Registration Dossier

Administrative data

Description of key information

A study was performed to evaluate the toxicity of trans-N1,N1,N4,N4-Tetrakis(2-hydroxyethyl)1,4-cyclohexanedicarboxamide after a repeated administration per inhalation to rats, according to OECD-Guideline 412/413 as far as useful for a dose range finder study.The No-observed-effect-level (NOEL) of trans-N1,N1,N4,N4-Tetrakis(2-hydroxyethyl)1,4-cyclohexanedicarboxamide dust was larger than 1.03 mg/L for rats after repeated exposure via inhalation for two weeks that represents the highest technically feasible concentration in the study.
A 90 day oral repeated dose toxicity study was performed in rats according to OECD 408. Daily oral (gavage) administration of the test item to Wistar rats for 90 consecutive days at 100, 300 and 1000 mg/kg bw/day was associated with minor effects at the highest dose level only. In conclusion, the NOAEL of the test item administered by oral gavage to Wistar rats for 90 consecutive days is considered to be 300 mg/kg bw/day based on a precautionary worst case approach.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2014-05-13 to 2014-09-09
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study; GLP study
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Version / remarks:
1998
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ORGANISMS: 
- Source: Charles River Deutschland, Sulzfeld
- Strain: CD / Crl: WI rats
- Age: young adult, approximately 8 weeks old at onset of treatment
- Body weight: males: 268 - 300 g, females: 196 - 239 g
- Housing: group-housed 2/3 animals/sex/cage (Type III polycarbonate)
- Diet: ad libitum, ssniff R/M "Autoclavable complete diet for rats and mice – breeding and maintenance" produced by ssniff Spezialdiäten GmbH,
D-59494 Soest, Germany; ad libitum,
- Water: ad libitum
- Acclimatisation period: 5/6 days
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.3 - 24.7° C
- Humidity (%): 32% - 65%
- Illumination: 12 hours artifical fluorescent light and 12 hours dark
- Air change: 15 - 20 air exchanges/hour
Route of administration:
oral: gavage
Vehicle:
water
Remarks:
distilled
Details on oral exposure:
ADMINISTRATION: 
- Frequency: once daily for 90 consecutive days
- Dose volume: 10 ml/kg b.w.
- Dose: 0, 100, 300 or 1000 mg/kg/bw , recovery group control, recovery group high dose
- Animals: 10 male and 10 female rats/dose, in conlusion: 60 male and 60 female rats
- DOSAGE PREPARATION:
- test item was formulated in the vehicle at the appropriate concentrations of 10, 30 and 100 mg/mL, according to the dose level and volume
selected, in the Central Dispensary of CiToxLAB Hungary Ltd.
- First day of dosing of each animal was regarded as Day 0. The individual volume of the treatment was based on the most recent individual body
weight of the animals.
- Formulations were prepared and used according to stability assessment results within 7 days while stored at room temperature.
- Stability of the test item in the vehicle was assessed in the conditions employed on the study according to CiToxLAB study code 13/372-316AN.
According to the results, the test item was stable in distilled water in concentration range of 1-150 mg/mL for 7 days at room temperature
(RT, 15-30ºC).
- Analysis of test item formulations for concentration and homogeneity was performed using a validated HPLC-UV method, in the Analytical
Laboratory of CiToxLAB Hungary Ltd. Duplicate top, middle and bottom samples were taken from test item formulations on three occasions (during
the first, sixth and the last weeks of treatment). One set was analyzed and one set was retained as a back-up, if required for any confirmatory
analyses. Similarly, one sample was taken in duplicate from the Group 1 (control) solution for concentration measurements.
- 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:
See Study Code: 13/372-316AN, details see below
All formulations were shown to be homogeneous. All formulations were found to be in the range of 99 to 106% of nominal concentrations. Based on
these results, test item formulations were considered suitable for the study purposes. The analytical results are summarized in the table below.
Duration of treatment / exposure:
90 consecutive days
Frequency of treatment:
daily
Remarks:
Doses / Concentrations:
0, 100, 300 and 1000 mg/kg bw/day
Basis:
other: Dose volume: 10 ml/kg b.w. oral per gavage
No. of animals per sex per dose:
10 per sex per dose,
10 per sex recovery control
10 per sex high dose recovery
Control animals:
yes, concurrent vehicle
Details on study design:
The rat is regarded as suitable species for toxicology studies. Wistar rat as a rodent is one of the standard strains for repeat-dose toxicity studies.
Dose levels were selected by the Sponsor in consultation with the Study Director, based on previous data available, including the results of a
preliminary dose range finding study in the rat (14 days).
Positive control:
not required
Observations and examinations performed and frequency:
IN-LIFE PROCEDURES
Mortality and clinical observations
- Mortality: twice daily (at the beginning and end of each working day)
- General clinical observations were made at least once a day at approximately the same time
- Detailed clinical observations were made on all animals outside the home cage at randomisation (Day -1), on Day 6 and at least once a week
afterwards.
- Observations were performed on the skin, fur, eyes, eyeballs and mucous membranes, autonomic activity (lachrymation, piloerection, pupil size,
respiratory pattern, occurrence of secretions and excretions), circulatory and central nervous system (tremor, convulsion, muscular contractions, etc.), somatomotor activity and behaviour pattern (changes in exploratory behaviour, ordinary behaviour including changes in grooming,
headshaking, gyration, etc., abnormal behaviour such as autophagia/self-mutilation, backward motion, abnormal vocalization, etc., aggression,
etc.), motor coordination, ambulatory abnormalities, changes in body position and posture (ex. hunchback posture, etc), gait, or response to
handling and to environmental stimulation. Particular attention was directed to observations for tremors, convulsions, salivation, diarrhoea,
lethargy, sleep and coma. No such clinical signs were noted during the study.
Neurological assessment (Functional Observation Battery)
- Towards the end of the treatment period, during week 13, each animal was subjected to the functional observation battery, including qualitative
assessment of the grip strength, and to measurements of the landing foot splay and motor activity assessment.
Examination of vaginal smears
- Prior to necropsy, the oestrus cycle of all females was determined by taking vaginal smears, which were prepared and stained with 1% aqueous
methylene blue solution
Ophthalmology evaluation
- Ophthalmoscopy was conducted in all main and recovers animals before treatment (Day -4), and in the Control Group 1 and High dose Group 4
animals, during Week 13 (Day 86).
Body weight measurement
- Body weight was recorded with a precision of 1 g at randomisation, on Day 0, prior to start of treatment, then at least weekly, including on Day 89
(last treatment day) and Day 117 (last day of recovery), and prior to necropsy (fasted, on Days 90 or 118).
Food consumption measurement
- Determination of food consumption was performed for the main and recovery groups once a week. The remaining, non-consumed food was
weighed at least weekly from Day 7 with a precision of 1 g. Weekly food consumption was calculated
CLINICAL PATHOLOGY
- At the end of the treatment or recovery periods, prior to scheduled necropsy on Day 90 or 118, clinical pathology investigations (haematology,
coagulation, clinical biochemistry and urinalysis) were conducted in all animals in main and recovery groups.
- After an overnight period of food deprivation of animals, 3 blood samples were collected by heart puncture under pentobarbital anaesthesia, for
haematology (in tubes with K3-EDTA as anticoagulant, 1.6 mg/mL blood), for blood clotting times (in tubes with sodium citrate as anticoagulant)
and one to obtain serum (in tubes with no anticoagulant) for clinical chemistry.
- Haematology:
RBC Red Blood Cell (erythrocyte) count, (1012/L) M/µL
WBC White Blood Cell (leukocyte) count, (109/L) K/µL
Hgb Haemoglobin concentration, (g/dL)
Hct Haematocrit (relative volume of erythrocytes) (%)
MCV Mean Corpuscular (erythrocyte) Volume (fL)
MCH Mean Corpuscular (erythrocyte) Haemoglobin, (pg)
MCHC Mean Corpuscular (erythrocyte) Haemoglobin Concentration, (g/dL)
RDW Red Cell (erythrocyte) volume (%)
Plt Platelet (thrombocyte) count (109/L) K/µL
MPV Mean Platelet Thrombocyte volume (fL)
RETIC % Reticulocyte count (%)
NE % Neutrophil (%)
LY % Lymphocyte (%)
MO % Monocyte (%)
BA % Basophil (%)
EO % Eosinophil (%)
LUC % Large Unstained Cells (%)
- Clinical chemistry:
Glucose Blood sugar concentration (mmol/L)
T-BIL Total Bilirubin concentration (μmol/L)
Urea Urea concentration (mmol/L)
Chol. Cholesterol concentration (mmol/L)
Creat. Creatinine concentration (μmol/L)
Phos. Phosphorus concentration (mmol/L)
Na+ Sodium concentration (mmol/L)
K+ Potassium concentration (mmol/L)
Ca++Calcium concentration (mmol/L)
Cl- Chloride concentration (mmol/L)
Tot. Prot. Total Protein concentration (g/L)
Alb. Albumin concentration (g/L)
A/G Alb/glob ration
AST/GOT Aspartate Aminotransferase activity (U/L)
ALT/GPT Alanine Aminotransferase activity (U/L)
ALKP Alkaline. Phosphatase – activity (U/L)
GGT Gamma Glutamyltransferase -activity (U/L)
Bile acids (µmol/L)
- Urinalysis
Urine collection was conducted over approximately 16 hours, during an overnight period of food deprivation of animals, which were placed in
metabolic cages. The evaluation of the urine samples was performed by observation (e.g. colour, appearance) or test strips as applicable. The
following parameters were evaluated:
LEU / Leukocyte
NIT / Nitrite
pH
PRO / Protein
GLU / Glucose
UBG / Urobilinogen
BIL / Bilirubin
KET / Ketones
BLD/ERY / Blood/Erythrocytes
SG / Specific Gravity
SED / Sediment
VOL / Volume
















