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Diss Factsheets

Toxicological information

Toxicity to reproduction

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Administrative data

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

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2020

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 443 (Extended One-Generation Reproductive Toxicity Study)
Version / remarks:
25 June 2018
GLP compliance:
yes (incl. QA statement)
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)

Test material

Constituent 1
Chemical structure
Reference substance name:
2,2'-dimethyl-4,4'-methylenebis(cyclohexylamine)
EC Number:
229-962-1
EC Name:
2,2'-dimethyl-4,4'-methylenebis(cyclohexylamine)
Cas Number:
6864-37-5
Molecular formula:
C15H30N2
IUPAC Name:
4-[(4-amino-3-methylcyclohexyl)methyl]-2-methylcyclohexan-1-amine
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.

Test animals

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 or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH
- Females nulliparous and non-pregnant: yes
- Age at 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

Administration / exposure

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).
Doses / concentrationsopen allclose all
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

Examinations

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

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

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 / performance (P0)

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.

Details on results (P0)

see executive summary

Effect levels (P0)

open allclose all
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

Target system / organ toxicity (P0)

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

Results: F1 generation

General toxicity (F1)

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 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.

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 generation parental animals, Cohort 1B
There were no test substance-related or spontaneous mortalities in any of the groups.

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 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.

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 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.

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 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.

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.

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 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.

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).

Developmental neurotoxicity (F1)

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 (F1)

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.

Effect levels (F1)

open allclose all
Key result
Dose descriptor:
NOAEL
Generation:
F1 (cohort 1A)
Effect level:
15 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: reproductive function (oestrus cycle) reproductive function (sperm parameters)
Remarks on result:
not determinable due to absence of adverse toxic effects
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

Target system / organ toxicity (F1)

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

Overall reproductive toxicity

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

Any other information on results incl. tables

see attachment: 90R0695_00R050_results and tables

Applicant's summary and conclusion

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.