Registration Dossier

Administrative data

Key value for chemical safety assessment

Toxic effect type:
dose-dependent

Effects on fertility

Description of key information

In a supporting study according OECD 422 the substance administered by oral gavage to Wistar rats resulted in signs of systemic toxicity at the highest dose of 15 mg/kg bw/d such as a reduction in food consumption and decrease in body weight (change), adverse changes in clinical pathology parameters and histopathology. No test-item related effects on oestrus cycle, sperm measures and reproductive performance were observed and therefore, the NOAEL for reproductive performance and fertility was set to 15 mg/kg bw/d for male and female Wistar rats. Furthermore, no treatment-related effects on the foetuses up to PND 13 were detected and the NOAEL for developmental toxicity was 15 mg/kg bw/d, the highest dose tested.

In the key study, an extended 1-generation reproduction toxicity study (OECD 443) the NOAEL (no observed adverse effect level) for general, systemic toxicity is the low dose of 1.5 mg/kg bw/d for the F0 and F1 animals. This was based on treatment-related, adverse effects such as a reduction in water and food consumption, decrease in body weight (change), altered clinical pathology parameters as well as histopathological changes in several organs, which were observed at the high- and mid-dose of 15 and 5 mg/kg bw/d. The NOAEL for fertility and reproductive performance for the F0 parental rats is 1.5 mg/kg bw/d, the lowest dose tested. This was based on the lower mean number of implantation sites and secondary decreased mean number of F1 pups delivered per dam in the high- and mid-dose groups. The NOAEL for developmental toxicity in the offspring is the mid-dose of 5 mg/kg bw/d, based on the decrease in pup body weight during lactation at the high-dose level of 15 mg/kg bw/d. This slight delay in development of the high-dose pups was observed in presence of maternal toxicity and, therefore, not assessed as independent effect. The NOAEL for developmental neurotoxicity in the F1 progeny is 15 mg/kg bw/d, the highest dose tested. Neuropathological findings observed in Cohort 2A adults of high- and mid-dose indicated a systemic, direct toxicity of the test substance and were not assessed as developmental neurotoxicological effects. The NOAEL for developmental immunotoxicity for the F1 progeny is 15 mg/kg bw/d, the highest dose tested. Lower mean and median anti-SRBC IgM antibody titers of the positive control group (4.5 mg/kg bw/d cyclophosphamide, oral) demonstrated that the test system worked properly.

Link to relevant study records

Referenceopen allclose all

Endpoint:
extended one-generation reproductive toxicity - with both developmental neuro- and immunotoxicity (Cohorts 1A, 1B without extension, 2A, 2B, and 3)
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:
OECD Guideline 443 (Extended One-Generation Reproductive Toxicity Study)
Version / remarks:
25 June 2018
GLP compliance:
yes (incl. certificate)
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 :
After an acclimatization period of at least 5 days, the F0 animals, with the exception of the controls, will receive the test substance daily by gavage according to the time schedule (exception: no administration to animals being in labor) for approximately 10 weeks prior to breeding and continuing through breeding (up to two weeks), and for a maximum of 6 post-mating weeks (males) or gestation (three weeks) and lactation (three weeks) for females.

- Basis for dose level selection :
Results from OECD TG 408 and 422 studies.

- Inclusion of developmental neurotoxicity Cohorts 2A and 2B : Yes

- Inclusion of developmental immunotoxicity Cohort 3 : Yes

- Route of administration : oral (gavage)

Specific details on test material used for the study:
Name of test substance: 2,2’-dimethyl-4,4’-methylenebis(cyclohexylamine)
Test substance No.: 00/0695-4
Batch identification: 69518616K0
Purity: 100 area-% (complex mixture of isomers)
Identity: Confirmed
Homogeneity: Given
Storage stability: Expiry date: 15 Jul 2019
The stability of the test substance under storage conditions over the test period was guaranteed by the sponsor, and the sponsor holds this responsibility.
Species:
rat
Strain:
Wistar
Remarks:
Crl:WI(Han)
Details on species / strain selection:
The rat is the preferred animal species for developmental and reproductive toxicity studies according to the various test guidelines. This Wistar rat strain (Crl:WI(Han)) is selected because extensive historical control data is available for these rats.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH
- Females nulliparous and non-pregnant: yes
- Age at study initiation: about 5 weeks
- Housing: Polysulfonate cages Typ 2000P
Exceptions:
From delivery to randomization (F0 animals), during mating, gestation, lactation, females after weaning, for functional observational battery and motor activity measurements: Polycarbonate cages type III
Polycarbonate cages : 1 animal (Exceptions during mating: 1 male/1 female per cage and during rearing up to PND 21/22: 1 dam with her litter)
- Diet: Ground Kliba maintenance diet mouse/rat “GLP”, Provimi Kliba SA, Kaiseraugst, Switzerland; ad libitum
- Water: Drinking water ad libitum
- 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
Route of administration:
oral: gavage
Vehicle:
CMC (carboxymethyl cellulose)
Remarks:
0.5% CMC suspension in drinking water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
For the test substance preparation, the specific amount of test substance will be weighed, topped up with 0.5% Sodium carboxymethyl cellulose (CMC) suspension in drinking water in a calibrated beaker and intensely mixed with a magnetic stirrer.
Before and during administration, the preparations will be kept homogeneous with a magnetic stirrer.

VEHICLE
- Justification for use and choice of vehicle: The test item is soluble in 0.5% CMC suspension in drinking water.
- Concentration in vehicle: 0.015 g/100 mL, 0.05 g/100 mL, 0.15 g/100 mL
- Amount of vehicle: 10 mL/kg bw/day
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: 14 days
- Proof of pregnancy: sperm in vaginal smear referred to as Day 0 of pregnancy
- After successful mating each pregnant female was caged in Polycarbonate cages (1 dam with her litter)
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The stability of the test substance in 0.5% CMC suspension in drinking water at room temperature over a period of 7 days had been verified prior to the start of the study in a similar batch.
Duration of treatment / exposure:
The F0 animals, with the exception of the controls, will receive the test substance daily by gavage according to the time schedule (exception: no administration to animals being in labor) for approximately 10 weeks prior to breeding and continuing through breeding (up to two weeks), and for a maximum of 6 post-mating weeks (males) or gestation (three weeks) and lactation (three weeks) for females. Selected F1 offspring (cohorts 1A, 1B, 2A, 3) will receive the test substance daily by gavage from PND 21 until one day before sacrifice.
Frequency of treatment:
Once daily
Details on study schedule:
F0 generation parental animals and F1 pups:
Male and female rats, aged about 4 weeks when supplied, were used as F0 generation parental animals. After an acclimatization period of at least 5 days, these rats were kept for at least 10 weeks. Then the F0 animals were paired. The female F0 animals were allowed to deliver and rear their pups (F1 generation pups) until postnatal days (PND) 4 or 21 or 22 (depending on the cohort). The male F0 generation parental animals were sacrificed during rearing. The female F0 generation parental animals were sacrificed after weaning of the F1 generation pups.
F1 pups and selection of cohorts:
Before weaning of the F1 generation pups on PND 21, 75 males and 75 females per group were randomly selected. Obvious runts (those pups whose body weight is equal to or greater than 25% below the mean body weight of the control group, separate for sexes) ere not included.
Cohorts:
Cohort 1A: One male and one female/litter (20/sex/group)
Cohort 1B: One male and one female/litter (25/sex/group)
Cohort 2A: One male or one female/litter (10/sex/group)
Cohort 2B: One male or one female/litter (10/sex/group)
Cohort 3: One male or one female/litter (10/sex/group)

Selected F1 offspring (except cohort 2B) received the test substance daily by gavage until one day before sacrifice. In addition, 10 male and 10 female pups were randomly selected from the control group to build test group 14 (positive control group).
Dose / conc.:
1.5 mg/kg bw/day (nominal)
Dose / conc.:
5 mg/kg bw/day (nominal)
Dose / conc.:
15 mg/kg bw/day (nominal)
No. of animals per sex per dose:
F0: 25/sex/group
Cohort 1A: 20/sex/group
Cohort 1B: 25/sex/group
Cohort 2A: 10/sex/group
Cohort 2B: 10/sex/group
Cohort 3: 10/sex/group
Control animals:
yes, concurrent vehicle
Positive control:
Yes, Cyclophosphamide monohydrate in cohort 3
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: once daily
signs of morbidity, pertinent behavioral changes and/or signs of overt toxicity

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once before the administration period (only F0 parents) on day 0 and subsequently once per week (as a rule in the morning)
- Parameters observed:
1. Abnormal behavior in handling
2. Fur
3. Skin
4. Posture
5. Salivation
6. Respiration
7. Activity/arousal level
8. Tremors
9. Convulsions
10. Abnormal movements
11. Gait abnormalities
12. Lacrimation
13. Palpebral closure
14. Exophthalmos (Protruding eyeball)
15. Assessment of the feces excreted during the examination (appearance/consistency)
16. Assessment of the urine excreted during the examination
17. Pupil size

BODY WEIGHT: Yes
- Time schedule for examinations: In general, the body weight of the male and female F0 parental animals, F1 rearing animals was determined once a week at the same time of the day (in the morning). The body weight of the F1 rearing animals was determined on the first day of test substance administration and then once a week at the same time of the day (in the morning).
The following exceptions are notable for the female parental animals:
• During the mating period of the F0 parental animals, the females were weighed on the day of positive evidence of sperm (GD 0) and on GD 7, 14 and 20.
• Females with litter were weighed on the day of parturition (PND 0) and on PND 1, 4, 7, 10, 14, 18 and 21.
Females without positive evidence of sperm, females without litter and females after weaning (PND 21/22) were weighed once a week together with the males

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
Generally, food consumption was determined once a week (over a period of 7 days) for the male and female F0 and F1 rearing animals, with the following exceptions:
• Food consumption will not be determined after the 10th premating week (male F0 animals) and during the mating period (male and female F0 animals).
• Food consumption of the F0 females with evidence of sperm was determined for GD 0-7, 7-14 and 14-20.
• Food consumption of the F0 females, which gave birth to a litter, was determined 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 and in the females without litter during lactation period.

WATER CONSUMPTION AND COMPOUND INTAKE: Yes
- Time schedule for examinations: Generally, water consumption was determined once a week (over a period of 3 days) for the male and female F0 and F1 rearing animals, with the following exceptions:
• Water consumption was not determined after the 10th premating week (male F0 animals) and during the mating period (male and female F0 parental animals).
• Water consumption of the F0 females with evidence of sperm was determined for GD 0-1, 3-4, 7-8, 10-11, 14-15, 17-18 and 19-20.
• Water consumption of the F0 females, which gave birth to a litter, was determined 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.
Oestrous cyclicity (parental animals):
For all F0 females, estrous cycle length and normality was evaluated by preparing vaginal smears during a minimum of 3 weeks prior to mating and throughout cohabitation until there is evidence of sperm in the vaginal smear.
In all cohort 1A females, vaginal smears was collected after vaginal opening until the first cornified smear (estrous) is recorded. The estrous cycle also was evaluated in cohort 1A and 1B females for 2 weeks around PND 75.
Additionally, on the day of scheduled sacrifice, the estrous status was determined in all female F0 animals and all females of cohorts 1A and 1B.
Sperm parameters (parental animals):
Parameters examined in all surviving male F0 parental generations and in all cohort 1A males:
After the organ weight determination, the following parameters were determined in the right testis or right epididymis sacrificed on schedule:
• Cauda epididymis sperm motility
• Sperm morphology
• Spermatid head count in the testis
• Sperm head count in the cauda epididymis

Initially, sperm morphology and sperm head count (cauda epididymis and testis) were evaluated for the control (00/10) and highest dose group (03/13), only.
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 10 pups/litter (5/sex/litter as nearly as possible); excess pups were killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
number and sex of pups, stillbirths, live births, body weight , physical or behavioural abnormalities, anogenital distance (AGD), presence of nipples/areolae in male pups, sexual maturity

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities; possible cause of death was/was not determined for pups born or found dead

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: Yes
Auditory startle response habituation in cohort 2A animals
On PND 24±1, the auditory startle response test was carried out in all animals of cohort 2A using the SR-LAB; STARTLE RESPONSE SYSTEM (San Diego Instruments, San Diego, CA, U.S.A.) in a randomized sequence. The examinations are started in the morning. Age-appropriate sized enclosures are used. The animals are given a 5 minute acclimation period in the response chamber with a 70 dBA background noise. Then the startle response is recorded in 50 trials at a startle stimulus sound level of 120 dBA with a 5 - 10 second variable interval between the trials. Response is recorded for 50 milliseconds. Measurement is carried out with the light and ventilator switched on in the measurement chambers; no feed or water is provided during the test. Data (maximum amplitude, latency to the peak of the response) are analyzed in 5 blocks of 10 trials each.

Functional observational battery (FOB) in cohort 2A animals
The FOB was carried out once, between PND 63-75, in all animals of cohort 2A. The examinations were generally started in the morning at about 10:00 h. The FOB was carried out in a randomized sequence. At least one hour before the start of the FOB the animals were transferred separately into polycarbonate cages (floor area about 800 cm2). Drinking water was provided ad libitum whereas no food will be offered during the measurements. The FOB was start with passive observations without disturbing the rats, followed by removal from the home cage, open field observations in a standard arena and sensory motor tests as well as reflex tests. The findings were ranked according to their degree or severity, if applicable.

The animals were observed for a short period (about 10-30 seconds) in their cages with the lids closed in the rack, while disturbing influences (touching of the cage and loud noises) are avoided.
- Parameters observed:
1. Posture
2. Tremors
3. Convulsions
4. Abnormal movements
5. Gait
6. Other findings

Open field observation
For observation, the animals were removed from their cages by the investigator and placed in a standard arena (50 × 50 × 25 cm). Besides noting other abnormalities, the following parameters were assessed:
1. Behavior on removal from the cage
2. Fur
3. Skin
4. Salivation
5. Nasal discharge
6. Lacrimation
7. Eyes/ pupil size
8. Posture
9. Palpebral closure
10. Respiration
11. Tremors
12. Convulsions
13. Abnormal movements/ stereotypes
14. Gait
15. Activity/ arousal level
16. Feces (consistency/color) excreted during the examination (2 minutes)
17. Urine (amount/color) excreted during the examination (2 minutes)
18. Rearing within 2 minutes
19. Other findings

Sensory-motoric test/Reflexes
The animals were removed from the open field and were subjected to the sensory motor and reflex tests listed below:
1. Reaction to an object being moved towards the face (Approach response)
2. Touch sensitivity (Touch response)
3. Vision (Visual placing response)
4. Pupillary reflex
5. Pinna reflex
6. Audition (Startle response)
7. Coordination of movements (Righting response)
8. Behavior during handling
9. Vocalization
10. Pain perception (Tail pinch)
11. Other findings
12. Grip strength of forelimbs
13. Grip strength of hindlimbs
14. Landing foot-splay test

Motor activity measurement in cohort 2A animals
The measurement of motor activity (MA) were carried out between PND 63-75, in all animals of cohort 2A, on the same day when FOB is conducted. The examinations were performed using the TSE Labmaster System supplied by TSE Systems GmbH, Bad Homburg, Germany. The animals were measured in individual clean polycarbonate cages with a small amount of bedding in randomized order (so that each session included males and females from different dose levels). Each cage is equipped with two sensor rings, the lower ring with 18 light beams and the upper ring (for counting of rearings) with 12 light beams. The number of beam interrupts and the rearing frequency were determined over 12 intervals, each lasting 5 minutes. On the respective testing days the measurement sessions will always be started at about 14:00 h, the individual starting time is staggered by the time needed to place the animals in the cages. Test sessions are one hour long for each animal and begin when the 1st beam is interrupted. No food or water was offered to the animals during these measurements. After the transfer of the last animal into the session, the measurement room is darkened.

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: Yes
Splenic lymphocyte subpopulation analysis
Ten males and females per group of cohort 1A were used to perform a splenic lymphocyte subpopulation analysis (CD4+ and CD8+ T lymphocytes, B lymphocytes, and natural killer cells) using one half of the spleen, the other half of the spleen being preserved for histopathological evaluation.

Cyclophosphamide dependent immune system response
Ten male and ten female offspring derived from test group 00 (as far as possible from different litters) will be selected at weaning to become a positive control group in this study. These animals will be treated with Cyclophosphamide monohydrate to prove the functional responsiveness of major components of the immune system of the rats against an immunosuppressant. The animals will be treated by daily oral gavage from PND 35 onwards, for about four weeks.

T-cell dependent antibody response
All males and females of cohort 3 and the positive control animals were used to assess the functional responsiveness of major components of the immune system to a T-cell dependent antigen, sheep red blood cells (SRBC). For this purpose, the Anti SRBC-IgM ELISA of Life Diagnostics Inc, West Chester, USA (cat. no. 4200-2), was performed. Each sample was diluted 1:500. SRBC-IgM concentrations outside the standard curve range was measured in a second test run with an appropriate dilution. Generally, two in-house controls were measured with each test run. The ELISA was measured with a Sunrise MTP-reader, Tecan AG, Maennedorf, Switzerland, and evaluated with the Magellan-Software of the instrument producer.

Immunization on PND 56+/-3 (in Reproduction Laboratory):
Route of administration: Intraperitoneal, using 1 mL tuberculin-syringes
Frequency of administration: twice (within one action)
Administration volume: 0.5 mL per animal, split into two portions of 0.25 mL
Six days after immunization blood samples were taken by puncturing the retrobulbar venous plexus following isoflurane anesthesia. Blood sampling and blood examinations were carried out in a randomized sequence.

BLOOD SAMPLING
Blood samples will be withdrawn from 10 surplus (culled) PND 4 offspring (as far as possible of different litters) per sex and group. PND 4 samples may be pooled per sex and litter if the available amount is not sufficient for a hormone analysis.
Blood samples will be withdrawn from 10 surplus PND 22 offspring per sex and group (as far as possible 1 male or 1 female of different litters).
The blood samples will be collected after decapitation (following isoflurane anesthesia).

HORMONE EVALUATIONS
The following hormones were determined in the serum samples:
1. T4 (thyroxine)
2. TSH
Postmortem examinations (parental animals):
SACRIFICE
All F0 parental animals and all cohort 1A and 1B animals were sacrificed by decapitation under isoflurane anesthesia. The exsanguinated animals were necropsied and assessed by gross pathology

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.

HISTOPATHOLOGY / ORGAN WEIGHTS (F0 and cohort 1A)
The following weights were determined in all animals sacrificed on schedule:
1. Anesthetized animals
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 gland (fixed)
16. Spleen
17. Thymus (fixed)
18. Thyroid glands (with parathyroid glands) (fixed)
19. Uterus (with cervix)
All paired organs were weighted together (left and right).

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

The left testis and left epididymis of all male F0 parental and cohort 1A animals sacrificed at scheduled dates were fixed in modified Davidson’s solution, whereas the right testis and epididymis were used for sperm parameters.
In case of macroscopic findings in the right testis or right epididymis, this testis as well as the corresponding epididymis were fixed for histopathological examination and the left testis and epididymis were used for sperm analysis.
For technical reasons, after about 24 hours fixation the ovaries of F0 and cohort 1A females of all test groups will be transferred to 70% ethanol.
The uteri of all cohabited female F0 parental animals will be 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 (1)). Then the uteri were rinsed carefully in physiologic salt solution (0.9 % NaCl).
Spleens of 10 animals per sex per group of cohort 1A were split in two comparable parts (transversally). One part of the spleen was fixed in 4% buffered formaldehyde and afterwards was embedded in paraplast. The other part of the spleen was frozen at -80°C, being used to perform a splenic lymphocyte subpopulation analysis (CD4+ and CD8+ T lymphocytes, B lymphocytes, and natural killer cells).

Reproductive organs of all F0 parental animals suspected of reduced fertility, or for which estrous cyclicity or sperm quality were affected, have been subjected to histopathological investigation. Organs demonstrating potential treatment–related changes were examined in the lower dose groups. A differential ovarian follicle count (DOFC) was conducted in test groups 10 and 13 (cohort 1A females) according to Plowchalk et.al.

CLINICAL PATHOLOGY
Clinical Pathology in F0 parental and cohort 1A animals
Samples were withdrawn from the first 10 surviving F0 parental (females with litter, corresponding males) and the first 10 surviving 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 ere individually transferred into metabolism cages (no food or drinking water provided); on the following morning, the individual urine specimens were examined in a randomized sequence (the list of randomization instructions was compiled with a computer).
The following parameters were examined in all animals:
Hematology:
1. Leukocytes
2. Erythrocytes
3. Hemoglobin
4. Hematocrit
5. Mean corpuscular volume (MCV)
6. Mean corpuscular hemoglobin (MCH)
7. Mean corpuscular hemoglobin concentration (MCHC)
8. Platelets
9. Differential blood count
10. Reticulocytes
11. Blood smear (only evaluated preparations will be archived)
12. Prothrombin time

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

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

Urinalysis
1. Volume
2. Color
3. Turbidity
4. pH value
5. Protein
6. Glucose
7. Ketones
8. Urobilinogen
9. Bilirubin
10. Blood
11. Specific gravity
12. Microscopy of sediment






Postmortem examinations (offspring):
SACRIFICE
On PND 4, as a result of standardization, selected F1 pups were sacrificed by decapitation under isoflurane anesthesia and blood was sampled for determination of serum thyroid hormone concentration. All other surplus F1 pups on PND 4 were sacrificed under isoflurane anesthesia with CO2. After sacrifice, all F1 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 werel sacrificed under isoflurane anesthesia with CO2 and were examined in the general pathology lab. The selected pups for hormone analyses were sacrificed by decapitation under isoflurane anesthesia in the pathology lab and blood were sampled for thyroid hormone analyses.

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.

HISTOPATHOLOGY / ORGAN WEIGHTS ( cohort 1B)
The following weights were determined in all animals sacrificed on schedule:
1. Anesthetized animals (terminal 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. Testes
10. Seminal vesicles including coagulating gland (fixed)
11. Uterus (with cervix)
All paired organs were weighted together (left and right).

The following organs or tissues were fixed in 4% formaldehyde solution or in modified Davidson’s solution:
1. All gross lesions
2. Adrenal glands
3. Cervix uteri
4. Coagulating glands
5. Epididymides (fixed in modified Davidson ´s solution)
6. Liver
7. Ovaries (fixed in modified Davidson´s solution)
8. Pituitary gland
9. Prostate
10. Seminal vesicles
11. Testes (fixed in modified Davidson ´s solution)
12. Uterus
13. Vagina

Reproductive organs of all cohort 1B animals for which estrous cyclicity is affected, were subjected to histopathological investigation.

HISTOPATHOLOGY / ORGAN WEIGHTS ( cohort 2A)
On postnatal day 77, cohort 2A animals were weighed and subjected to deep anesthesia (i.p. pentobarbital) and sacrificed by perfusion fixation.
SOERENSEN phosphate buffer was used as the rinsing solution, and a fixation solution according to KARNOVSKY was used as a fixative.
The perfusion fixed animals were necropsied with regard to the question of neuropathology, and the visible organs were assessed by gross pathology as accurately as is possible after a perfusion fixation. The cranial vault and the spinal cord were opened and the skin was removed from both hind extremities. In this state, the perfused animals were stored in a fixation solution according to KARNOVSKY for at least 48 hours.
Animals which die intercurrently or are sacrificed in a moribund state were necropsied as soon as possible after their death and assessed by gross pathology. Gross lesions were processed histotechnically and assessed by light microscopy.
Organ weights:
The following weights were determined (the brain will be weighed after its removal but before further preparation):
1. Brain (including olfactory bulb)
The terminal body weights were recorded to calculate the relative organ weights.
Length and width of brain
The length and maximum width of the brain was measured in all animals (length: on a line extending from the rostral end of the frontal lobe to the caudal medulla oblongata of the cerebellum; width: pituitary region).

The following organs/tissue specimens were carefully removed and examined:
1. All gross lesions
2. Brain with olfactory bulb
3. Eyes with retina and optic nerve
4. M. gastrocnemius
5. Nose (nasal cavity)
6. Pituitary gland
7. Sciatic nerve, proximal section
8. Spinal cord, cervical part (C1-C6)
9. Spinal cord, thoracic part (T5-8)
10. Spinal cord, lumbar part (L1-L4)
11. Spinal ganglia (C1-C6 [3x])
12. Spinal ganglia (L1-L4 [3x])
13. Tibial nerve (on the knee), proximal section
14. Tibial nerve (nerve branch in the lower leg muscles), distal section
15. Trigeminal ganglia
16. Root fibers, dorsal (C1-C6 and L1-L4)
17. Root fibers, ventral (C1-C6 and L1-L4)

Morphometry:
Thickness measurements of major brain layers (neocortex: frontal and parietal cortices, caudate nucleus/putamen, hippocampus, corpus callosum, cerebellum) was performed. Measurements were carried out bilaterally in the left and right halves of the brain with the exception of the corpus callosum and the cerebellum.
Selection of the planes:
• Measurements for the thickness of the neocortex, corpus callosum and caudate nucleus/putamen were carried out in a cross section which approximates the plane of section on page 88 in Sherwood and Timiras (1970 (3)).
• Measurements for the thickness of the hippocampus were carried out in a cross section which approximates the plane of section on page 110 in Sherwood and Timiras (1970 (4)).
• Measurements for the thickness of select folia of the cerebellum were carried out in a midsagittal section through the vermis of the cerebellum which approximates the plane of section on page 134 in Sherwood and Timiras (1970 (3)).
Conduct of the measurements:
• Neocortex (frontal and parietal cortices):
The width of the total cortical mantle (layers I-VI – from the surface of the pia mater to the white substance) was measured vertically to a tangent over a region of the frontal and parietal cortices determined beforehand.
• Caudate nucleus/putamen:
The largest lateral extension was measured.
• Corpus callosum:
The width was measured at the middle line of the cross section.
• Hippocampus:
The largest dorsoventral extension was measured.
• Cerebellum:
The width of a select folium (lobus vermis cerebelli No 8) was measured at the base of the folium from the secondary fissure to the prepyramidal fissure.

HISTOPATHOLOGY / ORGAN WEIGHTS ( cohort 2B) Pathological examinations of cohort 2B animals (Developmental Neuro-toxicity Cohort, weanlings):
On postnatal day 22, cohort 2B animals were weighed and subjected to deep anesthesia (i.p. pentobarbital) and sacrificed by perfusion fixation. SOERENSEN phosphate buffer was used as the rinsing solution, and neutrally buffered, 4% formaldehyde solution was used as a fixative.The perfusion fixed animals were necropsied with regard to the question of neuropathology, and the visible organs were assessed by gross pathology as accurately as is possible after a perfusion fixation. The cranial vault and the spinal cord were opened and the skin were removed from both hind extremities. In this state, the perfused animals were stored in neutrally buffered, 4% formaldehyde solution for at least 48 hours.

Organ weights
The following weights were determined (the brain will be weighed after its removal but before further preparation):
1. Brain (including olfactory bulb)
The terminal body weights were recorded to calculate the relative organ weights.

Length and width of brain
The length and maximum width of the brain was measured in all animals (length: on a line extending from the rostral end of the frontal lobe to the caudal medulla oblongata of the cerebellum; width: pituitary region).

Organ/Tissue fixation
The following organs/tissue specimens were carefully removed and processed histotechnically:
1. All gross lesions
2. Brain with olfactory bulb
3. Eyes with retina and optic nerve
4. Nose (nasal cavity)
5. Pituitary gland
6. Trigeminal ganglia

HISTOPATHOLOGY / ORGAN WEIGHTS ( cohort 3) Pathological examinations of cohort 3 animals (Immunotoxicity Cohort) and animals of the positive control
All cohort 3 animals and the animals of the positive control were sacrificed by decapitation under isoflurane anesthesia. The exsanguinated animals were necropsied and assessed by gross pathology. Animals which die intercurrently or are sacrificed in a moribund state were necropsied as soon as possible after their death and assessed by gross pathology.

Organ weights
The following weights were determined in all animals sacrificed on schedule:
1. Anesthetized animals
2. Spleen
3. Thymus (fixed)

Organ/ tissue fixation
The following organs or tissues were fixed in 4% buffered formaldehyde solution:
1. All gross lesions
2. Spleen
3. Thymus

HISTOPATHOLOGY / ORGAN WEIGHTS ( surplus F1 generation pups) Pathological examinations of surplus F1 generation pups on PND 22 (F1 weanlings not selected for cohorts)
All surplus F1 generation pups that were not used for the following organ weight determinations were sacrificed under isoflurane anesthesia with CO2. The selected pups for organ weight determination were sacrificed by decapitation under isoflurane anesthesia. All animals were necropsied and assessed by gross pathology with special emphasis on the reproductive organs.

Organ weights
The following weights were determined in up to 10 animals per sex per group sacrificed on schedule:
1. Anesthetized animals
2. Brain
3. Spleen
4. Thymus (fixed)

Organ/ Tissue fixation
The following organs or tissues of up to 10 animals per sex per group were be fixed in 4% buffered formaldehyde solution:
1. All gross lesions
2. Brain
3. Mammary gland (male and female)
4. Spleen
5. Thymus
6. Thyroid glands












Statistics:
see table 1
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Clinical observations for males and females (except gestation and lactation period)
In the high-dose group (15 mg/kg bw/d), one single high-dose male (No. 94) showed piloerection during study week 3 and transient salivation during study week 4 after the premating period. It is likely, that these temporary findings were induced by a bad taste of the test substance or local affection of the upper digestive tract. They are, however, not considered to be adverse toxicologically relevant findings.
In the high-dose group, eight high-dose females (Nos. 178, 183, 184, 190, 193, 195, 196 and 199) showed piloerection during study week 7 after the premating period. Since the finding was only transient during a short time period, it was not assessed as treatment-related and adverse.
One mid-dose female (No. 158) had a palpable mass during study weeks 6 - 7 after the premating period which is assessed as incidental and not related to treatment with the test compound. Two sperm negative control females (Nos. 101 and 110) and two sperm negative females of the high-dose group (Nos. 177 and 186) did not deliver F1 pups. This observation was not considered to be associated with the test compound.

Clinical observations for females during gestation of F1 litters
No treatment-related, adverse findings were observed in any of the the test groups.
One mid-dose female (No. 158) had a palpable mass during GD 20 – 21 and one sperm positive females of the low-dose group (No. 139 - 5 mg/kg bw/d) did not deliver F1 pups. Since the findings were not related to dose, they were not considered to be treatment-related.

