(2-hydroxy-1,1-dimethylethyl)ammonium toluene-4-sulphonate

Endpoint summary

Description of key information

OECD 422 DOSE RANGE FINDING STUDY (Provivo, 2020)

The aim of the study was to select the dose levels of the test item for the main reproduction/developmental toxicity study (OECD 422).

The rats were treated with 65, 200 or 600 mg test item/kg b.w. daily from test day 1 to test day 14. On test day 8 the high dose level was increased to 1000 mg test item/kg b.w. daily, as no signs of toxicity were noted.

None of the animals died prematurely.

No test item-related observations were noted in behaviour, the external appearance or the appearance of the faeces.

Body weight and food consumption of the male and female animals revealed no test item-related differences.

During the macroscopic inspection at necropsy mandibular lymph nodes with an increased size were noted for nearly all male and female animals of the intermediate and the high dose group (200 or 600/1000 mg test item/kg b.w./day).

After consideration of these data, the following dose levels are suggested for a reproduction/ developmental toxicity study in CD® rats based on OECD guideline 422:

Group 1:    Control (vehicle)

Group 2:    100 mg test item/ kg b.w./day

Group 3:    300 mg test item/ kg b.w./day

Group 4:    1000 mg test item/ kg b.w./day

 

 

OECD 422 MAIN STUDY (Provivo, 2022)

The aim of the study was to obtain information on possible effects of the test item on general toxicity, reproduction and/or development based on OECD guideline 422. The test item was administered orally to rats at dose levels of 100, 300 or 1000 mg/kg b.w./day.

No pathological lesions in the reproductive organs were related to the test item were noted during the histopathological examination in the dams that delivered live pups, regardless of the dose group (100, 300 or 1000 mg test item/kg b.w./day).
Histological differences were noted of the high dose females which lost all implantations. These included implantation sites appearing unphysiological, resorption in the uterus, poorly developed corpora lutea in the ovary and poorly developed mammary glands in 2 of 5 (2/5) females of Group 4. Poorly developed corpora lutea and mammary glands indicate the insufficient progesterone level necessary to maintain pregnancy. There were no abnormalities in the histology of the reproductive organs from the remaining three females of Group 4, as well as from 5 females/group of Groups 2 and 3, examined in this study.
Histopathological examination revealed pathological changes of the adrenal glands of the female high dose animals (1000 mg test item/kg b.w./day) in form of diffuse adrenocortical hypertrophy or multifocal to diffuse fatty vacuoles. Additionally, an increased group mean severity grade for thymus atrophy was observed for the female high dose animals. These results may indicate that the HD females, even in females successful of pregnancy, were under the stressful condition, although no major differences in the terminal body weights were detected. 

At the high dose level (1000 mg test item/kg b.w./day), a reduced number of implants per dam was noted. Additionally, 6 of 9 pregnant high dose females displayed a resorption of all of their implantations, resulting in an increased post-implantation loss and a decreased birth index. These effects were due to stress of the hig dose females.
No influence on the viability index was noted. Following the decreased birth index a reduction of the litter weight was noted for the high dose group (1000 mg test item/kg b.w./day). The implantation loss and the subsequently reduced birth index was also a consequence of the high stress level of the high dosed females.
No test item-related abnormalities (malformations or variations) were noted during the external macroscopic examination of the pups at necropsy.

The No Observed Adverse Effect Level (NOAEL) for systemic toxicity was considered to be 300 mg/kg/day for female rats because dosage of of 1000 mg/kg/day resulted in toxicologically significant histopathological findings in the uterus such as unphysiological, resorptions, poorly developed corpora lutea in the ovary and poorly developed mammary glands. Histopathological changes in the adrenal glands were noted for the female high dose animals  in form of diffuse adrenocortical hypertrophy or multifocal to diffuse fatty vacuoles. In addition, the group mean severity grade for thymus atrophy was increased in the high dose females. These are stress induced effects.

For male rats no toxicological effects could be observed up to the highest tested dose of 1000 mg/kg bw d.
 

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:

Study was conducted as OECD 422 as combined repeated dose toxicity and reproductive toxicity screening study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2020-09-30 (Date of experimental Initiation) to 2021-01-06 (End of in-life period)

Reliability:

1 (reliable without restriction)

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted 29 July 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Crj: CD(SD)

Remarks:

Rat / CD/ Crl:CD(SD) Charles River Laboratories, Research Models and Services, Germany GmbH Sandhofer Weg 7 97633 Sulzfeld Germany

Details on species / strain selection:

Selection of species: The rat is a commonly used rodent species for such studies.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH
- Number and sex of animals (on TD 15): 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.

- Age at first administration: Males: 99 days, Females: 99 days
- Weight at study initiation: (P) Males: 423.3 - 558.9 g; Females: 232.4 - 374.1g

- Housing: MAKROLON cages (type III plus) with a basal surface of approx. 39 cm x 23 cm and a height of approx. 18 cm

- Diet: ad libitum): ssniff® R/Z V1324, ssniff Spezialdiäten GmbH, 59494 Soest, Germany
- Water (e.g. ad libitum): Tap water was offered ad libitum.
- Acclimation period: 7 adaptation days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22°C ± 3°C (maximum range)
- Humidity (%): 55% ± 10% (maximum range)
- Air changes (per hr): 15 - 20
- Photoperiod (hrs dark / hrs light): 12 hours dark/12 hours light cycle

Route of administration:

oral: gavage

Details on route of administration:

Administration volume: 2 mL/kg b.w./day
Vehicle: Tap water

Vehicle:

water

Remarks:

tap water

Details on oral exposure:

Route of administration: Oral, by gavage
Frequency of administration: Once daily
Administration volume: 2 mL/kg b.w./day
Vehicle: Tap water

The administration formulations were freshly prepared every day.
The test item was dissolved in the vehicle to the appropriate concentrations and administered orally at a constant administration volume/kg b.w. once daily. The amount of test item was adjusted the animal's current body weight 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 the analysis of the test item-vehicle formulations, two (2) aliquots of approximately 3 mL each were taken at the following times and stored at -20°C ± 10% until analysis at the Test Facility:

At start of the treatment period (first dosing day):
Analysis of stability and concentration
Immediately after preparation of the administration formulation as well as after 8 and 24 hours storage at room temperature
(3 samples / dose level group)
Number of samples: 9 (18 aliquots)

Towards the end of the treatment period (when the majority of animals was dosed)
Analysis of concentration
During treatment always before administration to the last animal/dose level group
(1 sample / dose level group).
Number of samples: 3 (6 aliquots)

Sum of all samples: 12 (24 aliquots)

The samples were labelled with the study number, species, type of sample, concentration, aliquot number, test day, sampling time and date.
Only one aliquot was analysed; the second aliquot serves as back-up.
The analytical method was validated by Provivo. The following parameters were determined:
-Linearity
-Accuracy
-Precision
-Sensitivity
-Specificity
-Stability

Duration of treatment / exposure:

Duration of the study: 7 adaptation days and approximately 77 test days

Males: 35 treatment days (from TD 15 to TD 49; sacrifice on TD 50)

Females with litter and not mated females: 49 to 54 treatment days (from TD 15 or 21 to TD 63 or TD 69; sacrifice between TD 64 and TD 71)

Females with resorptions of all implants: 36 or 37 treatment days (from TD 15 to TD 55 or TD 56; sacrifice on TD 56 or TD 57 )

Frequency of treatment:

daily

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

control group

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

low dose group

Dose / conc.:

300 mg/kg bw/day (nominal)

Remarks:

intermediate dose group

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

high dose group

No. of animals per sex per dose:

10 males and 10 females per dose group

Control animals:

yes, concurrent vehicle

Details on study design:

Justification for dose selection
The dose levels of this study were selected in agreement with the Monitor based on the results of a 14-day dose-range finding study (LPT Study No. 38158, 2020).
The rats were treated with 65, 200 or 600 mg/kg b.w. daily from test day 1 to test day 14. On test day 8, the high dose level was increased to 1000 mg/kg b.w. daily, as no signs of toxicity were noted.
None of the animals died prematurely. No test item-related observations were noted in behaviour, the external appearance or the appearance of the faeces. Body weight and food consumption of the male and female animals revealed no test item-related differences. During the macroscopic inspection at necropsy, mandibular lymph nodes with an increased size were noted for nearly all male and female animals of the intermediate and high dose groups (200 or 600/1000 mg/kg b.w. daily).
Hence, doses of 100, 300 and 1000 mg/kg b.w. were suggested for this study.

