Registration Dossier

Diss Factsheets

Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2014-10-07 - 2015-01-29
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: OECD guideline study without deviations under GLP on the registered substance itself.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report date:
2015

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
3-cyclohexylaminopropane-1-sulphonic acid
EC Number:
214-492-1
EC Name:
3-cyclohexylaminopropane-1-sulphonic acid
Cas Number:
1135-40-6
Molecular formula:
C9H19NO3S
IUPAC Name:
3-cyclohexylaminopropane-1-sulphonic acid
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
- Name of test material (as cited in study report): 3-(cyclohexylamino)-propane sulfonic acid, 3-CAPS
- Substance type: pure substance
- Storage condition of test material: in a plastic container, 15-25°C

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: husbandry of laboratory animals of the Experimental Medicine Centre at the Medical University in Białystok
- Age at study initiation: approx. 8 weeks (range-finder), 7 weeks (main study)
- Weight at study initiation:
The body weight of individual animals was ± 20% of the average value for each sex. On the day of the
introduction to the dose range-finding study, average body weights of the animals were as follows:
- group 0: males 237.4 g; females 172.8 g
- group 1: males 237.2 g; females 173.0 g
- group 2: males 237.4 g; females 173.0 g
- group 3: males 237.2 g; females 173.0 g
On the day of the introduction to the main study, average body weights of the animals were as follows:
- group 0: males 236.0 g; females 177.3 g
- group 1: males 236.1 g; females 177.2 g
- group 2: males 236.1 g; females 177.5 g
- group 3: males 236.1 g; females 177.2 g
- group 0SAT: males 237.0 g; females 177.2 g
- group 3SAT: males 237.1 g; females 177.4 g

- Fasting period before study: no
- Housing: in air-conditioned rooms, in plastic cages covered with wire bar lids (58 x 37 x 21 cm (length x width x height)), 5 animals in one cage, each sex kept separately, UV-sterilized wood shavings used as bedding
- Diet (e.g. ad libitum): Murigran standard granulated laboratory fodder produced by Wytwórnia Koncentratów i Mieszanek Paszowych AGROPOL, Motycz (batch numbers: 3/14, 4/14, and 5/14), ad libitum
- Water (e.g. ad libitum): tap water ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 - 24°C
- Humidity (%): 35 - 80%
- Air changes (per hr): 16
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Remarks:
distilled
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:

VEHICLE
- Concentration in vehicle: 10, 40, 160 mg/mL
- Amount of vehicle (if gavage): 0.5 ml / 100g
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Chemical analyses of the concentration of the test item in the main study were conducted by BLIRT S.A. Laboratory, Trzy Lipy 3/1.38, 80-172 Gdańsk, Poland, which holds the Statement of GLP Compliance. Samples (aqueous solutions of three concentrations – doses) collected on each day of the study were frozen (about - 20oC) and sent to BLIRT S.A. (94 samples in two batches). The sample solutions stored in a freezer (-20±5oC) were stable for at least 20 days.
The concentrations of 3-CAPS in the sample solutions were within the following ranges:
9.816 - 11.177 mg/mL for the concentration of 10 mg/mL;
39.657 - 42.819 mg/mL for the concentration of 40 mg/mL;
165.839 - 173.370 mg/mL for the concentration of 160 mg/mL.
The results were within the range of 80 -120%.
Duration of treatment / exposure:
7 days (range-finder)
28 days (main study)
Frequency of treatment:
daily
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 50, 200, 800 mg/kg bw
Basis:
nominal in water
No. of animals per sex per dose:
5 / sex / dose (range-finder)
20 / sex/ dose (main study)
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: determined on the basis of the dose range-finding study
- Rationale for animal assignment (if not random): The animals were randomized to individual groups. Their sexes and body weights were taken into account.
- Post-exposure recovery period in satellite groups: 14 days
Positive control:
not required

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice a day or once a day (on days off)

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily. They involved the evaluation of skin, fur, eye and mucosa changes, the respiratory, circulatory, and autonomous and central nervous systems, somatic activity, and behaviour.

BODY WEIGHT: Yes
- Time schedule for examinations: twice a week

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No. Food intake was measured once a week during the dose range-finding study and the main study. Food intake/cage was measured. Then, it was converted into average food intake/100 g b.w.
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: On the days before the experiment and before euthanasia. An indirect ophthalmoscope was used. In case of the satellite groups, these examinations were also conducted after the end of the test item/medium administration.
- Dose groups that were examined: All

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at the end of treatment
- Anaesthetic used for blood collection: Yes, xylazine-ketamine mixture at a dose of 10 mg xylazine/kg b.w. and 100 mg ketamine/kg b.w. to collect blood samples from the heart.
- Animals fasted: Yes, for 18 hours
- How many animals: All

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at the end of treatment
- Animals fasted: Yes, for 18 hours
- How many animals: All

URINALYSIS: Yes
- Time schedule for collection of urine: 18 hours during the last day of the experiment
- Metabolism cages used for collection of urine: Yes
- Animals fasted: No data

