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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2017

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: liquid
Details on test material:
- Physical state: liquid
- Analytical purity: 54.7 corr. area% and 53.8 corr. area%
- Impurities (identity and concentrations): 34.3 g/100 g water
- Lot/batch 57012888Q0
- Stability under test conditions: stable until 16 Oct 2017, the stability of the test item under storage conditions over the study period was guaranteed by the sponsor,
and the sponsor holds this responsibility.
- Storage condition of test material: Room temperature

Test animals

Species:
rat
Strain:
Wistar
Details on species / strain selection:
The Wistra rat is a frequently used laboratory animal, and there is comprehensive experience with this animal species. Moreover, the rat has been proposed as a suitable animal species by the
OECD and the EPA for this type of study.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services GmbH, Sulzfeld, Germany
- Females (if applicable) nulliparous and non-pregnant: [yes]
- Age at study initiation: 42 ± 1 days
- Weight at study initiation: animals of comparable size and weight

The animals were housed together (5 animals per cage) in H-Temp polysulfonate cages
supplied by TECNIPLAST, Hohenpeißenberg, Germany (floor area about 2065 cm2). Motor
activity measurements were conducted in polycarbonate cages (floor area about 800 cm2)
supplied by TECNIPLAST, Hohenpeißenberg, Germany, with small amounts of bedding. The
cages were closed with wire covers from Ehret, Emmendingen, Germany. Dust-free wooden
bedding was used in this study (the present supplier is documented in the raw data). Wooden
gnawing blocks (Typ NGM E-022) supplied by Abedd® Lab. and Vet. Service GmbH, Vienna,
Austria and large play tunnels (Art. 14153) supplied by PLEXX B.V., Elst, Netherlands were
added for environmental enrichment. The animals were accommodated in fully air-conditioned
rooms in which central air conditioning resulted in uniform temperature range of 20-24°C, a
range of relative humidity of 30-70% and 15 air changes per hour. The day/night cycle was
12 hours (12 hours light from 06.00 h-18.00 h, 12 hours dark from 18.00 h-06.00 h).
Deviations from these ranges did not occur. The animal room was completely disinfected prior
to the study using a disinfector ("AUTEX", fully automatic, formalin-ammonia-based terminal
disinfector). The floor and the walls were cleaned once a week with water containing an
appropriate disinfectant.
The food used was ground Kliba maintenance diet mouse/rat “GLP”, meal, supplied by Provimi
Kliba SA, Kaiseraugst, Switzerland. Food and drinking water (from water bottles) were
available ad libitum.

Administration / exposure

Route of administration:
oral: gavage
Details on route of administration:
2-Propyn-1-ol, compd. with methyloxirane was applied as a solution. To prepare this solution,
the appropriate amount of test substance as supplied by the sponsor was weighed out depending on the desired
concentration. Then, ultrapure water was filled up to the desired volume, subsequently mixed
with a magnetic stirrer. The test-substance preparations were produced at least weekly and
stored in the refrigerator. The administration volume was 10 mL/kg body weight.
Vehicle:
water
Details on oral exposure:
Starting on the day of arrival the animals were accustomed to the environmental conditions of
the study for an adaptation period during which they received ground diet and drinking water
ad libitum. Prior to the first detailed clinical observation, the animals were distributed according
to weight among the individual test groups, separated by sex. The weight variation of the
animals used did not exceed 20 percent of the mean weight of each sex. The list of
randomization instructions was compiled with a computer.
The test substance was administered daily by gavage for 3 months. Control animals received
only the vehicle. All animals were sacrificed after a fasting period (withdrawal of food) of at
least 16 to 20 hours.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The analyses of the test-substance preparations were carried out at the Analytical Chemistry
Laboratory of Experimental Toxicology and Ecology of BASF SE, Ludwigshafen, Germany.
The stability of 2-Propyn-1-ol, compd. with methyloxirane in ultrapure water in the refrigerator
for a period of 8 days was proven before the start of the administration period (project
No. 01Y0334/04Y031).
Concentration control analyses of test-substance preparations were performed at the
beginning and towards the end of the administration period in all concentrations. A
homogeneity control analysis was not performed, because 2-Propyn-1-ol, compd. with
methyloxirane was administered as a solution in ultrapure water.
Duration of treatment / exposure:
3 months
Frequency of treatment:
once daily
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Dose / conc.:
5 mg/kg bw/day (actual dose received)
Remarks:
of the test material as supplied by the sponsor
Dose / conc.:
30 mg/kg bw/day (actual dose received)
Remarks:
of the test material as supplied by the sponsor
Dose / conc.:
150 mg/kg bw/day (actual dose received)
Remarks:
of the test material as supplied by the sponsor
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels were selected based on the results of the available OECD 422 study.
Positive control:
N/A

Examinations

Observations and examinations performed and frequency:
Mortality
A check for moribund and dead animals was made twice daily on working days and once daily
on Saturdays, Sundays and public holidays. If animals were in a moribund state, they were
sacrificed and necropsied.

Clinical observations
All animals were checked daily for any abnormal clinically signs before the administration as
well as within 2 hours and within 5 hours after the administration. Abnormalities and changes
were documented for each animal.

