Registration Dossier

Administrative data

Description of key information

OECD 408, NOAEL (male/female, systemic): 25 mg/kg bw/d -> adverse effects seen in hematological system (mid and high dose), liver, kidney and reproductive organs (high dose) (BASF, 2018) 

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Feb 2017 - May 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Version / remarks:
adopted 21 Sep 1998
Deviations:
no
Principles of method if other than guideline:
additional extended histophatological investigation of reproduction parameter, e.g. sperm motality etc ... see observation and examinations
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
further information available within analytical report 16L00577
Species:
rat
Strain:
Wistar
Details on species / strain selection:
Crl:WI(Han)
Supplier: Charles River Laboratories, Research Models and Services GmbH, Sulzfeld, Germany

The 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
- Age when supplied: 29 ± 1 days
- Age at the beginning of the administration period: 42 ± 1 days
- Fasting period before study: no
- Housing: The animals were housed together (5 animals per cage) in polysulfonate cages supplied by TECNIPLAST, Hohenpeißenberg, Germany (floor area about 2065 cm2). 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, The Netherlands, were added for environmental enrichment.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 13d

DETAILS OF FOOD AND WATER QUALITY: The food used was ground Kliba maintenance diet mouse/rat “GLP”, meal, supplied by Provimi Kliba SA, Kaiseraugst, Switzerland. The drinking water is regularly assayed for chemical contaminants by the municipal authorities of Frankenthal and by the Environmental Analytics Water/Steam Monitoring Department of BASF SE as well as for the presence of microorganisms by a contract laboratory.


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24°C
- Humidity (%): 45-65%
- Air changes (per hr): 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours

Route of administration:
oral: gavage
Vehicle:
water
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: The test substance was applied as a solution. Then, drinking 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 at refrigerator. The administration volume was 10 mL/kg body weight.

VEHICLE
- Justification for use and choice of vehicle (if other than water): commonly used vehicle
- Concentration in vehicle: up to 25%
- Amount of vehicle (if gavage): in total 4ml/kg bw were given (up to 25% TI content)
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 Competence Center Analytics of BASF SE, Ludwigshafen, Germany, as a part of this study. BASF project No. 17L00197

The 1H and 13C NMR spectra show the expected signals for the given chemical identity. The 13C spectrum shows additional signals of 2-propenyl-1-ol as side component (0.2%). The purity of the test item is 98.9% corrected area % a a mixture (exthoxylation degree 0-3, major 77% n=1, 1,7% n=0 ). The water content of the sample was measured as 0.3%/100g.

Concentration control analyses
The found concentrations (samples 3 - 5 and samples 8 - 10) of 2-Propyn-1-ol, polymer with ethylene oxide (>1 <2.5 mol EO) in drinking water were found to be in the range of 90 % - 110 % of the nominal concentrations. These results demonstrated the correctness of the concentrations of 2-Propyn-1-ol, polymer with ethylene oxide (>1 <2.5 mol EO) in drinking water

Duration of treatment / exposure:
90d
Frequency of treatment:
once daily
Dose / conc.:
400 mg/kg bw/day (actual dose received)
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
25 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale based on read across TNO 85R0334/04x029 and previously conduted range finder study
Positive control:
no
Observations and examinations performed and frequency:
CLINICAL EXAMINATIONS
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 high). 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. Impairment of gait
12. Lacrimation
13. Palpebral closure
14. Exophthalmus
15. Feces (appearance/ consistency)
16. Urine
17. Pupil size


Food consumption
Food consumption was determined weekly (as representative value over 7 days) for each cage and calculated as mean food consumption in grams per animal and day

Water consumption
Drinking water consumption was monitored by daily visual inspection of the water bottles for any 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 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 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 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 excreted within 2 minutes (appearance/ consistency)
17. Urine excreted within 2 minutes (amount/ color)
18. Rearing within 2 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 (auditory startle response)
7. Coordination of movements (righting response)
8. Behavior during handling
9. Vocalization
10. Pain perception (tail pinch)
11. Grip strength of forelimbs
12. Grip strength of hindlimbs
13. Landing foot-splay test
14. Other findings


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 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 (400 mg/kg bw/d) were examined for any changes using an ophthalmoscope (HEINE OPTOTECHNIK, Herrsching, Germany) after application of a mydriatic agent (Mydrum®, Bausch + Lomb GmbH, Berlin, Germany).


Estrous cycle determination
Estrous cycle length and normality were evaluated daily for all female animals for a minimum of 3 weeks prior to necropsy.


