Tetrahydro-4-methyl-2-(2-methylprop-1-enyl)pyran

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

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:

Reaction mass of (2S-cis)-tetrahydro-4-methyl-2-(2-methyl-1-propenyl)-2H-pyran and Tetrahydro-4-methyl-2-(2-methylpropen-1-yl)pyran (2R,4R) and Tetrahydro-4-methyl-2-(2-methylpropen-1-yl)pyran (2S,4S) and (2R-cis)-tetrahydro-4-methyl-2-(2-methyl-1-propenyl)-2H-pyran

EC Number:

939-429-1

Cas Number:

16409-43-1

Molecular formula:

C10H18O

IUPAC Name:

Reaction mass of (2S-cis)-tetrahydro-4-methyl-2-(2-methyl-1-propenyl)-2H-pyran and Tetrahydro-4-methyl-2-(2-methylpropen-1-yl)pyran (2R,4R) and Tetrahydro-4-methyl-2-(2-methylpropen-1-yl)pyran (2S,4S) and (2R-cis)-tetrahydro-4-methyl-2-(2-methyl-1-propenyl)-2H-pyran

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 study initiation: 42 ± 1 days
- Fasting period before study: no
- Housing: 5 animals per cage
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 6 days

DETAILS OF FOOD AND WATER QUALITY:
Food analyses:
On the basis of duration of use and the analytical findings with respect to chemical and microbiological contaminants, the diet was found to be suitable. Fed. Reg. Vol. 44, No. 91 of 09 May 1979, p. 27354 (EPA), served as a guideline for maximum tolerable chemical contaminants. The number of microorganisms did not exceed 1 × 10^5/g food.
Drinking water analyses:
On the basis of the analytical findings, the drinking water was found to be suitable. German “Trinkwasserverordnung” (Drinking Water Regulation) served as a guideline for maximum tolerable contaminants.

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:

other: microencapsulated in Alginate capsules

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:

DIET PREPARATION
For each concentration, the test substance was weighed out and mixed with a small amount of food. In order to obtain the desired concentrations, these premixes were added to the corresponding amounts of food, depending on test group.
The preparations were prepared in a frequency for which the stability in the carrier was demonstrated.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The study was carried out in compliance with the Principles of Good Laboratory Practice. The stability of the test substance in the diet for a period of 12 days in the refrigerator and up to 4 days at room temperature was proven before the start of the administration period.
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.

The various analyses confirmed:
• the stability of the test-substance preparations for a period for 12 days in the refrigerator up to 4 days at room temperature,
• the homogeneous distribution of the test substance in the vehicle,
• the correctness of the prepared concentrations (except the concentration control analyses for the low dose test group towards the end of the application period, which did not meet the specification limit of 90-110%).

Duration of treatment / exposure:

90 days

Frequency of treatment:

daily via feed

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

1 200 ppm

Remarks:

a.i. Rose oxide 90 (= 7500 ppm Rose oxide 90 encapsulated); Mean daily test substance intake: 73 mg/kg bw/d males; 93 mg/kg bw/d females.

Doses / concentrations 2

Dose / conc.:

4 000 ppm

Remarks:

a.i. Rose oxide 90 (= 25 000 ppm Rose oxide 90 encapsulated); Mean daily test substance intake: 394 mg/kg bw/d males; 512 mg/kg bw/d females.

Doses / concentrations 3

Dose / conc.:

12 000 ppm

Remarks:

a.i. Rose oxide 90 (= 75 000 ppm Rose oxide 90 encapsulated); Mean daily test substance intake: 1547 mg/kg bw/d males; 1620 mg/kg bw/d females.

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

yes, plain diet

Positive control:

not applicable

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
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
The animals were examined for evident signs of toxicity or mortality twice a day (in the morning and in the late afternoon) from Mondays to Fridays and once a day (in the morning) on Saturdays, Sundays and public holidays.

DETAILED CLINICAL OBSERVATIONS: Yes
Detailed clinical observations (DCO) were performed in all animals prior to the administration period and thereafter at weekly intervals.

BODY WEIGHT: Yes
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.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
Food consumption was determined weekly over a period of 3 days and calculated as mean food consumption in grams per animal and day.
The mean daily intake of test substance (group means) was calculated based upon individual values for body weight and mean food consumption per cage.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
Drinking water consumption was monitored by daily visual inspection of the water bottles for any changes in volume.

OPHTHALMOSCOPIC EXAMINATION: Yes
Prior to the start of the administration period on day 0 the eyes of all animals and on study day 91 the eyes of the control and high-dose animals were examined for any changes using an ophthalmoscope after administration of a mydriatic agent.

