Fatty acids, sunflower-oil, conjugated, maleated,reation products with diethanolamine, maleated tall-oil fatty acids and triethanolamine

Toxicity to reproduction

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

Study period:

09 Dec 2014 - 29 Jan 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 443 (Extended One-Generation Reproductive Toxicity Study) with the developmental neurotoxicity and immunotoxicity (DNT/DIT) cohorts but without the extension of Cohort 1B to mate the F1 animals to produce an F2 generation

GLP compliance:

yes

Limit test:

no

Justification for study design:

SPECIFICATION OF STUDY DESIGN FOR EXTENDED ONE-GENERATION REPRODUCTION TOXICITY STUDY WITH JUSTIFICATIONS [please address all points below]:

- Premating exposure duration for parental (P0) animals: 2 weeks
- Basis for dose level selection: dose-range finder similar or according to OECD 421
- Inclusion/exclusion of extension of Cohort 1B: yes
- Termination time for F2: no F2 generation was required
- Inclusion/exclusion of developmental neurotoxicity Cohorts 2A and 2B: inclusion of Cohorts 2A and 2B
- Inclusion/exclusion of developmental immunotoxicity Cohort 3: inclusion of Cohort 3
- Route of administration: raol via drinking water
- Other considerations, e.g. on choice of species, strain, vehicle and number of animals: regular strain in reproductive toxicity studies. The rat is the preferred animal species for reproduction studies according to test guidelines. This strain was selected since extensive historical control data were available for Wistar rats.

The design of the present OECD 443 Extended-one-genereation Reproductive Toxicity Study has been agreed with ECHA and eMSCA Germany in the according CORPA process and at MSC.

The objective of this multi-site study was to obtain general information on the possible effects of 2,2’-iminodiethanol on the integrity and performance of the male and female reproductive systems, including gonadal function, estrous cyclicity, mating behavior, conception, gestation, parturition, lactation and weaning, as well as on growth and development of the offspring. The study should also provide information about the effects on neonatal morbidity, mortality, target organs of the pups and preliminary data on prenatal and postnatal developmental toxicity. As part of this assessment, offspring were investigated for effects on the embryonic, fetal and preadult development of the nervous and immune systems as well as alterations in endocrine function (including thyroid pertubations). In addition choline was determined in blood plasma and livers of F1 adolescents.

Test material

Test material information

Constituent 1

Reference substance name:

2,2'-iminodiethanol

EC Number:

203-868-0

EC Name:

2,2'-iminodiethanol

Cas Number:

111-42-2

Molecular formula:

C4H11NO2

IUPAC Name:

2,2'-iminodiethanol

Details on test material:

- Name of test material (as cited in study report): 2,2’-iminodiethanol
- Physical state: Liquid/ colorless, clear
- Analytical purity: 99.9 area-% (see Analytical Report: 14L00128)
- Lot/batch No.: 000STD77L0 v. 14.05.14
- Expiration date of the lot/batch: 14 May 2015
- Stability under storage conditions: The stability of the test substance under storage conditions over the test period was guaranteed by the Sponsor /manufacturer, and the Sponsor /manufacturer holds this responsibility.
- Storage condition of test material: Room temperature

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 000STD77L0 v. 14.05.14 / Test substane number (internal): 14/0295-1
- Expiration date of the lot/batch: 14 May 2016
- Purity test date: regularly (also with respect to anaylsis of potential nitrosamine content)
- Purity: 99.9 corr. area-%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature. The stability of the test substance under storage conditions over the test period was guaranteed by the Sponsor, and the Sponsor holds this responsibility.
- Stability under test conditions: given, The stability of test substance in drinking water was demonstrated for a period of 9 days at room temperature
- Solubility and stability of the test substance in the solvent/vehicle: given (soluble in water)
Due to the fact that the test substance preparations were true solutions, it was not considered necessary to prove homogeneity through analytical procedures.