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: During the fourth week of the experiment, i.e. before the end of the experiment. In case of the satellite groups, these studies were performed during the fourth week of the experiment (measurement 1) and at the end of the sixth week of the experiment, i.e. before the end of the additional observation period (measurement 2)
- Dose groups that were examined: All
- Battery of functions tested: sensory activity / fore- and hindlimb grip strength / motor activity / other: open field observations
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
Gross examinations
All animals used in the dose range-finding study (DRF) and the main study were subjected to these examinations. They involved the observation of the external body surface, all natural apertures, and the cranial, thoracic, and abdominal cavities with their content.
Weights of internal organs
Weights of organs of all animals used in the main study were measured.
Absolute and relative weights of the following organs of the euthanized animals were determined:
brain, pituitary gland, thymus, heart, liver, spleen, kidneys, adrenal glands, ovaries, testicles, epididymides, and prostate with the seminal vesicles and coagulating glands.
Relative weights of internal organs were calculated on the basis of absolute weights with reference to body weights of living animals.

HISTOPATHOLOGY: Yes
These examinations were conducted only in the main study.
The following organs and tissues of all animals used in the main study were examined: brain with the cerebellum, eyeball with the optic nerve, pituitary gland, spinal cord, femur with the knee joint, muscle with the peripheral nerve, tongue, esophagus, salivary glands, lymph nodes, larynx, trachea, thyroid with the parathyroids, stomach, duodenum, jejunum, ileum, caecum, colon, liver (left and right lobes), pancreas, spleen, lungs, heart, aorta, thymus, kidneys with the ureters, adrenal glands, urinary bladder, ovaries, uterus with the cervix, oviducts, vagina, testicles, epididymides, accessory sex glands (prostate with the seminal vesicles and coagulating glands), skin, mammary gland, and Harderian gland.
All organs and tissues were fixed in a 10% aqueous solution of formalin.
The femur with the knee joint was fixed in a 10% aqueous solution of formalin and decalcified using the TBD-1 Rapid decalcifier (SHANDON).
The tissues were embedded in paraffin. The sections were stained using hematoxylin and eosin.
The slides were evaluated under a light microscope at 100x, 200x, and 600x magnification.
Statistics:
The results (average values and standard deviation) are presented in tables.
The treated groups, i.e. groups 1, 2, and 3 were compared to the control group (separately in the DRF study and the main study). The treated satellite group, i.e. 3SAT was compared to group 0SAT.
The clinical results were statistically analyzed using the Student’s t-test (p ≤ 0.05).
Food intake is summarized in tables. No statistical analyses were conducted, because the amount of data was insufficient (1 cage/group).
The clinical-chemical results were statistically analyzed using the one-way analysis of variance and Dunnet’s test (p ≤ 0.05).
Absolute and relative weights of internal organs were statistically evaluated using Dunnett’s test (p≤0.05).
The statistical analyses were conducted using the Microsoft Excel 2002 and 2010, Statistica 10 Pl, and Toksyk (software used at the Institute) software.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Description (incidence and severity):
Mortality and morbidity were not reported, pathological clinical signs were observed.
Mortality:
no mortality observed
Description (incidence):
Mortality and morbidity were not reported, pathological clinical signs were observed.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
average body weights of males and females from the treated groups (incl. satellite groups) did not differ statistically significantly from the ones of males and females in the control group
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Food intake by the treated and the control groups (incl. satellite groups) was similar
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Description (incidence and severity):
The ophthalmic examinations did not reveal any pathological changes.
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
No statistically significant differences between the treated and the control groups were found except a decrease in the number leukocytes in males and females from groups 2 & 3 and a decrease in the number of reticulocytes in females from group 2.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
No statistically significant changes between the treated and the control groups were observed, the only potentially relevant exemption is the increased concentration of chlorides in males from groups 2 and 3
Urinalysis findings:
no effects observed
Description (incidence and severity):
No statistically significant changes were observed.
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
All groups did not exhibit any involuntary clonic and tonic movements, changes in gait, or stereotypical behavior, no treatment-related effects on responses to visual, tactile, sound, and pain stimuli or locomotor activity.
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
The statistical analysis of absolute and relative weights of internal organs of males and females did not reveal any statistically significant changes.
Gross pathological findings:
no effects observed
Description (incidence and severity):
The gross examination of tissues and organs did not reveal any pathological changes in most euthanized rats.
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
The pathological studies showed no adverse effects of the test item on organs and tissues of the rats.
Histopathological findings: neoplastic:
not examined
Details on results:
CLINICAL SIGNS AND MORTALITY
DRF study: Mortality and morbidity were not reported (Table 1, see attachment). No pathological clinical signs were observed (Table 1).
Main study: Mortality and morbidity were not reported (Tables 6 and 7). The results of the clinical observations are shown in Tables 6 and 7. No pathological clinical signs were observed. The only exception was one female from group 3 which had scabs on the snout during the last week of the experiment.