Detailed clinical observations
Detailed clinical observations (DCO) were performed in all animals prior to the administration
period and thereafter at weekly intervals. The findings were ranked according to the degree of
severity, if applicable. The animals were transferred to a standard arena (50 × 37.5 cm with
sides of 25 cm height). The following parameters were examined:
1. Abnormal behavior when handled
2. Fur
3. Skin
4. Posture
5. Salivation
6. Respiration
7. Activity/ arousal level
8. Tremors
9. Convulsions
10.Abnormal movements
11.Gait abnormalities
12.Lacrimation
13.Palpebral closure
14.Exophthalmos
15.Assessment of the feces discharged during the examination (appearance/ consistency)
16.Assessment of the urine discharged during the examination
17.Pupil size

Food consumption
Food consumption was determined weekly (as representative value over 7 days) for each
cage. The average food consumption per cage was used to estimate the mean food
consumption in grams per animal and day.

Water consumption
Drinking water consumption was observed by daily visual inspection of the water bottles for
any overt changes in volume.

Body weight data
Body weight was determined before the start of the administration period in order to randomize
the animals. During the administration period the body weight was determined on study day 0
(start of the administration period) and thereafter at weekly intervals. The difference between
the body weight on the respective day of weighing and the body weight on study day 0 was
calculated as body weight change.

Functional observational battery
A functional observational battery (FOB) was performed in all animals at the end of the
administration period starting in the morning. At least one hour before the start of the FOB the
animals were transferred to single-animal polycarbonate cages. Drinking water was provided
ad libitum, but no food was offered during the measurements. The FOB started with passive
observations without disturbing the animals, followed by removal from the home cage, open
field observations in a standard arena and sensory motor tests as well as reflex tests. The
findings were ranked according to the degree of severity, if applicable. The observations were
performed at random.

Home cage observations:
The animals were observed in their closed home cages; during this period any disturbing
activities (touching the cage or rack, noise) were avoided during these examinations in order
not to influence the behavior of the rats. Attention was paid to:
1. Posture
2. Tremors
3. Convulsions
4. Abnormal movements
5. Gait
6. Other findings

Open field observations:
The animals were transferred to a standard arena (50 × 50 cm with sides of 25 cm height) and
observed for at least 2 minutes. The following parameters were examined:
1. Behavior on removal from the cage
2. Fur
3. Skin
4. Salivation
5. Nasal discharge
6. Lacrimation
7. Eyes/ pupil size
8. Posture
9. Palpebral closure
10. Respiration
11. Tremors
12. Convulsions
13. Abnormal movements/ stereotypes
14. Gait
15. Activity/ arousal level
16. Feces (consistency/ color) within two minutes
17. Urine (amount/ color) within two minutes
18. Rearing within two minutes
19. Other findings

Sensory motor tests/ reflexes:
The animals were then removed from the open field and subjected to following sensory motor
or reflex tests:
1. Reaction to an object being moved towards the face (Approach response)
2. Touch sensitivity (Touch response)
3. Vision (Visual placing response)
4. Pupillary reflex
5. Pinna reflex
6. Audition (Startle response)
7. Coordination of movements (Righting response)
8. Behavior during handling
9. Vocalization
10. Pain perception (Tail pinch)
11. Other findings
12. Grip strength of forelimbs
13. Grip strength of hindlimbs
14. Landing foot-splay test

Motor activity assessment
Motor activity (MA) was also measured in the early afternoon onwards on the same day as the
FOB was performed. The examinations were performed using the TSE Labmaster System
supplied by TSE Systems GmbH, Bad Homburg, Germany. For this purpose, the animals were
placed in new clean polycarbonate cages with a small amount of bedding for the duration of
the measurement. Eighteen beams were allocated per cage. The number of beam interrupts
was counted over 12 intervals for 5 minutes per interval. The sequence in which the animals
were placed in the cages was selected at random. On account of the time needed to place the
animals in the cages, the starting time was "staggered" for each animal. The measurement
period began when the 1st beam was interrupted and finished exactly 1 hour later. No food or
water was offered to the animals during these measurements and the measurement room was
darkened after the transfer of the last animal. The program requires a file name for the
measured data to be stored. This name consists of the reference number and a serial number.

Ophthalmoscopy
The eyes of all animals were examined prior to the start of the administration period. At the
end of the administration period, i.e. study day 91, the eyes of animals in test groups 0 (control)
and 3 (150 mg/kg bw/d) were examined for any changes using an ophthalmoscope (HEINE
OPTOTECHNIK, Herrsching, Germany) after application of a mydriatic agent (Mydrum,
Chauvin ankerpharm GmbH, Rudolstadt, Germany).

CLINICAL PATHOLOGY
In the morning blood was taken from the retro-bulbar venous plexus from fasted animals. The
animals were anaesthetized using isoflurane. The blood sampling procedure and subsequent
analysis of blood and serum samples were carried out in a randomized sequence. For
urinalysis the individual animals were transferred to metabolism cages (withdrawal of food and
water) and urine was collected overnight. Urine samples were evaluated in a randomized
sequence
The assays of blood and serum parameters were performed under internal laboratory quality
control conditions with reference controls to assure reliable test results.
The results of clinical pathology examinations were expressed in International System (SI)
units.
The following examinations were carried out in all animals per test group and sex at the end
of the administration period.