Statistics of clinical examinations
Means and standard deviations of each test group were calculated for several parameters
1. Body weight, body weight change
2. Rearing, grip strength forelimbs, grip strength hindlimbs, foot-splay test, motor activity, estrous cycle

CLINICAL PATHOLOGY
Blood samples on study day 15 for hematology were taken from not fasted animals by puncturing the retro-bulbar venous plexus under isoflurane anesthesia. Blood samples at the end of the administration period were taken from fasted animals by puncturing the retro-bulbar venous plexus under isoflurane anesthesia. Blood sampling and examination were carried out in a randomized sequence (the list of randomization instructions was compiled with a computer).
On the afternoon preceding the day fixed for urinalysis, the animals were transferred individually into metabolism cages (no food or drinking water provided). Urine was sampled overnight. On the following day, the samples were examined in a randomized sequence (the list of randomization instructions was compiled with a computer).

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.


Hematology
The following parameters were determined in blood with EDTA K3 as anticoagulant

1 Leukocyte count
(WBC)
2 Erythrocyte count
(RBC)
3 Hemoglobin
(HGB)
4 Hematocrit
(HCT)
5 Mean corpuscular volume
(MCV)
6 Mean corpuscular hemoglobin
(MCH)
7 Mean corpuscular hemoglobin concentration
(MCHC)
8 Platelet count
(PLT)
9 Differential blood count
10 Reticulocytes (RET)

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.

Clotting tests were carried out using a ball coagulometer
1 Prothrombin time (Hepato Quick’s test)
(HQT)

Clinical chemistry

Enzyme (systematic name and system number) parameters
1 Alanine aminotransferase
(ALT)
2 Aspartate aminotransferase
(AST)
3 Alkaline phosphatase
(ALP)
4 g-Glutamyltransferase
(GGT)

Blood Chemistry Parameter
1 Sodium
(NA)
2 Potassium
(K)
3 Chloride
(CL)
4 Inorganic phosphate
(INP)
5 Calcium
(CA)
6 Urea
(UREA)
7 Creatinine
(CREA)
8 Glucose
(GLUC)
9 Total bilirubin
(TBIL)
10 Total protein
(TPROT)
11 Albumin
(ALB)
12 Globulins
(GLOB)
13 Triglycerides
(TRIG)
14 Cholesterol
(CHOL)


Urinalysis

1 pH
2 Protein (PRO)
3 Glucose (GLU)
4 Ketones (KET)
5 Urobilinogen (UBG)
6 Bilirubin (BIL)
7 Blood
8 Specific gravity (SP.GR.) [g/L]
9 Sediment
10 Color, turbidity (COL, TURB)
11 Volume (VOL) [mL]

Sperm parameters
After the organ weight determination, the following parameters were determined in the right testis or right epididymis of all male on schedule:
1 Sperm motility
2 Sperm morphology
3 Sperm head count (cauda epididymis)
4 Sperm head count (testis)
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. Cauda epididymis
5. Epididymides
6. Heart
7. Kidneys
8. Liver
9. Ovaries
10. Pituitary gland
11. Prostate
12. Seminal vesicles with coagulation glands
13. Spleen
14. Testes
15. Thymus
16. Thyroid glands
17. 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, left (modified Davidson’s solution)
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. Testis, left (modified Davidson’s solution)
45. Thymus
46. Thyroid glands
47. Trachea
48. Urinary bladder
49. Uterus
50. Vagina

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.

For classification of observed pigment in the spleen of animals of test group 3 (400 mg/kg bw/d) a Turnbull stain (detection of iron) in the spleen of animals No. 1, 31, 41 and 71 was performed exemplarily.
Other examinations:
Histopathology
Fixation was followed by histotechnical processing, examination by light microscopy

For highest and control dose the following organs were examined:

Organs

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. Epididymis, left
12. Esophagus
13. Eyes with optic nerve
14. Female mammary gland
15. Heart
16. Ileum
17. Jejunum
18. Kidneys
19. Liver
20. Lung
21. Lymph nodes
(mesenteric and axillary lymph nodes)
22. Ovaries
23. Pancreas
24. Parathyroid glands
25. Peyer’s patches
26. Pituitary gland
27. Prostate
28. Rectum
29. Salivary glands
(mandibular and sublingual glands)
30. Sciatic nerve
31. Seminal vesicles
32. Skin
33. Spinal cord
(cervical, thoracic and lumbar cord)
34. Spleen
35. Stomach (forestomach and glandular stomach)
36. Testis, left
37. Thymus
38. Thyroid glands
39. Trachea
40. Urinary bladder
41. Uterus
42. Vagina
Statistics:
see below, different statistical test are used depending on the parameter
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
In test group 3 (400 mg/kg bw/d) the following clinical signs were observed within 2 hours after application: Slight to severe salivation, closed or semiclosed eyelids of both eyes as well as slight to severe poor general condition were seen in all males and all females intermittently over the entire study period. An abdominal position was observed in all males and all females on several days of the administration period starting on study day 0. Furthermore, piloerection was observed in 9 male and 7 female on several days over the entire study period. 8 males and 2 females showed colorless lacrimation in both eyes from study day 0 to 9. Red smeared fur in the nose region had 2 males and 4 females.One Male showed hypothermia on study day 2. Between 2 and 5 hours after treatment 5 males and 1 female of test group 3 (400 mg /kg bw/d) showed red smeared fur in the nose region. Smeared fur in the nose region occurred between study day 1 and 15.

Following clinical signs occurred in animals of test group 2 (100 mg/kg bw/d) within 2 hours after application: Slight to moderate salivation was observed in 8 male and 9 female animals starting on study day 14. Piloerection was seen in 3 male animals on single days of the administration period. Slight poor general condition was observed in 2 males and 1 female on study days 0 and 4. On study day 4 one male showed semiclosed eyelids and an abdominal position. Red smeared fur in the nose region was observed in 6 males and 3 females. Between 2 and 5 hours after treatment 5 males and 1 female of test group 2 (100 mg /kg bw/d) showed red smeared fur in the nose region.
Smeared fur in the nose region occurred from study day 0 to 2. All animals recovered from above mentioned clinical signs either within 5 hours after application or overnight.

Animals of test groups 0 and 1 (0 and 25 mg/kg bw/d) were without any clinical signs

The slight to sever salivation observed within 2 hours after administration but not later, was considered to be related to a bad test or slight irritant properties of the test substance preparation administrated by gavage. This finding is mediated by a local physiological activation of the parasympathetic nervous system. The observed lacrimation and smeared fur in the nose region were assessed as further consequences of it. The observation of smeared fur in the nose region with a higher incidence observed after 2-5 hours comparted to 0-2 hours after treatment was not considered as a late occurrence of this clinical finding. Since the first clinical observation after administration (0-2 hours) were made within half an hour after administration in general, it is most likely that a rhinorrhea got more often visible in smeared fur in the nose region slightly delayed and in its consequences still present after two hours. This finding was not observed before an administration. Therefore, the clinical findings salivation, lacrimation and smeared fur in the nose region were assessed as treatment-related but not adverse.
The clinical findings semi- and closed eyelid, piloerection, poor general conditions, and abdominal position in test group 3 (400 mg/kg bw/d) and 2 (100 mg/kg bw/d) were assessed as treatment-related and adverse.

Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Body weight was reduced in males of test group 3 (400 mg/kg bw/d) from study day 42 onwards up to -11.4% on study day 91. Furthermore, body weight gain was reduced in males over entire study period up to -19.1% on study day 91 (values on study days 21 and 28 were reduced not significant).
No test-substance related changes of mean body weights and mean body weight change values were observed in both sexes of test groups 1 and 2 (25 and 100 mg/kg bw/d) and in female animals of test group 3 (400 mg/kg bw/d).
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
no effects observed
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
After two weeks of compound administration in rats of both sexes of test groups 2 and 3 (100 and 400 mg/kg bw/d) hematocrit values were significantly decreased. Additionally, in rats of both sexes of test group 3 (400 mg/kg bw/d) and in males of test group 2 (100 mg/kg bw/d) hemoglobin values were significantly lower compared to controls. In females of the mentioned test groups 2 and 3 red blood cell (RBC) counts were significantly decreased. Absolute reticulocyte counts were significantly increased in male and female rats of test group 3 (400 mg/kg bw/d) and additionally in males of test groups 1 and 2 (25 and 100 mg/kg bw/d). However, in test group 1 this is the only changed hematology parameter among these individuals and therefore, in this test group this isolated alteration was regarded as treatment-related, but non-adverse (ECETOC Technical Report No. 85, 2002). In rats of both sexes of test group 3 (400 mg/kg bw/d) platelet counts were significantly decreased. All mentioned changes in this paragraph apart from higher reticulocyte counts in males of test group 1 were regarded as treatment-related and adverse.
After three months of administration in rats of both sexes of test group 3 (400 mg/kg bw/d) hemoglobin and hematocrit values as well as mean corpuscular volume (MCV) and mean corpuscular hemoglobin content (MCH) were significantly decreased. These changes were regarded as adverse.
At the end of administration in rats of both sexes of test group 3 (400 mg/kg bw/d), total white blood cell (WBC) counts as well as absolute neutrophils and eosinophils counts were significantly decreased. This was also true in males of test group 3 for significantly decreased lymphocyte counts. Relative monocyte counts were significantly increased in rats of both sexes of the mentioned test group. These mentioned alterations of the total white and differential cell counts were regarded as adverse.

Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
At the end of the administration period in rats of both sexes of test group 3 (400 mg/kg bw/d) total bilirubin values were significantly increased. Additionally, in males of the mentioned test group creatinine and cholesterol values were significantly decreased and triglyceride levels were significantly increased. In females of test group 3 inorganic phosphate levels were significantly higher compared to controls. In one male rat of test group 3 (no. 33) very high liver enzyme activities (alanine aminotransferase (ALT, aspartate aminotransferase (AST) and alkaline phosphatase (ALP)) were observed. These mentioned changes were regarded as treatment-related and adverse.
Urinalysis findings:
effects observed, non-treatment-related
Description (incidence and severity):
At the end of the administration period in male and female rats of test group 3 (400 mg/kg bw/d) urine pH values were decreased (in females not statistically significantly). Additionally, in males of test group 3 specific gravity of the urine was significantly increased and urine volume was decreased, the latter parameter not statistically significantly. The mentioned alterations of urine values were regarded as maybe treatment-related, especially regarding the lower pH-value, but they are not adverse because they do not compromise the normal function of the kidneys.
Behaviour (functional findings):
effects observed, treatment-related
Description (incidence and severity):
The following examinations were performed during FOB and have to be assessed individually:

Home cage observations:
No test substance-related adverse effects were observed.

Open field observations:
In 2 males of test group 3 (400 mg/kg bw/d) piloerection was observed during open field observations. This finding was assessed as treatment-related and adverse.
Male No. 40 of test group 3 (400 mg/kg bw/d) showed reddish nasal discharge. Additionally, in 4 male and 1 female animals of high dose group slight salivation was observed. These finding were assessed as treatment-related but not adverse

Sensorimotor tests/ reflexes:
No test substance-related effects were observed.

Quantitative parameters:
In females of test group 3 (400 mg/kg bw/d) rearing was decreased -47%. Additionally, rearing was decreased -35% in females of test group 2 (100 mg/kg bw/d). These findings were assessed as treatment-related. Furthermore, in females of mid dose group (100 mg/kg bw/d) mean value of food splay test was decreased -16.1%. This finding was observed without dose-dependency. Therefore, it was assessed as incidental and not related to treatment.

Motor activity measurement:
Single interval 1 in females of test group 3 (400 mg/kg bw/d) was decreased in motor activity. This finding was assessed as treatment-related.
Regarding the overall motor activity as well as single intervals, no treatment-related deviations were noted for rats in test groups 1 and 2 (25 and 100 mg/kg) and male rats of test group 3 (400 mg/kg bw/d).
Immunological findings:
not specified
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
The significantly reduced terminal body weight in males of test group 3 (400 mg/kg bw/day) was regarded as treatment-related. The significantly changed weight parameters of adrenal glands, epidiymides, kidneys, liver, ovaries, prostate, testes, thymus and thyroid glands of animals of test group 3 (400 mg/kg bw/day) and the liver weight of males of test group 2 (100 mg/kg bw/day) were regarded to be related to treatment. The significantly changed weight of the adrenal glands and kidneys of test group 2 males (100 mg/kg bw/day) were within historical control values and therefore assumed to be incidental.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Gross lesions were observed in Adreanl glands (reduced size), Epididymides (reduced size), Testis (reduced size, discoloration), Liver (enlarged) and Ovaries (enlarged) at highest dose group.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
In males of test group 3 (400 mg/kg bw/day) in single 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.

In the liver, a single male of test group 3 (400 mg/kg bw/day) showed the same intranuclear inclusions as described for the kidneys and an increase in single cell necrosis. These findings were also regarded to be adverse. Furthermore, males and females of this test group revealed minimal to slight centrilobular and/or diffuse hepatocellular hypertrophy. This was assumed to be the cause for the observed weight increase in both sexes and the macroscopically observed enlargement of the liver. In combination with the findings in clinical pathology, these liver findings were regarded to be treatment-related and adverse.