Clinical observations for females and offspring during lactation of F1 litters
No treatment-related, adverse findings were observed in any of the the test groups.
One mid-dose female (No. 158) had a palpable mass during the entire lactation period.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Description (incidence):
There were no test substance-related or spontaneous mortalities in any of the groups.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Mean body weights of the high-dose F0 males were statistically significantly below the concurrent control values on premating day 28 onwards till the end of the study (up to 22%).
Mean body weights were statistically significantly below the concurrent control values for the high-dose F0 females on premating day 28 onwards till the end of the study (up to 14%) and for the mid-dose F0 females during gestation (GD 0 and 20: up to 5%) and during lactation (PND 4 – 18: 7%).
Mean body weights were comparable to the concurrent control values in the mid-dose females during the premating period and in the low-dose males and females and mid-dose males during the entire study period. In males, body weight change was statistically significantly below the concurrent control values for the high-dose group during premating days 14 - 63, 0 - 63 (79%, 22%, respectively) and study weeks 0 - 2, 3 - 4 and 0 - 4 after the premating period (up to -2.2 g vs. 8.6 g in control).
For the mid-dose males, body weight change was decreased during premating days 21 - 28, 35 - 42 and study weeks 0 - 4 after the premating period (about 13%, 17% and 18%, respectively).
Low-dose males showed only a decrease during premating days 7 - 14 (about 11%) without relation to dose. Therefore, this was assessed as incidental. Body weight change was statistically significantly below the concurrent control values for the high-dose females during premating days 0 – 7, 28 – 35, 0 – 63, GD 7 – 20 and 0 - 20 (about 15%, 47%, 18%, 23% and 11%, respectively) and for the mid-dose females during GD 14 - 20 and PND 1 - 4 (about 13% and 48%, respectively).
Body weight change was comparable to the concurrent control values in the mid-dose females during the premating period and in the low-dose females during the entire study period.
The statistically significantly increased body weight change in the mid-dose females during PND 7 - 10 and in the high-dose females during PND 14 - 18 was considered to be spontaneous in nature and not treatment-related.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Food consumption of the high-dose F0 males was statistically significantly below the concurrent control values during the premating days 21 - 42, 56 - 69 and 0 - 69 (up to 12%, 15% and 9%, respectively).
Food consumption was statistically significantly below the concurrent control values for the high-dose F0 females during premating days 28 - 35, 42 - 49; GD 7 - 20 and the entire lactation period (up to 9%, 11%, 13% and 19%, respectively).
Mid-dose females showed a reduction in food consumption during lactation only (PND 1 – 18: up to 14%).
Food consumption was comparable to the concurrent control values in the mid-dose females during the premating and gestation period and in the low-dose males and females and middose males during the entire study period.
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Description (incidence and severity):
Water consumption was affected in males and females of the F0 high- and mid-dose groups:
In F0 males, mean water consumption values of the high- and mid-dose groups were statistically significantly below the concurrent control values during premating days 21 - 66 (up to 17) and 28 - 59 (up to 12%), respectively. In F0 females, water consumption was statistically significantly below the concurrent control values for the high- and mid-dose groups during premating, gestation and laction. For the high-dose group, the reduction was up to 21% below control during premating (days 21-51, 63-66), 18% during gestation (GD 14 - 15, 19 – 20) and 23% during lactation (PND 1 - 2 and 7 – 11). Mid-dose females showed a reduction up to 18% below control during premating (days 28 - 52, 63 – 66), 16% during gestation (GD 14 - 15, 19 – 20) and 19% during lactation (PND 1 – 2).

Water consumption of the low-dose F0 males and females was comparable to the concurrent control values throughout the entire study.
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
At the end of the administration period, in males and females of test group 03 (15 mg/kg bw/d) platelet counts were significantly increased. Additionally, in males of the mentioned test group absolute and relative eosinophil counts were significantly decreased, whereas in females of test group 03 absolute and relative monocyte counts were significantly increased. These changes were regarded as treatment-related and adverse.
In males of test groups 01, 02 and 03 (1.5, 5 and 15 mg/kg bw/d) red blood cell (RBC) counts were significantly decreased and in males of test groups 02 and 03 hemoglobin and hematocrit values were significantly lower compared to controls. However, all mentioned values were not dose-dependently changed, and they were within historical control ranges (F0 males, RBC 8.03-9.04 tera/L; hemoglobin 8.6-9.5 mmol/L; hematocrit 0.406-0.438 L/L). In males of test group 02 (5 mg/kg bw/d), absolute reticulocyte counts were significantly increased but the alteration was not dose dependent. Therefore, the mentioned changes in this paragraph were regarded as incidental and not treatment related.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
At the end of the administration period, in males and females of test group 03 (15 mg/kg bw/d) aspartate aminotransferase (AST) activities and inorganic phosphate levels were significantly increased. Additionally, in males of test group 03 triglyceride values were significantly higher compared to controls. These changes were regarded as treatment-related and adverse.
The following significant changes were regarded as incidental and not treatment-related, because the values were within historical control ranges: increased AST activities in males of test group 02 (5 mg/kg bw/d); decreased creatinine and glucose values in males and females of test group 03 (15 mg/kg bw/d); increased alanine aminotransferase (ALT) activities and urea values in males of test group 03; decreased calcium values in males of test groups 01, 02 and 03 (1.5; 5 and 15 mg/kg bw/d); decreased total bilirubin values in females of test groups 02 and 03 (F0 males, AST 1.37-2.21 μkat/L; creatinine 23.4-34.8 μmol/L; glucose 5.19-6.98 mmol/L; ALT 0.56-0.89 μkat/L; urea 3.75-6.08 mmol/L; calcium 2.48-2.62 mmol/L; F0 females, creatinine 28.0-41.3 μmol/L; glucose 5.04-6.01 mmol/L; total bilirubin 1.18-2.71 μmol/L).
The following significant changes were regarded as incidental and not treatment-related, because they were not dose-dependent: decreased total protein, albumin and globulin values in males of test group 02 (5 mg/kg bw/d); decreased glucose values in females of test group 01 (1.5 mg/kg bw/d).
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
In the urine sediment of males of test group 03, increased numbers of transitional epithelial cells were observed. This finding in combination with the histopathology findings in the kidneys is regarded as treatment-related and adverse.
At the end of the administration period, in male and female rats of test group 03 (15 mg/kg bw/d) and in males of test group 02 (5 mg/kg bw/d) urine pH values were significantly decreased. This change is probably treatment-related because of the excretion of acidic metabolites of the administered compound, but it is not regarded as adverse, per se.
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):
Treatment-related findings were observed in following organs: adrenal cortex, brain, esophagus, eyes with optic nerve, glandular stomach, heart, kidneys, left epididymis, liver, lungs, axillary and mesenteric lymph nodes, pancreas, pituitary gland, seminal vesicles and skeletal muscle. The main finding in all these target organs was a “microvesicular” type of cytoplasmic vacuolation, characterized by the presence of single to multiple vacuoles (depending on the organ) ranging from very fine to small (no larger than the nuclei of the cell). If the microvesicular vacuolation was so fine that vacuoles could not be distinguished individually within the cytoplasm, additional terms referred here as “ground glass” (finest vacuoles that cannot be individually differentiated), “foamy” or “granular” (vacuoles that can hardly be differentiated) were used for a better description. The cytoplasmic microvacuolation conferred the cells a very clear or transparent aspect and also increased their size. The finest type of vacuolation was frequently seen in epithelial cells. In the skeletal muscle, the vacuoles could be always individually visualized within the cells and had a particular birefringent aspect. Large cytoplasmic vacuoles, larger than the cell nuclei and often displacing them, were referred here as “macrovesicular” type of vacuolation and were only observed in the brain (choroid plexus) and seminal vesicles.

Adrenal cortex
In male animals of test group 03, the vacuolation of the zona fasciculata showed an increase in the incidence and grading. The vacuolation was characterized by the presence of a microvesicular pattern, giving the cytoplasm of the cells a foamy and pale aspect and often causing an increased size of the cells. This effect was clearly seen in males but not in females.

Axillary lymph node
The vacuolation of the high endothelial venules (HEV) was characterized by small vacuoles in the whole cytoplasm, which gave these vessels a conspicuous transparent aspect. Males were affected from test group 02 onwards and females only in test group 03.

Brain
The vacuolation was noted in the epithelial cells of the choroid plexus and was most frequently of microvesicular type. However, with increasing grading, macrovesicular vacuoles were also observed. A very tiny content was visible in these vacuoles, most likely representing membrane residues resulting from coalescence of smaller vacuoles. The choroid plexus of the lateral and dorsal third ventricles was affected most frequently. Only males and females in test group 03 were affected, with males showing a higher grading than females.

Esophagus
In the esophagus, the vacuolation was noted in the skeletal muscle layers of the wall. This finding was characterized by the presence of very tiny, transparent to birefringent microvacuoles ranging approximately from 2 – 4 μm in diameter within the muscle fibers. Males and females of test groups 02 and 03 appeared to be equally affected.

Eyes with optic nerve
A very fine microvesicular vacuolation was seen in the retinal pigment epithelium. The affected cells had ground-glass aspect and were minimally increased in size. This change was more manifested in the peripheral areas. Only animals in test group 03 were affected, with males having a slightly higher incidence and grading than females.

Glandular stomach
A microvesicular vacuolation (small vacuoles) was observed at the base of the glands of pyloric mucosa. Within the glandular cells, the vacuolation randomly displaced the nuclei from their basal position, giving the base of the glands a disorganized and paler aspect than normal. A dose-dependent increase from test group 02 onwards was noted in males and females.

Heart
The microvesicular vacuolation affected mainly the septum and left ventricular wall. It was characterized by multiple individual small vacuoles visible within the cardiomyocytes, without altering their shape or size. This change was not associated with visible degeneration or necrosis but conferred the cells a pale and disorganized aspect. Males appeared more affected than females.

Kidneys
In the kidneys the vacuolation was observed in the medulla (tubules of the inner stripe of the outer medulla) of both sexes, whereas the degeneration and/or regeneration was found in the cortex (proximal convoluted tubules) of males only. The vacuolation in the medulla was of a very fine microvesicular type, giving the epithelial cells a “ground glass” pale and swollen aspect. The tubular degeneration/regeneration was characterized by multifocal areas of convoluted tubules with microvesicular vacuolation, loss of normal architecture due to nuclear disorganization and crowding, single pyknosis and general light basophilia.

Left epididymis
The vacuolation was localized primarily in the ducts of approximately 2/3 of the distal corpus at the transition to the caudal region but did not include the cauda. The vacuolation of the epithelial cells ranged from small microvesicular vacuoles to vacuoles as large as the nuclei. The vacuoles were always located basal and lateral to the nuclei within the cytoplasm. A dosedependent increase in incidence and grading was noted from test group 02 onwards.

Liver
The vacuolation within the hepatocytes was characterized by very small cytoplasmic vacuoles of regular size distributed around the nuclei rather than in the periphery of the hepatocyte. This pattern was quite different from the “fatty change vacuolation” pattern, which is composed of vacuoles of different size. The mixed-cell inflammation observed particularly in females in test group 03, was composed predominantly of granulocytes and lymphocytes and was localized
in centrilobular areas affected by vacuolation. Often this type of inflammation was associated with apoptosis/single cell necrosis. Multinuclear hepatocytes in females were assumed to be the result of the coalescent damaged vacuolated hepatocytes. The vacuolation of the bile duct epithelium was of a “ground-glass” type affecting the whole cell and was observed in the portal bile ducts of large caliber. Their aspect was very pale, and the size of the epithelial cells was increased.

Lungs
The vacuolation was localized in the bronchial and bronchiolar epithelium and was characterized by a “foamy” aspect. In bronchi and large bronchioles, the vacuolation was mostly occupying the cytoplasm apical to the cell nuclei, whereas in the terminal bronchioles, the vacuolation was rather basal to the cell nuclei, displacing them to the apical region. In the bronchial associated lymphoid tissue (BALT), the high endothelial venules (HEV) showed the same type of vacuolation as described for the lymph nodes, with a noticeable pale aspect.

Mesenteric lymph node
Similarly, as observed in the axillary lymph nodes, the whole cells of the high endothelial venules (HEV) showed a microvesicular vacuolation (small vacuoles), conferring these vessels a very pale aspect. Macrophage aggregates were slightly increased in test groups 02 and 03 in males.

Pancreas
The cytoplasmic vacuolation of the acinar epithelium was of microvesicular. Within the acinar cells, very small vacuoles were localized in the apical cytoplasmic border adjacent to the zymogen granules (grade 1) or extended from the apical cell border to the cell nuclei (grade 2) accompanied by a reduction of zymogen granules. The ductal vacuolation was seen in the interlobular pancreatic ducts, with ground glass appearance of the epithelial cells accompanied by increase size.

Pituitary gland
The cytoplasmic vacuolation was localized in all cell types of the pars distalis. Within the cells the vacuolation was of a very fine microvesicular type involving the whole cytoplasm giving the cells a fine “granular” and pale aspect.

Seminal vesicles
The cytoplasmic vacuolation of the epithelial cells was characterized by small vacuoles of very regular size localized at the basal part of the cells displacing the nuclei to the apical border. The cells appeared wider and the nuclei lost their regular arrangement along the epithelium/basal membrane. Some animals showed single vacuoles of macrovesicular type. The vacuolation was dose-dependent from test group 02 onwards.

Skeletal muscle
As already described for the skeletal muscle of the esophageal wall, the vacuolation of the skeletal muscle was also characterized by the presence of very tiny intracytoplasmic microvacuoles (few μm in diameter) with a birefringent aspect and a multifocal distribution pattern within the muscle fiber. In addition, degeneration and /or regeneration of single myofibers was noted. The degenerating fibers revealed slightly altered staining features (basophilic or strong hyaline stain) and variable thickness (retracted or swollen aspect). Some of these degenerated fibers showed regeneration (reparative response) characterized by numerous central nuclei within the fibers. In test group 03 vacuolation was strongly associated with degeneration/regeneration, whereas in test group 02 only few animals showed vacuolation only.

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

Fertility
The female animals (Nos. 101, 110, 139, 177 and 186) that were not pregnant and the male mating partners (Nos. 1, 39 and 77) did not show relevant microscopic findings, whereas the male mating partners (Nos. 10 and 86) exhibited severe diffuse atrophy of the testes, and aspermia, debris and cribiform change in the epididymides. These findings were consistent with the macroscopic reduced size of the respective organs and explained the impaired fertility. No correlate was found for the reduced in size of the prostate and seminal vesicles found in animal No. 86. However, the seminal vesicles displayed a moderate microvesicular vacuolation.
Histopathological findings: neoplastic:
no effects observed
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
Thyroid hormones
In F0 males of test group 03 (15 mg/kg bw/d) significantly decreased T4 values were observed. This change was neither accompanied by any alteration of the TSH values nor by any histopathological change of the thyroids. The T4 and TSH values were within the historical control range (F0 males, T4 44.65-78.17 nmol/L; TSH 4.41-9.80 μg/L). No significant change of T4 and TSH values was observed in F0 females of test group 03. Therefore, this isolated change of the T4 values in males of test group 03 was regarded as incidental and not treatment related.
Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
Estrous cycle data, generated during the last 3 weeks prior to mating to produce the F1 litter, revealed regular cycles in the females of all test groups 01-03. The mean estrous cycle duration was comparable: 3.8 / 3.9 / 3.9 and 3.9 days in test groups 01-03, respectively.
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
Concerning motility of the sperms and the incidence of abnormal sperms in the cauda epididymidis as well as sperm head counts in the testis and in the cauda epididymidis no treatment-related effects were observed.
Reproductive performance:
effects observed, treatment-related
Description (incidence and severity):
Male reproduction data
For nearly all F0 parental males, which were placed with females to generate F1 pups, copulation was confirmed. Copulation was not confirmed for control males Nos. 1 and 10 paired with control females Nos. 101 and 110, respectively and for test group 03 males Nos. 77 and 86 paired with test group 03 females Nos. 177 and 186. Thus, the male mating index was 92% in control and test group 03 and 100% in test group 01 - 02.
Fertility was proven for most of the F0 parental males within the scheduled mating interval for F1 litter.
One low-dose male (No. 39) did not generate F1 pups.
Thus, the male fertility index ranged between 92% and 100% without showing a doseresponse. This reflects the normal range of biological variation inherent in the strain of rats used for this study.
The apparently infertile male rats (Nos. 1, 39 and 77) did not show histopathological findings that could explain infertility. The male rats (Nos. 10 and 86) had exhibited testes and epididymides of reduced size. Furthermore, the prostate and seminal vesicles were found reduced in size in male No. 86.

Female reproduction and delivery data
The female mating index calculated after the mating period for F1 litter ranged between 92% and 100% without showing a dose-response.
The mean duration until copulation was detected (GD 0) varied between 2.3 and 2.8 days without any relation to dosing.
All female rats delivered pups or had implants in utero with the following exception:
• Test group 00
female No. 101 (mated with male No. 1) did not become pregnant
female No. 110 (mated with male No. 10) did not become pregnant
• Test group 01
female No. 139 (mated with male No. 39) did not become pregnant
• Test group 03
female No. 177 (mated with male No. 77) did not become pregnant
female No. 186 (mated with male No. 86) did not become pregnant

The apparently infertile female rats did not show histopathological findings that could explain infertility. The fertility index ranged between 96% and 100% without showing any relation to dosing. The mean duration of gestation was comparable in all test groups (i.e. between 21.8 and 22.0 days).
The gestation index was 100% in in all test groups 00 - 03.
The mean number of implantation sites was statistically significantly below the concurrent control values in the high-dose group (12.3 / 12.1 / 11.2 and 10.3** [**= p ≤ 0.01] implants/dam in test groups 00 - 03, respectively). The mean value of the high-dose group was outside the historical control range (mean number of implantation sites per dam: range of 11.1 – 15.3).
There were no indications for test substance-induced intrauterine embryo-/fetolethality since the postimplantation loss did not show any statistically significant differences between the groups (0.6 / 0.6 / 1.1 and 0.8 in test groups 00 - 03, respectively) The mean number of F1 pups delivered per dam (average litter size) was statistically significantly below the concurrent control values in the mid- and high-dose groups (11.7 / 11.5 / 10.1** and 9.5** pups/dam, respectively in test groups 00 - 03). Both mean values of the high and mid-dose was outside the historical control range (mean number of delivered pups per dam, range 10.3-14.9).
The rate of liveborn pups was not affected by the test substance, as indicated by live birth index of 99% / 99% / 100% and 100% in test groups 00 - 03. Moreover, the number of stillborn pups was comparable between the groups.
see executive summary
Key result
Dose descriptor:
NOAEL
Effect level:
1.5 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
food consumption and compound intake
water consumption and compound intake
haematology
clinical biochemistry
histopathology: non-neoplastic
Key result
Dose descriptor:
NOAEL
Effect level:
1.5 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
histopathology: non-neoplastic
reproductive performance
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
5 mg/kg bw/day (nominal)
Organ:
heart
kidney
lungs
lymph node
pancreas
pituitary gland
seminal vesicle
stomach
other: esophagus, skeletal muscle, left epididymidis
Treatment related:
yes
Dose response relationship:
yes
Clinical signs:
no effects observed
Description (incidence and severity):
F1 generation parental animals, Cohort 1B
No clinical signs or changes of general behavior, which may be attributed to the test substance, were detected in any of the male and female animals in any of the groups.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Description (incidence):
F1 generation parental animals, Cohort 1B
There were no test substance-related or spontaneous mortalities in any of the groups.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
F1 generation parental animals, Cohort 1B
In males, mean body weights of the high- and mid-dose groups were statistically significantly below the concurrent control values during study days 14 - 49 and 42 - 49 (up to 12% and 6%), respectively. Body weight change was statistically significantly below the concurrent control values for the high-dose males during study days 14 - 49 and 0 - 49 (up to 36% and 14%, respectively) and for the mid-dose males during study days 14 - 28, 42 - 49 and 0 - 49 (up to 12%, 13% and 8%, respectively). Low-dose males showed a decreased body weight change value during study days 28 - 35 only, without relation to dose. Therefore, it was assessed as not treatment-related.
In females, mean body weights were statistically significantly below the concurrent control values for the high-dose females during study days 35 - 49 (up to 6%). Body weight change was statistically significantly below the concurrent control values for the high-dose females during study days 7 - 14 (about 10%). Both decreased body weight changes of the mid-dose females during study days 21 - 28 (about 19%) and the low-dose females during study days 7 - 14 (about 10%) were assessed as incidental and not treatment-related since they were not related to dose and occurred only during a short time period.
Mean body weight(s) (changes) were comparable to the concurrent control values in the low-dose males and low- and mid-dose females during the entire study.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
F1 generation parental animals, Cohort 1B
Food consumption of the high-dose males was statistically significantly below the concurrent control values during study days 42 - 49 (about 12%). High-dose females showed no statistically significant reduction. The reduction of food consumption in mid-dose females during study days 28 - 35 (about 7% below control) was assessed as not treatment-related since there was no relation to dose.
Food consumption was comparable to the concurrent control values in the low- and mid-dose males and in the low- and high-dose females during the entire study.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
effects observed, non-treatment-related
Description (incidence and severity):
F1 generation parental animals, Cohort 1B
Water consumption of the high-dose males was statistically significantly below the concurrent control values during study days 35 - 52 (up to 18%). Water consumption was statistically significantly below the concurrent control values for the
high-dose females during study days 35 - 38 (about 16%) and for the mid-dose females during study days 35 - 52 (up to 20%). Since the values for the mid-dose females were quite variable without relation to dose, the decrease was assessed as incidental and not-treatment related.
Water consumption was comparable to the concurrent control values in the low- and mid-dose males and in the low-dose females during the entire study.
Ophthalmological findings:
not examined
Haematological findings:
not specified
Description (incidence and severity):
see F1 generation
Clinical biochemistry findings:
not specified
Description (incidence and severity):
see F1 generation
Urinalysis findings:
not specified
Description (incidence and severity):
see F1 generation
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not specified
Description (incidence and severity):
see F1 generation
Gross pathological findings:
not specified
Description (incidence and severity):
see F1 generation
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not specified
Description (incidence and severity):
see F1 generation
Histopathological findings: neoplastic:
no effects observed
Other effects:
not specified
Description (incidence and severity):
see F1 generation
Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
F1 rearing animals, Cohort 1A
Estrous cycle data, generated during 2 weeks, revealed regular cycles in the females of all test groups 10 - 13. The mean estrous cycle duration was comparable between the groups: 4.0 / 4.0 / 4.1 and 4.0 days in test groups 10 - 13, respectively.

F1 generation parental animals, Cohort 1B
Estrous cycle data, generated during 2 weeks, revealed regular cycles in the females of all test groups 10 - 13. The mean estrous cycle duration in the different test groups was similar: 4.0 days in all groups (10 - 13).
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
F1 rearing animals, Cohort 1A
Concerning motility of the sperms and the incidence of abnormal sperms in the cauda epididymidis as well as sperm head counts in the testis and in the cauda epididiymidis, no treatment-related effects were observed.
Reproductive performance:
not examined
see executive summary
Key result
Dose descriptor:
NOAEL
Effect level:
1.5 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
Key result
Dose descriptor:
NOAEL
Effect level:
15 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reproductive function (oestrous cycle)
reproductive function (sperm measures)
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Critical effects observed:
no
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
F1 generation pups/litters
There were no test substance-related adverse clinical signs observed in any of the F1 generation pups of the different test groups.
For one female pup (No. 7) of test group 03 (dam No. 193) polydactyly (right hindlimb, one supernumerary digit) was recorded during PND 12 - 21. This observation was not considered to be associated with the test compound.

F1 rearing animals, Cohort 1A
No clinical signs or changes of general behavior, which may be attributed to the test substance, were detected in any of the male and female animals in any of the groups. One high-dose female animal (No. 376) showed swelling limbs (right hindlimb) during study days 16 - 44. This was assessed as incidental.

F1 rearing animals, Cohort 2A
No clinical signs or changes of general behavior, which may be attributed to the test substance, were detected in any of the male and female animals in any of the groups.

F1 rearing animals, Cohort 3
No clinical signs or changes of general behavior, which may be attributed to the test substance, were detected in any of the male and female animals in any of the groups.
Dermal irritation (if dermal study):
not examined
Mortality / viability:
no mortality observed
Description (incidence and severity):
F1 generation pups/litters
The viability index indicating pup survival during early lactation (PND 0 - 4) varied between 100% / 100% / 100% and 99% in test groups 00 – 03, showing no treatment-related effect. The lactation index indicating pup survival during PND 4 - 21 was 100% in all test groups. Thus, the test substance did not influence pup survival in any of the treated groups (01 - 03).

F1 rearing animals, Cohort 1A
There were no test substance-related mortalities in any of the groups. One female animal (No. 372) of test group 13 was found dead because of an accidental death on study day 12. This was assessed as incidental since no further animal was affected in any of the other cohorts.

F1 rearing animals, Cohort 2A
There were no test substance-related or spontaneous mortalities in any of the groups.

F1 rearing animals, Cohort 3
There were no test substance-related mortalities in any of the groups. One male animal (No. 1029) of test group 12 (5 mg/kg bw/d) was found dead on study day 23. As the animal was not investigated histopathologically, no cause of the death could be ascertained. A relationship to the treatment is not assumed since there was no relation to dose.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
F1 generation pups/litters
Mean body weights of the high-dose male and female pups and both sexes combined were statistically significantly below the concurrent control values during PND 7 - 21 (up to 11%, 10% and 10%, respectively). Mean body weight change of high-dose male and female pups and both sexes combined was statistically significantly below the concurrent control values during the entire lactation period (up to 16%, 14% and 15%, respectively).
No test compound-related influence on F1 pup body weight (change) were noted in all pups of the low- and mid-dose groups during the entire lactation period.

F1 rearing animals, Cohort 1A
Mean body weight (change) was affected in high-dose males and females. Mean body weights of the high-dose group males and females were statistically significantly below the concurrent control values during study days 42 - 56 and 49 - 56 (up to 11% and 6%, respectively). Body weight change was statistically significantly below the concurrent control values for the high-dose males during study days 21 - 56 and 0 - 56 (up to 49% and 14%, respectively). Body weight change was statistically significantly below the concurrent control values for the highdose females during study days 0 - 56 (about 8%, respectively). Mid- and low-dose males showed body weight change below control during study days 42 - 49 (about 24 and 21%, respectively). Since the decreases were only transient during a short time period, they were not assessed as treatment-related and adverse. Mean body weights were comparable to the concurrent control values in the low- and mid-dose males and females during the entire study period. Body weight change was comparable to the concurrent control values in the low- and mid-dose females during the entire study. The statistically significantly increased body weight change in the high-dose males during study days 7 - 14 was considered to be spontaneous in nature and not treatment-related since there was no relation to dose.

F1 rearing animals, Cohort 2A
The body weights of all test substance treated male animals was comparable to the concurrent control values throughout the entire study period. In males, body weight change was statistically significantly below the concurrent control values
for the high-dose males during study days 14 - 21 and 0 - 42 (about 11%, respectively). Mean body weights of the high-dose females were statistically significantly below the concurrent control values on study days 21, 35 and 42 (up to 8%). Consistently, body weight change was statistically significantly below the concurrent control values for the high-dose females during study days 0 - 42 (about 11%). Isolated decreased body weight changes in mid-dose males during study days 14 – 21 and low-dose females during study days 35 – 42 occurred only during short time points and were partly without relation to dose. Therefore, they were assessed as incidental and not treatmentrelated. Mean body weights in the low- and mid-dose females were comparable to the concurrent control values during the entire study period. Body weight change was comparable to the concurrent control values in the low-dose males and mid-dose females during the entire study.

F1 rearing animals, Cohort 3
The mean body weights and body weight change of all test substance-treated male animals were comparable to the concurrent control values throughout the entire study. Mean body weights were statistically significantly below the concurrent control values for the high-dose females during study days 21 - 28 (up to 9%). Body weight change was statistically significantly below the concurrent control values for the high-dose females during study days 0 - 28 (about 10%). The mean body weights and body weight change of low- and mid-dose female animals were comparable to the concurrent control values throughout the entire study.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
F1 rearing animals, Cohort 1A
Food consumption of all male and female animals of all test substance-treated groups was comparable to the concurrent control values throughout the entire study. The statistically significantly increased food consumption in the low-dose females during study days 0 - 14 and 21 - 28 was considered to be spontaneous in nature and not treatment-related since there was no relation to dose.

F1 rearing animals, Cohort 2A
Food consumption of the high-dose males was statistically significantly below the concurrent control values during study days 35 - 42 (about 10%) only. Food consumption was comparable to the concurrent control values in the low- and mid-dose
males and in all test substance treated females during the entire study.

F1 rearing animals, Cohort 3
Food consumption of all male and female animals of all test substance-treated groups was comparable to the concurrent control values throughout the entire study.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
effects observed, non-treatment-related
Description (incidence and severity):
F1 rearing animals, Cohort 1A
Water consumption of all male and female animals of all test substance-treated groups was comparable to the concurrent control values throughout the entire study.

F1 rearing animals, Cohort 2A
Water consumption of all test substance treated male and female animals was comparable to the concurrent control values throughout the entire study.

F1 rearing animals, Cohort 3
Water consumption of all test substance treated males was comparable to the concurrent control values throughout the entire study. Water consumption was statistically significantly below the concurrent control values for the high-dose females during study days 0 - 11 and for the mid-dose females during study days 14 – 18 only. However, these transient findings occurred only during a short time period. For the mid-dose females, it was not related to dose. Therefore, they were assessed as not related to treatment. Water consumption was comparable to the concurrent control values in the low-dose females during the entire study.
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
F1 rearing animals (cohort F1A)
At PND 90 in F1 males absolute and relative eosinophil counts were significantly decreased, whereas in females absolute and relative monocyte counts were increased (relative monocyte counts not statistically significantly). These alterations were regarded as treatment-related and adverse.
In males of test groups 11, 12 and 13 (1.5, 5 and 15 mg/kg bw/d) red blood cell (RBC) counts, hemoglobin and hematocrit values were significantly decreased. Hemoglobin and hematocrit levels in test groups 11 and 12 were not dose-dependently changed. All means were within historical control ranges (F1 males, RBC 7.50-8.51 Tera/L; hemoglobin 8.6-9.4 mmol/L; hematocrit 0.403-0.444 L/L). In females of test groups 11, 12 and 13 hemoglobin values, in females of test groups 11 and 13 additionally hematocrit values, and in females of test group 13 mean corpuscular hemoglobin concentration (MCHC) were significantly decreased. All values were within historical control ranges (F1 females, hematocrit 0.375-0.419 L/L; hemoglobin 8.2-9.2 mmol/L, MCHC 20.90-22.18 mmol/L). Therefore, these mentioned changes were regarded as incidental and not treatment related.
In male and female PND 90 F1 rats of test group 13 (15 mg/kg bw/d) and additionally in males of test group 12 (5 mg/kg bw/d) absolute reticulocyte counts were significantly increased above the historical control ranges (absolute reticulocytes, F1 males 102.1-184.0 Giga/L; F1 females 92.3-177.7 Giga/L). This was the only relevantly changed red blood cell parameter. No histopathological changes in the spleens of these individuals were observed. Therefore, the increased absolute reticulocyte counts were regarded as treatment-related, but non-adverse (ECETOC Technical Report No. 85, 2002).
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
F1 rearing animals (cohort F1A)
On PND90 in male and female F1 rats of test group 13 (15 mg/kg bw/d) aspartate aminotransferase (AST) activities were significantly increased. These alterations were regarded as treatment-related and adverse.
The following significant changes were regarded as incidental and not treatment-related, because the values were within historical control ranges: decreased AST activities in males of test group 11 (1.5 mg/kg bw/d); decreased creatinine and calcium values in males of test groups 11, 12 and 13 (1.5, 5 and 15 mg/kg bw/d); decreased total protein, albumin and globulin values in males of test group 13; decreased albumin values in males of test group 11; decreased glucose values in females of test group 13; decreased potassium values in females of test groups 12 and 13 (F1 males, AST 1.34-2.20 μkat/L; creatinine 19.6-30.9 μmol/L; calcium 2.50-2.68 mmol/L; total protein 58.40-63.79 g/L; albumin 33.74-40.01 g/L; globulins 21.53-29.13 g/L; F1 females, glucose 4.32-6.47 mmol/L; potassium 3.95-4.79 mmol/L).
The following significant alterations were regarded as incidental and not treatment-related, because they were not dose-dependent: decreased ALP activities in females of test groups 11 and 13 (1.5 and 15mg/kg bw/d); decreased creatinine values in females of test group 12 (5 mg/kg bw/d).
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
F1 rearing animals, Cohort 2A
No treatment-related, adverse changes among urinalysis parameters were observed.
At the end of the administration period, in F1 male and female rats of test group 13 (15 mg/kg bw/d) urine pH values were decreased (in females not statistically significantly). This change is probably treatment-related because of the excretion of acidic metabolites of the administered compound, but it is not regarded as adverse, per se.
Additionally, in males of test groups 12 and 13 (5 and 15 mg/kg bw/d) urine volume was significantly decreased whereas urine specific gravity was significantly increased. The changes were not dose dependent. They reflect the normal adaptation of the kidneys toward changed fluid income. Therefore, these alterations were regarded if ever treatment-related, as adaptive and non-adverse.
Sexual maturation:
effects observed, non-treatment-related
Description (incidence and severity):
F1 generation pups/litters
Vaginal opening
Each female F1 pup, which was selected to become a rearing female, was evaluated for commencement of sexual maturity. The first day when vaginal opening was observed was PND 27, the last was PND 37. The mean number of days to reach the criterion in the control and 1.5, 5 and 15 mg/kg bw/d test groups was 31.5; 31.3; 31.4 and 31.8 days, respectively. The mean body weight on the day, when vaginal opening was recorded, amounted to 93.8 g, 93.4 g, 92.7 g and 90.6 g in test groups 00 - 03. Neither a statistically significant nor a toxicologically relevant effect was noted in any of the treatment groups.

