in behaviour, the external appearance or the appearance of the faeces were noted for the male and female animals of the control group.
At the low dose level (250 mg/kg b.w./day) breathing sounds were noted for 1 of 5 male animals and salivation for 2 of 5 female animals.
At the high dose level (500/350 mg/kg b.w./day) salivation was noted for 2 of the 3 surviving male animals and for 3 of 5 female animals.
Furthermore, 2 of 5 female animals revealed breathing sounds. The observations of breathing sounds and salivation were considered to be test item-related.
Furthermore, piloerection and a haemorrhagic nose / snout were noted for the high dosed female animal no. 26 on the day of necropsy (test day 15).
However, these observations were considered to be a secondary effect of the bad health condition of animal no. 26 on test day 15 .

Duration of salivation:
Males:
For the 2 surviving male animals of the high dose group (500/350 mg/kg b.w./day) that showed salivation, salivation started immediately to 20 min after
administration and lasted between 20 and 60 min after administration. For the prematurely deceased animal no. 23 salivation started immediately to 5 min after
administration on test day 6 and was noted consistently during the day until death on test day 9.
Females:
For the 2 female animals of the low dose group (250 mg/kg b.w./day) that showed salivation, salivation started between 5 and 20 min after administration
and lasted consistently during the following days until test days 9 or test day 14.
At the high dose level (500/350 mg/kg b.w./day), the singular observations of salivation that were noted for 2 animals (nos. 28, 29) started 5 to 20 min after
administration and lasted between 20 and 60 min after administration. In case of animal no. 30, salivation started immediately to 5 min on test day 6 and lasted
consistently during the following days until test day 14.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, treatment-related

Description (incidence):

Males
No premature deaths were noted for the male animals of the control group and the low dose group (250 mg/kg b.w./day).
Two of 5 males of the high dose group (500/300 mg/kg b.w./day) were found dead in the morning of test day 5 (no. 25; one day before dose reduction) or
in the morning of test day 9 (no. 23; 3 days after dose reduction).
Both deaths were considered to be test item-related.
Females
None of the female animals of the control group or the treatment groups (250 or 500/350 mg/kg b.w./day) died prematurely.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Males
Test item-related reductions in body weight and body weight gain were noted at the low (250 mg/kg b.w./day) and the high dose level (500/350 mg/kg
b.w./day).

At the low dose level (250 mg/kg b.w./day) the mean body weight of the male animals was slightly but statistically significantly reduced on test day 14
(5.9% below the value of the control group; p ≤ 0.05).
At the high dose level (500/350 mg/kg b.w./day) the mean body weight of the male animals was statistically significantly reduced on test day 8 (15.8%
below the control value; p ≤ 0.01) and on test day 14 (11.7% below the control value; p ≤ 0.01).

Body weight gain
The mean percentage of body weight gain of the male animals was slightly reduced at the low dose level (250 mg/kg b.w./day) and markedly reduced at
the high dose level (500/350 mg/kg b.w./day).
Males Group1 Control Group 2 250 mg/kg Group 3 500/350 mg/kg
Test days 1 to 14 +17.7 % +12.1 % +6.1 %


In detail, the percentage of body weight gain from 4 of 5 low dosed animals was slightly below the lowest value of the control group (between 9.3% and 13.2% in
comparison to 15.6% as the lowest value of the control group). Only 1 of 5 low dose animals showed a percentage of body weight gain that was in the range of
the control group (no. 13 with 17.1% in comparison to the control range of 15.6% to 19.9%).
From the 3 surviving high dosed animals only 1 animal (no. 24 with 11.2%) showed a percentage of body weight gain that was only slightly below the range
of the control group. The percentages of body weight gain of the other 2 surviving high dosed males were markedly below the lowest value of the control group (nos.
21 and 22 with 4.2% or 3.0%)

Females
Test item-related reductions in body weight and body weight gain were noted at the low (250 mg /kg b.w./day) and the high dose level (500/350 mg/kg b.w./day).
At the low dose level (250 mg/kg b.w./day) the mean body weight on test day 8 was 20.8% (p ≤ 0.01) below the value of the control group. During the
second test week the mean body weight recovered. However, at the end of the study the mean body weight was still below the control value (8.4% below the
control value on test day 14; p ≤ 0.05).
The reduced mean body weight on test day 8 was due to 2 of the 5 low dose females (nos. 17 and 20). These 2 animals showed a marked body weight loss
between test days 1 and 8 (no. 17: 229.2 g on test day 1 to 199.3 g on test day 8; no. 20: 251.8 g on test day 1 to 169.7 g on test day 8). The body weight of
the remaining 3 low dose animals was only slightly below the lowest value of the control group on test day 8. During the second test week the body weights of
animal nos. 17 and 20 recovered and were in or only slightly below the range of the control group .
At the high dose level (500/350 mg/kg b.w./day) a statistically significantly reduced mean body weight was noted on test day 8 (14.5% below the control
value; p ≤ 0.05) and a less reduced body weight was noted on test day 14 (9.2% below the value of the control group; statistically not significant).
Hence, on test day 14, the body weights of the low and the high dosed females were nearly to the same extent (8.4% or 9.2%) reduced in comparison to the
control group.

Body weight gain
The mean percentages of body weight gain that were noted for the female animals of the low and the high dose group are nearly equal and reduced in comparison to
the control group.

Females Group1 Control Group 2 250 mg/kg Group 3 500/350 mg/kg
Test days 1 to 14 +14.3 % +6.4 % +7.0 %

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Males
No test item related influence on food consumption was noted at the low dose level (250 mg/kg b.w./day).
A test item-related reduction in food consumption was noted between test days 1 and 8 for the 4 surviving male animals of the high dose group (500/350 mg/kg b.w./day). During the second test week (between test days 8 and 14; after dose reduction) no difference of toxicological relevance was noted between
the 3 surviving male animals of the high dose group (350 mg/kg b.w./day) and the male animals of the control group.
In detail, a markedly reduced food consumption during the first test week was noted for the prematurely deceased animal no. 23 and moderately reduced food
consumptions during the first test week were noted for the 3 surviving high dosed animals.

Females
A test item-related reduction in food consumption was noted for the female animals of the low and the high dose group (250 or 500/350 mg/kg b.w./day) during the first test week, a little bit more pronounced at the low dose level than at the high dose level. During the second test week the food intake from
the female rats of the low and the high dose group completely recovered and no difference in comparison to the control group was noted anymore

Assessment of food consumption:
As the food consumption for the male and the female animals of the high and / or the low dose group recovered during the second test week, the reductions that
were noted during the first test week are not considered to be of toxicological relevance.

Food efficiency:

not examined

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

no effects observed

Description (incidence and severity):

Drinking water consumption (male and female)
No test item-related changes in the consumption of drinking water was noted for the male and female rats treated with 250 or 500/350 mg/kg b.w./day by
visual appraisal.

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Males and females
No test item-related changes for the examined haematological parameters were noted at the low and the high dose level (250 or 500/350 mg/kg b.w./day)
for the male and the female animals.
The statistical significant changes that were noted for the examined haematological parameters of the male and female animals are listed below.
However, changes in isolated parameters as those listed, that were furthermore due to only one or two animals (platelets, neutrophilic
granulocytes) or noted for a cell type with only a very small cell number (eosinophilic granulocytes) were considered to be spontaneous.
Critical findings were noted for the high dosed female no. 26 which revealed a low number of platelets and very low numbers of white blood cells and lymphocytes.
However, as these pronounced reductions were only noted for animal no. 26 they could be considered as a secondary effect of a bad general health condition of this
animal. An indication of such a generally poor health condition of animal no. 26 are the external observations at necropsy that were noted for animal no. 26.

Statistically significant changes in comparison to control that were noted for the haematological parameters.

Statistically significant changes in comparison to control [%]
- not considered to be test item-related -

Parameter Group 2 Group 3 #1
Males Platelets (PLT) +30.6 +51.6* (22)
Neutrophilic
granulocytes (abs.) +2.2 +93.2* (22)
Females Neutrophilic
granulocytes (abs.) +157.0* (18,19) +87.5 (29)
Eosinophilic
granulocytes (abs.) +5.3 - 70.2*

*/**: p ≤ 0.05 / 0.01, ANOVA/DUNNETT
#1: Animals that were mainly responsible for the observed changes are listed in brackets.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Males and females
No test item-related changes for the examined biochemical parameters were noted at the low and the high dose level (250 or 500/350 mg/kg b.w./day).
The statistical significant changes that were noted for the examined biochemical parameters of the male and female animals are listed below.
As the changes were only small, only noted for one sex or not of toxicological relevance (decreased alkaline phosphatase activity), they were all considered to be spontaneous and not test item-related.

Statistically significant changes in comparison to control that were noted
for the biochemical parameters.

Statistically significant changes in comparison to control [%]
- not considered to be test item-related -
Parameter Group 2 Group 3 #1
Males Globulin - 6.0 - 14.5*
Albumin/
Globulin ratio +2.5 +12.5**
Sodium - 0.3 - 1.5*
Alkaline
phosphatase (aP) - 9.2 - 24.6*
Females Chloride - 2.5 - 4.0*
*/**: p ≤ 0.05 / 0.01, ANOVA/DUNNETT

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

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

not examined

Dose descriptor:

other:

Sex:

male/female

Remarks on result:

other: no descriptor determined; dose range finding study for OECD 422

Critical effects observed:

yes

Lowest effective dose / conc.:

250 mg/kg bw/day (nominal)

System:

other: a reduced body weight, a decreased food consumption, breathing sounds, salivation

Critical effects observed:

yes

Lowest effective dose / conc.:

350 mg/kg bw/day (nominal)

System:

gastrointestinal tract

Organ:

intestine

stomach

Treatment related:

yes

Dose response relationship:

not specified

Relevant for humans:

not specified

Critical effects observed:

yes

Lowest effective dose / conc.:

350 mg/kg bw/day (nominal)

System:

other: mortality

Clinical signs:

not examined

Dermal irritation (if dermal study):

not examined

Mortality / viability:

not examined

Body weight and weight changes:

not examined

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):

not examined

Nipple retention in male pups:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

not examined

Histopathological findings:

not examined

Other effects:

not examined

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Conclusions:

The aim of the study was to select the dose levels of the test item for a reproduction/developmental toxicity study.
One male animal of the high dose group died at a dose level of 500 mg/kg b.w./day and another male animal after the reduction of the high dose level to 350 mg/kg b.w./day.
Changes in behaviour in the form of salivation (in many cases consistently during the whole day) and breathing sounds were noted for some male and female animals of the low dose group and / or the high dose group (250 or 500/350 mg/kg b.w./day)
There was a significantly reduced food consumption in both dose groups, which leads to significantly reduced body weight in the first week of application. As the food consumption recovered during the second test week, the reductions that were noted during the first test week are not considered to be of toxicological relevance.
Necropsy revealed test item related gastrointestinal changes for some male and female animals of the high dose group (500/350 mg/kg b.w./day).

Based on the results the following dose levels are recommended for the OECD 422 study (Combined repeated dose toxicity study with the reproduction/developmental toxicity screening test of the test item in rats by oral administration).
 30 mg/kg/b.w./day (low dose)
100 mg/kg/b.w./day (intermediate dose)
300 mg/kg/b.w./day (high dose)

Executive summary:

The aim of the study was to select the dose levelsof the test item for a reproduction/developmental toxicity study. The test item was administered orally to rats by gavage at dose levels of 250 or 500 mg/kg b.w./day for 2 weeks. Due to mortality and a reduced body weight, the high dose level was reduced to 350 mg/kg b.w./day on test day 6.

One male animal of the high dose group died at a dose level of 500 mg/kg b.w./day and another male animal after the reduction of the high dose level to 350 mg/kg b.w./day.

Changes in behaviour in the form of salivation (in many cases consistently during the whole day) and breathing sounds were noted for some male and female animals of the low dose group and / or the high dose group (250 or 500/350 mg/kg b.w./day).

Statistically significant reductions in body weight at the end of the first test week (test day 8) were noted for the female animals of the low dose group (250 mg/kg b.w./day) and for the male and female animals of the high dose group (500/350 mg /kg b.w./day). During the second test week a slight recovery of the body weight was noted for all affected sexes and dose levels. However, the values of body weight still remained below the control values.

Necropsy revealed test item related gastrointestinal changes for some male and female animals of the high dose group (500/350 mg/kg b.w./day).

There was a significantly reduced food consumption in both dose groups, which leads to significantly reduced body weight in the first week of application. As the food consumption recovered during the second test week, the reductions that were noted during the first test week are not considered to be of toxicological relevance.

Based on the above results the following dose levels are recommended for the OECD 422 study (Combined repeated dose toxicity study with the reproduction/developmental toxicity screening test of the test item in rats by oral administration).

30 mg/kg/b.w./day (low dose)

100 mg/kg/b.w./day (intermediate dose)

300 mg/kg/b.w./day (high dose)

Reference 3

Endpoint:

extended one-generation reproductive toxicity - basic test design (Cohorts 1A, and 1B without extension)

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

the extended one-generation reproductive toxicity study does not need to be conducted because there are no results from available repeated dose toxicity studies that indicate adverse effects on reproductive organs or tissues, or reveal other concerns in relation with reproductive toxicity

other:

Reproductive effects observed:

not specified

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Study duration:

subchronic

Species:

rat

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

Short description of key information:
No studies have been performed to explicitly address the question of reproductive effects in animals caused by 1,3,3-trimethyl-N-(-2-methylpropylidene)-5-[(2-methylpropylidene)amino]cyclohexanemethylamine. A data waiver is claimed.

Effects on developmental toxicity

Description of key information

No developmental toxicity/teratogenicity studies are available for 1,3,3-trimethyl-N-(-2-methylpropylidene)-5-[(2-methylpropylidene)amino]cyclohexanemethylamine.Due to the likely hydrolysis of the substance 1,3,3 -trimethyl-N-(2 -methylpropylidene)-5 -[(2 -methylpropylidene)amino]cyclohexanemethylamine during an in vivo study, to the hydrolysis products isophoron diamine and isobutyraldehyde, the studies on toxicity to reproduction will be waived and a supporting assessement for the hydrolysis products was chosen:

IPDA

Based on the results of a dose finding study, three groups of 24 mated female Sprague-Dawley rats received IPDA by daily oral administration (gavage) at 0 (water = control), 10, 50 and 250 mg/kg/day from day 6 to day 19 post-coitum inclusive (CIT 2002). The study was designed according to OECD TG 414. There was no treatment-related death in any of the dams. Clinical signs were not observed, except for ptyalism in most females of the 250 mg/kg/day group (from day 11, 12, 13 or 14 post-coitum until hysterectomy; effect not considered as adverse). Loud breathing and hold-up in the esophagus were recorded in 4 females of this group and a significantly lower body weight gain (-35%) was recorded after the first three days of treatment. Thereafter, the body weights were similar to that of the controls. Over the whole treatment period, the difference remained slight (-10 %, not statistically significant). The net body weight gain was also significantly lower at this dose-level (-25 %) when compared to the control group. In the 250 mg/kg/day group, a significant decrease in food consumption was recorded during the treatment period (-7%), with a more marked effect during the first three days of treatment (-21 %).Abortions or total resorptions were not observed in any of the groups, nor were there any macroscopic findings that were ascribed to treatment with the test item. No treatment related effects were observed on pre- or post-implantation loss, fetal weight or sex-ratio. With respect to the fetuses, no test item related external, soft tissue or skeletal malformations or variations were detected. There was an increase in fetal incidence of incomplete ossification of the 5th sternebra in the 250 mg/kg bw/day group (106/134 fetuses = 79.1 %, p < 0.01 were affected vs. 88/130 = 67.7 % in control group, statistically insignificant on a fetus/litter basis). Because these findings are of low concern and occur only in the presence of maternal toxicity, they are considered to be secondary. In the same group there was a statistically nonsignificant increase in fetal incidence of incomplete ossification of the rib(s) (9/137 fetuses = 6.7 % vs. 2/130 = 1.5 % in control group. When ossification was incomplete, cartilage was generally present, demonstrating that the skeletal variations recorded corresponded to slight fluctuations in the time of ossification rather than being a persistent alteration. In conclusion, these findings were considered to be incidental and of no toxicological significance. The NOEL for maternotoxicity was 50 mg/kg/day and the NOAEL for embryonic development was >250 mg/kg/day.