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:
- Protein
- Glucose
- Ketones
- Urobilinogen
- Bilirubin
- Blood
- Specific gravity
- Sediment
- Color, turbidity
- Volume
- pH


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
Estrous cycle length and normality were evaluated daily for all female animals from study day 63 until the day of sacrifice.

Sperm parameters
Immediately after necropsy and organ weight determination, the right testis and cauda epididymis were taken from all male animals. The right testis and right cauda epididymis were deep frozen at -20°C until evaluation of the sperm head count. Initially, sperm morphology and sperm head count (cauda epididymis and testis) were evaluated for the placebo control and high dose group, only.
- Sperm motility
- Sperm morphology
- Sperm head count (cauda epididymis)
- Sperm head count (testis)

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. The left testis and left epididymis of all animals sacrificed at scheduled dates were fixed in modified Davidson’s solution, whereas the right testis and epididymis were used for sperm parameters.
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. Epididymis, left
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. Testis, left
39. Thymus
40. Thyroid glands
41. Trachea
42. Urinary bladder
43. Uterus
44. Vagina

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)
WILCOXON-test with Bonferroni-Holm adjustment (with several dose groups): Sperm analysis parameters.

Results and discussion

Results of examinations

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

No test substance-related, adverse findings were observed for male and female animals of test groups treated with the test substance.

Different findings in 1 mid dose female were observed from study day 7 onwards, i.e. piloerection on study 7, tremors from study day 7
to 55 and unsteady gait from 7 onwards until the scheduled sacrifice with exception of study day 49. Because of the temporarily occurrence in this individual, a missing dose-response relationship and missing clinical findings in other animals of this test group, the changes were assessed to be incidental and not related to treatment.

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):

No significantly changes of mean body weight values were found in male and female animals of the low and mid dose test groups as well as in high dose females. In high dose males, the bw was significantly lower from study day 21 onwards with a maximum by -13% on study day 49.

No significantly changes of mean body weight change values were observed in low dose males and in all test substance treated females. In high dose males, bw changes were significantly lower over the entire study period with a maximum by -25% on study day 21. In mid dose males, bw changes were significantly decreased from study day 21 onwards with exception of study days 28, 70 and 91 and a maximum by -13% on study day 56.

A toxic effect of Rose oxide 90 resulting in reduced food consumption and lowered body weight development was considered less likely since no relevant changes were obtained for clinical pathology and pathology parameters. The reduced body weight parameters were considered to be related to palatability problems and reduced feed uptake.

Final body weights:
Significant reduction in high dose males:
1200 ppm: -2.1 vs. placebo ctrl.
4000 ppm: -7.1 vs. placebo ctrl.
12000 ppm: -11.6%** vs. placebo ctrl.
**p<=0.01 vs. placebo ctrl.

For further details, see background material attached.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

No test substance-related, adverse changes with regard to food consumption were observed in all test substance treated groups.
Because of intense food spilling by male and female animals of the mid and high dose groups, food consumption values could not be determined properly for these test groups. However, at least in female animals no significant changes were observed for body weight development during the course of the study and, given that, sufficient uptake of feed was considered.
For mid and high dose male animals, the feed uptake was considered to be reduced because of palatability problems. A toxic effect of Rose oxide 90 resulting in reduced food consumption and lowered body weight development was considered less likely since not relevant changes were obtained for clinical pathology and pathology parameters.

For further details, see background material attached.

Food efficiency:

not specified

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

no effects observed

Description (incidence and severity):

No test substance-related, adverse changes with regard to water consumption were observed.

For further details, see background material attached.

Ophthalmological findings:

no effects observed

Description (incidence and severity):

No treatment-related findings were observed. All apparent findings were assessed as being incidental in nature since they occurred in control as well as in treated animals and did not show a dose-response relationship.

For further details, see background material attached.

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

No treatment-related, adverse changes among hematological parameters were observed.
In high dose males hematocrit values were significantly increased at the end of the administration period (+4% vs. placebo ctrl.). This was the only significantly changed red blood cell parameter among these individuals. Therefore, this alteration was regarded as potentially treatment related but non-adverse.

For further details, see background material attached.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

No treatment-related, adverse changes among clinical chemistry parameters were observed.

In high dose males, creatinine values were significantly decreased at the end of the administration period (-8.5% vs placebo ctrl.). This was the only clinical chemistry value among these individuals which was significantly changed. Therefore, this alteration was regarded as potentially treatment-related, but non-adverse.