Hematology
The following parameters were determined in blood with EDTA-K3 as anticoagulant using a
particle counter (Advia 120 model; Bayer, Fernwald, Germany):
- Leukocyte count (WBC) =>giga/L [cytochemistry coupled with flow cytometry]
- Erythrocyte count (RBC) => tera/L [flow cytometric laserlight scattering]
- Hemoglobin (HGB) => mmol/L [cyanmethemoglobin method; according to ICSH]
- Hematocrit (HCT) => L/L [calculation: MCV x erythrocytes]
- Mean corpuscular volume (MCV) => fL [RBC/PLT method; mean of RBC volume distribution curve (histogram)]
- Mean corpuscular hemoglobin (MCH) =>fmol [calculation: hemoglobin, erythrocytes]
- Mean corpuscular hemoglobin concentration (MCHC) => mmol/L [calculation: hemoglobin, hematocrit]
- Platelet count (PLT) => giga/L [flow cytometric laserlight scattering]
- Differential blood count => % and giga/L [cytochemistry coupled with flow cytometry]
- Reticulocytes (RETA) => Giga/L [cytochemistry coupled with flow cytometry]
Furthermore, blood smears were prepared and stained according to WRIGHT without being
evaluated, because of non-ambiguous results of the differential blood cell counts measured by
the automated instrument. (reference: Hematology: Principles and Procedures, 6th Edition,
Brown AB, Lea & Febiger, Philadelphia, 1993, page 101).
Clotting tests were carried out using a ball coagulometer (AMAX destiny plus model; Trinity
biotech, Lemgo, Germany).

Parameter and method:
- Prothrombin time (Hepato Quick’s test) (HQT) => seconds [citrated blood with calcium thromboplastin]


Clinical chemistry
An automatic analyzer (Cobas c501; Roche, Mannheim, Germany) was used to examine the
clinicochemical parameters
Parameters and methods:
Enzyme:
- Alanine aminotransferase (ALT) (L-alanine: 2-oxoglutarate aminotransferase; EC 2.6.1.2.) => μkat/L [kinetic UV test, 340 nm; 37°C, (0.08 μkat/L)]
- Aspartate aminotransferase (AST) (L-aspartate: 2-oxoglutarate aminotransferase; EC 2.6.1.1.) => μkat/L [kinetic UV test, 340 nm; 37°C, (0.08 μkat/L)]
- Alkaline phosphatase (ALP) (orthophosphoric acid monoester phosphohydrolase; EC 3.1.3.1.) => μkat/L [kinetic color test, 415 nm, 37°C, (0.084 μkat/L)]
- gamma-Glutamyltransferase (GGT) (gamma-glutamyl) peptide: aminoacid-gamma-glutamyl-transferase; EC 2.3.2.2.) => nkat/L [kinetic color test, 415 nm, 37°C, (25 nkat/L)]

Blood Chemistry Parameters:
- Sodium (NA), Potassium (K) and Chloride (CL) => mmol/L [ion selective electrodes (ISE), (Na: 80, K: 1.5, Cl: 60 nmol/L)]
- Inorganic phosphate (INP) => mmol/L [molybdate reaction (0.1 mmol/L)]
- Calcium (CA) => mmol/L [o-cresolphthalein complex without deproteinization (0.2 mmol/L)]
- Urea (UREA) => mmol/L [enzymatic determination with the urease/ glutamate dehydrogenase method (0.5 mmol/L)]
- Creatinine (CREA) => μmol/L [enzymatic determination with the creatininase/ creatinase /sarcosinoxidase method (5 μmol/L)]
- Glucose (GLUC) => mmol/L [hexokinase/glucose-6-phosphate dehydrogenase method (0.11 mmol/L)]
- Total bilirubin (TBIL) => μmol/L [DPD method (0.56 μmol/L)]
- Total protein (TPROT) => g/L [biuret method (2 g/L)]
- Albumin (ALB) => g/L [bromocresol green method (3.2 g/L)]
- Globulins (GLOB) => g/L [difference between total protein and albumin]
- Triglycerides (TRIG) => mmol/L [enzymatic color test with lipase esterase/ glycerokinase/ glycerol-3-phosphate oxidase/4-aminophenazone (0.1 mmol/L)]
- Cholesterol (CHOL) => mmol/L [enzymatic determination with cholesterol esterase/ cholesterol oxidase/4-amino-phenazone (CHOD-PAP method) (0.1 mmol/L)]

Urinalysis
The dry chemical reactions on test strips (Combur-Test 10 M; Sysmex, Norderstedt, Germany)
used to determine urine constituents semiquantitatively were evaluated with a reflection
photometer (Miditron M; Sysmex, Norderstedt, Germany).
Parameters and methods:
- pH => methyl red and bromothymol blue
- Protein (PRO) => tetrabromophenol-phthaleinethylester (TBPE)
- Glucose (GLU) => GOD-POD reaction
- Ketones (KET) => sodium nitroprusside
- Urobilinogen (UBG) => p-methoxyaniline-diazonium-salt
- Bilirubin (BIL) => 2,5-dichloroaniline diazonium salt
- Blood => 2,5-dimethylhexane-2,5-dihydroperoxide, tetramethylbenzidine
- Specific gravity (SP.GR.) [g/L] => refractometer
- Sediment => microscopy
- Color, turbidity (COL, TURB) => by visual evaluation
- Volume (VOL) [mL] => graduated tubes


P
Sacrifice and pathology:
PATHOLOGY

Necropsy
The animals were sacrificed by decapitation under isoflurane anesthesia. The exsanguinated
animals were necropsied and assessed by gross pathology.