OTHER SPECIFICS:
- All mean values for 2,2’-iminodiethanol were in the expected range of the target concentrations (90 - 110%), demonstrating the correctness of the drinking water preparations
- Determination of total N-Nitrosamine content: No measurable content of total N-Nitrosamine (calculated as N-Nitrosodiethanolamine, NDELA) was detected in the test item preparations

Test animals

Species:

rat

Strain:

other: Crl:WI(Han)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH
- Age at study initiation: about 9 wks
- Housing: 1 animal per cage, polycarbonate cages type III with wooden gnawing blocks (Typ NGM E-022) and dust-free wooden bedding; during mating: 1 male/1 female per cage; during rearing up to weaning: 1 dam with her litter
- Diet: Ground Kliba maintenance diet mouse/rat “GLP”; ad libitum
- Water: Drinking water ad libitum
- Acclimation period: at least 5 days

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

Food analyses
With regard to the analytical findings of chemical and microbiological contaminants and the duration of application, the diet was found to be suitable. Fed. Reg. Vol. 44, No. 91 of 09 May 1979, p. 27354 (EPA), served as a guideline for maximum tolerable chemical contaminants. The concentration of microorganisms did not exceed 1*105/g feed.

Drinking water analyses
On the basis of the analytical findings, the drinking water was found to be suitable. German Drinking Water Regulation (Trinkwasserverordnung, Bundesgesetzblatt, 05 Dec 1990) served as a guideline for maximum tolerable contaminants.

Bedding and Enrichment analyses
On the basis of the analytical findings, bedding and cage enrichment were found to be suitable. Levels given in Lab Animal (Nov-Dec 1979, pp. 24-34) served as a guideline for maximum tolerable contaminants.

Administration / exposure

Route of administration:

oral: drinking water

Vehicle:

unchanged (no vehicle)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
For the test substance preparation, the specific amount of test substance and deionized water will be weighed in a vessel, depending on the dose group, and mixed until it is completely dissolved (details will be retained with the raw data).

Adjustment of pH: The pH of the administration solution of test groups 1-3 is adjusted to 7.3-7.5 by the addition of hydrochloric acid (1N, analytical grade) under pH meter control.

The test substance preparations will be prepared at intervals which guarantee that the test substance concentrations in the diet will remain stable. Stock solution will be stored at room temperature, protected from light and air.

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: until there is evidence of copulation or when a maximum period of 14 days has elapsed
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged (how): single

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The analytical investigations of the test substance preparations will be carried out at the Analytical Chemistry Laboratory of Experimental Toxicology and Ecology of BASF SE, Ludwigshafen, Germany, as a part of this study.

The analytical investigations will be performed according to the most recently authorized version of the control procedure 14/0295_1. The control procedure will be described in the raw data and the report.

The Analytical Report is included with the study report.

Duration of treatment / exposure:

The F0 animals will be offered drinking water preparations with test substance for approximately 2 weeks prior to breeding and continuing through breeding (up to two weeks), approximately 4 additional weeks (males) or gestation (three weeks) and lactation (three weeks) for females. Selected F1 offspring (cohorts 1A, 1B, 2A, 2B and 3) will be maintained on drinking water preparations with test substance until sacrifice.

Frequency of treatment:

daily

Details on study schedule:

F0 GENERATION PARENTAL ANIMALS AND F1 PUPS
Male and female rats, aged about 10 or 9 weeks when supplied, will be used as F0 generation parental animals. After an acclimatization period of at least 5 days, these rats will be kept for at least 2 weeks.
Then the F0 animals will be paired. The female F0 animals will be allowed to deliver and rear their pups (F1 generation pups) until postnatal days (PND) 4 or 21. The F0 generation parental animals will be sacrificed after weaning of the F1 generation pups. All F0 females will be sacrificed at about the same day after weaning (+/- 2 days).

F1 PUPS AND SELECTION OF COHORTS
Before weaning of the F1 generation pups on PND 21, 75 male and 75 females per group will be randomly selected, to be placed into cohorts. 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) will not be 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 will be maintained on drinking water preparations with test substance until sacrifice.