Positive control:

not needed

Observations and examinations performed and frequency:

DAILY OBSERVATIONS
Throughout the test period, each animal (parental animals and pups) was observed for clinical signs at least once daily. The frequency was increased when signs of toxicity were observed. Behavioural changes, signs of difficult or prolonged parturition, and all signs of toxicity were recorded. Mortality was recorded twice daily. Animals that were sacrificed prematurely were necropsied after sacrifice.
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.

Daily monitoring of vaginal lavages from female were continued throughout the pre-mating period until evidence of mating. A vaginal lavage was also taken in the morning of the day of scheduled necropsy.
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 F0 generation animals outside the home cage in a standard arena and at the same time, each time preferably by observers unaware of the treatment. Signs noted include 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 presence of clonic or tonic movements, stereotypies (e.g. excessive grooming, repetitive circling), difficult or prolonged parturition or bizarre behaviour (e.g. self-mutilation, walking backwards) would have been also be recorded.

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 was conducted as described on the following pages in five males and five females randomly selected from each group. The screening was conducted two hours after dosing and before any blood sampling for laboratory examinations:
5 male F0 animals per group: Shortly before scheduled sacrifice
5 female F0 animals per group: During lactation, shortly before scheduled sacrifice

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; for this outcome (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 clonic 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 manoeuvre
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 mounted on top of the home cage and any movements were measured for duration of 12 minutes 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 were weighed on each day of dosing for dose adjustment and at sacrifice; the individual body weights were recorded.
The report includes weekly values for the male animals (starting on test day 15) and the body weight on the day of sacrifice.
For the female animals, the body weights on the following days are given in the report:

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/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 (pre-mating and gestation) or treatment period (lactation).
Food residue (or total food left) was weighed and recorded as follows:

Study period Males Females
Pre-mating period TD 15, TD 22, TD 28 TD 15, TD 22, TD 28
Mating period None None
Gestation period Not applicable GD 7, GD 14, GD 20
Lactation period Not applicable LD 1, LD 7, LD 13

LABORATORY EXAMINATIONS
Blood samples were taken from the retrobulbar venous plexus under isoflurane anaesthesia from animals fasted overnight and collected into tubes as follows:
EDTA anticoagulant (whole blood).......for haematological examinations
Citrate anticoagulant (plasma).............for coagulation tests
Serum.....................................................for clinical chemistry
The following sampling times and animals were employed:
Sampling time
At study termination (before necropsy): 5 males and 5 females randomly selected from each group

HAEMATOLOGY
The following parameters were determined (Instrument: ADVIATM 120, Siemens Diagnostics GmbH, 35463 Fernwald, Germany):
Parameter (in blood)/ Unit
- Haemoglobin content (HGB)/ mmol/L
- Erythrocytes (RBC)/ 10^6/µL
- Leucocytes (WBC)/ 10^3/µL
- Differential blood count : - relative/ % - absolute/ 10^3/µL
- Reticulocytes (Reti)/ %
- Haematocrit value (HCT)/ %
- Platelets (PLT)/ 10^3/µL
- Mean corpuscular volume (MCV)/ fL
- Mean corpuscular haemoglobin (MCH)/ fmol
- 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.

Coagulation
The parameters listed below were determined (Instrument: Amax Destiny Plus™, TCoag Deutschland GmbH, 32657 Lemgo, Germany)
Parameter (in plasma)/ Unit
- Prothrombin time (PT)/ sec
- Activated partial thromboplastin time (aPTT)/ sec

CLINICAL CHEMISTRY
The parameters listed below were determined:
Parameter (in serum)/ Unit
Instrument: KONELAB 30i, Thermo Fisher Scientific, 63303 Dreieich, Germany
- Albumin/ g/L
- Bile acids/ µmol/L
- Bilirubin (total)/ µmol/L
- Cholesterol (total)/ mmol/L
- Creatinine/ µmol/L
- Glucose/ mmol/L
- Protein (total)/ g/L
- Blood urea nitrogen (BUN)/ mmol/L
- Calcium/ mmol/L
- Chloride/ mmol/L
- Potassium/ mmol/L
- Sodium/ mmol/L
- Alanine aminotransferase (ALAT)/ U/L
- Alkaline phosphatase (aP)/ U/L
- Aspartate aminotransferase (ASAT)/ U/L
- Lactate dehydrogenase (LDH)/ U/L
by substraction:
- Globulin/ g/L
by calculation:
- Albumin/globulin ratio/ non-dimensional
- Sodium/Potassium ratio/ non-dimensional
- BUN/creatinine ratio/ non-dimensional

THYROID HORMONE DETERMINATION T4
Blood samples were taken always at the same time of day, as scheduled below:
All evaluated dams At sacrifice 32 x 6 Samples Non-fasted Analysis: No Sample volume: 6 x 100 µL
All adult males At sacrifice 40 x 6 Samples Non-fasted Analysis: Yes Sample volume: 6 x 100 µL
The animals scheduled for blood sampling were anaesthetized and euthanized as follows:
Adults: sampling from the retrobulbar venus plexus under isoflurane anaesthesia; euthanized by carbon dioxide (CO2) inhalation.

LH AND PROGESTERONE DETERMINATION
Aliquots of blood samples of the dams were used for the determination of serum levels of LH and progesterone. Serum levels were determined by ELISA employing commercially available kits (IBL International GmbH, 22335 Hamburg; lot. nos: 16K041-2 (LH) and 23K110 (progesterone)).

Sacrifice and pathology:

GROSS NECROPSY
Vaginal lavages prepared on the day of necropsy were examined to determine the stage of the oestrous cycle and allowed correlation with the histopathology of the female reproductive organs.
The animals were euthanised by carbon dioxide (CO2) inhalation, exsanguinated by cutting the aorta abdominalis, weighed at the following times:
Males: On TD 50
Dams (surviving dams): On PND 13 or shortly thereafter


DISSECTION OF ALL ADULT ANIMALS
At the time of sacrifice or premature death during the study, the adult animals were dissected and 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 nerved. After ventral midline incision and skin reflection, all subcutaneous tissues were examined. The condition of the thoracic viscera was noted with due attention to the thymus, lymph nodes and heart.
The abdominal viscera were examined externally and by palpation. The gastro-intestinal tract was examined as a whole and the stomach and caecum were incised and examined. The lungs were removed and all pleural surfaces examined under suitable illumination.
The liver and the kidneys were examined. Any abnormalities in the appearance and size of gonads, adrenals uterus, intra-abdominal lymph nodes and accessory reproductive organs were recorded.
The weights of the organs listed below of all adult animals were determined before fixation, where applicable. Thyroid weight was determined after fixation. Paired organs were weighed individually and identified as left or right.