BODY WEIGHT AND WEIGHT GAIN
DRF study: Average body weights of the animals, measured at weekly intervals, are shown in Table 2 (males) and Table 3 (females).
During the dose range-finding study, average body weights of males and females in the treated groups did not differ statistically significantly from the ones of males and females in the control group.
Main study: Average body weights of the animals, measured at weekly intervals, are shown in Table 8 (males) and Table 9 (females).
During the main study, average body weights of males and females from the treated groups did not differ statistically significantly from the ones of males and females in the control group.
Average body weights of the animals from the satellite groups, measured at weekly intervals, are shown in Table 10 (males) and Table 11 (females).
During the main study, average body weights of males and females from the treated satellite group did not differ statistically significantly from the ones of males and females in the control satellite group.

FOOD CONSUMPTION
DRF study: Food intake/100 g b.w. is shown in Table 4 (males) and Table 5 (females).
Food intake by the treated and the control groups was similar. No statistical analyses of food intake were conducted, because the amount of data was insufficient (1 cage/group). Therefore, it was impossible to clearly determine the relationship between the treated and the control groups.
Main study: Food intake/100 g b.w. is shown in Table 12 (males) and Table 13 (females).
Food intake by the treated and the control groups was similar.
Food intake/100 g b.w. in the satellite groups is shown in Table 14 (males) and Table 15 (females).
Food intake by the treated and the control satellite groups was similar.
No statistical analyses of food intake were conducted, because the amount of data was insufficient. Therefore, it was impossible to clearly determine the relationship between the treated and the control groups.

OPHTHALMOSCOPIC EXAMINATION (main study)
The ophthalmic examinations did not reveal any pathological changes.

HAEMATOLOGY
DRF study: Peripheral blood examinations
The results of the hematological examinations of peripheral blood in the DRF study in groups 0, 1, 2, and 3 are shown in Table 40 (males) and Table 41 (females).
No statistically significant changes in the results of the hematological examinations of peripheral blood of the treated animals were observed. The only exception was a decrease in the number of leukocytes in males from group 3.
Main study:
Peripheral blood examinations
The results of the hematological examinations of peripheral blood in the main study in groups 0, 1, 2, and 3 are shown in Table 44 (males) and Table 45 (females).
No statistically significant differences between the treated and the control groups were found. The only exceptions were a decrease in the number leukocytes in males and females from groups 2 and 3 and a decrease in the number of reticulocytes in females from group 2.
The results of the hematological examinations of peripheral blood collected from the satellite groups (0SAT and 3SAT) in the main study are presented in Table 46 (males) and Table 47 (females).
No statistically significant differences between the treated and the control groups were noticed. The only exceptions were a decrease in the number of leukocytes and increased MCH values in females.
The results of the leukocytogram estimation in groups 0, 1, 2, and 3 are shown in Table 48 (males) and Table 49 (females).
The results of the leukocytogram estimation did not show any significant differences. The only exceptions were an increase in the number of neutrocytes and a decrease in the number of lymphocytes in females from group 3.
The results of the leukocytogram estimation in both satellite groups, i.e. 0SAT and 3SAT are presented in Table 50 (males) and Table 51 (females).
The results of the leukocytogram estimation did not reveal any statistically significant changes between the treated and the control groups.

Bone marrow examinations
The results of the erythrocyte system bone marrow examination conducted in groups 0, 1, 2, and 3 are shown in Table 52 (males) and Table 53 (females).
No statistically significant changes in the erythrocyte system bone marrow between the treated and the control groups were noticed. The only exception was an increase in the number of polychromatophilic erythroblasts in males from group 2.
The results of the erythrocyte system bone marrow examination conducted in the satellite groups, i.e. 0SAT and 3SAT are presented in Table 54 (males) and Table 55 (females).
No statistically significant changes in the erythrocyte system bone marrow of males and females from group 3SAT were observed.
The results of the leukocyte system bone marrow examination conducted in groups 0, 1, 2, and 3 are shown in Table 56 (males) and Table 57 (females).
No statistically significant changes in the leukocyte system bone marrow between the treated and the control groups were observed.
The results of the leukocyte system bone marrow examination conducted in the satellite groups, i.e. 0SAT and 3SAT are presented in Table 58 (males) and Table 59 (females).
No statistically significant changes in the leukocyte system bone marrow of males and females were stated.
The numbers of different cells in bone marrow, estimated in groups 0, 1, 2, and 3 are shown in Table 60 (males) and Table 61 (females).
No statistically significant changes in the numbers of different cells of bone marrow of males and females were noticed.
The numbers of different cells in bone marrow estimated in the satellite groups, i.e. 0SAT and 3SAT are illustrated in Table 62 (males) and Table 63 (females).
There were no statistically significant changes in the numbers of different cells in bone marrow between groups 3SAT and 0SAT.

CLINICAL CHEMISTRY
DRF study: Biochemical and enzymatic examinations
The results of the biochemical and enzymatic examinations of serum in the DRF study in groups 0, 1, 2, and 3 are shown in Table 42 (males) and Table 43 (females).
An increase in the concentration of urea nitrogen in males in group 1 and a decrease in the concentration of creatinine in females from group 3 were noticed.
The enzymatic examinations of males and females from the treated groups, i.e. 1, 2, and 3 and group 0 did not reveal any statistically significant changes.

