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Administrative data

Description of key information

As 1,3,3 -trimethyl-N-(2 -methylpropylidene)-5 -[(2 -methylpropylidene)amino]cyclohexanemethylamine hydrolises to the products isophorondiamin (IPDA) and isobutyraldehyde, those hydrolysis products are assessed.


In a subchronic drinking water study according to OECD TG 408 with rats, the administration of 150 mg/kg bw/day led to reduced absolute and relative kidney weights in male and female rats (histopathology being indicative for tubular nephrosis), while 59 mg/kg bw/day (males) and 62 mg/kg bw/day (females) were determined as a NOAEL (RCC 1986).    


In a dose range finding study for an OECD 422 study (LPT 2020), rats showed some clinical signs such as salivation and breathing sounds in the highest dose group of 500/350 mg/kg bw. Because one male animal died in first week, the highest dose was reduced from 500 to 350 mg/kg bw/d.
There was a significantly reduced food consumption in both (high) dose groups, which lead 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: 30 mg/ kg/b.w./day (low dose), 100 mg/ kg/b.w./day (intermediate dose), 300 mg/ kg/b.w./day (high dose).


The aim of the subsequently performed 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 (Provivo, 2022)


From two 14-day inhalative exposure studies with rats no NOAEL could be determined. At the first study’s LOAEL of 18 mg/m3, degeneration/necrosis in the olfactory epithelium of the nose were observed. Trachea, larynx and lungs were affected at 200 mg/m3 and above (degeneration/ necrosis, hyperplasia, squamous metaplasia) (DuPont, 1997). At the LOAEL of the follow-up study, i.e. at 2.2 mg/m³, reversible minimal to mild degeneration of respiratory nasal mucosa in the anterior dorsal nose was observed (DuPont, 2001). 


 


Isobutyraldehyde administered by inhalation (whole body exposure) for up to Thirteen Weeks or Two Years was a Respiratory Tract Toxicant but was not carcinogenic in F344/N Rats and B6C3F, Mice (Abdo 1998). Isobutyraldehyde was studied for toxicity and carcinogenicity by exposing male and female F344/N rats and B6C3F, mice. Animals were exposed to isobutyraldehyde vapors 6 h per day, 5 days per week for up to 13 weeks or 2 years. In the 13-week studies, groups of 10 male and 10 female F344/N rats and B6C3F, mice were exposed to concentrations of 0, 500, 1000, 2000, 4000, or 8000 ppm. Chemical-related body weight depression and deaths occurred in rats and mice exposed to 4000 and 8000 ppm. Necrosis of the epithelium accompanied with acute inflammatory reaction was observed in the nasal turbinate, larynx, and trachea of rats exposed to 8000 ppm. Exposure of rats to 4000 ppm resulted in metaplasia of the nasal respiratory epithelium, inflammation, degeneration of the olfactory epithelium, and osteodystrophy of the nasal turbinate bone. In the 13-week mouse study, exposure to 8000 ppm or 4000 ppm resulted in necrosis of the epithelium lining of the nasal turbinates. Osteodystrophy of the nasal turbinate bone and squamous metaplasia of the nasal respiratory epithelium were noted in mice exposed 4000 ppm. Degeneration of the olfactory epithelium was noted in males exposed 2000 ppm and in females exposed to 4000 ppm. In the 2-year studies, groups of 50 male and 50 male F344/ N rats and B6C3F1 were exposed to concentrations isobutyraldehyde vapors of 0, 500, 1000, or 2000 ppm 6 h per day, 5 days per week. There were no differences in survival rates or mean body weights between exposed groups and control rats. Survival of male mice exposed to 2000 ppm and mean body weights of female mice exposed to 1000 or 2000 ppm were lower than those of the of the controls. No increase in neoplasm incidence was observed in rats and mice in the 2-year studies that could be attributed to isobutyraldehyde exposure. Chemical-related nonneoplastic lesions were limited to the nose of rats and mice. They included squamous metaplasia of the respiratory epithelium (rats), suppurative inflammation (rats), and olfactory epithelial degeneration (rats and mice) at 1000 and 2000 ppm.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Referenceopen allclose all

Endpoint:
sub-chronic toxicity: oral
Data waiving:
other justification
Justification for data waiving:
other:
Critical effects observed:
not specified
Endpoint:
sub-chronic toxicity: oral
Remarks:
combined repeated dose and reproduction / developmental screening
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
Study started on 24th of July 2019, the end of in-life period was on 10 October 2019
and date of Final report was 31 March 2022.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
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
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
Details on route of administration:
Selection of administration route: According to OECD guideline 422
Vehicle:
water
Details on oral 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.
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
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.
Positive control:
not needed
Observations and examinations performed and frequency:
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.

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.
Sacrifice and pathology:
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.
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.
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.
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:
no effects observed
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
Description (incidence and severity):
Males and females: No test item-related differences were noted.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Males and females:
No macroscopic post mortem findings were noted for the male and female animals of the control group and no test item-related macroscopic post mortem findings were noted for the male and female animals of the low and the intermediate dose group (30 or 100 mg/kg b.w./day).

The following observations that were noted for one male and one female animal of the low dose group were considered to be incidental and not to be treatment-related:
Group 2:
- Small testes (bilateral) were noted for male no. 22.
- An adhesion between the spleen and the pancreas was noted for female no. 33.

At the high dose level (300/240 mg/kg b.w./day) kidney changes were noted for 2 of 8 surviving male animals. No findings were noted for the 7 surviving females.
Group 4:
- A dilatation of the renal pelvis was noted for the left kidney of male no. 61.
The dilatation was confirmed by microscopy.
- Pale kidneys (left and right kidney) with cysts were noted for animal no. 63.
No microscopic examination was performed, as no. 63 was not selected for microscopic examination.
The kidney changes listed above that were noted for male nos. 61 and 63 are not uncommon in animals of this strain. However, due to test item-related kidney changes that were observed during microscopic examination, a possible relationship with the treatment could not be denied
Neuropathological findings:
not examined
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
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
gross pathology
histopathology: non-neoplastic
mortality
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
240 mg/kg bw/day (nominal)
Organ:
kidney
stomach
Treatment related:
yes
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.
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.


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




















General toxicity



 



 



NOAEL (for systemic toxicity)



 



                                                                                            100 mg/kg b.w./day, p.o.


Endpoint:
repeated dose toxicity: oral, 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
Details on route of administration:
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:
water
Remarks:
Aqua ad injectabilia (Water for injection, batch 184928001, B. Braun, 34212 Melsungen, Germany)
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:

DIET PREPARATION
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.

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.