Sacrifice and pathology:
Phathology
- Terminal procedures
Necropsy and macroscopic examination were performed on all animals, at the end of treatment or recovery periods, on Days 90 or 118 (after the
sample collection for clinical pathology evaluation). The animals were euthanized by exsanguination under pentobarbital anaesthesia.
- Macroscopic evaluation
After exsanguinations, the external appearance of each rat was examined, cranium, thoracic and abdominal cavities were opened and the
appearance of the tissues and organs were observed macroscopically. Any abnormality was recorded with details of the location, colour, shape
and size, as applicable.
In addition, bone marrow smears from the femur of each animal were prepared at necropsy. The smears were fixed, then stained but not analysed.
The smears will be stored/archived at CiToxLAB Hungary Ltd.
- Organ weight measurement
The following organs were trimmed of fat and weighed in all animals (Main and Recovery Groups):
Brain, Epidiymides, Heart, Kidneys, Liver, Prostate, Seminal vesicles with coagulating glands, Spleen, Testes, Thymus, Uterus including cervix,
Adrenal glands, Ovaries, Thyroids with parathyroids glands, Pituitary
- Histopathology
On completion of the macroscopic examination the following tissues and organs were retained from all animals.
Gross findings:
Lungs with bronchi Skeletal muscle (quadriceps)
Adrenals Lymph node Small intestine
Animal identification Ovary Spinal cord
Aorta Oviduct Spleen
Brain Pancreas Sternum with marrow
Epididymis Pituitary Stomach
Eye with the optic nerve Prostate Testis
Oesophagus Salivary gland (including Thymus
Femur incl. cartilage with mandibular, sublingual and Thyroid with parathyroid gland
marrow parotid glands) Tongue
Heart
Kidney Sciatic nerve Trachea
Large intestine Seminal vesicle with Urinary bladder
Extraorbital lachrymal gland coagulating gland Uterus
Harderian gland Skin, subcutis with mammary Vagina
Liver gland (inguinal)