BREEDING OF THE F0 GENERATION PARENTAL ANIMALS
Male and female animals will be paired overnight in a 1:1 ratio until there is evidence of copulation or a maximum period of 14 days has elapsed. Throughout the mating period, each female will be paired with a predetermined male.

Normally, the female will be placed in the cage of her male partner about 16:00 h and separated from the male between 7:00 h and 9:00 h on the following morning. Deviations from the specified times are possible on Saturdays, Sundays and public holidays and will be documented in the raw data.

A vaginal smear will be prepared after each pairing and examined for sperm. If sperm are detected, pairing of the animals will be discontinued. The day on which sperm are detected will be referred to as gestation day (GD) 0 and the following day as GD 1.

STANDARDIZATION OF LITTERS (CULLING) OF F1 PUPS
On PND 4, all litters will be standardized to 10 pups per litter. Whenever possible, each litter contains 5 male and 5 female pups; as a rule, the first 5 surviving pups/sex in each litter will be used. If it is not possible to have 5 pups/sex in each litter, unequal numbers of males and females can be retained (e.g. 6 male and 4 female pups). Surplus animals will be sacrificed. Standardization of litters will not be carried out in litters with 10 pups or less.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 ppm (nominal)

Doses / concentrations 2

Dose / conc.:

100 ppm (nominal)

Remarks:

approx. 12.75 mg/kg body weight/day (mg/kg bw/d)

Doses / concentrations 3

Dose / conc.:

300 ppm (nominal)

Remarks:

approx. 37.68 mg/kg bw/d

Doses / concentrations 4

Dose / conc.:

1 000 ppm (nominal)

Remarks:

pprox. 128.35 mg/kg bw/d

No. of animals per sex per dose:

F0 parental animals: 30
F1 rearing animals, cohort 1A: 20
F1 rearing animals, cohort 1B: 25
F1 rearing animals, cohort 2A: 10
F1 rearing animals, cohort 2B: 10
F1 rearing animals, cohort 3: 10

Control animals:

yes, concurrent no treatment

Positive control:

The positive control substance (for immune system response) will be used with the given specifications of the producer (Sigma-Aldrich, Taufkirchen, Germany). No further analyses will be conducted.

Name of positive control substance: Cyclophosphamide monohydrate
CAS No.: 6055-19-2
Test-substance No.: 05/0012-6
Batch identification: SLBC0666V
Purity: 102.3% (according to supplier)
Homogeneity: Given (visually)
Stability: Expire date: March 2015
The stability of the positive control substance under storage conditions over the test period was guaranteed by the manufacturer and the manufacturer hold this responsibility.
Physical state/color: Solid/white
Storage conditions: Refrigerator (2-8°C)
The stability of Cyclophosphamide monohydrate (positive control substance) in the vehicle for 7 days at room temperature and 32 days frozen was verified in a comparable batch (Project No. 01Y0012/058064).

TEST SUBSTANCE PREPARATIONS AND ADMINISTRATION
Route of administration: Oral administration by gavage using 3 or 5 mL syringes.
Frequency of administration: Once daily
Volume to be administered: 10 mL/kg body weight; the body weight determined most recently will be used to calculate the administration volume.
Dose to be administered: 4.5 mg/kg body weight; the body weight determined most recently will be used to calculate the administration dose.
Preparation: Details on the technical procedure will be laid down in a description of the method and reported correspondingly.
Preparation frequency: At least once before the administration period.
Storage conditions of the test-substance preparations: The positive control substance preparations will be split in daily aliquots and stored closed in a freezer at -18°C.
Duration of the study: 4 weeks

Immunization and examinations (Anti SRBC-IgM ELISA) will be conducted at the same time and in exactly the same manner as with cohort 3 animals.

Examinations

Parental animals: Observations and examinations:

MORTALITY
A check for moribund and dead animals will be made twice daily from Mondays to Fridays and once daily on Saturdays, Sundays and public holidays.

