reaction mass of 3-methyl-4-(2,6,6-trimethylcyclohex-2-en-1-yl)but-3-en-2-one and 1-(2,6,6-trimethylcyclohex-2-en-1-yl)pent-1-en-3-one

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

OECD 443: Methylionon 70 (EC 942-741-0), oral, rat (BASF 2022)

- NOAEL (F0 general toxicity) = 40 mg/kg bw/d

- NOAEL (F1 general toxicity) = 150 mg/kg bw/d

- NOAEL (F0 and F1 fertility and reproductive performance) = 600 mg/kg bw/d

- NOAEL (F1 and F2 developmental toxicity) = 150 mg/kg bw/d

Link to relevant study records

Reference

Endpoint:

extended one-generation reproductive toxicity - with F2 generation (Cohorts 1A, and 1B with extension)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

19 Jan 2021 - 02 Jun 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 443 (Extended One-Generation Reproductive Toxicity Study)

Version / remarks:

25 June 2018

GLP compliance:

yes (incl. QA statement)

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: 10 weeks; according to ECHA Compliance Check Decision CCH-D-2114359360-53-01/F
- Basis for dose level selection: Based on a range finding study
- Inclusion of extension of Cohort 1B: yes; according to ECHA Compliance Check Decision CCH-D-2114359360-53-01/F
- Termination time for F2: terminal sacrifice of the F2 weanlings.
- Exclusion of developmental neurotoxicity Cohorts 2A and 2B: according to ECHA Compliance Check Decision CCH-D-2114359360-53-01/F
- Exclusion of developmental immunotoxicity Cohort 3: according to ECHA Compliance Check Decision CCH-D-2114359360-53-01/F
- Route of administration: oral (gavage): according to ECHA Compliance Check Decision CCH-D-2114359360-53-01/F
- Choice of species, strain: 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.

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Name of tst substance (used in the report): Methylionon 70
- Chemical Identity: Mixture of isomeres: 60-70% alpha-iso-methylionone, 17-20% alpha-n-methylionone, 1-10% beta-n-methylionone, 0-5% beta-iso-methylionone
- Batch number of test material: 00119477L0

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: under N2, room temperature
- Stability of the test material:
Expiry date: 15 Jun 2022
The stability of the test substance under storage conditions over the test period was guaranteed by the sponsor and the sponsor holds this responsibility.

Species:

rat

Strain:

Wistar

Remarks:

Crl:WI(Han)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source:
Charles River Laboratories, Research Models and Services, Germany GmbH
- Females nulliparous and non-pregnant: yes
- Age at start of the administration period: about 35 days
- Weight at study initiation: weight variation did not exceed 20 percent of the mean weight of each sex (P0)
- Fasting period before study: no
- Housing: up to 5 animals per sex and cage
From delivery to randomization, during overnight matings (male and female mating partners were housed together), gestation, lactation and females after weaning the animals were housed individually. Dams and their litters were housed together until PND 21/22.
- Diet: ad libitum; mouse and rat maintenance diet “GLP” (Granovit AG, Kaiseraugst, Switzerland)
- Water: ad libitum; tap water in water bottles
- Acclimation period: 7 days
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 45-65
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 26 Jan 2021 (TS administration) To: 12 Oct 2021 (sacrifice of F1 cohort 1B animals)

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Remarks:

0.5% Sodium carboxymethyl cellulose (CMC) suspension in deionized water (with 10 mg/ 100mL Cremophor)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:

VEHICLE
- Concentration in vehicle: 0.4, 1.5, 6.0 g/100 mL in low, mid and high dose, respectively
- Amount of vehicle (if gavage): 10 mL/kg bw/d

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: max. 2 weeks
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged: individually

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 0.5% CMC suspension indeionized water (with 10 mg/ 100 mL Cremophor) over a period of a maximum of 7 days at room temperature were verified prior to the start of the study in a comparable batch.

At the beginning (during premating) of the administration 3 samples each were taken from the lowest and highest concentration for potential homogeneity analyses. The 3 samples were withdrawn from the top, middle and bottom of the preparation vessel. These samples were used as a concentration control at the same time. At the above mentioned time point additionally one sample from the mid concentration was taken for concentration control analysis. Once during the middle and once during the end of the administration each 1 sample was taken from the low, mid and high concentration for a concentration control analysis.

Duration of treatment / exposure:

F0 males: 10 weeks (premating) + 2 weeks (mating) + max. 6 weeks (post-mating)
F0 females: 10 weeks (premating) + 2 weeks (mating) + approx. 6 weeks (pregnancy + lactation)
F1 males (Co 1B): 10 weeks (post-weaning/premating) + 2 weeks (mating) + 4 weeks (post-mating)
F1 males (Co 1B): 10 weeks (post-weaning/premating) + 2 weeks (mating) + approx. 6 weeks (pregnancy + lactation)
F1 animals (Co 1A): post weaning until an approx. age of 13 weeks

Frequency of treatment:

once daily

Details on study schedule:

F0 parental animals:
After a minimum of 10 weeks after the beginning of treatment, males and females from the same dose group were mated. 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 Cohorts 1A and 1B. Before weaning of the F1 pups the F0 generation parental male animals were sacrificed. After weaning of F1 pups the F0 generation parental female animals were sacrificed.

F1 (rearing animals):
Before weaning of the F1 generation pups on PND 21, 45 male and 45 females per group were randomly selected, to be placed into cohorts. Obvious runts (those pups whose body weight was >=25% below the mean body weight of the control group, separate for sexes) were not included:
- Cohort 1A: One male and one female/litter (20/sex/group)
- Cohort 1B: One male and one female/litter (25/sex/group)
Selected F1 offspring received the test substance daily by gavage until one day before sacrifice.

F1 parental animals (Cohort 1B):
After weaning, 25 male and 25 female F1 pups per test group became F1 generation parental animals. These animals were chosen by lot and each litter was taken into account as far as technically feasible. If fewer than 25 litters were available in a group or if one sex was missing in a litter, more animals were taken from the other litters of the respective test group to obtain the required number of animals to be paired. All selected animals were treated with the test substance at the same dose level as their parents, from post-weaning through adulthood. After a minimum of 10 weeks after assignment of the F1 generation parental animals, the males and females were mated, overnight. The partners were randomly assigned, mating of siblings was avoided. The females were allowed to deliver and rear their pups (F2 generation pups) until PND 4 (standardization) or PND 21. Before weaning of the F2 pups the F1 generation parental male animals were sacrificed. The F1 generation parental females were sacrificed, shortly after the F2 generation pups had been weaned.

Standardization of litters (F1 and F2 generation pups):
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). Surplus animals were sacrificed. Standardization of litters was not performed in litters with <= 10 pups.

Dose / conc.:

40 mg/kg bw/day (nominal)

Dose / conc.:

150 mg/kg bw/day (nominal)

Dose / conc.:

600 mg/kg bw/day (nominal)

No. of animals per sex per dose:

F0: 25 animals per sex per dose
F1A: 20 animals per sex per dose
F1B: 25 animals per sex per dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: based on range-finding study
- Fasting period before blood sampling for clinical biochemistry: yes, for about 16-20 hours

Positive control:

not applicable

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule:
Mortality: twice daily on working days or once daily (Saturday, Sunday or on public holidays).
Cage side observations: at least daily for any signs of morbidity, pertinent behavioral changes and signs of overt toxicity before the administration as well as within 2 hours and within 5 hours after the administration. The parturition and lactation behavior of the dams was generally evaluated in the mornings in combination with the daily clinical inspection of the dams. On weekdays (except Saturday, Sunday 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:
F0: animals once before the administration and subsequently once per week
F1 (Cohort 1A/1B): at weekly intervals during the administration period.

BODY WEIGHT: Yes
- Time schedule for examinations: once a week, except
• during the mating period of the F0 and F1 parental animals, the females were weighed on the day of positive evidence of sperm GD 0 and on GD 7, 14 and 20.
• F0 and F1 females with litter were weighed on the day of parturition PND 0 and on PND 1, 4, 7, 10, 14, 18 and 21.

FOOD CONSUMPTION:
- 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 7 days) for male and female F0 and F1 parental animals and F1 rearing animals, with the following exceptions:
• not determined after the 2nd premating week (male F0 animals) and after the 10th premating week (male F1 animals).
• determined with evidence of sperm of the F0 and F1 females for GD 0-7, 7-14 and 14-20.
• detemined for the F0 and F1 females, which gave birth to a litter for PND 1-4, 4-7, 7-10, 10-14, 14-18 and 18-21.
Food consumption was not determined in the females without positive evidence of sperm during mating and gestation periods, in the females without litter during lactation period and in the females after weaning.

WATER CONSUMPTION: Yes
- Time schedule for examinations: once a week for the male and female F0 and F1 parental animals and F1 rearing animals, with the following exceptions:
• not determined after the 10th premating week (male F1 parental animals) and during the mating period (male and female F0 parental animals)
• determined with evidence of sperm of the F0 and F1 females for GD 0-1, 3-4, 7-8, 10-11, 14-15, 17-18 and 19-20.
• determined for F0 and F1 females, which gave birth to a litter for PND 1-2, 4-5, 7-8, 10-11, 14-15, 17-18 and 20-21.
Water consumption was not determined in the females without positive evidence of sperm during mating and gestation periods and in the females without litter during lactation period and in the females after weaning.

CLINICAL PATHOLOGY
Samples were withdrawn from 10 F0 parental males and females per group at termination. Blood samples were taken from animals by puncturing the retrobulbar venous plexus following isoflurane anesthesia. In the afternoon preceding the day of urinalysis, the animals were individually transferred into metabolism cages (no food or drinking water provided); on the following morning, the individual urine specimens were examined.

Hematology
Parameters examined:
- Leukocyte count (WBC)
- Erythrocyte count (RBC)
- Hemoglobin (HGB)
- Hematocrit (HCT)
- Mean corpuscular volume (MCV)
- Mean corpuscular hemoglobin (MCH)
- Mean corpuscular hemoglobin concentration (MCHC)
- Platelet count (PLT)
- Differential blood count
- Reticulocytes (RETA)
- Prothrombin time (Hepato Quick’s test; HQT)

Clinical chemistry
Parameters examined:
- Alanine aminotransferase (ALT)
- Aspartate aminotransferase (AST)
- Alkaline phosphatase (ALP)
- gamma-Glutamyltransferase (GGT)
- Sodium (NA)
- Potassium (K)
- Chloride (CL)
- Inorganic phosphate (INP)
- Calcium (CA)
- Urea (UREA)
- Creatinine (CREA)
- Glucose (GLUC)
- Total bilirubin (TBIL)
- Total protein (TPROT)
- Albumin (ALB)
- Globulins (GLOB)
- Triglycerides (TRIG)
-Cholesterol (CHOL)

Hormones:
Parameters examined:
- Total thyroxine (T4)
- Thyroid stimulating hormone (TSH)
The concentrations of TSH were determined by radioimmunoassay (RIA), using commercially available RIA test kits. T4 was examined via ELISA.

Urinalysis
Parameters examined:
- pH
- Protein (PRO)
- Glucose (GLU)
- Ketones (KET)
- Urobilinogen (UBG)
- Bilirubin (BIL)
- Blood
- Specific gravity (SP.GR.)
- Sediment
- Color, turbidity (COL, TURB)
- Volume (VOL)

Oestrous cyclicity (parental animals):

evaluated by daily analysis of vaginal smear for all F0 female parental rats for all F1 female parental rats (= cohort 1B) 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 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 sacrificed on schedule:
- Sperm motility
- Sperm morphology
- Sperm head count (cauda epididymis)
- Sperm head count (testis)

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes

PARAMETERS EXAMINED
- Pup number and status at delivery
All pups delivered from the F0 parents (F1 litter) and the F1 parents (F2 litter) were examined as soon as possible on the day of birth to determine the total number of pups, the sex and the number of liveborn and stillborn pups in each litter. At the same time, the pups were also examined for macroscopically evident changes. Pups, which died before this initial examination, were defined as stillborn pups.

- Pup viability/mortality:
twice daily on workdays (once in the morning and once in the afternoon) or as a rule, only in the morning on Saturdays, Sundays or public holidays. The number and percentage of dead pups on the day of birth (PND 0) and of pups dying between PND 1-4, 5-7, 8-14 and 15-21 (lactation period) were determined; however, pups, which died accidentally or had to be sacrificed due to maternal death, were not included in these calculations. The number of live pups/litter was calculated on the day after birth, and on lactation days 4, 7, 14, and 21.

- Sex ratio
determined on the day of birth (PND 0) by observing the distance between the anus and the base of the genital tubercle; normally, the anogenital distance is considerably greater in male than in female pups. Later, during the course of lactation, this initial sex determination was followed up by surveying the external appearance of the anogenital region and the mammary line. The sex of the pups was finally confirmed at necropsy.

- Anogenital distance
Anogenital distance (AGD) is defined as the distance from the center of the anal opening to the base of the genital tubercle. AGD was determined in all live male and female pups on PND 1. These measurements were performed in randomized order, using a measuring ocular. They were conducted by technicians unaware of treatment group in order to minimize bias.

- Pup clinical observations
examined daily for clinical symptoms (including gross-morphological findings) during the clinical inspection of the dams.

- Nipple/areola anlagen
examined and counted in all surviving F1 and F2 male pups on PND 13 and on PND 20.

- Pup body weight data
on the day after birth (PND 1) and on PND 4 (before standardization), 7, 14 and 21.

- Vaginal opening
All female F1 pups selected to become the F1 parental generation females (cohort 1B) and F1 rearing animals (cohort 1A) were evaluated daily for vaginal patency beginning on PND 27. On the day of vaginal opening the body weights of the respective animals were determined.

- Preputial separation
All male F1 pups selected to become the F1 parental generation females (cohort 1B) and F1 rearing animals (chort 1A) were evaluated daily for preputial separation beginning on PND 38. On the day of preputial separation the body weights of the respective animals were determined.


CLINICAL PATHOLOGY (Cohort 1A):
Samples were withdrawn from 10 cohort 1A males and females per group at termination. Blood samples were taken from animals by puncturing the retrobulbar venous plexus following isoflurane anesthesia. In the afternoon preceding the day of urinalysis, the animals were individually transferred into metabolism cages (no food or drinking water provided); on the following morning, the individual urine specimens were examined.
Hematology
Parameters examined:
- Leukocyte count (WBC)
- Erythrocyte count (RBC)
- Hemoglobin (HGB)
- Hematocrit (HCT)
- Mean corpuscular volume (MCV)
- Mean corpuscular hemoglobin (MCH)
- Mean corpuscular hemoglobin concentration (MCHC)
- Platelet count (PLT)
- Differential blood count
- Reticulocytes (RETA)
- Prothrombin time (Hepato Quick’s test; HQT)

Clinical chemistry
Parameters examined:
- Alanine aminotransferase (ALT)
- Aspartate aminotransferase (AST)
- Alkaline phosphatase (ALP)
- gamma-Glutamyltransferase (GGT)
- Sodium (NA)
- Potassium (K)
- Chloride (CL)
- Inorganic phosphate (INP)
- Calcium (CA)
- Urea (UREA)
- Creatinine (CREA)
- Glucose (GLUC)
- Total bilirubin (TBIL)
- Total protein (TPROT)
- Albumin (ALB)
- Globulins (GLOB)
- Triglycerides (TRIG)
-Cholesterol (CHOL)

Urinalysis
Parameters examined:
- pH
- Protein (PRO)
- Glucose (GLU)
- Ketones (KET)
- Urobilinogen (UBG)
- Bilirubin (BIL)
- Blood
- Specific gravity
- Sediment
- Color, turbidity (COL, TURB)
- Volume (VOL)

Hormone analysis (F1; PND 4 and PND 22)
Blood samples were withdrawn from 10 surplus (culled) PND 4 offspring (as far as possible of different litters) per sex and group. PND 4 samples were pooled per sex and litter if the available amount is not sufficient for a hormone analysis. Blood samples were withdrawn from 10 surplus PND 22 offspring (as far as possible of different litters) per sex and group. The blood samples were collected after decapitation (following isoflurane anesthesia) from the Vena cava cranialis.
Parameters examined:
- Total thyroxine (T4)
- Thyroid stimulating hormone (TSH)

Hormone analysis (F1; Cohort 1A):
Samples were withdrawn from 10 cohort 1A males and females per group at termination. Blood samples were taken from animals by puncturing the retrobulbar venous plexus following isoflurane anesthesia.
Parameters examined:
- Total thyroxine (T4)
- Thyroid stimulating hormone (TSH)
The concentrations of TSH were determined by radioimmunoassay (RIA), using commercially available RIA test kits. T4 was examined via ELISA.


ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY:
no

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: no

Postmortem examinations (parental animals):

SACRIFICE
All F0 generation parental animals were 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.

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. Cauda epididymis
5. Epididymides
6. Heart
7. Kidneys
8. Liver
9. Ovaries
10. Pituitary gland (fixed)
11. Prostate (ventral and dorsolateral part together, fixed)
12. Testes
13. Seminal vesicles including coagulating glands (fixed)
14. Spleen
15. Thymus (fixed)
16. Thyroid glands (with parathyroid glands) (fixed)
17. Uterus with cervix
All paired organs were weighed together (left and right).


HISTOPATHOLOGY
The organs or tissues were fixed in 4% neutral-buffered formaldehyde solution except for epididymis, testis, eyes and ovaries (fixed in modified Davidson’s solution):
Organs/ tissues examined:
1. All gross lesions
2. Adrenal glands
3. Bone marrow (femur)
4. Brain
5. Cecum
6. Cervix
7. Coagulating glands
8. Colon
9. Duodenum
10. Epididymis, left
11. Esophagus
12. Eyes with optic nerve
13. Heart
14. Ileum
15. Jejunum
16. Kidneys
17. Liver
18. Lungs
19. Lymph nodes, axillary
20. Lymph nodes, mesenteric
21. Mammary gland (male and female)
22. Ovaries
23. Oviducts
24. Pancreas
25. Pituitary gland
26. Prostate
27. Peyer’s patches
28. Rectum
29. Sciatic nerve
30. Seminal vesicles
31. Skeletal muscle
32. Spinal cord (cervical, thoracic, lumbar)
33. Spleen
34. Stomach (forestomach and glandular stomach)
35. Testis, left
36. Thymus
37. Thyroid glands
38. Trachea
39. Urinary bladder
40. Uterus
41. Vagina
42. Vas deferens

The uteri of all cohabited female F0 generation parental animals were examined for the presence and number of implantation sites. The uteri of apparently nonpregnant animals or empty uterus horns were placed in 1% ammonium sulfide solutions for about 5 minutes in order to be able to identify early resorptions or implantations (SALEWSKI's method [Salewski, 1964]). Then the uteri were rinsed carefully in physiologic salt solution (0.9 % NaCl).

Postmortem examinations (offspring):

SPERM PARAMETERS (Cohort 1A)
After the organ weight determination, the following parameters were determined in the right testis or right epididymis of all male cohort 1A animals sacrificed on schedule:
- Sperm motility
- Sperm morphology
- Sperm head count (cauda epididymis)
- Sperm head count (testis)

DOFC (Cohort 1A)
A differential ovarian follicle count (DOFC) was conducted in control and high dose test groups according to Plowchalk et.al. (1993). In general, sections were prepared with 2 - 3 µm thickness and serial sections were taken every 100 µm to complete about 20 cut levels across the whole ovarian tissue. For the counting of primordial and growing follicles, H&E stained slides were prepared from all cut levels.


PATHOLOGY (F1 pups)
On PND 4, as a result of standardization, all surplus F1 pups were sacrificed by decapitation under isoflurane anesthesia and blood was sampled for determination of serum thyroid hormone concentrations. After sacrifice, these pups were examined externally, eviscerated and their organs were assessed macroscopically.

On PND 22, the surplus F1 generation pups that were not used for the formation of the cohorts or any investigations were sacrificed under isoflurane anesthesia with CO2 and were examined for pathology. The selected pups for hormone analyses were sacrificed by decapitation under isoflurane anesthesia blood was sampled for thyroid hormone analyses. All animals were necropsied and assessed by gross pathology with special emphasis on the reproductive organs.

- Organ weights (surplus F1 pups; PND 22)
The following weights were determined in up to 10 animals per sex per group sacrificed on schedule:
1. Anesthetized animals (final body weight)
2. Brain
3. Spleen
4. Thymus (fixed)


PATHOLOGY (Cohort 1A)
All F1 generation rearing animals, cohort 1A were sacrificed by decapitation under isoflurane anesthesia.

- Gross neropsy (Cohort 1A)
The exsanguinated animals were necropsied and assessed by gross pathology; special attention being given to the reproductive organs.

- Organ weights (Cohort 1A)
The following weights were determined in all animals sacrificed on schedule:
1. Anesthetized animals (final body weight)
2. Adrenal glands (fixed)
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 (fixed)
13. Prostate (ventral and dorsolateral part together, fixed)
14. Testes
15. Seminal vesicles including coagulating glands (fixed)
16. Spleen
17. Thymus (fixed)
18. Thyroid glands (with parathyroid glands) (fixed)
19. Uterus with cervix
All paired organs were weighed together (left and right).

