3,7-dimethyloctan-3-ol

Repeated dose toxicity: oral

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:

2019

Report date:

2019

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

GLP compliance:

yes (incl. QA statement)

Remarks:

BASF SE, Experimental Toxicology and Ecology, 67056 Ludwigshafen, Germany

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

3,7-dimethyloctan-3-ol

EC Number:

201-133-9

EC Name:

3,7-dimethyloctan-3-ol

Cas Number:

78-69-3

Molecular formula:

C10H22O

IUPAC Name:

3,7-dimethyloctan-3-ol

Details on test material:

- Name of test substance: Tetrahydrolinalool
- Batch: 00074277LO
- Purity: 99.9 cor. area-%
- Homogeneity: given
- Expiry date: 18 Dec 2019
- Date of production: 18 Dec 2017
- Physical state/ appearance: liquid/ colorless, clear
- Storage conditions: room temperature

Test animals

Species:

rat

Strain:

Wistar

Remarks:

Crl:WI(Han)

Details on species / strain selection:

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 or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services GmbH, Sulzfeld, Germany
- Age at the beginning of the administration period: 42 ± 1 days
- Fasting period before study: no
- Housing: 5 animals per cage
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 13 days

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

Administration / exposure

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Remarks:

mixed in food

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:

DIET PREPARATION
- Mixing appropriate amounts with (Type of food): Test substance was weighted out and mixed with a small amount of food (Premix). Premixes were added to the corresponding amounts of food, depending on test group. Mixing was carried out for about 10 minutes in a laboratory mixer. The stability of the test substance in the diet at room temperature up to 4 days and a stability of the test substance in the diet after a storage of 12 days in the freezer followed by 2 days at room temperature were demonstrated before the start of the administration period.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The stability in the diet was demonstrated. Homogeneity was verified in 3 samples in the highest and lowest concentration (was used as a concentration control at the same time at the beginning and towards the end of the administration period; additional concentration control analyses were done in the mid concentration.

Duration of treatment / exposure:

3 months

Frequency of treatment:

continuous via diet

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

1 500 ppm

Remarks:

Mean daily test substance intake: 94 and 113 mg/kg bw in males and females

Doses / concentrations 2

Dose / conc.:

5 000 ppm

Remarks:

Mean daily test substance intake: 316 and 384 mg/kg bw in males and females

Doses / concentrations 3

Dose / conc.:

15 000 ppm

Remarks:

Mean daily test substance intake: 982 and 1118 mg/kg bw in males and females

No. of animals per sex per dose:

10

Control animals:

yes, concurrent no treatment

Details on study design:

- Dose selection rationale: testing up to limit dose of 1000 mg/kg bw/d
- Rationale for animal assignment: 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.
- Fasting period before blood sampling for clinical biochemistry: yes, fasting period (withdrawal of food) of about 16 to 20 hours.

Positive control:

not applicable

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule:
A check for moribund and dead animals: twice daily on working days and once daily on Saturdays, Sundays and public holidays.
Clinical observations: daily for any abnormal clinically signs.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: in all animals prior to the administration period and thereafter at weekly intervals. 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. Feces (appearance/ consistency)
16. Urine
17. Pupil size

BODY WEIGHT: Yes
- Time schedule for examinations: day 0 (start of the administration period) and thereafter at weekly intervals.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

WATER CONSUMPTION: Yes
- Time schedule for examinations: monitored by daily visual inspection of the water bottles for any changes in volume.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Prior to the start of the administration period and at the end of the administration period, i.e. study day 91.
- Dose groups that were examined: test groups 0 (control) and 3 (15000 ppm)

HAEMATOLOGY: Yes
In the morning blood was taken from the retro-bulbar venous plexus from fasted animals anaesthetized using isoflurane.
The following parameters were determined in blood with EDTA K3 as anticoagulant:
- Leukocyte count
- Erythrocyte count
- Hemoglobin
- Hematocrit
- Mean corpuscular volume
- Mean corpuscular hemoglobin
- Mean corpuscular hemoglobin concentration
- Platelet count
- Differential blood count
- Reticulocytes
Clotting tests were carried out using a ball coagulometer: Prothrombin time (Hepato Quick’s test)