Organ weights
The following weights were determined in all animals sacrificed on schedule:
1. Anesthetized animals
2. Adrenal glands
3. Brain
4. Epididymides
5. Heart
6. Kidneys
7. Liver
8. Ovaries
9. Spleen
10. Testes
11. Thymus
12. Thyroid glands
13. Uterus with cervix

Organ/ tissue fixation
The following organs or tissues were fixed in 4% neutral-buffered formaldehyde solution or in
modified Davidson’s solution:
1. All gross lesions
2. Adrenal glands
3. Aorta
4. Bone marrow (femur)
5. Brain
6. Cecum
7. Cervix
8. Coagulating glands
9. Colon
10. Duodenum
11. Epididymides
12. Esophagus
13. Extraorbital lacrimal glands
14. Eyes with optic nerve (modified Davidson’s solution)
15. Femur with knee joint
16. Harderian glands
17. Heart
18. Ileum
19. Jejunum (with Peyer’s patches)
20. Kidneys
21. Larynx
22. Liver
23. Lungs
24. Lymph nodes (mesenteric and axillary lymph nodes)
25. Mammary gland (male and female)
26. Nose (nasal cavity)
27. Ovaries
28. Oviducts
29. Pancreas
30. Parathyroid glands
31. Pharynx
32. Pituitary gland
33. Prostate
34. Rectum
35. Salivary glands (mandibular and sublingual glands)
36. Sciatic nerve
37. Seminal vesicles
38. Skeletal muscle
39. Skin
40. Spinal cord (cervical, thoracic and lumbar cord)
41. Spleen
42. Sternum with marrow
43. Stomach (forestomach and glandular stomach)
44. Testes
45. Thymus
46. Thyroid glands
47. Trachea
48. Urinary bladder
49. Uterus
50. Vagina

Histopathology
Fixation was followed by histotechnical processing, examination by light microscopy and assessment of findings.
Oragns:
1. all animals affected/test group
2. - 17., 19., 21. - 45.: all animals in control and high dose group
18. and 20.: all animals in all groups (control, low, mid and high dose)

1. All gross lesions
2. Adrenal glands
3. Aorta
4. Bone marrow (femur)
5. Brain
6. Cecum
7. Cervix
8. Coagulating glands
9. Colon
10. Duodenum
11. Epididymides
12. Esophagus
13. Eyes with optic nerve
14. Female mammary gland
15. Heart
16. Ileum
17. Jejunum
18. Kidneys
19. Larynx
20. Liver
21. Lung
22. Lymph nodes (mesenteric and axillary lymph nodes)
23. Nose (nasal cavity, level 3)
24. Ovaries
25. Pancreas
26. Parathyroid glands
27. Peyer’s patches
28. Pharynx
29. Pituitary gland
30. Prostate
31. Rectum
32. Salivary glands (mandibular and sublingual glands)
33. Sciatic nerve
34. Seminal vesicles
35. Skin
36. Spinal cord (cervical, thoracic and lumbar cord)
37. Spleen
38. Stomach (forestomach and glandular stomach)
39. Testes
40. Thymus
41. Thyroid glands
42. Trachea
43. Urinary bladder
44. Uterus
45. Vagina