Main study:
Coagulation examinations
The results of the determination of PT and APTT in groups 0, 1, 2, and 3 are shown in Table 44 (males) and Table 45 (females).
No statistically significant differences between the treated and the control groups were observed.
The results of the PT and APTT determination in the satellite groups, i.e. 0SAT and 3SAT are illustrated in Table 46 (males) and Table 47 (females). There were no statistically significant changes between the treated and the control groups.

Biochemical examinations
The results of the blood serum biochemical examinations of serum in groups 0, 1, 2, and 3 are shown in Table 64 (males) and Table 65 (females).
No statistically significant changes between the treated and the control groups were observed. The only exceptions were:
- the decreased concentration of cholesterol in males from group 2,
- the increased concentration of chlorides in males from groups 2 and 3,
- the decreased concentration of creatinine in females from group 3,
- the decreased concentration of bilirubin in females from group 1.
The results of the biochemical examinations in the satellite groups, i.e. 0SAT and 3SAT are given in Table 66 (males) and Table 67 (females).

Enzymatic examinations in the main study
The results of the enzymatic examinations of serum in groups 0, 1, 2, and 3 are shown in Table 68 (males) and Table 69 (females).
No statistically significant changes were found.
Table 70 (males) and Table 71 (females) summarize the results of the serum enzymatic examinations in the satellite groups, i.e. 0SAT and 3SAT.
There were no differences in the activities of individual enzymes between the treated and the control groups.

URINALYSIS (main study)
General urinalysis:
The results of the urinalysis in groups 0, 1, 2, and 3 are shown in Table 72 (males) and Table 73 (females).
No significant changes were observed. Urine of all animals was yellow.
The results of the general urine examination in groups 0SAT and 3SAT are shown in Table 74 (males) and Table 75 (females).
No statistically significant changes were observed. Urine of all animals was yellow.
Urine sediment examinations:
The results of the urine sediment examinations in groups 0, 1, 2, and 3 are shown in Table 76 (males) and Table 77 (females).
No statistically significant changes were found.
The results of the urine sediment examinations in groups 0SAT and 3SAT are shown in Table 78 (males) and Table 79 (females).
There were not any statistically significant differences between group 3SAT (males and females) and the control group.

NEUROBEHAVIOUR (main study)
Open field observations
The results of the open field observations are presented in Table 16 (males) and Table 17 (females).
During these observations, males and females from groups 0, 1, 2, and 3 did not exhibit any involuntary clonic and tonic movements, changes in gait, or stereotypical behavior.
A slight decrease in arousal was noticed in one male from group 0.
The numbers of fecal boluses and urine pools left by males and females from groups 1, 2, and 3 did not differ statistically significantly from these left by the control ones.
Both horizontal and vertical locomotor activities of treated males was similar to these of the control ones (there were no statistically significant differences).
Statistically significantly higher horizontal locomotor activity of females in group 3 was observed.
The results of the open field observations of the animals from the satellite groups, made at the end of the treatment (4th week of the experiment - measurement 1) are shown in Table 18 (males) and Table 19 (females).
The animals from these groups (males and females) did not exhibit involuntary clonic and tonic movements, changes in gait, or stereotypical behavior.
A slight decrease in arousal was noticed in one male from group 3SAT. A slight increase in arousal was noticed in two males from group 3SAT.
The numbers of fecal boluses and urine pools left by males and females from group 3SAT did not differ statistically significantly from group 0SAT.
Locomotor activity (horizontal and vertical) of males and females from group 3SAT did not differ statistically significantly from group 0SAT.
The results of the open field observations of the satellite groups, made at the end of the additional observation (measurement 2) are shown in Table 20 (males) and Table 21 (females).
During these observations, no involuntary clonic and tonic movements, changes in gait, or stereotypical behavior were noticed.
A slight decrease in arousal was observed in two males from group 0SAT and one female from group 3SAT.
The number of fecal boluses left by males and females from groups 3SAT and 0SAT and the number of urine pools left by females from groups 3SAT and 0SAT did not differ statistically significantly. The number of urine pools left by males from group 3SAT was statistically significantly higher than in group 0SAT.
Locomotor activity (horizontal and vertical) of males and females from group 3SAT did not differ statistically significantly from group 0SAT.

Evaluation of sensorimotor responses to stimuli
There were no treatment-related effects on responses to visual, tactile, sound, and pain stimuli. Sensorimotor responses to stimuli are shown in Table 22 (males) and Table 23 (females).
The study aimed at observing reactions of the treated animals and the ones from group 0 (responses to objects, touching, and sound and pinna reflex) did not reveal any changes.
The latency of pain responses of males from groups 1 and 3 was similar to group 0. The latency of pain responses of males from group 2 was statistically significantly longer than in group 0. The latency of pain responses of females from the treated group was similar to group 0.
Responses to stimuli of the satellite groups, measured at the end of the treatment (measurement 1) are illustrated in Table 24 (males) and Table 25 (females).
No disturbances in case of males and females from group 3SAT were observed when compared to group 0SAT.
The latency of pain responses of males and females from group 3SAT was similar to group 0SAT.
Responses to stimuli of the satellite groups, measured at the end of the additional observation period (measurement 2) are given in Table 26 (males) and Table 27 (females).
As far as all the reactions mentioned above are concerned, no disturbances in case of males and females from group 3SAT and the control group were observed.
The latency of pain responses of males and females from group 3SAT was similar to group 0SAT.