- Histopathology (Cohort 1A)
The organs or tissues were fixed in 4% neutral-buffered formaldehyde solution except for epididymis, testis, eyes and ovaries (fixed in modified Davidson’s solution):
Organs/ tissues examined:
1. All gross lesions
2. Adrenal glands
3. Bone marrow (femur)
4. Brain
5. Cecum
6. Cervix
7. Coagulating glands
8. Colon
9. Duodenum
10. Epididymis, left
11. Esophagus
12. Eyes with optic nerve
13. Heart
14. Ileum
15. Jejunum
16. Kidneys
17. Liver
18. Lungs
19. Lymph nodes, axillary
20. Lymph nodes, mesenteric
21. Mammary gland (male and female)
22. Ovaries
23. Oviducts
24. Pancreas
25. Pituitary gland
26. Prostate
27. Peyer’s patches
28. Rectum
29. Sciatic nerve
30. Seminal vesicles
31. Skeletal muscle
32. Spinal cord (cervical, thoracic, lumbar)

33. Spleen
34. Stomach (forestomach and glandular stomach)
35. Testis, left
36. Thymus
37. Thyroid glands
38. Trachea
39. Urinary bladder
40. Uterus
41. Vagina
42. Vas deferens


PATHOLOGY (Cohort 1B)
All cohort 1B were sacrificed by decapitation under isoflurane anesthesia.

- Gross neropsy (Cohort 1B)
The exsanguinated animals were necropsied and assessed by gross pathology; special attention being given to the reproductive organs.

- Organ weights (Cohort 1B)
The following weights were determined in all animals sacrificed on schedule:
1. Anesthetized animals (final body weight)
2. Adrenal glands (fixed)
3. Cauda epididymis
4. Epididymides
5. Liver
6. Ovaries
7. Pituitary gland (fixed)
8. Prostate (ventral and dorsolateral part together, fixed)
9. Seminal vesicles including coagulating gland (fixed)
10. Testes
11. Uterus (with cervix)
All paired organs were weighed together (left and right).

- Histopathology (Cohort 1B)
The organs or tissues were fixed in 4% neutral-buffered formaldehyde solution except for epididymis, testis and ovaries (fixed in modified Davidson’s solution):
Organs/ tissues examined:
1. All gross lesions
2. Adrenal glands
3. Cervix uteri
4. Coagulating glands
5. Epididymides, left
6. Liver
7. Ovaries
8. Prostate
9. Pituitary gland
10. Seminal vesicles
11. Testes, left
12. Uterus
13. Vagina

The uteri of all cohabited female cohort 1B animals were examined for the presence and number of implantation sites. The uteri of apparently nonpregnant animals or empty uterus horns were placed in 1% ammonium sulfide solutions for about 5 minutes in order to be able to identify early resorptions or implantations (SALEWSKI's method [Salewski, 1964]). Then the uteri were rinsed carefully in physiologic salt solution (0.9 % NaCl).

- Splenic lymphocyte subpopulation analysis
10 males and 10 females of cohort 1A were used to perform a splenic lymphocyte subpopulation analysis using one half of the spleen.
The following parameters were analysed:
- B lymphocytes (B_SPL)
- T lymphocytes (T_SPL)
- CD4+ lymphocytes (CD4_SPL)
- CD8+ lymphocytes (CD8_SPL)
- Natural killer cells (NK_SPL)


PATHOLOGY (F2 pups)
After a similar standardization on PND 4, the surplus F2 pups were sacrificed under isoflurane anesthesia with CO2.
On PND 21, all F2 generation pups were sacrificed under isoflurane anesthesia with CO2. After sacrifice, these pups were examined externally, eviscerated and their organs were assessed macroscopically.

- Pup organ weights (F2 pups)
After the scheduled sacrifice, the brain, spleen and thymus of 1 pup/sex per F2 litter were weighed. Normally, the first male and the first female pups/litter were taken for these determinations. The corresponding in-life pup weights determined on PND 21 were used to calculate the relative organ weights.

Statistics:

- DUNNETT test (two-sided): Water consumption (parental and rearing animals), food consumption (parental and rearing animals), body weight and body weight change (parental animals, rearing animals and pups; for the pup weights, the litter means were used), gestation days, duration of sexual maturation (days to vaginal opening, days to preputial separation), anogenital distance, anogenital index.
- FISHER'S EXACT test (one-sided): Male and female mating indices, male and female fertility indices, gestation index, females mated, females pregnant, females delivering, females with liveborn pups, females with stillborn pups, females with all stillborn pups
- WILCOXON-test (one-sided): Urinalysis parameters (apart from pH, urine volume, specific gravity, color and turbidity)
- WILCOXON-test (one-sided) with BONFERRONI-HOLM adjustment: Spermanalysis parameters
- WILCOXON test (one-sided+) with BONFERRONI-HOLM adjustment: Mating days until day 0 pc, %postimplantation loss, pups stillborn, %perinatal Loss, nipple development
- WILCOXON test (one-sided-) with BONFERRONI-HOLM adjustment: Implantation sites, pups delivered, pups liveborn, live pups day x, viability index, lactation index
- WILCOXON-test (one-sided-): DOFC (differential ovarian follicular count); Follicles primordial, growing, primordial + growing
- WILCOXON test (two-sided): % live male day x, % live female day x
- KRUSKAL-WALLIS test (two-sided) + WILCOXON-test (two-sided): Number of cycles and Cycle Length, pup organ weights (absolute and relative), Organ weight parameters
- KRUSKAL-WALLIS test + WILCOXON-test (one or two-sided): Blood parameters, Urine pH, volume and specific gravity; splenic lymphocytes subpopulations

Reproductive indices:

Male reproduction data for F1 and F2 litters:
Male mating index (%) = number of males with confirmed mating / number of males placed with females x 100
Male fertility index (%) = number of males proving their fertility / number of males placed with females x 100

Female reproduction and delivery data for F1 and F2 litters:
Female mating index (%) = number of females mated / number of females placed with males x 100
Female fertility index (%) = number of females pregnant / number of females mated x 100
Gestation index (%) = number of females with live pups on the day of birth / number of females pregnant x 100

Offspring viability indices:

Live birth index (%) = number of liveborn pups at birth / total number of pups born x 100
Postimplantation loss (%) = number of implantations – number of pups delivered / number of implantations x 100
Viability index (%) = number of live pups on day 4* after birth / number of live pups on the day of birth x 100
Lactation index (%) = number of live pups on day 21 after birth / number of live pups on day 4* after birth x 100

*before standardization of litters (i.e. before culling)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Males (day 0-113)
- Test group 01 (40 mg/kg bw): no effects
- Test group 02 (150 mg/kg bw): salivation (25/25); plough nose-first into bedding (1/25)
- Test group 03 (600 mg/kg bw): salivation (25/25); unsteady gait (7/25); semiclosed eyelid (1/25); poor general condition, labored respiration and pilorection (2/25); plough nose-first into bedding (23/25)

Females (premating day 0-75)
- Test group 01 (40 mg/kg bw): salivation (3/25); eye: semiclosed eyelid, opacity and injury (1/25)
- Test group 02 (150 mg/kg bw): salivation (22/25); plough nose-first into bedding (1/25)
- Test group 03 (600 mg/kg bw): salivation (25/25); unsteady gait (18/25); semiclosed eyelid (2/25); pilorection (1/25); plough nose-first into bedding (21/25); reduced attention (1/25)
Females (mating day 0-14)
- Test group 01 (40 mg/kg bw): salivation (1/19)
- Test group 02 (150 mg/kg bw): salivation (9/20)
- Test group 03 (600 mg/kg bw): salivation (11/17); plough nose-first into bedding (3/17)
Females (gestation day 0-57)
- Test group 01 (40 mg/kg bw): saliation (5/24); eye injury, opacity, semiclosed eyelid and small eye (1/24)
- Test group 02 (150 mg/kg bw): salivation (20/25); plough nose-first into bedding (3/25)
- Test group 03 (600 mg/kg bw): salivation (23/24); plough nose-first into bedding (15/24)

- Observed salivation partly accompanied by plough nose-first into bedding in male and female animals was temporary and disappeared after a maximum of five hours which is why the effect was regarded to be test substance-induced but not adverse-

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

Males (day 0-113)
- Test group 01 (40 mg/kg bw): no deaths
- Test group 02 (150 mg/kg bw): no deaths
- Test group 03 (600 mg/kg bw): sacrificed moribund (2/25) due to poor general conditions

- the mortalities in the test group 03 were assessed to be not related to the test substance as the findings (mixed cell inflammation of pleura and pericardium, inflammatory cell infiltrates in the esophagus, inflammatory process in the thoracic cavity) are suspicious of a gavage error

Females (premating day 0-75, mating day 0-14, gestation day 0-57)
- Test group 01 (40 mg/kg bw/: no deaths
- Test group 02 (150 mg/kg bw): no deaths
- Test group 03 (600 mg/kg bw): no deaths

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Males (day 0-113)
- Test group 01 (40 mg/kg bw): no effects
- Test group 02 (150 mg/kg bw): body weight - no effects; body weight change – reduced from day 42-49 (-17.3%)
- Test group 03 (600 mg/kg bw): body weight - reduced from day 49-105 (max. -9.5% on day 105); body weight change - reduced from day 35-70 and day 98-105 (max. -38.6% on day 56)
- Observed effects on body weight and body weight change in male animals was considered test item-related but not adverse

Females (premating day 0-75)
- Test group 01 (40 mg/kg bw): no effects
- Test group 02 (150 mg/kg bw): no effects
- Test group 03 (600 mg/kg bw): no effects
Females (mating day 0-14)
- Test group 01 (40 mg/kg bw): no effects
- Test group 02 (150 mg/kg bw): no effects
- Test group 03 (600 mg/kg bw): no effects
Females (gestation day 0-56)
- Test group 01 (40 mg/kg bw): no effects
- Test group 02 (150 mg/kg bw): no effects
- Test group 03 (600 mg/kg bw): no effects
Females (lactation day 0-35)
- Test group 01 (40 mg/kg bw): no effects
- Test group 02 (150 mg/kg bw): body weight - no effect; body weight change - increased from PND 0-1 and PND 7-10 (1.5% on PND 1; 13% on PND 10), decreased from PND 4-7 (-1.2%)
- Test group 03 (600 mg/kg bw): body weight - no effect; body weight change - increased from PND0-1
- Observed effects on body weight change in female animals was considered incidental in nature, since there was no dose-dependency

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Males (day 0-113)
- Test group 01 (40 mg/kg bw): no effects
- Test group 02 (150 mg/kg bw): no effects
- Test group 03 (600 mg/kg bw): no effects

Females (premating day 0-75, gestation 0-56, lactation 0-35)
- Test group 01 (40 mg/kg bw): no effects
- Test group 02 (150 mg/kg bw): no effects
- Test group 03 (600 mg/kg bw): no effects

Food efficiency:

not examined

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

effects observed, treatment-related

Description (incidence and severity):

Males (day 0-113)
- Test group 01 (40 mg/kg bw): no effects
- Test group 02 (150 mg/kg bw): no effects
- Test group 03 (600 mg/kg bw): compared to control up to +25.0% (day 7-70)

Females (premating day 0-75)
- Test group 01 (40 mg/kg bw): no effects
- Test group 02 (150 mg/kg bw): no effects
- Test group 03 (600 mg/kg bw): compared to control up to +24% (day 7-63)
Females (gestation day 0-56)
- Test group 01 (40 mg/kg bw): no effects
- Test group 02 (150 mg/kg bw): compared to control up to +24% (GD 0-1) and +20% (GD19-20)
- Test group 03 (600 mg/kg bw): compared to control up to +41% (GD 1-20)
Females (lactation day 0-35)
- Test group 01 (40 mg/kg bw): no effects
- Test group 02 (150 mg/kg bw): no effects
- Test group 03 (600 mg/kg bw): no effects

- Observed increase in water consumption was regarded to be test substance-induced but not adverse

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Males
- Test group 01 (40 mg/kg bw): no effects
- Test group 02 (150 mg/kg bw): increased reticulocytes (138.6 giga/L)
- Test group 03 (600 mg/kg bw): shortened prothrombin time (32.0 sec), increased platelets count (771 giga/L), decreased hemoglobin (8.6 mmol/L), increased reticulocytes (171.1 giga/L), decreased absolute and relative eosinophil counts (0.10 giga/L; 2.0%)
- Observed effect on reticulocytes in male animals was assessed to be adaptive since a hypothetical decrease of the red blood cell parameters may be compensated which is confirmed by histological findings of extramedullary hematopoiesis in the spleens
- Observed effects on hemoglobin in male animals were within historical control range

Females
- Test group 01 (40 mg/kg bw): increased white blood cell count (2.99 giga/L), increased absolute lymphocyte count (2.15 giga/L)
- Test group 02 (150 mg/kg bw): shortened prothrombin time (33.0 sec), increased white blood cell count (2.88 giga/L)
- Test group 03 (600 mg/kg bw): shortened prothrombin time (31.7 sec), increased white blood cell count (3.80 giga/L), increased absolute lymphocyte count (2.73 giga/L), increased absolute large unstained cell counts (0.02 giga/L), decreased relative basophil count (0.3%)

- Observed effects on prothrombin time and platelets count in male and female animals were assessed to be test item-related and adverse
- Observed effects related to white blood cell count in male and female animals were within historical control range

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Males
- Test group 01 (40 mg/kg bw): decreased alanine aminotransferase (ALT) activities (0.64 µkat/L)
- Test group 02 (150 mg/kg bw): increased total protein (66.69 g/L), increased globulin (30.88 g/L), increased calcium levels (2.65 mmol/L), decreased total bilirubin level (1.15 µmol/L)
- Test group 03 (600 mg/kg bw): increased γ-glutamyl transferase (GGT) activities (69 nkat/L), increased total protein (70.04 g/L), increased globulin (38.02 g/L), increased calcium levels (2.67 mmol/L), decreased glucose levels (3.88 mmol/L), decreased alanine aminotransferase (ALT) activities (0.58 µkat/L), increased albumin (38.02 g/L) and potassium levels (5.00 mmol/L), decreased chloride levels (101. mmol/L)

Females
- Test group 01 (40 mg/kg bw): increased urea levels, decreased inorganic phosphate level
- Test group 02 (150 mg/kg bw): increased urea levels, increased sodium levels, decreased total bilirubin level
- Test group 03 (600 mg/kg bw): increased γ-glutamyl transferase (GGT) activities (40 nkat/L), increased total protein, increased globulin, increased calcium levels, decreased glucose levels, increased triglyceride values, decreased alkaline phosphatase (ALP) activities (0.58 µkat/L), decreased chloride levels, increased urea levels, increased albumin levels, increased sodium levels, decreased total bilirubin level

- Observed effects on total protein (male/female animals), globulin (male/female animals), calcium levels (male/female animals), GGT (male/female animals), glucose (male/female animals) and triglyceride (female animals) values were regarded as treatment-related and adverse
- Observed effects on ALT (male animals) and ALP (female animals) were regarded as treatment-related and adaptive (PSD Guidance Document: Toxicological Significance of Reduced Levels of Serum ALT and/or AST in Animal Studies, May 2007)
- Observed effects on albumin (male/female animals), potassium (male animals), chloride (male/female animals), urea (female animals) and sodium levels (female animals) were within historical control ranges
- Observed effects on total bilirubin level (male/female animals) and inorganic phosphate level (female animals) were regarded as incidental since these changes were not dose-dependent

Endocrine findings:

not specified

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

Males
- Test group 01 (40 mg/kg bw): no effects
- Test group 02 (150 mg/kg bw): decreased pH (6.2)
- Test group 03 (600 mg/kg bw): increased ketone body levels (2.0), decreased pH (6.2), increased incidences of granular and epithelial casts in urine sediment (0.8), increased urine volume (9.1 mL)
- Observed effects on ketone body levels and pH in male animals was regarded as treatment-related and adverse
- Observed effect on incidences of granular and epithelial casts in male animals was regarded as not human relevant as this is a finding typically observed in male rats of this age class in combination with an alpha-2u globulinuria which was confirmed histopathologically (Hard, G. C. Mechanisms of Rodent Renal Carcinogenesis Revisited. Toxicologic Pathology, 46 (8), 956-969 (2018))
- Observed effects on urine volume and urine specific gravity in male animals were assessed as adaptive mechanism of the kidneys to a changed fluid income

Females
- Test group 01 (40 mg/kg bw): no effects
- Test group 02 (150 mg/kg bw): no effects
- Test group 03 (600 mg/kg bw): decreased pH (5.3), increased urine specific gravity (1,054 g/L)
- Observed effects on urine specific gravity as well as change in urine pH were assessed as adaptive mechanism of the kidneys to a changed fluid income and acid-base homeostasis in blood, respectively 

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Male
Bile:
- Test group 03 showed an increased incidence and/or severity of diffuse bile duct hyperplasia => regarded as treatment-related
Liver:
- Single male animals of test groups 01 to 03 showed a minimal, diffuse hepatocellular hypertrophy with a minimally increased incidence in test groups 02 and 03. Although, a clear dose response relationship was not recognized. => regarded as treatment-related (in combination with increased liver weights)
Thyroid glands:
- test groups 02 and 03 showed a minimal to moderate diffuse follicular hypertrophy and hyperplasia => regarded as treatment-related (in combination with increased relative thyroid gland weights)
- test group 02 and 03 showed an increased amount of altered colloid in thyroid glands => regarded as treatment-related (in combination with increased relative thyroid gland weights)
Kidneys:
- test group 03 showed an increased incidence and severity of eosinophilic, intracytoplasmic droplets in proximal convoluted tubules, that was associated to granular casts in tubules at the junction of the outer and inner stripes of the outer medulla and an increased incidence and severity of basophilic tubules; in individual animals, eosinophilic droplets were exemplarily stained with an antibody against alpha 2u-Globulin and the globules were positive for alpha 2u-Globulin in all respective animals - therefore, the presence of alpha 2u-Globulin nephropathy is likely => regarded as treatment-related but not relevant for humans
- test group 02 showed a minimally increased severity of basophilic tubules and minimal granular casts in 3/20 animals => regarded as treatment-related (in combination with increased kidney weights)
Spleen:
- test group 02 and 03 showed an increased incidence and/or severity of extramedullary hematopoiesis => regarded as treatment-related (in combination with increased numbers of erythropoietic precursor cells)

Female
Bile:
- Test group 03 showed an increased incidence and/or severity of diffuse bile duct hyperplasia => regarded as treatment-related
Liver:
- Test groups 02 and 03 presented a minimal up to moderate centrilobular hepatocellular hypertrophy => regarded as treatment-related (in combination with increased absolute and relative liver weights)
Thyroid glands:
- test groups 02 and 03 showed a minimal to moderate diffuse follicular hypertrophy and hyperplasia => regarded as treatment-related (in combination with increased relative thyroid gland weights)
- test group 02 and 03 showed an increased amount of altered colloid in thyroid glands => regarded as treatment-related (in combination with increased absolute and relative thyroid gland weights)

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.

