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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
extended one-generation reproductive toxicity - basic test design (Cohorts 1A, and 1B without extension)
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:
2018
Report Date:
2018

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 443 (Extended One-Generation Reproductive Toxicity Study)
GLP compliance:
yes (incl. certificate)
Remarks:
BASF SE Experimental Toxicology and Ecology, 67056 Ludwigshafen, Germany
Limit test:
no
Justification for study design:
SPECIFICATION OF STUDY DESIGN FOR EXTENDED ONE-GENERATION REPRODUCTION TOXICITY STUDY WITH JUSTIFICATIONS:

- Premating exposure duration for parental (P0) animals:
According to OECD TG 443 and DECISION ON A TESTING PROPOSAL PURSUANT TO ARTIClE 40 OF REGULATION (EC) NO 1907/2006 for A mixture of: cis-tetrahydro-2-isobutyl-4-methylpyran-4-ol; trans-tetrahydro-2-isobutyl-4-methylpyran-4-ol (CAS 63500-71-0, EC 405-040-6), Decision number: TPE-D-2114322946-44-01/F.

- Basis for dose level selection:
Based on a modified one generation Reproduction Toxicity Study in Wistar Rats Range finding Study (BASF SE) and DECISION ON A TESTING PROPOSAL PURSUANT TO ARTIClE 40 OF REGULATION (EC) NO 1907/2006 for A mixture of: cis-tetrahydro-2-isobutyl-4-methylpyran-4-ol; trans-tetrahydro-2-isobutyl-4-methylpyran-4-ol (CAS 63500-71-0, EC 405-040-6), Decision number: TPE-D-2114322946-44-01/F.

- Inclusion/exclusion of extension of Cohort 1B:
Exclusion of extension of Cohort 1B based on the DECISION ON A TESTING PROPOSAL PURSUANT TO ARTIClE 40 OF REGULATION (EC) NO 1907/2006 for A mixture of: cis-tetrahydro-2-isobutyl-4-methylpyran-4-ol; trans-tetrahydro-2-isobutyl-4-methylpyran-4-ol (CAS 63500-71-0, EC 405-040-6), Decision number: TPE-D-2114322946-44-01/F.

- Inclusion/exclusion of developmental neurotoxicity Cohorts 2A and 2B
Exclusion based on the DECISION ON A TESTING PROPOSAL PURSUANT TO ARTIClE 40 OF REGULATION (EC) NO 1907/2006 for A mixture of: cis-tetrahydro-2-isobutyl-4-methylpyran-4-ol; trans-tetrahydro-2-isobutyl-4-methylpyran-4-ol (CAS 63500-71-0, EC 405-040-6), Decision number: TPE-D-2114322946-44-01/F.

- Inclusion/exclusion of developmental immunotoxicity Cohort 3
Exclusion based on the DECISION ON A TESTING PROPOSAL PURSUANT TO ARTIClE 40 OF REGULATION (EC) NO 1907/2006 for A mixture of: cis-tetrahydro-2-isobutyl-4-methylpyran-4-ol; trans-tetrahydro-2-isobutyl-4-methylpyran-4-ol (CAS 63500-71-0, EC 405-040-6), Decision number: TPE-D-2114322946-44-01/F.

- Route of administration
Oral route based on the DECISION ON A TESTING PROPOSAL PURSUANT TO ARTIClE 40 OF REGULATION (EC) NO 1907/2006 for A mixture of: cis-tetrahydro-2-isobutyl-4-methylpyran-4-ol; trans-tetrahydro-2-isobutyl-4-methylpyran-4-ol (CAS 63500-71-0, EC 405-040-6), Decision number: TPE-D-2114322946-44-01/F.

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Details on test material:
Purity: 99.7 % area%
Homogeneity:Given (visually)
Storage stability: Expiry date: 30 Jan 2019
The stability of the test substance under storage conditions over the test period was guaranteed by the sponsor, and the sponsor holds this responsibility.
Physical state/Appearance: Liquid / colorless clear
Storage conditions: Room temperature

Test animals

Species:
rat
Strain:
Wistar
Details on species / strain selection:
The rat is the preferred animal species for reproduction studies according to test guidelines.
This strain was selected since extensive historical control data were available for Wistar rats.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH
- Age at study initiation: The male and female rats were 28 (±1) days old when they arrived from the breeder. The 100 male and 100 female animals in the study were 34 (±1) days old at the beginning of treatment.
- Weight at study initiation: male animals 96.8 - 128.6 g, female animals 88.5 - 120.6 g
- Fasting period before study: no
- Housing: During the study period, the rats were housed together in Polysulfonate cages with the following exceptions:
• During overnight matings, male and female mating partners were housed together in Polycarbonate cages type III.
• Pregnant animals and their litters were housed together until PND 21 in Polycarbonate cages type III.
- Diet: Ground Kliba maintenance diet mouse/rat "GLP"; ad libitum
- Water: Drinking water ad libitum
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 30-70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:

DIET PREPARATION
- Rate of preparation of diet (frequency): study phase dependent; considering stability of test item in diet
- Storage temperature of food: Room temperature
Details on mating procedure:
In general, each of the male and female animals was mated overnight at a 1 : 1 ratio for a maximum of 2 weeks. Throughout the mating period, each female animal was paired with a predetermined male animal from the same dose group.