Preputial separation
Each male F1 pup, which was selected to become a rearing male, was evaluated for commencement of sexual maturity. The first day when preputial separation was observed was PND 38, the last was PND 50. The mean number of days to reach the criterion in the control and 1.5, 5 and 15 mg/kg bw/d test groups was 42.4, 42.3, 42.9 and 43.0 days, respectively. The mean body weight on the day, when preputial separation was recorded, amounted to 177.2 g, 177.6 g, 175.5 g and 169.0 g in test groups 00 - 03. Neither a statistically significant nor a toxicologically relevant effect was noted in any of the treatment groups. The statistically significantly decreased number of pups reaching the criteria on PND 39 in the high-dose males was assessed as not treatment-related since the mean number of days reaching preputial separation was comparable to controls. The statistically significantly increased number of pups reaching the criteria on PND 40 in the low-dose males was without relation to dose and, therefore, considered to be spontaneous in nature and not treatment related.
Anogenital distance (AGD):
no effects observed
Description (incidence and severity):
F1 generation pups/litters
The anogenital distance and anogenital index of all test substance treated male and female pups was comparable to the concurrent control values.
Nipple retention in male pups:
effects observed, non-treatment-related
Description (incidence and severity):
F1 generation pups/litters
The apparent number and percentage of male pups having areolae was not influenced by the test substance when examined on PND 13.
In the high- and mid-dose groups, the mean percentage of male pups reaching the criteria per litter was statistically significantly above the concurrent control values (83.8% / 85.7% / 89.6%* [*:p<=0.05] and 95.3%** [**:p<=0.01] in test groups 00 - 03, respectively) when examined on PND 13. Both mean values were outside the historical control range (HCD, mean percentage of male pups reaching criteria per litter: 8.7 – 84%). During the re-examination on PND 20 no nipples/areolae were detected in any male pups of all test groups.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
F1 rearing animals, cohort 1A
In test group 13, the significant terminal body weight decrease in males (-22%) and females (-7%) was below the historical control values and was regarded as treatment-related. The significant absolute weight increase of the liver in females (5.399 mg) of test group 13 was minimally above the historical control range (4.993 – 5.282 mg) and was assumed as treatment-related and most likely consistent with histopathological findings.The significant absolute weight increase of the kidneys in males of test group 13 (2.281 mg) was within the historical control range (2.156 – 2.304 mg) and was therefore not considered treatment-related. As a consequence of the terminal body weight decrease in test group 13, significant absolute weight decreases (thymus in males) and/or significant relative weight increases resulted in males (brain, cauda epididymis, epididymides, heart, liver, pituitary, spleen, testes and thyroid glands) and females (brain, heart, kidneys, liver, pituitary, spleen and thymus), that were considered secondary to the decreased terminal body weight.
In test group 12, the significant absolute weight decrease of the thymus and the significant relative weight increase of the kidneys in males were considered secondarily associated with the decrease of the terminal body weight (-4%). The same was true for the significant relative weight increases in the liver, kidneys and thymus in females.
In test group 11, the significant absolute weight decrease of the pituitary gland in males was not dose-dependent and therefore incidental. The significant relative weight increase of the liver in females was within the historical control range and regarded as incidental and not treatment-related. Significant absolute and/or relative weight increases observed in the uterus of females of test groups 11 and 13 occurred without a dose-dependency and were therefore considered incidental.
The significant absolute weight increases of the liver in females of test groups 11 and 12 were within the historical control values and were regarded as incidental and not treatment-related.

F1 rearing animals, cohort 1B
The significant decrease of the terminal body weight in males and females of test groups 12 (-7% males, -5% females) and 13 (-15% males, -8% females) was below the historical control values and was regarded as treatment-related. The absolute liver weight increase in females of test group 13 (5.558 g) was minimally above the historical control range (5.009 - 5.383 g) and was considered treatment related. The significant increase of the relative liver weight in these females was regarded as treatment-related but might be in part secondarily associated with the terminal body weight decrease. As a consequence of the terminal body weight decreases, significant absolute weight decreases and/or significant relative weight increases were seen in several organs, that were considered secondary to the decreased terminal body weights. This was true for the significant absolute weight decreases of the epididymides, prostate and testes in males of test groups 12 and 13, and the significant relative organ weight increases of the adrenal glands, cauda epididymis, epididymides, testes and liver in males of test groups 13. Similarly occurred in females showing significant relative weight increases in test groups 12 (adrenal glands, liver and ovaries) and 13 (adrenal glands, ovaries and pituitary), which were also attributed to the decreased terminal body weights. Besides this, the significant relative weight increases of the ovaries and the significant weight decrease of the uterus showed no dose-response and were regarded as incidental. Finally, in test group 11, the significant absolute weight decrease of the testes in males and the significant relative weight increase of the liver in females were regarded as incidental.

F1 animals, cohort 3
2,2’-dimethyl-4,4’-methylenebis(cyclohexylamine)
Absolute and relative organ weights
When compared to the control group 10 (set to 100%), the terminal body weight of females of test group 14 was significantly decreased (91%). All other mean absolute and relative weight parameters of males and females did not show significant differences when compared to the control group 10.

Cyclophosphamide monohydrate (positive control)
Absolute and relative organ weights
When compared to the control group 10 (set to 100%), the mean absolute and relative weight parameters of test group 14 (positive control) were significantly decreased. A significant decrease in the absolute and relative weights of the spleen and thymus was noted in the positive male and female control animals. These changes were expected to occur.

surplus F1 generation pups on PND 22 (F1 weanlings not selected for cohorts)
The decreased terminal body weight in males of test group 13 was considered treatment related. The decreased absolute brain and spleen weight in males of test group 13 as well as the relative brain weight increase in females of test group 13 was considered secondarily associated to the decreased terminal body weight.

Cohort 2A animals (Developmental Neurotoxicity Cohort, adults)
The decreased terminal body weights of test group 13 males (not statistically significant) and females (statistically significant) were regarded to be treatment-related. The increased relative brain weights in this group in both sexes were assessed as a secondary effect to the body weight decrease.

Cohort 2B animals (Developmental Neurotoxicity Cohort, weanlings)
The decreased terminal body weights of test group 13 males (statistically significant) and females (not statistically significant) were regarded to be treatment-related. The increased relative brain weights in this group in males was assessed as a secondary effect to the body weight decrease.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
F1 rearing animals, cohort 1A
All findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.

F1 rearing animals, cohort 1B
All findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.

Cohort 2A animals (Developmental Neurotoxicity Cohort, adults)
Findings were only recorded in the skin. They occurred individually and were considered to be incidental or spontaneous in origin and without any relation to treatment.

Cohort 2B animals (Developmental Neurotoxicity Cohort, weanlings)
No gross findings were recorded.

F1 animals, cohort 3
2,2’-dimethyl-4,4’-methylenebis(cyclohexylamine) (test item)
All findings occurred individually and were considered to be incidental or spontaneous in origin and without any relation to treatment.

Cyclophosphamide monohydrate (positive control)
Red and black foci (2 and 3 mm in diameter) were observed in 2 out of 10 females of test group 14. These findings were assumed to be background lesions.

surplus F1 generation pups on PND 22 (F1 weanlings not selected for cohorts)
No treatment-related gross changes were observed.
Histopathological findings:
effects observed, treatment-related
Description (incidence and severity):
F1 rearing animals, cohort 1A
The target organs and the histopathological findings in males and females of the cohort 1A were comparable to those observed in the in the parental F0 generation.

Adrenal cortex
In male animals of test group 13, the vacuolation of the zona fasciculata showed an increase in the incidence and grading. The vacuolation was characterized by the presence of a microvesicular pattern, giving the cytoplasm of the cells a foamy and pale aspect and often causing an increased size of the cells. This effect was clearly seen in males in test group 13. The vacuolation in test group 12 was considered incidental. Females were not affected.

Axillary lymph node
The vacuolation of the high endothelial venules (HEV) was characterized by small vacuoles in the whole cytoplasm, which gave these vessels a conspicuous transparent aspect. Males and females were affected from test group 12 onwards.

Brain
The vacuolation was noted in the epithelial cells of the choroid plexus and was most frequently of microvesicular type. However, with increasing grading, many macrovesicular vacuoles were also observed. A very tiny content was visible in these vacuoles, most likely representing membrane residues resulting from coalescence of smaller vacuoles. The affected choroid plexus was most clearly found in the lateral and dorsal third ventricles. Only males and females in test group 13 were affected, with males showing a higher grading than females.

Esophagus
In the esophagus, the vacuolation was noted in the skeletal muscle layers of the wall. This finding was characterized by the presence of very tiny, transparent to birefringent microvacuoles ranging approximately from 2 – 4 μm in diameter within the muscle fibers. Test groups 12 and 13 appeared to be equally affected in males and females.

Eyes with optic nerve
A very fine microvesicular vacuolation was seen in the retinal pigment epithelium. The affected cells had ground-glass aspect and were minimally increased in size. This change was more manifested in the peripheral areas. Males were affected in test groups 12 and 13 and females only in test group 13.

Glandular stomach
A microvesicular vacuolation (small vacuoles) was observed at the base of the glands of pyloric mucosa. Within the glandular cells, the vacuolation randomly displaced the nuclei from their basal position, giving the base of the glands a disorganized and paler aspect than normal. Only males and females of test group 13 were affected.

Heart
The microvesicular vacuolation affected mainly the septum and left ventricular wall. It was characterized by multiple individual small vacuoles visible within the cardiomyocytes, without altering their shape or size. This change was not associated with visible degeneration or necrosis but conferred the cells a pale and disorganized aspect. Males were affected from test group 12 onward and females only in test group 13.

Kidneys
In the kidneys the vacuolation was observed in the medulla (tubules of the inner stripe of the outer medulla) of both sexes, whereas the degeneration and/or regeneration was found in the cortex (proximal convoluted tubules) of males only. The vacuolation in the medulla was of a very fine microvesicular type, giving the epithelial cells a “ground glass” pale and swollen aspect. The tubular degeneration/regeneration was characterized by multifocal areas of convoluted tubules with microvesicular vacuolation, loss of normal architecture due to nuclear disorganization and crowding and general light basophilia.

Left epididymis
The vacuolation was localized primarily in the ducts of approximately 2/3 of the distal corpus at the transition to the caudal region but did not include the cauda. The vacuolation of the epithelial cells ranged from small microvesicular vacuoles to vacuoles as large as the nuclei. The vacuoles were always located basal and lateral to the nuclei within the cytoplasm. Only animals in test group 13 were affected.

Liver
The vacuolation within the hepatocytes was characterized by very small cytoplasmic vacuoles of regular size distributed around the nuclei rather than in the periphery of the hepatocyte. This pattern was quite different from the “fatty change vacuolation” pattern, which is composed of vacuoles of different size. The mixed-cell inflammation observed particularly in females in test group 13, was composed predominantly of granulocytes and lymphocytes and was localized in centrilobular areas affected by vacuolation. This type of inflammation was associated with apoptosis/single cell necrosis. Multinuclear hepatocytes in females as seen in females of the F0 generation were not present here. The vacuolation of the bile duct epithelium was of a “ground-glass” type affecting the whole cell and was observed in the portal bile ducts of large caliber. Their aspect was very pale, and the size of the epithelial cells was increased. All these findings affected males and females only in test group 13.

Lungs
The vacuolation was localized in the bronchial and bronchiolar epithelium and was characterized by a “foamy” aspect. In bronchi and large bronchioles, the vacuolation was mostly occupying the cytoplasm apical to the cell nuclei, whereas in the terminal bronchioles, the vacuolation was rather basal to the cell nuclei, displacing them to the apical region. In the bronchial associated lymphoid tissue (BALT), the high endothelial venules (HEV) showed the same type of vacuolation as described for the lymph nodes, with a noticeable pale aspect.

Mesenteric lymph node
Similarly, as observed in the axillary lymph nodes, the whole cells of the high endothelial venules (HEV) showed a microvesicular vacuolation (small vacuoles), conferring these vessels a very pale aspect.

Pancreas
The vacuolation of the acinar epithelium was of microvesicular type. Within the acinar cells, very small vacuoles were localized in the apical cytoplasmic border adjacent to the zymogen granules (grade 1) or extended from the apical cell border to the cell nuclei (grade 2) accompanied by a reduction of zymogen granules. The ductal vacuolation was seen in the interlobular pancreatic ducts, presenting a ground glass appearance of the epithelial cells accompanied by increase size.

Pituitary gland
The cytoplasmic vacuolation was localized in all cell types of the pars distalis. Within the cells the vacuolation was of a very fine microvesicular type involving the whole cytoplasm giving the cells a fine “granular” and pale aspect.

Seminal vesicles
The cytoplasmic vacuolation of the epithelial cells was characterized by small vacuoles of very regular size localized at the basal part of the cells displacing the nuclei to the apical border. The cells appeared wider and the nuclei lost their regular arrangement along the epithelium/basal membrane. Some animals showed single vacuoles of macrovesicular type. The vacuolation was dose-dependent from test group 02 onwards.

Skeletal muscle
As already described for the skeletal muscle of the esophageal wall, the vacuolation of the skeletal muscle was also characterized by the presence of very tiny intracytoplasmic microvacuoles (few μm in diameter) with a birefringent aspect and a multifocal distribution pattern within the muscle fiber. In addition, degeneration and /or regeneration of single myofibers was noted. The degenerating fibers revealed slightly altered staining features (basophilic or strong hyaline stain) and variable thickness (retracted or swollen aspect). Some of these degenerated fibers showed regeneration (reparative response) characterized by numerous central nuclei within the fibers. In test group 13 males, vacuolation was strongly associated with degeneration/regeneration, whereas in test group 12 no cytotoxicity was noted.

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.

Decedents
The female animal No.372 was found dead without showing any particular macroscopic or microscopic relevant finding.

Differential ovarian follicle count
The results of the differential ovarian follicle count (DOFC) – comprising the numbers of primordial and growing follicles, as well as the combined incidence of primordial plus growing follicles – did not reveal significant differences between the control group 10 and animals of test group 13.

Cohort 2A animals (Developmental Neurotoxicity Cohort, adults)
Treatment-related findings were observed in cervical and lumbar spinal dorsal root ganglia and trigeminus ganglia, gastrocnemius muscle, eyes with optic nerve, pituitary gland in males and females and choroid plexus in females of test group 13 and in eyes of test group 12 animals of both sexes

Eyes with optic nerves
In the eyes of animals of test groups 12 and 13 the retinal pigment epithelium was minimally expanded by tiny clear round vacuoles.

Spinal ganglia (cervical and lumbar dorsal root ganglia) and trigeminus ganglia
Cervical and lumbar ganglia were plastic embedded and stained with AMbF. With this method, lipids are preserved in the cell, which stain blue with AMbF. In this study, blue round structures were seen in the cytoplasm of the perikaryon of ganglion cells; they were recorded as “droplets”. In trigeminus ganglia, which were embedded in paraplast followed by Hematoxylin & Eosin stain, lipids are washed out during processing, leaving round, clear spaces in the cytoplasm of the perikaryon, recorded as “vacuolation”. “Droplets” and “vacuolation” constitute the same finding with different morphological manifestations depending on technical procedures (embedding and staining).

M. gastrocnemius
In test group 13 animals of both sexes, multifocal degeneration of the muscle fibers characterized by fragmented muscle fibers with influx of mixed inflammatory cells (degeneration) were noted. In a few animals, increased basophilia of the muscle fiber with centralization of nuclei (indicating regeneration) was also seen, but not recorded separately as degeneration was more prominent. Additionally, some muscle fibers showed very few tiny clear vacuoles. The degeneration seen in one test group 12 male and female was considered not treatment related as one control animal of each sex also showed this finding

Pituitary gland and choroid plexus
Findings in pituitary gland and choroid plexus were very minimal in this cohort in contrast to findings in F0 generation parental animals and F1 rearing animals, cohort 1A. This was assumed to be due to the different fixation techniques: F0 generation parental animals and F1 rearing animals, cohort 1A were immersion fixed while cohort 2A was perfusion fixed. Both pituitary and choroid plexus showed a much smaller (shrunken) cytoplasm in cohort 2A as opposed to F0 generation parental animals and F1 rearing animals, cohort 1A, which made the detection of vacuoles in the pituitary gland almost impossible. In the choroid plexus, there was additionally a moderate vacuolation also in control animals, which appeared morphologically indistinguishable from treated animals. Two females of test group 13, however, showed a very minimally increased number of vacuoles compared to controls. A comparison of the morphology in F0 generation parental animals and F1 rearing animals, cohort 1A and cohort 2A is presented in the photo documentation.

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.

Morphometry
Males
In test group 13 males, the measurements for frontal cortex left and right (measurements 1 and 2) were significantly (p <=0.01) smaller than controls. There is, however, no concordance with other parameters:
The other cortex measurements (parietal, measurements 5 and 6) were only unilaterally statistically significantly smaller (measurement 6), although the trend to a smaller size was visible on both sides. For a clear treatment-related effect, all cortex measurements would have been expected to change significantly, with lower test groups showing a trend in the same direction (test groups 11 and 12 show, however, a trend towards larger cortex measurements than controls). Gross measurements do not show an effect: the length is slightly (not statistically significantly) shorter than controls but the width is (again not statistically significantly) wider. The brain weight was comparable in control and treated groups. Concluding, there is no dose-response relationship for the cortex measurements or a change in the same direction as other parameters like weights, histopathological findings or gross measurements, nor even a consistent change within the four cortex measurements (1,2,5,6). The cortex measurements in males and females of test group 13 changed in opposite directions with a decrease in males and an increase in females, while in other parameters (histopathology, organ weights) both sexes were equally affected by treatment. Furthermore, there were no functional clinical findings in test group 13. The statistically changed cortex measurements (1,2,5,6) in test group 12 were considered incidental and not related to treatment due to a lack of dose-response. The statistically changed measurement 3 (Nucleus caudatus width left) in test groups 12 and 13 was considered incidental, as the change was unilateral only and did not show a doseresponse. Therefore, all changes are considered to be incidental and not related to treatment.

Females
The measurements of the frontal cortex showed higher values than controls in all treated test groups, however, without a dose-response relationship. There is, however, no concordance with other parameters:
The other cortex measurements (parietal, measurements 5 and 6) were only unilaterally statistically significantly changed in test groups 11 and 13 (measurement 6). For a clear treatment-related effect, all cortex measurements would have been expected to change significantly, with a clear dose-response-relationship. Gross measurements did not show an effect: The absolute brain weight was minimally decreased in treated groups, which is inconsistent with larger morphometric measurements. In histopathology, findings were following a clear dose response, in contrast to morphometry data which show highest values in test group 12. The cortex measurements in males and females changed in opposite directions with a decrease in males and an increase in female, while in other parameters (histopathology, organ weights) both sexes were equally affected by treatment. Furthermore, there were no functional clinical findings. Concluding, there is no dose-response relationship for the cortex measurements or a change in the same direction as other parameters like weights, histopathological findings or gross measurements, no consistent change within the four cortex measurements (1,2,5,6), no consistency between males and females and no functional clinical findings in test group 13. The statistically significantly increased measurements of the nucleus caudatus (3, 4) in test group 11 were regarded to be incidental as there was no dose-response relationship and they were not statistically significantly changed in test groups 12 and 13. Therefore, due to a lack of concordance with other parameters, and no dose-response relationship, the statistically significant brain measurements in test groups 11, 12 and 13 were regarded as incidental.

Cohort 2B animals (Developmental Neurotoxicity Cohort, weanlings)
No treatment-related histopathological findings were seen. All lesions are regarded as incidental and/or spontaneous.
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
F1 rearing animals (surplus pups PND4, PND22 and cohort 1A)
Thyroid hormones
In F1 PND 4 male and female pups (test groups 1, 2 and 3; 1.5; 5 and 15 mg/kg bw/d) no treatment-related alterations of T4 and TSH levels were observed.
In F1 PND 22 male and female pups (test groups 1, 2 and 3; 1.5; 5 and 15 mg/kg bw/d) no treatment-related alterations of T4 and TSH levels were observed.
In female PND 22 pups TSH values in test group 1 (1.5 mg/kg/ bw/d) were significantly increased, but the change was not dose dependent. Therefore, this alteration was regarded as incidental and not treatment related.
On PND 90, in F1 males of test groups 11 and 13 (1.5 and 15 mg/kg bw/d) TSH values were significantly decreased. However, this decrease was not dose-dependent and the TSH as well as the T4 values were within historical control ranges (F1 males, TSH 2.61-9.90 μg/L; T4 49.46- 88.73 nmol/L). No histopathological findings in the thyroids of these individuals were observed. Therefore, TSH alterations in F1 PND 90 males of test groups 11 and 13 were regarded as incidental and not treatment-related.
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
F1 rearing animals, Cohort 2A
Auditory Startle Response
No influence of the test substance on auditory startle habituation (maximum amplitude and latency) was observed in any male or female animal in all treated groups. The statistically significantly increased maxium amplitude in the high-dose males during measurement block 2 and the statistically significantly increased latency during measurement block 4 was considered as spontaneous in nature since they occurred only during one measurement and were covered by the historical control data (maximum amplitude = 684.8 + 515.9 and latency = 33.5 + 6.7).

Functional observational battery (FOB)
Home cage observations:
No test substance-related findings were observed in male and female animals of all test groups during the home cage observation. One male animal of test group 12 (No. 621) showed reduced exploration of the area. This observation was not considered to be associated with the test compound since it was not
related to dose.

Open field observations:
The open field observations did not reveal any test substance-related findings in male and
female animals of all test groups.

Sensorimotor tests/reflexes:
There were no test substance-related findings in male and female animals of all test groups. Spontanous, isolated findings in a few individuals such as approaching to object during the approach response test in single males and females of test group 10 and 11 and vocalization when touched were without relation to dose and assessed as not treatment-related.

Quantitative Parameters:
No test substance-related impaired parameters were observed in male and female animals of all test groups.

Motor activity measurement (MA)
Motor activity data was not influenced by the test substance of the cohort 2A animals of all test substance-treated groups in comparison to the concurrent control values. Overall activity levels and habituation to the test environment corresponded to the age of these animals at the specific testing date, if usual biological variation inherent in the strain of rats used for this experiment was considered.
Developmental immunotoxicity:
effects observed, non-treatment-related
Description (incidence and severity):
F1 rearing animals, Cohort 3
T-cell dependent antibody response
No treatment-related changes regarding the anti-SRBC IgM antibodies occurred in the F1 test groups at PND 63.
In females of test groups 11, 12 and 13 (1.5, 5 and 15 mg/kg bw/d) anti-SRBC IgM antibodies were significantly lower compared to study controls. However, the anti-SRBC IgM antibody titer in the study controls was above that of the historical control range whereas the corresponding titers in test groups 11, 12 and 13 were within this range (females, anti-SRBC IgM 8222-42129 U/mL). No changes in the splenic lymphocyte populations of these individuals were observed. Therefore, the significantly lower anti-SRBC IgM antibody titers in females of test groups 11, 12 and 13 were regarded as incidental and not treatment related. As expected, the anti-SRBC IgM antibody titers in males and females of the positive control (test group 14, 4.5 mg/kg bw/d cyclophosphamide) were significantly lower compared to the study controls.

F1 rearing animals, Cohort 1A
Lymphocyte subpopulations in spleen
No alterations in the absolute and relative lymphocyte subpopulation cell counts in the spleen tissue (B-, T-lymphocytes, CD4-, CD8-T-lymphocytes and natural killer (NK) cells) were observed in the F1 generation at PND 90 in both sexes.
Key result
Dose descriptor:
NOAEL
Generation:
F1 (cohort 1A)
Effect level:
1.5 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
histopathology: non-neoplastic
Key result
Dose descriptor:
NOAEL
Generation:
F1 (cohort 1B)
Effect level:
1.5 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
Key result
Dose descriptor:
NOAEL
Generation:
F1 (cohort 2A)
Effect level:
15 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
neuropathology
Key result
Dose descriptor:
NOAEL
Generation:
F1 (cohort 2B)
Effect level:
15 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
neuropathology
Key result
Dose descriptor:
NOAEL
Generation:
F1 (cohort 3)
Effect level:
15 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
developmental immunotoxicity
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
5 mg/kg bw/day (nominal)
Organ:
heart
kidney
lungs
lymph node
pancreas
seminal vesicle
other: eyes, esophagus
Treatment related:
yes
Dose response relationship:
yes
Clinical signs:
not examined
Dermal irritation (if dermal study):
not examined
Mortality / viability:
not examined
Body weight and weight changes:
not examined
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:
not examined
Anogenital distance (AGD):
not examined
Nipple retention in male pups:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
not examined
Histopathological findings:
not examined
Other effects:
not examined
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
Remarks on result:
not measured/tested
Critical effects observed:
no
Key result
Reproductive effects observed:
yes
Lowest effective dose / conc.:
5 mg/kg bw/day (nominal)
Treatment related:
yes
Relation to other toxic effects:
reproductive effects as a secondary non-specific consequence of other toxic effects
Dose response relationship:
yes

see attachment: 90R0695_00R050_results and tables

Conclusions:
Under the conditions of the present EOGRTS (extended 1-generation reproduction toxicity study) the NOAEL (no observed adverse effect level) for general, systemic toxicity is the low dose of 1.5 mg/kg bw/d for the F0 and F1 animals. This was based on treatment-related, adverse effects such as a reduction in water and food consumption, decrease in body weight (change), altered clinical pathology parameters as well as histopathological changes in several organs, which were observed at the high- and mid-dose of 15 and 5 mg/kg bw/d.

The NOAEL for developmental toxicity in the offspring is the mid-dose of 5 mg/kg bw/d, based on the decrease in pup body weight during lactation at the high-dose level of 15 mg/kg bw/d. This slight delay in development of the high-dose pups was observed in presence of maternal toxicity and, therefore, not assessed as independent effect.

The NOAEL for developmental neurotoxicity in the F1 progeny is 15 mg/kg bw/d, the highest dose tested. Neuropathological findings observed in Cohort 2A adults of high- and mid-dose indicated a systemic, direct toxicity of the test substance and were not assessed as developmental neurotoxicological effects.

The NOAEL for developmental immunotoxicity for the F1 progeny is 15 mg/kg bw/d, the highest dose tested. Lower mean and median anti-SRBC IgM antibody titers of the positive control group (4.5 mg/kg bw/d cyclophosphamide, oral) demonstrated that the test system worked properly.

The NOAEL for fertility and reproductive performance for the F0 parental rats is 1.5 mg/kg bw/d, the lowest dose tested. This was based on the lower mean number of implantation sites and secondary decreased mean number of F1 pups delivered per dam in the high- and mid-dose groups.
Executive summary:

2,2’-dimethyl-4,4’-methylenebis(cyclohexylamine) was administered to groups of 25 male and 25 female healthy young Wistar rats as test groups 00 - 03 as an aqueous preparation by stomach tube at different dosages (0, 1.5, 5 and 15 mg/kg body weight/day [mg/kg bw/d]):

F0 animals were treated at least for 10 weeks 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 5 different cohorts (1A, 1B, 2A, 2B and 3) which were subjected to specific postweaning examinations. The study was terminated with the terminal sacrifice of the F1 rearing animals of cohort 1A. Control animals were dosed daily with the vehicle (0.5% Sodium carboxymethyl cellulose [CMC] suspension in drinking water).

 

In general, analyses confirmed the prepared concentrations, the homogeneous distribution and the stability of the test substance in the vehicle.

 

Regarding clinical examinations, F0 and F1 males and females of the high- and mid-dose groups (15 and 5 mg/kg bw/d) showed signs of systemic toxicity consisting of treatment-related, adverse changes such as a reduction in food and water consumption and a decrease in body weight (change), the latter was the most affected parameter in F0 as well as in F1 animals.

 

In the F0 parental high-dose group, reduced water and food consumptions were observed in males (up to 17 and 15% below control, respectively) and females (up to 23 and 19% below control, respectively). In the mid-dose group, water consumption was reduced in males and females (up to 12 and 19% below control) whereas food consumption was reduced exclusively in females (up to 14% below control). Mean body weight was decreased in males and females of the high-dose (up to 22 and 14% below control) and females of the mid-dose group (up to 7% below control). Body weight change was decreased in both sexes of F0 high and mid-dose animals. In general, consistent findings across all F1 cohorts were observed. Mild, transient reductions in water and/or food consumption were observed in the high-dose groups of cohorts 1B and 2A. Body weight and/or body weight change were decreased mostly in both sexes in the high dose group of all cohorts. Body weight decreases were 11 and 6% below controls in males and females of cohort 1A, 12 and 6% in males and females of cohort 1B, 8 and 9% in females of cohorts 2A and 3, respectively. In almost all cases, body weight change was correspondingly decreased. The reduction in food and water consumption of high-dose F0 and F1 and mid-dose F0 animals in combination with the body weight decreases summed up to a treatment-related and adverse

assessment. In the F1 mid-dose, a decrease in body weight (change) was exclusively seen in males of cohort 1B (BW: 6% below control). However, corresponding decreased terminal body weights of males and females were confirmed by necropsy (see below). Therefore, this combination was assessed as treatment-related adverse.

 

All other parameters were not altered. No treatment-related adverse findings were observed in the low-dose (1.5 mg/kg bw/d) F0 and F1 animals.

 

Regarding clinical pathology, in F0 and F1A rats of both sexes in test groups 3 and 13 (15 mg/kg bw/d) stress was indicated by decreased absolute and relative eosinophil cells in males, but by increased absolute and relative monocyte counts in females. In the mentioned individuals no liver parameters were relevantly changed apart from higher triglyceride levels in F0 males, but aspartate aminotransferase (AST) activities and inorganic phosphate levels were increased. Additionally, in F1A females, alkaline phosphatase (ALP) activities were significantly increased. These altered parameters in combination with lower body weights of the rats were most probably due to a retarded growth affecting bones (inorganic phosphate, ALP) as well as skeletal muscle (AST).

 

F0 generation parental animals

Regarding pathology, terminal body weight was significantly decreased in males (-22%) and females (-12%) of test group 03. They were below the historical control values and were therefore considered treatment-related. Consequently, significant absolute weight decreases and/or significant relative weight increases were seen in several organs, that were considered secondary to the decreased terminal body weight.

Histopathology revealed treatment-related findings in following target organs of males and females: brain, esophagus, eyes with optic nerve, glandular stomach, heart, kidneys, liver, lungs, axillary and mesenteric lymph nodes, pancreas, pituitary gland, and skeletal muscle. Furthermore, the adrenal cortex, left epididymis and seminal vesicles were affected only in male animals.