Measurement of the fore- and hindlimb grip strength
The results of the measurement of the fore- and hindlimb grip strength of the animals are given in Table 28 (males) and Table 29 (females).
The fore- and hindlimb grip strength of the treated males did not differ statistically significantly from the control group (group 0). The forelimb grip strength of the treated females did not differ statistically significantly from the control ones (group 0). The hindlimb grip strength of females from group 1 was statistically significant lower than the one of the control animals.
As for the satellite groups, the results of the fore- and hindlimb grip strength measurement made at the end of the treatment (measurement 1) are presented in Table 30 (males) and Table 31 (females).
The fore- and hindlimb grip strength of males from group 3SAT did not differ statistically significantly from the control group. The hindlimb grip strength of females from group 3SAT did not differ statistically significantly from the control group. The forelimb grip strength of females from group 3SAT was statistically significantly higher than the one of group 0SAT.
The results of the satellite groups’ fore- and hindlimb grip strength measurement made at the end of the additional observation period (measurement 2) are illustrated in Table 32 (males) and Table 33 (females).
The fore- and hindlimb grip strength of males from group 3SAT did not differ statistically significantly from group 0SAT. The hindlimb grip strength of females from group 3SAT did not differ statistically significantly from group 0SAT. The forelimb grip strength of females from group 3SAT was statistically significant lower than the one of group 0SAT.

Measurement of locomotor activity
There were no treatment-related differences between the treated and the control groups. Table 34 (males) and Table 35 (females) illustrate locomotor activity of the animals.
Horizontal locomotor activity of males and females from the treated groups (0-30 minutes) was similar to group 0.
As far as individual stages of the experiment are concerned, i.e. 0-10 minutes, 10-20 minutes, and 20-30 minutes, horizontal motor activity of males and females from the treated and the control groups was similar.
Vertical locomotor activity of males and females from the treated groups (0-30 minutes) was also similar to group 0.
When it comes to individual stages of the experiment, i.e. 0-10 minutes, 10-20 minutes, and 20-30 minutes, vertical locomotor activity of males and females from the treated and the control groups was similar.
Table 36 (males) and Table 37 (females) present the results of the locomotor activity measurement in the satellite groups, made at the end of the treatment (measurement 1).
Locomotor activity (horizontal and vertical) of males and females from group 3SAT (0-30 minutes) did not differ statistically significantly from the control group.
At individual stages of the experiment, i.e. 0-10 minutes, 10-20 minutes, and 20-30 minutes, both vertical and horizontal locomotor activities of females from groups 3SAT and 0SAT were similar.
When it comes to females, their vertical locomotor activity was higher only at the first stage, i.e. 0-10 minutes. Their horizontal locomotor activity was similar to the control group.
Table 38 (males) and Table 39 (females) present the results of the locomotor activity measurement in the satellite groups, made at the end of the additional observation period (measurement 2).
Locomotor activity (horizontal and vertical) of males and females from group 3SAT (0-30 minutes) did not differ statistically significantly from the control group.
As far as individual stages of the experiment are concerned, i.e. 0-10 minutes, 10-20 minutes, and 20-30 minutes, locomotor activity (horizontal and vertical) of group 3SAT was similar to the control group.

ORGAN WEIGHTS (main study)
Weights of organs of males and females from groups 0, 1, 2, and 3 are summarized in Tables 81 and 82 (absolute weights) and Tables 83 and 84 (relative weights).
The results obtained in the groups treated with the test item (groups 1, 2, and 3) were compared with the ones obtained in the control group receiving distilled water (group 0).
The analysis of relative weights of internal organs of males showed only one statistically significant difference, i.e. a decreased heart weight in group 1. In the remaining organs, no statistically significant changes were discovered.
The statistical analysis of relative weights of internal organs of females and absolute weights of internal organs of males and females did not reveal any statistically significant changes.
Weights of organs of males and females from both satellite groups, i.e. 0SAT and 3SAT are summarized in Tables 85 and 86 (absolute weights) and Tables 87 and 88 (relative weights).
The results obtained in the group treated with the test item (group 3SAT) were compared with the ones obtained in the control group receiving distilled water (group 0SAT).
The statistical analysis of absolute and relative weights of internal organs of males and females did not reveal any statistically significant changes.

GROSS PATHOLOGY
DRF study: During the macroscopic examinations of all animals used in the DRF study, no lesions were found.
Main study: Table 80 contains a list of all gross changes observed in the animals from groups 0, 1, 2, and 3.
There were no gross changes in group 0. In the remaining groups, the following changes were observed:
group 1 – emphysema in the lungs of 1 rat (female),
group 2 – hyperemia in the lungs of 1 rat (male),
group 3 – hyperemia in the lungs of 1 rat (female).
There were not any pathological changes in tissues and organs of the rats from groups 0SAT and 3SAT.