In a prenatal developmental toxicity study according to OECD 414 in rabbits, the test item test item was administered orally to female rabbits at dose levels of 10, 25 or 75 mg/kg b.w./day from the 6th to 28th day of pregnancy (LPT 2020). Under the present test conditions, the no-observed-adverse-effect level (NOAEL) was 25 mg test item /kg b.w./day for the dams:

No test item-related premature death was noted for any of the dose groups. For the high dose group (75 mg test item /kg b.w./day), a reduction was noted for the body weight or the body weight gain. Also, a decrease was noted in food consumption at 75 mg test item /kg b.w./day. No changes were noted for the drinking water consumption. No changes in behaviour, external appearance or faeces were noted for the treatment groups. No test item-related pathological findings were noted during necropsy for the treatment groups. At 75 mg test item /kg b.w./day, a slight decrease was noted for the gravid uterus and the carcass weight.

The no-observed-adverse effect level (NOAEL) was above 75 mg test item /kg b.w./day for the fetuses and 25 mg test item /kg b.w./day for the rate of early resorptions/post-implantation loss: At the maternaltoxic dose level of 75 mg test item /kg b.w./day (reduced body weight, reduced body weight gain, reduced carcass weight and reduced food consumption), an increased number of early resorptions and accordingly an increased post-implantation loss were noted. Due to the increased number of early resorptions, the total resorption rate was slightly above the LPT historical control data (up to 1.3). The early resorption rate and the percentages of post-implantation loss were at the upper range but still within the LPT historical control data. No test item-related influence was noted on the mortality or body weight of the fetuses in the treatment groups. No test item-related deaths of the fetuses and no test item-related malformations, variations or retardations were noted.

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

The treatment with the test substance at 75 mg /kg b.w./day revealed increased incidences of early and total resorptions, and in consequence an increased percentage of post-implantation loss, which were statistically significantly different from the control. A treatment-related effect for the statistically significantly increased rate of early resorptions and the statistically significantly increased percentage of postimplantation loss cannot totally be excluded since both values were at the upper end of the LPT historical control data.

Although treatment-related and adverse effects cannot totally be excluded, the main reason for those increases is animal no. 82 that showed a total post-implantation loss. When assessing the number of live fetuses, the high dose group showed no remarkable difference to the control group (194 vs. 199 fetuses out of 20 dams with viable fetuses each). A feto-toxic effect of the test substance can be excluded, since no test item-related malformations, variations or retardations were evident.

 

Isobutyraldehyde

In a dose range finding study from BASF (1994), Wistar rats were daily exposed to vapor concentrations of 0 (control), 3, 7.5 and 12 mg/L from day 6 - day 15 post coitum for 6 hours per day. No clinical signs and no mortalities occurred during the study. Body weights and body weight gain were slightly reduced. Additionally, cholesterol was found to be slightly, but statistically significant reduced in the highest dose group. A NOAEC of 7.5 mg/L was found for systemic maternal toxicity. For local effects, the NOAEC was 3mg/L due to local effects in the nasal cavity (hyperplasia of the transitional epithelium, minimal degree) of some animals of the medium and high dose group. There were no signs of developmental toxicity.

In an OECD 414 guideline study pregnant female rats were exposed daily to vapor concentrations of 0 (control), 3, 7.5 and 12 mg/L from day 6 - 15 post coitum (BASF 1996). No clinical signs and no mortalities occurred. As already seen in the DRF study, also body weight and body weight gain were reduced in all dose groups. In medium and high dose group body weight gain is considered to be substance related decreased.  No substance-related effects on gestational parameters or fetuses were observed in all dose levels. The NOAEC for maternal toxicity was 3 mg/L due to body weight gain reduction and the NOAEC for teratogenicity was 12 mg/L (no teratogenic effects observed).

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Qualifier:

according to guideline

Guideline:

other: EC Commission Directive 87/302/EEC of Nov . 18, 1987 ; Part B : Methods for the determination of toxicity : Teratogenicity study (rodent and non-rodent)

Qualifier:

according to guideline

Guideline:

other: U .S . EPA, Pesticide Assessment Guidelines, Subdivision F, § 83-3 : Teratogenicity Study ; NTIS, Revised edition, pp . 126 - 130 (Nov . 1984 )

Qualifier:

according to guideline

Guideline:

other: Japan/MAFF : Testing Guidelines for Toxicology Studies : Teratogenicity Study, pp . 48 - 49 (1995)

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Dr. K . THOMAE GmbH, Biberach an der Riss,FRG,
- Age at study initiation: 75 - 84 days
- Weight at study initiation: approx . 231 g (mean)
- Housing: Singly in wire mesh cages type DK III of Becker & Co., Castrop-Rauxel, FRG (floor area about 800 cm2)). Underneath the cages, waste trays were fixed containing bedding material (typ 3/4 dust free embedding, supplied by SSNIFF, Soest, FRG) .
- Diet: Standard diet ad libitum (KLIBA rat/mouse/hamster laboratory diet 24-343-4, 10 mm pellets, Klingentalmühle AG, Kaiseraugst, Switzerland), ad libitum
- Water: Tap water, ad libitum
- Acclimation period: 8-11 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24°C
- Humidity (%): 30 - 70%
- Air changes (per hr): fully air-conditioned rooms
- Photoperiod (hrs dark / hrs light): 12 / 12

Route of administration:

inhalation: vapour

Type of inhalation exposure (if applicable):

whole body

Vehicle:

air

Details on exposure:

Animals were housed singly in wire cages located within a glass and steel 1.4 cubic meter inhalation chamber and were exposed to air for 5 days before the exposure period. Food and water was not available during exposure.
Generator system:
- piston metering pump (DESAGA)
- glass vaporizer with glass mixing stage, two component atomizer and thermostat
- two-component atomizer
- thermostat
Generation procedure: The test substance was supplied to the two-component atomizer at a constant rate by means of the metering pump. The generation was carried out using thermostated glass evaporators with downstream mixing units by means of spraying the substance with a two-component atomizer and compressed air into a counter current of ventilation air. Conditioned supply air (about 50% relative humidity, 22°C) and compressed air were used for the exposure in all test groups.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The nominal concentration was calculated from the study means of the amounts of substance dosed and the supply air flows.
The concentrations of the inhalation atmospheres were analyzed by gas chromatography. As a rule, daily means were calculated based on two measured samples. From these daily mean values mean concentrations and standard deviations for the entire study and for each replicate were derived. The constancy of concentrations in the inhalation chambers were monitored by means of non-calibrated total hydrocarbon analyzers.

Details on mating procedure:

Two untreated female rats were mated with one untreated male of the same breed . Mating took place from about 4 p.m. to about 7.30 a .m. on the following day. If sperm were detected microscopically in the vaginal smear in the morning, the animals were considered to be fertilized . This day was designated "day 0" (beginning of the study) and the following day "day 1" post coitum (p.c.).

Duration of treatment / exposure:

days 6 through 15 p.c.

Frequency of treatment:

daily, 6 hours per day

Duration of test:

until day 20 p.c.

Dose / conc.:

3 mg/L air

Dose / conc.:

7.6 mg/L air

Dose / conc.:

12 mg/L air

No. of animals per sex per dose:

25

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Based on dose-range finding study (see seperate IUCLID entry)

Maternal examinations:

CAGE SIDE OBSERVATIONS:
A check for dead animals was made daily. The behavior and state of health of the test animals were checked at least 3 times on exposure days and, as a rule, once a day during the preflow period and the post-exposure observation period . During only a groupwise examination was possible .

BODY WEIGHT:
The body weight of the animals was checked on day 0 (= day of detection of sperm) and on days 3, 6, 9, 12, 15, 17 and 20 p.c. As a rule, the animals were weighed at the same time of the day. The difference between the body weight on the day of weighing and the body weight on the previous weighing was calculated . Moreover the same was done for the 3 different study period: preflow period (days 0- 6 p.c.); exposure period (days 6- 15 p.c.); post-exposure observation period (days 15 - 20 p.c.). These values were defined as body weight change. Furthermore, the corrected body weight gain was calculated after terminal sacrifice (terminal body weight on day 20 p.c. minus weight of the unopened uterus minus body weight on day 6 p.c.).

POST-MORTEM EXAMINATIONS:
On day 20 p.c., the dams were sacrificed in randomized order by cervical dislocation and the fetuses dissected from the uterus. After the dams had been sacrificed, they were necropsied and assessed by gross pathology. The uterus and the ovaries were removed and the following data were recorded: Live fetuses, dead implantations: a) early resorptions (only decidual or placental tissues visible or according to Salewski (1964) from uteri from apparently non-pregnant animals and the empty uterus horn in the case of single-horn pregnancy) b) late resorptions (embryonic or fetal tissue in addition to placental tissue visible) c) dead fetuses (hypoxemic fetuses which did not breathe spontaneously after the uterus had been opened) Furthermore, calculations of conception rate and pre- and postimplantation losses were carried out.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes

Fetal examinations:

Fetuses were weighed, sexed, and examined macroscopically for external findings. Viability of each fetus, and the condition of the placenta, umbilical cord, and fetal membranes and fluids were also examined. Individual placental weights were also recorded. After fixation, approximately one half of the fetuses from all groups was subject to soft tissue examination; the skeletons of approximately half the fetuses were stained and examined microscopically for abnormalities.

Statistics:

DUNNETT's Test [Dunnett ; 1955, 1964] was used for a simultaneous comparison of several dose groups with the control. This test was performed two-sided and was used for the statistical evaluation of the following parameters: Body weight, body weight change, corrected body weight gain (net maternal body weight change), weight of the unopened uterus, number of corpora lutea, number of implantations, number of resorptions and number of live fetuses ; proportion of preimplantation loss, post-implantation loss, resorptions and live fetuses in each litter ; litter mean fetal body weight and litter mean placental weight .
FISHER's Exact Test [Siegel, 1956] was used for a pairwise comparison of each dose group with the control for the hypothesis of equal proportions. This test was performed one-sided and was used for female mortality, females pregnant at terminal sacrifice and the number of litters with fetal findings.
The WILCOXON-Test [Nijenhuis, 1978a ; Hettmansperger, 1984] was used for a comparison of each dose group. with the control for the hypothesis of equal medians. This test was performed one-sided and was used for the proportion of fetuses with malformations, variations, retardations and/or unclassified observations
in each litter.

Clinical signs:

no effects observed

Description (incidence and severity):

No abnormal signs and findings were detected throughout the study.

Mortality:

no mortality observed

Description (incidence):

No deaths were recorded throughout the study period.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

The body weight of the animals of test group 1, compared to the control group, was statistically significantly decreased on day 0 p.c. The body weight of the animals of test group 2, compared to the control group, was statistically significantly decreased on day 9 p.c. and 17 p.c. . The body weight of the animals of test group 3, compared to the control group, was statistically significantly decreased on day 0 p.c. and from day 9 p.c. until the end of the study (day 20 p.c.). The body weight change of the animals of test group 1, compared to the control group, was not statistically significantly different. The body weight changes of the animals of test groups 2 and 3, compared to the control group, were statistically significantly decreased from day 6 p.c. to day 9 p.c. and also if the entire exposure period (day 6 to day 15) was considered. This depression of body weight development in test groups 2 and 3 is considered to be substance related. The corrected body weight gain (terminal body weight on day 20 p.c. minus weight of the uterus before it was opened minus body weight on day 6 p.c.) was statistically significantly lower in the high and in the intermediate dose group (about 85% of the value of the concurrent control group). Furthermore the carcass weight (terminal body weight minus uterine weight) of the high dose group was statistically significantly decreased (95%), which is related to the test substance administration . All other differences between the groups are without any biological relevance.

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

The uterus weights of the animals were not influenced by the administration of the test substance. The differences between the groups are without biological relevance and do not show any dose-response relationship.

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

At necropsy, single animals of the control and test groups 1 and 2(3 or 7.5 mg/L) showed lungs with edema and/or marginal emphysema; these findings, which showed no relation to dosing, have to be related to the sacrifice of the animals . Moreover, one control dam and one female of the 7.5 mg/L group had a hydrometra; both animals did not become pregnant. The occurrence of hydrometra in these two animals is considered to be spontaneous in nature.

Histopathological findings: neoplastic:

no effects observed

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

The conception rate varied between 92% (test group 2: 7 .5 mg/L) and 100% (test group 1- 3 mg/L and test group 3 - 12 mg/L).
There were no substance-related and/or biologically relevant differences between the groups in conception rate, in the mean number of corpora lutea and implantation sites or in the values calculated for the pre- and the postimplantation losses, the number of resorptions and viable fetuses. The differences evident are considered to be incidental and within the normal range of deviations for animals of this strain and age for historical control data.

Dose descriptor:

NOAEC

Effect level:

3 mg/L air (analytical)

Based on:

test mat.

Basis for effect level:

body weight and weight gain

Fetal body weight changes:

no effects observed

Description (incidence and severity):

The mean placental weights in test groups 1, 2 and 3 were not influenced by the test substance exposure and were similar to the control values. The mean fetal weights in test groups 1, 2 and 3 were not influenced by the test substance exposure and there were no biologically relevant differences concerning the means of the control group and of the substance-exposed groups.

Changes in sex ratio:

no effects observed

External malformations:

no effects observed

Description (incidence and severity):

The external examination of the fetuses revealed three types of malformations in single fetuses of test groups 2 and 3. Anasarca was recorded for two fetuses of the intermediate dose group. The latter fetus showed also a kinky tail. Anasarca was also recorded for one high dose fetus which showed in addition a shortened tail. All three aforementioned external malformations were found at low incidences and without a clear dose-response relationship; moreover, the differences between the groups occurred without statistical significance and these malformations or very similar ones are also present at comparable ranges in the historical control data. Therefore, the observed external malformations at the intermediate and high dose level in the present study are considered coincidental and not biologically significant. No external variations were found in any group. So-called unclassified observations (placentae fused or necrobiotic) were recorded without a clear relation to exposure levels for single fetuses in all groups including the controls.

Skeletal malformations:

no effects observed

Description (incidence and severity):

Malformations of the fetal skeletons were seen in 5 out of 184 (= 2.7%) fetuses (in 5 out of 24 litters (= 21%)) of the control, in 10 out of 173 (= 5.8%) fetuses (in 8 out of 25 litters (= 36%)) of the 3 mg/L group, in 7 out of 162 (= 4.3%) fetuses (in 6 out of 23 litters (= 26%)) of the 7.5 mg/L group and in 13 out of 175 (= 7.4%) fetuses (in 9 out of 25 litters (= 36%)) of the 12 mg/L group. All skeletal malformations occurred without any statistically significant differences between the substance-exposed groups and the concurrent control group. Skeletal malformations, that were not correlates of external findings were related to the sternum (sternebra(e) bipartite, ossification centers dislocated or cleft sternum), the vertebral column (vertebral body/bodies dumbbell-shaped (asymmetrical) or bipartite (asymmetrical), vertebrae absent or vertebral arches fused), the skull (exoccipital bone(s) fused with atlas) and the ribs (fused ribs). These skeletal malformations or very similar ones can be found at comparable or even higher fetal/litter incidences in the historical control data. The other observed skeletal malformations (i.e. scapula(e) shortened or deformed; humerus, femur or radius/ulna shortened and bent); os ilium deformed) occurred exclusively in two fetuses of the 7 .5 mg/L group and high dose fetus. These fetuses showed a generalised edema (anasarca) at the external examination. As a consequence of the generalised accumulation of serum in the fetal bodies, the described skeletal findings occurred and thus are correlates of the external finding. The skeletal variations elicited were related to the ribs (shortened or absent 13th, accessory 14th, rudimentary cervical or wavy rib(s)), the sternum (sternebra(e) of irregular shape or bipartite), the skull (epactal bone between parietal and interparietal bones or splitting of skull bones) and the clavicula (deformed). All skeletal variations recorded appeared without a clear dose-response relationship, can be found in a similar frequency in the historical control data and/or the differences between the groups are without biological relevance. This includes the statistically significantly increased occurrence of affected fetuses/litter with rudimentary cervical rib(s) in test group 3. In all groups signs of skeletal retardations occurred substantiated by incomplete or missing ossification of skull (incl . hyoid) bones, vertebral column, sternebra(e), tibia/fibula and metatarsal bones. In total, the number of affected fetuses/litter with skeletal retardations was statistically significantly increased in test group 3. Since this value (61 .3%) is fully within the historical control range (41 .7 - 71 .5%) and thus is in the degree of variability for skeletal retardations, which has been observed in other studies using this strain of rats, it is not considered to be treatment-related. All other differences between the groups in respect to skeletal retardations appeared without statistical significance and/or without a clear dose-response relationship.