CLINICAL SIGNS
A cageside examination will be conducted at least once daily for any signs of morbidity, pertinent behavioral changes and/or signs of overt toxicity. If such signs occur, the animals will be examined several times daily. Abnormalities and changes will be documented for each animal.
The parturition and lactation behavior of the dams will generally be evaluated in the morning in combination with the daily clinical inspection of the dams. Only particular findings (e.g. inability to deliver or umbilical cord not cut) will be documented on an individual dam basis.
On weekdays (except Saturdays, Sundays and public holidays) the parturition behavior of the dams will be inspected in the afternoons in addition to the evaluations in the mornings.
The day of parturition is considered to be the 24-hour period from about 15:00 h of one day until about 15:00 h of the following day. Departures from this procedure may occur on Saturdays, Sundays and public holidays.

DETAILED CLINICAL OBSERVATIONS (DCO)
All F0 parental animals will be subjected to detailed clinical observations (DCO) outside their cages at the administration period (day 0) and subsequently once per week (in the morning) by the same trained technicians, whenever possible. For observation, the animals will therefore be removed from their cages and placed in a standard arena (50 × 37.5 × 25 cm). The scope of examinations and the scoring of the findings observed will be based on the current index of findings in GROSSE-Reprotox software and includes but is not limited to the following parameters listed:

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

FOOD CONSUMPTION
Generally, food consumption will be determined once a week for the male and female F0 parental animals, with the following exceptions:
• Food consumption will not be determined after the 2nd 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 will be determined for GD 0-7, 7-14 and 14-20.
• Food consumption of the F0 females, which gave birth to a litter, will be determined for PND 1-4, 4-7, 7-14 and 14-21.
Food consumption will not be 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
Generally, water consumption will be determined twice a week for male and female F0 parental animals.
• Additionally, after the 2nd premating week water consumption of the females during gestation (animals with evidence of sperm plugs) will be determined for GDs 0-1, 4-5, 7-8, 10-11, 14-15, 17-18 and 20-21.
• Water consumption of the F0 females, which gave birth to a litter, will be determined for PNDs 1-2, 4-5, 7-8, 10-11, 14-15, 17-18 and 20-21.
Water consumption will not be determined in the females without positive evidence of sperm during mating and gestation periods and in the females without litter during lactation period.

BODY WEIGHTS
In general, the body weight of the male and female F0 parental animals will be determined 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 will be weighed on the day of positive evidence of sperm (GD 0) and on GD 7, 14 and 20.
• Females with litter will be weighed on the day after parturition (PND 1) and on PND 4, 7, 14 and 21.
Body weight will not be determined in the females without positive evidence of sperm during mating and gestation periods and in the females without litter during lactation period.

CLINICAL PATHOLOGY IN F0 PARENTAL ANIMALS
Samples will be withdrawn from 10 F0 parental males and females per group at termination.

Blood samples will be taken from animals by puncturing the retrobulbar venous plexus following isoflurane anesthesia. Blood sampling and blood examinations will be carried out in a randomized sequence. The list of randomization instructions will be compiled with a computer.

In the afternoon preceding the day of urinalysis, the animals will be individually transferred into metabolism cages (no food or drinking water provided); on the following morning, the individual urine specimens will be examined in a randomized sequence (the list of randomization instructions will be compiled with a computer).

The following parameters will be 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  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
The following hormones will be determined in the serum samples:
1. T4 (thyroxine)
2. TSH
3.9.1.4. 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

Oestrous cyclicity (parental animals):

For all F0 females, estrous cycle length and normality will be evaluated by preparing vaginal smears during a minimum of 2 weeks prior to mating and throughout cohabitation until there is evidence of sperm in the vaginal smear.

In all cohort 1A and 1B females, vaginal smears will be collected after vaginal opening until the first cornified smear (estrous) is recorded. The estrous cycle also will be evaluated in cohort 1A and 1B females for 2 weeks around PND 75.

Additionally, on the day of scheduled sacrifice, the estrous status will be determined in all female F0 animals and all females of cohorts 1A and 1B.