Observations and examinations performed and frequency:
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
Sacrifice and pathology:
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.
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
Description (incidence and severity):
Organ weights (relative and absolute)

Males and females
No test item-related differences were noted between the male and female animals of the control group and the treatment groups (250 or 500/350 mg test item/kg b.w./day) for the absolute and relative weights of the examined organs.

Statistically significant differences that were noted for the organ weights
which were considered to be spontaneous.

Parameter Ref. Table Increase ↑ Group/ Test day Statistical Reason
no. Decrease ↓ sex significance
Kidney left (g/kg) 7-1 ↑ 3 m 15 p ≤ 0.05 A
Adrenal gland, left (g/kg) 7-1 ↑ 3 f 15 p ≤ 0.05 A
A: The slight increase was due to the reduced body weight at the high dose level. No increase or no statistically significant increase was noted for the absolute organ weight.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Macroscopic post mortem findings

Males
No observations were noted for the male animals of the control group, the low dose group (250 mg/kg b.w./day) and the surviving animals of the high dose
group (500/350 mg/kg b.w./day).
Females
No observations were noted for the female animals of the control group and the low dose group (250 mg/kg b.w./day).
At the high dose level (500/350 mg/kg b.w./day) gastrointestinal changes were noted for 3 of 5 animals.
As gastrointestinal changes were noted for 3 of 5 high dosed females and for the 2 prematurely deceased high dosed males, they were considered to be test itemrelated.


Test item-related gastrointestinal changes at the high dose level.
Test item-related gastrointestinal changes (females)
Animal Affected organ Observations
26 Stomach Light-yellow discoloured wall, inflated
Intestines Inflated
27 Stomach Reddened mucosa
29 Stomach Inflated
Intestines Inflated

The following observations that were noted for animal no. 26 were considered to be due to a bad health condition of this animal and not as a specific test itemrelated
effect:
- rough fur and a haemorrhagic nose/snout,
- a pale pancreas,
- a small spleen
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
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
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 (250or500/350 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 levels of 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 350mg/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)