Histopathological findings: neoplastic:

no effects observed

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Hormones:
Males
- Test group 01 (40 mg/kg bw): decreased T4 value (60.44 nmol/L), increased TSH value (not significant)
- Test group 02 (150 mg/kg bw): decreased T4 value (60.62 nmol/L)
- Test group 03 (600 mg/kg bw): decreased T4 value (48.34 nmol/L), increased TSH value (not significant)
- Observed effects on TSH and T4 in male animals were assessed as treatment-related and adverse in the test group 03 due to histopathological findings in the thyroids and increased relative thyroid weights
- Observed effect on T4 in male animals of test group 01 and 02 was regarded as maybe treatment related but not-adverse

Females
- Test group 01 (40 mg/kg bw): decreased TSH value (4.43 µg/L)
- Test group 02 (150 mg/kg bw): no effects
- Test group 03 (600 mg/kg bw): increased TSH value (7.82 µg/L)
- Observed effect on TSH in female animals of test group 01 were within historical control range and regarded as incidental and not treatment related
- Observed effect on TSH in female animals of test group 03 were assessed as treatment-related and adverse due to histopathological findings in the thyroids and increased absolute and relative thyroid weights

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

Females (last 3 weeks prior to mating)
- Test group 01 (40 mg/kg bw): no effects; mean 4.0
- Test group 02 (150 mg/kg bw): no effects; mean 4.1
- Test group 03 (600 mg/kg bw): no effects; mean 4.0

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

Males
- Test group 01 (40 mg/kg bw): no effects
- Test group 02 (150 mg/kg bw): no effects
- Test group 03 (600 mg/kg bw): no effects

Reproductive performance:

no effects observed

Description (incidence and severity):

Male
Mating Index:
- Test group 01 (40 mg/kg bw): 96%
- Test group 02 (150 mg/kg bw): 100%
- Test group 03 (600 mg/kg bw): 100%
Fertility index:
- Test group 01 (40 mg/kg bw): 92%
- Test group 02 (150 mg/kg bw): 100%
- Test group 03 (600 mg/kg bw): 100%
These values reflect the normal range of biological variation inherent in the strain of rats used for this study.
Histopathological findings:
- Test group 01 (40 mg/kg bw): one animal showed a reduced size of both testes and epididymides, that correlated to a diffuse massive degeneration/atrophy and to a massive oligospermia in testes and epididymides, respectively
- Test group 02 (150 mg/kg bw): no
- Test group 03 (600 mg/kg bw): no

Females
Mating index:
- Test group 01 (40 mg/kg bw): 96%
- Test group 02 (150 mg/kg bw): 100%
- Test group 03 (600 mg/kg bw): 96%
Mean duration until sperm was detected:
- Test group 01 (40 mg/kg bw): 2.3 days
- Test group 02 (150 mg/kg bw): 2.2 days
- Test group 03 (600 mg/kg bw): 2.2 days
All femals rats delivered pups or had implants in utero with the following exception:
- Test group 01 (40 mg/kg bw): 2 animals did not become pregnant.
- Test group 03 (600 mg/kg bw): 1 animal did not become pregnant.
Fertility index:
- Test group 01 (40 mg/kg bw): 96%
- Test group 02 (150 mg/kg bw): 100%
- Test group 03 (600 mg/kg bw): 100%
These values reflect the normal range of biological variation inherent in the strain of rats used for this study.
Histopathological findings:
- Test group 01 (40 mg/kg bw): no
- Test group 02 (150 mg/kg bw): no
- Test group 03 (600 mg/kg bw): no
Duration of gestation:
- Test group 01 (40 mg/kg bw): 22.2 days
- Test group 02 (150 mg/kg bw): 22.2 days
- Test group 03 (600 mg/kg bw): 22.2 days
Gestation index:
- Test group 01 (40 mg/kg bw): 100%
- Test group 02 (150 mg/kg bw): 96%
- Test group 03 (600 mg/kg bw): 100%
Implantation:
- Test group 01 (40 mg/kg bw): 12.8 implants/dam
- Test group 02 (150 mg/kg bw): 12.5 implants/dam
- Test group 03 (600 mg/kg bw): 12.0 implants/dam
Postimplantation loss:
- Test group 01 (40 mg/kg bw): 8.3 mean %
- Test group 02 (150 mg/kg bw): 10.2 mean %
- Test group 03 (600 mg/kg bw): 6.6 mean %
Mean number of pups:
- Test group 01 (40 mg/kg bw): 11.8 pups/dam
- Test group 02 (150 mg/kg bw): 11.4 pups/dam
- Test group 03 (600 mg/kg bw): 11.3 pups/dam
Rate of liveborn pups
- Test group 01 (40 mg/kg bw): 99%
- Test group 02 (150 mg/kg bw): 99%
- Test group 03 (600 mg/kg bw): 99%

Dose descriptor:

NOAEL

Remarks:

general systemic toxicity F0

Effect level:

40 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: clinical pathology and histopathological findings observed at 150 mg/kg bw/d

Dose descriptor:

NOAEL

Remarks:

general systemic toxicity, F1

Effect level:

150 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: clinical pathology and histopathological findings observed at 600 mg/kg bw/d

Dose descriptor:

NOAEL

Remarks:

fertility and reproductive performance

Effect level:

600 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no effects observed

Critical effects observed:

not specified

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Cohort F1A
Male (day 0-65)
Test group 11 (40 mg/kg bw): no effects
Test group 12 (150 mg/kg bw): salivation (16/20), plough nose-first into bedding (1/20)
Test group 13 (600 mg/kg bw): salivation (20/20), plough nose-first into bedding (19/20)

Female (day 0-65)
Test group 11 (40 mg/kg bw): salivation (2/20)
Test group 12 (150 mg/kg bw): salivation (18/20), plough nose-first into bedding (1/20), general poor condition + labored respiration + piloerection (1/20)
Test group 13 (600 mg/kg bw): salivation (20/20), plough nose-first into bedding (20/20)

- observed effect of salivation in male and female animals was reversible within in a maximum of two hours after administration and, thus, was regarded as treatment related but not adverse



Cohort F1B
Male (day 0-94)
Test group 11 (40 mg/kg bw): head injury
Test group 12 (150 mg/kg bw): salivation (23/25), plough nose-first into bedding (1/25),
Test group 13 (600 mg/kg bw): salivation (22/25); plough nose-first into bedding (20/25); piloerection, labored respiration, poor general conditions (1/25)

Female (day 0-75)
Test group 11 (40 mg/kg bw): semiclosed eyelid, closed eyelid, pale skin, poor general condition, hard abdomen, abdominal position, palpable in abdomen (1/25 – was sacrificed moribund)
Test group 12 (150 mg/kg bw): salivation (21/25), plough nose-first into bedding (3/25)
Test group 13 (600 mg/kg bw): salivation (25/25), plough nose-first into bedding (23/25)
Female (mating)
Test group 11 (40 mg/kg bw): no effects
Test group 12 (150 mg/kg bw): salivation (3/14)
Test group 13 (600 mg/kg bw): salivation (9/14)
Female (gestation)
Test group 11 (40 mg/kg bw): salivation (1/24)
Test group 12 (150 mg/kg bw): salivation (23/25)
Test group 13 (600 mg/kg bw): salivation (23/25), plough nose-first into bedding (21/25)
Female (lactation)
Test group 11 (40 mg/kg bw): salivation (6/22)
Test group 12 (150 mg/kg bw): salivation (22/25), supernumerary nipple (1/23)
Test group 13 (600 mg/kg bw): salivation (24/24), plough nose-first into bedding (23/24)

- salivation and accompanied plough nose-first into bedding temporary and were regarded test-item related but not adverse
- all other observed events were regarded to be not related to test item administration

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

Cohort F1A
Male
Test group 11 (40 mg/kg bw): no deaths
Test group 12 (150 mg/kg bw): no deaths
Test group 13 (600 mg/kg bw): no deaths

Female
Test group 11 (40 mg/kg bw): 1 accidental death
Test group 12 (150 mg/kg bw): 1 death after poor general condition
Test group 13 (600 mg/kg bw): no deaths
- observed mortalities in female animals was regarded as incidental and not test item related



Cohort F1B
Male
Test group 11 (40 mg/kg bw): no deaths
Test group 12 (150 mg/kg bw): no deaths
Test group 13 (600 mg/kg bw): no deaths

Female
Test group 11 (40 mg/kg bw): 1 animal showed a palpable mass in the abdomen, poor general state, paleness, semiclosed/closed eyelids, hard abdomen and abdominal position from inlife day 53 onwards and was sacrificed moribund on day 57. Histopathology indicated an agonal heart failure.
Test group 12 (150 mg/kg bw): no deaths
Test group 13 (600 mg/kg bw): no deaths
- observed event in single female of test group 11 was regarded as incidental

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Cohort F1A
Male (day 0-56)
Test group 11 (40 mg/kg bw): no effects
Test group 12 (150 mg/kg bw): no effects
Test group 13 (600 mg/kg bw): significantly reduced throughout total study duration with a maximum of -15.2% at day 63
- observed effect in male animals was regarded as treatment-related

Female (day 0-56)
Test group 11 (40 mg/kg bw): body weight change increased at day 56-63 (11.7+/-6.4)
Test group 12 (150 mg/kg bw): -7.2% at day 0
Test group 13 (600 mg/kg bw): -13.5% at day 0, -7.9% at day 7
- observed effect in female animals of test group 11 was regarded as incidental
- observed effect in female animals of test group 12 and 13 was regarded as test item-related



Cohort F1B
Male
Test group 11 (40 mg/kg bw): no effects
Test group 12 (150 mg/kg bw): no effects
Test group 13 (600 mg/kg bw): significantly reduced throughout d0-7 and d35-91 (maximum decrease of -10.5% on d77); body weight change of high-dose males was statistically reduced from inlife days 35-63, 70-77 and at the total interval days 0-91
- observed effect in male animals was regarded as treatment-related

Female
Test group 11 (40 mg/kg bw): decreased body weight change during d35-42
Test group 12 (150 mg/kg bw): increased body weight change during d21-28
Test group 13 (600 mg/kg bw): -8.8% at day 0, -5.8% at day 7
- observed effect in female animals of test group 11 and 12 were regarded as incidental
- observed effect in female animals of test group 13 was regarded as test item-related

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

Cohort F1A
Male (day 0-56)
Test group 11 (40 mg/kg bw): no effects
Test group 12 (150 mg/kg bw): no effects
Test group 13 (600 mg/kg bw): no effects

Female (day 0-56)
Test group 11 (40 mg/kg bw): +8.9% at day 42-49
Test group 12 (150 mg/kg bw): no effects
Test group 13 (600 mg/kg bw): no effects
- observed effect in female animals was regarded as incidental



Cohort F1AB
Male
Test group 11 (40 mg/kg bw): no effects
Test group 12 (150 mg/kg bw): no effects
Test group 13 (600 mg/kg bw): no effects

Female
Test group 11 (40 mg/kg bw): -9.0% at day 21-28
Test group 12 (150 mg/kg bw): no effects
Test group 13 (600 mg/kg bw): no effects
- observed effect in female animals was regarded as incidental

Food efficiency:

not examined

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

effects observed, treatment-related

Description (incidence and severity):

Cohort F1A
Male (day 0-56)
Test group 11 (40 mg/kg bw): no effects
Test group 12 (150 mg/kg bw): no effects
Test group 13 (600 mg/kg bw): +10.9% at day 10-14 and +12.6% at day 52-56
- observed effect in male animals was regarded as treatment-related

Female (day 0-56)
Test group 11 (40 mg/kg bw): +15.5% at day 52-56
Test group 12 (150 mg/kg bw): no effects
Test group 13 (600 mg/kg bw): +12.8% at day 10-14; +11.2% at day 17-21
- observed effect in female animals was regarded as unrelated to test item administration as no dose-dependency could be established



Cohort F1B
Male
Test group 11 (40 mg/kg bw): no effects
Test group 12 (150 mg/kg bw): no effects
Test group 13 (600 mg/kg bw): no effects

Female (pre-mating)
Test group 11 (40 mg/kg bw): no effects
Test group 12 (150 mg/kg bw): no effects
Test group 13 (600 mg/kg bw): no effects
Female (gestation)
Test group 11 (40 mg/kg bw): no effects
Test group 12 (150 mg/kg bw): increased on GD 10-11 (+18%) and 17-18 (+19%)
Test group 13 (600 mg/kg bw): increased during GD 7 to 21 (up to 44% on GD 21)
Female (lactation)
Test group 11 (40 mg/kg bw): no effects
Test group 12 (150 mg/kg bw): increased on PND 1-2 (+16%)
Test group 13 (600 mg/kg bw): increased on PND 1-2 (+24%) and 7-8 (+15%)
- observed effect in female animals was regarded as related to the test item

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Cohort F1A
No treatment-related, adverse changes among hematological parameters were observed
Male
Test group 11 (40 mg/kg bw): +20.12% white blood cell count
Test group 12 (150 mg/kg bw): no effects
Test group 13 (600 mg/kg bw): +23.4% reticulocytes
- observed effect on reticulocytes was regarded as adaptive/compensatory due to absence of any signs of anemia and histopathological findings (extramedullary hematopoiesis in the spleen)

Female
Test group 11 (40 mg/kg bw): +25.18% absolute lymphocyte count, +41.54% absolute monocyte count, +100% absolute basophile count, +60.0% basophile count
Test group 12 (150 mg/kg bw): no effects
Test group 13 (600 mg/kg bw): +33.32% absolute lymphocyte count, +44.64% absolute monocyte count
- observed effect on white blood cell count was regarded as maybe test item-related but non-adverse since this is the only relevantly changed differential blood cell fraction (ECETOC Technical Report No. 85, 2002)

- all other effects are regarded as incidental and not treatment-related as no dose-dependency could be established



Not performed in Cohort F1B

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Cohort F1A
Male
Test group 11 (40 mg/kg bw): decreased alanine aminotransferase (ALT)
Test group 12 (150 mg/kg bw): decreased bilirubin; increased total protein and albumin; increased calcium
Test group 13 (600 mg/kg bw): increased γ-glutamyl transferase (GGT); increased total protein, albumin and globulin; decreased glucose; decrease in alanine aminotransferase (ALT); decreased bilirubin; decreased chloride; increased calcium
- observed effect in test group 13 on GGT, total protein, albumin, globulin and glucose were regarded as treatment-related and adverse
- observed effect in test group 13 on ALT and in test group 13 and 13 on total bilirubin were regarded as treatment related but adaptive rather than adverse since it most probably occurred due to liver enzyme induction (PSD guidance, 2007) and an increased bilirubin conjugation followed by an accelerated release via the bile
- observed effects in total protein and albumin in test group 12 were regarded as non-adverse if at all treatment related (ECETOC Technical Report No. 85, 2002)
- all other findings were regarded as incidental since no dose-dependency could be established or values were within the historical control range

Female
Test group 11 (40 mg/kg bw): increased cholesterol and calcium levels
Test group 12 (150 mg/kg bw): increased urea; increased triglyceride
Test group 13 (600 mg/kg bw): no effects increased γ-glutamyl transferase (GGT); increased total protein, albumin and globulin; increased urea; decreased glucose values and increased globulin; decreased chloride
- observed effect in test group 13 on GGT, total protein, albumin and globulin were regarded as treatment-related and adverse
- all other findings were regarded as incidental since no dose-dependency could be established or values were within the historical control range



Not performed in Cohort F1B

Endocrine findings:

not specified

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

Cohort F1A
Male
Test group 11 (40 mg/kg bw): incidence of casts (0.6)
Test group 12 (150 mg/kg bw): incidence of transitional epithelial cells and granular and epithelial casts in urine sediment (1.7 and 1.9), increased pH (6.4)
Test group 13 (600 mg/kg bw): increased ketone body level (1.5), decreased pH (5.8), incidence of transitional epithelial cells and granular and epithelial casts in urine sediment (1.6 and 1.9), increased specific gravity (1.046 g/L)
- observed effect on ketone body level and decreased pH in test group 13 were regarded as treatment-related and adverse
- observed effect on casts was related to alpha-2u globulinuria which was confirmed histopathologically and is thus not regarded as relevant for humans (Hard et al., 2018)
- all other effects were regarded as isolated findings and as adaptation of the fluid income in the kidneys and an adaptive regulation of the acid-base homeostasis in blood

Female
Test group 11 (40 mg/kg bw): no effects
Test group 12 (150 mg/kg bw): no effects
Test group 13 (600 mg/kg bw): increased urine volume
- observed effect on increased urine volume was regarded as isolated findings and as adaptation of the fluid income in the kidneys



Not performed in Cohort F1B

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Cohort F1A
Terminal body weight
Male: -2.4% / -1.7% / -13.6%** versus control in test group 11 / 12 / 13, respectively => regarded as treatment related

Absolute organ weight
Male:
- Adrenal gland: -0.4% / -1.0% / -14.48%** versus control in test group 11 / 12 / 13, respectively => secondary to the decreased terminal body weight
- Heart: -4.4% / -5.0% / -10.7%** versus control in test group 11 / 12 / 13, respectively =>
- Kidneys: +1.9% / +7.6%* / +12.1%** versus control in test group 11 / 12 / 13, respectively => treatment related
- Liver: -0.2% / +13.8%** / +25.4%** versus control in test group 11 / 12 / 13, respectively => treatment related
- Thymus: -12.3% / -8.4% / -28.1%** versus control in test group 11 / 12 / 13, respectively => secondary to the decreased terminal body weight
Female
- Liver: +4.3% / +12.9%** / +49.7%** versus control in test group 11 / 12 / 13, respectively => treatment related
- Pituitary gland: +20.5%* / +15.%* / +8.9% versus control in test group 11 / 12 / 13, respectively => not treatment related, as there was no dose-response relationship or a correlating histopathological finding
- Thyroid glands: +4.4% / +7.0% / +26.6%** versus control in test group 11 / 12 / 13, respectively => treatment related

Relative organ weight
Male:
- Brain: +2.1% / +1.6% / +13.0%** versus control in test group 11 / 12 / 13, respectively => secondary to the decreased terminal body weight
- Cauda epididymides: -1.5% / -7.3%* / +13.3%* versus control in test group 11 / 12 / 13, respectively => secondary to the decreased terminal body weight
- Epididymides: -1.3% / -2.9% / +10.2%* versus control in test group 11 / 12 / 13, respectively => secondary to the decreased terminal body weight
- Heart: -2.2% / -3.6% / +3.0%* versus control in test group 11 / 12 / 13, respectively => secondary to the decreased terminal body weight
- Kidneys: +4.4% / +9.4%** / +29.8%** versus control in test group 11 / 12 / 13, respectively => treatment related
- Liver: +2.2% / +15.6%** / +45.1%** versus control in test group 11 / 12 / 13, respectively => treatment related
- Spleen: +4.8% / +5.5% / +21.0%** versus control in test group 11 / 12 / 13, respectively => secondary to the decreased terminal body weight
- Testes: 0.0% / -1.4% / +13.7%** versus control in test group 11 / 12 / 13, respectively => secondary to the decreased terminal body weight
- Thyroid glands: +.17% / +11.5%* / +22.2%** versus control in test group 11 / 12 / 13, respectively => treatment related
Female:
- Heart: -0.7% / +1.1% / +6.6%* versus control in test group 11 / 12 / 13, respectively => not treatment related (absolute weights not increased, no histopathological finding)
- Kidneys: +0.8% / +3.3%* / +10.3%** versus control in test group 11 / 12 / 13, respectively => treatment related for test group 13 (above historical control range)
- Liver: +3.2% / +13.1%** / +53.7%** versus control in test group 11 / 12 / 13, respectively => treatment related
- Pituitary glands: +18.4%* / +14.5%* / +10.8% versus control in test group 11 / 12 / 13, respectively => not treatment related, as there was no dose-response relationship or a correlating histopathological finding
- Spleen: +2.5% / -0.8% / +13.9%** versus control in test group 11 / 12 / 13, respectively => not treatment related (absolute weights not increased, no histopathological finding)
- Thyroid glands: +4.0% / +7.1%* / +29.3%** versus control in test group 11 / 12 / 13, respectively => treatment related for test group 13 (above historical control range)

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

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



Cohort F1B
Terminal body weight
Male: -3.0% / -1.9% / -12.0%** versus control in test group 11 / 12 / 13, respectively => regarded as treatment related

Absolute organ weight
Male:
- Liver: -2.6% / +14.8%** / +27.4%** versus control in test group 11 / 12 / 13, respectively => regarded as treatment related
Female
- Liver: +3.7% / +13.4%** / +49.4%** versus control in test group 11 / 12 / 13, respectively => regarded as treatment related
- Ovaries: +1.8% / +6.1% / +13.5%** versus control in test group 11 / 12 / 13, respectively => very unlikely to be test item related (ovaries of cohort F1A animals did not have a dose related weight increase or any histopathological change, or significant results of the DOFC)

Relative organ weight
Male:
- Cauda epididymides: +8.4%* / +4.3% / +13.8%** versus control in test group 11 / 12 / 13, respectively => secondary to the decreased terminal body weight for test group 13; incidental for test group 11 since no dose-response relationship
- Epididymides: +3.2% / 0.0% / +10.2%** versus control in test group 11 / 12 / 13, respectively => secondary to the decreased terminal body weight
- Liver: +0.6% / +17.1%** / +44.7%** versus control in test group 11 / 12 / 13, respectively => regarded as treatment related
- Seminal Vesicles: +1.6% / +4.0% / +16.4%** versus control in test group 11 / 12 / 13, respectively => secondary to the decreased terminal body weight
- Testes: +1.9% / -1.3% / +11.5%** versus control in test group 11 / 12 / 13, respectively => secondary to the decreased terminal body weight
Female:
- Adrenal glands: +1.6% / +3.6% / +15.2%** versus control in test group 11 / 12 / 13, respectively => not treatment related (absolute weights not increased, no histopathological finding, no similar effect in cohort F1A)
- Liver: +3.1%** / +14.0%** / +55.0%** versus control in test group 11 / 12 / 13, respectively => treatment related for test group 12 and 13; regarded as not treatment related for test group 11 (only relative weight parameters were significantly changed; weight increase was only minimal; absolute and relative liver weights lay below the range of historical controls)
- Ovaries: +1.3% / +6.8% / +17.2%** versus control in test group 11 / 12 / 13, respectively => very unlikely to be test item related (ovaries of cohort F1A animals did not have a dose related weight increase or any histopathological change, or significant results of the DOFC)

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

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

Surplus F1 generation pups on PND 22
Terminal body weight
Female: -1.1% / -2.2% / -9.0%* versus control in test group 11 / 12 / 13, respectively => regarded as treatment related

Absolute organ weights
Male:
No effects
Female:
- spleen: -3.5% / -10.8% / -15.1%* => related to the decreased terminal body weight

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

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

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Cohort F1A
Male
Test group 11 (40 mg/kg bw): no effects
Test group 12 (150 mg/kg bw): no effects
Test group 13 (600 mg/kg bw): enlarged kidney (2/20), enlarged spleen (1/20)
Female
Test group 11 (40 mg/kg bw): no effects
Test group 12 (150 mg/kg bw): no effects
Test group 13 (600 mg/kg bw): no effects
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.