The eyes with the optic nerve and the testes with epididymides were preserved in modified Davidson’s fixative; all other organs in 10% buffered
formalin solution.
The retained tissues and organs were embedded in paraffin wax, sections were cut at 4-6µ by microtome and transferred to slides. Tissue
sections were stained with haematoxylin-eosin/phloxine and examined by light microscope.
Full histopathology was performed in Groups 1 (Control) and 4 (High dose). In addition, organs or tissues with macroscopic finding in animals
from other groups were subjected to histological examination.
Statistics:
The statistical evaluation of the numerical data were performed with the program package SPSS PC+4.0 (SPSS Hungary Kft, Budapest).
Evaluation was made by comparing the data for each of the Groups 2 to 4, respectively, against the Control Group 1.
The heterogeneity of variance between groups was checked by Bartlett's homogeneity of variance test. Where no significant heterogeneity was
detected, a one-way analysis of variance was carried out.
If the obtained result was positive, Duncan's Multiple Range test was used to assess the significance of inter-group differences.
Where significant heterogeneity was found, the normal distribution of data was examined by Kolmogorov-Smirnov test.
In case of abnormal distribution, the non-parametric method of Kruskal-Wallis One-Way analysis of variance was applied.
If there was a positive result, the inter-group comparisons were performed using Mann-Whitney U-test.
The mean and standard deviations values, the frequency of clinical observations, macroscopic and microscopic findings were calculated as applicableRecovery groups were compared using Student T-test.
Data were collected using the software PROVANTIS v.7, (Instem LSS Ltd. UK) or were recorded on the appropriate forms from the relevant SOPs of
CiToxLAB Hungary Ltd., then tabulated using the software PROVANTIS v.7, Microsoft Office Word and/or Excel, as appropriate.
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
see details on result
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
see details on result
Urinalysis findings:
no effects observed
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
see details on result
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
not specified
Details on results:
RESULTS AND DISCUSSIONS
DOSE FORMULATION ANALYSIS
Test item concentration and homogeneity of the dosing formulations was determined three times during the treatment period (during the first, sixth and the last weeks of the treatment). No test item was detected in the Control solution samples. All formulations were shown to be homogeneous. All formulations were found to be in the range of 99 to 106% of nominal concentrations. Based on these results, test item formulations were considered suitable for the study purposes.
Table see below: Other information on results
MORTALITY
No animal mortality occurred during this study.
CLINICAL OBSERVATIONS
All animals were clinically normal and there were no toxicologically significant or test-item related findings during the study.
BODY WEIGHT MEASUREMENTS
There were no effects on bodyweight at any dose levels.
Minor, sporadic statistical differences to control were recorded in body weight or body weight gain, which were not considered treatment related and were without any toxicological significance.
FOOD CONSUMPTION MEASUREMENTS
The food consumption of the animals was not affected by the test item administration.
No toxicologically significant variations were recorded during the treatment period in any of the groups. Slightly higher mean values were recorded infemales at Mid dose group.
NEUROLOGICAL ASSESSMENT (FUNCTIONAL OBSERVATION BATTERY)
There were no treatment related effects.
There were no toxicologically significant changes in the animal behaviour, general physical condition, in the reactions to different type of stimuli in
the control or treated groups.
There was no effect of treatment noted during the assessment of landing foot splay, grip strength or motor activity.
EXAMINATION OF VAGINAL SMEARS
There were no test item related changes in the animal oestrus cycle evaluated prior to necropsy and the animals showed the normal distribution of
the oestrus phases.
OPHTHALMOLOGY
No test item related changes compared to pre-treatment were noted at ophthalmoscopy examination.
CLINICAL PATHOLOGY
Haematology
Variations were noted in a several parameters in the Main male animals, on occasion attaining statistical significance, including statistically higher
Mean Corpuscular (erythrocyte) Haemoglobin (MCH) in the Low and Mid dose (p<0.05), lower than control Red Blood Cell (erythrocyte) count (RBC) inthe Low and Mid dose (p<0.01 and p<0.05, respectively) and Monocyte percent (MONO %) in the Low dose (p<0.05). In the Recovery male animals
statistically higher Monocyte count (MONO, Abs.) and Large Unstained Cells count (LUC, Abs.) were noted (p<0.05). Although they were statistically
significant, were not dose-related, showed no consistent gender response and/or were within the normal historical control ranges.
In Main female animals statistically lower than control Red Blood Cell (erythrocyte) count (RBC) and Haemoglobin concentration (HGB) were measured (p<0.01) at High dose level and the Haematocrit value (Hct) at Low and High dose animals also was lower than the control value (p<0.05 and p<0.01, respectively). In the Recovery females, the RBC and HGB concentrations were still lower than the control (p<0.05), although both parameters
measured in the recovery animals, were within the historical control range. These changes were considered to be related to test item administration,
although they were very minor; it is noted that Reticulocytes were not affected.
Table see below: Other information on results
Coagulation parameters
There was no effect of treatment on coagulation parameters investigated, i.e. Activated Partial Thromboplastin Time (APTT) and Prothrombin
Time (PT). In recovery females, the Prothrombin Time (PT) was statistically higher than the control value (p<0.05), although all individual parameters were within the historical control range.
In summary there were no toxicologically significant effects in coagulation parameters at any dose levels
Clinical chemistry
Changes considered related to test item administration were noted in the clinical chemistry parameters examined under the conditions of this study.
On Day 90, Albumin (Alb.) concentration was slightly higher than control in the High dose males and females (7% increase in males and 19% in
females). The differences attained statistical significance and exceeding the historical control ranges in the 1000 mg/kg bw/day female group.
Consequently total protein concentration (Tot.Prot) and the Albumin to Globulin ratio (A/G) were also higher with attaining statistical significance in
both high dose male and female groups. These changes were fully reversed by the end of the recovery period.
Table see below: Other information on results
Variations were noted in a few other clinical chemistry parameters on occasion attaining statistical significance:
- in the Main male animals: statistically higher Urea concentration in the High dose (p<0.05) and Chloride concentration (Cl-) in the Mid dose
(p<0.05), lower than control Urea concentration in the Mid dose (p<0.05) and Bile acid in the Low and Mid dose (p<0.05). In the Recovery male
animals statistically higher Cholesterol (Chol) and Calcium concentration (Ca++) (p<0.05) and statistically lower Creatinine concentration (Creat)
were noted (p<0.01). All individual values were within the historical control range.
- in the Recovery females, statistically lower than control Total Bilirubin (p<0.05), Creatinine (p<0.01) and Phosphorus (p<0.01) concentrations were noted.
Although these variations were statistically significant, evaluation of the mean and individual results in correlation with the control and historical
haematology data did not reveal any test-item related cause of the changes noted, and/or no consistent dose or gender-related response was
observed. Therefore, these differences observed between the control and treated groups were considered to be incidental or individual findings,
which were not related to treatment and generally remained within the historical control ranges, or were with no toxicological significance.
Urinalysis
No effects were noted, which could be related to the test item.
All investigated parameters were comparable with the control. Results of sediment analysis revealed no significant differences between control and
treated animals.
PATHOLOGY EVALUATION AND ORGAN WEIGHTS
Macroscopic evaluation on Day 90
No test item-related macroscopic changes were recorded at dose levels of 100, 300 and 1000 mg/kg bw/day.
Dilation of oesophagus, brown discoloration of the perinasal/perioral fur, pale foci of the lungs, red discoloured mandibular lymph node, small
testes, enlarged left testis, red multifocal discoloration of the thymus, uterine dilatation were observed without meaningful incidence and were
considered to be incidental or a common background.
Macroscopic evaluation on Day 118
Single cyst of the right kidney, pale foci of the lungs, black focal discoloration of the glandular stomach mucosa, uterine dilatation seen at necropsy
were regarded as incidental or background.
Organ weights
At the high dose level, 1000 mg/kg bw/day, test item related differences were found in liver and kidney weights.
Evaluation of liver weight values revealed test item related differences at 1000 mg/kg bw/day (High dose) in females. The absolute liver weight was
higher by approximately 14%. Following the recovery period, the values were still slightly higher in females (approximately 7% for body weight relative values and 12-13% for absolute and brain relative values, respectively). But only the brain relative liver weight value was statistically higher in the
females after recovery.
On day 90 in males only the body weight relative liver value is statistically enhanced in high dose group compared to control. There is no statistically significant difference regarding the absolute liver weight and the brain relative liver value in high dose males compared to control on day 90. After
recovery, the liver values were comparable with the control in males.
These changes are consistent correlations with the clinical chemistry parameters (increased albumin, total protein and Albumin to Globulin ratio
concentration on day 90).
There were no toxicologically significant effects at the low or mid-dose levels.
Compared to control, slightly higher kidneys weights were recorded for both males and females in the high dose groups on Day 90. The differences
were approximately 7-23% (absolute value) and 6-21% (relative values), and were statistically significant for the body weight relative value (p<0.05) in males and for the absolute and relative values for females (p<0.01). Following the recovery period, the kidney values were still slightly higher in
females by 20% (absolute value) and up to 21% (relative values) and attained statistical significance (p<0.01). In males the values were comparable
with the controls.
There were no any other significant differences among groups in the weights of other organs measured when compared to controls.
Histopathology
There was no evidence of the test item-related microscopic findings.
Single cyst of the left adrenal (1/10 Control female), minimal uterine dilatation (2/10 High dose females), moderate reduced sperm content in the
epididymides (1/10 Control male), minimal focal mixed cellular infiltrate in the heart (1/10 Control and 1/10 High dose males), mild pyelitis with
lymphoid follicle formation of the kidney (1/10 High dose female), minimal to mild renal tubular basophilia (2/10 Control males and 2/10 High dose males), minimal to mild proteinaceous casts of the renal cortex (2/10 Control males and 1/10 High dose male, 1/10 High dose female), mild diffuse
congestion of the left kidney (1/10 High dose female) and minimal mixed mononuclear peripelvic infiltrate (1/10 High dose female), were observed
by light microscopy and considered to be incidental or background.

Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Critical effects observed:
not specified

 DOSE FORMULATION ANALYSIS

Analytical occasion

Nominal concentration (mg/mL)

Measured concentrations with the 95% confidence intervals (mg/mL)

Measured concentration in percentage of the nominal

13 May 2014

(First week)

10

9.96 ± 0.34

100

30

30.1 ± 1.16

100

100

105 ± 5.25

105

16 June 2014

(sixth week)

10

10.4 ± 0.10

104

30

31.4 ± 0.67

105

100

106 ± 3.49

106

05 August 2014

(Lastweek)

10

10.2 ± 0.23

102

30

30.8 ± 0.51

103

100

99.2 ± 3.32

99

CLINICAL PATHOLOGY,  

 Haematology

Females, Day 90

Dose (mg/kg bw/day)

 

Control

100

300

1000

 

Red Blood Cell (erythrocyte) count (RBC), M/mL

8.024

7.958

7.964

7.477**

DN

differences %

(-0.8)

(-0.7)

(-6.8)

 

Haemoglobin concentration (HGB), g/dL

15.00

14.65

14.98

14.07**

DN

differences %

(-2.3)

(-0.1)

(-6.2)

 

Haematocrit value (Hct),

%

43.59

42.18*

43.28

40.51**

DN

differences %

(-3.2)

(-0.7)

(-7.1)

 

 Clinical chemistry

day 90

Dose Group (mg/kg bw/day)

 

Control

100

300

1000

 

Males

 