The main finding in all these organs was a “microvesicular” type of cytoplasmic vacuolation, characterized by the presence of very few to multiple vacuoles, ranging from very fine to small vacuoles (no larger than the nuclei of the cell). Characteristically, if the cytoplasmic vacuolation was abundant, the cells were very clear or pale and increased in size. Vacuoles larger than the cell nuclei were referred here as “macrovesicular” type of vacuolation, which was observed in few organs (brain and seminal vesicles). In 13 out of 16 target organs the vacuolation occurred alone without additional signs of cytotoxicity. Only in 3 out of 16 target organs (kidneys, liver and skeletal muscle) the vacuolation was associated with signs of cytotoxicity (degeneration/regeneration, inflammation and apoptosis/single cell necrosis).

A summary of the histopathological treatment-related findings is given as follows:

In test group 03 (15 mg/kg bw/d), the adrenal cortex of males showed a microvesicular vacuolation of the zona fasciculata (minimal to moderate), giving the cytoplasm of the cells a foamy and pale aspect and often causing an increased size of the cells. This finding might have contributed to the significant relative weight increase of the adrenal glands (+41%). Females were not affected. In the left epididymis, the vacuolation was localized in the ducts of approximately 2/3 of the distal corpus and never included the cauda. The vacuolation (minimal to moderate) ranged from small microvesicular vacuoles to vacuoles as large as the nuclei. In the seminal vesicles, the vacuolation of the epithelial cells (minimal to moderate) was characterized by small vacuoles of very regular size localized at the basal part of the cells displacing the nuclei to the apical pole. Single vacuoles of macrovesicular type were also present in the most severe cases.

 

In following organs of test group 03, males and females were affected:

In the axillary and mesenteric lymph nodes, small vacuoles (minimal to slight) were seen in the cells of the high endothelial venules (HEV). In the brain, a microvesicular and macrovesicular vacuolation (minimal to moderate) was noted in the choroid plexus of the lateral and dorsal third ventricles. Males were more severely affected than females. In the esophagus, the vacuolation was seen in the skeletal muscle of the wall (minimal to slight), with similar characteristics as observed in the gastrocnemius muscle: very tiny, transparent to birefringent microvacuoles ranging approximately from 2 – 4μm in diameter within the fibers. A very fine vacuolation in the eyes (minimal to slight) was localized in the retinal pigment epithelium. The affected cells displayed a ground glass aspect and were minimally enlarged. In the glandular stomach, the vacuolation characterized by small vacuoles (minimal to slight) were observed at the base of the glands in the pyloric mucosa. The heart showed a microvesicular vacuolation affecting mainly the septum and left ventricular wall. This change was not associated with degeneration or necrosis but conferred the cells a damaged aspect. In the kidneys the vacuolation was observed in the medulla (tubules of the inner stripe of the outer medulla) in males and females, whereas only in males, degeneration/regeneration was noted in the cortex (proximal convoluted tubules) and was minimal to moderate. The vacuolation in the medulla was of a very fine microvesicular type (minimal to slight), giving the epithelial cells a “ground glass” pale and larger aspect. The more severe findings in the kidneys of males might have contributed to significantly increase the relative weight (+31%). The degeneration/regeneration was characterized by multifocal microvesicular vacuolation with loss of normal architecture due to nuclear disorganization and crowding, single pyknosis and general light basophilia. In the liver, the vacuolation in the hepatocytes was characterized by very small vacuoles of regular size (foamy aspect), which was more severe in females (minimal to moderate) than males (minimal to slight). An associated mixed-cell inflammation (minimal to moderate) and multinucleated hepatocytes were observed particularly in females and not in males. This type of inflammation was often associated with apoptosis/single cell necrosis (minimal to slight). Furthermore, the epithelium of the portal bile ducts of large caliber also showed a vacuolation with a ground-glass appearance in both sexes. In the lungs, the vacuolation (minimal to slight) was localized in the bronchial and bronchiolar epithelium and was characterized by a “foamy” aspect. In the bronchial associated lymphoid tissue (BALT), the high endothelial venules (HEV) showed the same type of vacuolation (minimal) as described in the lymph nodes. In the pancreas, a microvesicular vacuolation of the acinar epithelium was diffusely distributed along the organ (minimal to slight). Furthermore, a vacuolation was seen in the interlobular pancreatic ducts, presenting a ground glass appearance of the epithelial cells accompanied by increase size (minimal to slight). In the pituitary gland, the vacuolation was localized in the cells of the pars distalis. Within the cells the vacuolation was of a very fine microvesicular type, giving the cells a fine “granular” and pale aspect. It remains uncertain, if the vacuolation in the pituitary gland of females might have contributed to the significant relative weight increase of +41%. In the skeletal muscle (gastrocnemius muscle), as already described for the skeletal muscle of the esophageal wall, the vacuolation was characterized by very tiny microvacuoles with a birefringent aspect, ranging approximately from 2 – 4μm in diameter, with a multifocal distribution pattern within the muscle fiber. The vacuolation was minimal to slight in males and minimal in females. In addition, degeneration and /or regeneration of the myofibers was noted in males and females.

 

In test group 02 (5 mg/kg bw/d), the incidence and/or grading of the treatment-related vacuolation was generally lower and was not associated with additional signs of cytotoxicity Organs showing vacuolation in males only were: axillary and mesenteric lymph nodes, heart, left epididymis, pituitary gland and seminal vesicles. Organs with treatment-related vacuolation affecting both males and females were: esophagus, glandular stomach, kidneys, lungs, pancreas and skeletal muscle.

 

In test group 01 (1.5 mg/kg bw/d), no treatment-related findings were noted.

 

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

 

The morphological pattern and abundant distribution of the vacuolation in 16 target organs suggests that this finding represents most likely a systemic phospholipidosis (Nonoyama and Fukada, 2008; Rudmann et al, 2004). In a previous study (BASF project No.: 85R0695/00R047) with the same test substance, special stains (PAS, Oil-Red-Oil and Sudan black) revealed that the vacuolation most likely had a phospholipidic nature. Since a definitive diagnosis of phospholipidosis should rely on transmission electron microscopy or chemical analysis, a conclusive diagnosis for the presence of phospholipidosis cannot be done in this study. However, based on the systemic distribution of the vacuolation affecting organs involved in multiple vital functions (brain, eyes, heart, etc.), the cytoplasmic vacuolation associated or not with cytotoxicity in in males and females of test groups 03 and 02 was assessed as treatment-related and adverse.

 

F1 rearing animals, cohort 1A

Regarding pathology, terminal body weights were significantly decreased in males (-22%) and females (-7%) in test group 13, which was regarded as treatment-related. Consequently, significant absolute organ weight decreases, and relative organ weight increases were seen in several organs in males and females that were considered secondarily associated with the terminal body weight decreases. However, the significant absolute weight increase of the liver in females (+11%) of test group 13 was minimally above the historical control range and was regarded as treatment-related and most likely consistent with histopathological findings.

 

Histopathologic changes found in these organs might have contributed to increase their respective weights. Histopathology revealed the same target organs as described in F0 parental male and female animals: brain, esophagus, eyes with optic nerve, glandular stomach, heart, kidneys, liver, lungs, axillary and mesenteric lymph nodes, pancreas, pituitary gland and skeletal muscle. Furthermore, the adrenal cortex, left epididymis and seminal vesicles were affected only in male animals.

 

The main histopathological finding in all these organs, as described for the F0 generation, was a “microvesicular” cytoplasmic vacuolation, ranging from very fine to small vacuoles (no larger than the size of the nucleus of the cell). The distribution of the microvesicular cytoplasmic vacuolation within the cells of each target organ had the same morphologic features as in the F0 generation. Terms referred here as “ground glass” (finest vacuoles that cannot be individually differentiated), “foamy” or “granular” (vacuoles that can hardly be differentiated) were used for a better description. Vacuoles larger than the cell nuclei, referred here as “macrovesicular” type of vacuolation, were observed only in the brain and seminal vesicles, as described in the F0 parental animals. In 13 out of 16 target organs the vacuolation occurred alone. In 3 out of 16 organs: kidneys, liver and skeletal muscle, the same as in the F0 generation, signs of cytotoxicity (degeneration/regeneration or inflammation) were associated with the vacuolation.

 

In test group 13 (15 mg/kg bw/d), the histopathological treatment-related findings are summarized as follows:

In males only, the adrenal cortex showed vacuolation of the zona fasciculata (minimal to slight); the left epididymis displayed vacuolation of approximately the distal 2/3 portion of the corpus of (minimal), and in the seminal vesicles a vacuolation of the epithelium (minimal to moderate) was noted. In following organs of test group 13, males and females were always affected:

Vacuolation was seen in the cells of the high endothelial venules (HEV) in the axillary (minimal to moderate) and mesenteric (minimal) lymph nodes. In the brain, micro and macrovesicular vacuolation (minimal to moderate) was noted in the choroid plexus of the lateral and dorsal third ventricles. As observed in the F0 generation, males were more severely affected than females. In the esophagus, the vacuolation was seen in the skeletal muscle of the wall (minimal to slight), with similar characteristics as observed in the gastrocnemius muscle. In the eyes, a very fine and minimal vacuolation was found in the retinal pigment epithelium. The affected cells displayed a ground glass aspect and were minimally enlarged. In the glandular stomach, the vacuolation (minimal to slight) was observed at the base of the glands in the pyloric mucosa. In the heart, the vacuolation (minimal to slight) affected mainly cardiomyocytes in the septum and left ventricular wall and was not associated with degeneration. In the kidneys, the vacuolation was observed in the tubules of the inner stripe of the outer medulla in both sexes (minimal to slight), whereas degeneration/regeneration occurred in the cortex (proximal convoluted tubules) in males only (minimal to slight). In the liver, the vacuolation in the hepatocytes was characterized by very small vacuoles of regular size (foamy aspect) and was more sever in females (minimal to moderate) than males (minimal to slight). A minimal associated mixed-cell inflammation was observed in females and not in males. The more severe changes in females were consistent with the significant liver weight increases. The epithelium of the portal bile ducts of large caliber also showed a vacuolation (minimal to slight) with a ground-glass appearance in both sexes. In the lungs, the vacuolation (minimal to slight) was localized in the bronchial and bronchiolar epithelium and was characterized by a “foamy” aspect. In the bronchial associated lymphoid tissue (BALT), the high endothelial venules (HEV) showed the same type of vacuolation (minimal) as described in the lymph nodes. In the pancreas, a vacuolation of the acinar epithelium was diffusely distributed along the organ (minimal to moderate). Furthermore, a vacuolation was seen in the interlobular pancreatic ducts, presenting a ground glass appearance of the epithelial cells accompanied by increase size (minimal to slight). In the pituitary gland, the vacuolation (minimal to slight) was localized in the cells of the pars distalis, giving the cells a fine “granular” and pale aspect. In the skeletal muscle (gastrocnemius muscle), the vacuolation showed similar characteristics as in the skeletal muscle of the esophageal wall: very tiny microvacuoles with a birefringent aspect within the fibers. The vacuolation was minimal in males and females and was associated with degeneration and/or regeneration of the myofibers in both sexes (minimal to slight).

 

In test group 12 (5 mg/kg bw/d), the incidence and / or grading of the treatment-related vacuolation was lower and was not associated with signs of toxicity. Organs showing vacuolation in males only were the eyes, heart, kidneys, lungs, mesenteric lymph node, pancreas and seminal vesicles. Organs with treatment-related vacuolation affecting both males and females were the axillary lymph node and esophagus.

 

As already stated in the F0 generation, all these findings in males and females of test groups 12 and 13 were regarded as treatment-related and adverse.

 

In test group 11 (1.5 mg/kg bw/d), no treatment-related findings were noted.

 

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

 

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.

 

F1 rearing animals, cohort 1B

Regarding pathology, significant decreases of the terminal body weights were seen in males and females of test groups 12 (-7% males, -5% females) and 13 (-15% males, -8% females), which were below the historical control values and were regarded as treatment-related. Consequently, significant absolute weight decreases and/or significant relative weight increases were seen in several organs, that were considered secondary to the decreased terminal body weights. In females of test group 13, the liver showed a significant absolute (+12%) and relative (+23%) weight increase. The absolute weight increase was above historical control values and was regarded as treatment-related. The significant increase of the relative liver weight in these females was regarded as treatment-related but might be in part secondarily associated with the terminal body weight decrease.

 

Cohort 3 animals (Immunotoxicity Cohort) and animals of the positive control

Regarding pathology, females in test group 14 administered with the test item, showed a significant decrease in the terminal body weight (-9%), which was considered treatment-related. No other treatment-related findings were noted.

 

F1 generation pups on PND 22 (weanlings not selected for cohorts)

Regarding pathology, a significant terminal body weight was seen in males of test group 13 (-11%), which was considered treatment-related. No other treatment-related findings were noted.

 

Concerning fertility or reproductive performance of the F0 parental animals, the mean number of implantation sites was statistically significantly below the concurrent control values in the high-dose group (12.3 / 12.1 / 11.2 and 10.3** [**= p ≤ 0.01] implants/dam in test groups 00 - 03, respectively). Secondary to that finding, the mean number of F1 pups delivered per dam was affected (9.5 versus 11.7 pups/dam in control). In the mid-dose group, the mean number of implantation sites was slightly decreased (without statistical significance) and within the range of the historical control data. However, the mean value of delivered pups per dam (10.1 pups/dam) was outside the historical control range showing statistical significance. Due to the dose dependency, the findings in the high- and mid-dose groups were assessed as treatment-related and adverse.

Neither effects on ovarian follicle count (assessed in cohort 1A females) nor on several parameters in sperm analysis of F0 parental males could be observed. Fewer fertilized oocytes may have been implanted and caused these findings in F0 parental females. All other parameters such as estrous cycle data, mating behavior, conception, gestation, parturition and lactation were not affected.

 

Concerning developmental toxicity, a slight delay in development of the high-dose offspring was observed in presence of maternal toxicity. In male and female pups, body weight was statistically significantly decreased compared to control during the lactation period (both sexes combined during PND 7-21: up to 10% below control). Body weights of the respective dams were decreased correspondingly during lactation (up to 14%, terminal body weight: 12 % below control). The decrease in pup body weight is assessed as treatment-related and most likely secondary to maternal toxicity. It is not assessed as an independent effect. Body weights of the mid- and low-dose pups were not affected. Concerning nipple bearing animals, the mean percentage of male pups reaching the criteria per litter was statistically significantly above the concurrent control values (83.8%) in the high- and mid-dose groups (95.3% and 89.6%, respectively) on PND 13, slightly outside the historical control range. During the re-examination on PND 20, no nipples/areolae were detected in any male pups of all test groups. The deviations were rather marginal and recovered completely at end of lactation indicating a temporary delay in the development of the offspring.

All other parameters, such as postnatal survival and post-weaning development until puberty, were not affected in any of the test groups.

 

Measurement of thyroid hormones revealed no effect caused by the test item, neither in the F0 parental animals nor in the F1 offspring.

 

Anogenital distance and anogenital index of all test substance treated F1 did not exhibit any effect related to the treatment.

 

Vaginal opening and preputial separation are commonly used developmental markers for onset of puberty in laboratory rats. No delays beyond a normal range of biological variation in rat (multi)generation studies which might be attributable to the treatment were noted in any of the test substance-treated groups.

 

Concerning neuropathology, treatment-related findings were seen in Cohort 2A animals (adults, PND 77) in histopathology and weight parameters. In Cohort 2B (day 22, weanlings) only an effect on terminal body weight was observed.

 

Cohort 2A (day 77, adults)

Neurohistopathology

In the eyes of animals of test groups 12 and 13 the retinal pigment epithelium was minimally vacuolated. Cervical and lumbar ganglia of test group 13 animals of both sexes showed “droplets” in the cytoplasm of the perikaryon. In trigeminus ganglia of test group 13 animals of both sexes, “vacuolation” was noted. “Droplets” and “vacuolation” constitute the same finding with different morphological manifestations depending on different histotechnical processing in cervical and lumbar ganglia as opposed to trigeminus ganglia.

 

In test group 13 animals of both sexes, multifocal degeneration of the muscle fibers of the gastrocnemius muscle characterized by fragmented muscle fibers with influx of mixed inflammatory cells was observed. Additionally, some muscle fibers showed very few tiny clear vacuoles.

 

Findings in pituitary gland and choroid plexus were very minimal in cohort 2A in contrast to findings in F0 generation parental animals and F1 rearing animals, cohort 1A. This was assumed to be due to the different fixation techniques: F0 generation parental animals and F1 rearing animals, cohort 1A were immersion fixed while cohort 2A was perfusion fixed. Both pituitary and choroid plexus showed a much smaller (shrunken) cytoplasm in cohort 2A as opposed to F0 generation parental animals and F1 rearing animals, cohort 1A, which made the detection of vacuoles in the pituitary gland almost impossible. In cohort 2A in the choroid plexus, there was additionally a moderate vacuolation also in control animals, which appeared morphologically indistinguishable from treated animals. Two females of test group 13, however, showed a very minimally increased number of vacuoles compared to controls.

 

All histopathological findings were assessed as treatment-related and adverse.

 

Weight parameters

The decreased terminal body weights of test group 13 males (not statistically significant) and females (statistically significant) were regarded to be treatment-related. The increased relative brain weights in this group in both sexes were assessed as a secondary effect to the body weight decrease.

 

Cohort 2B (day 22, weanlings)

In Cohort 2B, there was only a treatment-related body weight decrease. The decreased terminal body weights of test group 13 males (statistically significant) and females (not statistically significant) were regarded to be treatment-related. The increased relative brain weights in this group in males was assessed as a secondary effect to the body weight decrease.

 

Brain weight determination, necropsy, length/width measurements of the brain, and morphometry did not reveal any neuropathological, treatment-related findings.

 

Concerning developmental neurotoxicity, the above-mentioned (neuro-)histopathological findings of Cohort 2A animals (adults, PND 77) were comparable to those observed in parental F0 animals. Cohort 2B weanlings (PND22) showed no neuropathological findings and all other parameters of Cohort 2 animals, e.g. motor activity and functional observation battery, were not affected. Therefore, the findings in Cohort 2A adults were assessed as a direct effect of the continuing treatment with the test substance indicating systemic toxicity. Thus, the test substance did not affect the development of the nervous system.

 

There was no evidence that the test substance produced any developmental immunotoxicity. Neither T-cell dependent anti-SRBC IgM antibody response, nor absolute and relative lymphocyte subpopulation cell counts in the spleen tissue (B-, T-lymphocytes, CD4-, CD8-T-lymphocytes and natural killer (NK) cells) displayed any treatment-related changes.

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
19 April 2017 - 29 November 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
29 Jul 2016
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
Wistar
Remarks:
Crl:WI(Han)
Details on species / strain selection:
The test guideline requires the rat to be used as the animal species. This rat strain was selected since extensive historical control data are available for Wistar rats.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH/ breeder: Charles River Laboratories,France
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 13-15 wks
- Weight at study initiation: Males: ca.365 g; Females: ca.230 g
- Housing:
During the pretreatment period, rats were housed together (up to 5 animals per sex and cage) in Polysulfonate cages Typ 2000P (H-Temp) supplied by TECNIPLAST,Hohenpeißenberg, Germany.
During the study period, rats were housed individually in Polycarbonate cages type III supplied by TECNIPLAST, Hohenpeißenberg, Germany and Becker & Co., Castrop-Rauxel, Germany, with the following exceptions:
• During overnight matings, male and female mating partners were housed together in Polycarbonate cages type III.
• Pregnant animals and their litters were housed together until PND 13 in Polycarbonate cages type III.
Pregnant females were provided with nesting material (cellulose wadding) toward the end of gestation.
- Diet: ad libitum, ground Kliba maintenance diet mouse/rat “GLP” meal, supplied by Provimi Kliba SA (new name Granovit AG), Kaiseraugst, Switzerland
- Water: ad libitum, tap water
- Acclimation period: about 3 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 30-70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Vehicle:
CMC (carboxymethyl cellulose)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test substance preparations in 0.5% CMC suspension in drinking water were prepared in intervals, which considered the analytical results of the stability verification. The preparations were kept at room temperature. For the test substance preparations, the specific amount of test substance was weighed in a calibrated beaker depending on the dose group, topped up with 0.5% CMC suspension in drinking water and intensely mixed with a magnetic stirrer. Before and during administration, the preparations were kept homogeneous with a magnetic stirrer.

Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: 2 weeks
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged: Pregnant animals and their litters were housed together until PND 13 in Polycarbonate cages type III. Pregnant females were provided with nesting material (cellulose wadding) toward the end of gestation.
- Any other deviations from standard protocol: no
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The measured values for test substance in the high dose (15 mg/kg bw/d) were in the expected range of the target concentrations (90 - 110%), demonstrating the correctness of the preparations. For the midand low-doses (5 and 1.5 mg/kg bw/d), the mean values of all measured samples were slightly above the tolerance range of the test facility (116 and 115 %). However, the test substance preparations were homogeneously distributed for all dose levels. Since the deviations in the mid and low doses are only minor, the measured values were assessed to be in an acceptable range of the nominal concentrations in this study.
Duration of treatment / exposure:
The duration of treatment covered a 2-week pre-mating and a mating period, approximately 1 day post-mating in males, and the entire gestation period as well as approximately 22 days of the lactation period in females.
Frequency of treatment:
daily
Dose / conc.:
1.5 mg/kg bw/day
Dose / conc.:
5 mg/kg bw/day
Dose / conc.:
15 mg/kg bw/day
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Positive control:
no
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily
- checked 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

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Detailed clinical observations (DCO) were performed in all animals once prior to the first administration (day 0) and at weekly intervals during the administration period. The examinations started in the morning. The findings were ranked according to the degree of severity, if applicable.
For observation, the animals were removed from their cages by the investigator and placed in a standard arena (50 × 37.5 × 25 cm). The following parameters listed were assessed:
1. Abnormal behavior in “handling”
2. Fur
3. Skin
4. Posture
5. Salivation
6. Respiration
7. Activity/arousal level
8. Tremors
9. Convulsions
10. Abnormal movements
11. Gait abnormalities
12. Lacrimation
13. Palpebral closure
14. Exophthalmos (Protruding eyeball)
15. Assessment of the feces excreted during the examination (appearance/consistency)
16. Assessment of the urine excreted during the examination
17. Pupil size

BODY WEIGHT: Yes
- Time schedule for examinations:
In general, the body weight of the male and female parental animals was determined once a week at the same time of the day (in the morning) until sacrifice.
The body weight change of the animals was calculated from these results.
• During the mating period the parental females were weighed on the day of positive evidence of sperm (GD 0) and on GD 7, 14 and 20.
• Females with litter were weighed on the day of parturition (PND 0) and on PND 4, 7, 10 and 13.
Females without positive evidence of sperm, without litter or waiting for necropsy, were weighed weekly.

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
Generally, food consumption was determined once a week for male and female parental animals, with the following exceptions:
• Food consumption was not determined after the 2nd premating week (male parental animals) and during the mating period (male and female F0 animals).
• Food consumption of the F0 females with evidence of sperm was determined on gestation days (GD) 0-7, 7-14 and 14-20.
• Food consumption of F0 females which gave birth to a litter was determined on PND 1-4, 4-7, 7-10 and 10-13.
Food consumption was not determined in females without positive evidence of sperm during the mating and the gestation period and in females without litter during the lactation period.

WATER CONSUMPTION AND COMPOUND INTAKE: Yes
- Time schedule for examinations:
Generally, water consumption was determined once a week for male and female parental animals, with the following exceptions:
• Water consumption was not determined after the 2nd premating week (male parental animals) and during the mating period (male and female F0 animals).
• Water consumption of the females with evidence of sperm was determined for GD 0-1, 6-7, 13-14 and 19-20.
• Water consumption of the F0 females, which gave birth to a litter was determined for PND 1-2, 3-4, 6-7, 9-10 and 12-13.
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.

OTHER: Functional observation battery
A functional observational battery (FOB) was performed in the first 5 surviving parental male and the first 5 surviving parental female animals with litter per group (in order of delivery) at the end of the administration period starting at about 10.00 h. The FOB started in a randomized sequence with passive observations without disturbing the animals, followed by removal from the home cage, open field observations in a standard arena and sensorimotor tests as well as reflex tests. The findings were ranked according to the degree of severity, if applicable. The observations were performed at random. A detailed description of the methods, the ranking and documentation system can be found in PART III (SUPPLEMENT).

Home cage observations
The animals were observed in their closed home cages (for a short period: about 10-30 seconds); any disturbing activities (touching the cage or rack, noise) were avoided during these examinations in order not to influence the behavior of the animals. Attention was paid to:
1. Posture
2. Tremors
3. Convulsions
4. Abnormal movements
5. Gait
6. Other findings

Open field observations
The animals were transferred to a standard arena (50 × 50 × 25 cm) and observed. The following parameters were examined:
1. Behavior on removal from cage
2. Fur
3. Skin
4. Salivation
5. Nasal discharge
6. Lacrimation
7. Eyes/pupil size
8. Posture
9. Palpebral closure
10. Respiration
11. Tremors
12. Convulsions
13. Abnormal movements/stereotypy
14. Gait
15. Activity/arousal level
16. Feces (appearance/ consistency) within 2 minutes
17. Urine (amount/color) within 2 minutes
18. Rearing within 2 minutes
19. Other findings

Sensory motor tests/Reflexes
The animals were removed from the open field and subjected to following sensory motor or
reflex tests:
1. Reaction to an object being moved towards the face (Approach response)
2. Touch sensitivity (Touch response)
3. Vision (Visual placing response)
4. Pupillary reflex
5. Pinna reflex
6. Audition (Startle response)
7. Coordination of movements (Righting response)
8. Behavior during handling
9. Vocalization
10. Pain perception (Tail pinch)
11. Other findings
12. Grip strength of forelimbs
13. Grip strength of hindlimbs
14. Landing foot-splay test

Motor activity measurement
The measurement of motor activity (MA) was carried out at the end of the administration period in the first 5 surviving parental males and the first 5 surviving females with litter (in order of delivery) per group. Motor activity (MA) was measured from 14:00 h onwards on the same day as the FOB was performed. The examinations were performed using the TSE Labmaster System supplied by TSE Systems GmbH, Bad Homburg, Germany. For this purpose, the animals were placed in new clean polycarbonate cages with a small amount of bedding for the duration of the measurement. Eighteen beams were allocated per cage. The number of beam interrupts were counted over 12 intervals for 5 minutes per interval. The sequence in which the animals were placed in the cages was selected at random. On account of the time needed to place the animals in the cages, the starting time was "staggered" for each animal. The measurement period began when the 1st beam was interrupted and was finished exactly 1 hour later. No food or water was offered to the animals during these measurements and the measurement room was darkened after the transfer of the last animal.

CLINICAL PATHOLOGY
The following parameters were examined in the first 5 surviving parental males per group at termination and in the first 5 females with litters (in order of delivery) per group at PND 14:
Hematology: 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: Alanine aminotransferase(ALT) (L-alanine: 2-oxoglutarate aminotransferase; Aspartate aminotransferase (AST) (L-aspartate: 2-oxoglutarate aminotransferase; Alkaline phosphatase (ALP) (orthophosphoric acid monoester phosphohydrolase; γ-Glutamyltransferase (GGT) (γ -glutamyl) peptide: aminoacid-γ-glutamyl-transferase; Sodium, Potassium (K), Chloride (CL), Inorganic Phospahte (INP), Calcium (CA), Urea, Creatinine (CREA), Glucose (GLUC), Total bilirubin (TBIL), Total protein (TPROT), Albumin (ALB), Globilins (GLOP), Triglycerides (TRIG), Cholesterol (CHOL), Bile acids (TBA)

Thyroid hormones:
Blood samples from all dams at PND 14 and all males at termination were taken by puncturing the retrobulbar venous plexus under isoflurane anesthesia. The adults were fastened before the blood sampling.
Blood samples from the adult males were assessed for serum levels for thyroid hormones (T4 and TSH)
Oestrous cyclicity (parental animals):
For a minimum of 2 weeks prior to mating estrous cycle length was evaluated by daily analysis of vaginal smear for all F0 female parental rats. Determination was continued throughout the pairing period until the female exhibited evidence of copulation.
At necropsy, an additional vaginal smear was examined to determine the stage of estrous cycle for each F0 female with scheduled sacrifice.
Sperm parameters (parental animals):
Parameters examined in F1male parental generations:
testis weight, epididymis weight, stages of spermatogenesis in the testes and histopathology of interstitial testicular cell structures
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 8 pups/litter (4/sex/litter as nearly as possible); excess pups were killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
- number and sex of pups, stillbirths, live births, postnatal mortality, anogenital distance (AGD), pup weight on the day of AGD, presence of nipples/areolae in male pups
- live pups were examined daily for clinical symptoms (including gross-morphological findings) during the clinical inspection of the dams and documented for each pup
- Thyroid hormones:
Blood samples were taken from all surplus pups per litter at PND 4 as well as one male and one female pup per litter at PND 13 by decapitation under isoflurane anesthesia. Blood samples from the PND 13 pups were assessed for serum levels for thyroid hormones (T4 and TSH).
- On PND 13, one selected male and one female pup per litter was sacrificed under isoflurane anesthesia by decapitation. Blood was sampled for determination of thyroid hormone concentrations. Thyroid glands/parathyroid glands were fixed in neutral buffered 4% formaldehyde solution and were transferred to the Pathology Laboratory, where no further examinations were conducted.

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: No

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: No
Postmortem examinations (parental animals):
SACRIFICE
All parental animals were sacrificed by decapitation under isoflurane anesthesia. The exsanguinated animals were necropsied and assessed by gross pathology, special attention being given to the reproductive organs.

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.

ORGAN WEIGHTS
The following weights were determined in all animals sacrificed on schedule:
1. Anesthetized animals (terminal body weight)
2. Epididymides
3. Ovaries
4. Prostate
5. Seminal vesicles with coagulating glands
6. Testes
7. Thyroid glands (fixed)
8. Uterus (with cervix)
The following weights were determined in 5 animals per sex/test group sacrificed on schedule (females with litters only, same animals as used for clinical pathological examinations):
1. Adrenal glands
2. Brain
3. Heart
4. Kidneys
5. Liver
6. Spleen
7. Thymus

HISTOPATHOLOGY
The following organs or tissues of all parental animals were fixed in 4% neutral-buffered formaldehyde or in modified Davidson’s solution:
1. All gross lesions
2. Adrenal glands
3. Aorta
4. Bone marrow (femur)
5. Brain
6. Cecum
7. Cervix
8. Coagulating glands
9. Colon
10. Duodenum
11. Eyes with optic nerve (modified Davidson’s solution)
12. Esophagus
13. Extraorbital lacrimal glands
14. Epididymides (modified Davidson’s solution)
15. Femur with knee joint
16. Heart
17. Ileum
18. Jejunum (with Peyer’s patches)
19. Kidneys
20. Larynx
21. Liver
22. Lungs
23. Lymph nodes (axillary and mesenteric)
24. Mammary gland (male and female)
25. Nose (nasal cavity)
26. Ovaries (modified Davidson’s solution)
27. Oviducts
28. Pancreas
29. Parathyroid glands
30. Pharynx
31. Pituitary gland
32. Prostate gland
33. Rectum
34. Salivary glands (mandibular and sublingual)
35. Sciatic nerve
36. Seminal vesicles
37. Skeletal muscle
38. Spinal cord (cervical, thoracic and lumbar cord)
39. Spleen
40. Sternum with marrow
41. Stomach (forestomach and glandular stomach)
42. Testes (modified Davidson’s solution)
43. Thymus
44. Thyroid glands
45. Trachea
46. Urinary bladder
47. Uterus (uteri of all apparently nonpregnant animals or empty uterus horns were stained according to Salewski E, 1964)
48. Vagina
METABOLOME ANALYSIS
In males, metabolite profiling (metabolomics) in serum samples was performed.
The purpose of the metabolome study is to apply MetaMap®Tox Profiler for the test substance to demonstrate the effect on rats. This phase was expected to perform the analysis on rat plasma with LC-MS/MS and GC-MS to deliver the metabolom data for the MetaMap®Tox. For sample preparation, 60 µLof the plasma samples were extracted with methanol/dichloromethan. Internal standards were added to each sample during extraction. The extract was separated into two aliquots for analysis using LC-MS/MS and GC-MS. The aliquot used for LC-MS/MS was separated without further sample processing by two different liquid chromatographic methods (reverse-phase chromatography and HILIC chromatography) and subsequently detected by MS/MS analysis. For gas chromatographic separation, the extract was separated in two phases by adding water (lipid and polar phase). The lipid phase was treated with methanolic hydrochloric acid. Subsequently, both phases were derivatized with O-methyl-hydroxylamine hydrochloride and N-methyl-N-(trimethylsilyl)trifluoroacetamide (MSTFA). A HP5-MS GC separation column was used to separate the lipid fraction (GC lipid) and a DB-XLB GC separation column was used to separate the polar fraction (GC polar). The results of all four analyses were evaluated using Genedata software. All GC data were normalized using the internal standards. Ultimately, all data are summarized and normalized using the pool samples.
Postmortem examinations (offspring):
SACRIFICE AND GROSS NECROPSY
On PND 4, as a result of standardization, the surplus pups were sacrificed under isoflurane anesthesia by decapitation. Blood was sampled for determination of thyroid hormone concentrations. After sacrifice, the pups were examined externally and eviscerated,
and the organs were assessed macroscopically.