The animals were paired by placing the female in the cage of the male mating partner from about 16.00 h until 6.30 - 9.00 h of the following morning. Deviations from the specified times were possible on weekends and public holidays and were reported in the raw data. A vaginal smear was prepared after each mating and examined for the presence of sperm. If sperm was detected, pairing of the animals was discontinued. The day on which sperm were detected was denoted " gestation day (GD) 0" and the following day "gestation day (GD) 1".
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analytical verifications of the stability of the test substance in the diet for a period of 13 days consisting of 10 days in a freezer followed by 3 days at ambient temperature was verified before the start of the study.
Duration of treatment / exposure:
After an acclimatization period rats were kept for at least 10 weeks. Then the F0 animals were paired. The females were allowed to deliver and rear their pups (F1 generation pups) until PND 4 (standardization) or PND 21 or 22 (depending on the cohort). Pups of the F1 litter were selected (F1 rearing animals) and assigned to 2 different cohorts which were subjected to specific postweaning examinations.
Frequency of treatment:
Continuous via diet
Details on study schedule:
After the acclimatization period, the test substance was administered to the parental animals as addition to the diet continuously throughout the entire study. After a minimum of 10 weeks after the beginning of treatment, males and females from the same dose group were mated. The female animals were allowed to deliver and rear their pups (F1 generation pups) until postnatal days (PND) 21 or 22. The F0 generation parental animals were sacrificed after weaning of the F1 generation pups.
Doses / concentrationsopen allclose all
Dose / conc.:
1 000 ppm (nominal)
Remarks:
During the lactation period the Pyranol concentrations in the diet of the F0 females were reduced to 50%. This dietary adjustment derived from historical body weight and food consumption data and maintained the dams at the desired target doses of Pyranol during this period of increased food intake.
Dose / conc.:
4 000 ppm (nominal)
Remarks:
During the lactation period the Pyranol concentrations in the diet of the F0 females were reduced to 50%. This dietary adjustment derived from historical body weight and food consumption data and maintained the dams at the desired target doses of Pyranol during this period of increased food intake.
Dose / conc.:
12 500 ppm (nominal)
Remarks:
During the lactation period the Pyranol concentrations in the diet of the F0 females were reduced to 50%. This dietary adjustment derived from historical body weight and food consumption data and maintained the dams at the desired target doses of Pyranol during this period of increased food intake.
No. of animals per sex per dose:
F0 Generation: 25/sex/group
F1 Generation:
Cohort 1A: One male and one female/litter (20/sex/group)
Cohort 1B: One male and one female/litter (25/sex/group)
Control animals:
yes, plain diet

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule:
A check for moribund and dead animals was made twice daily from Mondays to Fridays and once daily on Saturdays, Sundays and public holidays.
A cageside examination was conducted at least once daily for any signs of morbidity, pertinent behavioral changes and/or signs of overt toxicity. lf such signs occur, the animals were examined several times daily.
The parturition and lactation behavior of the dams was generally evaluated in the morning in combination with the daily clinical inspection of the dams. Only particular findings (e.g. inability to deliver or umbilical cord not cut) were documented on an individual dam basis.
On weekdays (except Saturdays, Sundays and public holidays) the parturition behavior of the dams was inspected in the afternoons in addition to the evaluations in the mornings.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule:
Detailed clinical observations were performed in all F0 parental animals and in cohorts 1A and 1B at weekly intervals during the administration period. The examinations started in the morning. The findings were ranked according to the degree of severity, if applicable.
For observation, the animals were removed from their cages by the investigator and placed in a standard arena (50 × 37.5 × 25 cm). The following parameters listed were assessed
1. Abnormal behavior in handling
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 (Protruding eyeball)
15. Assessment of the feces excreted du ring the examination (appearance/consistency)
16. Assessment of the urine excreted during the examination
17. Pupil size

BODY WEIGHT: Yes
- Time schedule for examinations:
The body weight change of the animals was calculated from these results.
• During pregnancy, body weight of the F0 females with evidence of sperm was determined weekly for GD 0-3, 3-6, 6-9, 9-12, 12-15, 15-18 and 18-21.
• During lactation, body weight of the F0 and F1 females, which gave birth to a litter was determined for PND 0-3, 3-6, 6-9, 9-12, 12-15, 15-18 and 18-21.

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
Generally, food consumption was determined once a week over a period of 3 days for male and female F0 parental animals and F1 rearing animals, with the following exceptions:
• Food consumption was not determined after the 10th premating week (male F0 animals) and during the mating period (male and female F0 parental animals).
• During pregnancy, food consumption of the F0 females with evidence of sperm was determined weekly for GD 0-3, 3-6, 6-9, 9-12, 12-15, 15-18 and 18-21.
• During lactation, food consumption of the F0 females, which gave birth to a litter was determined for PND 0-3, 3-6, 6-9, 9-12, 12-15, 15-18 and 18-21.

- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: yes

OTHER:
Clinical Pathology in F0 parental and cohort 1A animals
Samples were withdrawn from 10 F0 parental and cohort 1A males and females per group at termination. Blood samples were taken from animals by puncturing the retrobulbar venous plexus following isoflurane anesthesia. Blood sampling and blood examinations were carried out in a randomized sequence. Parameters:
Hematology
1. Leukocytes
2. Erythrocytes
3. Hemoglobin
4. Hematocrit
5. Mean corpuscular volume (MCV)
6. Mean corpuscular hemoglobin (MCH)
7. Mean corpuscular hemoglobin concentration (MCHC)
8. Platelets
9. Differential blood count
10. Reticulocytes
11. Prothrombin time

Clinical chemistry
1. Alanine aminotransferase
2. Aspartate aminotransferase
3. Alkaline phosphatase
4. Serum y-glutamyl transferase
5. Sodium
6. Potassium
7. Chloride
8. lnorg. phosphate
9. Calcium
10. Urea
11. Creatinine
12. Glucose
13. Total bilirubin
14. Total protein
15. Albumin
16. Globulins
17. Triglycerides
18. Cholesterol

Hormone evaluations
1. T4 (total thyroxine)
2. TSH

In the afternoon preceding the day of urinalysis, animals were individually transferred into metabolism cages (no food or drinking water provided); on the following morning, the individual urine specimens wer examined in a randomized sequence.
1. pH value
2. Protein
3. Glucose
4. Ketones
5. Urobilinogen
6. Bilirubin
7. Blood
8. Specific gravity
9. Microscopy of sediment
10. Color
11. Volume
Oestrous cyclicity (parental animals):
Estrous cycle length was evaluated by daily analysis of vaginal smear for all F0 female parental rats for a minimum of 3 weeks prior to mating. Determination was continued throughout the pairing period until the female exhibited evidence of copulation.
In all cohort 1A females, vaginal smears were collected after vaginal opening until the first cornified smear (estrous) was recorded. The estrous cycle also was evaluated in cohort 1A and 1B females for 2 weeks around PND 75.
At necropsy, an additional vaginal smear was examined to determine the stage of estrous cycle for each F0 female and cohort 1A and 1B female with scheduled sacrifice.
Sperm parameters (parental animals):
After the organ weight determination, the following parameters were determined in the right testis or right epididymis of all male F0 parental animals and all cohort 1A males sacrificed on schedule:
Sperm motility examinations were carried out in a randomized sequence.
Sperm morphology and sperm head count (cauda epididymis and testis) were evaluated for the control and highest dose groups, only.
Parameters:
• Sperm motility
• Sperm morphology
• Sperm head count in the cauda epididymis
• Spermatid head count in the testis
Litter observations:
STANDARDISATION OF LITTERS
On PND 4, the individual litters were standardized in such a way that, where possible, each litter contained 5 male and 5 female pups (always the first 5 pups/sex and litter were taken for further rearing). If individual litters did not have 5 pups/sex, the litters were processed in such a way that the most evenly distributed 10 pups per litter were present for further rearing (e.g., 6 male and 4 female pups). Standardization of litters was not performed in litters with <= 10 pups.

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
- Pup status (sex, live-born or stillborn) and litter size after birth
- Pup viability/mortality
- Clinical signs (including gross-morphological findings)
- Nipple/areola presence on PND 13 and one day prior to necropsy
- Anogenital distance (defined as the distance from the center of the anal opening to the base of the genital tubercle) on PND 1
- Pup body weights after birth (PND 1) and on PND 4, 7, 14 and 21
- Blood samples for clinical pathological investigations were withdrawn from 10 selected pubs per sex and group
- Blood samples were taken from a maximun of 10 surplus (culled) PND 4 pups per sex and group as well as from 10 surplus PND 22 pups per sex and group
- Hormone analysis (T4 (total thyroxine) and TSH) in PND 4 and 22 F1-offspring
Postmortem examinations (parental animals):
SACRIFICE
Sacrificed by decapitation under isoflurane anesthesia.

GROSS NECROPSY
The exsanguinated animals were necropsied and assessed by gross pathology, special attention being given to the reproductive organs. Animals which died intercurrently or were sacrificed in a moribund state were necropsied as soon as possible after their death 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. Lymph nodes, axillary (10 animals per sex per group, Cohort 1A animals only)
10. Lymph nodes, mesenteric (10 animals per sex per group, Cohort 1A animals only)
11. Ovaries
12. Pituitary gland
13. Prostate
14. Testes
15. Seminal vesicles including coagulating gland
16. Spleen
17. Thymus
18. Thyroid glands (with parathyroid glands)
19. Uterus (with cervix)

HISTOPATHOLOGY
The following organs or tissues were fixed in 4% formaldehyde solution or in modified Davidson's solution and assessed histopathologically:
1. All gross lesions
2. Adrenal glands
3. Bone marrow (femur)
4. Brain
5. Cecum
6. Cervix uteri
7. Coagulating glands
8. Colon
9. Duodenum
10. Epididymis, left (fixed in modified Davidson´s solution)
11. Esophagus
12. Eyes with optic nerve (fixed in modified Davidson's solution)
13. Heart
14. Ileum
15. Jejunum (with Peyer's patches)
16. Kidneys
17. Liver
18. Lungs
19. Lymph nodes, axillary
20. Lymph nodes, mesenteric
21. Mammary gland (male and female)
22. Ovaries (fixed in modified Davidson´s solution)
23. Oviducts
24. Pancreas
25. Pituitary gland
26. Prostate
27. Rectum
28. Sciatic nerve
29. Seminal vesicles
30. Skeletal muscle
31. Spinal cord (cervical, thoracic and lumbar cord)
32. Spleen
33. Stomach (forestomach and glandular stomach)
34. Testis, left (fixed in modified Davidson´s solution)
35. Thymus
36. Thyroid glands (with parathyroid glands)
37. Trachea
38. Urinary bladder
39. Uterus (uteri of all apparently nonpregnant animals or empty uterus horns were stained according to the method of Salewski E. (1964))
40. Vagina
41. Vas deferens
Postmortem examinations (offspring):
- COHORT 1A ANIMALS:

SACRIFICE
Sacrificed by decapitation under isoflurane anesthesia.