Albumin concentration, g/L

28.17

27.60

27.26

30.11**

DN

(differences%)

(-2.0)

(-3.2)

(6.9)

 

Total Protein concentration, g/L

53.57

51.88*

52.32

55.49*

DN

(differences%)

(-3.2)

(-2.3)

(3.6)

 

Albumin to Globulin ratio

1.10

1.14

1.08

1.19*

DN

(differences%)

(3.6)

(-1.8)

(8.2)

 

Females

 

Albumin concentration, g/L

32.30

31.69

34.33

38.45**

U

(differences%)

(-1.9)

(6.3)

(19.0)

 

Total Protein concentration, g/L

56.93

55.73

59.13

63.12**

U

(differences%)

(-2.1)

(3.9)

(10.9)

 

Albumin to Globulin ratio

1.32

1.32

1.39

1.56**

U

(differences%)

(0.0)

(5.3)

(18.2)

 

Conclusions:
In conclusion, the NOAEL of the test item administered by oral gavage to Wistar rats for 90 consecutive days is considered to be
300 mg/kg bw/day based on a precautionary worst case approach.
Executive summary:

The purpose of this study was to obtain information on the toxicity of the test item when administered daily for 90 days by oral gavage to Wistar rats of both sexes at dose levels of 100, 300 and 1000 mg/kg bw/day. The doses were selected by the Sponsor in consultation with the Study Director, based on previous data available, with the aim of inducing toxic effects but no death or suffering at the highest dose and a NOAEL at the lowest dose.

Male and female Wistar rats were treated according to the following experimental design: 

Gr. No.

Group Designation
Dose Level
(mg/kg bw/day)

Conc. (mg/mL)

Dose volume

(mL/kg bw)

Animal Numbers

Male

Female

Main

Recovery

Main

Recovery

1

Control

0

0

10

1001-1010

1011-1020

1501-1510

1511-1520

2

Low Dose

100

10

2001-2010

-

2501-2510

-

3

Mid Dose

300

30

3001-3010

-

3501-3510

-

4

High Dose

1000

100

4001-4010

4011-4020

4501-4510

4511-4520

The control group was treated concurrently with the vehicle only (distilled water).

 

Main animals were euthanized after 90 days of daily treatment, the first day of treatment was Day 0. Rats assigned to Recovery groups (10 male and 10 female in Control and High dose groups), were kept for a 28-day observation period following the completion of the 90-day treatment period, and then euthanized.

 

Analysis of test item formulations for concentration and homogeneity was performed three times during the treatment period (during the first, sixth and last week of treatment) using validated HPLC-UV method.

 

Parameters monitored during the study included mortality/morbidity, clinical signs, body weights and body weight gain, food consumption. Blood for haematology and clinical chemistry measurements was collected on Day 90 for the main animals and on Day 118 for the recovery animals. Urinalysis was also performed at the end of the treatment or recovery period. Animals were subjected to neurological assessments during the week 13. Examinations included functional observation battery (FOB), landing foot splay and grip strength measurements, and automated locomotor activity measurements (SMART). Ophthalmology was performed pre-treatment and at the end of the treatment period. Necropsy and macroscopic examination was performed on all animals at the end of treatment period on Day 90 and the end of the recovery period on Day 118, selected organs were weighed and preserved. Tissues from control and high dose groups were processed to slides and examined histopathologically.

Results

All formulations were found to be in the range of 99 to 106% of nominal concentration and were homogeneous. No test item was detected in the control samples. Based on these results, test item formulations were considered suitable for the study purposes.

There was no test item related mortality observed during the study.

There were no clinical signs or unscheduled mortality during the study.

 

There were no signs of toxicity observed on body weights, body weight gains or food consumption.

There were no toxicologically significant changes in the animal behaviour, general physical condition, in the reactions to different type of stimuli, grip strength or motor activity in the control or treated groups, at the evaluation performed on last week of the treatment.

 

There were no test item related changes in the animal oestrus cycle evaluated prior to necropsy and the animals showed the normal distribution of the oestrus phases.

No test item related changes were noted at ophthalmoscopy examination.

 

Evaluation of the haematological parameters revealed the following effects at 1000 mg/kg bw/day on Day 90: statistically lower than control Red Blood Cell (erythrocyte) count (RBC), Haemoglobin concentration (HGB) and Haematocrit value (Hct) in females. In the Recovery females, the RBC and HGB concentrations were still lower than the control (p<0.05), although both parameters measured in the recovery animals, were within the historical control range.

There were no toxicologically significant effects in coagulation parameters at any dose levels.

Evaluation of the clinical chemistry analysis revealed the following effects at 1000 mg/kg on Day 90:Albumin concentration was slightly higher in both sexes (7% increase in males and 19% in females). The differences attained statistical significance and exceeding the historical control ranges in females. Consequently, Total protein concentration and the Albumin to Globulin ratio were also higher attaining statistical significance in both high dose male and female groups. These changes were fully reversed by the end of the recovery periode.

No effects were noted at urinalysis which could be related to the test item.

There were no macroscopic findings considered related to test item administration under the conditions of this study.

Evaluation of liver weight values revealed test item related differences at 1000 mg/kg bw/day (High dose) in females. The absolute liver weight was higher by approximately 14%. Following the recovery period, the values were still slightly higher in females (approximately 7% for body weight relative values and 12-13% for absolute and brain relative values, respectively). But only the brain relative liver weight value was statistically higher in the females after recovery. On day 90 in males only the body weight relative liver value is statistically enhanced in high dose group compared to control. There is no statistically significant difference regarding the absolute liver weight and the brain relative liver value in high dose males compared to control on
day 90. After recovery, the liver values were comparable with the control in males. These changes are consistent correlations with the clinical chemistry parameters (increased albumin and total protein concentration).