Cohort F1B
Male
Test group 11 (40 mg/kg bw): no effects
Test group 12 (150 mg/kg bw): no effects
Test group 13 (600 mg/kg bw): macroscopically visible dark brown discoloration of the liver (4/25)
Female
Test group 11 (40 mg/kg bw): no effects
Test group 12 (150 mg/kg bw): no effects
Test group 13 (600 mg/kg bw): macroscopically visible dark brown discoloration of the liver (7/25)
- observed effect of discoloration in the liver was regarded as treatment related
- 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.

Surplus F1 generation pups on PND 22
Macroscopic findings were not observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Cohort F1A
Male
Bile:
- Test group 13 showed an increased incidence and/or severity of diffuse bile duct hyperplasia => regarded as treatment-related
Liver:
- Test groups 02 to 03 showed a minimal up to moderate centrilubolar hepatocellular hypertrophy => regarded as treatment-related (in combination with increased liver weights)
Thyroid glands:
- few animals of test group 13 showed a minimal to slight diffuse follicular hypertrophy and hyperplasia; animals of test group 12 and 13 presented additionally an increased amount of altered colloid => regarded as treatment-related (in combination with increased relative thyroid gland weights)
Kidneys:
- test groups 12 and 13 showed an increased incidence and severity of eosinophilic, intracytoplasmic droplets in proximal convoluted tubules, that was associated to increased incidence and severity of basophilic tubules; test group 13 showed additionally slight to moderate granular casts in tubules at the junction of the outer and inner stripes of the outer medulla which were composed of proteinaceous material admixed with cellular debris; in individual animals, eosinophilic droplets were exemplarily stained with an antibody against alpha 2u-Globulin and the globules were positive for alpha 2u-Globulin in all respective animals - therefore, the presence of alpha 2u-Globulin nephropathy is likely => regarded as treatment-related but not relevant for humans
- test group 11 showed likewise an increased incidence of eosinophilic droplets in proximal convoluted tubules, and of basophilic tubules=> regarded as treatment-related (within historical control range but increased incidence compared to the control group)
Spleen:
- test group 13 showed an increased incidence and severity of extramedullary hematopoiesis => regarded as not treatment-related (no clear dose response relationship)
Thymus:
- test group 13 males showed an increased incidence of increased cellularity of epithelial cells => incidental (no effect in females, no effects in P0 animals, level below female F0 control)

Female
Bile:
- Test group 13 showed an increased incidence and/or severity of diffuse bile duct hyperplasia => regarded as treatment-related
Liver:
- Test groups 02 to 03 showed a minimal up to moderate centrilubolar hepatocellular hypertrophy => regarded as treatment-related (in combination with increased liver weights)
- Test group 13 showed an increased incidence and severity of periportal vacuolation which upon ORO-stain turned out to be lipid droplets => treatment related
Thyroid glands:
- few animals of test group 13 showed a minimal to slight diffuse follicular hypertrophy and hyperplasia => regarded as treatment-related (in combination with increased relative thyroid gland weights)
- test group 11 and 13 presented additionally an increased amount of altered colloid => not treatment related (since no dose response relationship)

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


Differential ovarian follicle count in Cohort F1A
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 10 and animals of test group 13.



Not performed on Cohort F1B

Histopathological findings: neoplastic:

no effects observed

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Hormones
Surplus pups PND4 + PND22
Male
Test group 11 (40 mg/kg bw): no effects
Test group 12 (150 mg/kg bw): no effects
Test group 13 (600 mg/kg bw): no effects
Female
Test group 11 (40 mg/kg bw): no effects
Test group 12 (150 mg/kg bw): no effects
Test group 13 (600 mg/kg bw): no effects

Cohort F1A
Male
Test group 11 (40 mg/kg bw): decreased T4 (within historical control range)
Test group 12 (150 mg/kg bw): no effects
Test group 13 (600 mg/kg bw: decreased T4 (-34.0%; below historical control range)
- observed T4 decrease in males of test group 13 in combination with histopathologic findings in the thyroid and increased relative thyroid weights was regarded as treatment related and adverse whereas T4 decrease in males of test group 11 was regarded as incidental and not treatment related
Female
Test group 11 (40 mg/kg bw): no effects
Test group 12 (150 mg/kg bw): no effects
Test group 13 (600 mg/kg bw): increased TSH (marginally above historical control range)
- in combination with histopathologic findings in the thyroids and increased absolute and relative thyroid weights, higher TSH values in females of test group 13 were regarded as treatment related and adverse

Lymphocyte subpopulation in spleen
Cohort F1A
Male
Test group 11 (40 mg/kg bw): no effects
Test group 12 (150 mg/kg bw): no effects
Test group 13 (600 mg/kg bw): no effects
Female
Test group 11 (40 mg/kg bw): no effects
Test group 12 (150 mg/kg bw): no effects
Test group 13 (600 mg/kg bw): no effects



Hormones
Not performed in Cohort F1B

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

Cohort F1A
Female (generated for 2 weeks around PND75)
Test group 11 (40 mg/kg bw): no effects / 3.9 days
Test group 12 (150 mg/kg bw): no effects / 3.9 days
Test group 13 (600 mg/kg bw): no effects / 3.9 days



Cohort F1B
Female (during the last 3 weeks prior to mating)
Test group 11 (40 mg/kg bww): 4.0 days duration / 4.4 cycles
Test group 12 (150 mg/kg bw): 4.0 days duration / 4.6 cycles
Test group 13 (600 mg/kg bw): 4.0 days duration / 4.4 cycles
- observed reduced cycle number in test group 11 and 13 were regarded as incidental (no dose-dependency, absence of corroborating effect in cohort F1A)

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

Cohort F1A
Male
Test group 11 (40 mg/kg bw): no effects
Test group 12 (150 mg/kg bw): no effects
Test group 13 (600 mg/kg bw): no effects



Not examined for Cohort F1B

Reproductive performance:

no effects observed

Description (incidence and severity):

Not performed on Cohort F1A



Cohort F1B
Male
Mating Index:
- Test group 11 (40 mg/kg bw): 100%
- Test group 12 (150 mg/kg bw): 100%
- Test group 13 (600 mg/kg bw): 100%
Fertility index:
- Test group 11 (40 mg/kg bw): 96%
- Test group 12 (150 mg/kg bw): 92%
- Test group 13 (600 mg/kg bw): 92%
These values reflect the normal range of biological variation inherent in the strain of rats used for this study. All respective values are within the range of the historical control data of the test facility.
Histopathological findings:
- Test group 11 (40 mg/kg bw): no
- Test group 12 (150 mg/kg bw): no
- Test group 13 (600 mg/kg bw): no

Females
Mating index:
- Test group 11 (40 mg/kg bw): 100%
- Test group 12 (150 mg/kg bw): 100%
- Test group 13 (600 mg/kg bw): 100%
Mean duration until sperm was detected:
- Test group 11 (40 mg/kg bw): 2.3 days
- Test group 12 (150 mg/kg bw): 2.3 days
- Test group 13 (600 mg/kg bw): 2.2 days
All female rats delivered pups or had implants in utero with the following exception:
- Test group 10: 1 animal did not become pregnant
- Test group 11: 2 animals did not become pregnant
- Test group 12: 2 animals did not become pregnant
- Test group 13: 1 animal did not become pregnant
Fertility index:
- Test group 11 (40 mg/kg bw): 91.7%
- Test group 12 (150 mg/kg bw): 92.0%
- Test group 13 (600 mg/kg bw): 96.0%
Histopathological findings:
- Test group 11 (40 mg/kg bw): no
- Test group 12 (150 mg/kg bw): no
- Test group 13 (600 mg/kg bw): no
Duration of gestation:
- Test group 11 (40 mg/kg bw): 22.2 days
- Test group 12 (150 mg/kg bw): 22.0 days
- Test group 13 (600 mg/kg bw): 22.2 days
Gestation index:
- Test group 11 (40 mg/kg bw): 100%
- Test group 12 (150 mg/kg bw): 100%
- Test group 13 (600 mg/kg bw): 100%
Implantation:
- Test group 11 (40 mg/kg bw): 11.5 implants/dam
- Test group 12 (150 mg/kg bw): 11.7 implants/dam
- Test group 13 (600 mg/kg bw): 12.6 implants/dam
Postimplantation loss:
- Test group 11 (40 mg/kg bw): 5.6 mean %
- Test group 12 (150 mg/kg bw): 6.1 mean %
- Test group 13 (600 mg/kg bw): 3.8 mean %
Mean number of pups:
- Test group 11 (40 mg/kg bw): 11.0 pups/dam
- Test group 12 (150 mg/kg bw): 11.0 pups/dam
- Test group 13 (600 mg/kg bw): 12.2 pups/dam
Rate of liveborn pups
- Test group 11 (40 mg/kg bw): 99.2%
- Test group 12 (150 mg/kg bw): 98.0%
- Test group 13 (600 mg/kg bw): 96.6%

Dose descriptor:

NOAEL

Remarks:

general systemic toxicity F1

Effect level:

150 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: reduced body weights in males and clinical pathology and histopathological findings observed at 600 mg/kg bw/d:

Dose descriptor:

NOAEL

Remarks:

fertility and reproductive performance F1

Effect level:

600 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no effects observed

Critical effects observed:

not specified

Clinical signs:

no effects observed

Description (incidence and severity):

Test group 01 (40 mg/kg bw): no effects
Test group 02 (150 mg/kg bw): no effects
Test group 03 (600 mg/kg bw): no effects

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

Number of liveborn pups / percentage of stillborn pups:
Test group 00 (00 mg/kg bw): 11.4 / 1.0%
Test group 01 (40 mg/kg bw): 11.7 / 1.1%
Test group 02 (150 mg/kg bw): 11.3 / 0.7%
Test group 03 (600 mg/kg bw): 11.2 / 0.7%
Viability index (PND 0-4):
Test group 00 (00 mg/kg bw): 97.1%
Test group 01 (40 mg/kg bw): 95.0%
Test group 02 (150 mg/kg bw): 99.7%
Test group 03 (600 mg/kg bw): 97.8%
Lactation index (PND 4-21):
Test group 00 (00 mg/kg bw): 99.3%
Test group 01 (40 mg/kg bw): 100.0%
Test group 02 (150 mg/kg bw): 100.0%
Test group 03 (600 mg/kg bw): 100.0%
- the test substance did not influence pre-weaning F1-pup survival in all test groups

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Male:
Test group 01 (40 mg/kg bw): no effects
Test group 02 (150 mg/kg bw): no effects
Test group 03 (600 mg/kg bw): reduced on PND 14 (-7.6%), body weight change between PND 7-14
Female:
Test group 01 (40 mg/kg bw): no effects
Test group 02 (150 mg/kg bw): no effects
Test group 03 (600 mg/kg bw): reduced on PND 14 (-7.9%), body weight change between PND 7-14, reduced in the total interval PND 1-21

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

effects observed, non-treatment-related

Description (incidence and severity):

Sex ratio:
% live male pups PND 0 vs. PND 21 / % live female pups PND 0 vs. PND 21:
Test group 00 (00 mg/kg bw): 52.5 vs. 52.8 / 47.5 vs. 47.2
Test group 01 (40 mg/kg bw): 53.9 vs. 51.0 / 46.1 vs. 49.0
Test group 02 (150 mg/kg bw): 46.6 vs. 46.4 / 53.4 vs. 53.6
Test group 03 (600 mg/kg bw): 52.0 vs. 51.6 / 48.0 vs. 48.4
- no substantial difference; slight differences were regarded to be spontaneous in nature

Vaginal opening in females:
Days to reach criterion / mean body weight on that day:
Test group 00 (0 mg/kg bw): 31.6 d / 98.0 g
Test group 01 (40 mg/kg bw): 30.6 d / 91.5 g*
Test group 02 (150 mg/kg bw): 30.9 d / 91.7 g*
Test group 03 (600 mg/kg bw): 30.9 d / 87.6 g**
* p<=0.05, ** p <=0.01
- observed effect on body weights were within historical range of the strain and is, thus, considered to be not related to the compound

Preputial separation in males:
Days to reach criterion / mean body weight on that day:
Test group 00 (0 mg/kg bw): 41.2 / 173.4 g
Test group 01 (40 mg/kg bw): 41.5 d / 171.6 g
Test group 02 (150 mg/kg bw): 41.4 d / 172.4 g
Test group 03 (600 mg/kg bw): 42.2 d** / 165.8 g
** p <=0.01
- Although the time of preputial separation was slightly later in the high dose group, the animals of this dose group reached sexual maturity even at a slightly lower body weight when compared to controls. Therefore, the difference in timing of preputial separation is considered not to be a delay in commencement of sexual maturity, but due to lower postnatal body weight development. Additionally, all values for days and weights are well within the historical control range.

Anogenital distance (AGD):

no effects observed

Description (incidence and severity):

Males:
Test group 00 (00 mg/kg bw): 3.08 mm / 1.62 (Index Cubic Root)
Test group 01 (40 mg/kg bw): 3.12 mm / 1.65 (Index Cubic Root)
Test group 02 (150 mg/kg bw): 3.08 mm / 1.63 (Index Cubic Root)
Test group 03 (600 mg/kg bw): 3.04 mm / 1.58 (Index Cubic Root)
Females:
Test group 00 (00 mg/kg bw): 1.38 mm / 0.74 (Index Cubic Root)
Test group 01 (40 mg/kg bw): 1.41 mm / 0.76 (Index Cubic Root)
Test group 02 (150 mg/kg bw): 1.37 mm / 0.73 (Index Cubic Root)
Test group 03 (600 mg/kg bw): 1.39 mm / 0.74 (Index Cubic Root)

Nipple retention in male pups:

effects observed, non-treatment-related

Description (incidence and severity):

Males:
Nipple development (% / number) on day 13
Test group 01 (40 mg/kg bw): 40.9% */ 1.0
Test group 02 (150 mg/kg bw): 64.1%** / 1.9*
Test group 03 (600 mg/kg bw): 59.6%** / 2.2**
* p<=0.05, ** p <=0.01
Nipple development (% / number) on day 20
Test group 01 (40 mg/kg bw): 0.0 / 0.0
Test group 02 (150 mg/kg bw): 0.0 / 0.0
Test group 03 (600 mg/kg bw): 0.0 / 0.0
- effects observed show no dose-dependency and values are within historical control range
Within this context it has to be mentioned that there was an obvious negative correlation between the litter weight on PND 14 and the number of observed nipples / areola anlagen, i.e. the litters which had the highest incidences had also quite low litter weight. Thus, the observed differences are considered rather to be a consequence of delayed body weight development than to represent an antiandrogenic action of the test compound. This is also strengthened by the absence of any nipple / areola anlagen on PND 20.

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Males:
Test group 01 (40 mg/kg bw): post-mortem autolysis (2/87), general finding - not assessed (2/87)
Test group 02 (150 mg/kg bw): post-mortem autolysis (1/81), empty stomach (1/81)
Test group 03 (600 mg/kg bw): empty stomach (2/87)
Females:
Test group 01 (40 mg/kg bw): general finding - not assessed (1/75)
Test group 02 (150 mg/kg bw): post-mortem autolysis (1/94), general finding - not assessed (1/94)
Test group 03 (600 mg/kg bw): general finding - not assessed (3/75)
- these findings occurred without any relation to dosing and/or can be found in the historical control data at comparable or even higher incidences; therefore, these findings were not considered to be associated to the test substance

Histopathological findings:

not examined

Other effects:

not examined

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Dose descriptor:

NOAEL

Remarks:

developmental toxicity F1

Generation:

F1

Effect level:

150 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: reduced body weights of the offspring during lactation

Critical effects observed:

not specified

Clinical signs:

no effects observed

Description (incidence and severity):

Test group 11 (40 mg/kg bw): no effects
Test group 12 (150 mg/kg bw): no effects
Test group 13 (600 mg/kg bw): no effects

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

Number of liveborn pups / number of stillborn pups:
Test group 10 (00 mg/kg bw): 10.3 / 3.7 (1 animal had all pups stillborn, the respective values reflect the normal range of biological variation inherent in the strain used)
Test group 11 (40 mg/kg bw): 10.9 / 0.8
Test group 12 (150 mg/kg bw): 10.7 / 2.0
Test group 13 (600 mg/kg bw): 11.8 / 3.4
Viability index (PND 0-4):
Test group 10 (00 mg/kg bw): 99.6%
Test group 11 (40 mg/kg bw): 100.0%
Test group 12 (150 mg/kg bw): 99.6%
Test group 13 (600 mg/kg bw): 99.3%
Lactation index (PND 4-21):
Test group 10 (00 mg/kg bw): 100.0%
Test group 11 (40 mg/kg bw): 100.0%
Test group 12 (150 mg/kg bw): 100.0%
Test group 13 (600 mg/kg bw): 100.0%

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Male:
Test group 11 (40 mg/kg bw): no effects
Test group 12 (150 mg/kg bw): no effects
Test group 13 (600 mg/kg bw): reduced on PND 14-21 (max. -9.8%), reduced in the total interval PND 1-21
Female:
Test group 11 (40 mg/kg bw): no effects
Test group 12 (150 mg/kg bw): no effects
Test group 13 (600 mg/kg bw): reduced on PND 14-21 (max -9.5%), reduced in the total interval PND 1-21

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

no effects observed

Description (incidence and severity):

Sex ratio
% live male pups PND 0 vs. PND 21 / % live female pups PND 0 vs. PND 21:
Test group 00 (00 mg/kg bw): 50.5 vs. 50.8 / 49.5 vs. 49.2
Test group 01 (40 mg/kg bw): 48.4 vs. 47.5 / 51.6 vs. 52.5
Test group 02 (150 mg/kg bw): 52.1 vs. 51.3 / 47.9 vs. 48.7
Test group 03 (600 mg/kg bw): 47.8 vs. 48.8 / 52.2 vs. 51.2
- no substantial difference; slight differences were regarded to be spontaneous in nature

Anogenital distance (AGD):

no effects observed

Description (incidence and severity):

Males:
Test group 10 (00 mg/kg bw): 3.13 mm / 1.63 (Index Cubic Root)
Test group 11 (40 mg/kg bw): 3.13 mm / 1.64 (Index Cubic Root)
Test group 12 (150 mg/kg bw): 3.08 mm / 1.61 (Index Cubic Root)
Test group 13 (600 mg/kg bw): 3.03 mm / 1.60 (Index Cubic Root)
Females:
Test group 10 (00 mg/kg bw): 1.36 mm / 0.72 (Index Cubic Root)
Test group 11 (40 mg/kg bw): 1.36 mm / 0.72 (Index Cubic Root)
Test group 12 (150 mg/kg bw): 1.37 mm / 0.73 (Index Cubic Root)
Test group 13 (600 mg/kg bw): 1.34 mm / 0.71 (Index Cubic Root)

Nipple retention in male pups:

no effects observed

Description (incidence and severity):

Males:
Nipple development (% / number) on day 13
Test group 11 (40 mg/kg bw): 42.88% / 1.15
Test group 12 (150 mg/kg bw): 50.66% / 1.42
Test group 13 (600 mg/kg bw): 64.38% / 2.22
Nipple development (% / number) on day 20
Test group 11 (40 mg/kg bw): 0.0 / 0.0
Test group 12 (150 mg/kg bw): 0.0 / 0.0
Test group 13 (600 mg/kg bw): 0.0 / 0.0

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

Males:
Spleen (absolute): 0.2457 / 0.2350 / 0.2470 / 0.2072* versus in test group 10, 11 / 12 / 13, respectively
Thymus (absolute): 0.2411 / 0.2274 / 0.2540 / 0.2195* versus in test group 10, 11 / 12 / 13, respectively
Brain (relative): 102% / 102% / 108%** versus control in test group 11 / 12 / 13, respectively
Females:
Spleen (absolute): 0.2451 / 0.2366 / 0.2380 / 0.2053** versus in test group 10, 11 / 12 / 13, respectively
Brain (relative): 100% / 100% / 107%* versus control in test group 11 / 12 / 13, respectively
Males and females combined:
Spleen (absolute): 0.2454 / 0.2368 / 0.2425 / 0.2062** versus in test group 10, 11 / 12 / 13, respectively
Spleen (relative): 100% / 100% / 92%* versus control in test group 11 / 12 / 13, respectively
Brain (relative): 101% / 101% / 107%** versus control in test group 11 / 12 / 13, respectively
* or ** = p-level of significance ≤ 0.05 or ≤ 0.01, respectively
All weight changes in the high-dose group are considered to be a consequence of the reduced body weight of the corresponding pups at weaning, and may reflect a slight delay in the development of the immune system. This is further strengthened by the fact, that there was no evidence for immunotoxicity based on the examination of lymphocyte subpopulations in the spleen and by histopathology of the spleen and thymus in cohort F0 and F1 A animals.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Males:
Test group 11 (40 mg/kg bw): post-mortem autolysis (1/121)
Test group 12 (150 mg/kg bw): post-mortem autolysis (2/131), dilated renal pelvis (1/131)
Test group 13 (600 mg/kg bw): post-mortem autolysis (7/141), small liver lobe (1/141)
Females:
Test group 11 (40 mg/kg bw): no effects
Test group 12 (150 mg/kg bw): post-mortem autolysis (2/121), general finding - not assessed (1/121), empty stomach (1/121)
Test group 13 (600 mg/kg bw): post-mortem autolysis (3/151), general finding - not assessed (2/151), dilated renal pelvis (1/151)
- these findings occurred without any relation to dosing and/or can be found in the historical control data at comparable or even higher incidences; therefore, these findings were not considered to be associated to the test substance

Histopathological findings:

not examined

Other effects:

not examined

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Dose descriptor:

NOAEL

Remarks:

developmental toxicity F2

Generation:

F2

Effect level:

150 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: reduced body weights of the offspring during lactation

Critical effects observed:

not specified

Reproductive effects observed:

no

Conclusions:

Under the conditions of the present study the NOAEL (no observed adverse effect level) for general and systemic toxicity is 40 mg/kg bw/d for the F0 and 150 mg/kg bw/d for the F1 parental rats, based on reduced body weights in males at 600 mg/kg bw/d as well as clinical pathology and histopathological findings observed at 150 mg/kg bw/d (F0) and 600 mg/kg bw/d (F1).
The NOAEL for fertility and reproductive performance for the F0 and F1 parental rats is 600 mg/kg bw/d, the highest dose tested.
The NOAEL for developmental toxicity in the F1 and F2 progeny is 150 mg/kg bw/d, based on reduced body weights of the offspring during lactation at 600 mg/kg bw/d.