GROSS NECROPSY
The exsanguinated animals were necropsied and assessed by gross pathology, special attention being given to the reproductive organs. Animals which died intercurrently or were sacrificed in a moribund state were necropsied as soon as possible after their death 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. Lymph nodes, axillary (10 animals per sex per group, Cohort 1A animals only)
10. Lymph nodes, mesenteric (10 animals per sex per group, Cohort 1A animals only)
11. Ovaries
12. Pituitary gland
13. Prostate
14. Testes
15. Seminal vesicles including coagulating gland
16. Spleen
17. Thymus
18. Thyroid glands (with parathyroid glands)
19. Uterus (with cervix)

HISTOPATHOLOGY
The following organs or tissues were fixed in 4% formaldehyde solution or in modified Davidson's solution and assessed histopathologically:
1. All gross lesions
2. Adrenal glands
3. Bone marrow (femur)
4. Brain
5. Cecum
6. Cervix uteri
7. Coagulating glands
8. Colon
9. Duodenum
10. Epididymis, left (fixed in modified Davidson´s solution)
11. Esophagus
12. Eyes with optic nerve (fixed in modified Davidson's solution)
13. Heart
14. Ileum
15. Jejunum (with Peyer's patches)
16. Kidneys
17. Liver
18. Lungs
19. Lymph nodes, axillary
20. Lymph nodes, mesenteric
21. Mammary gland (male and female)
22. Ovaries (fixed in modified Davidson´s solution)
23. Oviducts
24. Pancreas
25. Pituitary gland
26. Prostate
27. Rectum
28. Sciatic nerve
29. Seminal vesicles
30. Skeletal muscle
31. Spinal cord (cervical, thoracic and lumbar cord)
32. Spleen
33. Stomach (forestomach and glandular stomach)
34. Testis, left (fixed in modified Davidson´s solution)
35. Thymus
36. Thyroid glands (with parathyroid glands)
37. Trachea
38. Urinary bladder
39. Uterus (uteri of all apparently nonpregnant animals or empty uterus horns were stained according to the method of Salewski E. (1964))
40. Vagina
41. Vas deferens

A differential ovarian follicle count (DOFC) was conducted in test groups 10 and 13 (Cohort 1A females) according to Plowchalk et.al. (1993).


- COHORT 1B ANIMALS:

SACRIFICE
Sacrificed by decapitation under isoflurane anesthesia.

GROSS NECROPSY
The exsanguinated animals were necropsied and assessed by gross pathology, special attention being given to the reproductive organs. Animals which died intercurrently or were sacrificed in a moribund state were necropsied as soon as possible after their death 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. Cauda epididymis
4. Epididymides
5. Liver
6. Ovaries
7. Pituitary gland
8. Prostate
9. Testes
10. Seminal vesicles including coagulating gland
11. Uterus (with cervix)

HISTOPATHOLOGY
The following organs or tissues were fixed in 4% formaldehyde solution or in modified Davidson's solution and assessed histopathologically:
1. All gross lesions
2. Adrenal glands
3. Cervix uteri
4. Coagulating glands
5. Epididymis, left (fixed in modified Davidson´s solution)
6. Liver
7. Ovaries (fixed in modified Davidson´s solution)
8. Pituitary gland
9. Prostate
10. Seminal vesicles
11. Testes, left (fixed in modified Davidson´s solution)
12. Uterus (uteri of all apparently nonpregnant animals or empty uterus horns were stained according to the method of Salewski E. (1964))
13. Vagina
Statistics:
DUNNETT test (two-sided): Food consumption, body weight and body weight change (parental animals, rearing animals and pups); estrous cycle length; mating days; duration of gestation; number of delivered pups per litter; developmental landmarks (days up to preputial separation or opening of the vagina), anogenital distance and index; implantation sites; postimplantation loss

FISHER's exact test: Number of live and dead pups and different indices (e.g. mating index, fertility index and gestation index) and number of litters with necropsy findings in pups; developmental landmarks (preputial separation or opening of the vagina)

KRUSKAL-WALLIS(-H) and WILCOXON test: Clinical pathology and sperm parameters; Weight of the anesthetized animals and absolute and relative organ weights; Absolute and relative pup organ weights; feces, rearing, grip strength forelimbs, grip strength hind limbs, landing foot-splay test, motor activity, startle response

WILCOXON test: Brain morphometry: linear measurements of selected brain regions; DOFC; Proportion of pups with necropsy findings per litter, presence of areolas/nipples

Dosed groups will be compared with the untreated control group.
Reproductive indices:
Male mating index (%) = number of males with confirmed mating / number of males placed with females * 100
Male fertility index (%) = number of males proving their fertility / number of males placed with females * 100
Female mating index (%) = number of females mated / number of females placed with males * 100
Female fertility index (%) = number of females pregnant / number of females mated * 100
Gestation index (%) = number of females with live pups on the day of birth / number of females pregnant * 100
Live birth index (%) = number of liveborn pups at birth / total number of pups born * 100
Postimplantation loss (%) = number of implantations – number of pups delivered / number of implantations * 100
Offspring viability indices:
Viability index (%) = number of live pups on day 4* after birth / number of live pups on the day of birth * 100
Lactation index (%) = number of live pups on day 21 after birth / number of live pups on day 4* after birth * 100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
no effects observed
Description (incidence and severity):
No clinical signs or changes of general behavior, which may be attributed to the test substance, were detected in any of the male and female F0 parental animals in any of the groups.