In the left testis of all males of test group 3 (400 mg/kg bw/day) severe diffuse degeneration of the seminiferous tubules was observed. This was also responsible for the decrease in testicular weight, reduction in organ size and discoloration observed. It led to the findings seen in the left epididymis (oligospermia, debris, cribriform change) of all males of the same test group. Again, this was the cause of reduced weight and reduced organ size. These findings in testis and epididymis were regarded to be treatment-related and adverse. The weight changes in prostate and seminal vesicles were seen as a consequence to the findings in the testis and therefore regarded be adverse.

In the ovaries of test group 3 (400 mg/kg bw/day) five females revealed a higher number of corpora lutea. This was also thought to have caused the weight increase and macroscopically observed enlargement. The meaning of this finding is not clear, especially as no findings in uterus or vagina were detected which often accompany findings in the ovary. As the meaning is not clear in pathology endpoints but an increased estrous cycle length and decrease number of cycles was observed, it was assumed to be treatment-related and adverse.
In the pituitary gland of six males of test group 3 (400 mg/kg bw/day) there was an increase in number of signet ring cells (so-called “castration cells”). Those cells are often observed after loss of the corresponding effective organ (e.g. after thyroidectomy, gonadectomy). With regard to the observed findings in the testes of all males of this test group, the formation of this cells was assumed to be secondary to the degeneration of seminiferous tubules. This finding was regarded to be treatment-related, secondary to the testicular findings and adverse.

In the spleen there was an increase in pigment storage in males of test group 3 (400 mg/kg bw/d) and females of test group 2 and 3 (100 and 400 mg/kg bw/d). With the positive Turnbull stain the pigment could be shown to be an increase in iron. In combination with the findings observed in clinical pathology (higher metabolism of red blood cells), this finding was regarded to be treatment-related and adverse.

The adrenal gland weights in males and females of test group 3 (400 mg/kg bw/day) were significantly decreased. Also, the organ size was decreased in nine males of test group 3 (400 mg/kg bw/day). There was no histopathologic finding, that could explain this weight and organ size reduction. Therefore, the finding was regarded to be treatment-related but not adverse.The same comes true for the thyroid glands in females of test group 3 (400 mg/kg bw/day). There was an increase in organ weight but no histopathologic correlate.
Histopathological findings: neoplastic:
no effects observed
Other effects:
effects observed, treatment-related
Description (incidence and severity):
At the end of the administration period in males of test group 3 (400 mg/kg bw/d) motility of the sperms and sperm head counts in the testis and in the cauda epididymidis were greatly decreased, and 99% of the sperms in the cauda epididymidis were abnormal. This is regarded as adverse.
In males of test group 2 (100 mg/kg bw/d) only the motility of the sperms in the cauda epididymidis was statistically significantly decreased, but the mean was within the historical control range (motility 79-93 %). All other sperm analysis parameters in males of test group 2 were comparable to those of the controls. Therefore, the lower motility of the sperms in males of test group 2 was regarded as incidental and not treatment-related.
Key result
Dose descriptor:
NOAEL
Effect level:
25 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
haematology
gross pathology
histopathology: non-neoplastic
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
100 mg/kg bw/day (nominal)
System:
other: hematological parameters
Organ:
blood
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
400 mg/kg bw/day (nominal)
System:
other: liver, kidney, reproductive tissues/parameters
Organ:
kidney
liver
ovary
pituitary gland
seminal vesicle
testes
Treatment related:
yes
Dose response relationship:
not specified

Absolute organ weights

 

Male animals

Female animals

Test group

(mg/kg bw/d)

1

(25)

2

(100)

3

(400)

1

(25)

2

(100)

3

(400)

Terminal body weight

100%

98%

88%**

 

 

 

Adrenal glands

92%

88%*

65%**

100%

86%

75%**

Brain

99%

98%

93%*

 

 

 

Cauda epididymis

93%

92%

47%**

 

 

 

Epididymides

101%

97%

47%**

 

 

 

Heart

104%

100%

92%*

92%*

97%

105%

Kidneys

 

 

 

97%

99%

114%**

Liver

 

 

 

97%

99%

133%**

Ovaries

 

 

 

105%

102%

135%*

Prostate

94%

94%

58%**

 

 

 

Seminal vesicles

103%

111%

76%**

 

 

 

Spleen

100%

101%

87%*

 

 

 

Testes

101%

100%

46%**

 

 

 

Thymus

95%

99%

72%**

 