Sperm parameters (parental animals):

After the organ weight determination, the following parameters will be determined in the right testis or right epididymis of all male F0 parental animals and cohort 1A males 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) will be evaluated for the control and highest dose group, only. The intermediate doses will only be evaluated if deemed necessary based on the obtained results or other findings.

On completion of the final report, the testis and epididymis samples that have not been processed will be discarded. Sperm morphology slides will be archived.

Litter observations:

MORTALITY
A check for moribund and dead animals will be made twice daily from Mondays to Fridays and once daily on Saturdays, Sundays and public holidays.

DETAILED CLINICAL OBSERVATIONS (DCO)
All F1 animals in cohorts 1A, 1B, 2A and 3 will be subjected to detailed clinical observations (DCO) outside their cages at the administration period (day 0) and subsequently once per week (in the morning) by the same trained technicians, whenever possible. For observation, the animals will therefore be removed from their cages and placed in a standard arena (50 × 37.5 × 25 cm). The scope of examinations and the scoring of the findings observed will be based on the current index of findings in GROSSE-Reprotox software and includes but is not limited to the following parameters listed:
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

FOOD CONSUMPTION
Generally, food consumption will be determined once a week for the F1 rearing animals.

WATER CONSUMPTION
Generally, water consumption will be determined twice a week for F1 rearing animals.

BODY WEIGHTS
In general, the body weight of the F1 rearing animals will be determined once a week at the same time of the day (in the morning).

AUDITORY STARTLE RESPONSE HABITUATION IN COHORT 2A ANIMALS
On PND 24±1, the auditory startle response test will be carried out in all animals of cohort 2A using the SR-LAB; STARTLE RESPONSE SYSTEM (San Diego Instruments, San Diego, CA, U.S.A.). 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.

On completion of measurement, the data will be printed. The printouts will be signed with the project No., date and name, and will be part of the raw data.

FUNCTIONAL OBSERVATIONAL BATTERY (FOB) IN COHORT 2A ANIMALS
The FOB will be carried out once, between PND 63-75, in all animals of cohort 2A. The examinations will generally start in the morning at about 10:00 h. The FOB will be carried out in a randomized sequence. The animals will not be transferred to new cages before the test, nor will food or drinking water be withdrawn. The FOB will 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 will be ranked according to their degree or severity, if applicable.

Home cage observation
The animals will be 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. While other abnormalities will be recorded, particular attention will be paid to the following parameters:
1. Posture
2. Tremors
3. Convulsions
4. Abnormal movements
5. Gait
6. Other findings

Open field observation
For observation, the animals will be removed from their cages by the investigator and placed in a standard arena (50 × 50 × 25 cm). Besides noting other abnormalities, the following parameters will be 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 will be removed from the open field and will be 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
The Measurement of motor activity (MA) will be carried out between PND 63-75, in all animals of cohort 2A. The MA will be measured from 14:00 h onwards on the same day as the FOB will be performed. The examinations will be performed using the TSE Labmaster System supplied by TSE Systems GmbH, Bad Homburg, Germany. For this purpose, the animals will be placed in clean polycarbonate cages with a small amount of bedding for the duration of the measurement. Eighteen beams are allocated per cage. The number of beam interrupts will be counted over 12 intervals for 5 minutes per interval. The sequence at which the animals are placed in the cages will be selected at random. Since the starting time of each animal will be vary by the time needed to place the animals in the cages, the measurement periods will be staggered accordingly, beginning at about 14:00 h for the first animal. The individual measurement period begins when the 1st beam is interrupted and finishes exactly 1 hour later. No food or water will be offered to the animals during these measurements. After the transfer of the last animal in each case, the measurement room will be darkened. The program requires a file name for the measured data to be stored. This name consists of the reference number and a serial number.

CLINICAL EXAMINATIONS OF F1 GENERATION PUPS
Pup status and litter size after birth
The status (sex, live-born or stillborn) and number of all pups delivered from the F0 parents will be determined as soon as possible after birth. At the same time, the pups will also be examined for gross-morphological changes.