On PND 13, one selected male and one female pup per litter was sacrificed under isoflurane anesthesia by decapitation. Blood was sampled for determination of thyroid hormone concentrations. Thyroid glands/parathyroid glands were fixed in neutral buffered 4%
formaldehyde solution and were transferred to the Pathology Laboratory.

The remaining pups were sacrificed under isoflurane by decapitation. After sacrifice, all pups were examined externally and eviscerated, and their organs were assessed macroscopically. All stillborn pups and all pups that died before weaning were examined externally, eviscerated and their organs were assessed macroscopically. All pups without notable findings or abnormalities were discarded after their macroscopic evaluation. Animals with notable findings or abnormalities were evaluated on a case-by-case basis, depending on the type of finding noted.
Statistics:
see table 1
Reproductive indices:
see table 2
Offspring viability indices:
see table 3
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
No clinical signs or changes of general behavior which may be attributed to the test substance were detected in any male or female F0 generation parental animals at dose levels of 1.5, 5 or 15 mg/kg bw/d during premating, mating, gestation, lactation and post-mating periods. One control female had a palpable mass in its right axillary region (<1.5 cm) during PND 17-19. One low-dose female (1.5 mg/kg bw/d) showed a labored respiration with sounds on PND 0, but recovered on the same day. Piloerection (PND 0) and a pale skin (PND 0-1) were recorded for one mid-dose female; furthermore, this animal had all pups stillborn (11 dead pups). One high-dose female had all pups stillborn (2 dead pups).Two sperm positive females of test group 1 (1.5 mg/kg bw/d) and two sperm positive females of test group 3 (15 mg/kg bw/d) did not deliver F1 pups and had no implants in the uterus.These observations were not considered to be associated with the test compound.
Mortality:
no mortality observed
Description (incidence):
There were no test substance-related or spontaneous mortalities in any of the groups.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
The body weights of the high-dose F0 parental males (15 mg/kg bw/d) were statistically significantly below the concurrent control on premating day 13 (-5%) and on mating days 7 and 14 (up to 8% below control). The body weights of the high-dose F0 parental females were statistically significantly reduced during lactation on PND 10 and 13 (up to 9% below control). There were no statistically significant effects on body weights during premating and gestation in this dose group. The body weights of the low- and mid-dose F0 males and females (1.5 and 5 mg/kg bw/d) were comparable to the concurrent control values throughout the entire study. A consistently lower body weight gain was noted in the high-dose F0 parental males (15 mg/kg bw/d) which became statistically significant during the entire premating and mating periods. A statistically significantly lower body weight gain was also noted for the low- and mid-dose F0 males (1.5 and 5 mg/kg bw/d) during premating. Since there was no effect on body weight gain in these test groups during the subsequent mating, the lower body weight gain of test groups 1 and 2 was assessed as treatment-related but not adverse. The body weight gain of the high-dose F0 parental females was reduced during lactation, attaining statistical significance when calculated for PND 0-13 (about 67% below the control value).The body weight gain of the low- and mid-dose F0 females was comparable to the concurrent control values throughout the entire study. The decrease in body weight (gain) of high-dose male and females was assessed as treatment-related and adverse.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
A statistically significantly reduced food consumption was recorded for the high-dose F0 males (15 mg/kg bw/d) during the entire premating period (up to 13% below the concurrent control group) and the mid-dose F0 males (5 mg/kg bw/d) during premating days 7-13 (about 8% below control). Since the statistically significant decrease in mid-dose males was only observable in a short time period (premating days 7-13), it was assessed as treatment-related but not as adverse. Food consumption of the low-dose males (1.5 mg/kg bw/d) was comparable to the concurrent control values. Food consumption of the F0 females in all dose groups (1.5, 5 and 15 mg/kg bw/d) was not statistically significantly altered throughout the entire study period. However, a decrease in food consumption was observable in the high-dose females during lactation (up to 14 % below control). The decrease in food consumption of the high-dose males and females was assessed as treatment-related and adverse, in combination with the decrease in body weight (gain).
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
effects observed, non-treatment-related
Description (incidence and severity):
Water consumption of all F0 parental male rats of all test groups was comparable to the concurrent control throughout the entire study. Water consumption of the female F0 rats in test groups 2 and 3 (5 and 15 mg/kg bw/d) was statistically significantly reduced during premating days 7-10 (about 22% in test group 2, about 27% in test group 3 in comparison to the concurrent control group). Water consumption mean values during gestation and lactation periods in these dose groups were not statistically significantly altered and showed a high variation partly without dose dependency. Since water consumption was only temporarily affected in one time period during the study, it was not assessed as adverse. Water consumption of the female F0 rats in test group 1 (1.5 mg/kg bw/d) was comparable to the concurrent control throughout the entire study.
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
After the administration period in males of test group 3 (15 mg/kg bw/d) total white blood cell (WBC) counts, absolute lymphocyte and monocyte counts as well as platelet counts were significantly increased. Additionally, in males of test group 2 and 3 (5 and 15 mg/kg bw/d) relative eosinophil counts were significantly decreased, but the values were within historical control ranges (males, relative eosinophils 1.3-2.7 %). Total WBC counts were also significantly higher compared to controls in males of test group 1 (1.5 mg/kg bw/d), but in this group WBC counts were not dose-dependently changed. Therefore, relative eosinophil count changes in males of test groups 2 and 3 as well as total WBC count alterations in males of test group 1 were regarded as incidental and not treatment-related.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
After the administration period, in male and female rats of test group 3 (15 mg/kg bw/d) aspartate aminotransferase (AST) activities were significantly increased (in females only). Additionally, in females of the mentioned test group inorganic phosphate levels were significantly higher compared to controls. In males of test group 2 (5 mg/kg bw/d) sodium levels were significantly decreased, but the values were not dose-dependently changed and, therefore, this alteration was regarded as incidental and not treatment-related.
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
No test substance-related or spontaneous findings were observed in male and female animals of all test groups during the home cage observation. The open field observations did not reveal any test substance-related findings in male and female animals of all test groups. No test substance-related impaired parameters were observed in male and female animals of all test groups. The statistically significantly increased FST value in the low-dose F0 males was considered as incidental since there was no relation to dose.
No statistically significant changes on motor activity data (summation of all intervals) was observed in all male and female animals of all dose groups in comparison to the concurrent control group.
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):
Treatment-related findings were observed in the liver, brain (choroid plexus), mesenteric and axillary lymph nodes (high endothelial venules = HEV) and glandular stomach (pyloric region) of males and females females.
Liver: The hepatocytes were characterized by a very fine cytoplasmic vacuolation (microvesicular pattern) which was centrilobular accentuated in males and revealed a diffuse distribution pattern in females. It proved to be PAS and Oil-Red-O negative. The Sudan Black stain was positive in the female with severity grade 3.
Brain: The epithelial cells of the choroid plexus, predominantly in the lateral ventricles, showed a microvesicular vacuolation comparable to that seen in the liver.
Axillary and mesenteric lymph nodes: The high endothelial venules (HEV) in the mesenteric as well as in the axillary lymph nodes showed a prominent, clear aspect due to a microvesicular vacuolation within the walls.
Glandular stomach: The epithelial cells at the base of the glands of the pyloric mucosa within the glandular stomach showed a prominent and clear aspect due to the presence of a microvesicular vacuolation pattern similar to that observed in the liver.

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 stages of spermatogenesis in the testes of males of the high dose (15 mg/kg bw/d) were comparable to those of the controls. In the ovaries of control and high dose females the different stages of functional bodies (especially corpora lutea) were present and normal.

The female animals, which were not pregnant and their male mating partners did not show relevant histopathological findings that could justify the lack of offspring.
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Description (incidence and severity):
Thyroid hormones:
In parental males of test group 3 (15 mg/kg bw/d) T4 levels were significantly decreased without any change of TSH values. T4 means were within the historical control range (males T4 44.87-88.29 nmol/L). Neither any thyroid weight change nor any histopathological alteration was found in these individuals. Therefore, the isolated decrease of T4 in males of test group 3 within historical control ranges was regarded as incidental and not treatment-related.
Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
Estrous cycle data, generated during the last 2 weeks prior to mating for the F1 litter, revealed regular cycles in the parental females of all test groups including the control. The mean estrous cycle duration in the different test groups (0-3) was between 3.8 and 3.9 days.
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
effects observed, non-treatment-related
Description (incidence and severity):
The female and male mating index calculated after the mating period for F1 litter was 100% in all test groups. The fertility index ranged between 80% and 100% without showing any relation to dosing. These values reflect the normal range of biological variation inherent in the strain of rats used for this study. The gestation index was 100% in test groups 0 and 1, 90% in test group 2 and 87.5% in test group 3. The mean value of test group 3 was at the lower end of the range of the historical control data (HCD; gestation index, range: 87.5 – 100 %). Implantation was not affected by the treatment since the mean number of implantation sites was comparable between all test substance-treated groups and the control, taking normal biological variation into account (13.3 / 13.6 / 14.0 and 11.9 implants/dam in test groups 0-3, respectively). Furthermore, there were no indications for test substance-induced intrauterine embryo-/fetolethality since the postimplantation loss did not show any significant differences between the groups, and the mean number of F1 pups delivered per dam remained unaffected (13.0 / 12.8 / 12.5 and 10.8 pups/dam in test groups 0-3, respectively). The rate of liveborn pups was also not affected by the test substance, as indicated by live birth indices of 99.2% / 100% / 89.6% and 96.5% in test groups 0-3, respectively. Moreover, the number of stillborn pups was not significantly different between the test groups. One female, each, of test groups 2 and 3 had all pups stillborn. This finding in test groups 3 and 2 can be found in the historical control at comparable incidences (HCD; number of females with all pups stillborn, range 0 - 1). These females had also a prolonged duration of gestation (24 and 23 days, respectively) compared to the group mean value of 22.1 days for both test groups. The prolonged duration of gestation may be the reason for the stillborn pups in the respective litters. Since all values of the parameters gestation index and number of females with all pups stillborn were within the historical control range, the findings were not assessed as treatment-related and adverse.
Key result
Dose descriptor:
NOAEL
Remarks:
systemic
Effect level:
5 mg/kg bw/day
Sex:
male/female
Basis for effect level:
body weight and weight gain
food consumption and compound intake
clinical biochemistry
histopathology: non-neoplastic
Key result
Dose descriptor:
NOAEL
Remarks:
reproductive performance and fertility
Effect level:
15 mg/kg bw/day
Sex:
male/female
Basis for effect level:
other: no adverse effects up to the highest dose tested
Critical effects observed:
no
Lowest effective dose / conc.:
15 mg/kg bw/day (nominal)
System:
hepatobiliary
Organ:
liver
Critical effects observed:
no
Lowest effective dose / conc.:
15 mg/kg bw/day (nominal)
System:
central nervous system
Organ:
brain
Critical effects observed:
no
Lowest effective dose / conc.:
15 mg/kg bw/day (nominal)
System:
gastrointestinal tract
Organ:
stomach
Critical effects observed:
no
Lowest effective dose / conc.:
15 mg/kg bw/day (nominal)
System:
immune system
Organ:
lymph node
Clinical signs:
no effects observed
Description (incidence and severity):
There were no test substance-related adverse clinical signs observed in any of the F1 generation pups of the different test groups.
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
The rate of liveborn pups was not affected by the test substance, as indicated by live birth indices of 99.2% / 100% / 89.6% and 96.5% in test groups 0-3, respectively. Moreover, the number of stillborn pups was not significantly different between the test groups. One female, each, of test groups 2 and 3 had all pups stillborn. This finding in test groups 3 and 2 can be found in the historical control at comparable incidences (HCD; number of females with all pups stillborn, range 0 - 1). These females had also a prolonged duration of gestation (24 and 23 days, respectively) compared to the group mean value of 22.1 days for both test groups. The prolonged duration of gestation may be the reason for the stillborn pups in the respective litters. Since all values of the parameters gestation index and number of females with all pups stillborn were within the historical control range, the findings were not assessed as treatment-related and adverse.
The viability index indicating pup survival during early lactation (PND 0-4) varied between 92.7%/ 97.8% / 98.3% and 97.8% in test groups 0-3, respectively. The survival index indicating pup survival during lactation (PND 4-13) was 100% in all test groups.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
No test substance-related influence on body weights and body weight change values of F1 pups were noted in test groups 1-3 (1.5, 5 and 15 mg/kg bw/d). One male runt was seen, each, in test groups 1 and 3; furthermore, one female runt was seen, each, in test groups 2 and 3.
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:
no effects observed
Description (incidence and severity):
In male and female pups at PND13 (1.5; 5 and 15 mg/kg bw/d), no treatment-related alterations of T4 and TSH levels were observed.
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
not examined
Anogenital distance (AGD):
no effects observed
Description (incidence and severity):
Neither on anogenital distance nor anogenital index test substance-related effects were noted in all treated F1 offspring (test groups 1-3; 1.5, 5 and 15 mg/kg bw/d).
Nipple retention in male pups:
effects observed, non-treatment-related
Description (incidence and severity):
The apparent number and percentage of male pups having areolae was not influenced by the test substance when examined on PND 13. The mean number of nipple / areolae in the respective male pups was statistically significantly increased in test group 2 (2.48 / 2.76 / 3.35* / 2.76 [p ≤ 0.05] in test group 0-3). Since the coherent incidence of nipple-bearing males in test group 2 was not affected and there was no relation to dose, this finding was not assessed as treatment-related.
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
A few pups showed spontaneous findings at gross necropsy, such as post mortem autolysis and partly cannibalized. These findings occurred without a relation to dosing and are considered to be spontaneous in nature. One mid-dose F1 pup had a small mouth and its lower jaw was absent; these findings were confirmed by a skeletal examination (comprising severely malformed skull bones). Since this finding showed no relation to dose, it was assessed as not treatment-related.
Histopathological findings:
not examined
Other effects:
no effects observed
Description (incidence and severity):
The mean number of delivered F1 pups per dam and the rates of liveborn, stillborn, found dead and cannibalized F1 pups were evenly distributed about the test groups. The respective values reflect the normal range of biological variation inherent in the strain used in this study.
The sex distribution and sex ratios of live F1 pups on the day of birth and PND 13 did not show substantial differences between the control and the test substance-treated groups; slight differences were regarded to be spontaneous in nature.
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
Key result
Dose descriptor:
NOAEL
Remarks:
developmental toxicity
Generation:
F1
Effect level:
15 mg/kg bw/day
Sex:
male/female
Basis for effect level:
other: no effects up to high dose tested
Critical effects observed:
no
Key result
Reproductive effects observed:
no

Table 1: The liver revealed the following findings:

Liver

Male animals

Female animals

Test group

0

1

2

3

0

1

2

3

(mg/kg bw/d)

(0)

(1.5)

(5)

(15)

(0)

(1.5)

(5)

(15)

No. of animals

10

10

10

10

10

10

10

10

Vacuolation,

0

0

0

7

0

0

0

10

hepatocellular

 

 

 

 

 

 

 

 

  Grade 1

 

 

 

5

 

 

 

7

  Grade 2

 

 

 

2

 

 

 

2

  Grade 3

 

 

 

 

 

 

 

1

 

Table 2: In the brain the following findings were seen:

Brain

Male animals

Female animals

Test group

0

1

2

3

0

1

2

3

(mg/kg bw/d)

(0)

(1.5)

(5)

(15)

(0)

(1.5)

(5)

(15)

No. of animals

10

10

10

10

10

10

10

10

Vacuolation, choroid

0

0

0

10

0

0

0

9

plexus

 

 

 

 

 

 

 

 

  Grade 1

 

 

 

3

 

 

 

5

  Grade 2

 

 

 

7

 

 

 

4

 

Table 3: The axillary and mesenteric lymph nodes revealed the following findings

Lymph nodes

Male animals

Female animals

Test group

(mg/kg bw/d)

0

(0)

1

(1.5)

2

(5)

3
(15)

0

(0)

1

(1.5)

2

(5)

3

(15)

No. of animals

10

10

10

10

10

10

10

10

Axillary lymph node

 

 

 

 

 

 

 

 

Vacuolation, HEV

0

0

0

8

0

0

0

9

  Grade 1

 

 

 

8

 

 

 

9

Mesenteric lymph node

 

 

 

 

 

 

 

 

Vacuolation, HEV

0

0

0

7

0

0

0

5

  Grade 1

 

 

 

7

 

 

 

5

 

Table 4: Glandular stomach

Glandular stomach

Male animals

Female animals

Test group

0

1

2

3

0

1

2

3

(mq/kq bw/d)

(0)

(1.5)

(5)

(15)

(0)

(1.5)

(5)

(15)

No. of animals

10

10

10

10

10

10

10

10

Vacuolation, glandular

0

0

0

7

0

0

0

8

  Grade 1

 

 

 

7

 

 

 

8

Table 5: Summary Estrous Cycles Report

Sex: Female - Phase: Pre-mating

 

 

Test Group 0/ F

Test Group 1/ F

Test Group 2/ F

Test Group 3/ F

 

 

0 mg/kg bw/d

1.5 mg/kg bw/d

5 mg/kg bw/d

15 mg/kg bw/d

Number of Cycles

Mean

2.9 k

3.0

2.8

2.8

 

S.d.

0.3

0.0

0.4

0.4

 

N

10

10

10

10

Cycles Length (days)

Mean

3.8 k

3.9

3.9

3.9

 

S.d.

0.2

0.1

0.2

0.1

 

N

10

10

10

10

Cycling Normally (3-6 Days)

N

10

10

10

10

 

%

100.0

100.0

100.0

100.0

Long Estrous (3 Days)

N

0

0

0

0

 

%

0.0

0.0

0.0

0.0

Long Diestrous (4 Days)

N

0

0

0

0

 

%

0.0

0.0

0.0

0.0

 Statistic Profile = Kruskal-Wallis + Wilcoxon test (two-sided), * p<=0.05, ** p <=0.01, X = Group excluded from statistics k=KRUSKALL-WALLIS

 

Table 6: Summary Mating Report

 

 

Test Group 0/ F

Test Group 1/ F

Test Group 2/ F

Test Group 3/ F

 

 

0 mg/kg bw/d

1.5 mg/kg bw/d

5 mg/kg bw/d

15 mg/kg bw/d

No. of females mated

N

10

10

10

10

- Inseminated

N

10 f-

10

10

10

Female mating index

%

100.0

100.0

100.0

100.0

-- Pregnant

N

10 f-

8

10

8

Female fertility index

%

100.0

80.0

100.0

80.0

No. of males mated

N

10

10

10

10

- With inseminated females

N

10 f-

10

10

10

Male mating index

%

100.0

100.0

100.0

100.0

- With pregnant females

N

10 f-

8

10

8

Male fertility index

%

100.0

80.0

100.0

80.0

Females with defined Day 0 pc

N

10

10

10

10

Mating days until Day 0 pc

Mean

1.8 X+

4.1 *

2.9

2.8

 

S.d.

1.1

3.3

1.3

2.1

 

N

10

10

10

10

Days 0 To 4

N

10

9

10

9

 

%

100.0

90.0

100.0

90.0

Days 5 To 9

N

0

0

0

1

 

%

0.0

0.0

0.0

10.0

Days 10 To 14

N

0

1

0

0

 

%

0.0

10.0

0.0

0.0

Statistic Profile = Fisher's exact test (one-sided-), Wilcoxon with Bonferroni-Holm (one-sided+), * p<=0.05, ** p <=0.01, X = Group excluded

from statistics f=FISHER-EXACT; x=WILCOX


 Table 7: Summary Pregnancy Status Report - Reproduction

Sex: Female

 

 

 

Test Group 0/ F

Test Group 1/ F

Test Group 2/ F

Test Group 3/ F

 

 

0 mg/kg bw/d

1.5 mg/kg bw/d

5 mg/kg bw/d

15 mg/kg bw/d

No. of females at start

N

10

10

10

10

No. of females mated

N

10

10

10

10

Without evidence of mating

N

0

0

0

0

-- Pregnant

N

0

0

0

0

- Not pregnant

N

0

0

0

0

Females with defined Day 0 pc

N

10

10

10

10

Pregnant

N

10

8

10

8

- sacrificed scheduled

N

10

8

10

8

Not pregnant

N

0

2

0

2

- sacrificed scheduled

N

0

2

0

2

Pregnant, not delivering

N

0

0

0

0

Delivering

N

10

8

10

8

-- With liveborn pups

N

10

8

9

7

 

%

100.0

100.0

90.0

87.5

-- With all pups stillborn

N

0

0

1

1

 

%

0.0

0.0

10.0

12.5

 

 

Table 8: Summary Delivery Report

Sex:Female

 

 

Test Group 0/ F

Test Group 1/ F

Test Group 2/ F

Test Group 3/ F

 

 

0 mg/kg bw/d

1.5 mg/kg bw/d

5 mg/kg bw/d

15 mg/kg bw/d

No. of females at start

N

10

10

10

10

No. of females mated

N

10 f-

10

10

10

 

%

100.0

100.0

100.0

100.0

Pregnant

N

10 f-

8

10

8

 

%

100.0

80.0

100.0

80.0

Without delivery

N

0

2

0

2

-- Pregnant

N

0-

0

0

0

- Not pregnant

N

0

2

0

2

Delivering

N

10 f-

8

10

8

 

%

100.0

100.0

100.0

100.0

-- With liveborn pups

N

10 f-

8

9

7

Gestation Index

%

100.0

100.0

90.0

87.5

Gestation days

Mean

22.3 n

22.4

22.1

22.1

 

S.d.

0.5

0.5

0.7

0.4

 

N

10

8

10

8

-- With stillborn pups

N

1 f+

0

3

2

 

%

10.0

0.0

30.0

25.0

-- With all pups stillborn

N

0 f+

0

1

1

 

%

0.0

0.0

10.0

12.5

Statistic Profile = Fisher's exact test (one-sided-), Dunnett test (two-sided), Fisher's exact test (one-sided+), * p<=0.05, ** p <=0.01,

X = Group excluded from statistics, f=FISHER-EXACT; n=DUNNETT

 

Table 9:Summary Litter Report - Pup Status

Sex: Female

 

 

 

Test Group 0/ F

Test Group 1/ F

Test Group 2/ F

Test Group 3/ F

 

 

0 mg/kg bw/d

1.5 mg/kg bw/d

5 mg/kg bw/d

15 mg/kg bw/d

Total Number of Pregnant Females

N

10

8

10

8

Total number of litters

N

10

8

10

8

-- With liveborn pups

N

10 f-

8

9

7

 

%

100.0

100.0

90.0

87.5

-- With stillborn pups

N

1 f+

0

3

2

 

%

10.0

0.0

30.0

25.0

-- With all pups stillborn

N

0 f+

0

1

1

 

%

0.0

0.0

10.0

12.5

Implantation Sites

N

133

109

140

95

 

Mean

13.3 X-

13.6

14.0

11.9

 

S.d.

3.2

3.1

1.5

4.9

 

N

10

8

10

8

Pups delivered

N

130

102

125

86

 

Mean

13.0 X-

12.8

12.5

10.8

 

S.d.

3.1

3.4

2.8

3.9

 

N

10

8

10

8

Postimplantation Loss

Mean%

2.1 X+

7.1

10.9

7.1

 

S.d.

3.4

8.2

16.7

9.7

 

N

10

8

10

8

 Statistic Profile = Wilcoxon with Bonferroni-Holm (one-sided-), Wilcoxon with Bonferroni-Holm (one-sided+), Wilcoxon test (two-sided),

Fisher's exact test (one-sided-), Fisher's exact test (one-sided+), * p<=0.05, ** p <=0.01, X = Group excluded from statistics

f=FISHER-EXACT; x=WILCOX

 

Table 10: Summary Litter Report - Pup Status

Sex: Female

 

 

 

Test Group 0/ F

Test Group 1/ F

Test Group 2/ F

Test Group 3/ F

 

 

0 mg/kg bw/d

1.5 mg/kg bw/d

5 mg/kg bw/d

15 mg/kg bw/d

Pups liveborn

N

129

102

112

83

 

%

99.2

100.0

89.6

96.5

 

Mean

12.9 X-

12.8

11.2

10.4

 

S.d.

3.0

3.4

4.8

4.5

 

N

10

8

10

8

Pups stillborn

N

1

0

13

3

 

%

0.8

0.0

10.4

3.5

 

Mean

0.1 X+

0.0

1.3

0.4

 

S.d.

0.3

0.0

3.4

0.7

 

N

10

8

10

8

Perinatal Loss

Mean%

0.7 X+

0.0

11.5

13.6

 

S.d.

2.1

0.0

31.2

35.0

 

N

10

8

10

8

 Statistic Profile = Wilcoxon with Bonferroni-Holm (one-sided-), Wilcoxon with Bonferroni-Holm (one-sided+), Wilcoxon test (two-sided),

Fisher's exact test (one-sided-), Fisher's exact test (one-sided+), * p<=0.05, ** p <=0.01, X = Group excluded from statistics x=WILCOX

Table 11:Summary Litter Report - Dead Pups

Sex: Female

 

 

 

Test Group 0/ F

Test Group 1/ F

Test Group 2/ F

Test Group 3/ F

 

 

0 mg/kg bw/d

1.5 mg/kg bw/d

5 mg/kg bw/d

15 mg/kg bw/d

Litters with liveborn pups

N

10

8

9

7

Pups delivered

N

130

102

125

86

found dead / Dead

N

0

0

1

1

 

%

0.0

0.0

0.8

1.2

stillborn / Dead

N

1

0

13

3

 

%

0.8

0.0

10.4

3.5

Alive / Alive

N

129

102

112

83

 

%

99.2

100.0

89.6

96.5

cannibalized / Dead

N

5

3

1

1

 

%

3.8

2.9

0.8

1.2

sacrificed scheduled / Dead

N

74

63

70

56

 

%

56.9

61.8

56.0

65.1

culled / Dead

N

50

36

40

25

 

%

38.5

35.3

32.0

29.1

Litters not surviving Day 13

N

0

0

1

1

 

%

0.0

0.0

10.0

12.5

 Statistic Profile = Wilcoxon with Bonferroni-Holm (one-sided-), Wilcoxon with Bonferroni-Holm (one-sided+), Wilcoxon test (two-sided),

Fisher's exact test (one-sided-), Fisher's exact test (one-sided+), * p<=0.05, ** p <=0.01, X = Group excluded from statistics

Table 12:Summary Litter Report - Pups Died

Sex: Female

 

 

 

Test Group 0/ F

Test Group 1/ F

Test Group 2/ F

Test Group 3/ F

 

 

0 mg/kg bw/d

1.5 mg/kg bw/d

5 mg/kg bw/d

15 mg/kg bw/d

Litters with liveborn pups

N

10

8

9

7

Pups delivered

N

130

102

125

86

Days 0 To 0

N

0

0

0

1

 

%

0

0

0

1.2

Days 1 To 4

N

5

3

2

1

 

%

3.8

2.9

1.6

1.2

Days 5 To 7

N

0

0

0

0

 

%

0

0

0

0

Days 8 To 13

N

0

0

0

0

 

%

0

0

0

0

Pups surviving days 0 To 4

N

124

99

110

81

Viability Index

Mean %

92.7 X-

97.8

98.3

97.8

 

S.d.

19.0

6.2

3.4

5.8

 

N

10

8

9

7

Pups surviving days 4 To 13

N

74

63

70

56

Survival Index

Mean %

100 NA

100.0

100.0

100.0

 

S.d.

0.0

0.0

0.0

0.0

 

N

10

8

9

7

Statistic Profile = Wilcoxon with Bonferroni-Holm (one-sided-), Wilcoxon with Bonferroni-Holm (one-sided+), Wilcoxon test (two-sided),

Fisher's exact test (one-sided-), Fisher's exact test (one-sided+), * p<=0.05, ** p <=0.01, X = Group excluded from statistics x=WILCOX;

NA=No Test Applicable

 

Table 13: Summary Litter Report - Live Pups/Litter

Sex: Female

 

 

 

Test Group 0/ F

Test Group 1/ F

Test Group 2/ F

Test Group 3/ F

 

 

0 mg/kg bw/d

1.5 mg/kg bw/d

5 mg/kg bw/d

15 mg/kg bw/d

Litters with liveborn pups

N

10

8

9

7

Pups delivered

N

130

102

125

86

Days 0

Mean

12.9 X-

12.8

12.4

11.9

 

S.d.

3.0

3.4

2.9

1.9

 

N

10

8

9

7

Days 4

Mean

12.4 X-

12.4

12.2

11.6

 

S.d.

3.8

3.0

2.9

1.8

 

N

10

8

9

7

Days 7

Mean

7.4 X-

7.9

7.8

8.0

 

S.d.

1.9

0.4

0.7

0.0

 

N

10

8

9

7

Days 13

Mean

7.4 X-

7.9

7.8

8.0

 

S.d.

1.9

0.4

0.7

0.0

 

N

10

8

9

7

 Statistic Profile = Wilcoxon with Bonferroni-Holm (one-sided-), Wilcoxon with Bonferroni-Holm (one-sided+), Wilcoxon test (two-sided),

Fisher's exact test (one-sided-), Fisher's exact test (one-sided+), * p<=0.05, ** p <=0.01, X = Group excluded from statistics x=WILCOX

 

Table 14: Summary Litter Report - Live Pups/Litter

Sex: Female

 

 

 

Test Group 0/ F

Test Group 1/ F

Test Group 2/ F

Test Group 3/ F

 

 

0 mg/kg bw/d

1.5 mg/kg bw/d

5 mg/kg bw/d

15 mg/kg bw/d

Litters with liveborn pups

N

10

8

9

7

Pups delivered

N

130

102

125

86

% Live male Day 0

Mean%

57.1 x

48.4

49.6

53.5

 

S.d.

11.3

16.7

14.0

16.8

 

N

10

8

9

7

% Live female Day 0

Mean%

42.9 x

51.6

50.4

46.5

 

S.d.

11.3

16.7

14.0

16.8

 

N

10

8

9

7

% Live male Day 13

Mean%

50.0 x

46.0

48.6

51.8

 

S.d.

0.0

8.8

4.2

11.2

 

N

10

8

9

7

% Live female Day 13

Mean%

50.0 x

54.0

51.4

48.2

 

S.d.