 

 

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

 

Relative organ weights

 

Male animals

Female animals

Test group

(mg/kg bw/d)

1

(25)

2

(100)

3

(400)

1

(25)

2

(100)

3

(400)

Adrenal glands

92%

90%*

74%**

103%

89%

74%**

Cauda epididymis

94%

94%

54%**

 

 

 

Epididymides

102%

99%

54%**

 

 

 

Heart

 

 

 

93%*

100%

104%

Kidneys

101%

106%*

108%**

99%

103%

113%**

Liver

104%

107%*

118%**

100%

102%

132%**

Ovaries

 

 

 

108%

106%

132%**

Prostate

93%

96%

66%**

 

 

 

Testes

102%

102%

52%**

 

 

 

Thyroid glands

 

 

 

104%

103%

122%*

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

 

Conclusions:
The administration of 2-Propyn-1-ol, polymer with ethylene oxide (>1 <2.5 mol EO) by gavage to male and female Wistar rats for 3 months caused signs of systemic toxicity. These findings had been observed in males and females of test group 3 (400 mg/kg bw/d) and 2 (100 mg/kg bw/d). Therefore, under the conditions of the present study the no observed adverse effect level (NOAEL) was 25 mg/kg bw/d for male and female rats.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
25 mg/kg bw/day
Study duration:
subchronic
Species:
rat
System:
other: hematological (first effect at LOAEL)
Organ:
blood
kidney
liver
ovary
seminiferous tubules
testes

Additional information

Justification for grouping of substances and read-across

In accordance with Article 13 (1) of Regulation (EC) No 1907/2006, "information on intrinsic properties of substances may be generated by means other than tests, provided that the conditions set out in Annex XI are met.” In particular for human toxicity, information shall be generated whenever possible by means other than vertebrate animal tests, which includes the use of information from structurally related substances (grouping or read-across).

Having regard to the general rules for grouping of substances and read-across approach laid down in Annex XI, Item 1.5, of Regulation (EC) No 1907/2006 whereby substances may be predicted as similar provided that their physicochemical, toxicological and ecotoxicological properties are likely to be similar or follow a regular pattern as a result of structural similarity.

Overview of repeated dose toxicity, oral

Substance

NOAEL (mg/kg bw/day)

Target substance

2-Propyn-1-ol, polymer with ethylene oxide

(CAS 25749-64-8)

 

OECD 408, NOAEL (male/female, systemic): 25 mg/kg bw/d -> adverse effects seen in hematological system (mid and high dose), liver, kidney and reproductive organs (high dose) (BASF, 2018)

Source substance (Read across)

2-Propyn-1-ol, compd. with methyloxirane

(CAS 38172-91-7)

 

OECD 422, NOAEL (male/female, systemic/fertility): 68.4 mg/kg bw (highest dose, a.i.)

-> no (adverse) effects

(BASF SE, 2013)

OECD 408, NOAEL (male/female, systemic):

16.4 mg/kg bw/d (corrected to a.i.)

-> adverse effects seen in hematological system, liver and kidney above (BASF, 2017)

 

The above mentioned substances are considered to behave similar on the basis of structural similarity resulting in similar properties and/or activities. Nevertheless as the read across was challenged by ECHA, OECD 408 studies are performed for both target and source substance.

All this information is taken into consideration here and in the analogue justification attached to underline the that read across was performed to the more detrimental substance and that the read across is further valid in accordance to Regulation (EC) No. 1907/2006 Annex XI, 1.5.

 

OECD guideline study:

Target substance (CAS 25749-64-8)

2-Propyn-1-ol, polymer with ethylene oxide (>1 <2.5 mol EO) was administered by gavage to groups of 10 male and 10 female Wistar rats at dose levels of 0 (test group 0, vehicle control), 25 (test group 1), 100 (test group 2) and 400 mg/kg bw/d (test group 3) over a period of 3 months according to OECD 408 under GLP (BASF 2018).

With regard to clinical examinations, the signs of general systemic toxicity were observed in following findings: In males and females of test group 3 (400 mg/kg bw/d) semiclosed or closed eyelid, general poor conditions, piloerection, hypothermia (male only), abdominal position in either sex and intermittently were observed over the entire study period. The body weight in males of test group 3 was significantly decreased. The estrous cycle length was increased and number of estrous cycles was decreased in females of test group 3. In test group 2 (100 mg/kg) only semiclosed eyelids, general poor conditions, piloerection, and abdominal position were observed intermittently up to study day 4. 