The following observations were not considered to be associated with the test compound: One mid-dose male animal (4000 ppm) showed skin lesion (neck region) during premating days 36 - 45. Two females (1000 ppm) of test group 01, one female (4000 ppm) of test group 02 and three females (12500 ppm) of test group 03 did not deliver F1 pups. One female of test group 01 had a palpable mass in the mammary line during PND 8 - 21.
Mortality:
no mortality observed
Description (incidence):
There were no test substance-related or spontaneous mortalities in any of the groups.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Body weights and body weight change of all test substance treated male and female F0 rats were essentially comparable to the concurrent control values throughout the entire study.

The final body weights of F0 males and females were marginally (approximately 3-5%) below control, but as the difference was not statistically significant and very small it cannot be attributed to the treatment without doubt.
There was, however, a number of episodes with statistically significantly lower or higher body weight change during several study sections: lower body weight change of the high-dose males during premating days 21 - 28 and 42 - 49 (about 13% and 23%, respectively); of the low-dose females during GD 9 - 12 (about 15%); and of the high-dose females during premating days 7 - 14 (about 22%) and GD 3 - 6 and 9 - 12 (about 25% and 23%, respectively) as well as higher body weight change in the low- and high-dose males during premating days 63 - 70. As none of these changes caused a consistent effect on body weights they were not regarded as of toxicological relevance.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Food consumption of the high-dose F0 male animals was statistically significantly below the concurrent control values during the premating days 0 - 3 (about 14%), however, the average food consumption of these animals during premating (day 0-73) was quite comparable to the control. Thus, the animals most likely adapted to the taste/smell of the medicated diet during the short episode of lower food consumption at the beginning of treatment and did not show signs of toxicity.
Food consumption of the high-dose F0 female animals was statistically significantly below the concurrent control values during the premating days 28 - 38 (up to 16%), during GD 0 - 6 (up to 8%) and during several parts of the lactation period (up to 9%). In contrast to the single episodes during premating and gestation the food consumption decrease was rather consistent during lactation, leading to an overall reduced food consumption by 7% in these females.

Food consumption of the low- and mid-dose F0 male and female rats was comparable to the concurrent control values throughout the entire study period.
Haematological findings:
no effects observed
Description (incidence and severity):
No treatment-related changes among hematological parameters were observed.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
No treatment-related, adverse changes among clinical chemistry parameters were observed.

At the end of the administration period in males of test group 03 (12500 ppm) urea values were significantly increased. The mean was marginally above the historical control range (males, urea 4.05-6.05 mmol/L). However, this was the only changed parameter among these individuals and therefore it was regarded as treatment-related but not adverse (ECETOC Technical Report No. 85, 2002).
In males of test group 01 (1000 ppm) inorganic phosphate levels were significantly increased. This alteration was not dose-dependent and therefore it was regarded as incidental and not treatment-related.
Urinalysis findings:
no effects observed
Description (incidence and severity):
No treatment-related changes among urinalysis parameters were observed.

In males of test groups 02 and 03 (4000 and 12500 ppm) urine pH values were significantly decreased and the incidences of epithelial and granular casts in the urine sediment were increased. Additionally, in males of test group 03 (12500 ppm) transitional epithelial cells were observed significantly more often in the urine sediment compared to controls. Lower urine pH values per se were regarded as maybe treatment-related but not adverse. Higher incidences of epithelial and granular casts and transitional epithelial cells which were observed in males only, in combination with the histopathologic finding of alpha-2-u-globulinuria is regarded as a rat specific effect with no relevance for humans (Hard et al., 1993).
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
see "Any other information on results incl. tables"
Other effects:
no effects observed
Description (incidence and severity):
In F0 rats of both sexes (test groups 01, 02 and 03; 1000, 4000 and 12500 ppm) no treatment-related alterations of T4 and TSH levels were observed.

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
The mean estrous cycle duration in the different test groups was similar: 3.9 days in control, 4.0 days in the low-dose group, 3.9 days in the mid-dose group and 3.9 days in the high-dose group.
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
The percentage of of abnormal sperms in the cauda epididymidis if males in test group 03 (12500 ppm) was slightly, but significantly higher compared to controls. However, the values were within the historical control range (males, abnormal sperms 5.0-6.8%) and therefore, this alteration was regarded as incidental and not treatment-related.
Reproductive performance:
no effects observed
Description (incidence and severity):
Male reproduction data:
For all F0 parental males, which were placed with females to generate F1 pups, copulation was confirmed. Thus, the male mating index was 100% in all test groups (00 - 03).
Fertility was proven for most of the F0 parental males within the scheduled mating interval for F1 litter. Two low-dose males (1000 ppm), one mid-dose male (4000 ppm) and three high-dose males (12500 ppm) did not generate F1 pups.
Thus, the male fertility index ranged between 88% and 100%, reflecting the normal range of biological variation inherent in the strain of rats used for this study. The apparently infertile male rats did not show histopathological findings that could explain infertility.

