3-aminomethyl-3,5,5-trimethylcyclohexylamine

Endpoint summary

Description of key information

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). 

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

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.

Reference 2

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)

Reference 3

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1985-08-05 - 1985-11-13

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

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

Details on species / strain selection:

This test system is recognized by the international regulatory agencies as a suitable rodent model for studies of this type.

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: Tap water at pretest and 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

Details on route of administration:

Oral, mixed with the drinking water. The water was available ad libitum. The control animals received drinking water without the test article.

Vehicle:

water

Details on oral exposure:

TEST ARTICLE PREPARATION
The test article was weighed into a glass beaker on a tared Mettler PK 300 balance and distilled water was added (g/g). The test article/drinking water mixtures were prepared daily during the first eleven days and twice weekly thereafter.

TEST ARTICLE ADMINISTRATION
METHOD
Oral, mixed with the drinking water. The water was available ad libitum. The control animals received drinking water without the test article.
Rationale: Proven efficacy for absorption of test article.

DOSE LEVELS (nominal)
Group 1, 0 mg/kg body weight/day
Group 2, 20 mg/kg body weight/day
Group 3, 60 mg/kg body weight/day
Group 4, 160 mg/kg body weight/day
Rationale: Based upon oral LD50 performed by the sponsor.

DURATION OF ACCLIMATION PERIOD
7 days

DURATION OF TREATMENT
13 weeks

VEHICLE
Tap water at pretest, distilled water during the treatment period.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The stability of the test article in the drinking water was performed during the first week of the study. Intercurrent sampling for concentration analyses were performed monthly. The analyses were performed in the analytical laboratories of RCC. The analyses were performed by capillary gas chromatography.
The mean concentrations in % of nominal and their standard deviations was found to be:
Group 2: 99.6 % (+/- 14.5 %)
Group 3: 94.2 % (+/- 5.6 %)
Group 4: 104.5 % (+/- 2.8 %)
The results of the stability test at room temperatureshowed that the test article was stable in drinking water for 96 hat least.

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

daily (continuously)

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

control group

Dose / conc.:

20 mg/kg bw/day (nominal)

Remarks:

low dose group
actual dose: 21.5 mg/kg bw/d for males; 22,6 mg/kg bw/d for females

Dose / conc.:

60 mg/kg bw/day (nominal)

Remarks:

intermediate group
actual dose: 59 mg/kg bw/d for males; 62 mg/kg bw/d for females

Dose / conc.:

160 mg/kg bw/day (nominal)

Remarks:

high dose group
actual dose: 150 mg/kg bw/d for males; 147 mg/kg bw/d for females

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:

OBSERVATIONS:
VIABILITY/ MORTALITY
Each animal was observed twice daily for viability.

SIGNS AND SYMPTOMS
Observations for clinical signs and symptoms of toxicity were performed twice daily. A description of any abnormality observed at any examination was recorded.

FOOD CONSUMPTION
The food consumption was recorded for 7-day periods and the mean daily food consumption calculated. These data were recorded weekly, using an on-line electronic recording system composed of a Mettler PK 4800 balance connected to a HewlettPackard 9835 desk computer.

WATER CONSUMPTION
The water consumption was recorded once weekly for a 24-hour period, using an on-line electronic recording system composed of a Mettler PK 4800 balance connected to a Hewlett-Packard 9835 desk computer.

BODY WEIGHTS
The body weight of each animal was recorded weekly, using an online electronic recording system composed of a Mettler PK 4800 balance connected to a Hewlett-Packard 9835 desk computer.

OPHTHALMOSCOPIC EXAMINATIONS
Ten animals per sex and group with the lowest identification numbers were examined for ocular abnormalities using a HeineBifocal ophthalmoscope (miraflex type) A description of any abnormality was recorded. Examinations were performed at pretest and at the end of study duration.

CLINICAL LABORATORY INVESTIGATIONS:
GENERAL
Blood samples for hematology and clinical biochemistry were collected from ten animals per group and sex with the highest identification numbers under light ether anesthesia. The animals were fasted for 18 hours before blood sampling but water was provided. Blood samples were collected from each animal between the hours of 7.00 and 8.30 a.m. to reduce biologic variation caused by circadian rhythm. Blood samples were drawn from the retro-orbital plexus.
Blood sampling, after 13 weeks November 11-12, 1985
The assays of blood parameters were performed under quality control conditions to assure reliable test results. The summary and individual tables were generated by a computer whose program limits the width of each column to 10 characters. Therefore, the names of some parameters have been abbreviated. Any nonstandard abbreviation has been defined in this section under "Parameter" in upper-case letters enclosed by parentheses. Clinical laboratory data are expressed in general accordance with the International System of Units (SI)
Remark code identification:
NV = no value
HEM = hemolytic sample