Pup viability/mortality
In general, a check will be made for any dead or moribund pups twice daily on workdays (once in the morning and once in the afternoon) or as a rule, only in the morning on Saturdays, Sundays or public holidays.

Pups, which died before the first determination of their status on the day of birth, will be defined as stillborn pups.

Clinical signs
All live pups will be examined daily for clinical symptoms (including gross-morphological findings) during the clinical inspection of the dams. If pups show particular findings, these will be documented with the dam concerned.

Nipple/areola presence
All surviving F1 male pups will be examined for the presence of nipples/areolas on PND 12 and again towards the end (either PND 20 or 21) of the lactation phase.

Anogenital distance
Anogenital distance (defined as the distance from the anus [center of the anal opening] to the base of the genital tubercle) measurements will be conducted in a blind randomized fashion, using a measuring ocular, on all live F1 pups on postnatal day 1.

Anogenital index
The anogenital index will be calculated according to the following formula:

anogenital index = anogenital distance [mm] / cubic root of pup weight [g]

Pup body weights
The pups will be weighed on the day after birth (PND 1) and on PND 4, 7, 14 and 21. Whenever possible, the individual weights will always be determined at the same time of the day (in the morning) and on PND 4 before standardization of the litters.

Additionally, the body weight of all F1 rearing animals will be determined on the day of vaginal opening and preputial separation.

Puberty onset
Developmental landmarks of puberty onset will be recorded on the following days in all cohort 1A, 1B, 2A, and 3 animals:
- From PND 27 onward: vaginal patency
- From PND 38 onward: balanopreputial separation

Recording will be completed when all animals are sexually mature or latest on PND 66 in males and in females.

CLINICAL PATHOLOGY IN COHORT 1A ANIMALS
Samples will be withdrawn from 10 cohort 1A males and females per group at termination.

Blood samples will be taken from animals by puncturing the retrobulbar venous plexus following isoflurane anesthesia. Blood sampling and blood examinations will be carried out in a randomized sequence. The list of randomization instructions will be compiled with a computer.

In the afternoon preceding the day of urinalysis, the animals will be individually transferred into metabolism cages (no food or drinking water provided); on the following morning, the individual urine specimens will be examined in a randomized sequence (the list of randomization instructions will be compiled with a computer).

The following parameters will be 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  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
The following hormones will be determined in the serum samples:
1. T4 (thyroxine)
2. TSH
3.9.1.4. 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

HORMONES IN PND 4 AND 22 F1-OFFSPRING
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 (as far as possible of different litters) per sex and group.
The blood samples will be collected after decapitation (following isoflurane anesthesia) from the Vena cava cranialis.

Hormone evaluations
The following hormones will be determined in the serum samples:
1. T4 (thyroxine)
2. TSH

BLOOD SAMPLING FOR CHOLINE DETERMINATION IN COHORT 1B ANIMALS
Blood will be sampled from all cohort 1B males and females shortly before sacrifice. Blood samples will be taken from non-fasted animals by puncturing the retrobulbar venous plexus under Isoflurane anesthesia. On the specific sampling days, samples will be withdrawn about 4 hours after lights turned on.

Blood samples (1 mL) will be collected in prefilled commercial EDTA tubes (e.g. Fa. Sarstedt). The samples will be centrifuged. The plasma will be separated. The preparation of the samples will be done under cooling. All samples will be stored at -80°C until analysis.

DEVELOPMENTAL IMMUNOTOXICITY EXAMINATIONS IN COHORT 3 ANIMALS
T-cell dependent antibody response
All males and females of cohort 3 will be 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), will be performed. Each sample will be diluted 1:500. SRBC-IgM concentrations outside the standard curve range will be measured in a second test run with an appropriate dilution. Generally, two in-house controls will be measured with each test run. The ELISA will be measured with a Sunrise MTP-reader, Tecan AG, Maennedorf, Switzerland, and evaluated with the Magellan-Software of the instrument producer.