Weight of organs determined:
- Adrenal gland (2)
- Brain
- Epididymis (2)
- Heart
- Kidney (2)
- Liver
- Ovary (2)
- Spleen
- Testicle (2)
- Thyroid (1) (including parathyroids)
- Thymus
- As a whole: prostate and seminal vesicles with coagulating glands
- Uterus including cervix

The organs or parts thereof listed below of all adult animals were preserved in the appropriate fixative.
Fixative: modified Davidson’s solution
- Epididymis (2)
- Testicle (2)

Fixative 7% neutral buffered formalin
- Gross lesions observed
- Ovary and oviduct (2)
- Prostate
- Seminal vesicles with coagulating glands
- Thyroid 1 (including parathyroids)
- Uterus including cervix
- Vagina

DISSECTION OF SELECTED ADULT ANIMALS
The organs listed below or parts thereof of the animal groups listed on the following page were preserved in a suitable fixative:
-All deceased or prematurely sacrificed animals
-5 male and 5 females randomly selected from each group

Fixative: Davidson's solution
- Eye with optic nerve (2)

Fixative: modified Davidson's solution
- Epididymis (2)
- Testicle (2)

Fixative: 7% neutral buffered formalin
- Adrenal gland (2)
- Bone
- Bone marrow (os femoris)
- Brain (cerebrum, cerebellum, pons)
- Gross lesions observed
- Heart (3 levels: right and left ventricle, septum)
- Intestine, small (duodenum, jejunum, ileum, including Peyer's patches, Swiss roll method)
-Intestine, large (colon, rectum)
- Kidney and ureter (2)
- Liver
- Lungs (with mainstem, bronchi and bronchioles), preserved by inflation with fixative and then immersion
- Lymph node (1, cervical)
- Lymph node (1, mesenteric)
- Mammary gland
- Muscle (skeletal)
- Nerve (sciatic)
- Oesophagus
- Ovary and oviduct (2)
- Pituitary
- Prostate
- Seminal vesicles with coagulating glands
- Spinal cord (3 sections)
- Spleen
- Stomach
- Thyroid (including parathyroids)
- Thymus
- Trachea (including larynx)
- Urinary bladder
- Uterus (including cervix)
- Vagina
Any other organs displaying macroscopic changes, possibly in particular mandibular lymph nodes were also preserved.

HISTOPATHOLOGY
Full histopathology was performed on the preserved organs of
-the selected parental animals of groups 1 and 4 and
-all deceased or prematurely sacrificed animals.
The organs listed above are examined histopathologically after preparation of paraffin sections and haematoxylin-eosin staining. Parathyroids could not always be identified macroscopically. They were examined microscopically if in the plane of section and in all cases where they were noted as grossly enlarged.
In addition, frozen sections of the heart, liver and on kidney were prepared, stained with Oil Red O, and examined.
Detailed histopathologic examinations with special emphasis on the qualitative stages of spermatogenesis and histopathology of interstitial testicular structure was performed on one testicle and one epididymis of the selected males of groups 1 and 4 following H E and PAS staining.
After observation of test item-related changes in group 4, the Sponsor was given sufficient notice and the organs listed below of the female animals of the low and intermediate dose groups (groups 2 and 3) were sectioned and examined additionally.

Organ (HE staining only) Number and sex of animals
Adrenal gland (2) 5 females per group
Ovary and oviduct (2) 5 females per group
Thymus 5 females per group
Uterus (including cervix) 5 females per group
Vagina 5 females per group

The blood smears prepared from all animals during the haematological examination are available for possible examination of pathological changes depending on necropsy findings and upon agreement with the Sponsor.

HISTOPATHOLOGICAL EXAMINATIONS
The histotechnique was performed by Provivo. All slides (labelled with study number, species, animal number, block number) prepared at Provivo were dispatched on 08 April 2021 (organs of groups 1 and 4) and on 01 June 2021 (organs of groups 2 and 3 females) to the Test Site 1 for histopathological evaluation. The transport of slides and tissues to the Test Site 1 was arranged by Provivo, whereas the return transport to the Test Facility was arranged by the Test Site 1.
Histopathological evaluation was performed by the Test Site 1 according to all relevant Test Site SOPs.
All observations upon final assessment were reported per animal and the findings considered relevant for the treatment in an incidence and occurrence table. All microscopic findings were recorded, reported and archived with the PathData system version 6.2e2.
The report of this phase of the study comprised a description of objective, materials and methods, results (including results of macroscopic and microscopic changes) and conclusions. The unaudited Draft Phase Report was presented electronically to the Study Director for comments/review. Comments was addressed and the Draft Final Phase Report of the histopathology phase of the study was presented to the TSQAU for auditing.
The audited Draft Final Phase Report of the histopathology of the study will be sent electronically to the Study Director who will provide documented approval on the study contents of the Phase Report by e-mail to the Principal Investigator. An original of the final, signed Phase Report will be sent (on paper and electronically) to the Study Director; a copy will be archived by the Test Site.
The Test Site Phase Draft Report can be found in Section 7 'Histopathology Report'.

Statistics:

A) The statistical evaluation of the parametrical values was done by Provantis (Provantis® integrated preclinical software, version 10.2.1.0, 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).
B) The following statistical methods were used for data not captured by Provantis software (reproductive data):
Homogeneity of variances was test using the BARTLETT's test. In case of homogeneity, intergroup comparison was performed by the DUNNETT's test (p ≤ 0.05 and p ≤ 0.01). In case of heterogeneity of variances, a stepwise comparison of the test groups with the control group was performed using a STUDENT's t-test (p ≤ 0.05 and p ≤ 0.01).
Statistically significantly different data are indicated in the summary tables and in the tables of the result sections.
The mean values and standard deviations were calculated to the highest possible degree of accuracy and then rounded to the reported number of decimals places. Hence, deviations to the last decimal place of up to ± 1 may occur caused by rounding.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

MALES
In the control group and in the intermediate dose group (300 mg test item/kg b.w./day), no changes in behaviour, external appearance or the faeces were noted.
In the low dose group (100 mg test item/kg b.w./day), the male animal no. 29 displayed moderate salivation on TD 21. Salivation was observed already before administration and disappeared 1 to 2 hours after administration.
The single and transient observation of salivation for one low dose animal on a single day was considered to be spontaneous.
In the high dose group (1000 mg test item/kg b.w./day), slight to moderate salivation was noted for 7 of 10 males for between 1 and 3 days per animal. For 2 animals, the saliva was red discoloured on one day each. Salivation started immediately to 5 minutes after administration or 5 to 20 minutes after administration and was observed not longer than 60 minutes after administration.
Due to only low numbers of occurrences (one to three times) of salivation per animal and due to the short duration, salivation was considered to be test item-related but not adverse.

FEMALES (Pre-mating period, gestation period, lactation period)
In the control group, females no changes in behaviour, external appearance or the faeces were noted.
In the low dose group (100 mg test item/kg b.w./day), the female animal no. 31 was noted with a slight discharge of red-discoloured saliva on TD 24. The single occurrence of one animal with salivation on day test day was considered to be spontaneous.
In the intermediate dose group (300 mg test item/kg b.w./day), the female animal no. 60 displayed slight salivation on TD 19 and animal no. 59 was noted with moderate salivation on GD 0. Both observations of salivation started immediately to 5 minutes after administration. For animal no. 60, salivation disappeared 20 to 60 minutes after administration. For female animal no. 59 salivation was no longer observed 1 to 2 hours after administration. However, the single occurrences of two animals with salivation on one day each were considered to be test item-related but not adverse.
Additionally in the intermediate dose group, animal no. 54 was noted with piloerection, a hemorrhagic vagina and was noted to be pale on the last day of its gestation period (GD 23) and on the first day of the lactation period (LD 1). Furthermore, also the female animal no. 60 was noted with a hemorrhagic vagina on day 9 of its pseudo-gestation period. However, as no observations of a hemorrhagic vagina were noted for the high dose group, the hemorrhagic vagina noted for two intermediate dose animals was considered to be not test item-related. The piloerection and the paleness of animal no. 54 were considered to be spontaneous.
In the high dose group (1000 mg test item/kg b.w./day), salivation was noted for 5 of 10 animals in the pre-mating/mating period and for 2 of 9 females during the gestation period for up to 2 days. Salivation started immediately to 5 minutes after administration and lasted until up to 60 minutes after administration. As salivation was noted for only up to 3 days per animal (female no. 78 displayed salivation for one day in the pre-mating/mating period and two days in the gestation period), salivation was considered to be test item-related but not adverse.