HEMATOLOGY
The following anticoagulants were used during blood collection, EDTA-K2 (hematology)
Sodium Citrate, 3.8 %. (coagulation, L10)
The following commercial reference controls were used to monitor the performance of the method:
Hematology:
Control Blood Plus - Level I (low range)
Control Blood Plus - Level II (normal range)
Control Blood Plus - Level III (high range)
(Merz & Dade AG, Duedingen/Switzerland)

Coagulation:
Ci-Trol-1 (normal range)
Ci-Trol-2 (high range)
(Merz & Dade AG, Duedingen/Switzerland)

The following methods were used to determine the values of the parameters listed:
Parameter // Method/ Instrument // Unit of measure
- Erythrocyte count (RBC) // Sysmex (TOA) CC-720 Multi-Parameter Automated Hematology Analyzer // T/ L
- Hemoglobin (HB) // Cyanmethemoglobin, Sysmex (TOA) CC-720 Multi-Parameter Automated Hematology Analyzer // mmol/ L
- Hematocrit (HCT) // Sysmex (TOA) CC-720 Multi-Parameter Automated Hematology Analyzer //L/ L
- Mean corpuscular volume (MCV) // Calculated value: HCT/RBCSysmex (TOA) CC-720 Multi Parameter Automated Hematology Analyzer // fl
- Mean corpuscular hemoglobin (MCH) // Calculated value: HB/RBC - Sysmex (TOA) CC-720 Multi Parameter Automated Hematology Analyzer // fmol
- Mean corpuscular hemoglobin concentration (MCHC) // Calculated value, HB/HCT - Sysmex (TOA) CC-720 Multi Parameter Automated Hematology Analyzer // mmol/ L
- Platelet count (PLATELETS) // Clay Adams Ultra Flo-100 Whole Blood Platelet Counter // G/ L
- Reticulocyte count (RETIC.) // Supravital staining with Brecher's New Methylene Blue solution - Manual count of 1000 erythrocytes of stained film, using a Leitz Laborlux 12 light microscope // 1
- Nucleated erythrocytes - normoblasts (NEN) // Reported as number of nucleated erythrocytes per 100 leukocytes in the differential count // NEN/100 WBC
- Total leukocyte count (WBC) // Sysmex /TOA) CC-720 MultiParameter Automated Hematology Analyzer // G/ L
- Differential leukocyte count (Diff. WBC Count) // Monocellular layer blood smears produced with a Perkin-Elmer Model 90 Coleman Uni-Smear Spinner Blood smear stained with a modified Wright's Eosin Methylene Blue solution (OMRON) using an Omron Microx AutoStainer Manual count of 100 leukocytes using a Leitz Laborlux 12 light microscope // 1 (rel.)
Cell classification:
BAND. = Band Neutrophil
SEG. = Segmented Neutrophil
ED. = Eosinophil
BASO. = Basophil
LYMPH. = Lymphocyte
MONO. = Monocyte
PLAS. = Plasma Cell
OTHER = Blast Cell (undifferentiated)
- Red cell morphology // By microscopic examination of stained blood smear. Erythrocytes that vary from the normal in size, shape and hemoglobin content, or contain greater amounts of nuclear remnants are indicated as abnormal erythrocytes, and are characterized as such // normal/ abnormal

COAGULATION:
Parameter // Method/ Instrument // Unit of measure
- Thromboplastin time (PT) // Quick's one stage method using Thromboplastin C (Merz & Dade) and a Amelung Digital Ball Coagulometer KC 10 // sec.
- Partial thromboplastin time (PTT) // Method using Actin Activated Cephaloplastin Reagent (Merz
& Dade) and a Amelung Digital Ball Coagulometer KC 10 // sec.

CLINICAL BIOCHEMISTRY
The following anticoagulant was used during blood collection, Lithium Heparin (140 U.S.P. Units). The following commercial reference controls were used to monitor the performance of the method:
Clinical Biochemistry
Seronorm (normal range)
Pathonorm L (low range)
Pathonorm H (high range)
(Nyegaard & Co., Oslo/Norway)
Moni-Trol I-E (normal range)
Moni-Trol II-E (abnormal range)
(Merz & Dade AG, Duedingen/Switzerland)
Protein Electrophoresis:
Seronorm Protein
(Nyegaard & Co., Oslo/Norway)