Endpoint:
sub-chronic toxicity: oral
Type of information:
other: supporting study about the hydrolysis product isophorone diamine
Adequacy of study:
key study
Study period:
1985-08-05 - 1985-11-13
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Version / remarks:
1981
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ORGANISMS
- Source: KFM Kleintierfarm Madoerin AG, CH
- Age: 6 weeks
- Weight at study initiation:
males 136-157 g, females 117-139 g
- Number of animals: Total 80 males, 80 females; 20 per sex and group
- Fasting period before study:
- Housing: groups of 5 in Makrolon type 4 cages
- Diet: ad libitum, Kliba 343 rat maintenance diet
- Water: Distilled water for the treatment period ad libitum
- Acclimation period: 7 days
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 2 °C
- Humidity (%): 55 +/- 10 %
- Air changes (per hr): 10 - 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 h/12h
Route of administration:
oral: drinking water
Vehicle:
water
Details on oral exposure:
ADMINISTRATION / EXPOSURE
- Duration of test/exposure: 13 weeks
- Type of exposure: oral, drinking water
- Post exposure period: -
- Vehicle: drinking water
- Doses:
Group 1 = control;
group 2 = 20 mg/kg bw/day;
group 3 = 60 mg/kg bw/day;
group 4 = 160 mg/kg bw/day
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The concentration and stability analysis of isophorone diamine were performed by gas chromatography.
The results of the stability test at room temperature showed that isophorone diamine was stable in drinking water for at least 96 h.
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
daily (continuously)
Remarks:
Doses / Concentrations:
20, 60, or 160 mg/kg bw d (nominal) = groups 2, 3, and 4
Basis:
nominal in water
Remarks:
Doses / Concentrations:
males: 0; 21.5; 59; 150 mg/kg bw per day / females: 0; 22.6; 62; 147 mg/kg bw per day
Basis:
other: mean daily dose received (calculated) over 13 weeks
No. of animals per sex per dose:
20
Control animals:
yes
Details on study design:
Post-exposure period: none
Positive control:
no positive control
Observations and examinations performed and frequency:
CLINICAL OBSERVATIONS AND FREQUENCY:
- Clinical signs: twice daily
- Mortality: twice daily
- Body weight: weekly
- Food consumption: weekly (7 day consumption)
- Water consumption: weekly (24 hours consumption)
- Ophthalmoscopic examination: at pretest and end of study;
10 animals per group (lowest ID numbers) and sex examined
- Haematology: end of study, 10 animals per group and sex
(highest ID numbers)
- Biochemistry: end of study, 10 animals per group and sex
(highest ID numbers)
Sacrifice and pathology:
ORGANS EXAMINED AT NECROPSY (MACROSCOPIC AND MICROSCOPIC):
- Macroscopic: all organs listed below under "Microscopic";
weights of adrenals, kidneys, liver, testes
- Microscopic: (all rats of groups 1 and 4 plus those which had gross lesions): adrenal glands, aorta (thoracic), bone (sternum), bone marrow
(sternum), brain, cecum, colon, duodenum, epididymides, esophagus, eyes, Harderian glands, heart, ileum, jejunum, kidneys, liver, lungs with
mainstem bronchi, lymph nodes (mandibular, mesenteric), mammary gland, ovaries, pancreas, pituitary gland, prostate, rectum, salivary gland
(mandibular, sublingual), sciatic nerve, seminal vesicles, sekletal muscle, skin, spinal cord, spleen, stomach, testes, thymus, thyroid gland, tongue,
trachea, urinary bladder, uterus
Other examinations:
see addendum to the pathology report:
In an attempt to further elucidate the toxicological significance of findings in the kidneys, a larger sample of renal tissue was re-examined from rats
of all groups. In addition to the already available sections, another set of histological slides was prepared from the wet tissue of rats of groups 1 and 4 which was matched by two sections from each rat of groups 2 and 3.
Statistics:
Univariate one-way analysis of variance for significance of intergroup differences;
Dunnett-test for comparison between treated groups and control group (if normal distribution assumed)
Steel-test (if normal distribution not assumed)
Fisher's exact test for spontaneous mortalities
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
no effects observed
Details on results:
NOAEL = 59 mg/kg bw d (males),
62 mg/kg bw d (females)
ACTUAL DOSE RECEIVED BY DOSE LEVEL BY SEX
males 21.5 / 59 / 150 mg/kg bw d
females 22.6 / 62 / 147 mg/kg bw d
TOXIC RESPONSE/EFFECTS BY DOSE LEVEL:
- Mortality and time to death:
No animal died during the study
- Clinical signs:
No treatment-related sign or symptom was noted.
- Body weight gain: Lower in group 4 than in other groups (statistically significant: males -16%; females -21%)
- Food/water consumption:
The mean food consumption of the group 4 animals was reduced when compared with that of the control rats (statistically significant for males at
week 9-13 and for females at week 8-10).
The mean water consumption was decreased when compared with the controls (statistically significant in group 4 animals):
Males group 2: -18.0%; group 3: -20.1%, group 4: -40.4%
Females group 2: -13.5%; group 3: -19.9%; group 4: -47.8%
- Ophthalmoscopic examination:
No treatment-related finding was observed in any animal.
- Clinical chemistry: The assessment of clinical biochemical data indicated no changes of toxicological significance. Some treatment-unrelated effects were noted and considered to be secondary. They were not supported by morphological findings. The following changes were statistically
significant:
urea level for group 4 males +40.4%
calcium level for group 3 females (-3.1%) and for group 4 males (-4.0%) and females (-7.1%)
phosphorus levels for group 3 males (-10.5%) and females (-15.4%) and for group 4 males (-18.7%) and females (-27.5%)
total protein levels for group 4 females -7.0%
albumin fraction (absolute) of the protein electrophoretic pattern for group 4 females -7.7%
alpha-1 globulin fraction (relative males -14.0%, females -10.4% and absolute males -18.1%, females -16.3%) of the protein electrophoretic pattern
for group 4 animals
alpha-2 globulin fraction (relative -18.6% and abolute-21.4%) for group 4 males
- Haematology: The assessment of hematological data indicated no changes of toxicological significance after 13 weeks of treatment. Some treatment unrelated effects were noted and considered to be secondary. They were not supported by morphological findings. The following changes were
statistically significant:
hemoglobin in group 4 females -6.2%
platelet count in group 4 males (+29.0%) and females (+12.5%)
reticulocyte count for group 4 females +46.7%
total leukocyte count for group 2 males (-20.2%), group 3 males (-20.2%) and females (-24.1%), and group 4 males (-22.9%) and females (-25.3%)
prolonged prothrombin time for group 4 females +4.7%
- Organ weights:
Kidney weights in group 4 males absolute +8.1% (not significant), relative +16.4% (statistically significant) and females absolute +13.5%, relative
+25.0% (both statistically significant)
Liver weights absolute +20.7%, relative +16.7% (both statistically significant) in group 3 males, absolute -13.8% (statistically significant), relative
-4.8 % (not significant) in group 4 females, absolute -3.3%, relative +3.6% (both insignificant) in group 4 males
Other absolute and relative organ weights were not affected significantly.
- Gross pathology: No treatment-related macroscopic findings were observed. A few spontaneous gross lesions were encountered in both control
and treated rats. Their incidence and severity are considered to be similar in all groups.
- Histopathology: In further examination of the kidneys (see addendum to the pathology report, Reference RCC 1989), isolated very small foci of
tubular atrophy were recorded in one organ only. The statistical analysis for positive trend with respect to dose rate yielded a significant result for males (Z=2.29, one-tailed P=0.01) and a negative one for females (Z=1.61, one-tailed P=0.55).
Under the conditions of the experiment, the test article produced morphological alterations in the kidneys of rats at 160 mg/kg bw/day (nominal; gr oup 4)
(see second addendum to the report, Reference RCC 2000). The findings consisted of an increased incidence in tubular basophilia (both sexes of
group 4), and tubular casts (both sexes of group 4) along with a higher incidence of lymphoid foci (both sexes of group 4). These changes are
indicative for tubular nephrosis. All findings were of minor severity degrees, but were statistically significant. The remainder of findings recorded
did not differ between controls and rats treated with the test article.
Frequency of findings treated (control)
-----------------------------------------------------------
Tubular basophilia males 17/20 (0/20), females 13/20 (6/20)
Tubular casts males 8/20 (1/20), females 11/20 (1/20)
Lymphoid foci males 16/20 (5/20), females 13/20 (4/20)
-----------------------------------------------------------
- Other: The remainder of findings recorded did not differ between controls and rats treated with the test article. They were considered to be within
the range of spontaneous background lesions which may be recorded in Wistar rats of this strain and age.
Dose descriptor:
NOAEL
Effect level:
60 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
other: histopathological alterations of kidneys
Dose descriptor:
LOAEL
Effect level:
160 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
other: histopathological alterations of kidneys
Dose descriptor:
NOAEL
Effect level:
59 mg/kg bw/day (actual dose received)
Sex:
male
Basis for effect level:
other: histopathological alterations of kidneys
Dose descriptor:
NOAEL
Effect level:
62 mg/kg bw/day (actual dose received)
Sex:
female
Basis for effect level:
other: histopathological alterations of kidneys
Dose descriptor:
NOAEL
Effect level:
> 160 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
other: effects on examined fertility organs
Critical effects observed:
not specified
no further remarks
Conclusions:
In this 13-week oral toxicity study according to OECD TG 408 isophorone diamine was administered in the drinking water to Wistar rats (20 animals/sex/dose) which received nominal daily doses of 0, 20, 60, and 160 mg/kg bw ( actual dose 21.5 / 59 / 150 mg/kg bw day for males, 22.6 / 62 / 147 mg/kg bw day for females). The NOAEL for isophorone diamine was 59 mg/kg bw/day for males and 62 mg/kg bw/day for females when administered orally in the drinking water to Wistar rats.
Executive summary:

In this 13-week oral toxicity study according to OECD TG 408 isophorone diamine was administered in the drinking water to Wistar rats (20 animals/sex/dose) which received nominal daily doses of 0, 20, 60, and 160 mg/kg bw ( actual dose 21.5 / 59 / 150 mg/kg bw day for males, 22.6 / 62 / 147 mg/kg bw day for females). No treatment-related clinical signs, symptoms or mortality were noted during the study. Food and water consumption and body weight gain were significantly reduced in high dose animals. In addition, animals of this group revealed higher absolute and relative liver and kidney weights. In the 60 mg/kg bw/day group there was a statistically significant decrease in total leukocyte count (-20.2 %) and increase in the absolute liver weights (+20.7 %) and the relative liver weights (+16.7 %) for males. Along with some other statistically significant hematological and clinical chemical findings in the higher dose groups, the decrease in total leukocyte count was considered to be secondary and not treatment-related, as these effects were in general not supported by morphological findings. The variations in liver weights were also considered to be not treatment related because of the lack of dose-dependency and supporting morphological findings.