0.0

8.8

4.2

11.2

 

N

10

8

9

7

Statistic Profile = Wilcoxon with Bonferroni-Holm (one-sided-), Wilcoxon with Bonferroni-Holm (one-sided+), Wilcoxon test (two-sided),

Fisher's exact test (one-sided-), Fisher's exact test (one-sided+), * p<=0.05, ** p <=0.01, X = Group excluded from statistics x=WILCON

Metabolome

Regarding metabolome analysis, 2,2'-dimethyl-4,4'-methylenebis (cyclohexylamine) induced only slight metabolome changes at the respective dose levels. At 15 mg/kg bw/d, 6.8% of metabolites were significantly changed, whereas at 5 mg/kg bw/d only 3.4% of the metabolites differed significantly from the control lying even below the false discover rate of 5%. The treatment with 2,2'-dimethyl-4,4'-methylenebis (cyclohexylamine) had only a weak impact on the plasma metabolome of male rats in this study. Based on the here obtained findings, no conclusion on the toxicity of the test substance can be drawn.

Effect on fertility: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
1.5 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
OECD TG 443
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

OECD 422 (BASF, 2019)

In a study according OECD TG 422 the substance was administered daily as an aqueous preparation to groups of 10 male and 10 female Wistar rats (F0 animals) by gavage at doses of 1.5, 5.0 and 15 mg/kg bw/day. Control animals (10 male and 10 female Wistar rats) were dosed daily with the vehicle only (0.5% Sodium carboxymethyl cellulose [0.5% CMC] suspension in drinking water). The duration of treatment covered a 2-week pre-mating and a mating period, approximately 1 day post-mating in males, and the entire gestation period as well as approximately 22 days of the lactation period in females.

After 2 weeks of premating treatment the F0 animals were mated to produce F1 generation pups. Mating pairs were from the same test group. Mating was discontinued as soon as sperm were detected in the vaginal smear. F0 animals were examined for their reproductive performance including determination of the number of implantation sites and the calculation of postimplantation loss for all F0 females.

A detailed clinical observation (DCO) was performed in all animals before the start of the administration period and, as a rule, thereafter at weekly intervals. Water consumption of the F0 parents was determined once weekly during premating (each time for a period of 3 days). In dams, water consumption was determined for gestation days (GD) 0-1, 6-7, 13-14 and 19-20 and lactation days (PND) 1-2, 3-4, 6-7, 9-10 and 12-13.

Food consumption of the F0 parents was determined once weekly during premating. In dams, food consumption was determined for GD 0-7, 7-14, 14-20 and PND 1-4, 4-7, 7-10 and 10-13. Body weights of F0 parents were determined once a week, in males throughout the study and in females during premating. During gestation and lactation period, F0 females were weighed on GD 0, 7, 14 and 20, on the day of parturition (PND 0) and on PND 4, 7, 10 and 13.

Estrous cycle data were evaluated for F0 generation females over a 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 pups were sexed and examined for macroscopically evident changes on PND 0. They were weighed on PND 1, 4, 7 and 13. Their viability was recorded. At necropsy on PND 13, all pups were sacrificed and examined macroscopically for external and visceral findings.

Anogenital distance measurements were conducted in a blind randomized fashion, using a measuring ocular on all live male and female pups on PND 1.

All surviving pups were examined for the presence or absence of nipple/areola anlagen on PND 13.

Blood samples were taken from all surplus pups at PND 4 as well as one male and one female pup per litter at PND 13 by decapitation under isoflurane anesthesia for hormone measurement.

Clinico-chemical and hematological examinations were performed in the first 5 surviving parental males per group at termination and the first 5 surviving parental females with litter (in order of delivery) per group at PND 14.

Blood samples from all dams at PND 14 and all males at termination were taken by puncturing the retrobulbar venous plexus under isoflurane anesthesia for hormone measurement.

In males, metabolite profiling (metabolomics) in serum samples was performed.

At the end of the administration period a functional observational battery was performed and motor activity was measured in 5 parental males and females per group.

All F0 parental animals were assessed by gross pathology. Weights of selected organs were recorded and a histopathological examination was performed.

Analytical measurements demonstrated the stability of the test substance preparations, confirmed the homogeneous distribution of the test substance in the vehicle and verified correct concentrations of the test substance preparations in the high dose. For the mid- and low-doses (5 and 1.5 mg/kg bw/d), the mean values were slightly above the tolerance range of the test facility (116 and 115 %, respectively). However, the test substance preparations were homogeneously distributed for all dose levels. Since the deviations in the mid and low doses are only minor, the measured values were assessed to be in an acceptable range of the nominal concentrations in this study.

The following results have been observed for the highest dose group animals:

A.   F0 parental generation (15 mg/kg bw/d)

-         Decrease in body weight (change) combined with a reduction in food consumption in both sexes

-         Increased total white blood cell (WBC), absolute lymphocyte and monocyte counts in males

-         Increased platelet counts in males

-         Increased aspartate aminotransferase (AST) activities in both sexes

-         Increased inorganic phosphate levels in females

-         Liver: Vacuolation, hepatocellular: 7 out of 10 males (minimal to slight) and all females (minimal to moderate)

-         Brain: Vacuolation, choroid plexus: all males and 9 out of 10 females (minimal to slight)

-         Axillary lymph nodes: Vacuolation, high endothelial venules: 8 out of 10 males and 9 out of 10 females (minimal)

-         Mesenteric lymph node: Vacuolation, high endothelial venules: 7 out of 10 males and 5 out of 10 females (minimal)

-         Glandular stomach: Vacuolation, glandular: 7 out of 10 males and 8 out of 10 females (minimal)

B.   F1 Pups (15 mg/kg bw/d)

-         No test substance-related, adverse findings were noted.

In the low-and mid-dose groups (1 or 5 mg/kg bw/d) no test substance-related, adverse findings were noted for the F0 parental animals and F1 pups.

In conclusion, under the conditions of the OECD 422 combined repeated dose toxicity study with the reproductive/developmental screening test in Wistar rats the oral administration of the test item by gavage to male and female Wistar rats resulted in signs of systemic toxicity at the highest dose of 15 mg/kg bw/d such as a reduction in food consumption and decrease in body weight (change), adverse changes in clinical pathology parameters and histopathology. Thus, the NOAEL for general systemic toxicity was 5 mg/kg bw/d for male and female Wistar rats. The NOAEL for reproductive performance, fertility and developmental toxicity was set to 15 mg/kg bw/d for male and female Wistar rats.

Discussion:

Regarding clinical examinations, adverse signs of toxicity were observed at the highest test group (15 mg/kg bw/d). Mid-dose males and females showed only minor, temporary and inconsistent changes in water consumption, food consumption and body weight during the whole study period which were assessed to be insufficient to proof adversity (see below).

Water consumption in high- and mid-dose females showed a minor reduction which was assessed as not adverse due to the high variation over the whole study period. Males of all test groups showed no treatment-related changes in water consumption.

A statistically significant decrease in body weight (change) (BW: up to 9 % below control) in combination with a (statistically significantly) reduced food consumption in test group 3 males and females (up to 14 % below control) was assessed as treatment-related and adverse.

Mid-dose F0 males showed a statistically significant reduction in food consumption (about 8% below control), only observable in a short time period (premating days 7-13) which was assessed as treatment-related but not adverse.

Mid- and low-dose F0 males showed a statistically significantly lower body weight gain during premating. Since there was no effect on body weight gain in these test groups during the subsequent mating, the lower body weight gain of test groups 1 and 2 was assessed as treatment-related but not adverse.

Mid- and low dose females showed no treatment-related changes in food consumption and body weight (change).

Regarding clinical pathology, increased total white blood cell counts, absolute lymphocyte and monocyte counts in males of test group 3 (15 mg/kg bw/d) indicated an acute phase reaction.

This may also involve the coagulation system and resulted in higher platelet counts in these individuals. Isolated higher aspartate aminotransferase (AST) activities in male and female rats of test group 3 (15 mg/kg bw/d) without any other change of liver parameters may be due to an affection of any other organ because this enzyme is regarded as not organ-specific in rats. A reason for the increased inorganic phosphate levels in females of test group 3 cannot be found.

Regarding metabolome analysis, 2,2'-dimethyl-4,4'-methylenebis (cyclohexylamine) induced only slight metabolome changes at the respective dose levels. At 15 mg/kg bw/d, 6.8% of metabolites were significantly changed, whereas at 5 mg/kg bw/d only 3.4% of the metabolites differed significantly from the control lying even below the false discover rate of 5%. The treatment with 2,2'-dimethyl-4,4'-methylenebis (cyclohexylamine) had only a weak impact on the plasma metabolome of male rats in this study. Based on the obtained findings, no conclusion on the toxicity of the test substance can be drawn.

Regarding pathology, target organs were the liver, brain, axillary and mesenteric lymph nodes and the pyloric mucosa of the glandular stomach. In females of test group 3 (15 mg/kg bw/d) the relative liver weight was statistically significantly increased (3.018%). Although this weight increase was within the historical control range (2.173 – 3.312%) a treatment-related vacuolation of the hepatocytes might have contributed to the weight increase. The vacuolation was characterized by a hepatocellular microvesicular cytoplasmic pattern, that was centrilobular in males (minimal to slight) and diffuse in females (minimal to moderate) at 15 mg/kg bw/d. Special stains performed in the liver to better characterize the nature of this vacuolation, excluded the presence of glycoproteins (PAS negative) or a fatty change (Oil-Red-O stained negative for neutral fat) suggesting that the vacuolation might be of a phospholipidic nature (Sudan Black stain positive together with Oil- Red-O negative stain). The same microvesicular pattern of vacuolation was observed in epithelial cells of other organs of male and female animals of test group 3, such as choroid plexus in the brain, high endothelial venules (HEV) in the axillary and mesenteric lymph nodes, and epithelial cells at the base of the glands of the pyloric mucosa. These changes, which were mostly minimal or slight, might also contribute to assume a treatment-related phospholipidosis. However, the definitive diagnosis is based on either phospholipid analysis or transmission electron microscopy as golden standard. Therefore, a conclusive diagnosis for the presence of phospholipidosis cannot be done in this study. Although no inflammatory or degenerative changes accompanied the vacuolation in examined organs, the presence of a fine vacuolation in the mentioned organs of males and females of test group 3 (15 mg/kg bw/d) was assessed as adverse.

Regarding fertility and reproductive performance, one female, each, of test groups 2 and 3 had all pups stillborn: No. 129 (5 mg/kg bw/d - 11 stillborn pups) and No. 138 (15 mg/kg bw/d – 2 stillborn pups). This finding in test groups 3 and 2 can be found in the historical control at comparable incidences (HCD; number of females with all pups stillborn, range 0 - 1). Female Nos. 129 and 138 had also a prolonged duration of gestation (24 and 23 days, respectively) compared to the group mean value of 22.1 days for both test groups. The prolonged duration of gestation may be the reason for the stillborn pups in the respective litters.

The gestation index was 100% in test groups 0 and 1, 90% in test group 2 and 87.5% in test group 3. The mean value of test group 3 was on the lower end of the range of the historical control data (HCD; gestation index, range: 87.5 – 100 %). Since all values of the parameters gestation index and number of females with all pups stillborn were within the historical control range, the findings were not assessed as treatment-related and adverse.

All parameters concerning mating and delivery were not adversely affected by the test substance.

Regarding F1 pups and litter data, pup status, viability, body weight and sex ratio showed no treatment-related, adverse findings. Neither determination of anogenital distance/index nor the count of nipple/areola anlagen revealed any treatment-related, adverse changes up to and including a dose of 15 mg/kg bw/d.

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.

OECD 443 (BASF, 2020)

2,2’-dimethyl-4,4’-methylenebis(cyclohexylamine) was administered to groups of 25 male and 25 female healthy young Wistar rats as test groups 00 - 03 as an aqueous preparation by stomach tube at different dosages (0, 1.5, 5 and 15 mg/kg body weight/day [mg/kg bw/d]). F0 animals were treated at least for 10 weeks 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 5 different cohorts (1A, 1B, 2A, 2B and 3) which were subjected to specific postweaning examinations. The study was terminated with the terminal sacrifice of the F1 rearing animals of cohort 1A. Control animals were dosed daily with the vehicle (0.5% Sodium carboxymethyl cellulose [CMC] suspension in drinking water).

 

The parents' and the pups' state of health was checked each day and parental animals were examined for their mating and reproductive performances. Water consumption of the F0 parents and F1 rearing animals (except of the positive control animals, test group 14) was determined regularly once weekly and weekly during gestation days (GD) 0-1, 3-4, 7-8, 10-11, 14-15, 17-18, 19-20 and lactation days (PND) 1-2, 4-5, 7-8, 10-11, 14-15, 17-18 and 20-21. Food consumption of the F0 parents and F1 rearing animals was determined regularly once weekly over a period of 7 days (except food consumption of the F0 males was determined after the 10th premating week) and weekly during GD 0-7, 7-14, 14-20 and PND 1-4, 4-7, 7-10, 10-14, 14-18 and 18-21. In general, body weights of F0 parents and F1 rearing animals were determined once weekly. However, during gestation and lactation F0 females were weighed on GD 0, 7, 14 and 20 and on PND 1, 4, 7, 10, 14, 18 and 21. A detailed clinical observation (DCO) was performed in all F0 parents and F1 animals in cohorts 1A, 1B, 2A and 3 at weekly intervals. Estrous cycle data were evaluated for F0 females over a three weeks period prior to mating until evidence of mating occurred. In all cohort 1A females, vaginal smears were collected after vaginal opening until the first cornified smear (estrous) was recorded. The estrous cycle also was evaluated in cohort 1A and 1B females for 2 weeks around PND 75. Moreover, the estrous stage of each female was determined on the day of scheduled sacrifice. An auditory startle response test was carried out in all animals of cohort 2A on PND 24. A functional observational battery (FOB) was performed in all animals of cohort 2A on PND 69. Motor activity was measured in all animals of cohort 2A on PND 69. 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, 14 and 21. Their viability was recorded. At necropsy, all pups were examined macroscopically. Date of sexual maturation, i.e. day of vaginal opening (females) or balanopreputial separation (males), of all F1 pups selected to become F1 rearing animals was recorded. All surviving pups were examined for the presence or absence of nipple/areola anlagen on PND 13. If nipple/areola anlagen were recorded, all surviving male pups were carefully reexamined on PND 20. The number of nipple/areola anlagen were counted. Anogenital distance (defined as the distance from the anus [center of the anal opening] to the base of the genital tubercle) measurements were conducted in a blind randomized fashion, using a measuring ocular on all live F1 pups on PND 1. Blood samples for clinical pathological investigations were withdrawn from 10 F0 parental and cohort 1A animals per sex and group. Furthermore blood samples were taken from 10 surplus (culled) PND 4 pups per sex and group as well as from 10 surplus PND 22 pups per sex and group. Various sperm parameters (motility, sperm head count, morphology) were assessed in the F0 and 1A males at scheduled sacrifice or after appropriate staining. All F0 parental 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 different pathological, neuro- and histopathological examinations.

 

Results:

The various analyses demonstrated the stability of the test substance preparations over a period of 7 days at room temperature and confirmed the homogeneous distribution of the test substance in 0.5% CMC suspension in drinking water. Moreover, the overall accuracy of the prepared test substance concentrations was confirmed.

 

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

15 mg/kg bw/d

F0 PARENTAL ANIMALS

CLINICAL EXAMINATIONS

- Decreased water consumption in males during premating (up to 17% below control) and in females during premating, gestation and lactation (up to 21, 18 and 23% below control, respectively)

- Decreased food consumption in males during premating (up to 15% below control) and in females during premating, gestation and lactation (up to 11, 13 and 19% below control, respectively)

- Decreased body weights in males during premating and study weeks 0 – 4 (up to 15 and 22% below control, respectively) and in females during premating, gestation and lactation (up to 9, 11 and 14% below control, respectively)

- Decreased body weight changes in males during premating and study weeks 0 – 4 (up to 79% below control) and in females during premating and gestation (up to 47%)

FERTILITY / REPRODUCTIVE PERFORMANCE

- Decreased mean number of implantation sites and secondary decreased mean number of delivered pups per dam

 

CLINICAL PATHOLOGY

- Increased platelet counts in males and females

- Decreased absolute and relative eosinophils in males

- Increased absolute and relative monocytes in females

- Increased aspartate aminotransferase (AST) activities and inorganic phosphate levels in males and females

- Increased triglyceride values in males

- Increased counts of transitional epithelial cells in the urine sediment

 

PATHOLOGY

Adrenal cortex

- Vacuolation, zona fasciculata: all males (minimal to moderate)

Axillary lymph nodes

- Vacuolation of high endothelial venules (HEV): 17 out of 20 males, 15 out of 20 females (minimal to slight)

Brain

- Vacuolation, choroid plexus: all males (slight to moderate) and all females (minimal to moderate)

Esophagus

- Vacuolation, skeletal muscle: 16 out of 20 males and 19 out of 20 females (minimal to slight)

Eyes with optic nerve

- Vacuolation, retinal pigmented epithelium: 7 out of 20 males (minimal to slight) and 5 out of 20 females (minimal)

Glandular stomach

- Vacuolation glandular: 14 out of 20 males and 19 out of 20 females (minimal to slight)

Heart

- Vacuolation, multifocal: 12 out of 20 males and 6 out of 20 females (minimal to slight)

Kidneys

- Vacuolation, tubular: 18 out of 20 males and 16 out of 20 females (minimal to slight)

- Degeneration/regeneration, tubular: 14 out of 20 males (minimal to slight)

Left epididymis

- Vacuolation, epithelial: 13 out of 22 males (minimal to moderate)

Liver

- Vacuolation, centrilobular: 17 out of 20 males (minimal to slight) and all females (minimal to moderate)

- Inflammation, multifocal: 13 out of 20 females (minimal to moderate)

- Apoptosis/single cell necrosis: 5 out of 20 females (minimal to slight)

- Multinucleated hepatocytes: 7 out of 20 females (minimal to slight)

- Vacuolation, bile duct: all males (minimal) and all females (minimal to slight)

Lungs

- Vacuolation, bronchial/bronchiolar epithelium (BALT): all males and all females (minimal to slight)

- Bronchial associated lymphoid tissue: Vacuolation, high endothelial venules: 12 out of 20 males and 10 out of 20 females (minimal

Mesenteric lymph node

- Vacuolation, high endothelial venules (HEV): 12 out of 20 males and 14 out of 20 females (minimal)

- Macrophage aggregate: 7 out of 20 males (minimal to slight)

Pancreas

- Vacuolation, acinar: all males and all females (minimal to slight)

- Vacuolation ductal: all males and all females (minimal to slight)

Pituitary gland

- Vacuolation, diffuse: 21 out of 22 males (minimal to slight) and 12 out of 22 females (minimal)

Seminal vesicles

- Vacuolation, epithelial: all males (minimal to moderate)

Skeletal muscle

- Vacuolation, fiber: 18 out of 20 males (minimal to slight) and 19 out of 20 females (minimal)

- Degeneration/regeneration: 17 out of 20 males (minimal to slight) and 13 out of 20 females (minimal)

- Necrosis, multifocal: 2 out of 20 males (minimal)

 

F1 PUPS

CLINICAL EXAMINATIONS

- Decreased pup body weight (PND 7 – 21: up to 10% below control) and body weight change (PND 1 – 21: up to 15% below control) in both sexes during lactation

 

SEXUAL MATURATION/ GROSS FINDINGS

- No test substance-related adverse findings

 

F1 REARING ANIMALS, COHORT 1A

CLINICAL EXAMINATIONS

- Decreased body weights in males during study days 42 - 56 (up to 11% below control) and in females during study days 49 - 56 (up to 6% below control)

- Decreased body weight change in males during major parts of the study period (up to 49% below control) and in females during study days 0 - 56 (about 8% below control)

 

CLINICAL PATHOLOGY

- Decreased absolute and relative eosinophils in males

- Increased absolute and relative monocytes in females

- Increased aspartate aminotransferase (AST) activities in males and females

 

PATHOLOGY

Adrenal cortex

- Vacuolation, zona fasciculata: 14 out of 20 males (minimal to slight)

Axillary lymph nodes

- Vacuolation of high endothelial venules (HEV): all males (minimal to slight), 19 out of 20 females (minimal to moderate)

Brain

- Vacuolation, choroid plexus: all males (slight to moderate) and 17 out of 20 females (minimal to slight)

Esophagus

- Vacuolation, skeletal muscle: all males and 18 out of 20 females (minimal to slight)

Eyes with optic nerve

- Vacuolation, retinal pigmented epithelium: 10 out of 20 males and 7 out of 20 females (minimal)

Glandular stomach

- Vacuolation glandular: 13 out of 20 males and 16 out of 20 females (minimal to slight)

Heart

- Vacuolation, multifocal: 11 out of 20 males (minimal to slight) and 13 out of 20 females (minimal)

Kidneys

- Vacuolation, tubular: 15 out of 20 males (minimal to slight) and 9 out of 20 females (minimal)

- Degeneration/regeneration, tubular: 3 out of 20 males (minimal to slight)

Left epididymis

- Vacuolation, epithelial: 12 out of 20 males (minimal)

Liver

- Vacuolation, centrilobular: 19 out of 20 males (minimal to slight) and 14 out of 20 females (minimal to moderate)

- Inflammation, multifocal: 2 out of 20 females (minimal)

- Vacuolation, bile duct: all males (minimal) and 17 out of 20 females (minimal to slight)

Lungs

- Vacuolation, bronchial/bronchiolar epithelium: all males (minimal to slight) and 19 out of 20 females (minimal)

- Bronchial associated lymphoid tissue (BALT): Vacuolation, high endothelial venules: 17 out of 20 males and 15 out of 20 females (minimal)

Mesenteric lymph node

- Vacuolation, high endothelial venules (HEV): 15 out of 20 males and 14 out of 20 females (minimal)

Pancreas

- Vacuolation, acinar: all males (minimal to moderate) and 19 out of 20 females (minimal to slight)

- Vacuolation ductal: all males and 19 out of 20 females (minimal to slight)

Pituitary gland

- Vacuolation, diffuse: 14 out of 20 males (minimal to slight) and 13 out of 20 females (minimal)

Seminal vesicles

- Vacuolation, epithelial: all males (minimal to moderate)

Skeletal muscle

- Vacuolation, fiber: 10 out of 20 males (minimal) and 3 out of 20 females (minimal)

- Degeneration/regeneration: 17 out of 20 males (minimal to slight) and 5 out of 20 females (minimal)

 

F1 REARING ANIMALS, COHORT 1B

CLINICAL EXAMINATIONS

- Decreased water consumption in males during study days 35 - 52 (up to 18% below control) and in females during study days 35 - 38 (up to 16% below control)

- Decreased food consumption in males during study days 42 - 49 (about 12% below control)

- Decreased body weights in males during study days 14 - 49 (up to 12% below control) and in females during study days 35 - 49 (up to 6% below control)

- Decreased body weight change in males during major parts of the study period (up to 36% below control) and in females during study days 7 - 14 (about 10% below control)

 

CLINICAL PATHOLOGY/ PATHOLOGY

- No test substance-related adverse findings

 

F1 REARING ANIMALS, COHORT 2A

CLINICAL EXAMINATIONS

- Decreased food consumption in males during study days 35 - 42 (about 10% below control)

- Decreased body weights in females on study day 21 and during 35 - 42 (up to 8% below control)

- Decreased body weight change in males during study days 14 - 21 and 0 - 42 (about 11% below control) and in females during study days 0 - 42 (about 11% below control)

 

NEUROPATHOLOGY

- Decreased terminal body weight in male and female animals

- Vacuolation of the retinal pigment epithelium of the eyes in 7/10 male and 6/10 female animals, graded minimal

- Droplets in the perikaryon of cervical ganglia in all male and female animals, graded minimal to slight in males and minimal in females

- Droplets in the perikaryon of lumbar ganglia in all male and female animals, graded minimal to slight in males and minimal in females

- Vacuolation in Trigeminus ganglia in all male and female animals, graded minimal in all animals

- Degeneration of muscle fibers of the gastrocnemius muscle in 9/10 animals of each sex, graded up to moderate.

- Vacuolation of muscle fibers of the gastrocnemius muscle in 9/10 male and 3/10 female animals, graded minimal

- Vacuolation of the choroid plexus of 2/10 female animals, graded minimal

- Vacuolation of the pituitary gland in 1/10 animals each of both sexes, graded minimal

 

F1 REARING ANIMALS, COHORT 2B (PND 22, weanlings)

NEUROPATHOLOGY

- Decreased terminal body weight in male and female animals

 

F1 REARING ANIMALS, COHORT 3

CLINICAL EXAMINATIONS

- Decreased water consumption in females during study days 0 - 11 (up to 15% below control)

- Decreased body weights in females during study day 21 - 28 (up to 9% below control)

- Decreased body weight change in females during study days 0 - 28 (about 10% below control)

 

CLINICAL PATHOLOGY/ PATHOLOGY

- No test substance-related adverse findings

 

SURPLUS F1 PUPS, PND 4, 22 AND 63 (NOT SELECTED FOR COHORTS)

CLINICAL PATHOLOGY/ PATHOLOGY

- No test substance-related adverse findings

 

5 mg/kg bw/d

F0 PARENTAL ANIMALS

CLINICAL EXAMINATIONS

- Decreased water consumption in males during premating days 28 - 59 (up to 12% below control) and in females during premating, gestation and during PND 1 - 2 (up to 18, 16 and 19% below control, respectively)

- Decreased food consumption in females during lactation (up to 14% below control)

- Decreased body weights in females during gestation and lactation (up to 5 and 7% below control, respectively)

- Decreased body weight change in males during premating and during study weeks 0 – 4 (up to 17 and 18% below control, respectively) and in females during gestation and lactation (about 13 and 48% below control, respectively)

 

FERTILITY / REPRODUCTIVE PERFORMANCE

- Decreased mean number of implantation sites and secondary decreased mean number of delivered pups per dam

 

PATHOLOGY

Axillary lymph nodes

- Vacuolation of high endothelial venules (HEV): 17 out of 20 males (minimal)

Esophagus

- Vacuolation, skeletal muscle: 19 out of 20 males (minimal) and 18 out of 20 females (minimal to slight)

Glandular stomach

- Vacuolation glandular: 3 out of 20 males and 2 out of 20 females (minimal)

Heart

- Vacuolation, multifocal: 2 out of 20 males (minimal)

Kidneys

- Vacuolation, tubular: 7 out of 20 males and 1 out of 20 females (minimal)

- Degeneration/regeneration, tubular: 1 out of 20 males (minimal)

Left epididymis

- Vacuolation, epithelial: 2 out of 20 males (minimal)

Liver

-Vacuolation, bile duct: 1 out of 20 males (minimal)

Lungs

-Vacuolation, bronchial/bronchiolar epithelium: 19 out of 20 males (minimal to slight) and 19 out of 20 females (minimal)

-Bronchial associated lymphoid tissue (BALT): Vacuolation, high endothelial venules: 1 out of 20 males (minimal)

Mesenteric lymph node

- Vacuolation, high endothelial venules (HEV): 4 out of 20 males (minimal)

- Macrophage aggregate: 6 out of 20 males (minimal to slight)

Pancreas

- Vacuolation, acinar: 17 out of 20 males (minimal to slight) and 1 out of 20 females (minimal)

- Vacuolation ductal: 17 out of 20 males and 5 out of 20 females (minimal)

Pituitary gland

- Vacuolation, diffuse: 4 out of 22 males (minimal)

Seminal vesicles

- Vacuolation, epithelial: 10 out of 20 males (minimal to slight)

Skeletal muscle

- Vacuolation, fiber: 6 out of 20 males and 2 out of 20 females (minimal)

 

F1 PUPS

CLINICAL EXAMINATIONS/ SEXUAL MATURATION/ GROSS FINDINGS

- No test substance-related adverse findings

 

F1 REARING ANIMALS, COHORT 1A

CLINICAL EXAMINATIONS/ CLINICAL PATHOLOGY/ PATHOLOGY

PATHOLOGY

Axillary lymph nodes

- Vacuolation of high endothelial venules (HEV): 15 out of 20 males (minimal to slight) and 1 out of 20 females (minimal)

Esophagus

- Vacuolation, skeletal muscle: 18 out of 20 males (minimal) and 19 out of 20 females (minimal to slight)

Eyes with optic nerve

- Vacuolation, retinal pigmented epithelium: 2 out of 20 males (minimal)

Heart

- Vacuolation, multifocal: 3 out of 20 males (minimal)

Kidneys

- Vacuolation, tubular: 1 out of 20 males and 1 out of 20 females (minimal)

Lungs

- Vacuolation, bronchial/bronchiolar epithelium: 3 out of 20 males (minimal)

- Bronchial associated lymphoid tissue (BALT): Vacuolation, high endothelial venules: 2 out of 20 males (minimal)

Mesenteric lymph node

- Vacuolation, high endothelial venules (HEV): 5 out of 20 males (minimal)

Pancreas

- Vacuolation, acinar: 13 out of 20 males (minimal)

- Vacuolation ductal: 17 out of 20 males and 11 out of 20 females (minimal)

Seminal vesicles

- Vacuolation, epithelial: 15 out of 20 males (minimal)

 

F1 REARING ANIMALS, COHORT 1B

CLINICAL EXAMINATIONS

- Decreased body weights in males during study days 42 - 49 (up to 6% below control)

- Decreased body weight change in males during several parts of the study period (up to 13% below control)

 

CLINICAL PATHOLOGY/ PATHOLOGY

- No test substance-related adverse findings

 

F1 REARING ANIMALS, COHORT 2A

CLINICAL EXAMINATIONS/ CLINICAL PATHOLOGY/ PATHOLOGY

- No test substance-related adverse findings

 

NEUROPATHOLOGY

- Vacuolation of the retinal pigment epithelium of the eyes in 3/10 male and 1/10 female animals, graded minimal

 

F1 REARING ANIMALS, COHORT 2B (PND 22, weanlings)

NEUROPATHOLOGY

- No test substance-related adverse findings

 

F1 REARING ANIMALS, COHORT 3

CLINICAL EXAMINATIONS/ CLINICAL PATHOLOGY/ PATHOLOGY

- No test substance-related adverse findings

 

SURPLUS F1 PUPS, PND 4, 22 AND 63 (NOT SELECTED FOR COHORTS)

CLINICAL PATHOLOGY

- No test substance-related adverse findings

 

1.5 mg/kg bw/d

F0 PARENTAL ANIMALS

CLINICAL EXAMINATIONS/ REPRODUCTIVE PERFORMANCE/ CLINICAL PATHOLOGY/ PATHOLOGY

- No test substance-related adverse findings

 

F1 PUPS

CLINICAL EXAMINATIONS/ SEXUAL MATURATION/ GROSS FINDINGS

- No test substance-related adverse findings

 

F1 REARING ANIMALS, COHORT 1A

CLINICAL EXAMINATIONS/ CLINICAL PATHOLOGY/ PATHOLOGY

- No test substance-related adverse findings

 

F1 REARING ANIMALS, COHORT 1B

CLINICAL EXAMINATIONS/ CLINICAL PATHOLOGY/ PATHOLOGY

- No test substance-related adverse findings

 

F1 REARING ANIMALS, COHORT 2A

CLINICAL EXAMINATIONS/ CLINICAL PATHOLOGY/ PATHOLOGY

- No test substance-related adverse findings

 

F1 REARING ANIMALS, COHORT 2B (PND 22, weanlings)

NEUROPATHOLOGY

- No test substance-related adverse findings

 

F1 REARING ANIMALS, COHORT 3

CLINICAL EXAMINATIONS/ CLINICAL PATHOLOGY/ PATHOLOGY

- No test substance-related adverse findings

 

SURPLUS F1 PUPS, PND 4, 22 AND 63 (NOT SELECTED FOR COHORTS)

CLINICAL PATHOLOGY

- No test substance-related adverse findings

 

Conclusion:

Under the conditions of the present extended 1-generation reproduction toxicity study the NOAEL (no observed adverse effect level) for general, systemic toxicity is the low dose of 1.5 mg/kg bw/d for the F0 and F1 animals. This was based on treatment-related, adverse effects such as a reduction in water and food consumption, decrease in body weight (change), altered clinical pathology parameters as well as histopathological changes in several organs, which were observed at the high- and mid-dose of 15 and 5 mg/kg bw/d.