During functional observation battery (FOB) examinations, decreased rearing was observed in females of test group 2 (100 mg/kg bw/d) and 3 (400 mg/kg bw/d) and decreased motor activity in a single interval (interval no. 1) in females of test group 3 while the motor activity over all 12 intervals was not significantly decreased. These findings were rather of unspecific character. The changes were assessed to be related to general toxicity and impaired well-being. 

Concerning clinical pathology, after two weeks of administration in male and female rats of test groups 2 and 3 (100 and 400 mg/kg bw/d) a regenerative, normochromic-normocytic anemia was observed (test group 3: decreased hemoglobin, hematocrit etc). After three months, the compensation of the anemia in test group 2 succeeded, but in test group 3 a normochromic-microcytic anemia developed. After three months of administration in rats of both sexes of test group 3 (400 mg/kg bw/d) a decrease of total white blood cell (WBC) counts due to a neutro- and eosinopenia was observed. Additionally, in males of this test group absolute lymphocyte counts were reduced and prothrombin time was prolonged in female and male rats of test group 3.

At the end of the study, liver cell metabolism seemed to be affected at least in males of test group 3 (400 mg/kg bw/d; decreased creatinine and cholesterol values and increased triglyceride values, increased liver enzyme activities).

At the end of the administration period in males of test group 3 (400 mg/kg bw/d)motility of the sperms and sperm head counts in the testis and in the cauda epididymidis were greatly decreased, and 99% of the sperms in the cauda epididymidis were abnormal.

Regarding pathology, kidneys, liver, testes, epididymides, ovaries, pituitary gland and spleen were target organs. Males of test group 3 (400 mg/kg bw/day) revealed a decrease in terminal body weight which was regarded to be treatment-related and adverse. The increase in kidney weight in males of test group 2 and 3 (100 and 400 mg/kg bw/day) and females of test group 3 (400 mg/kg bw/day) was regarded to be treatment-related but as no histopathologic finding could explain the weight increase it was regarded to be not adverse. Three males of test group 3 (400 mg/kg bw/day) revealed minimal intranuclear inclusions. This finding of males of test group 3 (400 mg/kg bw/day) was assumed to be treatment-related and adverse.

In the liver, a single male of test group 3 (400 mg/kg bw/day) showed the same intranuclear inclusions as described for the kidneys and an increase in single cell necrosis. Furthermore, males and females of this test group revealed minimal to slight centrilobular and/or diffuse hepatocellular hypertrophy. This was assumed to be the cause for the observed weight increase in both sexes and the macroscopically observed enlargement of the liver. In combination with the findings in clinical pathology, these liver findings were regarded to be treatment-related and adverse. The slight increase in relative liver weight (107%) in males of test group 2 (100 mg/kg bw/day) was assumed to be treatment-related, but due to missing clinical pathology and histopathology findings it was regarded to be not adverse.

In the left testis of all males of test group 3 (400 mg/kg bw/day) severe diffuse degeneration of the seminiferous tubules was observed. This was also responsible for the decrease in testicular weight, reduction in organ size and discoloration observed. It led to the findings seen in the left epididymis(oligospermia, debris, cribriform change) of all males of the same test group. These findings in testis and epididymis were regarded to be treatment-related and adverse. The weight changes in prostate and seminal vesicles were seen as a consequence to the findings in the testis and therefore regarded be adverse.

In the ovaries of test group 3 (400 mg/kg bw/day) five females revealed a higher number of corpora lutea. This was also thought to have caused the weight increase and macroscopically observed enlargement. The meaning of this finding is not clear, especially as no findings in uterus or vagina were detected which often accompany findings in the ovary. As the meaning is not clear in pathology endpoints but an increased estrous cycle length and decrease number of cycles was observed, it was assumed to be treatment-related and adverse.

In the pituitary gland of six males of test group 3 (400 mg/kg bw/day) there was an increase in number of signet ring cells (so-called “castration cells”). Those cells are often observed after loss of the corresponding effective organ (e.g. after thyroidectomy, gonadectomy). With regard to the observed findings in the testes of all males of this test group, the formation of this cells was assumed to be secondary to the degeneration of seminiferous tubules. This finding was regarded to be treatment-related, secondary to the testicular findings and adverse.

In the spleen there was an increase in pigment storage in males of test group 3 (400 mg/kg bw/d) and females of test group 2 and 3 (100 and 400 mg/kg bw/d). With the positive Turnbull stain the pigment could be shown to be an increase in iron. In combination with the findings observed in clinical pathology (higher metabolism of red blood cells), this finding was regarded to be treatment-related and adverse.