DETAILED CLINICAL OBSERVATIONS
Males and females
No test item-related observations were noted for the male animals of the dose groups (100, 300 or 1000 mg test item/kg b.w./day) and no changes were noted for the male and female animals of the control group and treated females.
Nevertheless, the male low dose animal no. 28 displayed eschar formation in the neck region in test week 4. In the intermediate dose group, male no. 49 revealed a wound at the right ear in test week 3 and eschar formation at the right ear in test week 4. These single occurrences of one male low and intermediate dose animal with a scratch wound and/or eschar formation were considered to be spontaneous.

Mortality:

no mortality observed

Description (incidence):

MALES
No prematurely deceased male animals were noted in the control group and in the dose groups.

FEMALES
No prematurely deceased female animal was noted for the control group and the low and intermediate dose groups.
The female animal no. 80 of the high dose group (1000 mg test item/kg b.w./day) was prematurely sacrificed on TD 19 after a misgavage. Necropsy revealed test item residuals as deposits in the right shoulder accompanied by edematous tissue at the right shoulder. No changes in behaviour, the external appearance or the faeces were noted in the days prior to the premature sacrifice.
This animal was excluded from the evaluation and replaced by animal no. 81.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

MALES
Body weight:
No test item-related differences for the body weight were noted between the control group and the dose groups (100, 300 or 1000 mg test item/kg b.w./day). The difference of the body weight between the control group and the dose groups ranged between 1.9% and +6.0%.
The animals were transferred to the study groups based on their body weight before start of treatment. Hence, the slightly higher body weight in the high dose group developed after transfer to the group and before start of treatment.
Body weight gain:
Accordingly, no test item-related changes in the body weight gain were noted between male animals of the control group and the males of the dose groups (100, 300 or 1000 mg test item/kg b.w./day)

FEMALES
Body weight:
No test item-related influence on body weight was noted for the dose groups (100, 300 or 1000 mg test item/kg b.w./day) during the pre-mating, the gestation and the lactation period.
During the pre-mating period and the first two weeks of the gestation period, no statistically significant differences were noted between the control group and the dose groups. On GD 20, the body weight of the high dose group was distinctly decreased (15.5% below the value of the control group, statistically significant at p < 0.01). However, on LD 1, the difference to the control group was only 1.5%. Further on, during the whole lactation period, no statistically significant differences were noted for the body weight between the control group and the dose groups. Therefore, the decreased body weight of the high dose group on GD 20 was considered to be the result of resorptions of all their implantations in 6 high dose females. Secondly, the resorption occurrence resulted in reduced pup litter weights due to only three litters that were born.

Body weight gain:
In accordance with the reduced body weight of the high dose group during the last week of gestation, also the body weight gain during the gestation period was decreased compared to the other groups. However, as mentioned above, the reduced body weight gain of the high dose group in the gestation period was considered to be due to the loss of all implantations for 6 females and the reduced weight of the three litters born and not due to a test item-related effect on the body weight of the dams.

BODY WEIGHT AT AUTOPSY
Males and females
No test item-related influence on the body weight at autopsy was noted for the male and female animals of any dose group (100, 300 or 1000 mg test item/kg b.w./day) in comparison to the control group.
The body weight at autopsy for the male dose groups ranged between +1.3% (intermediate dose) and +2.6% (low dose) in comparison to the control group and for the female animals of the dose groups, the body weight at autopsy ranged from -4.0% (high dose) to +5.7% (low dose) compared to the value of the control group.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Males and females
During the pre-mating period (TD 15 to TD 29), no statistically significant differences in food consumption were noted between the male rats of the control group and those of the dose groups (100, 300 or 1000 mg test item/kg b.w./day).
No test item-related differences in food consumption were noted between the control group and the treatment groups (100, 300 or 1000 mg test item/kg b.w./day) during the pre-mating, gestation and lactation period.

Food efficiency:

not examined

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

no effects observed

Description (incidence and severity):

No test item-related changes in the water consumption was were noted for the male and female rats of any dose group.

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

MALES
No test item-related differences between the control group and the dose groups (100, 300 or 1000 mg test item/kg b.w./day) for the haematological parameters of the male animals.
Statistically significantly decreased numbers of red blood cells were noted for the low ( 5.8%, p ≤ 0.05) and intermediate dose groups (6.8%, p ≤ 0.01 for the intermediate dose group). Additionally, a statistically significantly increased number of neutrophils was noted for the low dose group (+36%, p ≤ 0.05). However, as no dose-response relationship was present and the values were within the range of the Provivo background data, they were considered to be not test item-related.
In the high dose group, the mean values of the haematological ratio values haematocrit (HCT) and of the mean corpuscular volume (MCV) were slightly below the range of the Provivo background data. As both of these values are ratio values or rather the MCV is calculated from the HCT, it is no wonder that both values are slightly decreased. However, as no statistically significant difference were present, the values of the HCT and MCV below the background data range were considered to be not test item-related.

FEMALES
No test item-related differences for the haematological parameters were noted between the females of the control group and the low and intermediate dose groups (100 or 300 mg test item/kg b.w./day).
In the high dose group (1000 mg test item/kg b.w./day), a dose-dependent decrease was noted for the haemoglobin level (HGB), the number of red blood cells (RBC) and the haematocrit (HCT). For HGB and RBC, the mean values of all groups including the control group, and for HCT the mean values of the intermediate and high dose group were below the range of the Provivo background data. For the above mentioned parameters, the individual values for all of the three evaluated high dose females were below the Provivo background data range. Therefore, the dose-dependently decreased values for the haemoglobin levels, the number of red blood cells and for the haematocrit were considered to be test item-related. Yet, as only three animals could have been investigated and the decrease is only slightly no further conclusion should be drawn and this parameter should not be consulted for the NOAEL determination.
Additionally, mean values of the dose groups above the background data range were noted for the number of large unstained cells (LUC, groups 2 and 3), the activated partial thromboplastin time (aPTT, groups 2 and 4) and for the mean corpuscular volume (MCV, group 2). However, no dose-response relationship was noted for the mentioned parameters and therefore, the values above the background data range were considered to be spontaneous and not test item-related.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

MALES
No test item-related differences between the male animals of the control group and the animals of the dose groups (100, 300 or 1000 mg test item/kg b.w./day) were noted for the biochemical parameters.
Mean values slightly outside the Provivo background data range were noted for the levels of glucose, blood urea, sodium and LDH. However, in all cases, no dose-response relationship was present and also individual values of control males were slightly outside the background data range. Therefore, the mean values outside the background data range were considered to be not related to the test item.

FEMALES
No test item-related differences for the biochemical parameters were noted between the females of the control groups and the dose groups (100, 300 or 1000 mg test item/kg b.w./day).
Mean values of the dose groups slightly outside the Provivo background data were noted for the levels of total bilirubin, blood urea, potassium, alanine amino-transferase (ALAT) and alkaline phosphatase (aP). However, more individual values of control females than of the treated females were slightly outside the background data range. Secondly, no dose-response relationship was present. Therefore, the mean values outside the background data range for biochemical parameters of the female animals were considered to be spontaneous and not test item-related.

Endocrine findings:

no effects observed

Description (incidence and severity):

T4 LEVEL ANALYSIS
No test item-related differences for the serum T4 levels of the adult male animals were noted between the control group and the dose groups (100, 300 or 1000 mg test item/kg b.w./day).
The mean T4 levels of all four groups were below the range of the Provivo background data. However, as no statistical significance and no dose response-relationship were present, the T4 levels were considered to be not influenced by the test item.