For further classification of the pigment observed in the kidneys of some males of test group 3
(150 mg/kg bw/d), additional sections of the kidneys of animal No. 1 (control group) and animal
No. 31 (test group 3 [150 mg/kg bw/d]) were stained with Perl’s stain for detection of
hemosiderin (detection of Fe3+), Schmorls stain indicating structures that reduce Fe3+ to Fe2+
(e.g. lipofuscin, porphyrin), Periodic acid-Schiff reaction (PAS stain) for polysaccharides,
neutral muco-substances. Furthermore, unstained slides were taken from these two animals
for autofluorescence examination.
The organs were trimmed according to the “Revised guides for organ sampling and trimming
in rats and mice” (Ruehl-Fehlert et al., 2003; Kittel et al., 2004; Morawietz et al., 2004).
A correlation between gross lesions and histopathological findings was attempted.
Peer review
After completion of the histopathological assessment by the study pathologist an internal peer
review was performed by an additional pathologist including liver
and kidneys of males and females of control and all test groups. Results presented in this
report reflect the consensus opinion of the study pathologist and the peer review pathologist.
Statistics:
Detailed statistical analyses were conducted.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Salivation within 2 hours after treatment from slight to severe was observed in 7 male (Nos. 31
to 33, 36 and 38 to 40) and in all female (Nos. 71 to 80) animals of test group 3 (150 mg/kg
bw/d) from study day 10 onwards until the end of the administration period. Additionally, in
3 female animals (Nos. 62, 63 and 70) of test group 2 (30 mg/kg bw/d) slight salivation within
2 hours after treatment was observed. From the temporary, short appearance immediately
after dosing it was concluded that salivation was induced by a local affection of the upper
digestive tract by the test-substance preparation. This finding was considered by the authors
as treatment-related but not adverse.
No clinical findings were observed in male animals of test groups 1 and 2 (5 and 30 mg/kg
bw/d) and female animals of test group 1 (5 mg/kg bw/d).
Mortality:
no mortality observed
Description (incidence):
No animal died prematurely in the present study.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
In male animals of test group 3 (150 mg/kg bw/d) the body weight was significantly decreased
from study day 42 onwards until the end of the administration period with a maximum of -13.4%
on study day 84. On study day 91 the body weight of males in test group 3 differed by 13.2%
from control. Furthermore, body weight change values were significantly decreased in these
animals from study day 28 onwards until the end of the administration period with a maximum
of -22.4%. These findings were assessed as test substance-related.
No significant alteration of body weight was observed in females of all test groups. The body
weight changes were significantly increased in females of test group 1 (5 mg/kg bw/d) up to
31.5% and in females of test group 3 (150 mg/kg bw/d) up to 30.4% until study day 14. The
body weight change was not effected in females of test group 2 (30 mg/kg bw/d). Since no
dose-dependent effect was determined, these findings were assessed as incidental and not
treatment related.
No deviations were determined for body weight and body weight change data in male animals
of test groups 1and 2 (5 and 30 mg/kg bw/d) as well as in body weight data in females of all
test groups.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
No test-substance related findings were observed.
During administration period following deviations in food consumption values in comparison to
control group were observed: Food consumption was increased in males of test group 3
(150 mg/kg bw/d) between study days 7 and 14, 14 and 21 as well as 21 and 28 with a
maximum of 25.3%. Females of the same test group showed increased food consumption
between study days 0 and 7, 28 and 35, 35 and 42, 42 and 49, 56 and 63, 70 and 77 as well
as 84 and 91 with a maximum of 29.4%. In males of test group 2 (30 mg/kg bw/d) food
consumption was increased 13.3% between study days 28 and 35. In females of the same test
group food consumption was increased between study days 14 and 21, 21 and 28, 28 and 35,
56 and 63, 70 and 77, 77 and 84 as well as 84 and 91 with a maximum of 34.9%. In female
animals of test group 1 (5 mg/kg bw/d) food consumption was decreased -11.8% between
study days 49 and 56 and increased 12.4% between study days 84 and 91.
In cage 14 with females of test group 2 (30 mg/kg bw/d) spill of diet was determined between
study days 35 and 42 as well as 42 and 49. Therefore, for both observations intervals of
females of test group 2 only one food consumption value is given. Because the increased food
consumptions determined in females of test groups 2 and 3 (30 and 150 mg/kg bw/d) were not
related with body weight gain, the increased food consumption values were most likely caused
by playing with the diet. Therefore, all deviations in food consumption from control values were
assessed as treatment-related but not adverse.
Food efficiency:
not examined
Description (incidence and severity):
N/A
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
No test-substance related findings were observed.
Ophthalmological findings:
no effects observed
Description (incidence and severity):
There were no treatment-related findings.
All apparent findings were assessed as being incidental in nature since they occurred in
individual animals only and did not show a dose-response relationship.
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
After the administration period in rats of both sexes of test group 3 (150 mg/kg bw/d) mean
corpuscular volume (MCV) and mean corpuscular hemoglobin content (MCH) were
significantly decreased. Additionally, in males of the same test group red blood cell (RBC)
counts were significantly increased, and in females hemoglobin and hematocrit values were
significantly decreased. In males of test group 2 (30 mg/kg bw/d) RBC counts were already
significantly higher and MCV values were significantly lower compared to controls, but both
values were within historical control ranges (males RBC 8.31-9.08 Tera/L; MCV 47.8-51.1 fL).
Therefore, the changes in males of test group 2 were regarded as incidental and not treatmentrelated.
In females of test group 3 (150 mg/kg bw/d) total white blood cell (WBC) and absolute
monocyte and lymphocyte counts were significantly increased. Absolute large unstained cell
(LUC) counts were also significantly higher compared to controls, but they were within the
historical control range (females LUC 0.00-0.02 Giga/L). This increase was most probably due
to the inclusion of activated monocytes within this cell fraction. Therefore, it was regarded as
secondary to the increase of absolute monocytes and not as an independent adverse effect.
In males of test group 3 (150 mg/kg bw/d) platelet and absolute and relative monocyte counts
were significantly higher compared to controls, but the values were within historical control
ranges (platelets, relative monocyte counts) or marginally above the historical control range
(absolute monocyte counts) (males, platelet counts 643-836 Giga/L; absolute monocytes 0.07-
0.15 Giga/L; relative monocytes 1.5-2.8 %). Therefore, these alterations were regarded as
incidental and not treatment-related.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
After the administration period in males of test group 3 (150 mg/kg bw/d) alanine
aminotransferase (ALT), aspartate aminotransferase (AST) and alkaline phosphatase (ALP)
activities were significantly increased.
In rats of both sexes of test group 3 (150 mg/kg bw/d) inorganic phosphate levels were
significantly higher compared to controls. Additionally, in males of this test group creatinine
levels were significantly decreased and total bilirubin levels were significantly increased. In
females of test group 3 urea levels were significantly higher compared to controls.
In males of test group 3 (150 mg/kg bw/d) glucose and sodium values were significantly lower
and potassium levels significantly higher compared to controls. In males of test group 2
(30 mg/kg bw/d) creatinine values were already significantly decreased. However, all in this
paragraph mentioned parameters were within historical control ranges (males, glucose 5.19-
7.37 mmol/L; sodium 141.1-148.1 mmol/L; potassium 4.39-5.04 mmol/L; creatinine 24.0-
32.0 μmol/L). Therefore, these alterations were regarded as incidental and not treatmentrelated.
Urinalysis findings:
no effects observed
Description (incidence and severity):
No treatment-related changes among urinalysis parameters were observed.
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
Functional observational battery
Deviations from "zero values" were obtained in several animals. However, as most findings were equally distributed between test-substance treated groups and controls, were without a
dose-response relationship or occurred in single animals only, these observations were considered to have been incidental.
The following examinations were performed during FOB and have to be assessed individually:
Home cage observations:
No test-substance related effects were observed.
Open field observations:
No test-substance related effects were observed.
Sensorimotor tests/ reflexes:
No test-substance related effects were observed.
Quantitative parameters:
No test-substance related effects were observed.