The adrenal gland weights in males and females of test group 3 (400 mg/kg bw/day) were significantly decreased but without histopathologic finding and therefore regarded not adverse. The same comes true for thethyroid glads

in females of test group 3 (400 mg/kg bw/day).

There were no treatment related effects observed at 25mg/kg bw/d. Therefore the no observed adverse effect level (NOAEL) was 25 mg/kg bw/d for male and female Wistar rats.

 

Source substance (Read across) 2-Propyn-1-ol, compd. with methyloxirane (CAS 38172-91-7):

OECD 422:

The objective of this study was to provide initial data on the possible effects of the test item 2 - propyn-1-ol with methyloxirane (CAS 38172-91-7) on reproductive performance of Wistar rats and the development of pups consequent to daily oral administration of the test item via gavage at concentrations of 0, 5, 25 or 125 mg/kg (a.i. 54.7%) to male and female rats during a premating period of 2 weeks and during mating (1 week), up to a total of approximately 4 weeks for males and including gestation and lactation until postnatal day 4 (PN day 4) for females (up to a total of approximately 6 weeks). The study was performed according to OECD 422 protocol under GLP.

The 2-propyn-1-ol was homogeneously distributed in the gavage liquids.

Daily clinical observations during the premating, gestation and lactation period or neurobehavioural observations and motor activity assessment at the end of the study did not reveal any treatment-related changes in the animal’s appearance, general condition or behaviour.

No treatment-related effects were observed in mean body weight, body weight changes and food consumption in 2-propyn-1-ol compound with methyl oxirane-exposed animals throughout the study.

No treatment-related effects were observed on mating index, male and female fertility indices, gestation index, duration of gestation, number of corpora lutea, implantation sites, lost implantations. No effects were observed on litter size, pup sex and weight and pup survival.

For males and females the No Observed Adverse Effect Level (NOAEL) for systemic toxicity is established at the high dose level of 125 mg/kg bw 2-propyn-1-ol compound with methyl oxirane, based on the absence of adverse effects.

For males and females the NOAEL for fertility is established at the highest dose of 125 mg/kg bw, because no effects were seen on male/female fertility. For use as RA the NOAEL is converted using the purity of 54.7% to 68.4 mg/kg bw/d.

 

OECD 408:

2-Propyn-1-ol, compd. with methyloxirane (CAS 38172-91-7) 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 (BASF SE 2017). 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 thesubstance 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.

In conclusion 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. For use as RA the NOAEL is converted using the purity of 54.7% to 16.4 mg/kg bw/d.

There is no data available on the repeated dose toxicity after dermal application and inhalation.

 

Conclusions

In conclusion, the available data on repeated dose toxicity via the oral route shows a comparable picture from the OECD 408 for the source and the target substance. Anyhow as the target substance (CAS 25749-64-8) was now directly tested no read across is necessary concering this endpoint and the results can be directly used to derive DNELs for risk assessement.

Hence the NOAEL of 25 mg/kg bw/d is used as key result for further calculation. Based on the effects observed at 100 mg/kg bw/d additionally a classification is needed.

Due to the unclear testis effects which can also be happen in connection with general toxicity (as it only happens at dosages which are two stages above the NOAEL) further investigation concerning reproduction toxicity is needed.

As read across to a homologue substance (CAS 38172 -91 -7) with non maternal toxic dosages shows no such effects on testis and no effects on fertility it may be possible that the effects seen in testis here is only in connection with the generals toxicity. Nevertheless as this is currently unclear the substance will be classified in a precaution principle as H361: Suspected of damaging fertility or the unborn child, effect on testis at high doses.

Nevertheless data from the 90d studies underlines the validity of the read across as compareable effects can be observed and also NOEAL values are in the same range with a slight higher toxicity of source substance (this is already assumed in the analogue justification). Comparions of the new derived DNELs with the DNELs based on read across show no or only marginal changes implying that the risk was also adeqatley controlled using information from the read across substance.

 

 

Justification for classification or non-classification

From the available OECD 408 study conducted with the registred substance a LOAEL of 100mg/kg bw/d based on effects on the hämatological system can be deduced, concluding the substance is classified as STOT Rep. Exp. 2, H373: May cause damage to hämatopoetic system through prolonged or repeated exposure.

Labelling repeated dose toxicity:

STOT Rep. Exp. 2, H373: May cause damage to hematological system through prolonged or repeated exposure.