However, under the conditions of the experiment, the test article produced morphological alterations in the kidneys of rats at 160 mg/kg bw/day (nominal). The findings consisted of an increased incidence in tubular basophilia (both sexes), and tubular casts (both sexes) along with a higher incidence of lymphoid foci (both sexes). These changes are indicative for tubular nephrosis and may correspond to some of the clinical chemical findings, particularly the increased urea level for high dose males (+40.4 %). All findings were of minor severity degrees, but were statistically significant. The remainder of findings recorded did not differ between controls and rats treated with the test article. They were considered to be within the range of spontaneous background lesions which may be recorded in Wistar rats of this strain and age. The NOAEL for isophorone diamine was 59 mg/kg bw/day for males and 62 mg/kg bw/day

for females when administered orally in the drinking water to Wistar rats.
Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
59 mg/kg bw/day
Study duration:
subchronic
Species:
rat

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Referenceopen allclose all

Endpoint:
chronic toxicity: inhalation
Data waiving:
other justification
Justification for data waiving:
other:
Critical effects observed:
not specified
Endpoint:
chronic toxicity: inhalation
Remarks:
combined repeated dose and carcinogenicity
Type of information:
other: supporting study about the hydrolysis product Isobutyraldehyde
Adequacy of study:
supporting study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study according to good laboratory practice (GLP)
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies)
GLP compliance:
yes
Species:
rat
Strain:
Fischer 344
Sex:
male/female
Details on test animals or test system and environmental conditions:
Isobutyraldehyde was studied by exposing male and female F344/N rats and B6C3F mice
Route of administration:
inhalation: vapour
Type of inhalation exposure:
whole body
Details on inhalation exposure:
cited from the Abdo et al. (1998) publication:
Isobutyraldehyde vapor for the 13-week studies was generated by bubbling nitrogen gas through a column of the liquid maintained at a constant temperature in a water bath. The bubbler was continuously refilled via a side stainless steel tube and pressure stopcock to maintain a constant isobutyraldehyde liquid level in the bubbler. Diluting air was added to the nitrogen-borne vapor immediately above the bubbler to prevent condensation of isobutyraldehyde in the mainfold or delivery lines when it cooled to room temperature. Concentrations of isobutyraldehyde vapor were adjusted for the individual exposure chambers by altering either the nitrogen flow rate, the exposure chamber air flow rate, or the water bath temperature. Inhalation chambers (1,15 m3 each for teh 13-week and 2,3 m3 for the 2-year studies) of the Rochester design were used in the studies. The chamber ventilation system provided 12 to 15 charcoal- and HEPA-filtered air changes per hour and the internal design of the chamber afforded equal exposure to each animal. This flow rate was sufficient to meintain proper temperature and humidity, provide a uniform and reproducible test atmosphere, and remove ammonia. A small particle detector (Type CN, Gardner Associates, Schenectady, NY) was used with and without animals in the exposure chambers to ensure that isobutyraldehyde vapor , and not aerosol, was produced. No particle counts above the minimum resolvable level (approximately 200 particles/cm2) were detected.
Analytical verification of doses or concentrations:
not specified
Details on analytical verification of doses or concentrations:
cited from the Abdo et al. (1998) publication: The chamber concentrations of isobutyraldehyde in the 13-week studies were monitored 6 to 14 times per exposure period on a Wilkes model 80 infrared spectrophotometer. This method determines total aldehyde concentration. A method that specifically measures the chemical being is an NTP prerequisite for the 2-year studies. For this reason chamber concentrations in the 2-year studies were monitored foru times per hour using a 8-port stream select valve with two on-line gas chromatographs.
Duration of treatment / exposure:
Thirteen weeks and 2 years
Frequency of treatment:
Thirteen weeks treatment: 6h per day, 5 days per week
2 years: 6h per day, 5 days per week
Remarks:
Doses / Concentrations:
Thirteen weeks: 0, 500, 1000, 2000, 4000, 8000 ppm; 2 years: 0, 500, 1000, 2000 ppm
Basis:
nominal conc.
No. of animals per sex per dose:
Thirteen weeks: groups of 10 male and female rats and mice per dose
2 years: groups of 50 male and female rats and mice per dose
Control animals:
yes, concurrent no treatment
Details on study design:
cited from Abdo et al. (1998): Four-week old male and female F344/
Positive control:
no data
Observations and examinations performed and frequency:
Clinical observations were recorded once per week. Animals were weighed prior to initiation, and then weekly and at the end of the study. Necropsy was performed on all animals. The brain, heart, right kidney, liver, lungs, right testis, and thymus were weighed. Visible lesions and tissues masses were subject to microscopic examination.
Sacrifice and pathology:
Complete histopathologic examination was performed on control and 4000 ppm male rats, and control and 2000 ppm females.
The following tissues were examined during the gross necropsy. A complete gross necropsy is defined as external examination including body orifices and examination and fixation of these tissues: gross lesions, skin, mandibular lymph node, mammary glands, thigh muscle, sciatic nerve, sternum (including marrow), pancreas, spleen kidneys adrenals, urinary bladder, seminal vesicles, prostate, testes/epididymides/vaginal tunics of the testes and scrotal sac, esophagus, stomach, duodenum, tissue masses or suspect tumors and regional lymph nodes, ileum, colon, cecum, rectum, mesenteric lymph node, liver, costochondral junction (rib), thymus, oral cavity, larynx and pharynx, trachea, lungs and bronchi, heart and aorta, thyroid, parathyroids, ovaries, uterus, nasal cavity and nasal turbinates, jejunum, tongue, brain, pituitary, spinal cord, preputial or clitoral glands, eyes, Zymbal's glands (auditory sebaceous glands), vagina.
Other examinations:
Hematology, clinical chemistry, sperm morphology and vaginal cytology evaluation
Statistics:
method used for the evaluation of tumor incidence: logistic regression analysis, life table test, Fisher exact test and Cochran Armitage trend test
Clinical signs:
effects observed, treatment-related
Mortality:
mortality observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
not specified
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Haematological findings:
not specified
Clinical biochemistry findings:
not specified
Urinalysis findings:
not specified
Behaviour (functional findings):
not specified
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Chemical-related nonneoplastic lesions were limited to the nose of rats and mice
Histopathological findings: neoplastic:
no effects observed
Critical effects observed:
not specified

cited from Abdo et al. (1998):