Visceral malformations:

no effects observed

Description (incidence and severity):

The examination of the organs of the fetuses revealed two different malformations. Hydrocephaly was recorded for one fetus of test group 2 and dilatation of both heart ventricles (globular shaped heart) occurred in one fetus of test group 0 (control). Both malformations, which were not dose-related, are also present at a low incidence in the historical control data. Therefore, these malformations are considered to be spontaneous in nature. Soft tissue variations (dilated renal pelvis and/or hydroureter) were detected in all groups without any statistically significant and/or biologically relevant differences between the groups. Both findings are very common ones in the rat strain used and all respective values are fully in the range of biological variation. No so-called unclassified observations (like bloody imbibition of kidney(s)) were recorded during the soft tissue examination.

Key result

Dose descriptor:

NOAEC

Effect level:

>= 12 mg/L air (analytical)

Based on:

test mat.

Basis for effect level:

other: no adverse effects observed

Key result

Developmental effects observed:

no

Conclusions:

In an OECD 414 guideline study pregnant female rats were exposed daily to vapor concentrations of 0 (control), 3, 7.5 and 12 mg/L from day 6 - 15 post coitum (BASF 1996). Maternal toxicity was observed at 7.5 mg/L but no teratogenic effects in fetuses could be observed. Therefore the NOAEC for maternal toxicity was 3 mg/L due to body weight gain reduction and the NOAEC for teratogenicity was >=12 mg/L .

Executive summary:

In an OECD 414 guideline study pregnant female rats were exposed daily to vapor concentrations of 0 (control), 3, 7.5 and 12 mg/L from day 6 - 15 post coitum (BASF 1996).

No clinical signs and no mortalities occurred. As already seen in the DRF study, also body weight and body weight gain were reduced in all dose groups. In medium and high dose group body weight gain is considered to be substance related decreased.  No substance-related effects on gestational parameters or fetuses were observed in all dose levels. The NOAEC for maternal toxicity was 3 mg/L due to body weight gain reduction and the NOAEC for teratogenicity was >=12 mg/L (no teratogenic effects observed).

Reference 2

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with national standard methods

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

Dose Range Finder: examination of certain reproduction parameters inlcuding clinical chemistry, hematology and histophathology of the nasal cavities of the dams, but only gross external examination of the fetuses.

Principles of method if other than guideline:

In a range finding study, 3 groups of 10 pregnant Wistar rats were exposed to a vapor of the test substance at target concentrations of 3, 7.5 and 12 mg/L for 6 hours/day from day 6 to 15 post coitum (p.c.).

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Dr. K Thomae GmbH, Biberach, FRG
- Age at study initiation: 10-15 weeks
- Weight at study initiation: range of 200 - 300 gram
- Housing: Single housing in cages from DK III, EBECO, Becker & Co, Castrop-Rauxel, FRG
- Diet: Kliba laboratory diet, rat/ mouse/hamster maintenance diet, Kliba 24-343-4 10 mm pellets Klingentalmühle AG, Kaiseraugst, Switzerland
- Water: ad libitum
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24
- Humidity (%): 30 - 70
- Air changes (per hr): Central air conditioning
- Photoperiod (hrs dark / hrs light): 12 / 12

Route of administration:

inhalation: vapour

Type of inhalation exposure (if applicable):

whole body

Vehicle:

air

Details on exposure:

Generation of an Inhalation atmosphere (vapor): During exposure of the animals, an exposure mixture (Inhalation atmosphere: test substance in air) was generated continuously in such a way that it is as homogeneous and of a composition as constant as possible. By means of a metering pump constant amounts of the substance to be tested were supplied to a heated vaporizer, the vapors that develop were being mixed with a stream of fresh air and passed into the Inhalation system.

Exposure systems: The animals were housed singly in wire cages that are located in a glass-steel Inhalation chamber with a volume of ca. 1.1 m³. Control group 0: By means of an exhaust air system the pressure ratios in the Inhalation system were adjusted in such a way that the amount of exhaust air is lower (excess pressure). This was to ensure that no laboratory air reaches the control animals. Test groups 1 - 3: By means of an exhaust air system the pressure ratios in the inhalation system was adjusted in such a way that the amount of exhaust air is higher (pressure below atmospheric). This is to ensure that no contamination of the laboratory occurs as the result of leakages from the inhalation chambers.

Measurement and recording of technical conditions in the exposure systems: The air flow rates, pressure conditions within the chambers, as well as relative humidities and temperatures in the inhalation system were measured continuously by an automated measuring system and was monitored against preset limits and partially regulated. The generator parameters temperature and compressed air were also be recorded by means of this system Principles of recording with the automated measuring system.

The measured values of the various parameters were recorded at the appropriate measuring points with suitable measuring equipment (sensors, orifice plates etc.), standardized (0 - 20 or 4 20 mA) and transferred to the instrumentation consoles. There, the measured parameters were indicated in an analog way (where this is provided for) and some were used as actual value for regulating the specific parameter.

In addition the measured values were scanned about every 12 seconds, converted from analog to digital, transferred to a personal computer and displayed on the screen. The computer checks the arriving values against the preset threshold values, calculates daily mean and standard deviation for each parameter and generates daily protocols. Furthermore the start and the end of exposure was provided in the daily protocol. Malfunctions that lead to any exceeding of threshold values will cause reports on the daily protocol that indicate the type of disturbance, the affected measuring point as well as the duration and extreme value reached.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Absorption samples were taken adjacent to the animal's noses in order to determine the analytically measured concentration in the inhalation apparatus. The samples will be analyzed by gaschromatography. Sampling frequency: generally hourly

Details on mating procedure:

Up to 4 untreated female rats were mated with one untreated fertile male animal of the same strain. Mating took place from 16.00 h to 7.30 h of the following day. If sperms were detected microscopically in the vaginal smear in the morning, the animals were considered to be fertilized. This day was designated "day O" (beginning of the study}, the following day "day 1" post coitum (p c.).

Duration of treatment / exposure:

6 hours

Frequency of treatment:

daily, 6 hours per day

Duration of test:

from day 6 to 15 p. c.

Remarks:

Doses / Concentrations:
3, 7.5, 12 mg/L
Basis:
other: target conc.

Remarks:

Doses / Concentrations:
3.0, 7.5, 11.8 mg/L
Basis:
analytical conc.

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Maternal examinations:

Clinical examinations:
- Body weight: Days 0 (= day of sperm detection), 3, 6, 9, 12 and 15 p.c.
- General observations: Each workday, at least once during the preflow period and post-exposure observation and at least three times on exposure days
- Check for dead animals: Daily

Clinical chemistry and hematology:
Blood was taken in the morning after the last exposure by puncture of the retroorbital venous plexus. Blood samples were taken in a random order. State of the animals: non-fasted. In test groups 0 - 3 the following parameters weree examined in all the animals:
- Hematological examinations: Leucocytes, erythrocytes, hemoglobin, hematocrit, mean cell volume, hemoglobin content per erythrocyte, mean corpuscular hemoglobin concentration, platelets, differential blood count.
- Clotting analysis: Hepato Quick's test
- Enzymes: Alanine-aminotransferase, aspartate-aminotransferase, alkaline phosphatase, y-glutamyltransferase
- Clinicochemical examinations: Potassium, chloride, inorg. phosphate, calcium, urea, creatinine, glucose, total bilirubin, total protein, albumin, globulins, triglycerides, cholesterol, magnesium.

Pathological examinations:
- Macroscopic pathological examination,
- Determination of lung, liver and kidney weight
- Body weight of the killed dams for calculation of relative organ weights
- Asservation of head, lung, liver and kidneys in 4% formalin for possible histopathological examination

Ovaries and uterine content:

- Weight of intact uterus
- Number of corpora lutea
- Number of implants (differentiated for dead [early and late resorptions] and alive)
- Early resorptions (after SALEWSKI, 1964) in animals that seem to be non-pregnant or are pregnant in only one uterine horn
- Position of implants in the uterus

Fetal examinations:

Gross-pathological examination of the fetuses was possible only to a very limited extend at this stage of pregnancy; therefore, only a gross external assessment of the fetuses was carried out. Subsequently the fetuses were discarded.

Statistics:

As a rule arithmetic means and standard deviations were be calculated.
The following statistical tests were performed:
- KRUSKAL-WALLIS und MANN-WHITNEY-U-Test [Siegel, 1956]. All clinicochemical and hematological parameters except differential blond counts
- DUNNETT-Test [Dunnett 1955, 1964]. Body weight, body weight change, corrected body weight change, weight of intact uterus, organ weights, corpora
lutea, implants, pre- and post implantation loss, resorptions, living fetuses.
- FISCHER's Exact-Test [Siegel 1956]. Conception rate, mortality rate (of dams), fetal findings (per fetus and per litter).

Details on maternal toxic effects:

Maternal toxic effects:yes

Details on maternal toxic effects:
Lethality: No deaths occurred in any concentration group throughout the study.

Clinical signs and findings: Test group 0 (control), test group 1 (3 mg/L) and test group 2 (7 .5 mg/L): No abnormal signs and f findings were detected throughout the study. Test group 3 (12 mg/L): No abnormal signs and findings were detected during the preflow period (day 0 p.c. - day 5 p.c.). Only during the first exposure (day 6 p.c.) the animals showed apathy and salivation. From the second exposure onward no abnormal signs and findings were detected during exposure. On the day of killing no abnormal signs and findings were detected.

Body weight/Body weight change: The body weights of the animals of the test groups 1, 2 and 3 compared to the control group 0, were not statistically significantly different. The body weight change of the animals of the test groups 1 and 2, compared to the control group 0, were not statistically significantly different. The body weight change of the animals of the test group 3, compared to the control group 0, was statistically significantly decreased in the interval from day 6 p.c. to day 9 p.c. The results of the corrected body weight gain (terminal body weight on day 16 p.c. minus weight of the uterus before it was opened minus body weight on day 6 p.c.) of all exposure groups (3, 7.5 and 12 mg/L) did not show any statistically significant differences if compared to the controls. The respective value of the 12 mg/L group, however, was the lowest value (about 21% lower than the control value); thus, in connection with the impairments in body weight gain, this might be associated with the exposure of the dams.

Uterus weight: The uterus weights of the animals of test groups 1-3 (3.7, 5 or 12 mg/L) were not influenced by the test substance exposure. The differences between these groups and the control group are without biological relevance and do not show a clear relation to dosing.

Reproduction: The conception rate reached 80% in test group 1 (3 mg/L) and 100% in the control and in test groups 2 and 3 (7.5 and 12 mg/L). There were no substance-related and/or biologically relevant differences between the groups in conception rate, in the mean number of corpora lutea and implantation sites or in the values calculated for the pre- and the postimplantation losses, the number of resorptions and viable fetuses. The differences evident are considered to be incidental and within the normal range of deviations for animals of this strain and age.

Clinical chemistry and hematology: No exposure-related changes were observed in the results of hematology examinations. No exposure-related changes were observed in the results of clotting analysis. No exposure-related changes were observed in the results of serum enzyme examinations. Slightly, but statistically significantly decreased cholesterol concentrations were found in the sera of the animals of the high concentration group (12 mg/L). This isolated finding might be an exposure related effect since the cholesterol concentration level was beyond the normal range. No changes of toxicological concern were seen in the other blood chemistry parameters. There are some further statistically significant intergroup differences in the results of the clinical pathology testing. These deviations are marginal and, in most cases, there was no relationship to the concentration of the test compound. Accordingly, these findings are considered to be of no toxicological significance.

Pathology: There were no statistically significant differences of the terminal body weights and of the organ weights between control and treated dams. Gross lesions: Two animals of test group 1 (3 mg/L) were infertile. All others of test group 1 as well as all dams of test groups 0, 2 (7.5 mg/L) and 3 (12 mg/L) were pregnant.

Histopathology: In the anterior part of the nasal cavity (level I), in the lateral wall, a hyperplasia of the transitional epithelium was observed in six animals of test group 2 and in eight animals of test group 3. The degree of severity was minimal in all effected animals of test group 2 and in two of test group 3. In the other six effected animals of test group 3, the degree of severity was slight. This finding is regarded as substance-related and might be caused by an irritant effect of the test article. One animal of test group 3 showed a moderate focal atrophy of the olfactory epithelium in the dorsal meatus (level II). This finding occurred only in one single female and might have been developed spontaneously.

Dose descriptor:

NOAEC

Remarks:

systemic effects

Effect level:

7.5 mg/L air (analytical)

Based on:

test mat.

Basis for effect level:

body weight and weight gain

other: slightly, but significantly reduced cholesterol levels

Dose descriptor:

NOAEC

Remarks:

local effects

Effect level:

3 mg/L air

Based on:

test mat.

Basis for effect level:

other: hyperplasia of the nasal epithelium

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
External examination of the fetuses:
The external examination of the fetuses revealed no malformations, variations or unclassified observations in any group.

Dose descriptor:

NOAEC

Effect level:

>= 12 mg/L air

Based on:

test mat.

Developmental effects observed:

no

Conclusions:

In a DFR study Wistar rats were daily exposed to vapor concentrations of 0 (control), 3, 7.5 and 12 mg/L from day 6 - day 15 post coitum for 6 hours per day (BASF, 1996). Maternal toxicity could be observed at 12 mg/L (body weight and body weight gain decreased). There were no signs of developmental toxicity. Therefore the NOAEC for maternal toxicity is 7.5 mg/L and for developmental toxicity >=12 mg/L.

Executive summary:

In a dose range finding study from BASF (1994), Wistar rats were daily exposed to vapor concentrations of 0 (control), 3, 7.5 and 12 mg/L from day 6 - day 15 post coitum for 6 hours per day.

No clinical signs and no mortalities occurred during the study. Body weights and body weight gain were slightly reduced. Additionally, cholesterol was found to be slightly, but statistically significant reduced in the highest dose group. A NOAEC of 7.5 mg/L was found for systemic maternal toxicity. For local effects, the NOAEC was 3mg/L due to local effects in the nasal cavity (hyperplasia of the transitional epithelium, minimal degree) of some animals of the medium and high dose group. There were no signs of developmental toxicity.