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

According to the decision on a Compliance Check (CCH-D-2114359360-53-01/F) and based on Article 41 of Regulation (EC) No 1907/2006 (the ‘REACH Regulation’) the registrant is requested to submit information on a Extended one-generation reproductive toxicity study (Annex X, Section 8.7.3.; test method: EU B.56./OECD TG 443) in rats, oral route with the registered substance specified as follows:

- Ten weeks premating exposure duration for the parental (P0) generation;

- Dose level setting shall aim to induce some toxicity at the highest dose level;

- Cohort 1A (Reproductive toxicity);

- Cohort 1B (Reproductive toxicity) with extension to mate the Cohort 1B animals to produce the F2 generation.

 

A modified One-Generation Reproduction Toxicity Study in Wistar Rats was performed as a Range-Finding Study to ensure a sound dose selection for the main OECD 443 (BASF SE, 2021, 16R0397/09C164). The aim of this study was to obtain initial information on the possible effects of Methylionon 70 on F0 generation parental animals and F1 generation offsprings as well as on the integrity and performance of the male and female reproductive systems after oral administration by gavage. It also provides information about the effects on prenatal and postnatal developmental toxicity, up to approximately one week after weaning.

Methylionon 70 was given daily throughout the entire study to groups of 10 male and 10 female healthy young Wistar rats (F0 parental generation) by stomach tube at doses of 100, 300 and 1000 mg/kg body weight/day. Control animals were dosed daily with the vehicle only (0.5% Sodium carboxymethyl cellulose (CMC) suspension in deionized water (with 10 mg/ 100mL Cremophor)). After a 10 weeks premating period, F0 animals were mated to produce a litter (F1 rearing animals). Mating pairs were from the same dose group. The female F0 animals were allowed to deliver and rear their pups (F1 generation pups) until postnatal days (PND) 4 or 21, when the offspring was necropsied. The male F0 generation parental animals were sacrificed during rearing of the F1 generation pups. The female F0 generation parental animals were sacrificed after weaning of the F1 generation pups.

The parents' and the pups' state of health was checked each day, and parental animals were examined for their mating and reproductive performances. Food consumption of the F0 parental animals was determined regularly once weekly (over a period of 7 days) with the exceptions of male parental animals only during the premating phase, sperm-positive females on GD 0-7, 7-14 and 14-20 and females which gave birth to a litter on PND 1-4, 4-7, 7-10, 10-14, 14-18 and 18-21. Water consumption was determined once a week (over a period of 3 days) for the male and female parental animals, with the exceptions of male parental animals only during the premating phase, sperm-positive females on GD 0-1, 6-7, 13-14 and 19-20 and females which gave birth to a litter on PND 1-2, 3-4, 6-7 and 12-13 and 20-21. In general, body weights of F0 parental animals were determined once weekly. However, during gestation and lactation F0 females were weighed on gestation days (GD) 0, 7, 14 and 20 and on postnatal days (PND) 0, 1, 4, 7, 10, 14, 18 and 21. Estrous cycle data were evaluated for F0 generation females over a minimum of two-week period prior to mating until evidence of mating occurred. Moreover, the estrous stage of each female was determined on the day of scheduled sacrifice. The F1 pups were sexed on the day of birth (PND 0) and were weighed on the first day after birth (PND 1) as well as on PND 4, 7, 10, 14, 17 and 21. Their viability was recorded. The F1 rearing animals after weaning were weighed on following days after birth (PND) 0, 3 and 7. At necropsy, All F0 parental animals were assessed by gross pathology and subjected to a histopathological examination (including weight determinations of adrenal glands, kidneys, liver and spleen). Blood samples for hematology and clinical chemistry were withdrawn from fasted animals by puncturing the retrobulbar venous plexus following isoflurane anesthesia. After sacrifice, F1 rearing animals were examined externally, eviscerated and their organs were assessed macroscopically.

Maternal toxicity was manifested by clinical observations (semiclosed eyelids, piloerection, reduced attention) as well as decreases in body weights and body weight gain during/after premating in F0 males and females of dose group 03 (1000 mg/kg bw/d). On study day 91 body weight was 8.5% and body weight change was 12.6% below controls. At 300 mg/kg bw/d still significantly reduced terminal body weights were noted (6% below control). Furthermore, water consumption of males and females was increased up to 25.0% (males) and 24.3% (females). Changes in clinical chemistry were seen in both sexes in mid and high dose groups, i.e. an increase in gamma glutamyltransferase, total protein, albumin, inorganic phosphate and calcium as well as a decrease in glucose and triglycerides compared to controls. Absolute and relative organ weights of kidneys and liver were significantly increased in males and females of test group 03. A relative increase in liver weight was observed in test group 02 for males and females. No effects on fertility, gestation, parturition as well as mean number of delivered pups per dam (total and liveborn) became evident. Pup survival was not influenced. For the high dose group (1000 mg/kg bw/d), a significant reduction in pup weight (17.6% and 15.7% below controls, respectively) has been recorded for male and female pups as lactation progressed, while birth weights were unaffected. Accordingly, the pup body weight gain was decreased in the high dose groups (23.1% and 19.1% below controls, respectively). Under the conditions of the present modified one-generation reproduction toxicity study in Wistar Rats, range-finding study, the NOAEL for general, systemic toxicity is 100 mg/kg bw/d for males and females, based on adverse clinical signs, and adverse clinical pathology and pathology findings at 300 and 1000 mg/kg bw/d. In test group 1 (100 mg/kg bw/d) an increased liver weight was not accompanied with altered liver toxicity parameters in clinical pathology. Since not histopathology was performed in this study, and on the basis of the determined parameters, the weight change of the liver was assessed as treatment-related but not as adverse. The NOAEL for fertility and reproductive performance for the parental rats is 1000 mg/kg bw/d), the highest tested dose. The NOAEL for developmental toxicity in the F1 progeny is 100 mg/kg bw/d based on adverse clinical signs (poor general condition, semiclosed eyelids, piloerection, unsteady gait) and pup body weight effects. However, in test group 2 (300 mg/kg bw/d) clinical signs were only observed within the first 2 days after weaning, the observed lower body weight gain decreased over time and no significant effect on the body weight itself was observed. Therefore, these findings were assessed as treatment-related borderline toxicity.

 

 

In an EOGRTS according to OECD test guideline No. 443 and OECD Principles of Good Laboratory Practice (BASF SE, 2022, 90R0397/09C165) Methylionon 70 was administered to groups of 25 male and 25 female healthy young Wistar rats (F0 parental generation) as an aqueous preparation by stomach tube at dose levels of 0, 40, 150 and 600 mg/kg bw/day. F0 animals were treated at least for 75 days prior to mating to produce a litter (F1 generation). Mating pairs were from the same dose group. Pups of the F1 litter were selected (F1 rearing animals) and assigned to 2 different cohorts (1A and 1B) which were subjected to specific postweaning examinations. Groups of 25 males and 25 females, selected from F1 pups to become F1B parental generation, were treated with the test substance at dosages of 0; 40; 150 and 600 mg/kg body weight/day post weaning, and the breeding program was repeated to produce F2 litter. The study was terminated with the terminal sacrifice of the F2 weanlings and F1 adult animals. Control parental animals were dosed daily with the vehicle (0.5% CMC suspension in deionized water with 10 mg/100mL Cremophor).

Animals were assessed for their state of health, detailed clinical signs, body weights, food consumption, clinical pathological investigations (including thyroid hormone measurements) and urinalysis. Mating and reproductive performances were investigated in F0 and F1 animals and estrous cycling or sperm parameters were assessed. The offspring was sexed, monitored for their viability, body weights and the presence of nipple/areola anlagen and their sexual maturation was followed via vaginal opening and balanopreputial separation. All F0 parental and cohort 1A animals were assessed by gross pathology (including weight determinations of several organs) and subjected to an extensive histopathological examination; special attention being paid to the organs of the reproductive system. A quantitative assessment of primordial and growing follicles in the ovaries was performed for all control and high-dose F1 rearing females of cohort 1A. All F1 rearing animals were assessed by pathological examinations. In Cohort 1B, histotechnical processing and examinations by light microscopy was not performed.

 

Clinical observations were observed in most animals of the F0 and F1 animals of the mid and high dose group and consisted of transient salivation, which was often accompanied by ploughing the nose in the bedding. Salivation was also observed in occasional instances in the animals of the low dose group. These findings are considered as compound-related, but attributed to the taste/smell of the test compound and not as adverse. Additionally, unsteady gait was observed after the first dosing(s) at 600 mg/kg bw/d.

Water consumption was increased in the F0 animals of the high dose group (around 25%), and to a lesser extend also at the mid-dose level during gestation. Effects on water consumption were still observed in the cohorts F1 A and F1 B, but to a lesser extent. Food consumption was not influenced by the administration of the test compound in any cohort. Body weights were decreased in high-dose F0 males as well as high-dose F1A and F1B adolescents. The differences ranged from about 10% to 15% in the different age cohorts. Body weights were not remarkably changed in females and therefore did overall not constitute a consistent and noteworthy effect.

Mating performance, pre-coital interval, pregnancy, gestation length, parturition, litter size and postnatal survival of the pups were not affected by the administration of the test compound in the F0 and F1B generation. Body weights were slightly decreased in high-dose F1 and F2 pre-weaning offspring, the effect being up to approximately 10% at PND 14. Anogenital distance was not affected by the administration of the test compound in pups of cohorts F0 and F1 B. Numbers of retained nipples / areolae were higher in F1 pups of the mid and high dose groups, but the values were within the historical control range. Additionally, there was an obvious negative correlation between the litter weight on PND 14 and the number of observed nipples / areola anlagen. Thus, the observed differences are considered rather to be a consequence of delayed body weight development than to represent an antiandrogenic action of the test compound. This is also strengthened by the absence of any nipple / areola anlagen on PND 20. Nipple / areola anlagen in the F2 offspring were comparable among groups. No effects on vaginal opening were observed. Preputial separation was slightly later in male offspring at the high dose group (one day). However, the animals of this dose group reached sexual maturity even at a slightly lower body weight when compared to controls. Therefore, the difference in timing of preputial separation is considered not to be a delay in commencement of sexual maturity, but due to lower postnatal body weight development. Additionally, all values were well within the historical control range.

Regarding clinical pathology in parental (F0) males and females of test group 03 (600 mg/kg bw/d) as well as in the F1 generation of both sexes (F1A, test group 13, 600 mg/kg bw/d) a microsomal liver enzyme induction could be observed indicated by significantly increased γ-glutamyl transferase (GGT) activities and higher total protein and globulin levels (in F1A males of test group 13 additionally increased albumin levels). Higher (total) calcium levels in test group 03 (both sexes) were due to higher globulin levels among these individuals. Additionally, in F0 females of test group 03 and in F1A females of test group 13 triglyceride levels were significantly increased and the latter individuals showed also higher cholesterol levels. Reduced prothrombin time in F0 rats of both sexes in test group 03 can be explained by an increased synthesis of coagulation factors because of the liver enzyme induction. This happened also in F0 females of test group 02 (150 mg/kg bw/d). In F0 males of test group 03 the altered hemostasis situation led also to an increase of platelet counts due to an overreaching compensation of the consumption of platelets in the vessels by an increased release from the bone marrow. Slight alterations of the liver cell metabolism could already be observed in F0 males of test group 02 (150 mg/kg bw/d) by significantly increased total protein, globulin and calcium levels.

In parental rats of both sexes of test group 03 as well as in F1A males of test group 13, serum glucose values were significantly decreased showing a functional change in the energy metabolism. In parental as well as in F1A males of the high dose groups higher urinary levels of ketone bodies and a decreased pH value of the urine indicated a ketogenic metabolism.

Regarding thyroid hormone levels in F0 males of test group 03 (600 mg/kg bw/d) T4 level decreases and TSH value increases as well as T4 value decreases in adult F1A males of test group 13 (600 mg/kg bw/d) in combination with histopathologic findings in the thyroid and liver as well as the mentioned liver parameter alterations indicated a secondary hypothyroidism by increased clearance of T4 levels via the liver and at least in F0 males of test group 03 a compensatory increase of the TSH levels. In female F0 and F1A rats of test groups 03 and 13, only TSH values were significantly increased but in combination with histopathologic findings in the thyroids and increased absolute and relative thyroid weights an adverse effect on the thyroids cannot be excluded. In PND4 and PND22 pups no adverse T4 and TSH level changes could be observed.

Regarding pathology in F0 generation parental animals, treatment related findings were noted in the liver and thyroid gland of male and female animals, and in the kidneys and spleen of male animals. The significantly decreased terminal body weight in test group 03 males was regarded as treatment related. The liver of male animals of test groups 02 and 03 showed a minimally increased incidence of a diffuse hepatocellular hypertrophy. Although, a clear dose response relationship was not recognized. The diffuse hypertrophy in test groups 02 and 03 correlated to increased liver weights and to changed liver parameters in clinical chemistry, which were indicative of a microsomal liver enzyme induction. The hepatocellular hypertrophy and the increased liver weights in test group 03 animals were regarded as treatment related and adverse. The hepatocellular hypertrophy and the increased liver weights in test group 02 animals were regarded as treatment related and – in combination with clinical chemistry parameters – as adverse. The slightly increased liver weights in test group 01 males lay within the range of historical control values. A hepatocellular hypertrophy was only observed in a single animal of this test group. Liver parameters in clinical chemistry examination did not reveal any relevant change. Therefore, the weight increase, and the hepatocellular hypertrophy were assessed as not adverse, if treatment related at all. Female animals of test groups 02 and 03 presented a minimal up to moderate centrilobular hepatocellular hypertrophy, that correlated to increased absolute and relative liver weights and to changed liver parameters in clinical chemistry, which were indicative of a microsomal liver enzyme induction. The hepatocellular hypertrophy and the increased liver weights in test group 03 animals were regarded as treatment related and adverse. The hepatocellular hypertrophy and the increased liver weights in test group 02 animals were regarded as treatment related and – in combination with clinical chemistry parameters – as adverse.

Additionally, male and female animals of test group 03 showed an increased incidence and/or severity of diffuse bile duct hyperplasia, that was regarded as treatment related and adverse.

Thyroid glands of male and female animals in test groups 02 and 03 showed a minimal to moderate diffuse follicular hypertrophy and hyperplasia, accompanied by an increased amount of altered colloid in follicular lumens compared to the control group. Both findings were regarded as treatment related and correlated to significantly increased absolute thyroid gland weights in test group 03 females and significantly increased relative thyroid gland weights in test group 03 males and females. In combination with decreased T4 values and increased TSH values in test group 03 males and increased TSH values in test group 03 females, increased thyroid gland weights and follicular hypertrophy and hyperplasia were assessed as adverse in test group 03. A relation of increased thyroid gland weights and follicular hypertrophy/hyperplasia to a microsomal enzyme induction in the liver can be assumed. The microsomal enzyme induction in the liver may be responsible for the thyroid gland hypertrophy and hyperplasia of follicular cells due to an increased clearance of T4 (by T4-UDP-GT) and a compensatory increase of TSH levels.

The kidneys of male animals of test groups 02 and 03 showed an alpha 2u-Globulin nephropathy, characterized by an accumulation of eosinophilic droplets associated with granular casts and an increased incidence of basophilic tubules. Histopathological findings correlated to increased kidney weights in test groups 02 and 03 and to macroscopically enlarged kidneys in test group 03. The alpha 2u-Globulin nephropathy was regarded as treatment related and adverse. Alpha 2u-Globulin is a male and rat specific poor soluble protein synthesized in the male rat liver. Although alpha 2u-Globulin nephropathy is regarded as treatment-related and adverse, due to the tissue damage, this finding does not represent a risk for humans since they do not synthesize this protein (Durham and Swenberg, 2013) and is therefore of no human relevance. Significantly increased kidney weights in female animals of test group 03 lay above the range of historical control values and were regarded as treatment related. However, since no histopathological findings were noted in female kidneys, the weight increase was assessed as not adverse.

In the spleen of male animals, an increased incidence and/or severity of extramedullary hematopoiesis was observed in test groups 02 and 03. Hematological parameters presented slightly increased reticulocyte counts, but no changes, indicating an anemia. Therefore, the finding was regarded as treatment related and adaptive, but not adverse. 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.

The reproductive organs of one male animal suspected of reduced fertility showed a diffuse massive degeneration/atrophy of testicular tubules and a massive oligospermia in epididymides. These findings occurred only individually and had no relation to the treatment. The reproductive organs of all other mating pairs suspected of reduced fertility did not show histopathological findings that could explain the reduced fertility.

Regarding pathology in F1 generation, rearing animals, cohort 1A, treatment related findings were noted in the liver and thyroid glands of male and female animals, and in the kidneys and spleen of male animals.The significantly decreased terminal body weight in test group 13 males was regarded as treatment related.

The liver of male and female animals of test groups 12 and 13 presented a minimal up to moderate centrilobular hepatocellular hypertrophy, that correlated to increased absolute and relative liver weights. Male and female animals of test group 13 had remarkably increased liver weights and additional changes of clinical chemistry parameters indicative for a liver enzyme induction. Therefore, the weight increase, as well as the hepatocellular hypertrophy were regarded as treatment related and adverse. Test group 12 males and females had increased liver weights and a hepatocellular hypertrophy, but no change of clinical chemistry parameters. Hence, both findings were regarded as treatment related, but not adverse. Females of test group 13 showed an increased incidence and severity of periportal fatty change, that was regarded as treatment related but not adverse, since the finding was only slight. Additionally, the liver of male and female animals of test group 13 showed an increased incidence and/or severity of diffuse bile duct hyperplasia, that was regarded as treatment related and adverse.

A few thyroid glands of male and female animals of test group 13 showed a minimal to slight diffuse follicular hypertrophy and hyperplasia. Male animals of test groups 12 and 13 presented an increased amount of altered colloid compared to the control group. These findings were regarded as treatment related and correlated to significantly increased absolute and relative thyroid gland weights in test group 13 females and to significantly increased relative thyroid gland weights in test group 13 males. In combination with decreased T4 values in test group 13 males and increased TSH values in test group 13 females, increased thyroid gland weights and follicular hypertrophy and hyperplasia were assessed as adverse in test group 13. A relation of increased thyroid gland weights and follicular hypertrophy/hyperplasia to a microsomal enzyme induction in the liver can be assumed. The microsomal enzyme induction in the liver may be responsible for the thyroid gland hypertrophy and hyperplasia of follicular cells due to an increased clearance of T4 (by T4-UDP-GT) and, at least in test group 13 females, a compensatory increase of TSH levels.

The kidneys of male animals of test groups 12 and 13 showed an alpha 2u-Globulin nephropathy, characterized by an accumulation of eosinophilic droplets and an increased incidence and severity of basophilic tubules. The alpha 2u-Globulin nephropathy was associated to granular casts in test group 13. Histopathological findings correlated to increased kidney weights in test groups 12 and 13 and to macroscopically enlarged kidneys in test group 13. The alpha 2u-Globulin nephropathy was regarded as treatment related and adverse. Male animals of test group 11 showed likewise an increased incidence of eosinophilic droplets and basophilic tubules. The incidence of eosinophilic droplets and basophilic tubules of test group 11 males lay within the range of historical control values. Furthermore, this test group did not show significantly increased kidney weights. However, with respect to this study, there was at least with regard to basophilic tubules, a remarkably increased incidence compared to the control group. Therefore, the increased incidence and severity of eosinophilic droplets and basophilic tubules in test group 11 males was assumed to be treatment related and adverse. It was regarded as a very mild alpha 2u-Globulin nephropathy. Although alpha 2u-Globulin nephropathy is regarded as treatment-related and adverse, due to the tissue damage, this finding does not represent a risk for humans since they do not synthesize this protein (Durham and Swenberg, 2013) and is therefore of no human relevance. Significantly increased relative kidney weights in female animals of test group 13 were regarded as treatment related. However, since no histopathological findings were noted in female kidneys, the weight increase was assessed as not adverse.