Immunization:
Route of administration: Intraperitoneal, using 1 mL tuberculin-syringes
Preparation: Details of preparation of the sheep red blood cells will be described in the raw data (4×108 SRBC/mL)
Frequency of administration: twice (within one action)
Administration volume: 0.5 mL per animal, split into two portions of 0.25 mL
Five days after immunization blood samples will be taken by puncturing the retrobulbar venous plexus following isoflurane anesthesia. Blood sampling and blood examinations will be carried out in a randomized sequence. The list of randomization instructions will be compiled with a computer.

Splenic lymphocyte subpopulation analysis
Ten males and females per group of cohort 1A will be 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 10 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 following dose level of Cyclophosphamide monohydrate was selected to be sufficient to cause immunosuppressive activity as positive control substance:
4.5 mg/kg body weight/day: as dose level with expected immunosuppressive effects

CHOLINE DETERMINATION
Choline content will be determined in the liver of the blood-sampled PND 4 and 22 offspring.
Choline content will be determined in blood plasma and liver of cohort 1B animals. Details of the sampling are specified above, and in the section on pathology.

The choline analysis will be carried out at the Analytical Chemistry Laboratory of Experimental Toxicology and Ecology of BASF SE, Ludwigshafen, Germany, as a part of this study.

Methods of analysis
The analytical investigations will be performed according to the most recently authorized version of the control procedure. The control procedure will be described in the raw data and the report.

Postmortem examinations (parental animals):

NECROPSY
All F0 parental animals will be sacrificed by decapitation under isoflurane anesthesia. The exsanguinated animals will be necropsied and assessed by gross pathology, special attention being given to the reproductive organs. Animals which die intercurrently or are sacrificed in a moribund state will be necropsied as soon as possible after their death and assessed by gross pathology.

Organ weights
The following weights will be determined in all animals sacrificed on schedule:
1. Anesthetized animals
2. Adrenal glands
3. Brain
4. Cauda epididymis
5. Epididymides
6. Heart
7. Kidneys
8. Liver
9. Lymph nodes, axillary (10 animals per sex per group, Cohort 1A animals only)
10. Lymph nodes, mesenteric (10 animals per sex per group, Cohort 1A animals only)
11. Ovaries
12. Pituitary gland
13. Prostate
14. Testes
15. Seminal vesicles including coagulating gland
16. Spleen
17. Thymus
18. Thyroid glands (with parathyroid glands)
19. Uterus (with cervix)

Organ/Tissue fixation
The following organs or tissues will be 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 testes, epididymides, ovaries and eyes with optic nerve of animals that die or will have to be sacrificed intercurrently will be fixed in 4% buffered formaldehyde solution.

The left testis and left epididymis of all male F0 parental animals sacrificed at scheduled dates will be fixed in modified Davidson’s solution, whereas the right testis and epididymis will be used for sperm parameters.

In case of macroscopic findings in the right testis or right epididymis, this testis as well as the corresponding epididymis will be fixed for histopathological examination and the left testis and epididymis will be used for sperm analysis.

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 will be placed in 1% ammonium sulfide solutions for about 5 minutes in order to be able to identify early resorptions or implantations (SALEWSKI's method). Then the uteri will be rinsed carefully in physiologic salt solution (0.9 % NaCl). When the examinations are completed, the uteri will be transferred to the Pathology Laboratory for further processing.

Histopathology
Fixation will be followed by histotechnical processing, examination by light microscopy and assessment of findings.

Special attention will be given to stages of spermatogenesis in the male gonads.

Special attention will be given to the synchrony of the morphology in ovaries, uterus, cervix, and vagina to the estrous cycle status. Any morphological patterns of asynchrony will be reported.

Animals that die or are sacrificed in a moribund state will be processed histotechnically and assessed like control animals. Special stains of individual organs of individual animals will be prepared if required.

Reproductive organs of all low- and mid-dose F0 parental animals suspected of reduced fertility, or for which estrous cyclicity or sperm quality were affected, will be subjected to histopathological investigation. Organs demonstrating potential treatment–related changes will also be examined in the lower dose groups.

Further examinations or procedures will depend on the results obtained in the study.