Reference 3

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

June 2018 - October 2018

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:

2018
The 2018 update is to include rat-specific requirements in the TG 414 only; thus applies to rats and not to rabbits (for details see overall remarks/attachments below)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.31 (Prenatal Developmental Toxicity Study)

Version / remarks:

2008

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

Test species / Strain Rabbit / New Zealand White
Breeder Manfred Bauer Kaninchen, Lohe 7/1, 74632 Neuenstein, Germany
Age (on day 6 of gestation) 5 months

Body weight (on day 6 of gestation) 3.75 kg to 4.68 kg
Number of animals 128 treated females in groups 1 to 4:

Mated and treated animals:
Groups 1 + 2: 30 females per group
Groups 3 + 4: 34 females per group

Evaluated females with viable fetuses:
Group 1: 20 dams
Group 2: 20 dams
Group 3: 20 dams
Group 4: 20 dams

ENVIRONMENTAL CONDITIONS
DIET: ad libitum, Commercial diet ssniff® K-Z V2323 (ssniff Spezialdiäten GmbH, 59494 Soest, Germany)
WATER: ad libitum, tap water in bottles, Samples of drinking water are taken periodically by the Wasserwerk Wankendorf (24601 Wankendorf, Germany)

ENVIRONMENTAL CONDITIONS
Room temperature: 20°C ± 3°C (maximum range)
Relative humidity: 55% ± 10% (maximum range)
Illumination: 12-hour light/12-hour dark cycle
Ventilation rate: 15 - 20 air changes/hour

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

purified water (aqua ad injectabilia)

Details on exposure:

ADMINISTRATION
Frequency of administration Once daily, via gavage
Treatment period Day 6 to 28 of gestation
Vehicle Purified water
Administration volume 2 mL/kg b.w./day
Selection of route of administration According to OECD guideline 414

DOSAGE PREPARATION
The test item formulations were freshly prepared every day.
The test item was diluted in the vehicle to the appropriate concentrations 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.
In addition, the stability, homogeneity, and concentration of the test item mixture was monitored

Analytical verification of doses or concentrations:

yes

Remarks:

For the analysis of the application formulations of groups 2 to 4, samples of approximately 2 x 5 mL each were taken at the following times and stored at 20°C ± 10% until analysis

Details on analytical verification of doses or concentrations:

For the analysis of the application formulations of groups 2 to 4, samples of approximately 2 x 5 mL each were taken at the following times and stored at 20°C ± 10% until analysis:

At start of dosing

Analysis of stability and concentration
Immediately after preparation of the formula-tion as well as after 8 and 24 hours storage of the test item preparations at room temperature.
(3 samples / test item group; groups 2 - 4).
Number of samples: 3 x 3 = 9
(Date of sampling: 26 June 2018)

Homogeneity
At start of administration, during (middle) administration and before administration to the last animal of the dose group.
(3 samples / test item group; groups 2 - 4).
Number of samples: 3 x 3 = 9
(Date of sampling: 26 June 2018)

At end of the dosing period (at a time when the majority of animals was dosed)

Analysis of concentration
During treatment with the test item always before administration to the last animal of the group.
(1 sample / test item group; groups 2 - 4).
Number of samples: 1 x 3 = 3
(Date of sampling: 23 July 2018)

The samples were labelled with the study number, species, type of sample, concentration, sampling time and date.

The method of analysis was validated in LPT Study No. 36470. The following parameters were determined:
- Linearity
- Accuracy
- Precision
- Sensitivity
- Specificity
- Stability
The validation of the method revealed that the method employed was suitable for the determination and quantification of IPDA in the test item formulation samples.

Details on mating procedure:

For this experiment, sexually mature, purebred female artificially inseminated rabbits (New Zealand White) were used.

Duration of treatment / exposure:

Day 6 to 28 of gestation

Frequency of treatment:

Once daily

Duration of test:

On gestation day 29 (one day before the calculated date of parturition) the dams were laparotomised.

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

control group

Dose / conc.:

10 mg/kg bw/day (nominal)

Remarks:

low dose group

Dose / conc.:

25 mg/kg bw/day (nominal)

Remarks:

intermediate dose group

Dose / conc.:

75 mg/kg bw/day (nominal)

Remarks:

high dose group

No. of animals per sex per dose:

20 pregnant females per dose 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 two dose-range-finding studies which were conducted in pregnant rabbits with IPDA dosed at 5, 25 and 50 mg/kg b.w./day (LPT Study No. 35502) and in non-pregnant rabbits with IPDA dosed at 75 and 150 mg/kg b.w./day (LPT Study No. 36299).
LPT Report No. 35502 revealed no test item-related influences on any of the treatment groups.
In LPT Study No. 36299 the high dose group (150 mg IPDA/kg b.w./day) was noted with a distinctly reduced food consumption and body weight and therefore the animals were prematurely sacrificed on TD8. Necropsy revealed a severely reddened gastric mucosa in 3 of 3 examined animals.
The animals of the low dose group (75 mg IPDA/kg b.w./day) were noted with a reduced food consumption but no test item-related change for the body weight. A reddened gastric mucosa and ulcers were noted for 2 of 3 low dose animals during necropsy.
Based on complete lethality at 150 mg IPDA/kg b.w./day, 75 mg IPDA/kg b.w./day
were considered as the maximal tolerated dose (MTD).

The oral route was selected since this has been proven to be suitable for the detection of a toxicological hazard.

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.

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 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, 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 each working day and again in the afternoon to look for dead or moribund animals. This would have allowed post mortem exami-nations 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.

Body weight
The weight of each rabbit was recorded on the day of delivery (used for randomiza-tion), followed by daily weighings starting on GD 6 - always at the same time of the day. The body weight gain was also calculated in intervals (i.e. 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 gain 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.

Necropsy
One day before the calculated parturition, i.e. on gestation day 29, all surviving rabbits were sacrificed by lethal intravenous injection of 300 mg Pentobarbi-tal/kg b.w. and exsanguinated.
In order to check for possible drug effects, a dissection with macroscopic examina-tion 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 illumi-nation. The liver and the kidneys were examined. Any abnormalities in the appear-ance 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.

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
- mean % per group
- number of early resorptions (< 2 g)
- number of late resorptions (> 2 g)

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

Runts
- number per dam/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 variations7
- individual data per fetus
- type of variation: number and incidence (%) per group and litter
- number of affected fetuses per group8
Total variation rate [%] = fetuses per group with variations x 100
fetuses per group



Fetuses with retardations7
- individual data per fetus
- type of retardation: number and incidence (%) per group and litter
- number of affected fetuses per group8
Total retardation rate [%] = fetuses per group with retardations x 100
fetuses per group



 
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

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 with pentobarbital (60 mg/fetus, i.p.).

Each fetus was dissected:
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.

The head was removed from 50% of the fetuses and fixed in BOUIN'S solution. An examination according to WILSON was carried out, inspecting the internal head structures (e.g. eyes).
The cranium was opened for the remaining 50% of the fetuses and the brain was removed for external inspection in toto.
The eviscerated fetuses (intact and headless bodies) were dehydrated in ethanol and cleared in potassium hydroxide solution. The skeleton was stained with Alizarin red (according to DAWSON). The skeletal system was examined (determi-nation of the number and type of retardations, variations as well as malfor-mations).

Statistics:

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 and SHAPIRO-WILK 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 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 ≥ 100 (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 macroscopic 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 'Reproduction Data - Summary - Values per Group') was done using StatXact 4.0.1 software, as such a calculation is not possible in Provantis.

Indices:

Malformation rate, total variation rate, pre implantation loss index, post implantation loss index, viability index, retardations index

Historical control data:

LPT Background Data
Values of control groups (n = 18) and test groups (n = 55) not significantly influenced by any test compound; data taken from the years 2013 to September 2018

Clinical signs:

no effects observed

Description (incidence and severity):

No changes in behaviour, the external appearance or faeces were noted for the control group or the test item-treated groups (10, 25 or 75 mg IPDA/kg b.w./day).

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

No premature deaths were noted for the control group and the treatment groups (10, 25 or 75 mg IPDA/kg b.w./day).
However, one dam of the control group, two dams of the intermediate dose group and two dams of the high dose group were prematurely sacrificed after abortion. Macroscopic post mortem examination revealed no test item-related findings.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

BODY WEIGHT
No test item-related changes in body weight were noted in the dams after oral treatment with 10 mg IPDA/kg b.w./day.
In the intermediate dose group (25 mg IPDA/kg b.w./day), a not statistically significant but constantly lower body weight compared to the control group was noted from GD 9 until study termination on GD 29 (at maximum 2.4% below the value of the control group). Due to this slight reduction, the effect was considered to be not test item-related and within the normal range of variation.
At 75 mg IPDA/kg b.w./day, a more pronounced and constant reduction in body weight was noted from GD 9 until study termination on GD 29 (statistically signifi-cant between GD 9 and GD 23 at p ≤ 0.05 or 0.01) in comparison to the control group (at maximum 5.5% below the value of the control group on GD 16, p ≤ 0.05). The effect is considered a combination of two statistically non-significant effects, namely the reduced gravid uterus weight and the slightly reduced carcass weight. However, this distinctly and constantly lower body weight was dose dependent and therefore considered to be test item-related.

BODY WEIGHT GAIN
No test item-related differences between the control group and the low dose group (10 mg IPDA/kg b.w./day) was noted for the body weight gain.
A slightly but not statistically significantly lower body weight gain was noted for the intermediate dose group (25 mg IPDA/kg b.w./day). However, as the body weight, this small reduction was considered to be within the normal range of variation.
According to the lower body weight of the high dose group (75 mg IPDA/kg b.w./day), also the body weight gain was slightly lower but not statistically significantly different from the control group.
However, the distinctly and constantly lower body weight gain was dose dependent and therefore considered to be test item-related and adverse.

NET BODY WEIGHT GAIN FROM DAY 6
No differences of toxicological relevance between the control group and the treatment groups (10, 25 or 75 mg IPDA/kg b.w./day) were noted for the net body weight gain (without gravid uterus) between GD6 and GD29.

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 10 or 25 mg IPDA/kg b.w./day.
At 75 mg IPDA/kg b.w./day, a decreased food consumption was noted between GD8 and GD17 (at maximum 22.9% below the value of the control group on GD14 to GD15, p ≤ 0.05). This distinctly lower food consumption was considered to be test item-related and adverse (results see figure below).

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 the consumption of drinking water were noted for the dams treated with 10, 25 or 75 mg IPDA/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):

no effects observed

Description (incidence and severity):

Behaviour, external appearance, faeces
No changes in behaviour, the external appearance or faeces were noted for the control group or the test item-treated groups (10, 25 or 75 mg IPDA/kg b.w./day).

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Gravid uterus weight
No test item-related changes in the gravid uterus weight in comparison to the control group were noted for the dams of the low and the intermediate dose group (10 or 25 mg IPDA/kg b.w./day).
A slightly decreased mean value of the gravid uterus weight was noted at the high dose level (75 mg IPDA/kg b.w./day) (8.7% below the control value, statistically not significant). This finding was mainly due to the low uterine weight of animal no. 82 with a total implantation loss and was considered to be test item-related.

Carcass weight
No test item-related changes in the carcass weight in comparison to the control group were noted for the dams of all treatment groups (10, 25 or 75 mg IPDA/kg b.w./day).
A slightly reduced carcass weight was noted at the high dose level (3.3% below the value of the control group). The slight reduction was related to the reduction in body weight and considered to be test item-related.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No test item-related pathological findings were noted during macroscopic inspection of the dams treated with 10, 25 or 75 mg IPDA/kg b.w./day at necropsy.
In the low dose group there was one female with blck foci in the placenta and in the high dose group marked red discoloration in the stomach of one female was observed.
These single findings were considered to be spontaneous and not test item-related.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Number of abortions:

effects observed, non-treatment-related

Description (incidence and severity):

No test item-related abortions were noted for the treatment groups (10, 25 or 75 mg IPDA/kg b.w./day).
However, one control dam, two intermediate dose dams and two high dose dams were noted with spontaneous abortions. As abortions are known to occur spontaneously in rabbits of this strain and age and an abortion was also noted in the control group, the abortions noted in the intermediate and high dose group were considered to be not test item-related. Furthermore, the number of abortions is still in the range of LPT background data (see table below).

Pre- and post-implantation loss:

effects observed, non-treatment-related

Description (incidence and severity):

Animal no. 82 of the high dose group showed a total post-implantation loss.
Control 8.9 % showed pre-implantation loss & 2.9 % animals showed post implantation loss
Low dose 2.0% showed pre-implantation loss & 1% showed post implantation loss
Medium dose 4.6 % showed pre-implantation loss & 3.8% showed post implantation loss
High dose 2.6% showed pre-implantation loss & 13.8% showed post implantation loss

More details in attached table.

Total litter losses by resorption:

effects observed, treatment-related

Description (incidence and severity):

The treatment with the test substance at 75 mg IPDA/kg b.w./day revealed increased incidences of early and total resorptions, and in consequence an increased percentage of post-implantation loss, which were statistically significantly different from the control.
A treatment related effect for the statistically significantly increased rate of early resorptions and the statistically significantly increased percentage of post-implantation loss, cannot totally be excluded, since both values were at the upper end of the LPT historical control data.
Although treatment related and adverse effects cannot totally be excluded, the main reason for those increases is animal no. 82 that showed a total post-implantation loss. When assessing the number of live fetuses the high dose group showed no remarkable difference to the control group (199 vs. 194 out of 20 animals with viable fetuses each). A fetotoxic effect of the test substance can be excluded, since no test item-related malformations, variations or retardations were evident.

Early or late resorptions:

effects observed, treatment-related

Description (incidence and severity):

No test item-related influence was noted on the number of resorptions for the dams of the low and intermediate dose groups (10 or 25 mg IPDA/kg b.w./day).
The treatment with the test substance at 75 mg IPDA/kg b.w./day revealed increased incidences of early and total resorptions, and in consequence an increased percentage of post-implantation loss, which were statistically significantly different from the control.
A treatment related effect for the statistically significantly increased rate of early resorptions and the statistically significantly increased percentage of post-implantation loss, cannot totally be excluded, since both values were at the upper end of the LPT historical control data.
Although treatment related and adverse effects cannot totally be excluded, the main reason for those increases is animal no. 82 that showed a total post-implantation loss. When assessing the number of live fetuses the high dose group showed no remarkable difference to the control group (199 vs. 194 out of 20 animals with viable fetuses each). A fetotoxic effect of the test substance can be excluded, since no test item-related malformations, variations or retardations were evident.

Dead fetuses:

effects observed, non-treatment-related

Description (incidence and severity):

No dead fetus was noted in the control group and in the intermediate and high dose groups (25 or 75 mg IPDA/kg b.w./day).
At 10 mg IPDA/kg b.w./day, dam no. 30 was noted with two dead fetuses (nos. 30-8 and 30-9). As the two dead fetuses were only noted for one dam and no dose response-relationship was present, the two dead fetuses were considered to be spontaneous and not test item-related. Furthermore, the incidence of 2 dead fetuses in one of the treatment groups is in the range of the LPT background data (see table below).

Changes in pregnancy duration:

no effects observed

Changes in number of pregnant:

effects observed, non-treatment-related

Description (incidence and severity):

Control: From 30 mated and treated females in the control group 6 animals became not pregnant and 21 animals were pregnant. 3 animals were excluded and not examined.
Low dose group. From 30 animals 5 became not not pregnant and 20 animals were pregnant, 5 Animals were excluded from further examinations.
Medium dose group. From 34 mated and treated animals, 8 animals not became pregnatn and 22 animals were pregnant.
High dose group. From 34 mated and treated naimals 7 animals became not pregnant and 23 animals were pregnant.
Altogether in all dose groups including the control group, a poor pregnancy rate was observed for seasonal reasons. For details see also attached table.

Other effects:

no effects observed

Description (incidence and severity):

No test item-related differences for the placental weights were noted for any of the dosing groups (10, 25 or 75 mg IPDA/kg b.w./day) in comparison to the control group. The placental weights were within the normal variability.

Details on maternal toxic effects:

Mortality: No test item-related premature death was noted for the control group and the treatment groups (10, 25 or 75 mg IPDA/kg b.w./day).
Abortions: No test item-related abortions were noted for the treatment groups.
Clinical signs: No changes in behaviour and the external appearance were noted in the control group and the test item-treated groups.
Body weight and body weight gain: A test item-related reduced body weight was noted for the high dose dams treated with 75 mg IPDA/kg b.w./day from GD9 until study termination on GD29 (at maximum 5.5% below the value of the control group on GD16, p ≤ 0.05). Accordingly a lower body weight gain was noted for the high dose group (12.6% body weight gain for the high dose group compared to 14.1% in the control group).
Food and drinking water consumption: A test item-related decrease in food consumption was noted for the high dose group (75 mg IPDA/kg b.w./day) from GD8 to GD17 (at maximum 22.9% below the value of the control group on GD14 to GD15, p ≤ 0.05).
Necropsy findings: No test item-related observations were noted for the treatment groups during necropsy.
Uterus weight, carcass weight and net body weight gain:
At 75 mg IPDA/kg b.w./day a slight reduction in the gravid uterus weight was noted (8.7% below the control value, statistically not significant).
A slight reduction was noted for the carcass weight at the high dose level (3.3% below the control value, statistically not significant).

Key result

Dose descriptor:

NOAEL

Effect level:

25 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: maternal toxicity see below

Key result

Dose descriptor:

LOAEL

Effect level:

75 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

body weight and weight gain

early or late resorptions

food consumption and compound intake

organ weights and organ / body weight ratios

pre and post implantation loss

Fetal body weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

No test item-related influence was noted on the mean fetal weights after administration of 10, 25 or 75 mg IPDA/kg b.w./day.
No test item-related effect was noted for the number of runts. In total, 12 runts were noted at laparotomy: 5 runts were noted in the control group, 4 runts in the low dose group (10 mg IPDA/kg b.w./day), one runt in the intermediate dose group (25 mg IPDA/kg b.w./day) and two runts were noted in the high dose group (75 mg IPDA/kg b.w./day).