An increased incidence and severity of extramedullary hematopoiesis was observed in the spleen of test group 13 males that correlated to a macroscopically enlarged spleen in one case. Hematological parameters presented slightly increased reticulocyte counts, but no changes, indicating an anemia. Therefore, the finding was regarded as treatment related and adaptive, but not adverse.

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 – showed no significant differences between the control group 10 and animals of test group 13.

Regarding pathology in F1-generation, rearing animals, cohort 1B, treatment related findings were noted in the liver of male and female animals. The significantly decreased terminal body weight in test group 13 males was regarded as treatment related.

Significantly increased liver weights in male and female animals of test groups 12 and 13 were regarded as treatment related. They correlated to a macroscopically visible dark brown discoloration of the liver in test group 13. Since the weight increase in test group 13 animals was remarkable, it was assessed as an adverse finding. 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.

In conclusion, under the conditions of the present study the NOAEL (no observed adverse effect level) for general and systemic toxicity is 40 mg/kg bw/d for the F0 and 150 mg/kg bw/d for the F1 parental rats, based on reduced body weights in males at 600 mg/kg bw/d as well as clinical pathology and histopathological findings observed at 150 mg/kg bw/d (F0) and 600 mg/kg bw/d (F1).

The NOAEL for fertility and reproductive performance for the F0 and F1 parental rats is 600 mg/kg bw/d, the highest dose tested.

The NOAEL for developmental toxicity in the F1 and F2 progeny is 150 mg/kg bw/d, based on reduced body weights of the offspring during lactation at 600 mg/kg bw/d.

 

 

In addition, studies with the structural analogue substances pseudo-ionone (CAS 141-10-6) and mixed ionone isomers (CAS 8013-90-9) are available. These data are used to support the read-across assessment on developmental toxicity. For more information on the read-across approach, please refer to the read-across justification.

 

The reproduction toxicity was analyzed in an One-generation reproduction toxicity study with pseudo-ionone performed under GLP according to OECD guideline 415 (Beekhuizen, 2003). 96 male and 96 female Wistar rats were randomly assigned to four treatment groups which were administered by gavage with 0, 40, 120 or 360 mg/kg bw/d pseudoionone in maize/corn oil. Exposure length was 11 weeks prior to mating up to termination for males, giving the mean of 106 days, with a range from 104 to 108 days. The females were exposed for 2 weeks prior to mating up to termination; the mean duration of treatment was 60 days, with a range of 36 to 65 days. The offspring was not treated. Females were paired one-to-one with males from the same group and allowed to give birth normally. Pups were killed either at adjusting litters on day 4 post partum or at the end of the study at day 21 post partum. At the end of the study, all survivors were killed, the males after confirmation of the pregnancy or successful delivery of the female they were mated with, the females at day 21 post partum or shortly thereafter. After killing or natural death all parental animals were subjected to external examination and to macroscopic examination during dissection, with special attention to the reproductive organs. Also, pups were sexed and externally examined. Three unscheduled deaths occurred, which were considered incidental and therefore considered not to be treatment-related. Males and females of the 360 mg/kg bw/d group showed statistically significant increased absolute and relative liver and kidneys weight, which was also seen in the males of the 120 mg/kg bw/d group. In the absence of histopathological changes, both effects were considered not to be toxicologically relevant but rather manifestations of physiological adaptation to additional metabolic and excretionary loads.

Regarding reproductive parameters it was found that one female did not mate and one female was not pregnant in the 40 mg/kg bw/d group. In the 120 mg/kg bw/d group, one female showed delivery difficulties, and in the 360 mg/kg bw/d group, two females showed delivery difficulties, all of which were not considered related to the treatment but rather to the high number of foetuses. All other parameters, specifically mating performance, duration of gestation and fertility parameters including number of pups at birth were similar for the control and the treat­ment groups. In summary, the reproductive respectively fertility parameters were not affected up to 360 mg/kg bw/d. Thus, the general toxicological parental NOAEL was 120 mg/kg bw/d (NOEL 40 mg/kg bw/d) while the reproductive NOEL was 360 mg/kg bw/d based on the liver- and kidney-weight changes in males and salivation by all high dose animals.

 

Results of a two-generation study showed no adverse effects on reproduction, when 8-10 mg/kg bw/day of the structural analogue mixed ionone isomers (CAS 8013-90-9) were administered to the parental rats for 8 months (Sporn, 1963). Thereby, the female rats were studied through 3 reproduction cycles for number of pregnancies, weight and number of offspring, live pups, weight of pups at birth and after 7 and 21 days, and the viability of the pups after birth. The F1 generation (offspring) were allowed to reach maturity and were then treated with 15 mg/kg of ionone prior to being subject to reproductive toxicity testing. As a result, no effects on reproductive parameters and organs were observed in this study.

Effects on developmental toxicity

Description of key information

OECD 414: β-Ionone (CAS 79-77-6), oral, rat (BASF 2004)

- NOAEL (maternal toxicity) = 100 mg/kg bw/d

- NOAEL (prenatal developmental toxicity) ≥ 400 mg/kg bw/d

 

OECD 414: Methylionon 70 (EC 942-741-0), oral, rabbit (BASF 2021)

- NOAEL (maternal toxicity) = 150 mg/kg bw/d

- NOAEL (prenatal developmental toxicity) ≥ 500 mg/kg bw/d

 

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

GLP compliance:

yes (incl. QA statement)

Remarks:

Experimental Toxicology and Ecology, BASF AG

Limit test:

no

Specific details on test material used for the study:

- Name of test material (as cited in study report): lonon R
- Physical state: liquid / colorless - yellowish
- Analytical purity: 97.8% (Analytical Report: 02L00370)
- Lot/batch No.: continuous production
- Date of production: October 09, 2002
- Stability under test conditions: under storage conditions was confirmed by reanalysis
- Storage condition of test material: Refrigerator, protected from light
- Other: Analytical laboratory: Analytical Department, BASF Aktiengesellschaft, Ludwigshafen/Rhein, Germany

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Strain: Time-mated Wistar rats (CrIGIxBrIHan:WI)
- Source: Charles River Laboratories, Germany
- Animal identification: by ear tattoo
- Age at study initiation: 70 - 84 days
- Weight at study initiation: 148 .0 - 183.6 g
- Housing: singly from day 0 - 20 p .c. in type DK III stainless steel wire mesh cages supplied by BECKER & CO., Castrop-Rauxel, FRG (height : 15 cm, length: 37,5 cm, width: 21 cm; floor area about 800 cm2)
- Diet (e.g. ad libitum): ad libitum, ground Kliba maintenance diet rat/mouse/hamster meal (PROVIMI KLIBA SA, Kaiseraugst, Switzerland)
- Water (e.g. ad libitum): ad libitum, drinking water of tap water quality from water bottles
- Acclimation period: 6 days

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

ANALYSES
- Food: assayed for chemical as well as for microbiological contaminants
- Water: regularly assayed for chemical contaminants by the municipal authorities of Frankenthal and by Technical Services of BASF Aktiengesellschaft as well as for the presence of microorganisms by a contract laboratory

Route of administration:

oral: gavage

Vehicle:

olive oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:

DIET PREPARATION
- Rate of preparation of diet (frequency):prepared at the beginning of the administration period and thereafter at intervals, which took into account the analytical results of the stability verification .
- Mixing appropriate amounts with (Type of food): weighed in a graduated beaker (depending on the dose group), topped up with olive oil Ph .Eur./DAB, and subsequently thoroughly mixed using a magnetic stirrer.

VEHICLE
- Lot/batch no. (if required): Ph .Eur./DAB
- Amount of vehicle (if gavage): 5 ml/kg bw olive oil
- Concentration in vehicle: 500, 2000 and 8000 mg/100 ml

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples of the test substance solutions were sent to the analytical laboratory twice during the study period (at the beginning and towards the end) for verification of the concentrations.
Since the test substance preparations were true solutions, investigations concerning homogeneity were not necessary .

Details on mating procedure:

- Impregnation procedure: purchased timed pregnant:
The animals were mated by the breeder ("time-mated") and supplied on day 0 post coitum (= detection of vaginal plug / sperm). The animals arrived  on the same day (i.e. day 0 p.c.) at the experimental laboratory. The  following day was designed "day 1" post coitum (p.c.). Animals were  assigned to the test groups by taken random selection.

Duration of treatment / exposure:

day 6 through day 19 post coitum (p.c)

Frequency of treatment:

once daily

Duration of test:

On day 20  p.c., all surviving females were sacrificed

Dose / conc.:

25 mg/kg bw/day (nominal)

Dose / conc.:

100 mg/kg bw/day (nominal)

Dose / conc.:

400 mg/kg bw/day (nominal)

No. of animals per sex per dose:

25

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The following doses were chosen for the present full-scale toxicity study in Wistar rats. The oral route was selected since this has proven to be suitable for the detection of a toxicological hazard.

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes, check for mortality
- Time schedule: twice a day on working days or once a day (Saturday, Sunday or on public holidays) (days 0- 20 p .c.).

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at least once a day, or more often when clinical signs of toxicity were elicited (days 0- 20 p .c.).

BODY WEIGHT: Yes
- Time schedule for examinations: on days 0, 1, 3, 6, 8, 10, 13, 15, 17, 19 and 20 p.c.
The body weight change of the animals was  calculated from these results

CORRECTED BODY WEIGHT GAIN (net maternal body weight change): Yes
- Time schedule: calculated after terminal  sacrifice (terminal body weight on day 20 p.c. minus weight of the  unopened uterus minus body weight on day 6 p.c.)

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Time schedule: With the exception of day 0, the consumption of food  was determined on the same days as was body weight

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 20
- Organs examined: liver, uterus, ovaries

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other: dead fetuses

- Other: calculations of conception rate and pre- and postimplantation losses

Fetal examinations:

- External examinations: Yes: all per litter
- Soft tissue examinations: Yes:half per litter per litter
- Skeletal examinations: Yes: half per litter per litter
- Head examinations: Yes: all per litter

Statistics:

- two-sided Dunetts: Food consumption, body weight, body weight changes, corrected body weight gain (net maternal body weight change), carcass weight, weight of unopened uterus, number of corpora lutea, number of implantations, number of resorptions, number of live fetuses, proportion of preimplantation loss, proportions of postimplantation loss, proportions of resorptions, proportion of live fetuses in each litter, litter mean fetal body weight, litter mean placental weight
- two-sided Kruskal-Wallis test: liver weights
one-sided Fisher's Exact test: female mortality, females pregnant at terminal sacrifice, number of litters with fetal findings
- one-sided Wilcoxon test: proportions of fetuses with malformations, variations and/or unclassified observations in each litter

Historical control data:

The historical control data used for interpretation of findings refer to  the same test facility, the same rat strain and supplier of the animals  and cover a period of about 24 months (June 2001 - June 2003, 15 studies)

Key result

Dose descriptor:

NOAEL

Effect level:

100 mg/kg bw/day

Basis for effect level:

other: maternal toxicity

Dose descriptor:

LOAEL

Effect level:

400 mg/kg bw/day

Basis for effect level:

other: maternal toxicity

Dose descriptor:

NOAEL

Effect level:

400 mg/kg bw/day

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

no

Result: no influence on gestational parameters, no adverse signs of developmental  toxicity, no indications of teratogenic effects

TEST SUBSTANCE ANALYSES
The stability of the test substance suspensions over a period of 4 days  (at 4°C) and the correct concentration of the test substance in the test  preparation was demonstrated (always above 90% and below 110% of nominal  concentrations). 

MATERNAL TOXIC EFFECTS BY DOSE LEVEL
- Mortality and day of death: There were no substance-related or  spontaneous mortalities in any of the groups.
- Clinical examinations: Each test group including the controls contained  a sufficient number of females with implantation sites at necropsy (20 or  more).

Clinical symptoms: All high dose and the majority (22 out of 25) of the  mid dose animals showed transient salivation immediately after treatment on one or several days of the treatment period; however, the observed  salivation persisted in the respective females only for a few minutes  after the actual gavaging had taken place. After cessation of treatment  on day 19 p.c., salivation did not occur any longer. The observed  temporary salivation of the animals was considered to be  substance-induced. It is very likely, that this finding was induced by  bad taste of the test substance or local affection of the upper digestive  tract. Salivation itself is not assessed as an adverse or toxic effect. 

Additionally, 21 high dose dams showed dark-yellow discolored urine on  gestation days 12 - 20 p.c. which is probably related to a chemical  reaction of the test substance or its metabolites with the bedding or  with components of the air and does not represent a toxicologically  relevant finding. Comparable urine findings have been observed in a  4-week range finding study in Wistar rats with dietary administration of  the test substance, Project No.: 30S0449/02045 (BASF AG, 2003).
No indications for disturbances of the general behavior, however,  occurred in the control and low dose dams.

- Food consumption: The mean food consumption of the high dose dams was  statistically significantly reduced (9% below the concurrent control  value) at initiation of treatment (days 6 - 8 p.c.). On the following  days of the treatment period, however, food consumption of the high dose  rats reached or even exceeded control values. The food consumption of the  females of low and mid dose dams was unaffected and did not show any  statistically significant or biologically relevant differences in  comparison to the controls. 
The transient reductions in food consumption at 400 mg/kg bw were  accompanied by corresponding impairments in body weight gain of these  dams at initiation of dosing. 
 
- Body weight data: The mean body weights of the low, mid and high dose  rats were substantially similar to the concurrent control values. 
A statistically significant impairment in mean body weight gain (about  29% below the concurrent control value) occurred in high dose group on  treatment days 8 - 10 p.c.; on the other treatment days (i.e. days 6 - 8  and 10 - 19 p.c.) weight gains of the 400 mg/kg bw females were sometimes  below and sometimes above those of the corresponding controls without  attaining statistical significance. As the food consumption of these rats  was also transiently diminished and the corrected body weight gain was  also slightly decreased this was considered to be a substance-related  sign of maternal toxicity.
Body weight gains of the dams at 25 and 100 mg/kg bw/day) were similar to  those of the concurrent controls. 

- Corrected body weight gain (net maternal body weight change): The  corrected body weight gains (terminal body weight on day 20 p.c. minus  weight of the unopened uterus minus body weight on day 6 p.c.) of the  dams of the low and mid dose groups revealed no differences of any  biological relevance to the corresponding control group. The net weight  change of the 400 mg/kg bw rats, however, was about 17% below the  concurrent control value (not statistically significant). As food  consumption and body weight gain of this group was also temporarily  diminished, the effects on net body weight gain at the top dose are  considered to be substance-related, borderline signs of maternal toxicity.

EXAMINATION OF THE DAMS AT TERMINATION

- Uterus weight: The mean gravid uterus weights of the animals of all  test groups were not influenced by the administration of the test  substance.  

- Liver weight: Absolute and relative mean liver weights were  statistically significantly increased at the mid and high dose groups and  were about 9 or 29% (absolute) and 8 or 29% (relative) above control  values. These weight increases, which are considered to be  substance-induced, are indicative of hepatic changes primarily caused by  microsomal enzyme induction. Absolute and relative liver weights of the  low dose dams, however, were similar to the control values and did not  show any toxicologically significant changes.

- Necropsy findings: There occurred no substance-related observations at  necropsy in any of the dams of all test groups. Only very few spontaneous  findings were recorded for single low and mid dose rats (one hydrometra  in low dose female which consequently did not become pregnant,  hemorrhagic thymus in  one mid dose female). No association to the test  compound was assumed for these findings due to their scattered occurrence  without any relation to dosing.

- Reproduction data of dams: The conception rate reached 96% in the  controls, 92% in the low and the high dose groups, and 100% in the mid  dose. There were no substance-related and/or biologically relevant  differences between the test groups in the conception rate, in the mean  number of corpora lutea and implantation sites, the pre- and the  postimplantation losses, and the number of resorptions and viable fetuses  (see table below). The pre- and the postimplantation loss values in the  25 and 100 mg/kg groups, however, were above the upper ranges of the  historical control values and the mean number of live fetuses/low dose  dam was statistically significantly below the concurrent and the  historical control value. These differences appeared without any  dose-response relationship and thus are not considered to reflect any  substance-induced effect. They can well be explained by the fact that one  low dose dam and two mid dose dams resorbed all of their implants and  thus had no viable fetuses (the same was also observed for one control  dam). Moreover, one low dose, which had 7 implantation sites, resorbed 6  implants and had only one live fetus at terminal sacrifice. If these 5  rats are excluded from the calculation of the means, pre- and  postimplantation loss values as well as the mean number of live  fetuses/dam fit well into the historical control ranges with one  unimportant exception (preimplantation loss value at 25 mg/kg bw/day).

Table: Pre- and postimplantation loss (PRI and POI) values and mean  number of live fetuses per dam

Dose         Control Low    Mid   High    Historical
                            dose          control range


PRI          6.0     14.4   12.7   4.7    8.7 (3.5-12.2)
Corrected    4.8     12.9*   8.7   4.7    

POI          9.9     14.8   15.2   6.2    6.7 (3.7-11.3)
Corrected    6.0      7.3    7.9   6.2    

Live fetuses 8.7      7.4*   8.2   8.6    8.8 (7.9-9.8)
per dam                                       
Corrected    8.7      7.7    8.2   8.6

Corrected: exclusion of 5 dams,  *   P < = 0.05

EXAMINATION OF FETUSES

- Sex distribution of fetuses: The sex distribution of the fetuses in all  test groups was comparable with that of the control fetuses.
 - Weight of placentae: The mean placental weights in the  substance-treated groups did not show any differences with toxicological  relevance. The statistically significant increase of the mean placental  weight of the high dose male fetuses (0.45g versus 0.41g in the control  group; p <= 0.05) is not considered to demonstrate an adverse finding and  the respective value was fully within the historical control range  (0.32g-0.58g).
- Weight of fetuses: The mean fetal body weights in all test groups were  not influenced by the test substance administration and were very similar  to or even identical with concurrent control values.
- Fetal external, soft tissue and skeletal observations:
The scattered occurrence of the few observed external, soft tissue and  skeletal malformations in single fetuses of the controls, the low and mid  dose group without a consistent pattern, without any dose-response  relationship and/or at incidences, which are similar to historical  control rates did not suggest any substance-induced origin of these  findings. Most of the observed malformations were limited to one multiply  malformed mid dose fetus. The external examination of this fetus revealed  gastroschisis, anal atresia, malrotated left hindlimb, and a thread-like  tail. During its skeletal evaluation additional malformations like  severely malformed sternum and absent vertebrae were recorded. The other  malformations that occurred were anasarca (in one control fetus), situs  inversus (in one low dose fetus), and cleft sternum (in another control  fetus).

If all different types of malformations are summarized, in total 2 of the  200 examined control fetuses [= 1.0%] in 2 out of 23 litters [= 8.7%],  one of the 163 examined low dose fetuses [= 0.6%] in one out of 22  litters [= 4.5%], one of the 189 examined mid dose fetuses [= 0.5%] in  one out of 23 litters [= 4.3%] and none of the 197 examined high dose  fetuses (from 23 litters) showed malformations. The mean percentages of  affected fetuses/litter with total malformations amounted to 0.8, 0.5, 0.6, and 0.0% at 0; 25; 100 or 400 mg/kg bw/day respectively. These low, non dose-related incidences did not suggest any relationship to the test  substance.

External variations did not occur in any of the fetuses in this study.  Soft tissue variations, exclusively in the form of dilated renal pelvis  and/or ureters, and a broad range of skeletal variations occurred in all  test groups including the controls. All fetal and litter incidences for  these variations and the corresponding mean percentages of affected  fetuses/litter did not show a clear relation to dosing, were not  considered to be of any toxicological relevance and/or could be found at  a comparable frequency in the historical control data. This statement  included the statistically significantly increased occurrence of three  variations (i.e. supraoccipital holes, bipartite ossification of thoracic  centrum [with dumbbell-shaped cartilage of centrum] and supernumerary  14th rib [without cartilage]) at the mid dose group.

If all variations were summarized, in total 105 of the 200 examined  control fetuses [= 53%] in all 23 litters [= 100%], 88 of the 163  examined low dose fetuses [= 54%] in all 22 litters [= 100%], 99 of the  189 examined mid dose fetuses [= 52%] in all 23 litters [= 100%] and 97  of the 197 examined high dose fetuses [= 49%] in all 23 litters [= 100%]  showed variations. The mean percentages of affected fetuses/litter with  total variations amounted to 52.8, 56.2, 52.9, and 50.8% at 0; 25, 100 or  400 mg/kg bw/day respectively. These incidences did not suggest a  treatment-relationship, but reflect the usual biological variation  inherent in the strain of rats used for this experiment.

A spontaneous origin was also assumed for the few unclassified cartilage  observations which were recorded for several fetuses of all test groups.  Distribution and type of these findings do not suggest any relation to  treatment as the mean percentages of affected fetuses/litter with these  findings amounted to 17.0, 18.5, 15.4, and 13.6% at 0; 25; 100 or 400  mg/kg bw/day, respectively.