 

The NOAEL for developmental toxicity in the offspring is the mid-dose of 5 mg/kg bw/d, based on the decrease in pup body weight during lactation at the high-dose level of 15 mg/kg bw/d. This slight delay in development of the high-dose pups was observed in presence of maternal toxicity and, therefore, not assessed as independent effect.

 

The NOAEL for developmental neurotoxicity in the F1 progeny is 15 mg/kg bw/d, the highest dose tested. Neuropathological findings observed in Cohort 2A adults of high- and mid-dose indicated a systemic, direct toxicity of the test substance and were not assessed as developmental neurotoxicological effects.

 

The NOAEL for developmental immunotoxicity for the F1 progeny is 15 mg/kg bw/d, the highest dose tested. Lower mean and median anti-SRBC IgM antibody titers of the positive control group (4.5 mg/kg bw/d cyclophosphamide, oral) demonstrated that the test system worked properly.

 

The NOAEL for fertility and reproductive performance for the F0 parental rats is 1.5 mg/kg bw/d, the lowest dose tested. This was based on the lower mean number of implantation sites and secondary decreased mean number of F1 pups delivered per dam in the high- and mid-dose groups.

Final discussion

For the registered substance, there are two reproductive toxicity studies available: one OECD 422, which was used as a dose-range-finding study for the dose setting of an OECD 443 and the OECD 443 itself. Both studies showed DMDC to be capable to induce general, systemic toxicity, which is the most prominant finding after repeated exposure (see also chapter "repeated dose toxicity"). In the OECD 422, the oral administration of the substance resulted in signs of systemic toxicity at the highest dose of 15 mg/kg bw/d, such as a reduction in food consumption and decrease in body weight (change), adverse changes in clinical pathology parameters and histopathology. Thus, the NOAEL for general systemic toxicity was 5 mg/kg bw/d for male and female Wistar rats. No effects on the reproductive performance and developmental toxicity were seen in that study. In the OECD 443 treatment-related, adverse effects such as a reduction in water and food consumption, decrease in body weight (change), altered clinical pathology parameters as well as histopathological changes in several organs were observed at the high- and mid-dose of 15 and 5 mg/kg bw/d. Concerning developmental neurotoxicity and immunotoxicity, there were no findings up to the highest dose tested. In the mid dose group, a slight delay in development (decreased pub body weight during lactation) was seen, assessed as a non-independent, secondary effect as a consequence of maternal toxicity. Concerning fertility and reproductive performance for the F0 parental rats, the NOAEL was set to 1.5 mg/kg bw/d, the lowest dose tested. This was based on a lower mean number of implantation sites and secondary decreased mean number of F1 pups delivered per dam in the high- and mid-dose groups. Again, those effects were only observed in connection with pronounced systemic effects and therefore considered not relevant in terms of classification and labeling.

Effects on developmental toxicity

Description of key information

In an oral (gavage) OECD TG 414 and GLP study in rats the substance was neither teratogenic nor embryotoxic. The NOAEL for maternal toxicity (reduced corrected body weight gain) was 5 mg/kg bw/day, for fetotoxicity (slight retardation of ossification of skull bones) 15 mg/kg bw/day and for teratogenicity 45 mg/kg bw/day. Thus, the substance showed no test-item specific developmental toxicity, since, the only effect seen, slight fetotoxicity, was observed in the presence of maternal toxicity. In a further prenatal developmental toxicity study, the oral administration of test substance 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 the high- and mid-dose (9 and 3 mg/kg bw/d), such as reduced food consumption and body weights/body weight gain. Since there was no evidence for toxicologically relevant adverse effects of the test substance on fetal morphology at any dose, the NOAEL for prenatal developmental toxicity in rabbits is the highest dose of 9 mg/kg bw/d.

Link to relevant study records

Referenceopen allclose all

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
8th December 2000 - 2nd January 2001
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
Draft June 2000
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Version / remarks:
Nov. 18, 1987
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
CIT
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland GmbH, Sulzfeld, Germany
- Age at study initiation: 10-11 weeks old
- Weight at study initiation: a mean body weight of 242 g (range: 198 g to 293 g).
- Housing: individual
- Acclimation period: 5 days
- Diet: ad libitum
- Water: ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 2
- Humidity (%): 50 ± 20
- Air changes (per hr): about 12 cycles/hour of filtered, non-recycled air.
- Photoperiod (hrs dark / hrs light): 12/12


Route of administration:
oral: gavage
Vehicle:
CMC (carboxymethyl cellulose)
Details on exposure:
There was a total of four groups of 25 female rats of the Sprague-Dawley strain, which received the test or control substances once a day by oral administration from day 6 to day 19 post-coitum.
Analytical verification of doses or concentrations:
yes
Remarks:
HPLC analysis
Details on analytical verification of doses or concentrations:
The results of the analyses confirmed the homogeneity and concentrations of the samples of the dosage forms taken on the first day and on the last day of treatment.
Details on mating procedure:
Mating: females were mated at the breeder's facilities. The day of confirmed mating (detection of a vaginal plug) was designated as day 0 post-coitum.
Duration of treatment / exposure:
Each animal was given the dosage forms once a day, at approximately the same time, from day 6 to day 19 post-coitum
Frequency of treatment:
once/day
Duration of test:
day 0 to day 20 post-coitum
Dose / conc.:
5 mg/kg bw/day (nominal)
Dose / conc.:
15 mg/kg bw/day (actual dose received)
Dose / conc.:
45 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
25
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The doses used in this study were selected based on the results of a preliminary range finding maternal toxicity study. Groups of 10 mated female Sprague-Dawley rats received the test substance in 0.5% aqueous carboxymethylcellulose at dose levels of 0 (vehicle control group), 50, 100, and 200 mg/kg bw/d. The test substance was administered by gavage on days 6 through 19 of gestation inclusive. The animals were sacrificed on day 20 of gestation, with exception of the high dose dams; these rats were sacrificed on day 10 post coitum due to marked toxicity.
Conclusions of the dose-finding study: The test substance produced slight maternotoxic effects when administered to pregnant rats (days 6 to 19 post coitum) at a dose level of 50 mg/kg bw/d. At 100 mg/kg bw/d, marked maternotoxicity was recorded, with forestomach, stomach and liver as target organs. The 200 mg/kg bw/d dose level was dramatically toxic to the pregnant rats. All surviving dams were sacrificed in a moribund condition before schedule on day 10 post coitum.

- Rationale for animal assignment: the animals were allocated to the groups, according to a stratification procedure, so that the average body weight of each group was similar.
Maternal examinations:
Mortality
Each animal was checked for mortality or signs of morbidity:at least twice a day during treatment period, at least once a day on other days.

Clinical symptoms
Each animal was observed for clinical signs (including evidence of abortion/resorption) at approximately the same time.

Food consumption
The quantity of food consumed by each female was recorded for the following intervals: 2-6, 6-9, 9-12, 12-15, 15-18 and 18-20 post-coitum.

Body weight data
The body weight of each female was recorded on days 2, 6, 9, 12, 15, 18 and 20 post-coitum.

After hysterectomy, the females were subjected to a macroscopic post-mortem examination of the principal thoracic and abdominal organs.
A gross evaluation of placentas was also performed.
Ovaries and uterine content:
The weight of the gravid uterus was recorded for each pregnant female at hysterectomy (with at least one live fetus).
The ovaries and uterus of females were examined to determine:
number of corpora lutea,
number and distribution of dead and live fetuses,
number and distribution of early and late resorptions,
number and distribution of implantation sites.
Fetal examinations:
- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: No data

Examination of the fetuses after dissection from the uterus
Each fetus was submitted to a detailed external examination, which included the observation of all visible structures, surfaces and orifices. Dead fetuses were then discarded. The body weight of each live fetus was recorded. The live fetuses were killed by subcutaneous injection of thiopental sodium.

Soft tissue examination of the fetuses
Approximately half of the live fetuses per litter were fixed with Harrisson's fluid. A detailed soft tissue examination was performed according to a free-hand sectioning technique (Wilson technique) which included the observation of all the organs and structures of the head, neck, thorax and
abdomen.

Skeletal and cartilage examination of the fetuses
The remaining fetuses per litter were fixed in ethyl alcohol. A detailed examination of the skeleton was performed after staining with alizarin red S and alcian blue. This examination included the observation of all the bone structures and cartilage of the head, spine, rib cage, pelvis and limbs.
In particular, cartilage was specifically examined where a bony structure was altered.

Sex of fetuses
The sex of each live fetus was determined at the time of evisceration (after fixation in alcohol) or at the time of the Wilson sectioning.
Statistics:
Mean values were compared by one-way analysis of variance and Dunnett test (mean values being considered as normally distributed and variances being considered as homogeneous).
Percentage values were compared by the Fisher exact probability test.
Indices:
The conception rate (%) was calculated as follows:
Number of pregnant females/Number of females mated x 100

The pre-implantation loss was calculated as follows:
Number of corpora lutea - Number of implantation sites/Number of corpora lutea x 100

The post-implantation loss was calculated as follows:
Number of implantation sites - Number of live fetuses/Number of implantation sites x 100
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
No substance-related clinical symptoms were observed in the control and the treated groups, the few findings occasionally observed being of no toxicological significance.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
There were no deaths in the control and the treated groups except for one female in the 15 mg/kg/day which was found dead on day 20 of gestation prior to the terminal sacrifice. No clinical signs were observed prior to the death. Food consumption and body weight gain were slightly lower than the group mean values but still within normal range. Foamy content and dilatation of the lungs were recorded at necropsy. The reason for this death is not known but a relation to treatment with the test substance can be ruled out since this single mortality was observed in the mid-dose group
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
During the period of treatment (day 6 to day 19 post-coitum), the group mean body weight gain was similar in the control and the 5 mg/kg/day groups. In the 15 and 45 mg/kg/day treated groups, the group mean body weight gain was slightly (-8%, not statistically significant) to clearly (-13%, p<0.05) reduced during the period of treatment, with a more marked effect at the beginning (first 3 days of treatment). In these treated groups, the mean values of terminal body weight did not statistically differ from that of the control group, but were lower by -4% and -6%, respectively.
The corrected body weight gain (terminal body weight on day 20 post-coitum minus the weight of gravid uterus minus body weight on day 6 post-coitum) was minimally reduced in the 5 mg/kg/day group (-17%, not statistically significant). This finding was considered to be fortuitous because it was the only effect recorded in this group (no difference in gross body weight gain or food consumption). The corrected body weight gain was slightly (-23%, not significant) to markedly (-44%, p<0.001) reduced in the 15 and 45 mg/kg/day groups respectively. Because this effect was dose-related and correlated with a lower food consumption (in the 45 mg/kg/day group), it was considered to be related to the treatment with the test substance.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
The food consumption was similar in the control and the 5 and 15 mg/kg/day groups. In the 45 mg/kg/day group, there was a notable decrease in food consumption during the treatment period (-7%, not statistically significant), with a more marked effect during the first week of treatment.
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
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 value of gravid uterus weight was similar in the control and the treated groups, the slight fluctuations recorded correlating with the mean litter size.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
There was no remarkable observation in the 5 and 15 mg/kg/day treated groups. However, in the 45 mg/kg/day treated group, 6/25 females presented paleness, accentuated lobular pattern and/or whitish area on the liver. These necropsy findings might be related to the test substance administration.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed
Changes in pregnancy duration:
not examined
Changes in number of pregnant:
not examined
Description (incidence and severity):
There were no substance-related and /or biological relevant differences between the different test groups in conception rate, in the mean number of corpora lutea and implantation sites or in the pre and post-implantation losses and the number of resorptions. There was only a clearly higher number of live fetuses/dam in the 45 mg/kg/day group: 13.7 fetuses per litter on average, versus 12.6 fetuses per litter in the control group. This difference was considered to be incidental as both values are fully within the historical control range. However, the increased mean number of live fetuses/dam in the highdose group may well account for the slight delays in ossification of the fetal skeletons at this dose-level.
Key result
Dose descriptor:
NOAEL
Effect level:
5 mg/kg bw/day
Basis for effect level:
body weight and weight gain
Key result
Abnormalities:
effects observed, non-treatment-related
Fetal body weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
The body weight of the fetuses was similar in the control and the treated groups. Quite large variations were observed between the litters, probably because of a broad range in the time of overnight mating, but the distribution of this range was similar in all the groups.
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Description (incidence and severity):
The sex distribution was similar in the control and the treated groups.
Changes in litter size and weights:
no effects observed
Changes in postnatal survival:
not examined
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
No malformations were observed at external examination of the fetuses from the control and the 5 and 45 mg/kg/day treated groups. In the 15 mg/kg/day, 1/293 fetuses (= 0.3% of the examined fetuses) from one out of 23 litters (= 4% of the examined litters) displayed a thread-like tail. This external malformation can be also observed occasionally in control fetuses and thus is considered to be of spontaneous occurrence.
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
There was a low incidence of malformations in each group:
Control group: one fetus (= 0.7% of the examined fetuses) from one out of 22 litters
(= 4.5% of the examined litters) affected
-1/146 fetuses with misshapen 11th pair of ribs.
5 mg/kg/day group: two fetuses (= 1.5% of the examined fetuses) from two out of
19 litters (= 10.5% of the examined litters) affected
-2/133 fetuses with misshapen rib(s) (11th pair),
15 mg/kg/day group: two fetuses (= 1.6% of the examined fetuses) from two out of
18 litters (= 11.1 % of the examined litters) affected
-1/122 fetuses with fused ribs (4th 5th 6th right ribs); this fetus had a slightly lower body weight,
-1/122 fetuses with misshapen ribs (7th to 11th pair).
45 mg/kg/day: four fetuses (= 2.7% of the examined fetuses) from four out of 21 litters (= 19.0% of the examined litters) affected
-3/148 fetuses with misshapen rib (one to five ribs affected, unilaterally),
-1/148 fetuses with misshapen, enlarged 6th sternebra.

Statistical significance was not reached for any of the recorded skeletal malformations, either for fetal incidence, litter incidence or mean percentage of affected fetuses per litter. In addition, there was no clear dose-relationship between the dose-groups: incidence in the mid-dose group is lower than in the low-dose group for misshapen rib; incidence in the mid-dose group is higher than in the low and high-dose groups for fused ribs. Consequently, concerning fetal skeletal malformations, a relationship to treatment with the test substance was ruled out.

In the 5 and 15 mg/kg/day treated groups the incidence and nature of the fetal skeletal variations were similar to those recorded in the control group: the slight differences observed were occasional, not dose-related, and/or not statistically significant.
In addition, the values were within the range of CIT historical control data. The single exception was the incomplete ossification of the frontal bone, which was recorded at a slightly higher incidence in the 15 mg/kg/day group when compared to the contemporary controls and the historical data. However, the difference was slight and the actual control value was already slightly above the highest historical control value. Consequently, this single finding was not considered to be of toxicological significance.
In the 45 mg/kg/day treated group, there was a higher incidence of a number of skeletal variations representing incomplete ossification of skull bones and absence of ossification of 5th sternebra. The differences were greater than those recorded at lower dose-levels, and/or were occasionally outside the range of CIT historical control data. When comparing fetal incidences, statistical significance was reached for three out of five findings. The slightly increased occurrence of delays in ossification at the high dose is, however, not considered to be substance-induced for the following reasons:
-the mean number of live fetuses per litter was distinctly higher in the high-dose
group than in the concurrent control group (13.7 fetuses/dam at 45 mg/kg vs. 12.6 control fetuses/dam - see also § 4.2.2.3). It is very likely, that this is the actual cause
for the transient delays in ossification (although the mean fetal body weights are rather similar in all groups),
-statistical significance for these findings was not reached when comparing the number of affected fetuses on a per litter basis,
-moreover, the overall incidence for fetal skeletal variations was not increased in the high-dose group (93.9%) in comparison to the control group (94.5%).

Special comments on additional skeletal examinations:
A total of 16 litters, accounting for 105 fetuses were also examined for skeletal and cartilage findings. However, since the labeling of the flasks containing the fetuses was accidentally lost, it was not possible to identify which experimental group each litter belonged to. However, a global analysis showed that:
-malformation was confined to a single fetus displaying misshapen ribs (7th to 10th right ribs),
-variations were similar in nature and incidence to that recorded in the properly identified fetuses.
In conclusion, although the skeletal findings recorded in these 105 fetuses cannot be properly attributed to each of the four experimental groups, their incidence and nature did not differ from that observed in the 549 evaluated fetuses and thus do not change the final assessment which is summarized below.

Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
The only soft tissue variations recorded were the dilatation of renal pelvis and/or ureter. These common variations were recorded at a similar incidence in the control and treated groups and did not suggest any treatment- or dose-relationship. In addition, the incidence was within the range of CIT historical control data for this strain of rats.
Key result
Dose descriptor:
NOAEL
Effect level:
45 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: highest dose tested
Key result
Abnormalities:
effects observed, non-treatment-related
Key result
Developmental effects observed:
no

Table 1: Skeletal variations

Mean incidence of notable skeletal variations

Dose-level (mg/kg/day)

0

5

15

45

Historical

control range

Interparietal: incomplete ossification

fetal incidence (%)

6.8

12.8

16.4*

29.1***

8.7
(1.5-19.1)

affected fetuses/litter (%)

7.0

12.5

17.7

29.1

Parietal: incomplete ossification

fetal incidence (%)

1.4

3.8

4.9

16.2***

2.7
(0.0-6.9)

affected fetuses/litter (%)

1.6

3.9

5.1

16.2**

Supraoccipital: incompleteossification

fetal incidence (%)

2.7

3.0

2.5

10.1*

4.1
(0.0-17.6)

affected fetuses/litter (%)

3.1

3.0

3.0

9.8

Frontal: incomplete ossification

fetal incidence (%)

6.8

2.3

9.0

13.5

2.2
(0.0-6.3)

affected fetuses/litter (%)

6.3

2.3

8.4

12.2

5th sternebra: unossified

fetal incidence (%)

14.4

12.0

18.9

30.4**

23.7
(9.9-40.5)

affected fetuses/litter (%)

16.0

12.3

17.1

28.6

Total of skeletal variations

fetal incidence (%)

94.5

94.7

91.8

93.9

X

affected fetuses/litter (%)

94.2

94.6

91.2

92.8

*: p<0.05        **: p<0.01       ***: p<0.001 (statistics calculated using absolute values) underlined values: outside CIT historical data.

 

Table 2: HYSTERECTOMY DATA (Summary table)

Dose-level (mg/kg/day)

 

0

5

15

45

Pregnant Females Alive at Term

N

25

24

23

24

with Total Resorptions

N

0

0

0

0

with all DeadFetuses

N

0

0

0

0

with Live Fetuses

N

25

24

23

24

 

 

 

 

 

 

Corpora Lutea

Total

353

363

332

357

No. per animal

Mean

14.1 d

15.1

14.4

14.9

 

S.d.

2.4

2.6

3.0

2.7

 

 

 

 

 

 

Implantation Sites

Total

322

330

299

336

No. per animal

Mean

12.9 d

13.8

13.0

14.0

 

S.d.

2.6

1.8

2.4

2.1

 

 

 

 

 

 

Preimplantation Loss

Total

31 f

33

33

21

 

%

8.8

9.1

9.9

5.9

 

 

 

 

 

 

Fetuses

N

316

321

293

329

No. per animal

Mean

12.6 d

13.4

12.7

13.7

 

S.d.

2.7

2.0

2.3

2.5

Alive

%

100.0

100.0

100.0

100.0

Dead

%

0.0

0.0

0.0

0.0

 

 

 

 

 

 

Live Fetuses

N

316 f

321

293

329

% of implantation sites

 

98.1

97.3

98.0

97.9

No. per animal

Mean

12.6 d

13.4

12.7

13.7

 

S.d.

2.7

2.0

2.3

2.5

 

 

 

 

 

 

Dead Fetuses

N

0 f

0

0

0

% of impantation sites

 

0.0

0.0

0.0

0.0

No. per animal

Mean

0.0

0.0

0.0

0.0

 

S.d.

0.0

0.0

0.0

0.0

 

 

 

 

 

 

Resorptions + Scars

N

6 f

9

6

7

% of impantation sites

 

1.9

2.7

2.0

2.1

No. per animal

Mean

0.2 d

0.4

0.3

0.3

 

S.d.

0.6

0.8

0.6

0.8

 

 

 

 

 

 

Implant Scars

N

0 f

0

0

0

% of impantation sites

 

0.0

0.0

0.0

0.0

No. per animal

Mean

0.0

0.0

0.0

0.0

 

S.d.

0.0

0.0

0.0

0.0

 

 

 

 

 

 

Resorptions: early

N

6 f

9

4

7

% of impantation sites

 

1.9

2.7

1.3

2.1

No. per animal

Mean

0.2 d

0.4

0.2

0.3

 

S.d.

0.6

0.8

0.5

0.8

 

 

 

 

 

 

Resorptions: late

N

0 f

0

2

0

% of impantation sites

 

0.0

0.0

0.7

0.0

No. per animal

Mean

0.0 d

0.0

0.1

0.0

 

S.d.

0.0

0.0

0.3

0.0

 

 

 

 

 

 

Postimplantation Loss

Total

6 f

9

6

7

% of impantation sites

 

1.9

2.7

2.0

2.1

No. per animal

Mean

0.2 d

0.4

0.3

0.3

 

S.d.

0.6

0.8

0.6

0.8

 

 

 

 

 

 

Male Fetuses

N

157 f

147

138

174

 

%

49.7

45.9

47.1

52.9

Female Fetuses

N

159 f

173

155

155

 

%

50.3

54.1

52.9

47.1

 

 

 

 

 

 

Fetal Body Weight (g)

Mean

4.53 d

4.72

4.48

4.49

 

S.d.

0.81

0.88

0.74

0.90

 

 

 

 

 

 

Male Fetuses

Mean

4.65 d

4.84

4.62

4.62

 

S.d.

0.82

0.91

0.79

0.97

 

 

 

 

 

 

Female Fetuses

Mean

4.42 d

4.60

4.37

4.32

 

S.d.

0.84

0.86

0.71

0.85

Statistical key: d=ANOVA + Dunnett-test       f=Fishers exact test

15 mg/kg/day: 1/24 pregnant females died just prior to the hysterectomy

Table 3: SUMMARY OF FETAL EXTERNAL MALFORMATIONS

 

Dose-level (mg/kg/day)

 

0

5

15

45

Litters Evaluated

N

25

24

23

24

Fetuses Evaluated

N

316

321

293

329

Live

N

316

321

293

329

Dead

N

0

0

0

0

 

 

 

 

 

 

Tail

Litter Incidence

N

0

0

1

0

Fetal Incidence

N

0

0

1

0

 

 

 

 

 

 

THREAD-LIKE TAIL

Fetal Incidence

N

0 f

0

1

0

 

%

0.0

0.0

0.3

0.0

Litter Incidence

N

0 f

0

1

0

 

%

0.0

0.0

4.3

0.0

 

 

 

 

 

 

Affected Fetuses/Litter

Mean%

0.0 d

0.0

0.4

0.0

 

S.d.

0.0

0.0

1.7

0.0

 

 

 

 

 

 

TOTAL FETAL EXTERNAL MALFORMATIONS

Fetal Incidence

N

0 f

0

1

0

 

%

0.0

0.0

0.3

0.0

Litter Incidence

N

0 f

0

1

0

 

%

0.0

0.0

4.3

0.0

 

 

 

 

 

 

Affected Fetuses/Litter

Mean%

0.0 d

0.0

0.4

0.0

 

S.d.

0.0

0.0

1.7

0.0

 Statistical key: d=ANOVA + Dunnett-test       f=Fishers exact test

 

Table 4: SUMMARY OF FETAL EXTERNAL VARIATIONS

Dose-level (mg/kg/day)

 

0

5

15

45

Litters Evaluated

N

25

24

23

24

Fetuses Evaluated

N

316

321

293

329

Live

N

316

321

293

329

Dead

N

0

0

0

0

 

 

 

 

 

 

TOTAL FETAL EXTERNAL VARIATIONS

Fetal Incidence

N

0 f

0

0

0

 

%

0.0

0.0

0.0

0.0

Litter Incidence

N

0 f

0

0

0

 

%

0.0

0.0

0.0

0.0

 

 

 

 

 

 

Affected Fetuses/Litter

Mean%

0.0

0.0

0.0

0.0

 

S.d.

0.0

0.0

0.0

0.0

 Statistical key: f=Fishers exact test

Table 5: SUMMARY OF FETAL SOFT TISSUE MALFORMATIONS  

Dose-level (mg/kg/day)

 

0

5

15

45

Litters Evaluated

N

25

24

23

24

Fetuses Evaluated

N

149

155

141

160

 

 

 

 

 

 

TOTAL FETAL SOFT TISSUE MALFORMATIONS

Fetal Incidence

N

0 f

0

0

0

 

%

0.0

0.0

0.0

0.0

Litter Incidence

N

0 f

0

0

0

 

%

0.0

0.0

0.0

0.0

 

 

 

 

 

 

Affected Fetuses/Litter

Mean%

0.0

0.0

0.0

0.0

 

S.d.

0.0

0.0

0.0

0.0

Statistical key: f=Fishers exact test

Table 6: SUMMARY OF FETAL SOFT TISSUE VARIATIONS

Dose-level (mg/kg/day)

 

0

5

15

45

Litters Evaluated

N

25

24

23

24

Fetuses Evaluated

N

149

155

141

160

 

 

 

 

 

 

KIDNEYS

Litter Incidence

N

7

2

3

2

Fetal Incidence

N

10

2

6

8

 

 

 

 

 

 

DILATED RENAL FELVIS

Fetal Incidence

N

10 f

2

6

8

 

%

6.7

1.3

4.3

5.0

Litter Incidence

N

7 f

2

3

2

 

%

28.0

8.3

13.0

8.3

 

 

 

 

 

 

Affected Fetuses/Litter

Mean%

6.1 d

1.4

4.3

5.0

 

S.d.

11.1

4.7

13.4

20.6

 

 

 

 

 

 

URETER

Litter Incidence

N

1

0

2

0

Fetal Incidence

N

2

0

3

0

 

DILATED URETER

Fetal Incidence

N

2 f

0

3

0

 

%

1.3

0.0

2.1

0.0

Litter Incidence

N

1 f

0

2

0

 

%

4.0

0.0

8.7

0.0

 

 

 

 

 

 

Affected Fetuses/Litter

Mean%

1.1 d

0.0

1.7

0.0

 

S.d.

5.7

0.0

5.7

0.0

 

 

 

 

 

 

TOTAL FETAL SOFT TISSUE VARIATIONS

Fetal Incidence

N

10 f

2

6

8

 

%

6.7

1.3

4.3

5.0

Litter Incidence

N

7 f

2

3

2

 

%

28.0

8.3

13.0

8.3

 

 

 

 

 

 

Affected Fetuses/Litter

Mean%

6.1 d

1.4

4.3

5.0

 

S.D.

11.1

4.7

13.4

20.6

 Statistical key: d=ANOVA + Dunnett-test       f=Fishers exact test

Table 7: SUMMARY OF FETAL SKELETAL MALFORMATIONS

Dose-level (mg/kg/day)

 

0

5

15

45

Litters Evaluated

N

22

19

18

21

Fetuses Evaluated

N

146

133

122

148

 

 

 

 

 

 

STERNEBRA

Litter Incidence

N

0

0

0

1

Fetal Incidence

N

0

0

0

1

 

 

 

 

 

 

MISSHAPEN STERNEBRA (E)

Fetal Incidence

N

0 f

0

0

1

 

%

0.0

0.0

0.0

0.7

Litter Incidence

N

0 f

0

0

1

 

%

0.0

0.0

0.0

4.8

 

 

 

 

 

 

Affected Fetuses/Litter

Mean%

0.0 d

0.0

0.0

1.0

 

S.d.

0.0

0.0

0.0

4.4

 

 

 

 

 

 

RIB

Litter Incidence

N

1

2

2

3

Fetal Incidence

N

1

2

2

3

 

MISSHAPEN RIB

Fetal Incidence

N

1 f

2

1

3

 

%

0.7

1.5

0.8

2.0

Litter Incidence

N

1 f

2

1

3

 

%

4.5

10.5

5.6

14.3

 

 

 

 

 

 

Affected Fetuses/Litter

Mean%

0.6 d

1.5

0.9

2.4

 

S.d.

3.0

4.7

3.9

6.2

 

 

 

 

 

 

FUSED RIBS

Fetal Incidence

N

0 f

0

1

0

 

%

0.0

0.0

0.8

0.0

Litter Incidence

N

0 f

0

1

0

 

%

0.0

0.0

5.6

0.0

 

 

 

 

 

 

Affected Fetuses/Litter

Mean%

0.0 d

0.0

0.6

0.0

 

S.D.

0.0

0.0

2.6

0.0

 

TOTAL FETAL SKELETAL MALFORMATIONS

Fetal Incidence

N

1 f

2

2

4

 

%

0.7

1.5

1.6

2.7

Litter Incidence

N

1 f

2

2

4

 

%

4.5

10.5

11.1

19.0

 

 

 

 

 

 

Affected Fetuses/Litter

Mean%

0.6 d

1.5

1.5

3.4

 

S.D.

3.0

4.7

4.6

7.2

Statistical key: d=ANOVA + Dunnett-test       f=Fishers exact test

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
5th April 2016 - 2nd November 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
22 Jan 2001
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Version / remarks:
Aug 1998
Deviations:
no
Qualifier:
according to
Guideline:
other: Commission Regulation (EC) No 440/2008 laying down test methods pursuant to Regulation (EC) No 1907/2006 (REACH), Part B: Methods for the deter: Prenatal Developmental Toxicity Study; Official Journal of the European Union, No. L 142
Version / remarks:
30 May 2008
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rabbit
Strain:
New Zealand White
Remarks:
Crl:KBL(NZW)
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, France
- Age at study initiation: 16-17 weeks
- Weight at study initiation: Based on the pregnant animals the body weight on GD 0 varied between 3385 – 4337 g.
- Housing: singly in Type 4X03B700CP cages supplied by TECNIPLAST, Hohenpeißenberg, Germany
- Diet: ad libitum, pelleted Kliba maintenance diet rabbit and guinea pig “GLP”, supplied by Provimi Kliba SA, Kaiseraugst, Switzerland
- Water: ad libitum, tap water
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20±2
- Humidity (%): 30-70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:
oral: gavage
Vehicle:
CMC (carboxymethyl cellulose)
Remarks:
0.5% CMC in water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
For the test substance preparation, the specific amount of the test substance was weighed, topped up with 0.5% Carboxymethylcellulose suspension in drinking water in a calibrated beaker and intensely mixed with a magnetic stirrer. During administration, the preparations were kept homogeneous with a magnetic stirrer. The test substance was administered to the animal orally by gavage from implantation to one day prior to the expected day of parturition (GD 6-28) always at approximately the same time of day.


Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The results of the analyses of the test substance preparations in 0.5% Carboxymethylcellulose suspension in drinking water confirmed the correctness of the prepared concentrations. The mean measured concentrations of the samples of treatment groups corresponded to the expected values within the limits of the analytical method, i.e. were above 90% and below 110% of the nominal concentrations.
.
Details on mating procedure:
- Impregnation procedure: artificial insemination
A synthetic hormone (0.2 mL), which stimulates release of LH and FSH from the anterior pituitary lobe (Receptal®) was injected intramuscularly to the female rabbits about 1 hour before insemination. The ejaculate samples used for the artificial insemination were obtained from male New Zealand White rabbits of the same breed as the females. Each female was inseminated with the sperm of a defined male donor as documented in the raw data. The male donors were kept under conditions (air conditioning, diet, water) comparable to those of the females participating in this study.
The day of insemination was designated as GD 0 (beginning of the study) and the following day as GD 1.
Duration of treatment / exposure:
GD 6-28 (23 applications)
Frequency of treatment:
once daily
Dose / conc.:
1 mg/kg bw/day
Remarks:
low dose
Dose / conc.:
3 mg/kg bw/day
Remarks:
mid dose
Dose / conc.:
9 mg/kg bw/day
Remarks:
high dose
No. of animals per sex per dose:
25
Control animals:
yes, concurrent vehicle
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: once daily
- checked for any signs of morbidity, pertinent behavioral changes and signs of overt toxicity. If such signs occurred, the animals were examined several times daily (GD 0-29).
During the administration period (GD 6-28) all animals were checked daily for any abnormal clinical signs before the administration as well as within 5 hours after the administration

BODY WEIGHT: Yes
- Time schedule for examinations: All animals were weighed on GD 0, 2, 4, 6, 9, 11, 14, 16, 19, 21, 23, 25, 28 and 29. The body weight change of the animals was calculated based on the obtained results. Furthermore, the corrected body weight gain was calculated after terminal sacrifice (terminal body weight on GD 29 minus weight of the unopened uterus minus body weight on GD 6).

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
The consumption of food was recorded daily during GD 0-29.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 29
On GD 29, the surviving does were sacrificed in randomized order by intravenous injection of pentobarbital (Narcoren®; dose 2 mL/animal) and later, fetuses were removed from the uterus

All prematurely dead or sacrificed females were examined following the same procedures as for females sacrificed on schedule with the exception that no uterine weights were determined. After the does had been sacrificed, they were necropsied and were assessed by gross pathology in randomized order to minimize bias. The uteri and the ovaries were removed and the following data were recorded:
- Weight of the unopened uterus
- Number of corpora lutea
- Number and distribution of implantation sites classified as:
• Live fetuses
• Dead implantations:
a) Early resorptions (only decidual or placental tissues visible or according to SALEWSKI from uteri from apparently non-pregnant animals and the empty uterus horn in the case of single horn pregnancy)
b) Late resorptions (embryonic or fetal tissue in addition to placental tissue visible)
c) Dead fetuses (hypoxemic fetuses which did not breathe spontaneously after the uterus had been opened)
After the weight of the uterus had been determined, all subsequent evaluations of the does and the gestational parameters were conducted by technicians unaware of treatment group in order to minimize bias. For this purpose animal numbers were encoded.
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
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
Statistics:
see table 1
Indices:
see table 2
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Two females of the high-dose group (Nos. 92, 94 - 9 mg/kg bw/d) had blood in bedding on GD 16-19, respectively. In total, reduced defecation was observed in six control, three low-dose, four mid-dose and eleven high-dose females (0, 1, 3 and 9 mg/kg bw/d). Incidence and distribution of this finding do not indicate a relationship to the test substance. There were no clinical findings in the other does in the study.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
One control female (No. 24 - 0 mg/kg bw/d) and one high-dose female (No. 92 - 9 mg/kg bw/d) were sacrificed after abortion ahead of schedule (GD 20 and 18, respectively). Spontaneous abortions in single does are not uncommon findings in the strain of rabbits used for this study. Thus, this was considered to be a spontaneous incident. One control female (No. 17) was sacrificed moribund on GD 14 after an accidental fracture of maxilla and dental root. Furthermore, one female of test group 2 (No. 74 - 3 mg/kg bw/d) died after a gavage error.
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 does (9 mg/kg bw/d) were statistically significantly reduced on GD 16-29 (BW), on GD 9-11 and GD 14-16 (BWC), which is considered to be treatment-related. If calculated for the entire treatment period (GD 6-28) these does gained 82% less body weight (with statistical significance) than the concurrent control rabbits. No statistically significant difference was observed for the BW of the mid-dose does (3 mg/kg bw/d) when compared to the concurrent control group. However, from GD 16 onwards the mid-dose body weights were slightly lower than the control values. Also, the BWC of these rabbits was statistically significantly reduced on GD 9-11. If calculated for the entire treatment period (GD 6-28), the does of the mid-dose group gained 50% less body weight than the control does. The mean body weights and the average body weight gain of the low-dose group (1 mg/kg bw/d) were generally comparable to the concurrent control group throughout the entire study period.
Food consumption and compound intake (if feeding study):
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 (9 mg/kg bw/d) was reduced during major parts of the treatment period, attaining statistical significance on GD 14-20. If calculated for the entire treatment period (GD 6-28) or the entire study period (GD 0-29), the high-dose does consumed 19% or 14% less food than the concurrent control does (without statistical significance). The food consumption of the low- and mid-dose rabbits (1 and 3 mg/kg bw/d) was comparable to the concurrent control (0 mg/kg bw/d) throughout the entire study period.
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
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
The mean gravid uterus weight of the rabbits of test groups 2 and 3 (3 and 9 mg/kg bw/d) was statistically significantly reduced in comparison to the control group (test group 2: about 20% below control; test group 3: about 19% below control), which was likely to be a subsequent effect of a lower number of implants in these test groups. Thus, is not considered to be an independent effect. The mean gravid uterus weight of the rabbits of test group 1 (1 mg/kg bw/d) was not influenced by the test substance. The difference between this group and the control group showed no dose-dependency and was assessed to be without biological relevance.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Some spontaneous findings were noted in individual females of test groups 0-3 (0, 1, 3 and 9 mg/kg bw/d). These gross findings were:
• Infarct of liver in one control doe (No. 7 - 0 mg/kg bw/d)
• Erosion(s) in stomach in one control doe (No. 5 - 0 mg/kg bw/d) and one low-dose doe (No. 28 - 1 mg/kg bw/d)
• Watery feces in two high-dose does (Nos. 77 and 79 - 9 mg/kg bw/d)
• Absence of uterine horn(s) in one mid-dose doe (No. 65 - 3 mg/kg bw/d)

Two additional findings were noted in test groups 0 and 2. Both were associated with unscheduled
maternal death or sacrifice:
• Fracture of maxilla and dental root in control doe No. 17 (sacrificed moribund on GD 14)
• Findings after gavage error (i.e. thoracic cavity filled with blood) in mid-dose doe No. 74 (died after gavage error on GD 18)
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed
Changes in pregnancy duration:
not examined
Changes in number of pregnant:
no effects observed
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
Female rabbits were placed into the study in four cohorts. The conception rate was 80% in the mid-dose group (3 mg/kg bw/d), 88% in the low-dose group (1 mg/kg bw/d), 92% in the high-dose group (9 mg/kg bw/d) and 96% in the control group (0 mg/kg bw/d). A sufficient number (approximately 20, but not fewer than 16 females with implantation sites) of pregnant females was available for the purpose of the study.
There were no test substance-related and/or biologically relevant differences between the different test groups in conception rate, mean number of corpora lutea or in the values calculated for the pre- and the postimplantation losses and the number of resorptions.
The number of implantation sites and subsequently of live fetuses (live litter size) was statistically significantly lower than the concurrent control in the mid- and high-dose groups (3 and 9 mg/kg bw/d). This apparent decrease in the mid- and high-dose group needs, however, to be considered in view of the unusually high number of implants and litter size of the concurrent control group. These were distinctly above the upper range of the historical control data (implantation sites: 10.9 vs 7.1 – 10.3, litter size: 10.2 vs 6.6 - 9.7). On the other hand the mean implant/litter size values of the mid- and high-dose group were close to the mean of the historical control ranges (8.7 and 8.1, respectively). In addition, no relation to the dose was observed for any of the two apparent effects. Therefore, they were not considered to be treatment-related.
All other differences observed are considered to reflect the normal range of fluctuations for animals of this strain and age.
Key result
Dose descriptor:
NOAEL
Effect level:
1 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
body weight and weight gain
food consumption and compound intake
Key result
Abnormalities:
no effects observed
Fetal body weight changes:
no effects observed
Description (incidence and severity):
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.
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
no effects observed
Changes in postnatal survival:
not examined
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
External malformations occurred in test groups 0 and 2 (0 and 3 mg/kg bw/d) only. One control fetus had multiple external malformations associated with multiple skeletal malformations, while one mid-dose fetus had an open eye and associated soft tissue malformations. The distribution of the findings about the dose groups does not indicate an association to the treatment. No statistically significant differences between the groups were noted.
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Skeletal malformations were detected in test groups 0, 1 and 2 (0, 1 and 3 mg/kg bw/d). One control, one low-dose and one mid-dose fetus had associated external and/or soft tissue malformations. No skeletal malformations were observed in the high-dose group (9 mg/kg bw/d). No dose-response relationship was observed and, therefore, all these findings were assessed as not-treatment related.
For the test groups 0-3, 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 skeleton and appeared in the majority of cases without a relation to dosing. Although the rate of affected fetuses per litter was statistically significantly increased in the low-, mid- and high-dose groups (1, 3 and 9 mg/kg bw/d), the incidence of test group 3 was well within the historical control range, while the control, low- and mid-dose values were below the historical control range.
Some isolated cartilage findings without impact on the respective bony structures, which were designated as unclassified cartilage observations, occurred in test groups 0-3. The observed unclassified cartilage findings did not show any relation to dosing and were considered to be spontaneous in nature.
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Soft tissue malformations occurred in all test groups including control (0, 1, 3 and 9 mg/kg bw/d). The distribution of the findings about the dose groups does not indicate an association to the treatment and no statistically significant differences between the groups were noted. A number of observations affecting the spleen were noted in individual litters. Asplenia was observed in two low-dose litters (fetuses Nos. 36-05 F and 39-02 F) and in one mid-dose litter (fetuses Nos. 51-09 F and 51-10 F). Additionally, small spleens were observed in 12 fetuses of the same mid-dose litter (No. 51) and another small spleen in a different litter. These spleen dysplasia were not observed in the high-dose group and showed, therefore, no relation to dosing. Furthermore, the gestational parameters, in particular the resorption rate, do not indicate any embryofetal mortality in-utero which could have masked the occurence of potentially malformed offspring in the high-dose group. All other malformations can either be found in the historical control data or were single cases (e.g. hydronephrosis in the low dose) without any relation to dosing. Therefore, they were considered as not treatment related.
The examinations of the organs revealed a broad variety of soft tissue variations, i.e. dilated cerebral ventricle, malpositioned carotid branch and absent lung lobe (lobus inferior medialis) in fetuses of all test groups including the control (0, 1, 3 and 9 mg/kg bw/d). The incidences of these variations were neither statistically significantly different from control nor dosedependent and, therefore, not considered biologically relevant. All of them can be found in the historical control data at comparable incidences.
Only one unclassified soft tissue observation was recorded: discolored kidney in one middose fetus (3 mg/kg bw/d). This single incidence was not considered to be biologically relevant.
Details on embryotoxic / teratogenic effects:
There were noted external, soft tissue and skeletal malformations in seven control, four low-dose, eighteen mid-dose and two high-dose litters. The distribution of total malformations about the litters and groups was not related to dose and they were, therefore, considered as not treatment-related.
Nine fetuses were multiple-malformed. For control female fetus No. 10-11 an absent thoracic vertebra and fused ribs were recorded, while the findings in male control fetus No. 11-01 consisted of an absent lumbar vertebra and a branched rib. Female control fetus No. 12-05 had multiple external malformations, i.e. protruding tongue, absent tooth, no bony structure palpable in lower jaw and bent forelimbs, associated with multiple skeletal malformations, i.e. shortened incisive bones, shortened mandible, bent ribs, bent radius and ulna, bent humerus, bent scapula, bent tibia and fibula and bent femur. Furthermore, for male control fetus No. 16-04 a lumbar hemivertebra, a misshapen lumbar vertebra and a branched rib were recorded, while male low-dose fetus No. 28-09 (1 mg/kg bw/d) had a hydronephrosis and a hydroureter. Multiple visceral malformations were seen in female low-dose fetus No. 36-04 (1 mg/kg bw/d), i.e. diaphragmatic hernia, displaced and irregular shaped stomach, absent esophagus, indistinct cartilage rings of trachea, small gallbladder and malpositioned kidney. Female low-dose fetus No. 39-02 (1 mg/kg bw/d) had asplenia combined with malpositioned and bipartite sternebra. For female mid-dose fetus No. 58-09 (3 mg/kg bw/d) an open eye and multiple visceral malformations, i.e. diaphragmatic hernia, cardiomegaly, dilated pulmonary trunk, absent descending aorta and absent subclavian, were recorded, associated with severely fused sternebrae (bony plate) and misshapen caudal vertebra. Finally, female middose fetus No. 64-09 (3 mg/kg bw/d) showed severely fused sternebrae (bony plate) and a misshapen cervical vertebra.
A number of observations affecting the spleen were noted in individual litters. Asplenia was observed in two low-dose litters (fetuses Nos. 36-05 F and 39-02 F) and in one mid-dose litter (fetuses Nos. 51-09 F and 51-10 F). Additionally, small spleens were observed in 12 fetuses of the same mid-dose litter (No. 51) and another small spleen in a different litter. These spleen abnormalities were not observed in the high-dose group and showed, therefore, no relation to dosing. Furthermore, the gestational parameters, in particular the resorption rate, do not indicate any embryofetal mortality in-utero which could have masked the occurence of potentially malformed offspring in the high-dose group. The distribution of these observations in only very few litters and the absence of any dose response suggest a non-treatment related origin of the spleen findings. Other malformations, such as absent gallbladder, absent subclavian, thoracic hemivertebra and malpositioned kidney were scattered observations in individual fetuses of test groups 0, 2 and 3.
In general, neither the incidence of multiple-malformed offspring nor the individual malformations were dose-related and most of them can be found in the historical control data at comparable or higher frequency. No ontogenetic pattern is recognizable for the individual malformations nor was there any cluster of any of these individual malformations seen in the other offspring of any test group. An association of these findings to the treatment is not assumed.
No external variations were seen in any of the fetuses in this study. A spontaneous origin is assumed for the soft tissue variations and the broad range of skeletal variations which were observed in fetuses of all test groups including the controls. Although the total incidence of skeletal variations was statistically significantly increased in the low-, mid- and high-dose groups (1, 3 and 9 mg/kg bw/d), the incidence in test group 3 was well within the historical control range, while the control, low- and mid-dose values were below. Generally, all skeletal variations are equally distributed about the different test groups including controls, if normal biological variation is taken into account. The changes which were significantly different from the concurrent control were either not related to dose or can be found in the historical control data at a comparable or higher frequency. Although the rates of affected fetuses per litter were statistically significantly increased in test groups 1, 2 and 3 (1, 3 and 9 mg/kg bw/d), the overall incidence of all classified fetal variations in test group 3 was well within the historical control range, while the values in test groups 0, 1 and 2 were below. Thus, an association to the treatment is not assumed.
No unclassified external observations were seen in any of the fetuses in this study. A spontaneous origin is assumed for 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 a dose of 9 mg/kg bw/d.
Key result
Dose descriptor:
NOAEL
Effect level:
9 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse effects observed
Key result
Abnormalities:
effects observed, non-treatment-related
Key result
Developmental effects observed:
no

Tab. 1: Total external malformations

 

 

Test group 0

0 mg/kg bw/d

Test group 1

1 mg/kg bw/d

Test group 2

3 mg/kg bw/d

Test group 3

9 mg/kg bw/d

Litter

Fetuses

N
N

22

225

22

206

19

160

22

185

Fetal incidence

N (%)

2 (0.9)

0.0

1 (0.6)

0.0

Litter incidence

N (%)

2 (9.1)

0.0

1 (5.3)

0.0

Affected

fetuses/litter

Mean%

0.9

0.0

0.5

0.0

mg/kg bw/d = milligram per kilogram body weight per day; N = number; % = per cent

 

Tab. 2: Total soft tissue malformations

 

 

Test group 0

0 mg/kg bw/d

Test group 1

1 mg/kg bw/d

Test group 2

3 mg/kg bw/d

Test group 3

9 mg/kg bw/d

Litter

Fetuses

N
N

22

225

22

206

19

160

22

185

Fetal incidence

N (%)

1 (0.4)

4 (1.9)

16 (10)

2 (1.1)

Litter incidence

N (%)

1 (4.5)

3 (14)

3 (16)

2 (9.1)

Affected

fetuses/litter

Mean%

0.4

2.4

6.3

1.2

mg/kg bw/d = milligram per kilogram body weight per day; N = number; % = per cent

Tab. 3: Total soft tissue variations

 

 

Test group 0

0 mg/kg bw/d

Test group 1

1 mg/kg bw/d

Test group 2

3 mg/kg bw/d

Test group 3

9 mg/kg bw/d

Litter

Fetuses

N
N

22

225

22

206

19

160

22

185

Fetal incidence

N (%)

6 (2.7)

5 (2.4)

2 (1.3)

1 (0.5)

Litter incidence

N (%)

3 (14)

5 (23)

2 (11)

1 (4.5)

Affected

fetuses/litter

Mean%

2.4

2.5

1.2

0.5

mg/kg bw/d = milligram per kilogram body weight per day; N = number; % = per cent

 

  Tab. 4: Total soft tissue unclassified observations

 

 

Test group 0

0 mg/kg bw/d

Test group 1

1 mg/kg bw/d

Test group 2

3 mg/kg bw/d

Test group 3

9 mg/kg bw/d

Litter

Fetuses

N
N

22

225

22

206

19

160

22

185

Fetal incidence

N (%)

0.0

0.0

1 (0.6)

0.0

Litter incidence

N (%)

0.0

0.0

1 (5.3)

0.0

Affected

fetuses/litter

Mean%

0.0

0.0

0.5

0.0

mg/kg bw/d = milligram per kilogram body weight per day; N = number; % = per cent

 

Tab. 5: Total skeletal malformations

 

 

Test group 0

0 mg/kg bw/d

Test group 1

1 mg/kg bw/d

Test group 2

3 mg/kg bw/d

Test group 3

9 mg/kg bw/d

Litter

Fetuses

N
N

22

225

22

206

19

160

22

185

Fetal incidence

N (%)

5 (2.2)

1 (0.5)

3 (1.9)

0.0

Litter incidence

N (%)

5 (23)

1 (4.5)

2 (11)

0.0

Affected

fetuses/litter

Mean%

2.4

0.8

1.6

0.0

mg/kg bw/d = milligram per kilogram body weight per day; N = number; % = per cent

Tab. 6: Total fetal skeletal variations

 

 

Test group 0

0 mg/kg bw/d

Test group 1

1 mg/kg bw/d

Test group 2

3 mg/kg bw/d

Test group 3

9 mg/kg bw/d

Litter

Fetuses

N
N

22

225

22

206

19

160

22

185

Fetal incidence

N (%)

188 (84)

188 (91)

147 (92)

173 (94)

Litter incidence

N (%)

22 (100)

22 (100)

19 (100)

22 (100)

Affected

fetuses/litter

Mean%

83.2

90.0*

91.0*

94.7**

mg/kg bw/d = milligram per kilogram body weight per day; N = number; % = per cent

* = p ≤ 0.05 (Wilcoxon-test [one-sided]) ** = p ≤ 0.01 (Wilcoxon-test [one-sided])

 

Tab. 7: Occurrence of statistically significantly increased fetal skeletal variations

(expressed as mean percentage of affected fetuses/litter)

Finding

Test group 0

0 mg/kg bw/d

Test group 1

1 mg/kg bw/d

Test group 2

3 mg/kg bw/d

Test group 3

9 mg/kg bw/d

HCD

Mean%

(range)

Splitting of skull bone

0.0

1.5*

0.6

2.8

0.7

(0.0 – 2.4)

Irregular ossification of interparietal

0.6

0.6

1.2

3.5*

0.9

(0.0 – 3.8)

Supernumerary thoracic

vertebra

7.8

17.0

20.1

20.5*

20.6

(12.6 – 29.9)

Bipartite ossification of sternebra;

unchanged cartilage

0.0

0.9

1.8*

2.5**

1.5

(0.0 – 3.0)

 mg/kg bw/d = milligram per kilogram body weight per day; HCD = Historical control data; % = per cent

* = p ≤ 0.05 (Wilcoxon-test [one-sided])

** = p ≤ 0.01 (Wilcoxon-test [one-sided])

 

Tab. 8: Total unclassified cartilage observations

 

 

Test group 0

0 mg/kg bw/d

Test group 1

1 mg/kg bw/d

Test group 2

3 mg/kg bw/d

Test group 3

9 mg/kg bw/d

Litter

Fetuses

N
N

22

225

22

206

19

160

22

185

Fetal incidence

N (%)

14 (6.2)

18 (8.7)

22 (14)

12 (6.5)

Litter incidence

N (%)

8 (36)

11 (50)

10 (53)

9 (41)

Affected

fetuses/litter

Mean%

6.6

8.6

12.2

7.0

mg/kg bw/d = milligram per kilogram body weight per day; N = number; % = per cent

 

Tab. 9: Total fetal malformations

 

 

Test group 0

0 mg/kg bw/d

Test group 1

1 mg/kg bw/d

Test group 2

3 mg/kg bw/d

Test group 3

9 mg/kg bw/d

Litter

Fetuses

N
N

22

225

22

206

19

160

22

185

Fetal incidence

N (%)

7 (3.1)

4 (1.9)

18 (11)

2 (1.1)

Litter incidence

N (%)

7 (32)

3 (14)

4 (21)

2 (9.1)

Affected

fetuses/litter

Mean%

3.2

2.4

7.4

1.2

mg/kg bw/d = milligram per kilogram body weight per day; N = number; % = per cent

Tab. 10: Total fetal variations

 

 

Test group 0

0 mg/kg bw/d

Test group 1

1 mg/kg bw/d

Test group 2

3 mg/kg bw/d

Test group 3

9 mg/kg bw/d

Litter

Fetuses

N
N

22

225

22

206

19

160

22

185

Fetal incidence

N (%)

189 (84)

189 (92)

147 (92)

174 (94)

Litter incidence

N (%)

22 (100)

22 (100)

19(100)

22 (100)

Affected

fetuses/litter

Mean%

83.6

90.4*

91.0*

95.2**

mg/kg bw/d = milligram per kilogram body weight per day; N = number; % = per cent

* = p ≤ 0.05 (Wilcoxon-test [one-sided])

** = p ≤ 0.01 (Wilcoxon-test [one-sided])

Tab. 11: Summary of Reproduction Data

 

 

Test group 0

0 mg/kg bw/d

Test group 1

1 mg/kg bw/d

Test group 2

3 mg/kg bw/d

Test group 3

9 mg/kg bw/d

Females Mated

N

25

25

25

25

Pregnant
           Conception Rate

N
%

24

96

22

88

20

80

23

92

Aborted

N

1

0

0

1

Premature Births

N

0

0

0

0

Dams with Viable Fetuses

N

22

22

19

22

Dams with all Resorptions

N

0

0

0

0

 

 

 

 

 

 

Female Mortality

N

%

2Fi

8.0

0

0.0

1

4.0

1

4.0

 

 

 

 

 

 

Pregnant at Terminal Sacrifice

N

%

22fi

88

22

88

19

76

22

88

 

 

 

 

 

 

Corpora Lutea

Mean

11.1 D

11.0

9.8

10.0

 

S.D.

1.23

2.24

2.74

2.34

 

Total

244

241

186

221

 

 

 

 

 

 

Implantation Sites

Mean

10.9 D

10.5

8.8*

9.0*

 

S.D.

1.23

2.54

2.59

2.82

 

Total

240

230

168

199

 

 

 

 

 

 

Preimplantation Loss

Mean%

1.6 D

4.8

9.6

10.1

 

S.D.

3.44

11.14

12.09

19.20

 

 

 

 

 

 

Postimplantation Loss

Mean%

5.9 D

9.4

4.2

6.4

 

S.D.

8.68

12.77

5.94

8.88

 

 

 

 

 

 

Statistics:        D=Dunnett-test (two sided)     F=Fisher’s exact test (one sided)

* = p ≤ 0.05    ** = p ≤ 0.01

Tab. 12: Summary of Reproduction Data

 

 

Test group 0

0 mg/kg bw/d

Test group 1

1 mg/kg bw/d

Test group 2

3 mg/kg bw/d

Test group 3

9 mg/kg bw/d

Pregnant at Terminal Sacrifice

N

22

22

19

22

 

 

 

 

 

 

Resorptions: Total

Mean

0.7 D

1.1

0.4

0.6

 

S.D.

1.04

1.48

0.61

0.90

 

Total

15

24

8

14

 

 

 

 

 

 

 

Mean%

5.9 D

9.4

4.2

6.4

 

S.D.

8.68

12.77

5.94

8.88

 

 

 

 

 

 

Resorptions: Early

Mean

0.3 D

0.4

0.3

0.4

 

S.D.

0.70

0.66

0.48

0.67

 

Total

6

8

6

9

 

 

 

 

 

 

 

Mean%

2.2 D

3.4

3.2

4.3

 

S.D.

5.53

6.41

5.07

6.96

 

 

 

 

 

 

Resorptions: Late

Mean

0.4 D

0.7

0.1

0.2

 

S.D.

0.73

1.20

0.32

0.75

 

Total

9

16

2

5

 

 

 

 

 

 

 

Mean%

3.7 D

6.0

1.0

2.1

 

S.D.

6.71

9.67

2.90

7.04

 

 

 

 

 

 

Dead Fetuses

N

0

0

0

0

Statistics:             D=Dunnett-test (two sided)            

* = p ≤ 0.05        ** = p ≤ 0.01

Tab. 13: Summary of Reproduction Data

 

 

Test group 0

0 mg/kg bw/d

Test group 1

1 mg/kg bw/d

Test group 2

3 mg/kg bw/d

Test group 3

9 mg/kg bw/d

Dams with Viable Fetuses

N

22

22

19

22

 

 

 

 

 

 

Live Fetuses

Mean

10.2 D

9.4

8.4*

8.4*

 

S.D.

1.27

2.32

2.34

2.65

 

Total

225

206

160

185

 

 

 

 

 

 

 

Mean%

94.1 D

90.6

95.8

93.6

 

S.D.

8.68

12.77

5.94

8.88

 

 

 

 

 

 

Females

Mean

5.0 D

4.77

5.1

4.2

 

S.D.

1.59

2.03

2.15

2.18

 

Total

111

104

96

93

 

 

 

 

 

 

 

Mean%

46.0 D

45.8

57.1

48.6

 

S.D.

14.08

18.30

14.51

20.90

 

 

 

 

 

 

Males

Mean

5.2 D

4.6

3.4**

4.2

 

S.D.

1.68

1.94

1.42

1.97

 

Total

114

102

64

92

 

 

 

 

 

 

 

Mean%

48.1 D

44.8

38.8

45.0

 

S.D.

16.77

15.75

15.61

20.90

 

 

 

 

 

 

PER CENT LIVE FEMALES

49.3

50.5

60.0

50.3

PER CENT LIVE MALES

50.7

49.5

40.0

49.7

Statistics:        D=Dunnett-test (two sided)     

* = p ≤ 0.05    ** = p ≤ 0.01

Tab. 14: Summary of Reproduction Data

 

 

Test group 0

0 mg/kg bw/d

Test group 1

1 mg/kg bw/d

Test group 2

3 mg/kg bw/d

Test group 3

9 mg/kg bw/d

PLACENTALWEIGHTS UNITS: GRAMS

Of all Viable Fetuses

Mean

4.9 D

4.8

4.8

4.9

 

S.D.

0.90

0.73

1.08

0.92

 

N

22

22

19

22

 

 

 

 

 

 

Of Male Fetuses

Mean

5.0 D

4.9

4.8

4.9

 

S.D.

0.96

0.76

1.03

0.91

 

N

22

22

19

21

 

 

 

 

 

 

Of Female Fetuses

Mean

4.9 D

4.7

4.8

4.9

 

S.D.

0.95

0.78

1.14

0.92

 

N

22

22

19

22

 

 

 

 

 

 

FETAL WEIGHTS UNITS: GRAMS

Of all Viable Fetuses

Mean

35.3 D

34.4

37.1

35.5

 

S.D.

4.38

4.79

7.52

5.47

 

N

22

22

19

22

 

 

 

 

 

 

Of Male Fetuses

Mean

34.8 D

35.2

36.9

34.5

 

S.D.

5.26

5.08

7.49

4.70

 

N

22

22

19

21

 

 

 

 

 

 

Of Female Fetuses

Mean

35.2 D

34.5

37.2

35.3

 

S.D.

4.55

5.29

7.72

5.29

 

N

22

22

19

22

Statistics:        D=Dunnett-test (two sided)     

* = p ≤ 0.05    ** = p ≤ 0.01

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
9 mg/kg bw/day
Study duration:
subacute
Species:
rabbit
Quality of whole database:
OECD TG 414
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

In a study according OECD TG 414 the test substance was administered to pregnant Sprague-Dawley rats daily by stomach tube from implantation to one day prior to the expected day of parturition (days 6-19 post-coitum). 45 mg of the test substance/kg body weight/day elicited some maternal toxicity, substantiated by reduced food consumption (about 7% below controls), lower mean

terminal body weights, clear impairment in absolute (about 13% below controls) and corrected (about 44% below controls) body weight gain and macroscopic changes in the liver (i.e. paleness, accentuated lobular pattern and/or whitish areas). At the mid dose-level (15 mg/kg bw/day), maternal toxicity was slight, substantiated only by reduced absolute (about 8% below controls) and corrected (about 23% below controls) body weight gain. No signs of substance-induced maternal toxicity occurred at the low dose-level (5 mg/kg bw/day). There were no substance-induced, dose-related influences on the gestational parameters and no signs of prenatal developmental toxicity, especially no substance-induced indications of teratogenicity, up to and including the high dose-level (45 mg/kg bw/day).

Based on these results, the no observed adverse effect level (NOAEL) for maternal toxicity is 5 mg/kg/day, while it is 45 mg/kg bw/day for prenatal developmental toxicity (BASF AG, 2001).

In a further study according to OECD TG 414, the test item was administered to pregnant New Zealand White rabbits daily by stomach tube from implantation to one day prior to the expected day of parturition (GD 6-28) at dose levels of 1, 3 and 9 mg/kg bw/day (BASF SE, 2016). Analyses confirmed the correctness of the prepared concentrations, their homogeneous distribution and the stability of the test substance in the vehicle CMC (0.5%).

Generally, clinical observations including food consumption and body weight gain revealed no toxicologically relevant effects at the low dose of 1 mg/kg bw/d (OECD 414 and GLP). At the mid- and the highdose (3 and 9 mg/kg bw/day) signs of maternal toxicity were observed such as a dosedependent reduction of body weights/body weight gain, and reduced food consumption in the high-dose group, only. No differences of toxicological relevance between the control and the treated groups (1, 3 and 9 mg/kg bw/day) were determined for any reproductive parameters, such as conception rate, mean number of corpora lutea, mean number of implantations, as well as pre- and postimplantation loss. Similarly, no adverse effect of the test substance on fetal weight and sex distribution of the fetuses was noted at any dose. Overall, there were no toxicologically relevant adverse effects of the test substance on fetal morphology at any dose (BASF, 2016).

Toxicity to reproduction: other studies

Description of key information

no data available

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008
The available information are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Based on available experimental information, the test substance is not classified for toxicity to reproduction or developmental toxicity according to Regulation (EC) No 1272/2008 (CLP), as amended for the twelfth time in Regulation (EU) 2019/521.