HISTOPATHOLOGY: NON-NEOPLASTIC (main study)
Histopathological changes found in groups 0, 1, 2, and 3 are shown in Table 89.
They are also listed below.
Group 0:
lungs
- erythrocytorrhagia in 1 male
liver
- fine-cell infiltration in 1 female
kidneys
- small cyst in 1 female
pancreas
- hyperplasia of the islets of Langerhans in 1 male
ileum
- Peyer’s path hyperplasia in 1 male
colon
- nodular lymphoid hyperplasia in 1 male

Group 1:
lungs
- hyperemia in 1 male and 1 female
- edema in 1 male
- emphysema in 1 female
liver
- hyperemia in 3 females
- erythrocytorrhagia in 1 male
- fine-cell infiltration in 1 male
kidneys
- fine-cell infiltration in 1 female
thyroid
- epithelial hyperplasia in 1 male

Group 2:
lungs
- hyperemia in 2 males
liver
- hyperemia in 1 male and 3 females
- fine-cell infiltration in 1 female
adrenal glands
- cortical hyperemia in 1 male

Group 3:
lungs
- hyperemia in 1 females
- erythrocytorrhagia in 1 male and 2 females
- edema in 2 females
liver
- hyperemia in 1 female
kidneys
- partial atrophy of the glomeruli in 1 female
pancreas
- hyperplasia of the islets of Langerhans in 1 female
duodenum
- lymphocytic infiltrations in 1 female
colon
- nodular lymphoid hyperplasia in 1 male and 1 female
mandibular lymph nodes
- hyperemia in 1 male
- hyperplasia in 1 male
thymus
- hyperemia in 1 male
Harderian gland
- small encysted foci of glandular cell degeneration in 1 female
uterus
- endometrial hyperplasia in 1 female

Table 90 summarizes all histopathological findings observed in the satellite groups (0SAT and 3SAT).
The changes were in the following organs:

Group 0SAT
lungs
- hyperemia in 2 males
- edema in 2 males
- emphysema in 2 males
liver
- hyperemia in 2 males
- hyperplasia of the Browicz-Kupffer cells in 2 males
- fine-cell infiltrations in 4 females
kidneys
- erythrocytorrhagia in 1 female
prostate
- lymphocytic infiltrations in 1 male
Harderian gland
- lymphocytic infiltrations in 1 male and 1 female

Group 3SAT:
lungs
- hyperemia in 2 males
liver
- hyperemia in 1 males
- hyperplasia of the Browicz-Kupffer cells in 1 females
- fine-cell infiltrations in 2 males
- thickening of the walls of the blood vessels and bile ducts in 1 male
spleen
- partial atrophy of the white pulp in 1 male
pancreas
- hyperplasia of the islets of Langerhans in 3 males
- lymphocytic infiltrations around the cysts in 1 male
- two small cysts in 1 male
- hyperplasia of the ducts in 1 male
colon
- nodular lymphoid hyperplasia in 1 male and 2 females
prostate
- lymphocytic infiltrations in 1 male
pituitary gland
- hyperemia in 1 male
thyroid
- exfoliated epithelial cells in 1 male
No histopathological lesions were found in the remaining organs.

Effect levels

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Key result
Dose descriptor:
NOAEL
Effect level:
50 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: haematology; clinical chemistry
Key result
Dose descriptor:
NOAEL
Effect level:
50 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: haematology

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
The study was conducted under GLP according to OECD guideline 407 on the registered substance itself. The method is to be considered scientifically reasonable with no deficiencies in documentation and performance. Hence, the results can be considered as reliable to assess the repeated dose oral toxicity (short-term) in rats.

Following administration of 3-(Cyclohexylamino)-propane sulfonic acid (daily over 28 days) at doses of 0, 50, 200 and 800 mg/kg b.w. to 10 rats per sex per dose (and additionally 2 satellite groups dosed with 0 and 800 mg/kg b.w./day and two weeks recovery), a NOAEL of 50 mg/kg and consequently indicating a LOAEL of 200 mg/kg was found. Generally, according to Regulation (EC) No. 1272/2008, this LOAEL may trigger a classification as STOT RE Cat. 2 as it is generally below the guidance value of 300 mg/kg for subacute studies.

Target organ toxicity (repeated exposure), as defined by the Regulation, “means specific, target organ toxicity arising from a repeated exposure to a substance or mixture. All significant health effects that can impair function, both reversible and irreversible, immediate and/or delayed are included. … Classification for target organ toxicity (repeated exposure) identifies the substance as being a specific target organ toxicant and, as such, it may present a potential for adverse health effects in people who are exposed to it”. Furthermore, “substances are classified as specific target organ toxicants following repeated exposure by the use of expert judgement”, and “3.9.2.7. Effects considered to support classification for specific target organ toxicity following repeated exposure...
... (c) any consistent and significant adverse change in clinical biochemistry, haematology, or urinalysis parameters;”
But also: “3.9.2.8. Effects considered not to support classification for specific target organ toxicity following repeated exposure
3.9.2.8.1. It is recognised that effects may be seen in humans and/or animals that do not justify classification. Such effects include, but are not limited to: ...
... (b) small changes in clinical biochemistry, haematology or urinalysis parameters and/or transient effects, when such changes or effects are of doubtful or minimal toxicological importance; ...
... (d) adaptive responses that are not considered toxicologically relevant;”