Motor activity measurement
Regarding the overall motor activity as well as single intervals, no test substance-related
deviations were noted for male and female rats.
In comparison to values of control group single interval 2 was significantly decreased and
single interval 12 was significantly increased in male animals of test group 3 (150 mg/kg bw/d).
These two isolated alterations in opposite direction were regarded as incidental and not
treatment-related.
Immunological findings:
no effects observed
Description (incidence and severity):
No treatment-related changes were observed.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
See table in section "any other information on results incl. tables"
Gross pathological findings:
no effects observed
Description (incidence and severity):
All findings occurred individually. They were considered to be incidental or spontaneous in
origin and without any relation to treatment.
Neuropathological findings:
no effects observed
Description (incidence and severity):
No treatment-related changes were observed (see also section: Behaviour (functional findings).
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
See table in section "any other information on results incl. tables"
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed

Effect levels

open allclose all
Key result
Dose descriptor:
NOAEL
Effect level:
30 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects observed at this dose level.
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Dose descriptor:
LOAEL
Effect level:
150 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
haematology
clinical biochemistry
organ weights and organ / body weight ratios
histopathology: non-neoplastic

Target system / organ toxicity

Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
150 mg/kg bw/day (actual dose received)
System:
other: Liver and Kidney
Organ:
kidney
liver
Treatment related:
yes
Dose response relationship:
no
Relevant for humans:
not specified

Any other information on results incl. tables

Absolute organ weights

When compared with control group 0 (=100%), the following mean absolute weights were significantly changed (statistically significant changes printed in bold):

 

 

Male animals

Female animals

Test group

(mg/kg bw/d)

1

(5)

2

(30)

3

(150)

1

(5)

2

(30)

3

(150)

Terminal body weight

100%

96%

85%**

 

 

 

Adrenal glands

95%

98%

82%**

 

 

 

Kidneys

 

 

 

105%

107%*

121%**

Liver

 

 

 

107*%

103%

140%**

Thymus

78%**

85%

76%**

 

 

 

* : p <= 0.05, **: p <= 0.01

 

All other mean absolute weight parameters did not show significant differences when compared to the control group 0.

Relative organ weights

When compared with control group 0 (=100%), the following mean relative organ weights were significantly changed (statistically significant changes printed in bold):

 

 

Male animals

Female animals

Test group

(mg/kg bw/d)

1

(5)

2

(30)

3

(150)

1

(5)

2

(30)

3

(150)

Brain

101%

103%

113%**

 

 

 

Epididymides

103%

104%

118%**

 

 

 

Heart

104%

104%

108%**

 

 

 

Kidneys

108%*

107%

121%**

102%

109%*

117%**

Liver

100%

107%*

131%**

104%

105%

136%**

Testes

100%

103%

118%**

 

 

 

Thymus

79%**

89%

90%

 

 

 

* : p <= 0.05, **: p <= 0.01

 

All other mean relative weight parameters did not show significant differences when compared to the control group 0.

The reduced terminal body weight of males in test group 3 (150 mg/kg bw/d) was outside the historical control data (see Appendix) and regarded to be treatment-related. The increase in absolute and relative liver weight in females of test group 3 (150 mg/kg bw/d) was outside the range of historical control data and regarded to be treatment-related. Whereas the slight but significant increase of absolute liver weight in test group 1 females (5 mg/kg bw/d) was thought to be incidental due to a missing dose response-relationship and unchanged relative organ weight. In males of test group 3 (150 mg/kg bw/d) the significant increase in relative liver weight was above historical control values and therefore regarded to be treatment-related (see Appendix). The significant increase in relative liver weight of males of test group 2 (30 mg/kg bw/d) was regarded not to be treatment-related due a missing histopathologic correlate and the organ weight was within the historical control values (see Appendix). The significantly increased relative kidney weight in test group 3 males (150 mg/kg bw/d) were above historical control values and was therefore also regarded to be treatment-related (see Appendix). The significantly increased absolute and relative kidney weight in females of test group 3 (150 mg/kg bw/d) was above historical control data (see Appendix) and therefore regarded to be treatment-related. The significant increase in absolute and relative kidney weight of females of test group 2 (30 mg/kg bw/d) was within historical control values (see Appendix) and there were no microscopic findings observed in this test group for which reason it was assumed not to be treatment-related.