Compared to control, slightly higher absolute kidneys weights, body relative and brain relative kidney weight values were recorded for females in the High dose groups on Day 90. Following the recovery period, the kidney values were still slightly higher in females. In males, the values were comparable with the controls except for a higher body relative kidney weight value on Day 90. Following the recovery period, the kidney values were comparable with the respective controls in males.

No clear test item related microscopic changes were seen at histopathology.

Conclusion

A daily oral (gavage) administration of the test item to Wistar rats for 90 consecutive days at 100, 300 and 1000 mg/kg bw/day was associated with minor effects at the highest dose level only. High dose group findings included slight anaemia with lower RBC and HGB concentrations (females only), increased Albumin and consequently increased Total Protein concentrations, and increased A/G ratio in both sexes with association to statistically significant increased liver weight values (including the brain and body weight relative values) in females and statistically significant increased body weight relative values in males. Statistically significant increased kidney weight values (including the brain and body weight relative values) in females and statistically significant increased body weight relative values in males were observed after 90 days exposure without relevant histopathological changes. Although signs of reversibility were observed in recovery animals, the changes in female liver (brain weight relative value only), kidneys and haematology parameters persisted to some extent. No effects of toxicological relevance were observed at the low or mid dose levels.

In conclusion, the NOAEL of the test item administered by oral gavage to Wistar rats for 90 consecutive days is considered to be

300 mg/kg bw/day based on a precautionary worst case approach.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
300 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
GLP study; Klimisch 1

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:
2011-06-01 to 2011-06-21
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study; GLP study without deviations
Qualifier:
according to
Guideline:
OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)
Version / remarks:
2009
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
Fischer 344
Sex:
male/female
Details on test animals and environmental conditions:
- Source: Charles River Laboratories, Sulzfeld (Germany)
- Strain: rats, F344, SPF
- Age: approx. 7 weeks when supplied
- Animals: 20 males, 20 femals
- Acclimatisation: at least 5 days
- Weight at study initiation: male: 198.8g - 201.0g, female: 140.0g - 142.0g
- Diet: ad libitum, ssniff R/M-H V 1534-300
- Water: tap water ad libitum
- Temperature (°C): 20.8 - 22.3 °C, mean: 21.4 °C
- Humidity: about 37 - 77 %, mean 61.7 &
- Illumination: 12 hours artifical fluorescent light and 12 hours dark
- Air exchange: 12 per hour
Route of administration:
inhalation: dust
Type of inhalation exposure:
nose/head only
Vehicle:
other: conditioned air
Remarks on MMAD:
MMAD / GSD: The mass median aerodynamic diameter of the dust particles was 3.13, 3.20 and 3.55 µm for the low, mid and high concentration. This is slightly higher than the recommended range of 1 to 3 µm. But although the test substance was ground before producing the dust and a separator for larger particles was used, a smaller aerodynamic diameter was not obtainable.
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: see attachment, Head Nose Only Exposure Unit from TSE-Systems GmbH, Bad Homburg, Germany
- Method of holding animals in test chamber:
- Source and rate of air:
- Method of conditioning air:
- System of generating particulates/aerosols: test substance is milled in a ball mill and pressed to a dust cake. In the dust generator a scrapper
removes parts of this cake. The dust is removed from the scrapper and dispersed by the flow inside the outlet nozzle into the inhalation unit.
The mass median aerodynamic diameter (MMAD) of the dust in the breathing zone shall be in the range of 1 to 3 µm with a geometric standard
deviation of 1.5 to 3.0. A slightly increased MMAD will be accepted as otherwise the desired dust concentration cannot be produced.
- Temperature, humidity in air chamber: 18.9 - 20.9 °C, humidity: lower than 5%,
- Air flow rate:
- Air change rate:
- Method of particle size determination: cascade impactor (Berner-Impaktor Type LPI4/0,06/2 from Hauke KG, Gmunden, Austria).
- Treatment of exhaust air:

TEST ATMOSPHERE
- Analyses of dust particleize: three times during study
- Graimetric analyses of dust concentration: three times per day


Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The dust concentration in the breathing zone was determined gravimetrically. An accurately measured volume of air from the inhalation devices was sucked through a pre-weighed filter with cotton wool. The filters were dried before and after the sampling by pressing dry air through them. From the weight difference and the volume the actual dust concentration was calculated.
Duration of treatment / exposure:
5 days per week for 2 weeks
Frequency of treatment:
5 days/week x 6 hours/day
Remarks:
Doses / Concentrations:
0.12, 0.36, 1.03 mg/l
Basis:
analytical conc.
No. of animals per sex per dose:
5
20 males and 20 females
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Based on the technical possibilities of the dust generation and the absence of toxic actions at one preliminary exposure.
In preliminary experiments the highest dust concentration which could be produced with acceptable particle size distribution was 1.1 mg/L. One
animal was exposed to this dust concentration for 6 hours and showed no noteworthy signs of toxicity. Therefore this concentration was the target
for the high concentration group. The low concentration was 1/10 of the high one and the mid concentration was the geometric mean.
Positive control:
not nesessary
Observations and examinations performed and frequency:
Animal observations: All animals, before and after the exposure, on days without exposure once a day.
Body weights: All animals, twice per week.
Feed consumption: All animals, for weekly periods.
Sacrifice and pathology:
Necropsy with gross pathological examination: All animals on Day 15.
Organ weight determination: All animals, fresh weights of lungs, kidneys, livers and spleens, at necropsy.
Histopathological examination: As no significant effects of the test substance exposure was seen, no histopathological examinations were performed.
Other examinations:
no other examinations
Statistics:
- Analysis of variance followed by the Scheffé-test: all data with means and standard deviations determined, comparison of more than two groups
t-test
- t-test: all data with means and standard deviations determined, for comparison of two groups only
- H-test of Kruskal and Wallis followed by the test of Nemenyi: counted events with scoring or in cases where the requirements for the analysis of
variance were not fulfilled
- Chi2-test: counted events
- Fisher's exact test: counted events, if the Chi2-Test was not applicable
Clinical signs:
no effects observed
Description (incidence and severity):
Chromodacryorrhea occurred in animals of all groups during the inhalation exposure. It was probably caused by the restraining in the inhalation tubes.
Mortality:
no mortality observed
Description (incidence):
Chromodacryorrhea occurred in animals of all groups during the inhalation exposure. It was probably caused by the restraining in the inhalation tubes.
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Details on results:
- Mortality: There was no mortality prior to scheduled sacrifice.
- Observations in life: Chromodacryorrhea occurred in animals of all groups during the inhalation exposure. It was probably caused by the
restraining in the inhalation tubes.
- Body weights and feed consumption: The body weights, weight gains, and feed consumptions were similar in all groups. There was no significant
difference between the control and any dosed group.
- Necropsy with gross pathological examination: There were no spontaneous findings made at the gross examination during the necropsy in any of the animals.
- Organ weights: There were no significant differences in the organ weights or the organ weight / body weight ratios between the control group and
any of the dosed groups.
Dose descriptor:
NOEL
Effect level:
> 1.03 mg/L air
Sex:
male/female
Critical effects observed:
not specified