The following substance-related findings were obtained:

Test group 3 (400 mg/kg body weight/day):

• transient salivation in all rats immediately after gavaging on days 6 - 19 p.c.

• discolored urine in a total of 21 out of 25 dams (days 12 - 20 p.c.)

• statistically significantly reduced food consumption on days 6 - 8 p.c. (about 9% below controls)

• statistically significant impairments in absolute body weight gain on days 8 -10 p.c.(about 29% below controls)

• lower corrected body weight gain (about 17% below controls) without attaining statistical significance

• statistically significantly increased absolute and relative liver weights (about 29% above controls)

• no substance-related effects on gestational parameters or fetuses

Test group 2 (100 mg/kg body weight/day):

• transient salivation in 22 out of 25 rats immediately after gavaging between treatment days 10 - 19 p.c.

• statistically significantly increased absolute and relative liver weights (about 8 or 9% above controls)

• no substance-related effects on gestational parameters or fetuses

Test group 1 (25 mg/kg body weight/day):

• no substance-related adverse effects on dams, gestational parameters or fetuses

Reference 2

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

25 June 2008

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

Version / remarks:

Aug 2008

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Batch No.of test material: 00073177LO
- Expiration date of the lot/batch: 09 Jun 2019
- Physical state/appearance: Liquid/ yellowish, clear

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature
- Stability under test conditions: Guaranteed by the Sponsor
- Stability of the test substance in the solvent/vehicle: Verified prior the start of the study (at room temperature over a period of 7d)

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing:
The specific amount of test substance were weighed, topped up with 0.5% Sodium carboxymethyl cellulose (CMC) suspension in deionized water (with 10 mg/ 100 mL Cremophor EL) in an Erlenmeyer flask and intensely mixed with a magnetic stirrer.
Before and during administration, the preparations were kept homogeneous with a magnetic stirr.

Species:

rabbit

Strain:

New Zealand White

Remarks:

Crl:KBL(NZW)

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services,
Germany GmbH/ Charles River Laboratories, France
- Housing: Single housing
- Fasting period before study: Not applicable
- Diet (e.g. ad libitum): ad libitum; pelleted Kliba maintenance diet rabbit and guinea pig “GLP” (Granovit AG, Kaiseraugst/Switzerland)
- Water (e.g. ad libitum): Drinking water ad libitum
- Acclimation period: At least 5 d before artificial insemination

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17-21
- Humidity (%): 45-65
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12 hours light (06:00 h - 18:00 h); 12 hours darkness (18:00 h - 06:00 h)

Route of administration:

oral: gavage

Vehicle:

other: 0.5% CMC suspension in deionized water (with 10 mg/ 100 mL Cremophor EL)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
- The specific amount of test substance was weighed, topped up with 0.5% CMC suspension in deionized water (with 10 mg/ 100 mL Cremophor EL) in an Erlenmeyer flask and intensely mixed with a magnetic stirrer. Before and during administration, the preparations was kept homogeneous with a magnetic stirrer. The test substance preparations were prepared at intervals which guaranteed that the test substance concentrations in the vehicle remained stable.

VEHICLE
- Concentration in vehicle: 0, 0.5, 1.5, 5.0 g/100 mL
- Amount of vehicle (if gavage): 10 mL/kg body weight

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Concentration control analysis at the beginning of administration. The samples of the mid and end of the study are analyzed only if any imprecision occurs during the analysis of the samples from the start of the study.
The stability of the test substance in the vehicle at room temperature over a period of 7 days had been verified prior to the start of the study.

Details on mating procedure:

- Impregnation procedure: Artificial insemination (GD 0)
- 0.2 mL of a synthetic hormone (Receptal, Intervet Deutschland GmbH, Unterschleißheim, Germany), which releases LH and FSH from the anterior pituitary lobe, was injected intramuscularly into the female rabbits about 1 h before insemination
- The samples of the ejaculate used for the artificial insemination were derived from male New Zealand White rabbits of the same strain as the females. The male donors were maintained under the same conditions (air conditions, feed and water) as the females used in the study. The day of insemination was referred to as GD 0.

Duration of treatment / exposure:

GD 6-28

Frequency of treatment:

daily

Dose / conc.:

50 mg/kg bw/day (nominal)

Dose / conc.:

150 mg/kg bw/day (nominal)

Dose / conc.:

500 mg/kg bw/day (nominal)

No. of animals per sex per dose:

25

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
In a three-week test study (BASF project No.: 01R0397/09R146), Methylionon 70 was administered to each three non-pregnant New Zealand White rabbits per test group at dose levels of 1000, 300 and 0 (control) mg/kg bw/d. The animals of test group 2 (1000 mg/kg bw/d) showed decreased food consumption (up to -90% compared to control) and body weight reduction since the beginning of the substance administration. Therefore, the dose was reduced to 800 mg/kg bw/d from SD 8 onward resulting in a higher food consumption and a slight recovery of body weights after SD 9 (comparable body weight change from SD 9 onwards compared to control group). At 300 mg/kg bw/d, mean food consumption values were reduced up to SD 6 bot recovered towards the end of the study. No effects on mean body weight and body weight chance were observed compared to the control group.

In a following maternal toxicity range-finding study (BASF project No.: 20R0397/09R156), Methylionon 70 was administered daily to each five pregnant New Zealand White rabbits per test group from implantation to one day prior to the expected day of parturition (GD 6-28). Dose levels of 800 and 300 mg/kg bw/d were used. One animal each of the control and high dose group were not pregnant. At the high-dose level of 800 mg/kg bw/d, the following effects were observed: no feces in four out of five animals, a pronounced reduction in mean food consumption (GD 6-28: 52% below control), a body weight loss (bw up to 15% below control on GD 21), a decreased body weight change. Two animals of this test group aborted and were sacrificed on GD 24. At the next lower dose of 300 mg/kg bw/d, mean food consumption values were slightly reduced in the beginning of the study but recovered to control values from GD 6 onwards. No further relevant changes were observed at that dose level.

Therefore, based on the above-mentioned range-fining study, the following doses were chosen for the present prenatal developmental toxicity study in New Zealand White rabbits: 0, 50, 150 and 500 mg/kg bw/day.

- Rationale for animal assignment: During the acclimatization period the animals were assigned to the different test groups according to a randomization plan (NIJENHUIS and WILF) and on the basis of their body weights.

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule:
- At least once daily for any signs of morbidity, pertinent behavioral changes and/or signs of overt toxicity
- A check for moribund and dead animals twice daily from Mondays to Fridays and once daily on Saturdays, Sundays and public holidays (GD 0 to 29)
- During administration period (GD 6-28): all animals were checked daily for abnormal clinical signs before administration as well as within 5 hours after the administration. Abnormalities and changes are documented for each animal

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: GD 0, 2, 4, 6, 9, 11, 14, 16, 19, 21, 23, 25, 28 and 29

FOOD CONSUMPTION: Yes
- Time schedule for examination: daily during GD 0-29

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 29
- Organs examined: Gross-pathological examination

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Other: Site of implantations in the uterus; Gross-pathological examination

Blood sampling:

Blood samples were collected for external research projects beyond the scope of this study and without GLP status. The results of the study were not influenced by this procedure because blood sampling occurred just prior to sacrifice. The sampling procedures did not affect the outcome and compliance of the GLP study. The data from these research projects do not affect the outcome, assessment and compliance of this GLP study.

Fetal examinations:

- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: all per litter
- Skeletal examinations: Yes: all per litter
- Head examinations: Yes: half per litter (and the heads of any fetus which revealed severe findings during the external examination, e.g. anophthalmia, microphthalmia or hydrocephalus)

Statistics:

Means and standard deviations were calculated. In addition, the following statistical analyses were carried out:

- DUNNETT’s test: Food consumption, body weight, body weight change, corrected body weight gain, carcass weight,
weight of the unopened uterus, weight of the placentas and fetuses, corpora lutea, implantations, pre- and postimplantation losses, resorptions and
live fetuses
- FISHER's exact test: Number of pregnant animals at the end of the study, mortality rate (of the does) and number of litters with fetal findings
- WILCOXON test: Proportion of fetuses with findings per litter

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

One female of the low-dose group (50 mg/kg bw/d) had blood in bedding before/after treatment on GD 17-19, as mentioned above this doe aborted on GD 20. Furthermore, one high-dose female (500 mg/kg bw/d) had vaginal hemorrhage before/after treatment on GD 20-21.
No defecation was observed in two control females, one low-dose, two mid-dose and six high-dose females. Reduced defecation was observed in five control, four low-dose, eight mid-dose and sixteen high-dose females (0, 50, 150 and 500 mg/kg bw/d). Although frequency of defecation usually shows great variability among individual rabbits across treatment groups during a study, the distinctly higher incidence of these observations in the high-dose group, along with reduced food consumption indicates a treatment-related effect.
There were no further clinical findings in the other does in this study.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

One female, each, of the test groups 1, 2 and 3 (50, 150 and 500 mg/kg bw/d) were sacrificed after abortion ahead of schedule. Spontaneous abortions in single does are not uncommon findings in the strain of rabbits used for this study. Thus, these were considered to be spontaneous incidents.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

The mean body weights (BW) and the average body weight gain (BWC) of the high-dose rabbits (500 mg/kg bw/d) were distinctly reduced and statistically significant on GD 14-16, GD 21-23, GD 28-29 (BW, up to 6% below control) and on GD 6-9 (BWC, -37.2g vs. 47.0 g in control) and GD 25-28 (-20.1 g vs. 55.6 g). Body weight of the high-dose does at term (GD 29) was 6% below control. During the treatment period (GD 6-28), the high-dose does gained approx. 77% less weight (attaining statistically significance) than the concurrent control does.
During a short period in mid-pregnancy (GD 16-19), the rabbits in test group 3 lost significantly less weight than the other rabbits, however, this is assessed as incidental and not treatment-related.
The mean body weights and the average body weight gain of the low- and mid-dose groups (50 and 150 mg/kg bw/d) were generally comparable to the concurrent control group throughout the entire study period.

Mean carcass weight of the does in test group 3 (500 mg/kg bw/d) was statistically significantly reduced (-5%) in comparison to the concurrent control group. Furthermore, the corrected body weight change (terminal body weight on GD 29 minus weight of the unopened uterus minus body weight on GD 6) was significantly lower in the high-dose group (-366.9 g) in comparison to the concurrent control group (-199.7 g).
The mean carcass weights and the corrected body weight gain were comparable between test groups 0-2 (0, 50 and 150 mg/kg bw/d).

Food efficiency:

effects observed, treatment-related

Description (incidence and severity):

In comparison to the control group, the mean food consumption of the does in test group 3 (500 mg/kg bw/d) was consistently reduced during the treatment period, attaining statistical significance on GD 6-12, GD 13-17, GD 19-20 and GD 27-29 (up to -61% below control). Overall, the high-dose does consumed 25% less food than the concurrent control does during the treatment period (GD 6-28).
The food consumption of the low- and mid-dose rabbits (50 and 150 mg/kg bw/d) was comparable to the concurrent control (0 mg/kg bw/d) throughout the entire study period.

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

The mean gravid uterus weight of the rabbits of test groups 1-3 (50, 150 or 500 mg/kg bw/d) was not influenced by the test substance. The differences between these groups and the control group showed no dose-dependency and were assessed to be without biological relevance.
Uterus weight:
- Test group 0 (control): 477.7 g
- Test group 1 (50 mg/kg bw/d): 483.7 g (101%)
- Test group 2 (150 mg/kg bw/d): 502.4 g (105%)
- Test group 3 (500 mg/kg bw/d): 427.0 g (89%)

Gross pathological findings:

no effects observed

Description (incidence and severity):

Some spontaneous findings were noted in individual females of all test groups (0, 50, 150 and 500 mg/kg bw/d):
• intestines bloated and a granulated surface of liver in one control doe,
• multiple erosions in stomach combined with watery feces in one mid-dose doe,
• agenesia of gallbladder in one high-dose doe,
• watery feces in the large intestine in one mid-dose doe and one high-dose doe,
• in general, watery feces in one control doe, one low-dose doe and three high-dose does.
Additional findings were noted. They were associated with unscheduled maternal death or sacrifice:
• watery feces in one mid-dose doe (sacrificed after abortion on GD 29),
• pale heart and pale kidneys (bilateral), enlarged gallbladder and watery feces in the large intestine in one high-dose doe (sacrificed after abortion on GD 25).

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Number of abortions:

no effects observed

Description (incidence and severity):

There were no test substance-related and/or biologically relevant differences between the different test groups in the number of does with abortion. All differences observed are considered to reflect the normal range of fluctuations for animals of this strain and age.

Resorptions:
Test group 0 (control): 0
Test group 1 (50 mg/kg bw/d): 1
Test group 2 (150 mg/kg bw/d): 1
Test group 3 (500 mg/kg bw/d): 1

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

There were no test substance-related and/or biologically relevant differences between the different test groups in the values calculated for pre- and post-implantation losses. All differences observed are considered to reflect the normal range of fluctuations for animals of this strain and age.

Preimplantation loss
- Test group 0 (control): 5.9%
- Test group 1 (50 mg/kg bw/d): 5.4%
- Test group 2 (150 mg/kg bw/d): 6.4%
- Test group 3 (500 mg/kg bw/d): 6.6%

Postimplantation loss
- Test group 0 (control): 11.3%
- Test group 1 (50 mg/kg bw/d): 5.2%
- Test group 2 (150 mg/kg bw/d): 6.5%
- Test group 3 (500 mg/kg bw/d): 11.1%

Total litter losses by resorption:

no effects observed

Description (incidence and severity):

There were no test substance-related and/or biologically relevant differences between the different test groups in the number of does with a total litter loss by resorption. All differences observed are considered to reflect the normal range of fluctuations for animals of this strain and age.

Does with all resorptions
- Test group 0 (control): 1
- Test group 1 (50 mg/kg bw/d): 0
- Test group 2 (150 mg/kg bw/d): 0
- Test group 3 (500 mg/kg bw/d): 1

Early or late resorptions:

no effects observed

Description (incidence and severity):

There were no test substance-related and/or biologically relevant differences between the different test groups in the number of resorptions. All differences observed are considered to reflect the normal range of fluctuations for animals of this strain and age.

Early / late / total mean resorptions
- Test group 0 (control): 0.6 / 0.3 / 0.8
- Test group 1 (50 mg/kg bw/d): 0.3 / 0.2 / 0.6
- Test group 2 (150 mg/kg bw/d): 0.4 / 0.3 / 0.8
- Test group 3 (500 mg/kg bw/d): 0.4 / 0.4 / 0.8
8

Dead fetuses:

no effects observed

Description (incidence and severity):

There were no test substance-related and/or biologically relevant differences between the different test groups in the number of viable fetuses. All differences observed are considered to reflect the normal range of fluctuations for animals of this strain and age.

Dead fetuses
Test group 0 (control): 0
Test group 1 (50 mg/kg bw/d): 0
Test group 2 (150 mg/kg bw/d): 0
Test group 3 (500 mg/kg bw/d): 0

Changes in pregnancy duration:

no effects observed

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

The conception rate was 84% in the mid-dose group (150 mg/kg bw/d), 88% in
the low-dose group (50 mg/kg bw/d) and 96% in the control and the high-dose groups (0 and 500 mg/kg bw/d). A sufficient number (approximately 20, but no fewer than 16 females with implantation sites) of pregnant females was available

Key result

Dose descriptor:

NOAEL

Effect level:

150 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

body weight and weight gain

food consumption and compound intake

Key result

Abnormalities:

no effects observed

Reduction in number of live offspring:

no effects observed

Description (incidence and severity):

The mean number of live fetuses in test groups 1-3 (50, 150 and 500 mg/kg bw/d) was comparable to the control fetuses. Any observable differences were without biological relevance.

Mean live fetuses:
Test group 0 (control): 9.7
Test group 1 (50 mg/kg bw/d): 9.1
Test group 2 (150 mg/kg bw/d): 10.0
Test group 3 (500 mg/kg bw/d): 9.2

Changes in sex ratio:

no effects observed

Description (incidence and severity):

The sex distribution of the fetuses in test groups 1-3 (50, 150 and 500 mg/kg bw/d) was comparable to the control fetuses. Any observable differences were without biological relevance.

Mean sex distribution females/males
Test group 0 (control): 4.8/4.9
Test group 1 (50 mg/kg bw/d): 4.7/4.4
Test group 2 (150 mg/kg bw/d): 5.2/4.8
Test group 3 (500 mg/kg bw/d): 4.6/4.5

Changes in litter size and weights:

no effects observed

Description (incidence and severity):

No dead fetuses was observed in any test group.

Mean litter size:
Test group 0 (control): 9.7
Test group 1 (50 mg/kg bw/d): 9.1
Test group 2 (150 mg/kg bw/d): 10.0
Test group 3 (500 mg/kg bw/d): 9.2


The mean fetal weights of test groups 1, 2 and 3 were not influenced by the test substance and did not show any biologically relevant differences in comparison to the control group.

Mean fetal weights of all viable fetuses:
Test group 0 (control): 35.7 g
Test group 1 (50 mg/kg bw/d): 36.9 g
Test group 2 (150 mg/kg bw/d): 34.9 g
Test group 3 (500 mg/kg bw/d): 33.3 g

External malformations:

no effects observed

Description (incidence and severity):

One external malformation was recorded for a control fetus (0 mg/kg bw/d) which had an associated skeletal malformation. Furthermore, one external malformation occurred in one high-dose fetus (500 mg/kg bw/d). These individual external findings are considered to be incidental.

Individual fetal external malformations:
Test group 0 (control): one female with short tail
Test group 1 (50 mg/kg bw/d): none
Test group 2 (150 mg/kg bw/d): none
Test group 3 (500 mg/kg bw/d): one female with umbilical hernia

Skeletal malformations:

no effects observed

Description (incidence and severity):

Skeletal malformations were detected in single fetuses of test groups 0, 1 and 2 (0, 50 and 150 mg/kg bw/d). One control fetus had an associated external malformation. No statistically significant differences between the test substance-treated groups and the control were noted. The overall incidences were well within the historical control range of the test facility.

Individual fetal skeletal malformations:
Test group 0 (control): one male with fused rib, one male with severely malformed vertebral column and/or ribs, one female with absent caudal vertebra
Test group 1 (50 mg/kg bw/d): one female with cleft sternum
Test group 2 (150 mg/kg bw/d): one male with lumbar hemivertebra
Test group 3 (500 mg/kg bw/d): none

Visceral malformations:

no effects observed

Description (incidence and severity):

Soft tissue malformations occurred in fetuses of test groups 1 and 2 (50 and 150 mg/kg bw/d). One female low-dose fetus had multiple visceral malformations, such as small thymus, small lung, retroesophageal aortic arch, dilated pulmonary trunk, absent subclavian, large heart, thin ventricular septum and absent gallbladder. The addition of those individual findings resulted in a slightly increased affected
fetuses per litter-incidence of soft tissue malformations in test group 1 attaining statistical significance. In absence of a dose-relationship and of a specific morphologic pattern, none of these malformations are assessed as treatment related.

Individual fetal soft tissue malformations:
Test group 0 (control): none
Test group 1 (50 mg/kg bw/d): one female with atretic aortic arch, one female with absent gallbladder, 1 female with multiple visceral malformations
Test group 2 (150 mg/kg bw/d): one male with absent gallbladder
Test group 3 (500 mg/kg bw/d): none

Other effects:

no effects observed

Description (incidence and severity):

Fetal external variations
One external variation (paw hyperflexion) was recorded in one control fetus. This finding is considered not biologically relevant, it can be found in the historical control data.


Fetal external unclassified observations
One unclassified external observation was recorded in test group 3 (500 mg/kg bw/d), i.e. placentae discolored. This isolated finding is considered to be not related to treatment.


Fetal soft tissue variations
The examinations of the soft tissues revealed malpositioned carotid branches and an absent lung lobe (Lobus inferior medialis) in all test groups including the control (0, 50, 150 and 500 mg/kg bw/d). A cystic dilatation of the brain and a dilated renal pelvis occurred in one control fetus, each.
The incidences of these variations were neither statistically significantly different from control nor dose-dependent and, therefore, considered not biologically relevant. Most of them can be found in the historical control data of the test facility at comparable incidences.

Soft tissue variations (affected fetuses/litter):
Test group 0 (control): 4.8%
Test group 1 (50 mg/kg bw/d): 3.0%
Test group 2 (150 mg/kg bw/d): 2.3%
Test group 3 (500 mg/kg bw/d): 3.3%


Fetal soft tissue unclassified observations
Two unclassified soft tissue observations were recorded: a discolored spleen was seen in one single fetus of test group 3 (500 mg/kg bw/d), furthermore, a blood coagulum around urinary bladder was recorded in twelve control, six low-dose, three mid-dose and five high-dose fetuses. These findings were considered not biologically relevant.