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

Detailed clinical observations
Males and females
No test item-related observations were noted for the male animals of the dose groups (100, 300 or 1000 mg test item/kg b.w./day) and no changes were noted for the male and female animals of the control group and treated females.
Nevertheless, the male low dose animal no. 28 displayed eschar formation in the neck region in test week 4. In the intermediate dose group, male no. 49 revealed a wound at the right ear in test week 3 and eschar formation at the right ear in test week 4. These single occurrences of one male low and intermediate dose animal with a scratch wound and/or eschar formation were considered to be spontaneous.

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

Males and females
No test item-related changes were noted for the relative and absolute organ weights of the dose groups (100, 300 or 1000 mg test item/kg b.w./day) in comparison to the control group.
In the low dose group, a statistically significantly decreased relative weight was noted for the prostate and seminal vesicles. However, as no dose-response relationship was noted, the decreased relative prostate and seminal vesicle weight was considered to be not test item-related.
Furthermore, dose-dependently increased or decreased absolute organ weights were noted for the liver, thymus and left thyroid of the male animals and for the left ovary, thymus and left thyroid of the female animals. However, as no statistical significance was observed, the dose-dependently increased or decreased absolute organ weights were considered to be of no toxicological relevance.

Gross pathological findings:

no effects observed

Description (incidence and severity):

MALES
No test item-related pathological changes were noted for any dose group (100, 300 or 1000 mg test item/kg b.w./day).
In the control group, the male animal no. 3 was noted with an oesophagus being perforated in the thorax region, the thorax filled with yellow-white liquid and the lungs partly stuck to the diaphragm pointing to a possible misgavage.
In the low dose group (100 mg test item/kg b.w./day), the male animal no. 21 was noted with the left testis and epididymis to be reduced in size and with a soft consistency. However, these single occurrences were considered to be not test item-related but spontaneous.
No pathological changes were noted for the male animals of the control group and the intermediate and high dose group (300 or 1000 mg test item/kg b.w./day).

FEMALES
No test item-related changes were noted for the female animals of the dose groups (100, 300 or 1000 mg test item/kg b.w./day) during the macroscopic examination during necropsy.
In the control and the low dose group, a few dark-red foci were observed for the lungs of the female animal no. 14 and 36, respectively.
In the high dose group (1000 mg test item/kg b.w./day), two animals (nos. 72 and 79) were observed with a thickened uterus. Additionally, no. 74 was noted with a dark-red discoloured thymus. The prematurely sacrificed no. 80 with edematous tissue of the right shoulder. However, these single occurrences were considered to be spontaneous.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

MALES
No pathological lesions that were related to the test item were noted during the histopathological examination of the males animals of the high dose group (1000 mg test item/kg b.w./day).
There was no histological evidence of toxicity in the male reproductive organs including testes, epididymides, prostate gland, seminal vesicles and coagulating glands of the high dose males in this study.
Testes were checked on completeness of cell populations and stages, while taking into account the interstitial cell structure and presence/absence of any degenerative changes. As a result, no treatment-related effects on the testicular histomorphology were observed.
Focal and segmental seminiferous tubular degeneration was found unilaterally in the testis of one animal (no. 61) from group 4. This was within the range of normal background lesions that may be observed in this study type and animals of this strain and age, and therefore, was deemed to be incidental changes not related to treatment with the test item.
In males, there were no differences in the incidence and severity of histologic findings recorded in the adrenal glands and thymus, between groups 1 and 4.
All remainder of findings noted for the male reproductive organs and the other organs were within the range of normal background lesions that may be observed in this study type and animals of this strain and age. Therefore, the observed findings were deemed to be incidental changes and not related to treatment with the test item.

FEMALES
No pathological lesions in the reproductive organs that were related to the test item were noted during the histopathological examination in the dams that delivered live pups, regardless of their dose group (100, 300 or 1000 mg test item/kg b.w./day).
Histologically, there were differences in the histology of the female reproductive organs between groups 1 and 4, but the differences were limited to the high dose (HD) females with loss of visible implantations in spite of Salewski staining positive in the gross observations. These included implantation sites appearing unphysiological, resorption in the uterus, poorly developed corpora lutea in the ovary and poorly developed mammary glands in 2 of 5 (2/5) females of group 4. Poorly developed corpora lutea and mammary glands indicate the insufficient progesterone level necessary to maintain pregnancy. In these animals, the ovaries showed histology similar to proestrus and metoestrus stage, and the vagina appeared mucosal figures corresponding to diestrus/proestrus or metestrus/diestrus stages.
Whereas females gotten normally pregnant showed marked to extensive glandular proliferation in the mammary glands as a physiological alteration. Furthermore, the vaginal mucosa of the female high dose animals with a total loss of implantation sites was noted to be in the di- or proestrus or in the met- or dioestrus. Whereas the vaginal mucosa of the females which delivered live pups was in the lactational dioestrus. The ovaries of these animals showed histology similar to proestrus or metoestrus stage.
Meanwhile, the implantation sites of the uterus in the other three HD females (3 of 5 females examined, nos. 75, 77 and 78) without macroscopic abnormalities showed normal histologic appearance like the control females. These females had well-developed (enlarged) corpora lutea in the ovary. The vaginal mucosa was in lactational dioestrus and the mammary glands developed well. Thus, there were no qualitative differences in histology of ovaries, vagina and mammary glands between these three females and the control females, and the reproductive organs and mammary glands of the HD females without macroscopic abnormalities appeared histological figures corresponding normal pregnant females. There were no abnormalities in the female reproductive organs of animals examined in groups 2 and 3.
Thus, except for failure of maintenance of pregnancy, the microscopic findings recorded in 2/5 HD females were histomorphology corresponding to the pregnant status. There were no abnormalities in the histology of the reproductive organs from the remaining three females of group 4, as well as from 5 females of groups 2 and 3, examined in this study.
In order to investigate if there was a failure of maintenance of pregnancy that led to the high number resorptions, serum progesterone and LH level were measured. In the high dose group, lower serum levels of progesterone were noted for the females which did not delivedered live pups and showed poorly developed corpus lutea. (-55.5%, not statistically significant). However, the progesterone levels were measured at wrong time point e.g. at necropsy.
In addition, pathological changes were noted in the adrenal glands in form of hypertrophy for 4 of 5 examined high dose females (none in the control animals) in which animals with loss of grossly visible implantations were included. In addition, multifocal to diffuse fatty vacuoles were found in 3/5 HD females including two animals with loss of grossly visible implantations, and the incidence and group mean severity grade of this change slightly increased in females of group 4. In the examined female animals of groups 2 and 3, no hypertrophy and no fatty changes were observed in the adrenal glands.
In the thymus, group mean severity grade of atrophy slightly increased in females of group 4. Atrophy was also noted in the thymus of all examined group 2 and 3 females. However, the severity grade was comparable to that of the control females. These findings in adrenals and thymus indicate that the high dose females, even in females successful of pregnancy, were under a stressful condition, although no major differences in the terminal body weights were detected

Histopathological findings: neoplastic:

no effects observed

Key result

Dose descriptor:

NOAEL

Remarks:

general toxicity

Effect level:

300 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

histopathology: non-neoplastic

Key result

Dose descriptor:

NOAEL

Remarks:

general toxicity

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Remarks on result:

not determinable due to absence of adverse toxic effects

Critical effects observed:

yes

Lowest effective dose / conc.:

1 000 mg/kg bw/day (nominal)

System:

female reproductive system

Organ:

mammary gland

ovary

uterus

Treatment related:

not specified

Dose response relationship:

not specified

Relevant for humans:

not specified

Conclusions:

The aim of the study was to obtain information on possible effects of the test item on general toxicity, reproduction and/or development based on OECD guideline 422. The test item was administered orally to rats at dose levels of 100, 300 or 1000 mg/kg b.w./day.
The NOAEL for general toxicity is considered to be 300 mg/kg bw/d for the female animals because histological differences were noted of the high dose females which lost all implantations. These included implantation sites appearing unphysiological, resorption in the uterus, poorly developed corpora lutea in the ovary and poorly developed mammary glands in 2 of 5 (2/5) females of Group 4. Poorly developed corpora lutea and mammary glands indicate the insufficient progesterone level necessary to maintain pregnancy.
For the male animals the general NOAEL is considered to be above 1000 mg/kg bw/d due to the absence of any toxicological effects.