CLINICAL CHEMISTRY: Yes
In the morning blood was taken from the retro-bulbar venous plexus from fasted animals anaesthetized using isoflurane.
The following parameters were determined:
- Alanine aminotransferase
- Aspartate aminotransferase
- Alkaline phosphatase
- gamma-Glutamyltransferase
- Sodium
- Potassium
- Chloride
- Inorganic phosphate
- Calcium
- Urea
- Creatinine
- Glucose
- Total bilirubin
- Total protein
- Albumin
- Globulins
- Triglycerides
- Cholesterol
- Bile acids
- HDL & LDL Cholesterol
- TSH, T3, T4
Thyroid hormones: The concentrations of TSH were determined by radioimmunoassay (RIA), using commercially available RIA test kits and a Gamma-Counter. T3 and T4 was determined via an Elisa.

URINALYSIS: Yes
Individual animals were transferred to metabolism cages (withdrawal of food and water) and urine was collected overnight.
The following parameters were determined:
- pH
- Protein
- Glucose
- Ketones
- Urobilinogen
- Bilirubin
- Blood
- Specific gravity
- Sediment
- Color, turbidity
- Volume

NEUROBEHAVIOURAL EXAMINATION: Yes
A functional observational battery (FOB) was performed in all animals at the end of the administration period starting at about 10:00 h. 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.
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 from 14:00 h onwards on the same day as the FOB was performed. 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.

OTHER:
Estrous cycle determination
Vaginal smears for terminal vaginal cytology examinations were prepared in the morning of the day of sacrifice.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
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 (final body weight)
2. Adrenal glands (fixed)
3. Brain
4. Epididymides
5. Heart
6. Kidneys
7. Liver
8. Ovaries (fixed)
9. Pituitary gland (fixed)
10. Prostate (ventral and dorsolateral part together, fixed)
11. Spleen
12. Seminal vesicles including coagulating glands (fixed)
13. Testes
14. Thymus (fixed)
15. Thyroid glands (with parathyroid glands) (fixed)
16. Uterus with cervix

HISTOPATHOLOGY: Yes (see table)
Organs/tissues were fixed in 4% neutral-buffered formaldehyde solution. Testes, epididymides and eyes with optic nerve were fixed in modified Davidson’s solution.
Histopathological examination was performed in:
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. Femur with knee joint
15. Heart
16. Ileum
17. Jejunum
18. Kidneys
19. Liver
20. Lung
21. Lymph nodes (mesenteric and axillary lymph nodes)
22. Mammary gland (female)
23. Ovaries
24. Pancreas
25. Parathyroid glands
26. Peyer’s patches
27. Pituitary gland
28. Prostate
29. Rectum
30. Salivary glands (mandibular and sublingual glands)
31. Sciatic nerve
32. Seminal vesicles
33. Skeletal muscle
34. Skin
35. Spinal cord (cervical, thoracic and lumbar cord)
36. Spleen
37. Stomach (forestomach and glandular stomach)
38. Testes
39. Thymus
40. Thyroid glands
41. Trachea
42. Urinary bladder
43. Uterus
44. Vagina

For clear evaluation of eosinophilic droplets in the cortex of male kidneys, a CAB stain was made and evaluated by light microscopy of all male animals. For further classification, an immunohistochemical stain against alpha 2µ was performed.
Special attention was given for the synchrony of the morphology of the estrous cycle in ovaries, uterus, cervix, and vagina.
Special attention was given for the male reproductive organs, especially the stage of seminiferous tubules.