Female reproduction and delivery data:
The female mating index calculated after the mating period for F1 litter was 100% in all test groups.
The mean duration until sperm was detected (GD 0) varied between 1.9 and 3.2 days.
All female rats delivered pups or had implants in utero with the following exception: 2 females of Test goup 1, 1 female of Test group 2 and 3 females of Test group 3 did not become pregnant. The apparently infertile female rats did not show histopathological findings that could explain infertility. The fertility index ranged between 88% and 100% reflecting the normal range of biological variation inherent in the strain of rats used for this study. The mean duration of gestation values varied between 21.7 and 22.3 days without any relation to dosing. The gestation index was 100% in in all test groups.
Implantation was not affected by the treatment since the mean number of implantation sites was comparable between all test substance-treated groups and the control, taking normal biological variation into account (12.7 / 12.9 / 13.0 and 11.5 implants/dam in test groups 00 - 03, respectively). Furthermore, there were no indications for test substance-induced intrauterine embryo-/fetolethality since the postimplantation loss did not show any statistically significant differences between the groups (5.8% / 4.1% / 8.6% and 6.1% in test groups 00 – 03, respectively), and the mean number of F1 pups delivered per dam remained unaffected (12.0 / 12.4 / 11.9 and 11.0 pups/dam, respectively in test groups 00 - 03).
The rate of liveborn pups was also not affected by the test substance, as indicated by live birth indices of 100% / 96% / 100% and 99% in test groups 00 - 03. Moreover, apart from test group 1, there were only single cases of stillborn pups noted in the high-dose group and the control.
The higher number of stillborn pups and subsequently statistically significantly reduced number of liveborn pups in test group 01 were solely caused by one single dam with 7 stillborn pups. This single case was not dose-related and was considered to be spontaneous in nature and not treatmend related.

Effect levels (P0)

open allclose all
Dose descriptor:
NOAEL
Remarks:
general, systemic toxicity
Effect level:
4 000 ppm (nominal)
Sex:
male/female
Basis for effect level:
food consumption and compound intake
Dose descriptor:
NOAEL
Remarks:
fertility and reproductive performance
Effect level:
12 500 ppm (nominal)
Sex:
male/female
Basis for effect level:
other: highest dose tested

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Description (incidence and severity):
No clinical signs or changes of general behavior, which may be attributed to the test substance, were detected in any of the male and female animals in any of the groups.

One control male animal showed a palpable mass (throat, enlarged, size >1.5 cm) during study days 43 - 67 and one control female animal showed long teeth on study day 7 and anomaly of teeth during study days 8 - 71.
Mortality / viability:
no mortality observed
Description (incidence and severity):
There were no test substance-related or spontaneous mortalities in any of the groups.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
The body weights of the high-dose male rats were statistically significantly below the concurrent control values on study days 28 - 56 (up to 7%). The body weights of the high-dose female rats were statistically significantly below the concurrent control values during study days 42 - 49 and on study day 63 (up to 6%).

The body weight change of the high-dose males was below the concurrent control in most study sections, gaining statistical significance during study days 7 – 28 (about 11% below control). Overall the males gained significantly less weight (7% between day 0 and 63). Likewise, the body weight change of the high-dose females was below the concurrent control in most study sections, gaining statistical significance during study days 14 – 21 and 35 – 42 (about 14 and 35% below control). All single decreases add up to a statistically significantly lower female weight gain (7% between day 0 and 63).

The body weights/body weight change of the low- and mid-dose male and female rats were comparable to the concurrent control values throughout the entire study.
Several episodes of increased or decreased body weights/body weight change were regarded as sporadic events: increased body weights in the mid-dose males on study day 0, in the mid-dose females during study days 0 – 7; increased body weight change in the high-dose females during study days 49 - 56; decreased body weight change in the mid-dose females during study days 28 - 35 and 56 – 63.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Food consumption of all test substance treated male and female rats (1000, 4000 and 12500 ppm) was comparable to the concurrent control values throughout the entire study.
Haematological findings:
no effects observed
Description (incidence and severity):
No treatment-related changes among hematological parameters were observed.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
No treatment-related changes among clinical chemistry parameters were observed.
Urinalysis findings:
no effects observed
Description (incidence and severity):
No treatment-related, adverse changes among urinalysis parameters were observed.

In males of test group 13 (12500 ppm) urine pH-values were significantly decreased and the incidence of transitional epithelial cells and granular and epithelial casts in the urine sediment were significantly increased. Higher counts of casts were already found in males of test group 2 (4000 ppm). Lower urine pH values per se were regarded as maybe treatment-related but not adverse. Higher incidences of epithelial and granular casts and transitional epithelial cells which were observed in males only, in combination with the histopathologic finding of alpha-2-u-globulinuria is regarded as a rat specific effect with no relevance for humans (Hard et al., 1993).

In males of test group 2 (4000 ppm) specific gravity of the urine was significantly higher compared to controls, but the alteration was not dose-dependent and therefore it was regarded as incidental and not treatment-related.
Sexual maturation:
no effects observed
Description (incidence and severity):
Estrous cycle data, generated during 2 weeks, revealed regular cycles in the females of all test groups including the control. The mean estrous cycle duration in the different test groups was similar: 4.0 days in control, 4.0 days in the low-dose group, 4.2 days in the mid-dose group and 4.2 days in the high-dose group.

No alterations of the percentage of abnormal sperms occurred among the F1A males.
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
For tables please refer to "Any other information on results incl. tables"

COHORT 1A:
In test group 13 (12500 ppm), the significant absolute and relative weight increase of the adrenal glands in males (absolute / relative: 77.35 mg / 0.025%) and females (88.45 mg / 0.046%) were above the historical control values and was regarded as a treatment-related effect. The significant relative weight increase of the kidneys in males (0.717%) was marginally above the historical control values and had a histopathological correlate and was regarded as treatment-related.
The significant relative liver weight increase of animals of test group 13 (males: 3.082%, females: 2.812%) and 12 (males: 2.976%, females: 2.784%) were above the historical control range values. Although no histopathological correlate could be identified, a possible treatment-related effect cannot be excluded.
The significant relative weight increases of the seminal vesicles and testes in males of test group 13, were within (seminal vesicles) or marginally above (testes) historical control values. The absence of a histopathological correlate suggested, that the weight increases were rather incidental and not treatment-related.
All other mean absolute and relative weight parameters did not show significant differences when compared to the control group 10.