Based on the available information, a classification as STOT RE Cat. 2 is not considered necessary as will be outlined below.
The LOAEL of 200 mg/kg is based on the following effects:
- A statistically significant decrease in the number of leukocytes in males and females in groups 2 and 3
- An increase in the concentration of chlorides in males from groups 2 and 3, detected via biochemical examinations

Hyperchloremia is commonly caused e.g. by renal tubular acidosis or some kidney diseases in general, an overactivity of parathyroid glands or diarrhoea and vomiting. Besides various infections and diseases, Leukopenia may be caused by some tumors or an enlargement of the spleen.
A certain consistency and persistence of the effect, i.e. decreased lymphocytes in males and females is given as it can be found also in the 3SAT group. However, there are findings relativizing this effect.

The clinical observations performed during the study involved the evaluation of general condition of the animals and detailed clinical observations. No vomiting or diarrhea was reported. Physical appearance and behavior of the treated animals did not differ from the ones of the control group, and the animals showed no pathological clinical signs during the entire experiment.

The results of the leukocytogram estimation did not show any significant differences. The only exceptions were an increase in the number of neutrocytes and a decrease in the number of lymphocytes in females from group 3. Similar tendencies, although not statistically significant, can be observed for males, too. This slight change in the leukocytogram may be indicative for an adaptive response of the immune system by transferring leukocytes from the blood into tissue. The bone marrow examinations included i.a. the leukocyte system: myeloblasts, promyelocytes, orthochromatophilic and acidophilic myelocytes, orthochromatophilic and acidophilic metamyelocytes, rod neutrophils and rod eosinophils, filamented neutrophils, filamented eosinophils, and basophils. No statistically significant changes in the leukocyte system bone marrow between the treated and the control groups were observed. It can be concluded that hence no effect on the bone marrow as the generating organ can be detected and the decrease in leukocytes may be only seen as a minor adaptive response.

Gross examinations did not reveal any visual neoplastic lesions, which was however to expect based on the short exposure duration.

Weights of organs of all animals used in the main study were measured, absolute and relative weights of i.a. thymus and kidneys were determined. The statistical analysis of relative weights of internal organs and absolute weights of internal organs did not reveal any statistically significant changes with regard to these organs, actually no dose-related changes at all were seen.

The test item did not affect any parameters of urine.

The histopathological examinations included i.a. femur with the knee joint, spleen, thymus and kidneys. There were no relevant or dose-related histopathological changes found. Generally, kidney issues are a known and common problem in male rats, related to α2u-globulin. This effect does not occur in female rats and is not relevant for humans. Potentially such an effect could have occurred undetected and may be responsible for the increase in chloride.

In summary, this could be seen as a non-consistency which does not trigger classification. Based on expert judgement, the observed effects do not suffice for and justify a classification of 3-(cyclohexylamino)-propane sulfonic acid as STOT RE Cat. 2.
Executive summary:

In a subacute oral toxicity study according to OECD guideline 407, 3-(cyclohexylamino)-propane sulfonic acid was administered daily by gavage in distilled water over 28 days to each 10 Wistar rats (Crl: WI(Han); outbred) per sex and per dose at dose levels of 0, 50, 200 and 800 mg/kg b.w. Also, 0 and 800 mg/kg b.w. were administered similarly to two satellite groups with a 14-day postobservation period. The following observations were made:

Clinical studies:

All animals survived the experiment. One female in group 3 had scabs on the snout during the last week of the experiment. The remaining animals did not exhibit any changes. The ophthalmic examinations did not reveal any changes. No statistically significant differences in body weights between the treated and the control groups were observed. Food intake by the treated and the control groups was similar (no statistical analysis).

No treatment-related effects were observed during the detailed behavioral studies. On the basis of the detailed clinical observations, made during and after the treatment, and the open field observations, no muscarinic symptoms (weakness, lacrimation,salivation, anxiety), nicotine symptoms(diarrhea, motor coordination difficulties), or central symptoms were noticed. There was also no delayed neurotoxicity in the form of paralysis of the hindlimbs, clumsiness, sensory disturbances, and general paralysis. The evaluation of responses to stimuli showed no effects of the test item.

On the basis of the fore- and hindlimb grip strength measurement, the detailed clinical observations, and the open field observations (body posture, gait, locomotor activity), it can be concluded that the test item at the doses of 50, 200 mg/kg b.w. (males and females), and 800 mg/kg b.w. (males) does not cause any miotoxic changes. When considering the results of the measurement of the forelimb grip strength of females (dose of 800 mg/kg b.w.), it is difficult to clearly state whether the test item at the dose of 800 mg/kg b.w. affects the nervous system or muscles.

On the basis of the clinical observations and the body weight and food intake measurements, it may be concluded that the test item at the doses of 50, 200, and 800 mg/kg b.w. had no adverse effects.