Thirteen-Week Studies Rats: All rats exposed to 8000 ppm died before the end of the study. Three male rats and six female rats in the 4000 ppm groups and one female in the 500 ppm group died before the end of the study. The final body mean weight of 4000 ppm male and the mean body weight gains of 4000 ppm males and females were significantly less than those of the controls. Clinical findings in rats exposed to 4000 or 8000 ppm included abnormal respiratory sounds, decreased activity, nasal discharge, prostration, and slowed respiration. No biologically significant organ weight changes were observed. Gross lesions that could be attributed to isobutyraldehyde exposure were not evident ant necropsy. Male and female rats exposed to 8000 ppm had congestion and severe necrosis of the epithelium, and occasionally of the entire mucosa, of the nasal turbinates accompanied by acute inflammation and accumulation of serous or fibropurulent exudate within the nasal passages. Male and female rats exposed to 4000 ppm had mild epithelial hyperplasia of the mucosa of the nasal cavity and nasopharynx. Increased incidences of squamous metaplasia and mild acute (suppurative) inflammation occurred in male and female rats exposed to 4000 ppm. In addition , male rats exposed to 4000 or 8000 ppm and females exposed to 4000 ppm hat mild osteodystrophy in the bones of the maxillo- and nasoturbinates characterized by decreased numbers of osteoblasts, increased numbers of osteoclasts, decreased bone densitiy, and increased amounts of periosteal connective tissue. These changes were accompanied by inflammation of the overlying mucosa. Minimal to mild degeneration of the olfactory epithelium characterized by reduced thickness and loss of sensory cell nuclei was observed in all male rats exposed to 2000 or 4000 ppm and in three female rats exposed to 2000 ppm. In the larynx and trachea , the incidences of necrosis/degeneration were increased in male rats exposed to 8000 ppm.

Mouse study: One male in the control group, one male in the 1000 ppm group, nine males in the 4000 ppm group, and all males in the 8000 ppm group died before the end of the study. All female mice in the 4000 and 8000 ppm groups died before the end of the study. Final mean body weights and mean body weight gains of mal mice were similar to those of the controls. The final mean body weight and mean body weight gain of female mice in the 1000 ppm group were signficantly lower than those of the controls. The absolute and relative kidney weights of males in the 1000 and 2000 ppm groups were significantly greater thatn those of the chamber controls. The absolute liver weight of 1000 ppm females and absolute and relative liver weights of 500 ppm females were signficantly less than those of the chamber controls. The absolute thymus weight of 1000 ppm females and the absolute and relative thymus weight of 2000 ppm females were sgnificantly less than those of the chamber controls. Because of lack of dose response and the absence of lesions related to exposure to isobutyraldhyde in these organs, these variations in organ weights are not considered to be chemically related. There were no gross lesions observed at necropsy that could be associated with isobutyraldehyde exposure. Increased incidences of nonneoplastic lesions of the nasal cavity were observed in male and female mice exposed to 1000 ppm or greater; these lesions were histologically similar to those that occured in rats. These lesions included necrosis, inflammation, hyperplasia, and squamos metaplasia of the epithelium; serous and suppurative exudate within the nasal passages; olfactory epithelial degeneration; and osteodystrophy of the turbinate bone.

Two-Year mouse study: Estimates of 2-year survival probabilities for male and female rats are shown in the Kaplan-Meier survival curves (Fig. 1). There were no significant differences in survival rates between exposed and control male or female rats. The mean body weights of male and female rats were generally similar to those of the controls throughout the study (Fig. 2). No clinical findings that could be attributed to isobutyraldehyde exposure were observed. Pathology. No increase in incidence of neoplasms was observed in rats of either sex that could he attributed to isobutyraldehyde administration (NTP, 1997). However three primary nasal neoplasms were observed in male and female rats exposed to isobutyraldehyde. One polypoid adenoma was present in the anterior nose section of a male rat exposed to 1000 ppm, one adenoma of the vomeronasal organ was noted in a 2000 ppm male, and an undifferentiated malignant neoplasm, classified as a sarcoma (mesenchymal origin), was present in the most posterior section of the nose in a 500 ppm female (Table 3). Exposure-related nonneoplastic lesions in the nose consisted of squamous metaplasia of the respiratory epithelium, degeneration of the olfactory epithelium, and suppurative inflammation. The incidences of minimal to mild squamous metaplasia in 1000 and 2000 ppm males and females and in 500 ppm females were significantly greater than those in the controls (Table 3). This lesion occurred most frequently in the median septum and the medial aspect of the maxillary turbinates and was characterized by replacement of the cuboidal and columnar ciliated epithelium and some keratinization in the surface cells. The incidences of minimal to mild degeneration of the olfactory epithelium in male and female rats exposed to 2000 ppm were significantly greater than those in the controls. Olfactory epithelial degeneration occurred in the dorsal wall in Level II of the nasal cavity. The affected olfactory epithelium had fewer layers of sensory cells and the remaining cells were flattened and irregular (Plates 3 and 4). The incidences of suppurative inflammation (rhinitis) in male and female rats exposed to 2000 ppm were increased compared to the controls. This lesion was most notable in the anterior nasal section and occurred sometimes, but not always, in association with squamous metaplasia of the respiratory epithelium. The inflammation tended to be focal and was occasionally associated with foreign material.

Survival, body weights, and clinical findings. Estimates of 2-year survival probabilities for male and female rats are shown in the Kaplan-Meier survival curves (Fig. 1). There were no significant differences in survival rates between exposed and control male or female rats. The mean body weights of male and female rats were generally similar to those of the controls throughout the study (Fig. 2). No clinical findings that could be attributed to isobutyraldehyde exposure were observed. Pathology. No increase in incidence of neoplasms was observed in rats of either sex that could he attributed to isobutyraldehyde administration (NTP, 1997). However three primary nasal neoplasms were observed in male and female rats exposed to isobutyraldehyde. One polypoid adenoma was present in the anterior nose section of a male rat exposed to 1000 ppm, one adenoma of the vomeronasal organ was noted in a 2000 ppm male, and an undifferentiated malignant neoplasm, classified as a sarcoma (mesenchymal origin), was present in the most posterior section of the nose in a 500 ppm female (Table 3). Exposure-related nonneoplastic lesions in the nose consisted of squamous metaplasia of the respiratory epithelium, degeneration of the olfactory epithelium, and suppurative inflammation. The incidences of minimal to mild squamous metaplasia in 1000 and 2000 ppm males and females and in 500 ppm females were significantly greater than those in the controls (Table 3). This lesion occurred most frequently in the median septum and the medial aspect of the maxillary turbinates and was characterized by replacement of the cuboidal and columnar ciliated epithelium and some keratinization in the surface cells. The incidences of minimal to mild degeneration of the olfactory epithelium in male and female rats exposed to 2000 ppm were significantly greater than those in the controls. Olfactory epithelial degeneration occurred in the dorsal wall in Level II of the nasal cavity. The affected olfactory epithelium had fewer layers of sensory cells and the remaining cells were flattened and irregular (Plates 3 and 4). The incidences of suppurative inflammation (rhinitis) in male and female rats exposed to 2000 ppm were increased compared to the controls. This lesion was most notable in the anterior nasal section and occurred sometimes, but not always, in association with squamous metaplasia of the respiratory epithelium. The inflammation tended to be focal and was occasionally associated with foreign material. Downloaded from http://toxsci.oxfordjournals.org/ by guest on October 16, 2012