Statistics:

DUNNETT's test: Body weight, body weight change
KRUSKALWALLIS test; Posttest WILCOXON: Rearing, grip strength forelimbs, grip strength hindlimbs, footsplay test, motor activity, Blood parameters, Urine pH, volume and specific gravity, organ weights.
WILCOXON test: Urinalysis parameters (apart from pH, urine volume, specific gravity, color and turbidity)

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Description (incidence and severity):

No test substance-related findings were observed. For further details, see background material attached.

Mortality:

no mortality observed

Description (incidence):

No animal died prematurely in the present study. For further details, see background material attached.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Mean body weight: No significantly changes observed.
Mean body weight change: significantly decreased in high dose male animals (15000 ppm) on study day 7 (-16%) and in high dose female animals (15000 ppm) on study days 56 (-13%) and 63 (-11%). These observations were found in the high dose group of both sexes, therefore, these findings were assessed as treatment-related and adverse. No effects seen in low and mid dose group animals of both sexes.

For further details, see background material attached.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

No test substance-related, adverse changes were observed. For further details, see background material attached.

Food efficiency:

not examined

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

no effects observed

Description (incidence and severity):

No test substance-related changes were observed.

Ophthalmological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related findings were observed. All apparent findings were assessed as incidental in nature since they occurred in control as well as in treated animals and did not show a dose-response relationship.
Blood vessels only visible blurred were seen in the right eye of one high dose female animal (15000 ppm). This isolated finding in one animal was assessed as incidental and not related to treatment.
For further details, see background material attached.

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Prothrombin time (Hepatoquick’s test, HQT) was significantly shortened in high dose rats of both sexes (15000 ppm). This change was regarded as treatment-related and adverse.
For further details, see background material attached.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

- Glucose (-14.9% deviation vs ctrl.) and Chloride (-1.9% deviation vs ctrl.) levels were significantly decreased in high dose males (15000 ppm).
- Potassium levels were significantly increased (+3.2% deviation vs ctrl.) in high dose males (15000 ppm).
These alterations in combination were regarded as treatment-related and adverse.
For further details, see background material attached.

- Urea levels were significantly increased (+13.85% deviation vs ctrl.) in high dose females (15000 ppm).
- Chloride levels were significantly decreased (-1.0% deviation vs ctrl.) in low dose males (1500 ppm).
These alterations were isolated in the respective individuals of those test groups and therefore, they were regarded as maybe treatment-related, but non-adverse (ECETOC Technical Report No. 85, 2002).

- increased aspartate aminotransferase activities in mid dose males (5000 ppm).
- increased inorganic phosphate levels in low dose males (1500 ppm).
- decreased globulin levels in low dose females (1500 ppm).
- decreased total bilirubin values in low dose females (1500 ppm)
These significant alterations were not dose-dependent and therefore, they were regarded as incidental and not treatment-related.

Other significantly altered values (decreased total bilirubin, aspartate aminotransferase; increased calcium, globulin, inorganic phosphate) were within historical control ranges and therefore, the changes were regarded as incidental and not treatment-related.

THYROID HORMONES
For T3 and TSH in treated male and females rats and for T4 in all treated females, no significant changes of the hormone values were observed. In low and mid test group males (1500 and 5000 ppm) T4 values were statistically significantly increased, but the alteration was not dose-dependent. Therefore, this change was regarded as incidental and not treatment-related.

T3 males: +8.47%, +10.05%, +10.71% deviation vs ctrl in low, mid and high dose animals (1500, 5000, 15000 ppm)
T3 females: -3.41%, -2.70%, -9.39% deviation vs ctrl in low, mid and high dose animals (1500, 5000, 15000 ppm)

T4 males: +12.03%*, +11.54%*, +3.08% deviation vs ctrl in low, mid and high dose animals (1500, 5000, 15000 ppm)
T4 females: +8.50%, +6.31%, +1.93% deviation vs ctrl in low, mid and high dose animals (1500, 5000, 15000 ppm)

TSH males: -15.62%, +4.42%, +12.24% deviation vs ctrl in low, mid and high dose animals (1500, 5000, 15000 ppm)
TSH males: -12.03%, -16.17%, -11.47% deviation vs ctrl in low, mid and high dose animals (1500, 5000, 15000 ppm)

*p<=0.05

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

No treatment-related, adverse changes with human relevance among urinalysis parameters were observed.