no remarks

Conclusions:
The No-observed-effect-level (NOEL) of trans-N1,N1,N4,N4-Tetrakis(2-hydroxyethyl)1,4-cyclohexanedicarboxamide dust was therefore larger than 1.03 mg/L.
Executive summary:

This study was performed to evaluate the toxicity of trans-N1,N1,N4,N4-Tetrakis(2-hydroxyethyl)1,4-cyclohexanedicarboxamide after a repeated administration per inhalation to rats, according to OECD-Guideline 412/413, 7 September 2009, as far as useful for a dose range finder study.

The used dust concentration of the high dose group, 1.03 mg/L, was the highest technically feasible dust concentration. Nevertheless no toxic effect was observed in this study after a two week exposure to this dust. The body weight, the feed consumption, the behaviour, and selected organ weights were not changed in the exposed groups.

The No-observed-effect-level (NOEL) of trans-N1,N1,N4,N4-Tetrakis(2-hydroxyethyl)1,4-cyclohexanedicarboxamide dust was therefore larger than 1.03 mg/L for rats after repeated exposure via inhalation for two weeks.

.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEC
1 030 mg/m³
Study duration:
subacute
Species:
rat
Quality of whole database:
GLP study, Klimisch1

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:
2011-06-01 to 2011-06-21
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study; GLP study without deviations
Qualifier:
according to
Guideline:
OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)
Version / remarks:
2009
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
Fischer 344
Sex:
male/female
Details on test animals and environmental conditions:
- Source: Charles River Laboratories, Sulzfeld (Germany)
- Strain: rats, F344, SPF
- Age: approx. 7 weeks when supplied
- Animals: 20 males, 20 femals
- Acclimatisation: at least 5 days
- Weight at study initiation: male: 198.8g - 201.0g, female: 140.0g - 142.0g
- Diet: ad libitum, ssniff R/M-H V 1534-300
- Water: tap water ad libitum
- Temperature (°C): 20.8 - 22.3 °C, mean: 21.4 °C
- Humidity: about 37 - 77 %, mean 61.7 &
- Illumination: 12 hours artifical fluorescent light and 12 hours dark
- Air exchange: 12 per hour
Route of administration:
inhalation: dust
Type of inhalation exposure:
nose/head only
Vehicle:
other: conditioned air
Remarks on MMAD:
MMAD / GSD: The mass median aerodynamic diameter of the dust particles was 3.13, 3.20 and 3.55 µm for the low, mid and high concentration. This is slightly higher than the recommended range of 1 to 3 µm. But although the test substance was ground before producing the dust and a separator for larger particles was used, a smaller aerodynamic diameter was not obtainable.
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: see attachment, Head Nose Only Exposure Unit from TSE-Systems GmbH, Bad Homburg, Germany
- Method of holding animals in test chamber:
- Source and rate of air:
- Method of conditioning air:
- System of generating particulates/aerosols: test substance is milled in a ball mill and pressed to a dust cake. In the dust generator a scrapper
removes parts of this cake. The dust is removed from the scrapper and dispersed by the flow inside the outlet nozzle into the inhalation unit.
The mass median aerodynamic diameter (MMAD) of the dust in the breathing zone shall be in the range of 1 to 3 µm with a geometric standard
deviation of 1.5 to 3.0. A slightly increased MMAD will be accepted as otherwise the desired dust concentration cannot be produced.
- Temperature, humidity in air chamber: 18.9 - 20.9 °C, humidity: lower than 5%,
- Air flow rate:
- Air change rate:
- Method of particle size determination: cascade impactor (Berner-Impaktor Type LPI4/0,06/2 from Hauke KG, Gmunden, Austria).
- Treatment of exhaust air:

TEST ATMOSPHERE
- Analyses of dust particleize: three times during study
- Graimetric analyses of dust concentration: three times per day


Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The dust concentration in the breathing zone was determined gravimetrically. An accurately measured volume of air from the inhalation devices was sucked through a pre-weighed filter with cotton wool. The filters were dried before and after the sampling by pressing dry air through them. From the weight difference and the volume the actual dust concentration was calculated.
Duration of treatment / exposure:
5 days per week for 2 weeks
Frequency of treatment:
5 days/week x 6 hours/day
Remarks:
Doses / Concentrations:
0.12, 0.36, 1.03 mg/l
Basis:
analytical conc.
No. of animals per sex per dose:
5
20 males and 20 females
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Based on the technical possibilities of the dust generation and the absence of toxic actions at one preliminary exposure.
In preliminary experiments the highest dust concentration which could be produced with acceptable particle size distribution was 1.1 mg/L. One
animal was exposed to this dust concentration for 6 hours and showed no noteworthy signs of toxicity. Therefore this concentration was the target
for the high concentration group. The low concentration was 1/10 of the high one and the mid concentration was the geometric mean.
Positive control:
not nesessary
Observations and examinations performed and frequency:
Animal observations: All animals, before and after the exposure, on days without exposure once a day.
Body weights: All animals, twice per week.
Feed consumption: All animals, for weekly periods.
Sacrifice and pathology:
Necropsy with gross pathological examination: All animals on Day 15.
Organ weight determination: All animals, fresh weights of lungs, kidneys, livers and spleens, at necropsy.
Histopathological examination: As no significant effects of the test substance exposure was seen, no histopathological examinations were performed.
Other examinations:
no other examinations
Statistics:
- Analysis of variance followed by the Scheffé-test: all data with means and standard deviations determined, comparison of more than two groups
t-test
- t-test: all data with means and standard deviations determined, for comparison of two groups only
- H-test of Kruskal and Wallis followed by the test of Nemenyi: counted events with scoring or in cases where the requirements for the analysis of
variance were not fulfilled
- Chi2-test: counted events
- Fisher's exact test: counted events, if the Chi2-Test was not applicable
Clinical signs:
no effects observed
Description (incidence and severity):
Chromodacryorrhea occurred in animals of all groups during the inhalation exposure. It was probably caused by the restraining in the inhalation tubes.
Mortality:
no mortality observed
Description (incidence):
Chromodacryorrhea occurred in animals of all groups during the inhalation exposure. It was probably caused by the restraining in the inhalation tubes.
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Details on results:
- Mortality: There was no mortality prior to scheduled sacrifice.
- Observations in life: Chromodacryorrhea occurred in animals of all groups during the inhalation exposure. It was probably caused by the
restraining in the inhalation tubes.
- Body weights and feed consumption: The body weights, weight gains, and feed consumptions were similar in all groups. There was no significant
difference between the control and any dosed group.
- Necropsy with gross pathological examination: There were no spontaneous findings made at the gross examination during the necropsy in any of the animals.
- Organ weights: There were no significant differences in the organ weights or the organ weight / body weight ratios between the control group and
any of the dosed groups.
Dose descriptor:
NOEL
Effect level:
> 1.03 mg/L air
Sex:
male/female
Critical effects observed:
not specified

no remarks

Conclusions:
The No-observed-effect-level (NOEL) of trans-N1,N1,N4,N4-Tetrakis(2-hydroxyethyl)1,4-cyclohexanedicarboxamide dust was therefore larger than 1.03 mg/L.
Executive summary:

This study was performed to evaluate the toxicity of trans-N1,N1,N4,N4-Tetrakis(2-hydroxyethyl)1,4-cyclohexanedicarboxamide after a repeated administration per inhalation to rats, according to OECD-Guideline 412/413, 7 September 2009, as far as useful for a dose range finder study.

The used dust concentration of the high dose group, 1.03 mg/L, was the highest technically feasible dust concentration. Nevertheless no toxic effect was observed in this study after a two week exposure to this dust. The body weight, the feed consumption, the behaviour, and selected organ weights were not changed in the exposed groups.

The No-observed-effect-level (NOEL) of trans-N1,N1,N4,N4-Tetrakis(2-hydroxyethyl)1,4-cyclohexanedicarboxamide dust was therefore larger than 1.03 mg/L for rats after repeated exposure via inhalation for two weeks.

.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEC
1 030 mg/m³
Study duration:
subacute
Species:
rat
Quality of whole database:
GLP; Klimisch 1

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

Additional information

A study was performed to evaluate the toxicity of trans-N1,N1,N4,N4-Tetrakis(2-hydroxyethyl)1,4-cyclohexanedicarboxamide after a repeated administration per inhalation to rats, according to OECD-Guideline 412/413 as far as useful for a dose range finder study.

The used dust concentration of the high dose group, 1.03 mg/L, was the highest technically feasible dust concentration. Nevertheless no toxic effect was observed in this study after a two week exposure to this dust. The body weight, the feed consumption, the behaviour, and selected organ weights were not changed in the exposed groups.

The No-observed-effect-level (NOEL) of trans-N1,N1,N4,N4-Tetrakis(2-hydroxyethyl)1,4-cyclohexanedicarboxamide dust was therefore larger than 1.03 mg/L for rats after repeated exposure via inhalation for two weeks.

A 90 day oral repeated dose toxicity study was performed in rats according to OECD 408. Daily oral (gavage) administration of the test item to Wistar rats for 90 consecutive days at 100, 300 and 1000 mg/kg bw/day was associated with minor effects at the highest dose level only. High dose group findings included slight anaemia with lower RBC and HGB concentrations (females only), increased Albumin and consequently increased Total Protein concentrations, and increased A/G ratio in both sexes with association to statistically significant increased liver weight values (including the brain and body weight relative values) in females and statistically significant increased body weight relative values in males. Statistically significant increased kidney weight values (including the brain and body weight relative values) in females and statistically significant increased body weight relative values in males were observed after 90 days exposure without relevant histopathological changes. Although signs of reversibility were observed in recovery animals, the changes in female liver (brain weight relative value only), kidneys and haematology parameters persisted to some extent. No effects of toxicological relevance were observed at the low or mid dose levels.

In conclusion, the NOAEL of the test item administered by oral gavage to Wistar rats for 90 consecutive days is considered to be 300 mg/kg bw/day based on a precautionary worst case approach.


Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
Relevant key study for this endpoint

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

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

Repeated dose toxicity: via oral route - systemic effects (target organ) cardiovascular / hematological: other; digestive: liver; urogenital: kidneys

Justification for classification or non-classification

According to the criteria of EC Directive 67/548/EEC and EC Regulation 1272/2008 and subsequent regulations on classification, labelling and packaging of substances and mixtures and based on the results of the studies trans-N1,N1,N4,N4-Tetrakis(2-hydroxyethyl)1,4-cyclohexanedicarboxamide is not classified.