Two-Year Mouse Study

Survival, body weights, and clinical findings. Estimates of rates among male mice decreased with increasing exposure 2-year survival probabilities for male and female mice are concentration. The survival rate of males exposed to 2000 ppm was marginally reduced relative to the controls. There were no Mean body weights are given in Fig. 4. The mean body significant differences in survival rates between exposed and weights of male mice were generally similar to those of the control females. controls throughout the study. The mean body weights of female mice exposed to 1000 or 2000 ppm were lower than those of the controls during the second year of the study. No clinical findings that could be attributed to isobutyraldehyde exposure were observed. No increase in tumor incidence was observed in mice (NTP, 1997). The incidences of olfactory epithelial degeneration in 1000 and 2000 ppm males and females were significantly greater than in the controls (Table 4). Degeneration of the olfactory epithelium was minimal to mild in severity and occurred in the dorsal meatus of Level II; in a few mice, Level HI was also involved. Affected olfactory epithelium was characterized by fewer layers of sensory cells, which were often disorganized, and was irregular in thickness. In some mice, only sustentacular and basal cell layers persisted, or ciliated, columnar, respiratory-like epithelium replaced areas of the olfactory epithelium. In some areas, only a thin layer of fusiform cells covered the surface; in others, the basal cells remained. Two 1000 ppm females, one 2000 ppm male, and one 2000 ppm female had necrosis of the olfactory epithelium. Necrotic olfactory epithelium had pyknotic or karyorrhectic nuclei and dense eosinophilic cytoplasm.

Conclusions:
Isobutyraldehyde administered by inhalation (whole body exposure) for up to thirteen weeks or two years was a respiratory tract toxicant but was not carcinogenic in F344/N Rats and B6C3F, Mice.
NOAEL systemic effects: ca 2.9 mg Isobutyraldehyde/l ; LOAEC: ca 5.8 mg Isobutyraldehyde/l
(based on conversation factor 1 ppm= 2.9 mg/m3, see NTP report TR-472)
Executive summary:

Isobutyraldehyde administered by inhalation (whole body exposure) for up to Thirteen Weeks or Two Years Was a Respiratory Tract Toxicant but Was Not Carcinogenic in F344/N Rats and B6C3F, Mice. Abdo, K. M, Haseman, J. K., and Nyska, A. (1998). Toxicol. Sci. 42, 136-151. Isobutyraldehyde (a chemical structurally related to formaldehyde and used as a flavoring agent) was studied for toxicity and carcinogenicity by exposing male and female F344/N rats and B6C3F, mice. Animals were exposed to isobutyraldehyde vapors 6 h per day, 5 days per week for up to 13 weeks or 2 years. In the 13-week studies, groups of 10 male and 10 female F344/N rats and B6C3F, mice were exposed to concentrations of 0, 500, 1000, 2000, 4000, or 8000 ppm. Chemical-related body weight depression and deaths occurred in rats and mice exposed to 4000 and 8000 ppm. Necrosis of the epithelium accompanied with acute inflammatory reaction was observed in the nasal turbinate, larynx, and trachea of rats exposed to 8000 ppm. Exposure of rats to 4000 ppm resulted in metaplasia of the nasal respiratory epithelium, inflammation, degeneration of the olfactory epithelium, and osteodystrophy of the nasal turbinate bone. In the 13-week mouse study, exposure to 8000 ppm or 4000 ppm resulted in necrosis of the epithelium lining of the nasal turbinates. Osteodystrophy of the nasal turbinate bone and squamous metaplasia of the nasal respiratory epithelium were noted in mice exposed 4000 ppm. Degeneration of the olfactory epithelium was noted in males exposed 2000 ppm and in females exposed to 4000 ppm. In the 2-year studies, groups of 50 male and 50 male F344/ N rats and B6C3F1 were exposed to concentrations isobutyraldehyde vapors of 0, 500, 1000, or 2000 ppm 6 h per day, 5 days per week. There were no differences in survival rates or mean body weights between exposed groups and control rats. Survival of male mice exposed to 2000 ppm and mean body weights of female mice exposed to 1000 or 2000 ppm were lower than those of the of the controls. No increase in neoplasm incidence was observed in rats and mice in the 2-year studies that could be attributed to isobutyraldehyde exposure. Chemical-related nonneoplastic lesions were limited to the nose of rats and mice. They included squamous metaplasia of the respiratory epithelium (rats), suppurative inflammation (rats), and olfactory epithelial degeneration (rats and mice) at 1000 and 2000 ppm.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEC
3 000 mg/m³
Study duration:
subchronic
Species:
rat

Repeated dose toxicity: inhalation - local effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: inhalation
Type of information:
other: supporting study about the hydrolysis product isophorone diamine
Adequacy of study:
supporting study
Study period:
not reported
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: published study results; acceptable for assessment
Principles of method if other than guideline:
see Test sections "Test materials", "Test animals" and "Administration/exposure"
GLP compliance:
not specified
Limit test:
no
Species:
rat
Strain:
not specified
Sex:
male
Details on test animals or test system and environmental conditions:
details not reported
Route of administration:
inhalation
Type of inhalation exposure:
nose only
Vehicle:
air
Remarks:
aerosol/vapour atmosphere
Remarks on MMAD:
MMAD / GSD: no data
Details on inhalation exposure:
- Concentrations (substance concentrations in aerosol/vapour atmosphere): 18 (low dose group) / 200 (medium dose group) / 550 mg/m3 (high dose group)
- Doses: 9 inhalation nose-only exposures for 6 hours per day in low and medium dose groups; exposure of high dose group ended after 4 nose-onl y exposures due to unexpected mortality of 4 rats
Analytical verification of doses or concentrations:
not specified
Details on analytical verification of doses or concentrations:
no data
Duration of treatment / exposure:
low and medium dose group: 9 exposures (1 exposure/day; 6hours/day )
high dose group: 4 exposures (1 exposure/day; 6hours/day)
Frequency of treatment:
each group: 1exposure /day (6hours/day)
Remarks:
Doses / Concentrations:
18; 200; 550 mg/m3
Basis:
no data
No. of animals per sex per dose:
10
Control animals:
other: use of control rats reported but no specific information provided
Details on study design:
- Concentrations (substance concentrations in aerosol/vapour atmosphere): 18 (low dose group) / 200 (medium dose group) / 550 mg/m3 (high dose group)