The following methods were used to determine the values of the
parameters listed:
Parameter // Method/ Instrument // Unit of measure
- Glucose // GOD/PAP - Greiner Selective Analyzer G-300 // mmol/ L
- Urea // Urease/Berthelot - Greiner Selective Analyzer G-300 // mmol/ L
- Creatinine // Jaffe-kinetic reaction - Greiner Selective Analyzer G-300 // µmol/ L
- Bilirubin, total (BILI. T.) // Diazotized 2,4-dichloroaniline - Greiner Selective Analyzer G-300 // µmol/ L
- Cholesterol, total (CHOLEST. T. ) // CHOO/ PAP - Greiner Selective Analyzer G-300 // mmol/ L
- Aspartate aminotransferase (ASAT (GOT)) // MOH/ NAOH coupled reaction, optimized method based on Scand. Soc. for Clin. Chem. recommendations - Greiner Selective Analyzer G-300 // µkat/ L (37 °C)
- Alanine aminotransferase (ALAT (GPT)) // LDH / NADH coupled reaction, optimized method based on Scand. Soc. for Clin. Chem. recommendations - Greiner Selective Analyzer G-300 // µkat/ L (37 °C)
- Lactate dehydrogenase (LDH) // Direct NADH coupled reaction using pyruvate as substrate (PYR --> LAC reaction), optimized method based on German Soc. for Clin. Chem. recommendations - Greiner Selective Analyzer G-300 // µkat/ L (37 °C)
- Alkaline phosphatase (ALP) // p-nitrophenylphosphate./ diethanolamine (PNPP/DEA),
optimized method based on Scand. Soc. for Clin. Chem. recommmendations - Greiner Selective Analyzer G-300 // µkat/ L (37 °C)
- Calcium // Reaction with o-cresolphthalein complexone - Gilford Analyzer, System 203 // mmol/ L
- Phosphorus // Unreduced phosphomolybdate complex without deproteinization (360 nm) - Gilford Analyzer, System 203 // mmol/ L
- Sodium // Flame photometry with internal Lithium standard - Greiner Selective Analyzer G-300 (IL-743, OEM) // mmol/ L
- Potassium // Flame photometry with internal Lithium standard - Greiner Selective Analyzer G-300 (IL-743, OEM) // mmol/ L
- Chloride // Mercuric thiocyanate - Greiner Selective Analyzer G-300 // mmol/ L
- Protein, total // Biuret end point - Greiner Selective Analyzer G-300 // g/ L
- Protein electrophoresis (PROT. ELECTROPH.) // Horizontal electrophoresis, using agarose gel film and a Bio-Rad (s) horizontal electrophoresis system (Model 1415 Horizontal Electrophoresis Cell and Model 500 Constant Power Supply), and a Beckman CDS-200 Computing Densitometer // 1 (rel.), g/ L (abs.)
Electrophoretic fractions:
ALBUMIN = Albumin
A1-GLOB. = Alpha 1-globulin
A2-GLOB. = Alpha 2-globulin
SB-GLOB. = Summ of beta globulins
G-GLOB. = Gamma globulin
Calculated value based on peak area: A/G RATIO= Albumin to Globulin ratio.

Sacrifice and pathology:

ORGAN WEIGHTS
The following organ weights of all animals necropsied after 13 weeks were recorded, Adrenals, kidneys, liver and testes.

NECROPSY AND HISTOPATHOLOGY
Necropsy of all rats was performed by RCC. Animals were killed by exsanguination following the induction of anesthesia by intraperitoneal injection of sodium pentobarbitone. Tissue samples were fixed in neutral phosphate buffered 4 % formalin, processed and embedded in paraffin wax. Sections were cut at an approximate thickness of 4 micrometers and stained with hematoxylin and eosin. Sections of the following organs and tissues were examined microscopically from all rats of groups 1 and 4 and from those which had gross lesions (number of sections per organ), Adrenal glands (2), aorta (thoracic; 1), bone (sternumi 1), bone marrow (sternum1 1), brain (3), cecum (1), colon (1), duodenum (1), epididymides (2), esophagus (1), eyes (2), Harderian glands (2), heart (1), ileum (1), jejunum (1), kidneys (2), liver (2), lungs with mainstem bronchi (2), lymph nodes (mandibular, mesenteric; 1), mammary gland (1), ovaries (2), pancreas (1), pituitary gland (1), prostate (1), rectum (1), salivary gland (mandibular, sublinguali 1), sciatic nerve (1), seminal vesicles (2), skeletal muscle (1), skin (1), spinal cord (1), spleen (1), stomach (1), testes (2), thymus (1), thyroid gland (2), tongue (1), trachea (1), urinary bladder (1), uterus (3), and all macroscopic abnormalities.