COHORT 1B:
In males and females of test group 13 (12500 ppm), the significant decrease of the terminal body weight (males: 327.852 g, females: 196.392 g) was within the historical control values and was regarded as incidental and not treatment-related. The significant absolute and relative weight increase of the adrenal glands in males (absolute / relative: 81.4 mg / 0.025%) and females (96.04 mg / 0.049%) were above the historical control values.
The significant absolute and relative liver weight increase in test groups 12 and 13 males and test group 12 and 13 females were above the historical control values. All these changes were assumed to be treatment-related.
All other mean absolute and relative weight parameters did not show significant differences when compared with the control group 10.

PATHOLOGY EXAMINATIONS OF SURPLUS F1 GENERATION PUPS ON PND 22 (F1 WEANLINGS NOT SELECTED FOR COHORTS)
Absolute organ weights
When compared with the control group 01 (set to 100%), the spleen of male animals in test group 03 was significantly decreased (86%). These change was regarded as incidental, since the relative spleen weight showed no significant changes. All other mean absolute weight parameters showed no significant differences.
Relative organ weights
All mean relative weight parameters of male and female animals showed no significant differences when compared with the respective control groups.
Gross pathological findings:
no effects observed
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.
Histopathological findings:
no effects observed
Description (incidence and severity):
For tables please refer to "Any other information on results incl. tables"

COHORT 1A:
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.
Reproductive organs of all cohort 1A male and female animals showed no histopathological changes. No treatment-related alterations of estrous cyclicity were observed.
Other effects:
no effects observed
Description (incidence and severity):
In F1A rats of both sexes (test groups 11, 12 and 13; 1000, 4000 and 12500 ppm) no treatment-related alterations of T4 and TSH levels were observed.

No alterations of the percentage of abnormal sperms occurred among the F1A males.

Details on results (F1)

Differential ovarian follicle count:
The results of the differential ovarian follicle count (DOFC) – comprising the numbers of primordial and growing follicles, as well as the combined incidence of primordial plus growing follicles – did not reveal significant differences between the control group 11 and animals of test group 13

Effect levels (F1)

open allclose all
Dose descriptor:
NOAEL
Remarks:
general, systemic toxicity
Generation:
F1
Effect level:
4 000 ppm (nominal)
Sex:
male/female
Basis for effect level:
body weight and weight gain
food consumption and compound intake
Dose descriptor:
NOAEL
Remarks:
developmental toxicity
Generation:
F1
Effect level:
12 500 ppm (nominal)
Sex:
male/female
Basis for effect level:
other: highest dose tested
Dose descriptor:
NOAEL
Remarks:
developmental immunotoxicity
Generation:
F1
Effect level:
10 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
other: highest dose tested

Overall reproductive toxicity

Key result
Reproductive effects observed:
no

Any other information on results incl. tables

PATHOLOGY of F0 GENERATION, PARENTAL ANIMALS

Absolute organ weights

When compared with the control group 00 (set to 100%), the mean absolute weights of following organs were significantly increased in one or more test groups (statistically significant changes printed in bold):

F0

Male animals

 

 

Female animals

 

 

Test group

(ppm)

01

(1000)

02

(4000)

03

(12500)

01

(1000)

02

(4000)

03

(12500)

Adrenal glands

105%

111%*

134%**

98%

106%

118%**

Kidneys

100%

101%

108%**

 

 

 

Liver

105%

109%*

119%**

 

 

 

Seminal vesicles

106%

108%*

114%**

 

 

 

Thyroid glands

 

 

 

103%

106%

114%**

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

 

Relative organ weights

When compared with the control group 00 (set to 100%), the mean relative weights of following organs were significantly increased in one or more test groups (statistically significant changes printed in bold):

 

F0

Male animals

Female animals

Test group

(ppm)

01

(1000)

02

(4000)

03

(12500)

01

(1000)

02

(4000)

03

(12500)

Adrenal glands

106%

114%**

140%**

98%

105%

121%**

Kidneys

100%

103%

113%**

 

 

 

Liver

105%

112%**

124%**

100%

106%*

106%**

Seminal vesicles

106%

111%*

118%**

 

 

 

Thyroid glands

 

 

 

102%

106%

117%**

Spleen

89%

103%

111%*

0

 

 

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

Histopathology

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

Adrenal cortex 

F0

Male animals

 

 

 

Test group

(ppm)

00

(0)

01

(1000)

02

(4000)

03

(12500)

No. of animals

20

22

21

23

Hyperplasia, cortical, diffuse

0

0

0

5

·          Grade 1

 

 

 

5

This finding was characterized by a minimal diffuse increase in the number of cortical cells of the zona fasciculata, resulting in an increased cortical thickness.