Reduction in number of live offspring:

effects observed, non-treatment-related

Description (incidence and severity):

No dead fetus was noted in the control group and in the intermediate and high dose groups (25 or 75 mg IPDA/kg b.w./day).
At 10 mg IPDA/kg b.w./day, dam no. 30 was noted with two dead fetuses (nos. 30-8 and 30-9). As the two dead fetuses were only noted for one dam and no dose response-relationship was present, the two dead fetuses were considered to be spontaneous and not test item-related. Furthermore, the incidence of 2 dead fetuses in one of the treatment groups is in the range of the LPT background data.

Changes in sex ratio:

no effects observed

Description (incidence and severity):

The sex distribution of the fetuses in the dose groups (10, 25 or 75 mg IPDA/kg b.w./day) was comparable to the control fetuses. Slight differences observed were within the biological variability.
Sex ratio in study groups(male/female):
Control 0.88
Low dose 0.76
Medium dose 1.06
High dose 1.06

Changes in litter size and weights:

no effects observed

Changes in postnatal survival:

no effects observed

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

No visible gross alteration (malformation or variation) was noted for fetuses of the low and intermediate dose groups (10 or 25 mg IPDA/kg b.w./day).
In the high dose group (75 mg IPDA/kg b.w./day), the fetus no. 84-9 was noted with multiple malformations in form of an absent upper jaw and cranial roof as well as a small brain and a raschisis in the neck region. This malformation complex confirmed the external observation and was considered to be not test item-related but spontaneous according to type and incidence.

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

No alterations in the form of malformations were noted during skeletal examina-tions of the fetuses according to DAWSON at the low and intermediate dose level (10 or 25 mg IPDA/kg b.w./day).
At the high dose level (75 mg IPDA/kg b.w./day), the fetus no. 84-9 was noted with a malformation of the skull in form of an absent cranium and maxilla as well as a partly ossified temporal bone and cervical vertebral arches being partly reduced in size and misaligned. However this single occurrence was considered to be sponta-neous and not test item-related.

The skeletal variations observed during examination according to DAWSON were related to the caudal vertebral bodies (fused), to the lumbar vertebral bodies (misaligned), to the thoracic vertebral arches (fused), to the sternum (sternebra(e) bipartite, fused, misaligned, misshapen) or the rib(s) (accessory 13th ribs (uni- or bilateral), short, less than 12 ribs ossified).
No test item-related influences were noted on the incidence of the above men-tioned skeletal variations in the treatment groups (10, 25 or 75 mg IPDA/kg b.w./day).

The retardations observed during skeletal examination (according to DAWSON) were related to the skull (incomplete ossification), the lumbar vertebral arches (reduced in size), the lumbar vertebral bodies (reduced in size), the sternum (sternebra(e) unossified, incompletely ossified or reduced in size), the talus (unossified), the thoracic vertebral arches (unossified) and the thoracic vertebral bodies (reduced in size or unossified).
No test item-related influences were noted on the incidence of the above men-tioned skeletal retardations in the treatment groups (10, 25 or 75 mg IPDA/kg b.w./day).
However, statistically significant increases were noted for the incidences of the incomplete ossification of the skull in the intermediate dose group (p ≤ 0.05) as well as for the incidences of unossified sternebra(e) and the total retardations in the low dose group (for both p ≤ 0.01).
As no dose response relationship was noted and the values were within the range of the LPT background data ; these 3 statistically significantly increased incidences were considered to be not test item-related.

For details see attached table.

Visceral malformations:

effects observed, non-treatment-related

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 examination of the fetuses of the control group and the dose groups (10, 25 or 75 mg IPDA/kg b.w./day). The gross inspection of the brain in 50% of the fetuses revealed no changes for any of the fetuses after opening of the cranium and removal of the brain.

No alterations in the form of malformations were noted during soft tissue examina-tions of the fetal head according to WILSON at any tested dose level (10, 25 or 75 mg IPDA/kg b.w./day).
No test item-related influence was noted in the number of soft tissue variations of the fetal head compared to the control at any tested dose level (10, 25 or 75 mg IPDA/kg b.w./day).
The observed and classified soft tissue variations that were noted during the examination of the fetal head according to WILSON were in the form of dilatations of the 4th cerebral ventricle, subdural haemorrhages in the meninges and haemor-rhages in the cerebrum as well as cystic or semicircular cystic areas in the cerebral hemisphere. No test item-related differences in the incidences of the observed variations of the fetal head were noted between the control group and the test groups.
The observed cystic or semicircular cystic areas in the cerebrum noted in altogeth-er 6 fetuses of the test item groups are known to be fixation-induced artefacts .

Other effects:

no effects observed

Details on embryotoxic / teratogenic effects:

One abortion was noted for the control group (no. 1) and two abortions each were noted for the intermediate dose group (nos. 58 and 63) and the high dose group (nos. 76 and 90) treated with 25 or 75 mg IPDA/kg b.w./day). As abortions are known to occur spontaneously in rabbits of this strain and age, these single incidences were considered to be an incidental finding and hence, are judged to be not test item-related.
Two dead fetuses were noted for dam no. 30 (nos. 30-8 and 30-9) treated with 10 mg IPDA/kg b.w./day. As no dose dependence-relationship was noted and because of the low incidence, the occurrence of one dam with two dead fetuses was considered to be not test item-related.
No test item-related increase was noted for the incidence of runts at any tested dose level.
No test item-related malformations or variations were noted during the macro-scopic external examination and the macroscopic gross inspection of the internal organs at laparotomy and the skeletal examination according to DAWSON or the soft tissue examination of the fetal heads according to WILSON for the treatment groups (10, 25 or 75 mg IPDA/kg b.w./day).
Furthermore, the skeletal examination according to DAWSON revealed no test item-related retardations.

Key result

Dose descriptor:

NOAEL

Effect level:

> 75 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no prenatal developmental toxicity effects observed up to the highest tested dose

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

no

Table: Overview on abortions of the study animals

Group	Dam no.	Day of abortion (GD)	Time of abortion	Day of sacrifice	Abortion rate (Aborted animals / evaluated pregnant females; row 5 in text table 4-1)
1 Control	1	22	during the day	22	1 / 21 = 4.8%
2 10 mg/kg	-	-	-	-	0 / 20 = 0.0%
3 25 mg/kg	58	20	during the night	21	2 / 22 = 9.1%
	63	26	during the night	27
4 75 mg/kg	76	26	during the night	27	2 / 23 = 8.7%
	90	21	during the night	22

 

Table: Comparison of the abortion rate with theLPTbackground data.

Abortions		Abortion rates in this study (LPT Report 35503)		LPT Background Data#^1- Values of control groups (n = 18) and test groups (n = 55) not significantly influenced by any test compound; data taken from the years 2013 to September 2018   [Mean value from the mean values of the groups used] [Range of mean values of the individual groups used]
Abortion rate (%)		Control:	4.8	4.13 ± 3.81 0.0 - 9.5	control groups
		Group 2:	0.0
		Group 3:	9.1	3.32 ± 3.65 0.0 - 10.0	test groups
		Group 4:	8.7
#^1:	not audited by QAU

Table:Summary of the mean body weight gain from GD6 to GD29

Mean Body Weight Gain
Group	Time interval Gestation day 6 - 29 (whole study period)
	Gain in kg	Gain in %	Difference to control %
Control	0.60	14.1	n.a.
Group 2 (10 mg/kg)	0.64	14.7	+6.6
Group 3 (25 mg/kg)	0.53	12.8	-10.8
Group 4 (75 mg/kg)	0.52	12.6	-12.8

 

Table:Comparison of the resorption rates and the post-implantation loss with the LPT background data.

Parameter		Values observed in this study (LPT Report 35503)   [fetal incidence in % per group]		LPT Background Data#^1- Values of control groups (n = 18) and test groups (n = 55) not significantly influenced by any test compound; data taken from the years 2013 to September 2018   [Mean value from the mean values of the groups used] [Range of mean values of the individual groups used]
Total resorptions mean % per dam		Control:	0.3	0.70 ± 0.39 0.1 - 1.3	control groups
		Group 2:	0.0
		Group 3:	0.4	0.79 ± 0.41 0.0 - 1.6	test groups
		Group 4:	1.5** #^2
Early resorptions mean % per dam		Control:	0.1	0.39 ± 0.30 0.0 - 1.0		control groups
		Group 2:	0.0
		Group 3:	0.3	0.45 ± 0.32 0.0 - 1.3		test groups
		Group 4:	1.0** #^2
Late resorptions mean % per dam		Control:	0.3	0.33 ± 0.22 (0.0 - 0.7)	control groups
		Group 2:	0.0
		Group 3:	0.1	0.36 ± 0.23 (0.0 - 0.9)	test groups
		Group 4:	0.4
Post-implantation loss % per group		Control:	2.9	7.71 ± 4.66 (0.5 - 16.5)	control groups
		Group 2:	1.0 #^3
		Group 3:	3.8	8.83 ± 4.36 (1.0 - 17.4)	test groups
		Group 4:	13.8** #^3
Post-implantation loss mean % per dam		Control:	2.0	7.85 ± 4.92 (0.4 - 16.9)	control groups
		Group 2:	1.0
		Group 3:	3.9	8.71 ± 4.30 (1.0 - 15.7)	test groups
		Group 4:	15.1** #^2
#^1:	not audited by QAU
#^2*/**:	(p ≤ 0.05/0.01) Dunnett test
#^3*/**:	(p ≤ 0.05/0.01) Chi^2test

Table: :Summary of the reproduction data

Parameter					Group 1 Control (n=20)	Group 2 10 mg/kg (n=20)	Group 3 25 mg/kg (n=20)	Group 4 75 mg/kg (n=21) #^1
Corpora lutea			total mean per dam		225 11.3	199 10.0	195 9.8	231 11.0
Implantation sites			total mean per dam		205 10.3	195 9.8	186 9.3	225 10.7
Total resorptions			total mean per dam		6 0.3	0 0.0	7 0.4	31 1.5 ** #^1
Early resorptions			total mean per dam		1 0.1	0 0.0	5 0.3	22 1.0 ** #^1
Late resorptions			total mean per dam		5 0.3	0 0.0	2 0.1	9 0.4
Fetuses (alive + dead)			total mean per dam		199 10.0	195 9.8	179 9.0	194 9.7 #²
Live fetuses			total mean per dam		199 10.0	193 9.7	179 9.0	194 9.7 #²
Dead fetuses			total mean per dam		0 0.0	2 0.1	0 0.0	0 0.0 #²
Post-implantation loss			per group		2.9	1.0	3.8	13.8 **
[%]			mean per dam		2.0	1.0	3.9	15.1 **
	* / **	Significantly different from the controls at p ≤ 0.05 / p ≤ 0.01, Chi^2test, only performed for the group values of the pre- and post-implantation loss (see table7-1).
	* / **	Significantly different from the controls at p ≤ 0.05 / p ≤ 0.01, Dunnett test, performed for the mean values per group (see table7-2).
	#^1	A total post-implantation loss (9 early resorptions) was noted in 1 of 21 litters.
	#^2	The calculation was based on 20 litters with viable fetuses (see table7-2).

 

Table :Comparison of the number of dead fetuses with the LPT background data.

Parameter		Values observed in this study (LPT Report 35503)		LPT Background Data#^1- Values of control groups (n = 18) and test groups (n = 55) not significantly influenced by any test compound; data taken from the years 2013 to September 2018
Dead fetuses (total number per group)		Control:	0	0 - 1 #^2	control groups
		Group 2:	2
		Group 3:	0	0 - 5	test groups
		Group 4:	0
Dead fetuses (mean per dam)		Control:	0.0	0.01 ± 0.02 #^4 (0.0 - 0.1) #^3	control groups
		Group 2:	0.1 ± 0.4
		Group 3:	0.0	0.06 ± 0.09 (0.0 - 0.4)	test groups
		Group 4:	0.0
#^1:	not audited by QAU
#^2	Range of the total number of dead fetuses in the control groups.
#^3	Range of the mean number of dead fetuses per dam in the cpntrol groups.
#^4	Mean value of the mean numbers of dead fetuses per dam in the control groups.

Table: :Overview of the runts per group

IPDA
Group 1: Control		Group 2: 10 mg/kg		Group 3: 25 mg/kg		Group 4: 75 mg/kg
Dam no.	Runt Fetus no.	Dam no.	Runt Fetus no.	Dam no.	Runt Fetus no.	Dam no.	Runt Fetus no.
7	6 m	40	5 m	56	10 f	81	7 m
	7 f		7 f	-	-	120	5 f
12	5 f	44	5 f	-	-	-	-
18	2 f		8 f	-	-	-	-
	7 f	-	-	-	-	-	-

Table :Background data of statistically significant incidences of skeletal retardations

Skeletal retardations		Values observed in this study (LPT Report 35503)   [fetal incidence in % per group]		LPT Background Data#^1- Values of control groups (n = 18) and test groups (n = 55) not significantly influenced by any test compound; data taken from the years 2013 to September 2018   [fetal incidence in % per group] [Mean value ±SD (range)]
Sternebra(e) unossified		Control:	24.1	12.8 ± 7.8 2.2 - 25.7	control groups
		Group 2:	38.5 ** #²
		Group 3:	19.0	13.5 ± 7.8 4.0 - 39.0	test groups
		Group 4:	25.3
Skull incompletely ossified		Control:	0.0	0.37 ± 0.85 0.0 - 3.4		control groups
		Group 2:	1.0
		Group 3:	1.7 * #²	0.53 ± 0.84 0.0 - 2.9		test groups
		Group 4:	1.5
Total fetal skeletal retardations		Control:	68.3	56.4 ± 14.9 (20.7 - 77.1)	control groups
		Group 2:	80.0 ** #^2
		Group 3:	66.5	53.6 ± 13.9 (25.2 - 80.5)	test groups
		Group 4:	73.7
#^1:	not audited by QAU
#^2:	Considered to be not test item-related as withinLPTbackground data range.
*/**:	(p ≤ 0.05/0.01) Fisher or Chi^2- test

Conclusions:

Under the conditions of the study, 3-Aminomethyl-3,5,5-trimethylcyclo-hexanamine did not show any teratogenic potential.

Executive summary:

Under the conditions of the study, IPDA did not show any teratogenic potential.In this prenatal developmental toxicity study according to OECD 414 (version 2001), the test item 3-Aminomethyl-3,5,5-trimethylcyclo-hexanamine was administered orally to 128 inseminated female rabbits at dose levels of 0, 10, 25 or 75 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 (purified water) without test substance. The body weight of the 5-month-old animals at day 6 of gestation was 3.75 kg to 4.68 kg. 20 dams per dose group were examined and evaluated at the end of the study.

 

Examination of the dams         

Mortality:                       No test item-related premature death was noted for the control group and the treatment groups (10, 25 or 75 mg IPDA/kg b.w./day).

Abortions:                     No test item-related abortions were noted for the treatment groups.

Clinical signs:                  No changes in behaviour and the external appearance were noted in the control group and the test item-treated groups.

Body weight:                 A test item-related reduced body weight was noted for the high dose dams treated with 75 mg IPDA/kg b.w./day from GD9 until study termination on GD29 (at maximum 5.5% below the value of the control group on GD16, p ≤ 0.05). Accordingly, a lower body weight gain was noted for the high dose group (12.6% body weight gain for the high dose group compared to 14.1% in the control group).

Food and DW

Consumption:                A test item-related decrease in food consumption was noted for the high dose group (75 mg IPDA/kg b.w./day) from GD8 to GD17 (at maximum 22.9% below the value of the control group on GD14 to GD15, p ≤ 0.05).

 

Necropsy findings:        No test item-related observations were noted for the treatment groups during necropsy.

Uterus weight, carcass weight and net body weight gain:           

At 75 mg IPDA/kg b.w./day a slight reduction in the gravid uterus weight was noted (8.7% below the control value, statistically not significant).