Executive summary:

The aim of the study was to obtain information on possible effects of the test item on general toxicity, reproduction and/or development based on OECD guideline 422. The test item was administered orally to rats at dose levels of 100, 300 or 1000 mg/kg b.w./day.

 

General toxicity

Parental male and female animals

No test item-related prematurely deceased animals were observed. One female animal in the high dose group was killed due to animal welfare reasons and replaced.

No changes in behaviour, external appearance or the faeces considered to be adverse were noted for any dose group.

No test item-related changes were noted for the body weight, the body weight gain and for the food consumption.

Decreases in the number of red blood cells, the level of haemoglobin and the haematocrit were noted for the three investigated female high dose animals (1000 mg test item/kg b.w./day).

No test item-related differences were noted for the examination of the biochemical parameters and the T4 levels.

No test item-related significant differences were noted for the body weight at autopsy and the absolute and relative organ weights and no pathological changes considered to be test item-related were observed during necropsy.

No pathological lesions that were related to the test item were noted during the histopathological examination of the male animals of the high dose group (1000 mg test item/kg b.w./day).

No pathological lesions in the reproductive organs were related to the test item were noted during the histopathological examination in the dams that delivered live pups, regardless of the dose group (100, 300 or 1000 mg test item/kg b.w./day).

Histological differences were noted of the high dose females which lost all implantations. These included implantation sites appearing unphysiological, resorption in the uterus, poorly developed corpora lutea in the ovary and poorly developed mammary glands in 2 of 5 (2/5) females of Group 4. Poorly developed corpora lutea and mammary glands indicate the insufficient progesterone level necessary to maintain pregnancy.  There were no abnormalities in the histology of the reproductive organs from the remaining three females of Group 4, as well as from 5 females/group of Groups 2 and 3, examined in this study. The implantation loss was considered to be induced by stress of the high dosed female animals.

Histopathological examination revealed pathological changes of the adrenal glands of the female high dose animals (1000 mg test item/kg b.w./day) in form of diffuse adrenocortical hypertrophy or multifocal to diffuse fatty vacuoles. Additionally, an increased group mean severity grade for thymus atrophy was observed for the female high dose animals. These findings in adrenals and thymus may indicate that the high dose females, even in females successful of pregnancy, were under a stressful condition, although no major differences in the terminal body weights were detected.

 

General toxicity - Parental animals
Mortality (test item-related)	Males and females No test item-related premature deaths were noted for the male and female animals of any dose group (100, 300 or 1000 mg test item/kg b.w./day).
Clinical signs	Males and females Non-adverse salivation was noted for 2 females of the intermediate dose group (300 mg test item/ kg b.w./day) and for 7 males and 7 females of the high dose group (1000 mg test item/kg b.w./day). Additionally, in the intermediate dose group, animal no. 54 was noted with piloerection, a hemorrhagic vagina and the animal was noted to be pale on the last day of its gestation period (GD 23) and on the first day of the lactation period (LD 1). Furthermore, also the female animal no. 60 was noted with a hemorrhagic vagina on one day of its pseudo-gestation period.
Observational Screening (TD 48/49 for the males, TD 63 (LD 8-12) for the females)	Males and females No test item-related influence was noted.
Grip strength (TD 48/49 for the males, TD 63 (LD 8-12) for the females)	Males and females No test item-related influence was noted.
Spontaneous motility (TD 48/49 for the males, TD 63 (LD 8-12) for the females)	Males and females No test item-related influence was noted.
Body weight and body weight gain	Males and females No test item-related influence on the body weight and body weight gain was noted.
Food consumption		Males and females No test-item related differences were noted.
Drinking water consumption		Males and females No test-item related changes were noted by visual appraisal.
Haematology		Males No test item-related differences were noted. Nevertheless, statistically significantly decreased numbers of red blood cells were noted for the low (‑5.8%; p ≤ 0.05) and intermediate dose groups (‑6.8%; p ≤ 0.01). In addition, a statistically significantly increased number of neutrophils was noted for the low dose.   Females In the high dose group (1000 mg test item/kg b.w./day), consisting of three female animals, decreases were noted for the levels of red blood cells and haemoglobin and for the haematocrit (6.5%, 6.1% and 7.9% below the value of the control group, not statistically significant)
Clinical biochemistry	Males and females No test item-related differences were noted.
T4 level analysis	Males and females No test item-related differences were noted. However, the mean T4 levels of all four groups were below the range of the Provivo background data. Yet, there was not statistically significant change or dose dependency noticed.
Examination at termination
Body weight at autopsy		Males and females No influence on body weight at autopsy was noted.
Macroscopic post mortem examination		Males and females No test item-related pathological changes were noted.
Oestrous stage at necropsy		No test item-related differences were noted.
Organ weights		Males and females No test item-related differences were noted.
Histopathological examinations		Males No test item-related pathological changes were noted.   Females No pathological lesions in the reproductive organs that were related to the test item were noted during the histopathological examination in the dams that delivered live pups. The two investigated high dose females with a total loss of implantations showed some changes such as implantation sites appearing unphysiological, resorption in the uterus, poorly developed corpora lutea in the ovary and poorly developed mammary glands in 2 of 5 (2/5) females of Group 4. Poorly developed corpora lutea and mammary glands indicate an insufficient progesterone level, which is necessary to maintain pregnancy.  There were no abnormalities in the histology of the reproductive organs from the remaining three females of Group 4, as well as from 5 females/group of Groups 2 and 3, examined in this study. Histopathological changes in the adrenal glands were noted for the female high dose animals (1000 mg test item/kg b.w./day) in form of diffuse adrenocortical hypertrophy or multifocal to diffuse fatty vacuoles. In addition, the group mean severity grade for thymus atrophy was increased in the high dose females. These findings in adrenals and thymus may indicate that the high dose females, even in females successful of pregnancy, were under a stressful condition, although no major differences in the terminal body weights were detected.

 

Reference 2

Endpoint:

short-term repeated dose toxicity: oral

Remarks:

Dose-Range-Finding Study for OECD 422 in rats

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

23.06.2020 to 07.07.2020

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Remarks:

Short term study with reduced scope of examination

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.

Version / remarks:

2016

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:
24 animals (12 males and 12 females)
Oral administration by gavage
2 mL/kg b.w./day , vehicle: water
single housing in MAKROLON cages
temperature 22°C ± 3°C and the relative humidity 55% ± 10%

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

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

GLP compliance:

no

Limit test:

no

Species:

rat

Strain:

other: CD/ Crl:CD(SD)

Details on species / strain selection:

Selected because of its proven suitability in toxicology and reproduction studies and its compliance with regulatory requirements for testing in a rodent animal species.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH Sandhofer Weg 7, 97633 Sulzfeld, Germany
- Strain: Rat / CD / Crl:CD(SD)
- Sex: male and female
- Age: 62 days
- Body weight (at 1st administration): Males: 340,6 g - 369,9 g, Females: 216,3 g - 249,0 g
- Housing: singly in MAKROLON cages (type III plus) with a basal surface of approx. 39 cm × 23 cm and a height of approx. 18 cm, Granulated textured wood (Granulat A2, J. Brandenburg, 49424 Goldenstedt, Germany) was used as bedding material for the cages. The cages were changed and cleaned once a week
- Diet (ad libitum): ssniff® R/Z V1324, ssniff Spezialdiäten GmbH, 59494 Soest, Germany
- Water (ad libitum): tap water
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS:
- Temperature: 22°C ± 3°C
- Humidity: 55% ± 10%
- Photoperiod: 12 hours dark/12 hours light

Route of administration:

oral: gavage

Details on route of administration:

Selection of route of administration according to international guideline 422.