 

Liver

F0

Male animals

Female animals

Test group

(ppm)

00

(0)

01

(1000)

02

(4000)

03

(12500)

00

(0)

01

(1000)

02

(4000)

03

(12500)

No. of animals

20

20

20

20

20

20

20

20

Foci of cellular alteration

0

1

1

6

2

7

4

16

·          Basophilic

tigroid

0

0

1

4

2

7

4

16

·          Eosinophilic

 

0

0

0

2

0

0

0

2

·          Basophilic (NOS*)

0

1

0

0

0

0

0

0

Vasculitis /

perivasculitis

0

1

1

4

1

0

0

0

·          Grade 1

 

 

1

4

1

 

 

 

·          Grade 2

 

1

 

 

1

 

 

 

* = not otherwise specified

 

The increased incidence of foci of cellular alteration in males and females of test group 03 (12500 ppm) was assumed to be treatment-related. Almost all foci were single and small (up to 8 -15 cells) and of basophilic tigroid type. In addition, two males and two females of the high dose group showed additional single eosinophilic foci.

Minimal perivasculitis and /or vasculitis was noted in 4 males of the high dose test group. This lesion is a well-known background finding in the liver of rats. Because of the low incidence (20%) and grading they were judged as incidental and not treatment-related.

 

Kidneys

F0

Male animals

Test group

(ppm)

00

(0)

01

(1000)

02

(4000)

03

(12500)

No. of animals

20

20

20

20

Eosinophilic droplets

16

16

19

20

·          Grade 1

6

6

5

3

·          Grade 2

5

4

5

7

·          Grade 3

5

6

9

9

 

An accumulation of eosinophilic droplets was present in all test groups, with a subtle tendency to increase in severity in test group 02 and 03.These droplets proved to be positive in the immunohistochemical stain, revealing that the protein accumulation was alpha-2u-globulin. A possible treatment-related effect cannot be excluded, but has no human relevance as it is a species-specific effect in the male rat. 

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.

PATHOLOGY F1 REARING ANIMALS, COHORT 1A

Absolute organ weights

When compared with the control group 10 (set to 100%), the mean absolute weights of following organs were significantly increased in one or more test groups (statistically significant changes printed in bold):

F1A

Male animals

Female animals

Test group

(ppm)

11

(1000)

12

(4000)

13

(12500)

11

(1000)

12

(4000)

13

(12500)

Adrenal glands

99%

97%

116%**

100%

104%

113%**

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

 

Relative organ weights

When compared with the control group 10 (set to 100%), the mean relative weights of following organs were significantly increase in one or more test groups (statistically significant changes printed in bold):

F1A

Male animals

Female animals

Test group

(ppm)

11

(1000)

12

(4000)

13

(12500)

11

(1000)

12

(4000)

13

(12500)

Adrenal glands

100%

101%

125%**

102%

107%

119%**

Kidneys

98%

103%

110%**

 

 

 

Liver

96%

110%*

114%**

106%

112%*

113%**

Seminal vesicles

104%

111%

115%**

 

 

 

Testes

101%

99%

108%*

 

 

 

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

Histopathology

Treatment-related findings were observed in the adrenal cortex and kidneys of males, with incidences and grading according to the table below:

Adrenal cortex 

F1A

Male animals

Test group

(ppm)

10

(0)

11

(1000)

12

(4000)

13

(12500)

No. of animals

20

20

20

20

Hyperplasia, cortical, diffuse

0

0

0

2

·          Grade 1

 

 

 

2

The finding in the adrenal cortex was characterized as in the F0 generation by a minimal diffuse increase in the number of cortical cells of the zona fasciculata, resulting in an increased cortical thickness.

 

Kidneys 

F1A

Male animals

Test group

(ppm)

10

(0)

11

(1000)

12

(4000)

13

(12500)

No. of animals

20

20

20

20

Eosinophilic droplets

16

16

17

20

·          Grade 1

8

12

8

1

·          Grade 2

8

4

9

8

·          Grade 3

 

 

 

11

Casts, tubular granular

0

0

0

3

·          Grade 1

 

 

 

3

Tubules, basophilic

10

13

18

18

·          Grade 1

10

13

18

16

·          Grade 2

 

 

 

2

An accumulation of eosinophilic droplets in cortical tubules was present in all test groups, however, the severity was clearly increased in males of test group 13. These droplets proved to be positive in the immunohistochemical stain, revealing that the protein accumulation was alpha-2u-globulin. This was accompanied by minimal granular casts and a tendency to increased basophilic tubules in test group 13, which represent secondary effects to the protein accumulation. Changes in test group 13 were regarded as treatment-related, but of no human relevance as this finding is specific only for the male rat.

 

PATHOLOGY F1 REARING ANIMALS, COHORT 1B

Absolute and relative organ weights

When comparedwith the control group 10 (set to 100%), the mean absolute weights of following organs were significantly increased or decreased in one or more test groups (statistically significant changes printed in bold):

F1B

Male animals

Female animals

Test group

(ppm)

11

(1000)

12

(4000)

13

(12500)

11

(1000)

12

(4000)

13

(12500)

Terminal body weight

101%

100%

95%*

100%

100%

95%*

Adrenal glands

101%

106%

124%**

97%

103%

120%**

Liver

101%

110%*

109%*

98%

106%*

104%

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

Relative organ weights

When compared with the control group 10 (set to 100%), the mean relative weights of following organs were significantly increased in one or more test groups (statistically significant changes printed in bold):

F1B

Male animals

Female animals

Test group

(ppm)

11

(1000)

12

(4000)

13

(12500)

11

(1000)

12

(4000)

13

(12500)

Adrenal glands

100%

106%

130%**

97%

103%

127%**

Liver

100%

110%**

115%**

97%

105%*

109%**

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

Applicant's summary and conclusion