The reduction of absolute thymus weight in males of test group 1 (5 mg/kg bw/d) and test group 3 (150 mg/kg bw/d) was thought to be incidental in test group 1 (5 mg/kg bw/d) due to a missing dose response relationship and as it was still within the range of historical control data (see Appendix). In test group 3 (150 mg/kg bw/d) it was regarded to be caused by the decrease in terminal body weight and therefore secondary and no direct effect of the test substance. Also the reduced absolute weights in adrenal glands in test group 3 (150 mg/kg bw/d) males was regarded to be secondary to the reduced body weight as in addition no histopathological findings were observed that could explain the weight reduction. The significant increase in relative weight of brain, epididymides, heart and testes in males of test group 3 (150 mg/kg bw/d) was a consequence to the reduced terminal body weight and therefore no direct effect on the organ weights and regarded to be non-adverse.

Histopathology

 

Treatment-related findings were observed in the kidneys and liver of male and female animals with incidences and grading according to the table below:

 

Liver

Male animals

Female animals

Test group

(mg/kg bw/d)

0

(0)

1

(5)

2

(30)

3

(150)

0

(0)

1

(5)

2

(30)

3

(150)

No. of animals

10

10

10

10

10

10

10

10

Hypertrophy, diffuse

0

0

0

5

0

0

0

0

·        Grade 3

 

 

 

3

 

 

 

 

·        Grade 4

 

 

 

2

 

 

 

 

Hypertrophy, intermediate

0

0

0

5

0

0

0

5

·        Grade 2

 

 

 

 

 

 

 

4

·        Grade 3

 

 

 

4

 

 

 

1

·        Grade 4

 

 

 

1

 

 

 

 

Hypertrophy, centrilobular

0

0

1

0

0

0

0

5

·        Grade 1

 

 

1

 

 

 

 

1

·        Grade 2

 

 

 

 

 

 

 

2

·        Grade 3

 

 

 

 

 

 

 

2

Hyperplasia oval/ Kupffer cells

0

0

0

10

0

0

0

0

·        Grade 1

 

 

 

7

 

 

 

 

·        Grade 2

 

 

 

2

 

 

 

 

·        Grade 3

 

 

 

1

 

 

 

 

Karyomegaly

0

0

0

10

0

0

0

10

·        present

 

 

 

10

 

 

 

10

Inclusions, nuclear

0

0

0

10

0

0

0

10

·        Grade 1

 

 

 

 

 

 

 

1

·        Grade 2

 

 

 

 

 

 

 

3

·        Grade 3

 

 

 

2

 

 

 

5

·        Grade 4

 

 

 

8

 

 

 

1

Necrosis, single cell

0

0

0

3

0

0

0

0

·        Grade 2

 

 

 

3

 

 

 

 

 

The liver all test group 3 animals (150 mg/kg bw/d) revealed liver cell hypertrophy which was either, diffuse, intermediate or centrilobular. Males were more severely affected than females. The single male animal of test group 2 (30 mg/kg bw/d) showing a minimal centrilobular hypertrophy was regarded to be incidental as no other findings were observed and no dose-response relationship was present. In addition, male animals of test group 3 (150 mg/kg bw/d) revealed oval cell and/or Kupffer cell hyperplasia. Furthermore, three male animals of this test group had a single cell necrosis which means an increase in liver cell death. All males and all females of test group 3 (150 mg/kg bw/d) revealed intranuclear inclusions comparable to those observed in the kidneys. The nuclei were often enlarged (karyomegaly), the chromatin was marginalized had a condensed and star-shaped form caused by the inclusions, and showed an irregular or oval shape compared to the round nuclei in control animals. All these findings were regarded to be treatment-related.

 

Kidneys

Male animals

Female animals

Test group

(mg/kg bw/d)

0

(0)

1

(5)

2

(30)

3

(150)

0

(0)

1

(5)

2

(30)

3

(150)

No. of animals

10

10

10

10

10

10

10

10

Inclusions, nuclear

0

0

0

9

0

0

0

2

·        Grade 1

 

 

 

4

 

 

 

2

·        Grade 3

 

 

 

4

 

 

 

 

·        Grade 4

 

 

 

1

 

 

 

 

Necrosis single cells

0

0

0

1

0

0

0

0

·        Grade 1

 

 

 

1

 

 

 

 

Pigment storage, tubular

0

1

3

5

0

0

1

0

·        Grade 1

 

1

3

4

 

 

1

 

·        Grade 2

 

 

 

1

 

 

 

 

 

In males and females of test group 3 (150 mg/kg bw/d) in tubular epithelial cells of the cortex intranuclear inclusions were observed. These inclusions were mostly not membrane-bound, granular, eosinophilic and caused a marginalization of the chromatin. This finding was regarded to be treatment-related. One male animal of test group 3 (150 mg/kg bw/d) showed a minimal increase in single cell death. This was regarded to be a consequence to the intranuclear inclusions and therefore treatment-related.