Low and medium dose groups (10 male rats/group):
- Doses: 9 inhalation nose-only exposures for 6 hours per day in low and medium dose groups;
- end of exposure period: 5 rats/dose group were sacrified for pathological examinations after collection of blood and urine samples and the remaini g 5 rats/dose group undergone a 20 day recovery period. After 20 day recovery period the rats were used for pathology examinations

High dose group (10 male rats/group):
- exposure of high dose group ended after 4 nose-only exposures (6 hours/day) due to unexpected mortality of 4 rats
- remaining 6 rats were killed and necropsied one day after fourth exposure
Positive control:
no data
Observations and examinations performed and frequency:
Daily examinations during exposure period:
- weighing of rats
- observation for clinical signs of toxicity
Sacrifice and pathology:
Sacrifice:
- no detailed information available: see "Details on study design"
Pathology:
- microscopic examination of nose, trachea, larynx and lungs
Other examinations:
not reported
Statistics:
not reported
Clinical signs:
effects observed, treatment-related
Mortality:
mortality observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
not specified
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Haematological findings:
not specified
Clinical biochemistry findings:
not specified
Urinalysis findings:
not specified
Behaviour (functional findings):
not specified
Organ weight findings including organ / body weight ratios:
not specified
Gross pathological findings:
not specified
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
not specified
Details on results:
TOXIC RESPONSE/EFFECTS BY DOSE LEVEL:
- Mortality and time to death: 1 during 3rd exposure, 2 during 4th exposure and 1 after 4th exposure in high dose group
- Mean body weight and mean body weight gain: significantly reduced in high dose group during period of exposure
-Respiratory system: dose and compound-related microscopic alterations were observed in the nose, trachea, larynx and lungs of the rats in the med ium dose group (200 mg/m3) and in the high dose group (550 mg/m3) and only in the nose of rats of the low dose group (18 mg/m3). In all dose g roups after exposure period these alterations were in general characterized by necrosis but also repair (hypertrophy and hyperplasia) was evident at tis time. Lungs and trachea of rats of the low (18 mg/m3) and medium (200 mg/m3) dose group were normal at the end of recovery period (20 da ys) and tissue repair was still in progress in nose and larynx in these dose groups. Hence the authors expect close to full microscopic restitution.
Dose descriptor:
LOEC
Effect level:
18 mg/m³ air
Sex:
male
Basis for effect level:
other: substance releated effects in the nose (considered to be reversible)
Critical effects observed:
not specified

no further information

Conclusions:
The substance isophorone diamine was examined in an inhalation repeated toxicity study (nose-only) using male rats (10/dosage group) exposed to aerosol/vapour concentrations of 18, 200, and 550 mg/m³ (6 hours/day for 9 exposures with low and medium dose groups and for 4 exposures with the high dose group). Dose-dependent histopathology was identified in the respiratory tract. A NOEL was not reported in the published study information. The LOEC is 18 mg/m3 air.
Executive summary:

The substance isophorone diamine was examined in an inhalation repeated toxicity study (nose-only) using male rats (10/dosage group) exposed to aerosol/vapour concentrations of 18, 200, and 550 mg/m³ (6 hours/day for 9 exposures with low and medium dose groups and for 4 exposures with the high dose group). Treatment with 550 mg/m³ was associated with mortality, significantly reduced mean body weights and significantly reduced mean body weight gains. Dose-dependent histopathology was identified in the respiratory tract. In the low and medium dose groups these microscopic alterations in the respiratory tract are considered to be reversible. A NOEL was not reported in the published study information. The LOEC is 18 mg/m3 air.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
LOAEC
18 mg/m³
Study duration:
subchronic
Species:
rat

Repeated dose toxicity: dermal - systemic effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: dermal
Data waiving:
exposure considerations
Justification for data waiving:
other:
Critical effects observed:
not specified
Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: dermal
Data waiving:
exposure considerations
Justification for data waiving:
other:
Critical effects observed:
not specified
Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

As 1,3,3 -trimethyl-N-(2 -methylpropylidene)-5 -[(2 -methylpropylidene)amino]cyclohexanemethylamine hydrolises to the products isophorondiamin and isobutyraldehyde, those hydrolysis products are assessed. This is due to the fact that with repeated dose application the substance will hydrolise before it will be bioavailable as such. The following discussion is based on the findings for isophorone diamin CAS 2855 -13 -2


 


Studies in Animals


 


Inhalation exposure


Cited from SIAR for SIAM 18 (Paris, April 2004):


"3-Aminomethyl-3,5,5-trimethylcyclohexylamine was evaluated for repeated inhalation toxicity in male CD(SD)BR rats (10/dosage group) exposed nose-only to nominal aerosol/vapour concentrations of 18, 200, and 550 mg/m³, 6 hours/day for two weeks (4 - 5 days/week). In total, there were 9 exposures in the low and medium dose groups, while the high dose group was ended after 4 exposures due to unexpected mortality of four rats, the other six rats being necropsied the next day. Following the last exposure, 5 rats from each group (except high dose) were sacrificed and necropsied, while the remaining were sacrificed after a subsequent 20-day recovery period. Treatment with 550 mg/m³ was associated with significantly reduced bodyweights (-7.8 % within 4 days vs +0.7 % in control group). Dose-dependent histopathology was identified in the nose (18, 200, 550 mg/m³), trachea (200, 550 mg/m³), larynx (200, 500 mg/m³), and lungs (200, 550 mg/m³). Observed effects were: Degeneration/necrosis in the olfactory epithelium (nose: 5/6 “minimal” at 18 mg/m3; 5/5 “mild” at 200 mg/m3; 1/10 “minimal”, 8/10 “mild”, 1/10 “moderate” at 550 mg/m3) and in the respiratory epithelium (nose, trachea, and larynx: 3/5 “minimal” at 200 mg/m3; 1/10 “minimal”, 6/10 “mild”, 3/10 “moderate” at 550mg/m3); hyperplasia/squamous metaplasia (nose and larynx: 5/5 “mild”, only nose at 18 mg/m3; 2/5 “mild”, 3/5 “moderate” at 200mg/m3; 1/10 “minimal”; 8/10 “mild” at 550mg/m3), and hypertrophy/hyperplasia (trachea and lungs: 2/5 “minimal” at 200mg/m3; 3/10 “minimal”, 4/10 “mild” at 550mg/m3). By the end of the 20-day recovery period, the lungs and trachea of rats exposed to 18 and 200mg/m3 were within normal limits. Tissue repair was still in progress in the nose and larynx of these same rats; however, close to full microscopic restitution was expected. A NOEL for this study could not be determined (DuPont, 1997).