In the urine sample of high dose males (15000 ppm), pH-values were significantly decreased. This is a compensation mechanism of the potential metabolic acidosis in this dose group. Therefore, this alteration is regarded as adaptive rather than adverse.
Additionally, in the urine sediment of males of this test group, the incidences of transitional epithelial cells and granulated and epithelial cell casts were increased. These changes occurring only in the male sex of this age indicated an alpha-2µ-globulinuria, which was observed also histopathologically and which is regarded as species-specific finding in male rats without human relevance (Hard et al., 1993).

For further details, see background material attached.

Behaviour (functional findings):

effects observed, non-treatment-related

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.
- 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 for all treated males and low, high dose group females of test groups (1500 and 15000 ppm). The grip strength of the hindlimbs was significantly increased by 24.9% in mid dose females (5000 ppm) versus controls. Because this finding showed no dose-dependency, it was assessed as incidental and not related to treatment.

Motor activity measurement
Regarding the overall motor activity as well as single intervals, no test substance-related deviations to the control animals were noted for all treated male and female animals. The shape of the habitation curves was comparable to control
for all test groups of both sexes. Single interval No. 10 in mid dose male animals (5000 ppm) was significantly decreased. In female low and high dose animals (1500 and 15000 ppm), a significantly decrease of beam interruptions was recorded for interval 11. These single occurrences did not demonstrate a dose-dependent decrease of motor activity and was assessed as being incidental and not related to treatment.

For further details, see background material attached.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

Absolute organ weights: All mean absolute weight parameters did not show significant differences when compared to the control group.
Relative organ weights:
Liver - males: -2.0%, +1.2%, +13.4%** deviation vs ctrl in low, mid and high dose animals (1500, 5000, 15000 ppm)
Liver - females: +0.9%, +4.3%, +10.3%** deviation vs ctrl in low, mid and high dose animals (1500, 5000, 15000 ppm)
**: p <= 0.01

The statistically significant increase in relative liver weight in high dose group animals was regarded to be mainly due to the decrease in terminal body weight in this test group. Furthermore, there were no histopathologic findings observed, that could explain the weight increase. It was therefore regarded to be secondary and no direct effect of the test substance on the liver.

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

For further details, see background material attached.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

All 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.
For further details, see background material attached.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

In the kidneys of treated and control male animals, eosinophilic globules and occasionally granular casts (treated animals, only) were observed. For a better visualization, a CAB stain in the kidneys of all males was performed. There were no clear differences between control and treated animals and the globules could be shown to be alpha 2µ globulin with a positive immunohistochemical stain. This result was regarded to be not related to treatment.

Males, Kidney - Eosinophilic droplets in 4/10, 1/10, 1/10 and 6/10 animals of the control, low, mid and high dose group (0, 1500, 5000, 15000 ppm)
Males, Kidney - Cast(s) in 2/10, 0/10, 2/10 and 4/10 animals of the control, low, mid and high dose group (0, 1500, 5000, 15000 ppm)

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.
For further details, see background material attached.

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Effect levels

Dose descriptor:

NOAEL

Effect level:

5 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

clinical biochemistry

haematology

Remarks on result:

other: 316 and 384 mg/kg bw in males and females

Target system / organ toxicity

Critical effects observed:

no

Any other information on results incl. tables

Test substance analyses

The stability of the test substance in the diet at room temperature for a period of 4 days was demonstrated during the study. An additional stability analysis revealed a stability of the test substance in the diet after a storage of 12 days in the freezer followed by 2 days at room temperature.