Single males of all test groups and one female of test group 2 (30 mg/kg bw/d) revealed a light brown to orange pigment within the cytoplasm of tubular epithelial cells. This pigment was negative for PAS and Schmorl stain and did not show autofluorescence when excited by UV light. In males this finding was regarded to be treatment-related, in the single female it was regarded to be incidental due to a missing dose-response relationship.

 

All other findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.

 

Applicant's summary and conclusion

Conclusions:
The administration of 2-Propyn-1-ol, compd. with methyloxirane by gavage to male and female
Wistar rats for 3 months caused signs of systemic toxicity at the highest dose level tested
(150 mg/kg bw/d). The target organs were liver and kidney for both genders.
Therefore, under the conditions of the present study the no observed adverse effect level
(NOAEL) was 30 mg/kg bw/d for male and female Wistar rats.
Executive summary:

2-Propyn-1-ol, compd. with methyloxirane was administered by gavage to groups of 10 male

and 10 female Wistar rats at dose levels of 0 (test group 0, vehicle control), 5 (test group 1),

30 (test group 2) and 150 mg/kg bw/d (test group 3) over a period of 3 months.

The test substance was tested as delivered by the sponsor without further adjustments as

all constituents are regarded being part of the test substance (including ca. 34 % water content).

With regard to clinical examinations, signs of general systemic toxicity were only observed in

males of test group 3 (150 mg/kg bw/d) manifested in decreased body weight observable from

study day 42 onwards. At the end of the study the body weight of males in this test group were

decreased by 13.2% in comparison to control.

Regarding clinical pathology in females of test group 3 (150 mg/kg bw/d) a microcytic anemia

was observed, because of decreased hemoglobin, hematocrit and mean corpuscular volume

(MCV) values. In males of the same test group the anemia was compensated by the release

of more red blood cells in the circulation. Additionally, in females of test group 3, higher total

white blood cell (WBC) and absolute monocyte and lymphocyte counts indicated an acute

phase reaction.

Regarding clinical chemistry, the liver is the target organ because of increased liver enzyme

values in males of test group 3 (150 mg/kg bw/d; increased alanine and aspartate

aminotransferase (ALT; AST) as well as alkaline phosphatase (ALP) activities). There was also

a dysregulation of the protein metabolism in the liver cells because of lower creatinine values

in males, but higher urea levels in females of test group 3 (150 mg/kg bw/d). The reason for

higher total bilirubin values in males of this test group may be a higher metabolism of

hemoglobin as consequence of the compensated anemia or also a dysregulation of the liver

cells in these individuals.

Regarding pathology, findings were seen in the kidneys and the liver. In the kidneys of nine

males and two females of test group 3 (150 mg/kg bw/d) there were intranuclear inclusions

observed. One male of test group 3 (150 mg/kg bw/d) had an increase of single cell necrosis.

In addition, males of all test groups revealed an intracytoplasmatic stored light brown to orange

pigment in tubular epithelial cells which was negative with all special stains performed. In the

single female of test group 2 (30 mg/kg bw/d), the single male of test group 1 (5 mg/kg bw/d),

and the three males of test group 2 (30 mg/kg bw/d) the pigment storage was only minimal

and the only finding with regard to the findings mentioned above. It was therefore thought to

be non-adverse if related to treatment at all.

For mice and rats it is described in literature that diverse substances can cause these

intranuclear inclusions in tubular epithelial cells and also pigment storage within the cytoplasm

(Dietrich et al., 2008; Radi et al. 2013). In the described cases it was mostly protein

accumulation within the nucleus and it was regarded to be a stress-reaction of the cell to the

substance administered. In this study no further determination of the inclusions was performed

and the pigment could not be determined by the special investigations undertaken. Therefore,

and with the information from literature, these findings of animals of test group 3 (150 mg/kg

bw/d) were regarded to be treatment-related and adverse.

In the liver of all males and females of test group 3 (150 mg/kg bw/d) different forms of liver

cell hypertrophy were observed (either diffuse, intermediate or centrilobular). Furthermore, the

nuclei revealed similar inclusions as described for the kidney. Whenever “Inclusion, nuclear”

was diagnosed, there was in addition karyomegaly and abnormal chromatin

condensation/formation detected which was regarded to have been a consequence to the

inclusions. The inclusions were fine granular and eosinophilic and in most cases did not have

a visible surrounding membrane. In addition, three males of test group 3 (150 mg/kg bw/d)

showed an increase in numbers of single cell death which was regarded a consequence to the

above mentioned findings. All males revealed hyperplasia of oval cells and/or Kupffer cells.

This was regarded to be a reaction to the degenerative/hepatotoxic insult where the liver tried

to “repair” the cell loss or damaged hepatocytes. In literature it is described that stress protein

synthesis was observed prior to overt hepatic injury (Goering et al., 1993). Therefore, these

findings might be a reaction to a hepatotoxic substance and were regarded to be adverse.

The decrease of the terminal body weight in males of test group 3 (150 mg/kg bw/d) as well as

the increase in kidney and liver weight in males and females of test group 3 (150 mg/kg bw/d)

were regarded to be a cause of the test substance administration and a consequence to the

above mentioned findings in this organs. Therefore, it was regarded to be adverse.