Vehicle:

water

Details on oral exposure:

Frequency of administration: Once daily for 14 days
Administration volume: 2 mL/kg b.w./day
Vehicle: Tap water
Dose levels : Group 1: Control (vehicle), Group 2: 65 mg/kg b.w./day, Group 3: 200 mg/kg b.w./day, Group 4: 600/1000 mg/kg b.w./day
The test item formulations were 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:

no

Details on analytical verification of doses or concentrations:

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

Duration of treatment / exposure:

14 days

Frequency of treatment:

once daily

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

control

Dose / conc.:

65 mg/kg bw/day (nominal)

Remarks:

low dose level

Dose / conc.:

200 mg/kg bw/day (nominal)

Remarks:

intermediate dose level

Dose / conc.:

600 mg/kg bw/day (nominal)

Remarks:

high dose level until test day 8

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

high dose level after test day 8

No. of animals per sex per dose:

3

Control animals:

yes, concurrent vehicle

Details on study design:

The rat was selected because of its proven suitability in toxicology and reproduction studies and its compliance with regulatory requirements for testing in a rodent animal species.

The dose levels were selected by the Monitor based on available data. The initial high dose level of 600 mg/kg b.w./day was selected due to an oral LD50 value of 918 mg/kg b.w./day. The initial high dose level of 600 mg/kg b.w./day was increased to 1000 mg/kg b.w./day as of test day 8 onwards as none of the animals of the high dose group revealed any signs of toxicity during the first test week employing 600 mg/kg b.w./day.

Positive control:

no

Observations and examinations performed and frequency:

CLINICAL SIGNS
Each animal was observed before and after dosing at each time of dosing for any signs of behavioural 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. by cageside observations. On Saturdays and Sundays, the animals were checked regularly from 7.00 a.m. to 11.00 noon with a final check performed at approximately 3.30 p.m. Cageside 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 disappearance of clinical signs of each animal 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 would have 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 body weight of each rat was recorded at the time of group allocation and daily thereafter during the treatment period for dose adjustment. Weekly values are stated in the report.

FOOD AND DRINKING WATER CONSUMPTION
The quantity of food left by individual animals was recorded on a weekly basis throughout the experimental period. The food intake per animal (g/animal/week) was calculated using the total amount of food given to and left by each animal in each group on completion of a treatment week.
The relative food consumption (in g/kg b.w./day) was determined using the following formula: (total food given [g] - total food left [g]) / number of animal days x body weight [kg])

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

Sacrifice and pathology:

On test day 15 (approximately 24 hours after the last administration) all 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. The cranial roof was 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 conditions of the thoracic viscera were noted with due attention to the thymus, lymph nodes, and heart.
The abdominal viscera were examined before and after removal. The urinary bladder was examined externally and by palpation. The gastro-intestinal tract was examined as a whole, and the stomach and caecum were incised and examined. The lungs were removed and all pleural surfaces were examined under suitable illumination. The liver and the kidneys were examined. Any abnormalities in the appearance and size of the gonads, adrenal glands, uterus, intra-abdominal lymph nodes, and accessory reproductive organs were recorded.
The weights of the following organs of all animals were determined before fixation:
Adrenal gland (2)
Brain
Epididymis (2)
Heart
Kidney (2)
Liver
Ovary (2)
Spleen
Prostate and seminal vesicle with coagulating glands (as a whole)
Testicle (2)
Thymus
Uterus including cervix
Paired organs were weighed individually and identified as left or right. Special attention was paid to the organs of the reproductive system.
The following organs or parts of organs of all animals were fixed in 7% buffered formalin except for the testes and epididymides, which were preserved in modified Davidson's solution:
Epididymis (2)
Organs showing macroscopic lesions (if any)
Ovary (2)
Prostate and seminal vesicle with coagulating glands (as a whole)
Testicle (2)
Uterus including cervix
Vagina

Statistics:

Toxicology and Pathology data were captured, as far as possible, using the departmental computerised systems (Provantis® Integrated preclinical software, Instem LSS Ltd., version 10.2.1). Raw data not fully compatible with the computerized systems were maintained on paper according to appropriate SOPs.
The following comparison was carried out:
The test item groups 2 to 4 were compared to the control group 1 using the following statistical method:
Test: Multiple t-test based on DUNNETT, C. W.
New tables for multiple comparisons with a control. Biometrics, 482-491 (September 1964)
Parameter: Body weight / Food consumption / Relative and absolute organ weights (p ≤ 0.05 and p ≤ 0.01)
The following settings were used for the statistical evaluation of the parametrical values captured by Provantis: 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 was made by the DUNNETT's test (p ≤ 0.01 and p ≤ 0.05). These statistical procedures were used for all data. Significantly different data are indicated in the table section of this report.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

No test item-related changes in behaviour, the external appearance were noted during the daily cage side observations of the male and female animals that were treated with 65, 200 or 600/1000 mg test item/kg b.w./day.
However, slight salivation was observed for the high dosed male no. 21 on test day 11 in the morning at work start before dosing, lasting up to 60 min after dosing. Further, a decreased water consumption was noted for 1 of 3 females of the low dose group, and for 2 of 3 females each of the intermediate and the high dose group on test day 6. Both observations (salivation and decreased water consumption) were regarded to be incidental and of no toxicological relevance. Since these changes were noted only on a single test day during the 14-day treatment period. The faeces of all animals showed a normal consistency during the course of the study.

Mortality:

no mortality observed

Description (incidence):

No premature deaths were noted for the male and female animals treated with 65, 200 or 600/1000 mg test item/kg b.w. daily for 14 days.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

No test item-related difference in body weight and body weight gain were noted between the control group and the treatment groups (65, 200 or 600/1000 mg test item/kg b.w./day). No test item-related difference in the body weight at autopsy.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

No test item-related difference in food consumption was noted between the control group and the treatment groups (65, 200 or 600/1000 mg test item/kg b.w./day).

Food efficiency:

not examined

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

effects observed, non-treatment-related

Description (incidence and severity):

Male: The visual appraisal of the drinking water consumption did not reveal any test item related influence on the water intake of the male animals at any tested dose level.
Female: A decreased water consumption was noted for 1 of 3 females of the low dose group and for 2 of 3 females each of the intermediate and the high dose group on one test day. The visual appraisal of the drinking water consumption on the other test days did not reveal any test item-related influence on the water intake of the animals at any tested dose level.

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

Slight salivation was observed for the high dosed male no. 21 on test day 11 in the morning at work start before dosing, lasting up to 60 min after dosing. Further, a decreased water consumption was noted for 1 of 3 females of the low dose group, and for 2 of 3 females each of the intermediate and the high dose group on test day 6.