In a follow-up study, the effects of repeated inhalation exposure to lower concentrations of 3- aminomethyl-3,5,5-trimethylcyclohexylamine were investigated. Again, there were 9 nose-only inhalation 6 h/day exposures within two weeks (4 or 5 per week) of male Sprague-Dawley rats (10 per group). The test concentration was 2 mg/m3 (nominal); 2.2 +/- 0.25 (range 1.0 - 3.0) mg/m3 (analytical). Each five rats per group were sacrificed and necropsied the day after the last exposure and after a two week recovery period, respectively. Microscopic studies focussed on the respiratory tract: Lungs, trachea, pharynx/lanrynx, nose (first sacrifice); nose (recovery sacrifice). The only exposure related findings were minimal (2/5) to mild (3/5) degeneration of respiratory nasal mucosa in the anterior dorsal nose. They were reversible and had resolved by the end of the recovery period (DuPont, 2001)."


 


 


 


Oral exposure


 


Partly cited from SIAR for SIAM 18 (Paris, April 2004):


"In a 13-week oral toxicity study according to OECD TG 408 (RCC Research and Consulting Company, 1986), 3-aminomethyl-3,5,5-trimethylcyclohexylamine was administered in the drinking water to Wistar rats (20 animals/sex/dose) which received nominal daily doses of 0, 20, 60, and 160 mg/kg bw ( actual dose 21.5 / 59 / 150 mg/kg bw day for males, 22.6 / 62 / 147 mg/kg bw day for females). No treatment-related clinical signs, symptoms or mortality were noted during the study. Food and water consumption and body weight gain were significantly reduced in high dose animals. In addition, animals of this group revealed higher absolute and relative liver and kidney weights" ..."In the 60 mg/kg bw/day group there was a statistically significant decrease in total leukocyte count (-20.2 %) and increase in the absolute liver weights (+20.7 %) and the relative liver weights (+16.7 %) for males. Along with some other statistically significant hematological and clinical chemical findings in the higher dose groups, the decrease in total leukocyte count was considered to be secondary and not treatment-related, as these effects were in general not supported by morphological findings. The variations in liver weights were also considered to be not treatment related because of the lack of dose-dependency and supporting morphological findings. However, under the conditions of the experiment, the test article produced morphological alterations in the kidneys of rats at 160 mg/kg bw/day. The findings consisted of an increased incidence in tubular basophilia (both sexes), and tubular casts (both sexes) along with a higher incidence of lymphoid foci (both sexes). These changes are indicative for tubular nephrosis and may correspond to some of the clinical chemical findings, particularly the increased urea level for high dose males (+40.4 %). All findings were of minor severity degrees, but were statistically significant. The remainder of findings recorded did not differ between controls and rats treated with the test article. They were considered to be within the range of spontaneous background lesions which may be recorded in Wistar rats of this strain and age (RCC Research and Consulting Company, 2000). The NOAEL was 59 mg/kg bw/day for males and 62 mg/kg bw/day for females."



Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
The substance 1,3,3-trimethyl-N-(2-methylpropylidene)-5-[(2-methylpropylidene)amino]cyclohexanemethylamine will rapidly hydrolize to the reaction products isophoron diamine (CAS No. 2855-13-2) and isobutyraldehyde (CAS No. 78-84-2) in the rats after oral application due to the low pH value in the rat stomach.The substance is therefore hydrolyzed before it will be bioavailable. For this reason a supporting assessment of the hydrolysis reaction products isophorone diamine and isobutyraldehyde has been chosen. (Annex XI, chapter I). For the hydrolysis product isophorone diamine a subchronic oral repeated dose study (90 days) is available.

Justification for selection of repeated dose toxicity inhalation - systemic effects endpoint:
The substance 1,3,3-trimethyl-N-(2-methylpropylidene)-5-[(2-methylpropylidene)amino]cyclohexanemethylamine will rapidly hydrolize to the reaction products isophoron diamine (CAS No. 2855-13-2) and isobutyraldehyde (CAS No. 78-84-2) in the rats after inhalation. The substance is therefore hydrolyzed before it will be bioavailable. For this reason a supporting assessment of the hydrolysis reaction products isophorone diamine and isobutyraldehyde has been chosen. (Annex XI, chapter I). For both hydrolysis products subchronic inhalation repeated dose studies (90 days) are available. The NOAEC for isobutyraldehydes is 3000 mg/m3. However the predominant effect are the local effects in the lung caused by isophorone diamine with an LOEC of 18 mg/m3. The LOEC for isophorone diamine for local effects in the lung is much lower than that for isobutyraldehyde and will therefore be used for further risk assessment.

Justification for selection of repeated dose toxicity inhalation - local effects endpoint:
The substance 1,3,3-trimethyl-N-(2-methylpropylidene)-5-[(2-methylpropylidene)amino]cyclohexanemethylamine will rapidly hydrolize to the reaction products isophoron diamine (CAS No. 2855-13-2) and isobutyraldehyde (CAS No. 78-84-2) in the rats after inhalation.The substance is therefore hydrolyzed before it will be bioavailable. For this reason a supporting assessment of the hydrolysis reaction products isophorone diamine and isobutyraldehyde has been chosen. (Annex XI, chapter I). For both hydrolysis products subchronic repeated dose studies (90 days) are available.

Justification for selection of repeated dose toxicity dermal - systemic effects endpoint:
The substance 1,3,3-trimethyl-N-(2-methylproylidene)-5-[(2-methylpropylidene)amino]cyclohexanemethylamine is a skin sensitizer. Any dermal exposure has to be avoided. Hence dermal exposure is considered not relevant for human and because of column 2 of Annex IX (8.6.1 and 8.6.2) of REACH regulation a dermal repeated dose toxicity study is not needed.

Justification for selection of repeated dose toxicity dermal - local effects endpoint:
The substance 1,3,3-trimethyl-N-(2-methylproylidene)-5-[(2-methylpropylidene)amino]cyclohexanemethylamine is a skin sensitizer. Any dermal exposure has to be avoided. Hence dermal exposure is considered not relevant for human and because of column 2 of Annex IX (8.6.1 and 8.6.2) of REACH regulation a dermal repeated dose toxicity study is not needed.

Justification for classification or non-classification

Because of the results of repeated dose toxicity studies for the two hydrolysis products for the substance, the substance

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

is not classified according to 67/548/EEC and CLP regulation (1272/2008).