On the basis of the behavioral studies, it may be concluded that the test item at the doses of 50, 200 mg/kg b.w. (males and females), and 800 mg/kg b.w. (males) does not cause any miotoxic changes or affect the nervous system. When considering the results of the measurement of the forelimb grip strength of females (dose of 800 mg/kg b.w.), it is difficult to clearly state whether the test item at the dose of 800 mg/kg b.w. affects the nervous system or muscles of these animals.

 

Clinical-chemical examinations:

Statistical analyses of the results of the clinical-chemical examinations showed a few statistically significant changes. Some of them can be regarded as accidental, whereas the others should be perceived as caused by the test item.

The hematological examinations (examinations of peripheral blood, leukocytogram, and bone marrow) revealed the following changes:

● the decreased number of leucocytes in males and females in groups 2 and 3,

● the increased number of neutrocytes and the decreased number of lymphocytes in females in group 3,

● the decreased number of reticulocytes in females in group 2,

● the increased number of polychromatophilic erythroblasts in bone marrow in males in group 2.

These hematological changes except for the number of reticulocytes and the increased number of polychromatophilic erythroblasts can be perceived as caused by the test item.

During the coagulation examinations, no statistically significant changes were observed.

During the biochemical examinations, the following changes were found:

● the decreased concentration of cholesterol in males in group 2,

● the increased concentration of chlorides in males in groups 2 and 3,

● the decreased concentration of creatinine in females in group 3,

● the decreased concentration of bilirubin in females in group 1.

The decreased concentrations of cholesterol and bilirubin may be considered as accidental, because they were not observed in the group treated with the test item at a higher dose. The decreased concentration of creatinine, which was also observed in the DRF study, was not diagnostically significant.

The enzymatic examinations, general urinalysis, and examinations of urine sediment did not reveal any statistically significant changes.

 

For the satellite group, no statistically significant changes in the investigated parameters were observed after the 14-day break in the treatment. The only exceptions were the decreased number of leucocytes, the increased MCH value in females, and the increased concentration of globulin in males.

The increased the MCH value and concentration of globulin might be accidental, whereas the decreased number of leucocytes in females could be considered as a stable sign, because it was observed after the 14-day break in the treatment.

 

On the basis of the clinical-chemical examinations, it can be stated that 28-day administration of the test substance at the doses of 200 and 800 mg/kg b.w.may cause a decrease in the number of leucocytes in blood and an increase in the chloride concentration in males. The decreased number of leucocytes in females could be considered as a stable sign.

On the basis of the results of the clinical-chemical examinations at the dose of 50 mg/kg b.w.no treatment-related changes were observed.

 

Post-mortem examinations

There were no gross changes in group 0. In the remaining groups, the following changes were observed:

group 1 – emphysema in the lungs of 1 rat (female),

group 2 – hyperemia in the lungs of 1 rat (male),

group 3 – hyperemia in the lungs of 1 rat (female).

There were not any pathological changes in tissues and organs in groups 0SAT and 3SAT.

The analysis of absolute and relative weights of internal organs showed some statistically significant changes, i.e. a decrease of heart relative weights in males in group 1.

There were no statistically significant changes in absolute and relative weights of the remaining organs in any group.

The histopathological examination of organs and tissues revealed a few changes in the Harderian gland, thyroid, mandibular lymph nodes, thymus, lung, liver, spleen, pancreas, intestines (duodenum, jejunum, and colon), kidneys, adrenal glands, prostate, and uterus. The histopathological changes observed in these organs were blood circulation disorders (pulmonary edema and hyperemia in the lung, liver, mandibular lymph nodes, thymus, and adrenal cortex, and erythrocytorrhagia in the lungs, liver, and kidneys), inflammatory lesions (lymphocytic infiltration in the Harderian gland, prostate, pancreas, and duodenum, fine-cell infiltration in the liver and kidney, Peyer's patches hyperplasia in the jejunum, nodular lymphoid hyperplasia in the colon, and mandibular lymph node hyperplasia), regressive changes (partial atrophy of the glomeruli, partial atrophy of the white pulp of the spleen, and small encysted foci of glandular cell degeneration in the Harderian gland), single cases of emphysema, hyperplasia, and exfoliated cells of the thyroid follicular epithelium, hyperplasia of the Kupffer-Browicz cells, hyperplasia and thickening of the bile ducts in the liver, pancreatic ductal hyperplasia, hyperplasia of the islets of Langerhans, uterine endometrial hyperplasia, and single cases of small cysts in the Harderian gland, kidneys, and pancreas.

The pathological studies of the rats exposed to the test item, i.e. 3-(cyclohexylamino)-propane sulfonic acid at the doses of 50, 200, and 800 mg/kg for 28 days involved the macroscopic and histological examinations of organs and tissues, the evaluation of the immune system, and the statistical evaluation of absolute and relative weights of organs.

The pathological studies showed no adverse effects of the test item on organs and tissues of the rats.

 

The LOAEL is 200 mg/kg bw/day, based on changes in haematology and clinical chemistry (males only), the NOAEL is 50 mg/kg bw/day.

 

This subacute toxicity study in the rat is acceptable and satisfies the guideline requirement for a subacute oral study according to OECD TG 407.