Immunological findings:

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

No test item-related difference for the organ weights was noted between the control group and the treatment groups (65, 200 or 600/1000 mg test item/kg b.w./day). Decreased absolute and relative organ weights were noted for the adrenal glands and the thymus of the male animals (statistically not significant), whereas increased absolute and relative weights of the adrenal glands and the thymus were noted for the female animals (statistically not significant). Furthermore, an increased organ weight was noted for the absolute and the relative spleen weight of the male animals (statistically significant for the relative spleen weight), whereas no changes were noted for the absolute and relative spleen weight of the female animals. Due to the opposite effect in male and female animals the observed changes are most likely spontaneous and due to the small number of examined animals per group.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Treatment with 200 or 600/1000 mg test item/kg b.w./day led to an enlarged mandibular lymph node size in 3 of 3 male and female animals each of the intermediate dose group, and in 2 of 3 male and 3 of 3 female animals of the high dose group.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Dose descriptor:

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

Sex:

male/female

Critical effects observed:

yes

Lowest effective dose / conc.:

200 mg/kg bw/day (nominal)

Organ:

lymph node

Treatment related:

yes

Dose response relationship:

not specified

Relevant for humans:

not specified

Text table 3‑1:      Group size / Dose levels

Group	Propan-1-ol, 2-amino-2-methyl-, 4-methyl- benzenesulphonate dose [mg/kg b.w./day, p.o.]	No. and sex of animals	Animal number
1	0 (control)	3 m 3 f	1 - 3 4 - 6
2	65 (low dose)	3 m 3 f	7 - 9 10 - 12
3	200 (intermediate dose)	3 m 3 f	13 - 15 16 - 18
4	600 / 1000^# (high dose)	3 m 3 f	19 - 21 22 - 24

m:   male

f:     female

#:    The initial high dose level of 600 mg/kg b.w./day was increased to 1000 mg/kg b.w./day as of test day 8 onwards as none of the animals of the high dose group revealed any signs of toxicity during the first test week employing 600 mg/kg b.w./day.

 

Text table 3‑2:      Weighed organs

Weighed organs
Adrenal gland (2) Brain Epididymis (2) Heart Kidney (2) Liver	Ovary (2) Spleen Prostate and seminal vesicle   with coagulating glands   (as a whole) Testicle (2)	Thymus Uterus including cervix

 

Text table 3‑3:      Preserved organs

Preserved organs
Epididymis (2) Organs showing macroscopic lesions (if any) Ovary (2) Prostate and seminal vesicle   with coagulating glands (as a whole)	Testicle (2) Uterus including cervix Vagina

 

Text table 3‑4:      Statistics

Test	Parameter
Multiple t-test based on DUNNETT, C. W. New tables for multiple comparisons with a control. Biometrics, 482-491 (September 1964)	Body weight / Food consumption / Relative and absolute organ weights (p ≤ 0.05 and p ≤ 0.01)

 

Text table 4‑1:      Body weight gain of male animals during the treatment period

Males		Group 1 Control	Group 2 65 mg/kg	Group 3 200 mg/kg	Group 4 600/ 1000 mg/kg
Body weight gain # (test day 1 to test day 14)		+13.7 %	+14.9 %	+16.9 %	+14.4 %
#:	Values taken from table 3-1 'Body Weight Gain - Summary - Males'

 

Text table 4‑2:      Body weight gain of female animals during the treatment period

Females		Group 1 Control	Group 2 65 mg/kg	Group 3 200 mg/kg	Group 4 600/ 1000 mg/kg
Body weight gain # (test day 1 to test day 14)		+7.1 %	+11.3 %	+10.7 %	+8.6 %
#:	Values taken from table 3-2 'Body Weight Gain - Summary - Females'

 

Text table 4‑3:      Macroscopic post mortem findings

Macroscopic organ changes (number of animals affected / examined)
Organ / Finding	Group 1 Control		Group 3 200 mg/kg		Group 4 600/1000 mg/kg
	m	f	m	f	m	f
Mandibular lymph nodes   enlarged	0 / 3	0 / 3	3 / 3	3 / 3	2 / 3	3 / 3

 

Text table 4‑4:      Organs with noticeable changes in comparison to control; statistically significant from control or not (not considered to be test item-related)

			Changes in comparison to the control group [%]
			Group 2	Group 3	Group 4
Males
Adrenal gland (left)		(g) #1	- 4.3	- 8.6	- 19.0
		(g/kg) #2	- 1.6	- 4.7	- 16.0
Adrenal gland (right)		(g) #1	- 16.9	- 11.9	- 28.8
		(g/kg) #2	- 14.5	- 7.8	-26.1
Thymus		(g) #1	-15.3	-16.0	-22.2
		(g/kg) #2	- 13.3	- 12.7	- 19.1
Spleen		(g) #1	+13.6	+11.4	+4.5
		(g/kg) #2	+16.5**	+15.9*	+8.3
Females
Adrenal gland (left)		(g) #1	+19.3	+27.2	+12.3
		(g/kg) #2	+16.0	+27.5	+10.3
Testis left
Adrenal gland (right)		(g) #1	+20.6	+23.4	+8.4
		(g/kg) #2	+17.6	+24.3	+6.3
Thymus		(g) #1	+65.0	+28.0	+51.0
		(g/kg) #2	+66.6	+33.4	+53.1
Spleen		(g) #1	+0.6	- 8.4	- 2.4
		(g/kg) #2	- 0.8	- 7.8	- 3.3
*/**:	Statistically significant from control at p ≤ 0.05 / 0.01 (Anova / Dunnett test).
#1:	Values taken from table 8-1 'Absolute Organ Weights - Sum. - Males' and table 8-2 'Absolute Organ Weights - Sum. - Females'
#2:	Values taken from table 7-1 'Relative Organ Weights - Sum. - Males' and table 7-2 'Relative Organ Weights - Sum. - Females'

 

Conclusions:

The aim of the study was to select the dose levels of Propan-1-ol, 2 amino-2-methyl-, 4-methyl-benzenesulphonate for a reproduction/developmental toxicity study (OECD 422). The rats were treated with 65, 200 or 600 mg Propan-1-ol, 2 amino-2-methyl-, 4-methyl-benzenesulphonate/kg b.w. daily from test day 1 to test day 14. On test day 8 the high dose level was increased to 1000 mg Propan-1-ol, 2 amino-2-methyl-, 4-methyl-benzenesulphonate/kg b.w. daily, as no signs of toxicity were noted.

Executive summary:

The aim of the study was to select the dose levels of Propan-1-ol, 2 amino-2-methyl-, 4-methyl-benzenesulphonate for a reproduction/developmental toxicity study (OECD 422).

The rats were treated with 65, 200 or 600 mg Propan-1-ol, 2 amino-2-methyl-, 4-methyl-benzenesulphonate/kg b.w. daily from test day 1 to test day 14. On test day 8 the high dose level was increased to 1000 mg Propan-1-ol, 2 amino-2-methyl-, 4-methyl-benzenesulphonate/kg b.w. daily, as no signs of toxicity were noted.

None of the animals died prematurely.

No test item-related observations were noted in behaviour, the external appearance or the appearance of the faeces.

Body weight and food consumption of the male and female animals revealed no test item-related differences.

During the macroscopic inspection at necropsy mandibular lymph nodes with an increased size were noted for nearly all male and female animals of the intermediate and the high dose group (200 or 600/1000 mg Propan-1-ol, 2 amino-2-methyl-, 4-methyl-benzenesulphonate/kg b.w./day).

After consideration of these data, the following dose levels of Propan-1-ol, 2 amino‑2-methyl-, 4-methyl-benzenesulphonate are suggested for a reproduction/ developmental toxicity study in CD® rats based on OECD guideline 422:

Group 1:    Control (vehicle)

Group 2:    100 mg Propan-1-ol, 2 amino-2-methyl-, 4-methyl-benzenesulphonate/ kg b.w./day

Group 3:    300 mg Propan-1-ol, 2 amino-2-methyl-, 4-methyl-benzenesulphonate/ kg b.w./day

Group 4:    1000 mg Propan-1-ol, 2 amino-2-methyl-, 4-methyl-benzenesulphonate/ kg b.w./day

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Study duration:

subchronic

Species:

rat

Quality of whole database:

Klimisch 1

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on the results of the performed studies OECD 422 DRF study (Provivo, 2020) and the main OEC D422 study (Provivo 2022), the test material has not be classied according to the criteria of EC Regulation 1272/2008.