A slight reduction was noted for the carcass weight at the high dose level (3.3% below the control value, statistically not significant).

Summary table:Overview of the animals (total numbers)

IPDA		Group 1 Control	Group 2 10 mg/kg	Group 3 25 mg/kg	Group 4 75 mg/kg
1	Mated and treated females	30 #	30 #	34 #	34 #
2	Not examined females (excluded/spare animals)	3	5	4	4
3	Non-pregnant females	6	5	8	7
4	Deceased animals	none	none	none	none
5	Evaluated pregnant females	21	20	22	23
6	Evaluated dams without viable fetuses (total post implantation loss)	none	none	none	1
7	Evaluated dams with abortion	1	none	2	2
8	Evaluated litters with viable fetuses	20	20	20	20

 

#      Further animals were included to yield 20 evaluable litters with viable fetuses.

        A poor pregnancy rate was observed for seasonal reasons.

 

Examination of the fetuses      

In the high dose group (75 mg IPDA/kg b.w./day), a test item-related increase was noted for the number of early resorptions (22 early resorptions in the high dose group in comparison to 1 early resorption in the control group). Accordingly, also the post-implantation loss was increased in the high dose group (13.8% in the high dose group compared to 2.9% in the control group).

No test item-related deaths of fetuses were noted.

No test item-related increase was noted for the incidence of runts in the test item groups in comparison to the control group.

Sex distribution:             No test item-related differences were noted.

Fetal weights:                No test item-related influence on the fetal weights was noted for the treatment groups.

Placental weights:         No test item-related differences were noted.

Fetal alterations            

Malformations:             No test item-related malformation was noted during the external examination at laparotomy, the gross inspection of the internal organs, the skeletal examination according to DAWSON and the soft tissue examination of the fetal head according to WILSON.

Variations:                      The external examination at laparotomy, the gross inspection of the internal organs, the skeletal examination according to DAWSON and the soft tissue examination of the fetal head according to WILSON revealed no test item-related variations.

Retardations:                 Examination according to DAWSON revealed no test item-related delays in the ossification.

 

 

Analysis of test item formulations

The measured actual concentrations of the test item in the test item vehicle-mixtures were between 102.7% and 106.5% of the nominal concentrations, indicating correctly prepared, stable and homogeneous formulations.

Conclusion 

Under the present test conditions, the no-observed-adverse-effect level (NOAEL) was 25 mg IPDA/kg b.w./day for the dams:

No test item-related premature death was noted for any of the dose groups.

For the high dose group (75 mg IPDA/kg b.w./day), a reduction was noted for the body weight or the body weight gain. Also a decrease was noted in food consumption at 75 mg IPDA/kg b.w./day.

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

No test item-related pathological findings were noted during necropsy for the treatment groups.

At 75 mg IPDA/kg b.w./day, a slight decrease was noted for the gravid uterus and the carcass weight.

 The no-observed-adverse effect level (NOAEL) was above 75 mg IPDA/kg b.w./day for the fetuses and 25 mg IPDA/kg b.w./day for the rate of early resorptions/postimplantation loss:

At the maternotoxic dose level of 75 mg IPDA/kg b.w./day (reduced body weight, reduced body weight gain, reduced carcass weight and reduced food consumption), an increased number of early resorptions and accordingly an increased post-implantation loss were noted. Due to the increased number of early resorptions, the total resorption rate was slightly above the LPT historical control data (up to 1.3). The early resorption rate and the percentages of post-implantation loss were at the upper range but still within the LPT historical control data.

No test item-related influence was noted on the mortality or body weight of the fetuses in the treatment groups.

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

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

 

Reference 4

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2001-06-05 - 2001-06-28

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:

1981

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Remarks:

Crl CD® (SD) IGS BR, Caesarian Obtained, Barrier Sustained-Virus Antibody Free, (COBS-VAF®).

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, L'Arbresle (France)
- Strain and sanitary status: Crl CD® (SD) IGS BR, Caesarian Obtained, Barrier Sustained-Virus Antibody Free, (COBS-VAF®).
- Age: 10-11 weeks
- Weight at study initiation: 206-301, mean 245 g. Mean weights in the four groups were similar.
- Housing: The animals were housed individually in suspended wire-mesh cages (43.0 x 21.5 x 18.0 cm). A metal tray, containing autoclaved sawdust (SICSA, Alfortville, France), was placed under each
cage.
- Diet: Pelleted maintenance diet, ad libitum
- Water: tap water, ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 2 °C
- Humidity (%): 50 +/- 20 %
- Air changes (per hr): 12 cycles/hour
- Photoperiod (hrs dark / hrs light): 12h/12h

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

VEHICLE
The vehicle was purified water, obtained by reverse osmosis using a Milli-Ro 8 plus apparatus (Millipore SA, Saint-Quentin en Yvelines, France).

DOSAGE FORM PREPARATION
The test item was administered as a solution in the vehicle. The test item was mixed with the required quantity of vehicle in order to achieve the concentrations of 1, 5 and 25 mg/mL and then homogenized using a magnetic stirrer. The test item dosage forms were made for up to 9 days according to the stability results obtained for the study and were stored at +4 °C and protected from light prior to use. The dosage forms were delivered each day to the animal room.

TREATMENT GROUPS
The dose-levels were determined in agreement with the Sponsor, following the results of a
previously conducted preliminary study. In this study, minor to marked matemotoxicity was recorded at 20, 70 and 250 mg/kg/day dose-levels. Consequently, it was decided, in agreement with the Sponsor, to lower the low and intermediate dose-levels in order to obtain a NOAEL at the low dose.
Group // Number of animals // Dose-level (mg/kg/day)
1 // 24 mated females* // 0
2 // 24 mated females* // 10
3 // 24 mated females* // 50
4 // 24 mated females* // 250
*: only the first 20 pregnant females were taken into consideration for fetal examinations.

ADMINISTRATION
The oral route was selected since it was one of the routes of exposure in Man. The test item or control dosage forms were administered by gavage using a plastic syringe fitted with a metal gavage tube. The quantity of the dosage form administered to each animal was adjusted according to the most recently recorded body weight. A constant dosage-volume of 10 mL/kg/day was used. The dosage forms were stirred continuously throughout the dosing procedure.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Chemical analysis of the dosage forms:
Before the start of treatment, the suitability of the proposed formulation procedure was
determined by the analysis of concentration and stability of dosage forms which were prepared
using the procedure.
During the treatment period, a check of the concentration was performed on dosage forms
prepared for use in the study.

Stability
Two dosage forms were prepared at the lowest and highest concentrations used in the study
(1 and 25 mg/mL). Each dosage form was protected from light and sampled after O Gust after
preparation), 4 and 9 days storage at +4 °C.
The aliquots sampled just after preparation ( day 0) were analyzed immediately and those
sampled on day 4 were stored frozen at -20°C pending analysis on last sampling occasion (day 9)
when all samples were assayed. -> The results of the chemical analyses demonstrated a satisfactory stability of both dosage forms prepared over a nine-day period at +4°C (away from light).

Concentration
The concentration of samples taken from each dosage form (including the control) prepared for
use on the first day of treatment and on the last day of treatment was determined. -> Throughout the study, a satisfactory agreement was observed between the nominal and actual concentrations of the test material in the administered dosage forms since the deviations from nominal concentration were in an acceptable range of± 6 %.

Details on mating procedure:

Females were mated at the breeder's facilities. The day of confirmed mating (detection of a vaginal plug) was designated as day 0 post-coitum.

Duration of treatment / exposure:

day 6 to day 19 post-coitum inclusive

Frequency of treatment:

daily

Duration of test:

until day 20 post-coitum

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

control group

Dose / conc.:

10 mg/kg bw/day (nominal)

Remarks:

low dose group

Dose / conc.:

50 mg/kg bw/day (nominal)

Remarks:

intermediate dose group

Dose / conc.:

250 mg/kg bw/day (nominal)

Remarks:

high dose group

No. of animals per sex per dose:

24 (females only); only the first 20 pregnant females were taken into consideration for fetal examinations

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: dose levels were determined based on the results of a preliminary study

Maternal examinations:

CLINICAL EXAMINATIONS
MORBIDITY AND MORTALITY
Each animal was checked for mortality or signs of morbidity:
- at least twice a day during treatment period,
- at least once a day on other days.
Female found dead on day 16 was subjected to a macroscopic post-mortem examination. The number of corpora lutea and implantation sites was recorded.

CLINICAL SIGNS
Each animal was observed for clinical signs (including evidence of abortion/resorption) at approximately the same time.

FOOD CONSUMPTION
The quantity of food consumed by each female was recorded for the following intervals: 2-6, 6-9, 9-12, 12-15, 15-18 and 18-20 post-coitum.
Food intake per animal and per day was calculated.

BODY WEIGHT
The body weight of each female was recorded on days 2, 6, 9, 12, 15, 18 and 20 post-coitum. The net body weight was calculated (gross body weight minus gravid uterine weight), as well as the gross and net body weight change.

SACRIFICE AND TERMINAL EXAMINATIONS
These examinations were carried out in all females.
HYSTERECTOMIES
On day 20 post-coitum, all the females were killed by asphyxiation using carbon dioxide. The weight of the gravid uterus was recorded for each pregnant female at hysterectomy (with at least one live fetus). The ovaries and uterus of females were examined to determine:
- number of corpora lutea,
- number and distribution of dead and live fetuses,
- number and distribution of early and late resorptions,
- number and distribution of implantation sites (or uterine scars).
Early resorptions refer to evidence of implantation without recognizable embryo or fetus; late resorptions refer to dead embryo or fetus with external degenerative changes; scars refer to evidence of implantation without recognizable structure (embryo, fetus or placenta). In apparently non-pregnant females, the presence of implantation scars on the uterus was checked using the Salewski staining technique.

MACROSCOPIC POST-MORTEM EXAMINATION
After hysterectomy, the females were subjected to a macroscopic post-mortem examination of the principal thoracic and abdominal organs. A gross evaluation of placentas was also performed. The number of corpora lutea and implantation sites was recorded in any female that died.

Ovaries and uterine content:

- Examination of uterine content: Weight of gravid uterus, number or corpora lutea, number and distrubution of implantation sites (or uterine scars), number and distrubution of early and late resorptions, number and distribution of dead and live fetuses

Fetal examinations:

These examinations were carried out only in the first 20 litters (females with at least one live fetus). The other litters were discarded without further investigation. All the fetal examinations were conducted without knowledge of treatment group in order to minimize bias. The fetal findings were described according to the glossary of the International Federation of Teratology Societies (IFTS) and classified as malformation or variation: malformation refers to a permanent structural change that is likely to adversely affect the survival or health, variation refers to a change that occurs within the normal population under investigation and is unlikely to adversely affect survival or health (this might include a delay in growth or morphogenesis that has otherwise followed a normal pattern of development).

BODY WEIGHT
The body weight of each live fetus was recorded.

EXTERNAL EXAMINATION
Each fetus was submitted to a detailed external examination, which included the observation of all visible structures, surfaces and orifices. The fetuses were then killed by a subcutaneous injection of thiopental sodium.

SOFT TISSUE EXAMINATION
Approximately half of the live fetuses per litter were fixed with Bouin's fluid. A detailed soft tissue examination was performed according to a free-hand sectioning technique (Wilson technique) which included the observation of all the organs and structures of the head, neck, thorax and abdomen.

SKELETAL EXAMINATION
The remaining live fetuses per litter were fixed in ethyl alcohol. A detailed examination of the skeleton was performed after staining with alizarin red S and alcian blue. This examination included the observation of all the bone structures and cartilage of the head, spine, rib cage, pelvis and limbs.

SEX OF FETUSES
The sex of each live fetus was determined at the time of the evisceration ( after fixation in alcohol) or at the time of the Wilson sectioning.

Statistics:

Mean values were compared by one-way analysis of variance and Dunnett test (mean values being considered as normally distributed and variances being considered as homogeneous). Percentage values were compared by the Fisher exact probability test.

Indices:

Data are expressed as group mean values ± standard deviation (maternal body weight and food consumption, fetal body weight and number of corpora lutea, implantations, fetuses and
resorptions) or as proportions (pre-implantation loss, post-implantation loss and fetal findings).
Whenever necessary, the experimental unit of comparison was the litter.
The pre-implantation loss was calculated as follows:
((Number of corpora lutea - Number of implantation sites) / Number of corpora lutea) x 100
The post-implantation loss was calculated as follows:
((Number of implantation sites - Number of live fetuses) / Number of implantation sites) x 100
Fetal findings were analyzed as follows:
(Number of fetuses with a particular finding / Total number of fetuses examined) x 100
In addition, the mean percentage of affected fetuses/litter was calculated.

Historical control data:

historical control data avaiable for fetal soft tissue variation, fetal skeletal malformations and fetal skeletal variation

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

There were no clinical signs in the control and the 10 and 50 mg/kg/day groups.
At the 250 mg/kg/day dose-level, ptyalism was recorded in most females of this group from
day 11, 12, 13 or 14 post-coitum until hysterectomy. Loud breathing and regurgitation were
recorded in 4 females of this group. All these findings might be the consequence of the corrosive
properties of the test item and the high pH of the dosage forms (pH between 1 0 and 12).

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, treatment-related

Description (incidence):

There were no deaths in the control, 10 and 50 mg/kg/day treated groups.
In the 250 mg/kg/day treated group, one female was found dead on day 16 of pregnancy. Loud
breathing caused by regurgitation of the test item was recorded the day before death. No changes
of body weight were noted before death. Whitish foci on the lung were recorded at necropsy.
Consequently, this death was attributed to regurgitation following gavage and was not
considered to be related to the toxicity of the test item.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

The body weight gain was similar in the control and the 10 and 50 mg/kg/day groups throughout
the treatment period.
In the 250 mg/kg/day group, a significantly lower body weight gain (-35%, p<0.001) was
recorded after the first three days of treatment. Thereafter, the body weight was similar to that of
the controls. Over the whole treatment period, the difference remained slight (-10%, not
statistically significant).
The net body weight gain was also significantly lower at this dose-level (-25%, p<0.0 1) when
compared to the control group.
Because the reduction in food consumption correlated with a reduction in body weight gain at
the 250 mg/kg/day dose-level, these significant changes were considered to be related to the
treatment with the test item.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

The food consumption was similar in the control and the 10 and 50 mg/kg/day groups.
In the 250 mg/kg/day group, a slightly significant decrease in food consumption was recorded
during the treatment period (-7%, p<0.001 ), with a more marked effect during the first three days
of treatment (-21 %, p<0.001).

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

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment related findings were observed at any dose level. An exception are whitish foci on the lung in the decedent female.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Other effects:

not examined

Number of abortions:

no effects observed

Description (incidence and severity):

There were no abortions in any group.

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

Pre-implantation loss: The number of corpora lutea and implantation sites, and therefore the pre-implantation loss were
similar in the control and the treated groups. The fluctuations between the groups including a
slightly lower pre-implantation loss in the high-dose group were among those commonly
recorded for rats of this strain and age. Consequently, because the treatment occurred after the
implantation of the ovum, this was considered to be fortuitous.
Post-implantation loss: The number and rate of resorptions (late and early) were low and similar in the control and the treated groups.

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Description (incidence and severity):

No total resorption in any group

Dead fetuses:

no effects observed

Changes in pregnancy duration:

no effects observed

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

Number pregnant per dose level: Control 24; low- and high-dose 23; mid-dose 22

Other effects:

no effects observed

Details on maternal toxic effects:

Maternal toxic effects:yes

Details on maternal toxic effects:
- Mortality and day of death: One female of the high-dose group was found dead on day 16. This death was attributed to an effect of hold-up in the
esophagus following gavage. No other mortalities were observed.
- Clinical signs: The only treatment-related clinical sign was ptyalism in most females of the 250 mg/kg bw/day group from day 11, 12, 13, or 14 until hysterectomy. Loud breathing and presence of material in the mouth, probably due to hold-up in the esophagus, were recorded in 4 females. These observations might be the consequence of the corrosive properties of the test item (pH between 10 and 12).
- Number pregnant per dose level:
Control 24; low- and high-dose 23; mid-dose 22
- Number aborting: No abortion in any group
- Number of resorptions: No total resorption in any group
- Pre and post implantation loss: No treatment related findings were observed at any dose level.
- Body weight gain: Not affected in low and mid dose groups. A significant decrease (-35 %) was observed in the high-dose group after the first three days of treatment. Thereafter, the body weight was similar to that of the controls. The net body weight gain was also significantly lower (-25 %) at
this dose level.
- Food/water consumption: Not affected in low and mid dose groups; a slightly significant decrease was observed in the high dose group during the
treatment period.
- Gross pathology incidence and severity: No treatment related findings were observed at any dose level. An exception are whitish foci on the lung in
the decedent female.