In high dose females, globulin levels were significantly decreased (-6.7% vs placebo ctrl.). However, the mean value (23.41 g/L) was within the historical control range (females, globulins 23.07-30.98 g/L).
In low dose females, chloride levels were significantly lower compared to the placebo controls, whereas in low dose males, alkaline phosphatase (ALP) activities and inorganic phosphate values were significantly increased. However, these values were not dose-dependently changed. Therefore, all mentioned changes were regarded as incidental and not treatment-related.

For further details, see background material attached.

Urinalysis findings:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related changes among urinalysis parameters were observed.
In mid dose males, significantly higher incidences of granular and epithelial casts were observed in the urine sediment at the end of the administration period (also observed in high dose males, but without gaining statistical significance). This change was isolated without any other finding in the urine or among blood renal parameters and it was hardly dose dependent. Therefore, it was regarded as incidental and not treatment-related.

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. Unsteady gait (1 mid dose female), injury in the left ear region (1 placebo ctrl. male) and an injury in the throat (1 female untreated ctrl.) were assessed as being incidental and spontaneous in nature.
- 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 to the control animals were noted for male and female animals of all test substance test groups.

For further details, see background material attached.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

The final body weight was significantly decreased in high dose males resulting in secondarily significant decreases of the mean absolute brain, prostate, and thyroid glands weights and in significant increases of the mean relative weights of epididymides, kidneys, liver, and testes. There were no treatment-related histopathological findings in all these organs.

The mean increased relative kidney and liver weights in mid dose males were related to the slightly but not significantly decreased final body weight (-7.1%) in this test group. As there was no dose-response relationship, the increased mean relative liver weight in low dose males and mid dose females were considered to be incidental.

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

Absolute organ weights: Significant changes
Males - 12000 ppm:
Brain: -3.77%* vs. placebo ctrl.
Prostate: -14.53%** vs. placebo ctrl.
Thyroid glands: -20.16%** vs. placebo ctrl.
*: p ≤ 0.05, **: p ≤ 0.01

Relative organ weights: Significant changes vs. placebo ctrl
Epididymides (males):
1200 ppm: -2.82%
4000 ppm: +6.03%
12000 ppm: +8.87%*

Testes (males):
1200 ppm: -1.43%
4000 ppm: +7.17%
12000 ppm: +9.19%*

Kidneys (males):
1200 ppm: +0.69%
4000 ppm: +6.66%*
12000 ppm: +12.55%*

Liver (males / females):
1200 ppm: +10.16%* / +6.01%
4000 ppm: +5.84%* / +9.35%*
12000 ppm: +13.09%* / +5.67%

*: p ≤ 0.05, **: p ≤ 0.01

For further details, see background material attached.

Gross pathological findings:

no effects observed

Description (incidence and severity):

All findings were single observations. 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):

No treatment-related findings were observed in males and females. All findings occurred either individually or were biologically equally distributed over placebo control group and treatment groups (e.g. eosinophilic droplets in the kidneys of male animals) or over untreated control group , placebo control group and treatment groups (e.g. mucification of vaginal/cervical epithelium correlating with prolonged diestrus). They were considered to be without any relation to treatment.

Eosinophilic droplets in the kidneys (males): Animals affected/ Animals examined
Placebo ctrl.: 10/10
1200 ppm: 10/10
4000 ppm: 10/10
12000 ppm: 10/10

Increased mucification in vagina (females): Animals affected/ Animals examined
Untreated ctrl.: 6/10
Placebo ctrl.: 1/9
1200 ppm: 2/10
4000 ppm: 5/10
12000 ppm: 4/10

For further details, see background material attached.

Histopathological findings: neoplastic:

not examined

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

Estrous cycle
Estrous cycle data revealed regular cycles of all test groups including the controls.

Sperm analysis
Concerning motility of the sperms and the incidence of abnormal sperms in the cauda epididymidis as well as sperm head counts in the testis and in the cauda epididymidis no treatment-related effects were observed.

Motile sperm in the cauda epididymidis [%]
Placebo ctrl.: 86 (6)
1200 ppm: 86 (6)
4000 ppm: 87 (4)
12000 ppm: 88 (6)

Abnormal sperm in the cauda epididymidis [%]
Placebo ctrl.: 5.6 (0.8)
12000 ppm: 5.6 (2.1)

Sperm head counts in testis / cauda epididymidis [Mio/g]
Placebo ctrl.: 105 (11) / 604 (123)
12000 ppm: 114 (12) / 571 (144)

Thyroid hormones
In all test substance treated male and female animals, no treatment related alterations of T3, T4 and TSH levels were observed after the administration period.
In mid dose males T4 values were significantly decreased (-14% vs. placebo ctrl.), but the change was not dose-dependent. Therefore, this alteration was regarded as incidental and not treatment-related.