Soft tissue unclassified observations (affected fetuses/litter):
Test group 0 (control): 7.8%
Test group 1 (50 mg/kg bw/d): 3.5%
Test group 2 (150 mg/kg bw/d): 1.3%
Test group 3 (500 mg/kg bw/d): 2.5%


Fetal skeletal variations
For all test groups, skeletal variations of different bone structures were observed, with or without effects on corresponding cartilages. The observed skeletal variations were related to several parts of fetal skeletons and appeared without relation to dosing in the majority of cases. The overall affected fetuses/litter incidence of skeletal variations was statistically significantly increased in test group 3 (500 mg/kg bw/d), while the litter incidence was the same as for control. However, in all test groups, the overall incidence of skeletal variations was covered by the historical control range (mean%: 91.3 [83.2 - 95.4]).

Fetal skeletal variations (affected fetuses/litter):
Test group 0 (control): 88.5%
Test group 1 (50 mg/kg bw/d): 85.5%
Test group 2 (150 mg/kg bw/d): 85.3%
Test group 3 (500 mg/kg bw/d): 95.4%


Fetal skeletal unclassified cartilage observations
Some isolated cartilage findings without impact on the respective bone structures, which were designated as unclassified cartilage observations, occurred in all test groups. The observed unclassified cartilage findings were related to the vertebral column, sternum and the ribs and did not show any relation to dosing. Therefore, they are assessed as not treatment-related.

Unclassified cartilage observations (affected fetuses/litter):
Test group 0 (control): 8.4%
Test group 1 (50 mg/kg bw/d): 4.6%
Test group 2 (150 mg/kg bw/d): 9.8%
Test group 3 (500 mg/kg bw/d): 6.1%

Key result

Dose descriptor:

NOAEL

Effect level:

500 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects up to the highest dose tested

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

no

Only pregnant does were used for the calculations of mean maternal food consumption, body weight and body weight change. Only pregnant does with scheduled sacrifice (GD 29) were used for the calculation of mean gravid uterine weights, mean organ weights, corrected (net) body weight gain and summary of reproduction data.
The following females were excluded from the above-mentioned calculations:
Test group 0 (0 mg/kg bw/d):
• female No. 21 - not pregnant
Test group 1 (50 mg/kg bw/d):
• females Nos. 26, 28, 44 - not pregnant
• female No. 40 - sacrificed after abortion
Test group 2 (150 mg/kg bw/d):
• females Nos. 56, 59, 64, 71 - not pregnant
• female No. 51 - sacrificed after abortion
Test group 3 (500 mg/kg bw/d):
• female No. 76 - not pregnant
• female No. 85 - sacrificed after abortion

 

There were noted external, soft tissue and skeletal malformations in all test groups (0, 50, 150 or 500 mg/kg bw/d). The distribution of total malformations about the groups was not related to dose.

A spontaneous origin is assumed for the single external variation, four soft tissue variations and the majority of skeletal variations which were observed in all test groups including controls.

If all different types of variations are summarized, the incidence of total fetal variations is increased in test group 3 attaining statistical significance. However, the high-dose incidence covered by the historical control range (mean%: 91.6 [83.6 - 95.4]). The apparent increase in the high-dose group is driven by a higher incidence of skeletal variations, which is, in turn, increased by two variants discussed below.

A spontaneous origin is assumed for the unclassified external, unclassified soft tissue and unclassified skeletal cartilage observations which were observed in several fetuses of test groups 0, 1, 2 and 3. The distribution and type of these findings do not suggest any relation to treatment.

Finally, fetal examinations revealed that there is no adverse effect of the compound on the respective morphological structures up to the highest dose tested (500 mg/kg bw/d).

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Toxicity to reproduction: other studies

Additional information

First species:

Methylionon 70 (EC 942-741-0) was tested for its prenatal developmental toxicity in New Zealand White rabbits according to OECD 414 (BASF, 2021). The test substance was administered as an aqueous suspension to groups of 25 inseminated female New Zealand White rabbits orally by gavage in doses of 50, 150 and 500 mg/kg body weight/day (mg/kg bw/d) on gestation days (GD) 6 through 28. The vehicle control group, consisting of 25 females, was dosed with the vehicle (0.5% Sodium carboxymethyl cellulose [CMC] suspension in deionized water (with 10 mg/100 mL Cremophor EL) in parallel.

At terminal sacrifice on GD 29, 20-24 females per group had implantation sites.

Food consumption and body weight of the animals were recorded regularly throughout the study period. The state of health of the animals was checked each day. On GD 29, all females were sacrificed and assessed by gross pathology (including weight determinations of the unopened uterus and placentas). For each doe, corpora lutea were counted and number and distribution of implantation sites (differentiated between resorptions, live and dead fetuses) were determined. The fetuses were removed from the uterus, sexed, weighed and further investigated for any external, soft tissue and skeletal (inclusive cartilage) findings.

The following test substance-related adverse effects/findings were noted:

Test group 3 (500 mg/kg bw/d):

Does

• Increased number of rabbits with ‘no defecation’ (6 vs. 2 in control) and ‘reduced defecation’ (16 vs. 5 in control),


• Reduced food consumption during major parts of the gestation period (up to 61% below control); during the entire treatment period (GD 6-28) the rabbits consumed 25% less food than the control,


• Reduced body weight and body weight gain; during the entire treatment period (GD 6-28) the rabbits gained 77% less body weight than the control,


• Lower carcass weights (-5% in comparison to the control) and corrected (net) body weight gain (-366.9 g vs. -199.7 g in control).

Fetuses

• No test substance-related adverse effects on fetuses.

Test group 2 (150 mg/kg bw/d) and Test group 1 (50 mg/kg bw/d):

• No test substance-related adverse effects on does, gestational parameters or fetuses.

Under the conditions of this prenatal developmental toxicity study, the oral administration of Methylionon 70 to pregnant New Zealand White rabbits from implantation to one day prior to the expected day of parturition (GD 6-28) caused evidence of maternal toxicity at a dose of 500 mg/kg bw/d, such as reduced or no defecation, reduced food consumption as well as reduced gross and net body weight/weight gain.

In conclusion, the no observed adverse effect level (NOAEL) for maternal toxicity is 150 mg/kg bw/d.

The no observed adverse effect level (NOAEL) for prenatal developmental toxicity is 500 mg/kg bw/d, the highest dose tested.

 

 

In addition to support the read across approach (for more information please refer to the read-across justification), information on prenatal developmental toxicity in the rabbit is available for the structural similar compound β-Ionone. In a study performed according to OECD Guideline 414 and GLP (BASF SE, 2014) 22 female New Zealand White rabbits/group received Beta Ionon R by inclusion in the diet on day 6 through day 29 post coitum (p.c.) at target dose levels of 50, 200 and 1000 mg/kg bw/day (Groups 2, 3 and 4, respectively). The same diet without the test substance was administered to rabbits of the control group. Group 4 showed severely reduced food intake after introduction of the test diet. As no recovery occurred up to Day 10 post-coitum, it was decided to stop treatment, remove the high dose group from the study without further examination and to add an extra group of 22 mated females treated at a target dose level of 17 mg/kg bw/day (Group 5). On day 29 p.c., all animals were sacrificed and subjected to an external, thoracic and abdominal examination as well as a laparo hysterectomy. The uteri, placentae and ovaries were examined, and the numbers of fetuses, early and late resorptions, total implantations and corpora lutea were recorded. Gravid uterine weights were recorded, and corrected body weights (changes) were calculated. The fetuses were weighed, examined for external and visceral malformations and developmental variations, and sexed (by internal examination). Soft cephalic tissue examinations were done for approximately half of the fetuses of all groups and skeletal examinations were performed for all fetuses.

Following dietary treatment at 200 mg/kg bw/day, reduced food consumption (absolute and relative to body weight) were noted during the entire treatment period (days 6-29 p.c.), except for the period from days 10-14 p.c. during which temporally recovery towards normal values was noted. In addition, reduced body weights, lower body weight gain and/or body weight loss were recorded. Body weight gain between days 6 and 19 p.c. was only 42 g at 200 mg/kg bw/day, compared to 335 g in controls. Furthermore, several animals had reduced faeces production. Although the latter finding was observed in all groups, including control animals, there was a trend towards a longer period of reduced faeces production following dietary treatment at 200 mg/kg bw/day as compared to the lower dose levels. The treatment did not result in mortality or gross findings at necropsy. No maternal toxicity was observed in the 17 and 50 mg/kg bw/day groups.

Fetal body weights were slightly lower in males and females at 200 mg/kg bw/day, reaching statistical significance for males only. This change was considered to be secondary to the reduced food intake and markedly decreased body weight gain of the dams. No toxicologically relevant effects on viability, litter size or sex ratio were noted up to 200 mg/kg bw/day. There were no fetal morphology findings (external, visceral and skeletal) up to 200 mg/kg bw/day that were considered to be toxicologically relevant. The incidence of unossified metacarpals and/or metatarsals was slightly higher in the fetuses at 200 mg/kg bw/day, reflecting the slightly lower fetal weights observed in this dose group.

Based on the results in this prenatal developmental toxicity study the maternal and developmental No Observed Adverse Effect Level (NOAEL) for Beta Ionon R was established as being 50 mg/kg bw/day.

 

 

Second species:

As stated in the Decision on a Compliance Check (decision number: CCH-D-2114359360-53-01/F) the registrant is reminded “that before performing a pre-natal developmental toxicity study in a second species you must consider the specific adaptation possibilities of Annex X, Section 8.7.2., column 2 and general adaptation possibilities of Annex XI. If the results of the test in the first species enable such adaptation, testing in the second species should be omitted and the registration dossier should be updated containing the corresponding adaptation statement.” Therefore, all available data on developmental toxicity on the registered substance, on single constituents of the reaction mass as well as on structural similar compounds have to be taken into account for the purpose of classification and labelling and risk assessment. This procedure enables a final decision on further animal testing with the registered substance in terms of additional information for hazard or risk assessment needed.

 

Relevant information on prenatal developmental toxicity in the rat is available for the structural similar compound β-ionone (for more information please refer to the read-across justification).

Developmental toxicity was evaluated in a study with β-ionone (CAS 79-77-6) performed according to OECD Guideline 414 (BASF AG, 2004). The test substance was administered as a solution in olive oil to 25 time-mated female Wistar rats/group by stomach tube at doses of 25, 100 and 400 mg/kg bw on day 6 through day 19 post coitum (pc). On day 20 post coitum, all females were sacrificed and assessed by gross pathology including the uterus and the placentae where corpora lutea were counted and number and distribution of implantation sites (differentiated as resorptions, live and dead fetuses) were determined. The fetuses were removed from the uterus, sexed, weighed, and further investigated for any external findings. Thereafter, nearly one half of the fetuses of each litter were examined for soft tissue findings and the remaining fetuses for skeletal findings. As a result, administration of 400 mg/kg bw/d elicited substance-induced effects on the dams including signs of maternal toxicity like reduced body weight gain (-29%). The dosage of 100 mg/kg bw/d resulted in some substance-related findings (i.e. temporary salivation, marginally increased liver weights), which are not considered to be adverse, but mirror some adaptive responses of the animals. At the low dose (25 mg/kg bw/d) no substance-induced effects on the dams occurred.

The test substance β-ionone  had no influence on gestational parameters and induced no adverse signs of developmental toxicity up to and including the high dose level (400 mg/kg bw/d); especially, no indications of teratogenic effects occurred which could be causally related to the test substance administration. Based on these results, the no observed adverse effect level (NOAEL) for maternal toxicity is 100 mg/kg bw/d. The NOAEL for prenatal developmental toxicity could be fixed at 400 mg/kg bw/d.

 

 

As furthermore stated in the Decision on a Compliance Check (decision number: CCH-D-2114359360-53-01/F), “ECHA considers that the extended one-generation reproductive toxicity study provides relevant information on two aspects, namely on sexual function and fertility in P1 and F1 generations (further referred to as “sexual function and fertility”) and on developmental toxicity observable peri- and postnatally in F1 generation (further referred to as “post-natal developmental toxicity”)”. Therefore, results of the ongoing OECD TG 443 (rat) with the registered substance has to be taken into account for the assessment of developmental toxicity.

In an EOGRTS according to OECD test guideline No. 443 and OECD Principles of Good Laboratory Practice (BASF SE, 2022, 90R0397/09C165) Methylionon 70 was administered to groups of 25 male and 25 female healthy young Wistar rats (F0 parental generation) as an aqueous preparation by stomach tube at dose levels of 0, 40, 150 and 600 mg/kg bw/day. F0 animals were treated at least for 75 days prior to mating to produce a litter (F1 generation). Mating pairs were from the same dose group. Pups of the F1 litter were selected (F1 rearing animals) and assigned to 2 different cohorts (1A and 1B) which were subjected to specific postweaning examinations. Groups of 25 males and 25 females, selected from F1 pups to become F1B parental generation, were treated with the test substance at dosages of 0; 40; 150 and 600 mg/kg body weight/day post weaning, and the breeding program was repeated to produce F2 litter. The study was terminated with the terminal sacrifice of the F2 weanlings and F1 adult animals. Control parental animals were dosed daily with the vehicle (0.5% CMC suspension in deionized water with 10 mg/100mL Cremophor).

Animals were assessed for their state of health, detailed clinical signs, body weights, food consumption, clinical pathological investigations (including thyroid hormone measurements) and urinalysis. Mating and reproductive performances were investigated in F0 and F1 animals and estrous cycling or sperm parameters were assessed. The offspring was sexed, monitored for their viability, body weights and the presence of nipple/areola anlagen and their sexual maturation was followed via vaginal opening and balanopreputial separation. All F0 parental and cohort 1A animals were assessed by gross pathology (including weight determinations of several organs) and subjected to an extensive histopathological examination; special attention being paid to the organs of the reproductive system. A quantitative assessment of primordial and growing follicles in the ovaries was performed for all control and high-dose F1 rearing females of cohort 1A. All F1 rearing animals were assessed by pathological examinations. 

For details on general and systemic toxicity in F0 and F1 parental animals please refer to the section "Toxicity to reproduction". 

Mating performance, pre-coital interval, pregnancy, gestation length, parturition, litter size and postnatal survival of the pups were not affected by the administration of the test compound in the F0 and F1B generation. Body weights were slightly decreased in high-dose F1 and F2 pre-weaning offspring, the effect being up to approximately 10% at PND 14. Anogenital distance was not affected by the administration of the test compound in pups of cohorts F0 and F1 B. Numbers of retained nipples / areolae were higher in F1 pups of the mid and high dose groups, but the values were within the historical control range. Additionally, there was an obvious negative correlation between the litter weight on PND 14 and the number of observed nipples / areola anlagen. Thus, the observed differences are considered rather to be a consequence of delayed body weight development than to represent an antiandrogenic action of the test compound. This is also strengthened by the absence of any nipple / areola anlagen on PND 20. Nipple / areola anlagen in the F2 offspring were comparable among groups. No effects on vaginal opening were observed. Preputial separation was slightly later in male offspring at the high dose group (one day). However, the animals of this dose group reached sexual maturity even at a slightly lower body weight when compared to controls. Therefore, the difference in timing of preputial separation is considered not to be a delay in commencement of sexual maturity, but due to lower postnatal body weight development. Additionally, all values were well within the historical control range.

Regarding pathology in F1-generation, rearing animals, cohort 1B, treatment related findings were noted in the liver of male and female animals. The significantly decreased terminal body weight in test group 13 males was regarded as treatment related.

The decreased terminal body weight in female pups was regarded as treatment related. No other treatment related findings were noted in PND 22 pups.

In conclusion, under the conditions of the present study the NOAEL (no observed adverse effect level) for general and systemic toxicity is 40 mg/kg bw/d for the F0 and 150 mg/kg bw/d for the F1 parental rats, based on reduced body weights in males at 600 mg/kg bw/d as well as clinical pathology and histopathological findings observed at 150 mg/kg bw/d (F0) and 600 mg/kg bw/d (F1).

The NOAEL for developmental toxicity in the F1 and F2 progeny is 150 mg/kg bw/d, based on reduced body weights of the offspring during lactation at 600 mg/kg bw/d.

 

The registered substance (EC 942-741-0) is a multi-constituent substance consisting approx. 67% of the main constituent of α-iso-methylionone [3-methyl-4-(2,6,6-trimethylcyclohex-2-en-1-yl)but-3-en-2-one, EC 204-846-3, CAS 127-51-5]. Therefore, available data on this single constituent are taken into account to access the hazard and risk properties of the registered substance.

In a GLP-study according to FDA guideline alpha-iso-methylionone (CAS 127-51-5) was evaluated for developmental toxicity in Sprague-Dawley rats (Politano 2007). One hundred presumed-pregnant rats were randomly assigned to four dose groups (25/group). The rats were dosed orally (gavage) at 0, 3, 10, or 30 mg/kg/day alpha-iso-methylionone on days 7-17 of gestation (GDs 7 through 17). The dosage volume was 10 ml/kg and the vehicle was corn oil. Rats were observed for viability, clinical signs, abortions, premature delivery, deaths, body weights, and feed consumption. On GD 21, all rats were Caesarean-sectioned and a gross necropsy was performed on the thoracic, abdominal and pelvic viscera. Pregnancy occurred in 21-25 rats in each dose group. Uteri were examined for number and distribution of implantations, live and dead fetuses, and early and late resorptions. Numbers of corpora lutea were also recorded. Fetuses were weighed and examined for gender and gross external changes. Approximately one-half of the fetuses in each litter were examined for soft tissue alterations and the remaining for skeletal alterations. No deaths occurred. No treatment-related clinical signs were observed. Feed consumption, body weight gains, gross tissue changes at necropsy, and Caesarean-section and litter parameters, as well as fetal developmental morphology, were unaffected by dosages of alpha-iso-methylionone as high as 30 mg/kg/day. Based on these data, maternal and developmental no-observable-adverse-effect-levels (NOAELs) of more than 30 mg/kg/day were established for alpha-iso-methylionone.

 

 

Further information on prenatal developmental toxicity in the rat is available for the structural similar compound pseudo-ionone (for more information please refer to the read-across justification).

Developmental toxicity was analyzed in a One-generation reproduction toxicity study with pseudo-ionone performed under GLP according to OECD guideline 415 (Beekhuizen, 2003). 96 male and 96 female Wistar rats were randomly assigned to four treatment groups which were administered by gavage with 0, 40, 120 or 360 mg/kg bw/d pseudoionone in maize/corn oil. Exposure length was 11 weeks prior to mating up to termination for males giving the mean of 106 days, with a range from 104 to 108 days. The females were exposed for 2 weeks prior to mating up to termination; the mean duration of treatment was 60 days, with a range of 36 to 65 days. The offspring was not treated. Females were paired one-to-one with males from the same group and allow give birth normally. Pups were killed either at adjusting litters on day 4 post-partum or at the end of the study at day 21 post-partum. They were sexed and externally examined. Although the mean bodyweights of the pups in the 120 and 360 mg/kg bw/d groups, were slightly but significantly reduced (90.6% respect­ively 95.6% of concurrent controls), these values were within the range of historical data, so that this finding was not toxicologically relevant. The number of dead and living pups at first litter check, of living pups on day 4 post-partum, of breeding losses during days 5–21 post-partum, of living pups on day 21 post-partum and the weaning index were similar for control and all treated groups. However, postnatal deaths were significantly increased at 360 mg/kg bw/d during days 0–4 post-partum, due to which the viability index was decreased in this group (91.0 compared to 96.6 control). Thus, the reproductive toxicity NOAEL was set at 120 mg/kg bw/d. The development of the pups itself was unaffected by treatment up to 360 mg/kg bw/d and no teratogenic malformations are reported. Hence, the developmental NOEL was set at 360 mg/kg bw/d.

 

 

Additional in silico information on toxicokinetics show that ionones can be divided into two groups - α-ionones and β-ionones - with high structural and metabolic similarity within these groups (for details please refer to the attached document and read across document).

 

 

Specific adaptation possibilities of Annex X, Section 8.7.2., column 2 and general adaptation possibilities of Annex XI have to be considered before performing a pre-natal developmental toxicity study in a second species with the registered substance. The available results on animal testing summarized above are adequate and reliable for the purpose of classification and labelling and risk assessment, have adequate and reliable coverage of the key parameters addressed in the corresponding test method referred to in Article 13(3), cover an exposure duration comparable to or longer than the corresponding test method referred to in Article 13(3), and adequate and reliable documentation of the applied method is provided. Therefore, the available data enable such adaptation and testing in the second species has to be omitted.

 

No additional information on a Pre-natal developmental toxicity study (OECD TG 414) in a second species (rat) is needed to conclude on the developmental toxicity of the registered substance. For more information please refer to the read-across justification.

Taken together all available information on the reaction mass (EC 942-741-0) and structural similar substances, the registered substance had no influence on gestational parameters and did not induce adverse signs of developmental toxicity or teratogenic effects at dose levels tested in two species (i.e. the rabbit and the rat). It is not anticipated that further animal tests would have an impact on the hazard or risk profile of the registered substance.

Justification for classification or non-classification

The present data on reproductive toxicity do not fulfill the criteria laid down in Regulation (EC) No. 1272/2008, and therefore, a non-classification is warranted.