Key result

Dose descriptor:

NOEL

Effect level:

50 mg/kg bw/day

Basis for effect level:

other: maternal toxicity

Fetal body weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

The fetal weight was similar in the control and the treated groups. There was only a statistically
significantly higher fetal weight in the 50 mg/kg/day group: 4.10 g vs. 3.81 g (p<0.05). However,
since this difference was not dose-related, a relationship to treatment with the test item was ruled
out.

Reduction in number of live offspring:

no effects observed

Description (incidence and severity):

The number of live fetuses per female was similar in the control and the treated groups.

Changes in sex ratio:

no effects observed

Description (incidence and severity):

No treatment related findings were observed at any dose level.

Changes in litter size and weights:

no effects observed

Description (incidence and severity):

No treatment related findings were observed at any dose level.

Changes in postnatal survival:

no effects observed

External malformations:

no effects observed

Description (incidence and severity):

No treatment related external malformations or variations were observed at any dose level.

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Fetal skeletal malformation:
There was no malformation in the 10 and 250 mg/kg/day treated groups.
One malformation was recorded in the control group (1/130 fetuses with supernumerary lumbar
vertebra) and in the 50 mg/kg/day treated groups (1/132 fetuses with splitted sternebra).
Because of the low incidence of these findings, which was within the range of historical control
data and the absence of dose-relationship, the malformations were not considered to be related to
the treatment with the test substance.

Fetal skeletal variation:
All the findings were recorded without clear dose-relationship and/or statistical significance
and/or occurred at incidences consistent with historical control background data.
The two exceptions were the increase in fetal incidence of incomplete ossification of
5th stemebra in the 250 mg/kg/day group: 106/134 fetuses (79.l %, p<0.0 1) were affected, versus
88/130 (67.7%) in the control group. However, when expressed on a fetus/litter basis, it was not
statistically significant.
There was also a non statistically significant increase in fetal incidence of incomplete
ossification of rib(s) in the same group: 9/134 fetuses (6.7%) were affected, versus 2/130 (1.5%).
The litter incidence was also slightly increased in this group when compared to the control but
there were no statistical differences.

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

There were no malformations in the control and the 10 and 50 mg/kg/day groups.
In the 250 mg/kg/day treated group, misshapen heart was recorded in 1/126 fetuses.
Because this finding was the only soft tissue malformation recorded in the study, it was
considered to be fortuitous in origin. Only two kind of common findings (dilated renal pelvis and dilated ureter) were recorded in the
control or the treated groups. Because the incidence of this finding was not dose-related, not
statistically significant and found among CIT historical control data, it was not considered to be
related to the treatment with the test item.

Other effects:

no effects observed

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
- Litter size and weights: No treatment related findings were observed at any dose level.
- Sex ratio: No treatment related findings were observed at any dose level.
- External abnormalities: No treatment related external malformations or variations were observed at any dose level.
- Soft tissue abnormalities: No treatment related soft tissue malformations or variations were observed at any dose level.
- Skeletal abnormalities: No statistically significant treatment related skeletal malformations or variations were observed at any dose level.
There was a statistically insignificant increase in fetal incidence of incomplete ossification of the 5th sternebra in the 250 mg/kg bw/day group
(106/134 fetuses = 79.1%, p<0.01 were affected vs. 88/130 = 67.7% in control group).
In the same group there was a statistically nonsignificant increase in fetal incidence of incomplete ossification of the
rib(s) (9/137 fetuses = 6.7 % vs. 2/130 = 1.5% in control group.
When ossification was incomplete, cartilage was generally present, demonstrating that the skeletal variations recorded corresponded to slight
fluctuations in the time of ossification rather than being a persistent alteration. In conclusion, these findings were considered to be incidental and of no toxicological significance.

Key result

Dose descriptor:

NOAEL

Effect level:

> 250 mg/kg bw/day

Basis for effect level:

other: embryotoxicity

Key result

Dose descriptor:

NOAEL

Effect level:

> 250 mg/kg bw/day

Basis for effect level:

other: fetotoxicity

Key result

Dose descriptor:

NOAEL

Effect level:

> 250 mg/kg bw/day

Basis for effect level:

other: teratogenicity

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

no

No teratogenic or embryofetotoxic effects were recorded at any dose level.

Conclusions:

The test item did not show any teratogenic or embryofetotoxic effects in a gavage study with rats performed in accordance with OECD TG 414 (2001) up to and including the highest tested dose level of 250 mg/kg bw/day. The NOEL for maternal toxicity was 50 mg/kg bw/day, effects at 250 mg/kg bw/day were reduced food consumption and reduced body weight gain. The NOAEL for developmental toxicity is > 250 mg/kg bw/day

Executive summary:

Based on the results of a dose finding study, three groups of 24 mated female Sprague-Dawley rats received 3-aminomethyl-3,5,5-trimethylcyclohexylamine by daily oral administration (gavage) at 0 (water = control), 10, 50 and 250 mg/kg/day from day 6 to day 19 post-coitum inclusive. On day 20 post-coitum, the dams were sacrificed and subjected to macroscopic examination. The study was designed according to OECD TG 414.There was no treatment-related death in any of the dams. Clinical signs were not observed, except for ptyalism in most females of the 250 mg/kg/day group (from day 11, 12, 13 or 14 post-coitum until hysterectomy; effect not considered as adverse). Loud breathing and hold-up in the esophagus were recorded in 4 females of this group and a significantly lower body weight gain (-35%) was recorded after the first three days of treatment. Thereafter, the body weights were similar to that of the controls. Over the whole treatment period, the difference remained slight (-10 %, not statistically significant). The net body weight gain was also significantly lower at this dose-level (-25 %) when compared to the control group. In the 250 mg/kg/day group, a significant decrease in food consumption was recorded during the treatment period (-7%), with a more marked effect during the first three days of treatment (-21 %).Abortions or total resorptions were not observed in any of the groups, nor were there any macroscopic findings that were ascribed to treatment with the test item. No treatment related effects were observed on pre- or post-implantation loss, fetal weight or sex-ratio. With respect to the fetuses, no test item related external, soft tissue or skeletal malformations or variations were detected. There was an increase in fetal incidence of incomplete ossification of the 5th sternebra in the 250 mg/kg bw/day group (106/134 fetuses = 79.1 %, p < 0.01 were affected vs. 88/130 = 67.7 % in control

group, statistically insignificant on a fetus/litter basis). Because these findings are of low concern and occur only in the presence of maternal toxicity, they are considered to be secondary. In the same group there was a statistically nonsignificant increase in fetal incidence of incomplete ossification of the rib(s) (9/137 fetuses = 6.7 % vs. 2/130 = 1.5 % in control group. When ossification was incomplete, cartilage was generally present, demonstrating that the skeletal variations recorded corresponded to slight fluctuations in the time of ossification rather than being a persistent alteration. In conclusion, these findings were considered to be incidental and of no toxicological significance. The NOEL for maternotoxicity was 50 mg/kg/day and the NOAEL for embryonic development was >250 mg/kg/day.

Reference 5

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

2018-04-12 to 2018-06-19

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

This OECD 414 study is used as dose range finding study for the OECD 414 main study with rabbits (LPT, 2020).

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; and -OECD Principles of Good Laboratory Practice' Document Nos. 1, 8 and 13

Limit test:

no

Specific details on test material used for the study:

The test item was diluted in the vehicle to the appropriate concentration.

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ORGANISMS: 
- Species: Rabbit
- Source: Manfred Bauer Kaninchen, Lohe 7/1, 74632 Neuenstein, Germany
- Strain: New Zealand White
- Age: 5 months
- body weight: 3.55 - 4.30 kg
- Diet: ad libitum, Commercial ssniff K-Z V2323 (ssniff Spezialdiäten GmbH, 59494 Soest, Germany,
- Water: ad libitum
- Adaptation period: 20 days
-Housing: Except during the mating period, the dams were kept separately in breeding cages with wire floors
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 °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:

water

Remarks:

Purified water (aqua ad injectabilia)

Details on exposure:

ADMINISTRATION: 
- Frequency: once daily, day 6 to 28 of gestation
- Dose volume: 2 ml/kg b.w.
- Dose: 0, 5, 25, 50 mg/kg/bw
- Animals: 16 females in groups 1 to 4
Test item preparation:
The test item formulations were freshly prepared every day.
The test item was diluted 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

Details on analytical verification of doses or concentrations:

For each test or reference item that is mixed with a vehicle, test by appropriate analytical methods will be carried out for the main study to determine concentration, homogeneity and, if needed, stability of the test item in the formulations.

Details on mating procedure:

Sexually mature ('proved') male rabbits 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 in one cage in the forenoon. Successful copulation was ascertained by observation.The day of copulation was considered as day 0 of pregnancy. Only animals with positive signs of copulation were included in the study.

Duration of treatment / exposure:

23 administration days from gestation day 6 to 28

Frequency of treatment:

Once daily

Duration of test:

Laparotomy on gestation day 29

Dose / conc.:

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

Remarks:

Vehicle control

Dose / conc.:

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

Remarks:

Low dose

Dose / conc.:

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

Remarks:

Intermediate dose

Dose / conc.:

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

Remarks:

High dose

No. of animals per sex per dose:

4 female rabbits/dose
Evaluated litters: at least 12 pregnant females, 3 litters per dose

Control animals:

yes, concurrent vehicle

Details on study design:

Principle
Pregnant rabbits were treated with the test item starting from the 6th and lasting until the 28th day of pregnancy (the 'critical' phase of organogenesis and the fetal development). The development of the pregnant rabbits was observed during the gestation period (day of mating is day 0 of pregnancy). One day before the calculated date of birth (29 days after mating) the dams were laparotomised and examined for corpora lutea, implantation sites, resorptions in the uterus and for the condition of the fetuses.

Dose selection rationale:
The dose levels were selected in agreement with the Sponsor based on available toxicological data.

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 rec-orded. In case of changes, the animals were observed until thesymptoms 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.
No abortion or premature delivery occurred during the 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. These measurements were also used for calculating the daily amount of test item to be administered.
The body weight gain was calculated in intervals (i.e. gestation day 6-9, 9 12, 12-15, 15-18, 18-21, 21-24, 24-27, 27-29) and for the period after the start of treatment until necropsy (gestation day 6-29).

Furthermore the carcass weight and the net weight change from day 6 are given.
Carcass weight: Carcass weight = Terminal body weight minus uterine weight

Net weight change from day 6 = Carcass weight minus body weight on day 6

These values are stated in the report.


Food and drinking water consumption
The quantity of food consumed by each rabbit was recorded daily. 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.

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:
One day before the calculated parturition, i.e. on gestation day 29, all surviving rabbits were sacrificed by lethal intravenous injection of 300 mg Pentobarbital/kg b.w. and exsanguinated.
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.
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.

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, % per litter
- distributions in the uterine horns
- absolute number per group
- mean per group
- mean % per group
- early resorptions < 2 g
- late resorptions > 2 g

Weight of placentae
- individual weight per fetus (alive and dead)
- mean placental weight per dam (placentae from male and female fetuses alone or combined)
- mean placental weight per dam and group (litter mean per group)

Weight of fetuses
- individual weight per fetus (alive and dead)
- mean fetal weight per dam (male and female fetuses alone or combined)
- mean fetal weight per dam and group (litter mean per group)

Fetuses
- absolute number per dam and group (alive and dead, male and female fetuses alone or combined)
- mean number of fetuses per dam and group (alive and dead, male and female fetuses alone or combined)
- sex ratio per litter
- distribution in the uterine horns

Runts
- number per dam
- mean per group

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

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

Fetuses with variations
- individual data per fetus
- type of variation
- total number and incidence (%) of affected fetuses and litters per group8

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.
(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 a lethal intraperitoneal injection of 60 mg Pentobarbi-tal/kg b.w..

Each fetus was dissected:
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.

Statistics:

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 ≥ 100 (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).
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 was done using StatXact 4.0.1 software.

Clinical signs:

no effects observed

Description (incidence and severity):

No changes in behaviour, the external appearance or faeces were noted for the control group or the test item-treated groups (5, 25 or 50 mg test item/kg b.w./day).

Mortality:

no mortality observed

Description (incidence):

No premature death was noted for the control group and the treatment groups (5, 25 or 50 mg test item/kg b.w./day).

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

No test item-related changes in body weight or body weight gain were noted between the dams of the control group and the dams of the treatment groups (5, 25 or 50 mg test item/kg b.w./day).

Body weight gain:
The changes in body weight gain after the start of treatment on gestation day 6 until necropsy on gestation day 29 are given in the table below:

Body weight gain (mean %) # Group 1 Control Group2, 25 mg/kg Group 3, 25 mg/kg Group 4, 50 mg/kg
Gestation day 6 to gestation day 29 4.5% 7.8% 7.4% 7.4%

Food consumption and compound intake (if feeding study):

effects observed, non-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 5, 25 or 50 mg test item/kg b.w./day.
However, a statistically significantly increased food consumption in comparison to the control group was noted on several test days between gestation day 19 and gestation day 24 for the dams of the treatment groups (5, 25 or 50 mg test item/kg b.w./day; at maximum 241.5% above the value of the control group at the intermediate dose level between gestation days 23 and 24). This was due to a low food intake of the control dams on several test days. Hence, the statistically significantly increased food consumption that was noted for the treatment groups was considered to be spontaneous.

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 the consumption of drinking water were noted for the dams treated with 5, 25 or 50 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):

Gravid uterus and carcass weight
No test item-related changes in the gravid uterus weight and the carcass weight in comparison to the control group were noted for the dams of the treatment groups (5, 25 or 50 mg test item/kg b.w./day).

Net body weight gain from day 6
No test item-related changes between the control group and the treatment groups (5, 25 or 50 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.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No test item-related pathological findings were noted during macroscopic inspection of the dams of the control group and dams treated with 5, 25 or 50 mg test item/kg b.w./day.
One animal of the high dose group revealed changes in the liver and the stomach. However, as only one animal was affected, these changes were considered to be spontaneous and not test item-related

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Details on results:

General toxicity maternal animals: no effects

Number of abortions:

no effects observed

Description (incidence and severity):

No test item-related influences on the number of resorptions and the number of live fetuses were noted for the dams of the treatment groups (5, 25 or 50 mg test item/kg b.w./day).

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

No test item-related influences on the number of resorptions and the number of live fetuses were noted for the dams of the treatment groups (5, 25 or 50 mg test item/kg b.w./day).
The values of pre- and post-implantation loss were in the range of normal variability.

Total litter losses by resorption:

no effects observed

Description (incidence and severity):

No test item-related influences on the number of resorptions and the number of live fetuses were noted for the dams of the treatment groups (5, 25 or 50 mg test item/kg b.w./day).

Early or late resorptions:

no effects observed

Description (incidence and severity):

No test item-related influences on the number of resorptions and the number of live fetuses were noted for the dams of the treatment groups (5, 25 or 50 mg test item/kg b.w./day).

Dead fetuses:

no effects observed

Description (incidence and severity):

No dead fetus was noted in the control group and in the treatment groups (5, 25 or 50 mg test item/kg b.w./day).

Changes in pregnancy duration:

no effects observed

Description (incidence and severity):

No effects observed

Changes in number of pregnant:

no effects observed

Details on maternal toxic effects:

Maternal toxic effects:no effects

Key result

Dose descriptor:

NOAEL

Effect level:

> 50 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: no maternal toxicity

Key result

Abnormalities:

no effects observed

Fetal body weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

No test item-related influence was noted on the fetal weight after administration of 5, 25 or 50 mg test item/kg b.w./day.
One runt (no. 15-7) was noted in the high dose group (50 mg/kg b.w./day). This was in the normal range of variability and considered to be spontaneous.