In placebo ctrl. males, TSH mean values were significantly lower compared to the untreated diet control values. Although higher, no significant difference of the TSH values from test substance treated males towards the low placebo control group were observed. Therefore, it can be assumed that there is also no significant difference of the TSH values in the mentioned treatment groups to TSH of the diet control which are more similar values.

Mean T3 [nmol/L] (SD) of Males / Females
Placebo ctrl.: 0.90 (0.14) / 0.76 (0.10)
1200 ppm: 0.98 (0.27) / 0.83 (0.08)
4000 ppm: 0.90 (0.18) / 0.70 (0.08)
12000 ppm: 0.87 (0.17) / 0.74 (0.10)

Untreated ctrl.: 0.98 (0.30) / 0.73 (0.13)


Mean T4 [nmol/L] (SD) of Males / Females
Placebo ctrl.: 60.23 (5.95) / 38.26 (6.37)
1200 ppm: 58.92 (5.85) / 37.91 (7.98)
4000 ppm: 51.81 (6.83)* / 38.31 (6.09)
12000 ppm: 52.13 (8.77) / 38.43 (4.18)
*p<=0.05 vs. placebo ctrl.
Untreated ctrl.: 57.72 (4.95) / 38.07 (5.90)

Mean TSH [µg/L] (SD) of Males / Females
Placebo ctrl.: 3.83 (0.94) / 2.69 (0.42)
1200 ppm: 5.37 (2.30) / 2.56 (0.56)
4000 ppm: 5.55 (2.44) / 2.74 (0.33)
12000 ppm: 5.12 (1.12) / 2.82 (0.71)

Untreated ctrl.: 5.33 (1.61) / 2.95 (0.84)

For further details, see background material attached.

Effect levels

Dose descriptor:

NOAEL

Effect level:

12 000 ppm

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

other: no adverse test substance related effects observed

Remarks on result:

other: a.i. Rose oxide 90 (= 75 000 ppm Rose oxide 90 encapsulated); Mean daily test substance intake: 1547 mg/kg bw/d males; 1620 mg/kg bw/d females.

Target system / organ toxicity

Critical effects observed:

no

Applicant's summary and conclusion

Executive summary:

Rose oxide 90 (encapsulated) was administered via the diet to groups of 10 male and 10 female Wistar rats at concentrations of 0 ppm (pure maintenance diet), 0 ppm (Placebo Alginate – without Rose oxide 90), 7500 ppm, 25000 ppm and 75000 ppm over a period of 3 months. Based on a content of about 16% pure Rose oxide 90 in the alginate capsule material, the effective concentrations of Rose Oxide 90 (a.i.) were 0 ppm, 0 ppm, 1200 ppm, 4000 ppm and 12000 ppm, respectively. To achieve a constant concentration of alginate capsules in the diet of test substance groups, Placebo Alginate without Rose oxide 90 was supplemented.

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.

With regard to clinical observations, no findings of toxicological relevance were obtained. For mid and high dose male animals the feed uptake was assumed to be reduced because of palatability problems. A toxic effect of Rose oxide 90 resulting in reduced food consumption and lowered body weight development was considered less likely since no relevant changes were obtained for clinical pathology and pathology parameters. Concerning clinical pathology, no treatment-related, adverse effects were observed up to a concentration in the diet of Rose oxide 90 (a.i.) of 12000 ppm (i.e. encapsulated Rose oxide 90 of 75000 ppm). Regarding pathology, there were neither substance-related organ weight deviations nor gross lesions. Histopathological findings occurred either individually or were biologically equally distributed over untreated control, placebo and treatment groups. They were considered to be without any relation to treatment.

In conclusion, the administration of Rose oxide 90 via the diet to male and female Wistar rats for 3 months did not cause any test substance-related, adverse signs of toxicity in male and female animals up to a concentration in the diet of encapsulated Rose oxide 90 of 75000 ppm (i.e. 12000 ppm Rose Oxide 90 as active ingredient). Therefore, under the conditions of the present study the no observed adverse effect level (NOAEL) was 75000 ppm (12000 ppm a.i.) in male (1547 mg/kg bw/d) and female (1620 mg/kg bw/d) Wistar rats.