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:

The following statistical methods were used to analyze the body weights, water and food consumption, organ weights, mortality, and clinical laboratory data:
Univariate one-way analysis of variance was used to assess the significance of intergroup differences.
If the variables could be assumed to follow a normal distribution, the Dunnett-test (many to one t-test) based on a pooled variance estimate was applied for the comparison between the treated groups and the control groups.
The Steel-test (many-one rank test) was applied when the data could not be assumed to follow a normal distribution.
For the overall spontaneous mortality data, the Fisher's exact test for 2x2 tables was applied.
Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables.
Individual values, means, standard deviations and statistics were rounded off before printing. For example, test statistics were calculated on the basis of exact values for means and pooled variances and then rounded off to two decimal places. Therefore, two groups may display the same printed means fot a given parameter, yet display different test statistics values.

Clinical signs:

no effects observed

Description (incidence and severity):

No treatment-related sign of toxicity or clinical symptom was noted in any animal of any group.

Mortality:

no mortality observed

Description (incidence):

No animal died during the study.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Body weight gain: Lower in group 4 than in other groups (statistically significant: males -16%; females -21%)

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

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).

Food efficiency:

not examined

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

effects observed, treatment-related

Description (incidence and severity):

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%

Ophthalmological findings:

no effects observed

Description (incidence and severity):

No treatment-related finding was observed in any animal.

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

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%

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

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
- alpha-2 globulin fraction (relative -18.6% and abolute-21.4%) for group 4 males

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

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 pathological findings:

no effects observed

Description (incidence and severity):

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.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

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; group 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)

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Description (incidence and severity):

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.

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:

LOAEL

Effect level:

160 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

food consumption and compound intake

other: histopathological alterations of kidneys

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male

Basis for effect level:

other: no treatment related adverse effects were seen in the intermediate dose group

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

female

Basis for effect level:

other: no treatment related adverse effects were seen in the intermediate dose group

Critical effects observed:

not specified

no further remarks

Conclusions:

In this 13-week oral toxicity study according to OECD TG 408 the test item 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 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 the test item 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

Quality of whole database:

The study is reliable without restrictions (Klimisch 1)

System:

urinary

Organ:

kidney

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: inhalation

Type of information:

experimental study

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)

Dose / conc.:

0 mg/m³ air

Remarks:

control group

Dose / conc.:

18 mg/m³ air

Remarks:

low dose group

Dose / conc.:

200 mg/m³ air

Remarks:

intermediate dose group

Dose / conc.:

550 mg/m³ air

Remarks:

high dose group

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

Description (incidence):

1 during 3rd exposure, 2 during 4th exposure and 1 after 4th exposure in high dose group

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Mean body weight and mean body weight gain: significantly reduced in high dose group during period of exposure

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

Description (incidence and severity):

Dose and compound-related microscopic alterations were observed in the nose, trachea, larynx and lungs of the rats in the medium 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 groups 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 days) and tissue repair was still in progress in nose and larynx in these dose groups

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:

no adverse effect observed

Study duration:

subacute

Species:

rat

Quality of whole database:

The study is reliable with restrictions (Klimisch 2)

System:

other: All systemic effects occuring during the inhalation study can be considered to be secondary to the local effects based on the corrosive properties of the substance.

Repeated dose toxicity: inhalation - local effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: inhalation

Type of information:

experimental study

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)

Dose / conc.:

0 mg/m³ air

Remarks:

control group

Dose / conc.:

18 mg/m³ air

Remarks:

low dose group

Dose / conc.:

200 mg/m³ air

Remarks:

intermediate dose group

Dose / conc.:

550 mg/m³ air

Remarks:

high dose group

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

Description (incidence):

1 during 3rd exposure, 2 during 4th exposure and 1 after 4th exposure in high dose group

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Mean body weight and mean body weight gain: significantly reduced in high dose group during period of exposure

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

Description (incidence and severity):

Dose and compound-related microscopic alterations were observed in the nose, trachea, larynx and lungs of the rats in the medium 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 groups 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 days) and tissue repair was still in progress in nose and larynx in these dose groups

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

2.2 mg/m³

Study duration:

subacute

Species:

rat

Quality of whole database:

The study is reliable with restrictions (Klimisch 2)

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

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."

 

The aim of a dose range finding study at LPT (2020) 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)

The aim of the subsequently performed OECD 422 study (Provivo 2022) was to obtain information on possible effects of the test item on general toxicity, reproduction and/or development. 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) was established for systemic toxicity: 100 mg/kg bw/d.

 

 

Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
valid OECD 408 guideline study

Justification for selection of repeated dose toxicity inhalation - systemic effects endpoint:
valid repeated dose inhalation study

Justification for selection of repeated dose toxicity inhalation - local effects endpoint:
valid repeated dose inhalation study

Justification for selection of repeated dose toxicity dermal - systemic effects endpoint:
The substance isophorone diamine 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 isophorone diamine 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 the substance isophorone diamine is not classified according to CLP regulation (1272/2008).