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 treatment groups (5, 25 or 50 mg test item/kg b.w./day).

Changes in sex ratio:

no effects observed

Description (incidence and severity):

The sex distribution of the fetuses in the control group and in the treatment groups (5, 25 or 50 mg test item/kg b.w./day) was inside the range of normal biological variability.

Changes in litter size and weights:

no effects observed

Description (incidence and severity):

No test item-related influences on the number of resorptions and the number of live fetuses were noted for the dams of the treatment groups (5, 25 or 50 mg test item/kg b.w./day).

Changes in postnatal survival:

not examined

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

No external malformation was noted for the fetuses of the control group and the treat-ment groups (5, 25 or 50 mg test item/kg b.w./day).
External variations in the form of a hyperflexion of the extremities were noted for 3 fetuses from 2 dams of the low dose group (5 mg test item/kg b.w./day). As no extremities with a hyperflexion were noted for the fetuses of the intermediate and the high dose group (25 or 50 mg test itemkg b.w./day) the observations at the low dose level were considered to be spontaneous.

Skeletal malformations:

not examined

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Internal gross alterations
A macroscopic internal examination was performed to detect gross alterations of the internal organs. No internal malformation or variation was noted for the fetuses of the treatment groups (5, 25 or 50 mg test item/kg b.w./day).
At the control group a spontaneous variation in the form of a malpositioned left kidney was noted for fetus no. 4-8.

Other effects:

no effects observed

Description (incidence and severity):

One runt each was noted in the intermediate dose group (300 mg test item/kg b.w./day) and in the control group.

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects
No dead fetuses were noted in the litters of the control group and the treatment groups (treated orally with 5, 25 or 50 mg /kg b.w./day).
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 (5, 25 or 50 mg test item/kg b.w./day).

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Basis for effect level:

other: no developmental toxicity

Key result

Developmental effects observed:

no

Summary of animals examined

Test item	Group 1 Control	Group 2 5 mg/kg	Group 3 25 mg/kg	Group 4 50 mg/kg
Treated females	4	4	4	4
Not examined females (excluded/spare animals)	none	none	none	1
Non-pregnant females	1	1	1	none
Deceased animals	none	none	none	none
Evaluated pregnant females	3	3	3	3
Dams without viable fetuses (total post implantation loss)	none	none	none	none
Dams with abortion	none	none	none	none
Evaluated litters with viable fetuses	3	3	3	3

Reproduction data of the dams

Parameter					Group 1 Control (n=3)	Group 2 5 mg/kg (n=3)	Group 3 25 mg/kg (n=3)	Group 4 50 mg/kg (n=3)
Corpora lutea			total mean per dam		31 10.3	33 11.0	29 9.7	39 13.0
Implantation sites			total mean per dam		31 10.3	30 10.0	27 9.0	35 11.7
Total resorptions			total mean per dam		1 0.3	1 0.3	2 0.7	0 0.0
Early resorptions			total mean per dam		1 0.3	1 0.3	2 0.7	0 0.0
Late resorptions			total mean per dam		0 0.0	0 0.0	0 0.0	0 0.0
Fetuses (alive + dead)			total mean per dam		30 10.0	29 9.7	25 8.3	35 11.7
Live fetuses			total mean per dam		30 10.0	29 9.7	25 8.3	35 11.7
Dead fetuses			total mean per dam		0 0.0	0 0.0	0 0.0	0 0.0
Pre-implantation loss			per group		0.0	9.1	6.9	10.3
[%]			mean per dam		0.0	7.1	6.7	8.9
Post-implantation loss			per group		3.2	3.3	7.4	0.0
[%]			mean per dam		3.3	3.0	7.2	0.0
* / **	Significantly different from the controls at p ≤ 0.05 / p ≤ 0.01, Chi^2test, only performed for the group values of the pre- and post-implantation loss
* / **		Significantly different from the controls at p ≤ 0.05 / p ≤ 0.01, Dunnett test, performed for the mean values per group

Body weight gain

Body weight gain (mean %)#	Group 1 Control	Group 2 5 mg/kg	Group 3 25 mg/kg	Group 4 50 mg/kg
Gestation day 6 to gestation day 29	4.5%	7.8%	7.4%	7.4%

Sex distribution of fetuses

Parameter	Sex ratio observed in this study (male / female)
Sex ratio of fetuses (male/female)	Group 1:	0.76
	Group 2:	0.71
	Group 3:	0.92
	Group 4:	1.33

Conclusions:

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

Based on the data obtained in this dose-range-finding study, it is not possible to derive dose levels for main study. Therefore, an additionally tolerance test was conducted (7.5.1 LPT_DRF_21 days_Rabbits_2018, LPT Study No. 36299).
Based on data obtained in both dose range finding studies, the following dose levels are suggested for LPT Study No. 35503 (Prenatal developmental toxicity study in rabbits):
Group 1: Control
Group 2: 10 mg test item/kg b.w./day, p.o
Group 3: 25 mg test item/kg b.w./day, p.o
Group 4: 75 mg test item/kg b.w./day, p.o

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 rabbits when administered orally during the critical period of organogenesis and the fetal development (6th to 28th day of gestation).

 

In this prenatal developmental toxicity study, the test item was administered orally to female rabbits at dose levels of 5, 25 or 50 mg/kg b.w./day from the 6th to 28th day of pregnancy.

Findings:

Examination of the dams
Mortality	No premature death was noted for the control group and the treatment groups (5, 25 or 50 mg test item/kg b.w./day).
Clinical signs	No changes in behavior, the external appearence and the consistency of the faeces were noted in the control group and the test item-treated groups.
Body weight and body weight gain	No test item-related difference was noted for the body weight and the body weight gain.
Food consumption	No test item-related difference was noted for the food consumption.
Drinking water consumption	No test item-related influence was noted for the drinking water consumption at any of the tested dose levels (visual assessment).
Necropsy findings	No test item-related observations were noted for the treatment groups during necropsy.
Uterus and carcass weights	No test item-related influence on the uterus and carcass weight was noted for the treatment groups.
Examination of the fetuses	No test item-related influence was detected on the prenatal fetal development at 5, 25 or 50 mg test item/kg b.w./day with respect to the incidence of resorptions, number of live fetuses and the values calculated for the pre- and post-implantation loss.
	Nodead fetuswas noted in the test item groups or the control group.
	Sex distribution
	No test item-related differences were noted.
	Fetal weights
	No test item-related influence on the fetal weights was noted for the treatment groups.
	Placental weights
	No test item-related influence was noted.

Fetal alterations
Malformations	No malformation was noted for the fetuses treated with 5, 25 or 50 mg test item/kg b.w./day during the inspection at laparotomy, including a macroscopic external examination and a macroscopic gross inspection of the internal organs.
Variations	The macroscopic inspection at laparotomy revealed no test item-related external variation for the fetuses treated with 5, 25 or 50 mg test item/kg b.w./day. No variation was noted during the gross inspection of the internal organs.

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

None of the animals died prematurely.

No changes were noted in behaviour, the external appearance and the consistency of the faeces.

No test item-related effect was noted for the body weight, body weight gain or food consumption.

Necropsy revealed no test item-related pathological findings.

The no-observed-adverse effect level (NOAEL) for the fetal organism was also above 50 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 any of the treatment groups.

No test item-related influence was noted on the body weight of the fetuses in the treatment groups.

No dead fetuses, no malformation and no test item-related variations were noted.

Based on the data obtained in this dose-range-finding study, it is not possible to derive dose levels for main study. Therefore, an additionally tolerance test was conducted (LPTStudy No. 36299). Based on data obtained in both dose range finding studies, the following dose levels are suggested forLPTStudy No.35503 (Prenatal developmental toxicity study in rabbits):

Group 1:	Control
Group 2:	10 mg test item/kg b.w./day, p.o
Group 3:	25 mg test item/kg b.w./day, p.o
Group 4:	75 mg test item/kg b.w./day, p.o

Reference 6

Endpoint:

developmental toxicity

Remarks:

14 d Dose Range Finding Study for OECD 414

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

May 2018

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

no guideline required

Principles of method if other than guideline:

14-day preliminary oral DRF study in rabbits, as tolerance test for main study OECD 414.
Based on the data obtained in the dose-range-finding study OECD 414, it is not possible to derive dose levels for main study. Therefore, this additionally tolerance test was conducted.

GLP compliance:

no

Limit test:

no

Species:

rabbit

Strain:

New Zealand White

Remarks:

only females used

Details on test animals or test system and environmental conditions:

TEST ORGANISMS: 
- Species: Rabbit (only females uses)
- Source: Manfred Bauer Kaninchen, Lohe 7/1, 74632 Neuenstein, Germany
- Strain: New Zealand White
- Age: 4 months
- body weight: 4.37 - 5.49 kg

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

Purified water (aqua ad iniectabilia)

Details on exposure:

ADMINISTRATION: 
- Frequency: once daily
- Dose volume: 2 ml/kg b.w.
- Dose: 75, 150 mg/kg/bw
- Animals: 6 females in groups 1 to 2

Analytical verification of doses or concentrations:

no

Details on mating procedure:

not needed

Duration of treatment / exposure:

Group 1, 75 mg/kg b.w.: dosing extended to 21 test days
Group 2, 150 mg/kg b.w.: dosing terminated on test day 7 (humane sacrifice)

Frequency of treatment:

one daily

Duration of test:

21 days

Dose / conc.:

75 mg/kg bw/day (nominal)

Dose / conc.:

150 mg/kg bw/day (nominal)

Control animals:

no

Details on study design:

3

Maternal examinations:

Clinical observations: Twice daily (incl. mortality, systemic tolerance); food and drinking water consumption
Body weight: On each dosing day

Terminal examinations:
Group 1: day 22
Group 2: day 8
Macropathology of all animals, preservation of all organs showing gross abnormalities/ lesions

Ovaries and uterine content:

not needed as study was only a preliminary tolerance test

Fetal examinations:

not needed as study was only a preliminary tolerance test

Indices:

not needed as study was only a preliminary tolerance test

Historical control data:

no

Clinical signs:

no effects observed

Description (incidence and severity):

No test item-related changes were noted from test day 1 until terminal sacrifice on test day 22 (group 1: 75 mg test item/kg b.w./day or until humane sacrifice on test day 8 (group 2: 150 mg test item/kg b.w./day).

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

None of the animals died prematurely.
However, the three high dose animals (group 2: 150 mg test item/ kg b.w./day) were sacrificed prematurely on test day 8 due to refusal of food intake from start of dosing onwards.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

No test item-related influence was noted on the body weight or the body weight gain of the low dose animals (group 1: 75 mg test item/kg b.w./day).
The body weight development of the high dose animals (group 2: 150 mg test item/kg b.w./day) was retrograde due to refusal of food intake from start of dosing onwards.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

A reduction in food consumption was noted for the low dose animals (group 1: 75 mg test item/kg b.w./day) from test days 4-5 onwards, being up to 44% below the start value on test days 5-6. This finding was obviously due to the distinctly low food intake of one of 3 animals (no. 2 f). However, food consumption improved from test day 9 onwards.
No or only minimal food intake was noted for the high dose animals (group 2: 150 mg test item/kg b.w./day) from start of dosing onwards until premature sacrifice on test day 8 due to animal welfare reasons.

Food efficiency:

not examined

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

no effects observed

Description (incidence and severity):

No test item-related influence was noted for the drinking water consumption by visual appraisal during daily clinical observations.

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:

not examined

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Necropsy on test day 22 revealed a reddened gastric mucosa and ulcer(s) in 2 of 3 low dose animals (21-day dosing of 75 mg test item/kg b.w./day).
Necropsy after premature sacrifice on test day 8 revealed a severely reddened gastric muscosa in 3 of 3 high dose animals treated with 150 mg test item/kg b.w./day. Furthermore, a pale liver was noted in 2 of 3 animals and a recessed periphery of the liver was additionally noted in one of them.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Details on maternal toxic effects:

no other information

Dose descriptor:

LOAEL

Effect level:

75 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

body weight and weight gain

food consumption and compound intake

gross pathology

mortality

Abnormalities:

effects observed, treatment-related

Localisation:

other: stomach

Description (incidence and severity):

Reddened gastric mucosa and ulcer(s) in 2 of 3 low dose animals

no other information

Conclusions:

Based on data obtained in both dose range finding studies, the following dose levels are suggested for LPT Study No. 35503 (Prenatal developmental toxicity study in rabbits):
Group 1: Control
Group 2: 10 mg test item/kg b.w./day, p.o
Group 3: 25 mg test item/kg b.w./day, p.o
Group 4: 75 mg test item/kg b.w./day, p.o

Executive summary:

These preliminary oral study in rabbits, was conducted as additionally tolerance test for main study OECD 414.

The test item was administered orally to two groups with 3 female rabbits once daily at dose levels of 75 mg/kg b.w test item and 150 mg/kg b.w. test item. Group 1 dosing was extended to 21 test day, group 2 dosing was terminated on test day 7 because of humane sacrifice.

Examined Parameters are: Clinical observations, twice daily (incl. mortality, systemic tolerance); food and drinking water consumption and body weight on each dosing day.

Terminal examinations are: Macropathology of all animals and preservation of all organs showing gross abnormalities/ lesions.

 

No test item-related changes of systemic tolerance were noted from test day 1 until terminal sacrifice on test day 22 (group 1: 75 mg test item/kg b.w./day or until humane sacrifice on test day 8 (group 2: 150 mg test item/kg b.w./day).

None of the animals died prematurely.

However, the three high dose animals (group 2: 150 mg test item/ kg b.w./day) were sacrificed prematurely on test day 8 due to refusal of food intake from start of dosing onwards.

No test item-related influence was noted on the body weight or the body weight gain of the low dose animals (group 1: 75 mg test item/kg b.w./day).

The body weight development of the high dose animals (group 2: 150 mg test item/kg b.w./day) was retrograde due to refusal of food intake from start of dosing onwards.

A reduction in food consumption was noted for the low dose animals (group 1: 75 mg test item/kg b.w./day) from test days 4-5 onwards, being up to 44% below the start value on test days 5-6. This finding was obviously due to the distinctly low food intake of one of 3 animals (no. 2 f). However, food consumption improved from test day 9 onwards.

No or only minimal food intake was noted for the high dose animals (group 2: 150 mg test item/kg b.w./day) from start of dosing onwards until premature sacrifice on test day 8 due to animal welfare reasons.

No test item-related influence was noted for the drinking water consumption by visual appraisal during daily clinical observations.

Necropsy on test day 22 revealed a reddened gastric mucosa and ulcer(s) in 2 of 3 low dose animals (21-day dosing of 75 mg test item/kg b.w./day).

Necropsy after premature sacrifice on test day 8 revealed a severely reddened gastric muscosa in 3 of 3 high dose animals treated with 150 mg test item/kg b.w./day. Furthermore, a pale liver was noted in 2 of 3 animals and a recessed periphery of the liver was additionally noted in one of them.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

Due to the likely hydrolysis of the substance 1,3,3 -trimethyl-N-(2 -methylpropylidene)-5 -[(2 -methylpropylidene)amino]cyclohexanemethylamine during an in vivo study, to the hydrolysis products isophoron diamine and isobutyraldehyde, the studies on toxicity to reproduction will be waived and a supporting assessement for the hydrolysis products was chosen (see IUCLID Chapter 7.8.1, and 7.8.2).

Justification for selection of Effect on developmental toxicity: via oral route:
For the hydrolysis product from 1,3,3 -trimethyl-N-(2 -methylpropylidene)-5 -[(2 -methylpropylidene)amino]cyclohexanemethylamine, i.e. Isophorone diamine, did not show any teratogenic or embryofetotoxic effects in a gavage study with rats performed in accordance with OECD TG 414 (2001) up to and including the highest tested dose level of 250 mg/kg bw/day. The NOEL for maternal toxicity was 50 mg/kg bw/day, effects at 250 mg/kg bw/day were reduced food consumption and reduced body weight gain. The NOAEL for developmental toxicity is > 250 mg/kg bw/day

Justification for classification or non-classification

Regarding toxicity to reproduction 1,3,3-trimethyl-N-(-2-methylpropylidene)-5-[(2-methylpropylidene)amino]cyclohexanemethylamine is not classified according to the criteria of EC Regulation 1272/2008.