Considering the low relative standard deviation in the homogeneity analyses ranging from 0.2% to 4.1%, it can be concluded that Tetrahydrolinalool was distributed homogeneously in in the diet.

At the beginning of the administration period, the (mean) concentrations of the test substance in the diet were found to be in the range of 96.5% to 101.3% of the nominal concentration. Towards the end of the administration period, the concentrations determined in the dose groups 1500, 5000 and 15000 ppm were 87.4%, 81.8% and 90.5%, respectively. Thus, low and mid dose groups were below the lower limit of the specification of the test facility (90%). Retained reserve samples of these test groups showed a mean concentration for the three samples of 90.5% in the low dose group and 86.5% in the mid dose group. During the administration period, further samples (taken after 8 weeks of the administration period of the test substance in case of inconsistent results determined according to the test guideline) were analyzed. The result was 97.7% of the nominal concentration. Accordingly, the mean value of three samples analyzed for the mid dose group was 93.4%. Therefore, the concentrations of the mid dose test substance preparations were considered to be within the specification of the test facility. Furthermore, the value for the mid dose group towards the end of the administration period was still within the international accepted specification for formulation in solids like the diet, i.e. 80-120% (Whitmire ML 2010). Overall, the results were assessed to demonstrate the correctness of the concentrations of the test substance in the diet.

Applicant's summary and conclusion

Executive summary:

The test substance was administered orally via the diet to groups of 10 male and 10 female Wistar rats at concentrations of 0, 1500, 5000 and 15000 ppm over a period of 3 months.

Food consumption and body weight were determined weekly. The animals were examined for signs of toxicity or mortality at least once a day. Detailed clinical examinations in an open field were conducted prior to the start of the administration period and weekly thereafter. Ophthalmological examinations were performed before the beginning and towards the end of the administration period. Beside this, a functional observational battery as well as measurement of motor activity were carried out towards the end of the administration period. Clinico-chemical and hematological examinations as well as urinalyses were performed

towards the end of the administration period. After the administration period all animals were sacrificed and assessed by gross pathology. Organ weights were determined followed by histopathological examinations.

The various analyses confirmed a homogeneous distribution of the test substance in the diet, a general correctness of the prepared concentrations and the stability at room temperature for a period of 4 days during the study. In addition, stability analyses revealed a stability of the test substance in the diet after a storage of 12 days in the freezer followed by 2 days at room temperature.

With regard to clinical examinations, signs of general systemic toxicity were observed in decreased body weight changes at 15000 ppm in male (982 mg/kg bw/d) and female (1118 mg/kg bw/d) animals, the highest concentration tested. Body weight changes were decreased in males (-15.6% on study day 7) and females (up to -12.7% on study day 56).

 

Regarding clinical pathology in high dose rats of both sexes (15000 ppm) a shortened prothrombin time (Hepatoquick’s test, HQT) indicated an increased synthesis of coagulation factors by the liver cells. In males of the mentioned test group, lower glucose values combined with lower chloride levels and higher potassium levels were most probably due to a changed energy metabolism, coupled with a potential metabolic acidosis. This metabolic acidosis is partly compensated by renal excretion of acids lowering the pH value of the urine.

Increased incidences of transitional epithelial cells and granulated and epithelial casts were observed in the urine sediment of high dose males (15000 ppm). These changes - occurring only in the male sex of this age - indicated an alpha-2u-globulinuria, which was observed also in histopathological examinations and which is regarded as species-specific finding in male rats without human relevance.

Regarding pathology, all 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.

 

No treatment-related, adverse effects were observed in Clinical Examinations, Clinical Pathology and Pathology for the low and mid dose group animals (1500 and 5000 ppm).

In conclusion, the oral administration of the test substance via diet over a period of 3 months revealed adverse effects in male and female Wistar rats. Under the condition of this study the no observed adverse effect level (NOAEL) was 5000 ppm in males (316 mg/kg bw/d) and females (384 mg/kg bw/d) based mainly on effects in clinical pathology.