Postmortem examinations (offspring):

POSTMORTEM EXAMINATION OF F1 GENERATION PUPS
Pups sacrificed on schedule
On PND 4, all surplus F1 pups as a result of standardization will be sacrificed by decapitation under isoflurane anesthesia and blood will be sampled for determination of serum thyroid hormone concentrations. After sacrifice, these pups will be examined externally, eviscerated and their organs will be assessed macroscopically.

On PND 22, the surplus F1 generation pups that will not be used for the formation of the cohorts will be sacrificed by decapitation under isoflurane anesthesia with CO2 and blood will be sampled for thyroid hormone analyses.

Pups showing clinical symptoms or gross-morphological findings may be further examined using appropriate methods. Organs/tissues with gross-morphological findings may be preserved in a suitable manner for potential histopathological examination.

All F1 pups not used for other purposes without any notable findings will be discarded after their macroscopic evaluation.

Prematurely dead or sacrificed pups
Pups that die or are sacrificed in a moribund state will be eviscerated and examined for possible defects and/or the cause of death using appropriate methods. These animals will be preserved for this purpose, if necessary.

Liver sampling for choline determination in PND 4 pups
Livers of 10 male and 10 female culled PND 4 offspring per group (as far as possible of different litters) will be sampled.

As soon as possible after dissection of the animals, the livers will be weighed in toto. The weight of the livers will be recorded but will not be reported. Immediately after weighing, the liver samples will be frozen in liquid nitrogen. Until analysis, the samples will be stored at -80°C.

NECROPSY
COHORT 1A
All cohort 1A animals will be sacrificed by decapitation under isoflurane anesthesia. The exsanguinated animals will be necropsied and assessed by gross pathology, special attention being given to the reproductive organs. Animals which die intercurrently or are sacrificed in a moribund state will be necropsied as soon as possible after their death and assessed by gross pathology.

Organ weights
The following weights will be determined in all animals sacrificed on schedule:
1. Anesthetized animals
2. Adrenal glands
3. Brain
4. Cauda epididymis
5. Epididymides
6. Heart
7. Kidneys
8. Liver
9. Lymph nodes, axillary
10. Lymph nodes, mesenteric
11. Ovaries
12. Pituitary gland
13. Prostate
14. Testes
15. Seminal vesicles including coagulating gland
16. Spleen
17. Thymus
18. Thyroid glands (with parathyroid glands)
19. Uterus (with cervix)

Organ/Tissue fixation
The following organs or tissues will be 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 testes, epididymides, ovaries and eyes with optic nerve of animals that die or will have to be sacrificed intercurrently will be fixed in 4% buffered formaldehyde solution.
The left testis and left epididymis of all male animals sacrificed at scheduled dates will be fixed in modified Davidson’s solution, whereas the right testis and epididymis will be used for sperm parameters.
In case of macroscopic findings in the right testis or right epididymis, this testis as well as the corresponding epididymis will be fixed for histopathological examination and the left testis and epididymis will be used for sperm analysis.
Spleens of 10 animals per sex per group of cohort 1A will be split in two comparable parts (transversally). One part of the spleen will be fixed in 4% buffered formaldehyde and afterwards be embedded in paraplast. The other part of the spleen will be frozen at -80°C, being used to perform a splenic lymphocyte subpopulation analysis (CD4+ and CD8+ T lymphocytes, B lymphocytes, and natural killer cells).

Histopathology
Fixation will be followed by histotechnical processing, examination by light microscopy and assessment of findings.
Special attention will be given to stages of spermatogenesis in the male gonads.
Special attention will be given to the synchrony of the morphology in ovaries, uterus, cervix, and vagina to the estrous cycle status. Any morphological patterns of asynchrony will be reported.
Animals that die or are sacrificed in a moribund state will be processed histotechnically and assessed like control animals. Special stains of individual organs of individual animals will be prepared if required.
A differential ovarian follicle count (DOFC) will be conducted in test groups 10 and 13 (Cohort 1A females) according to Plowchalk et.al..
Further examinations or procedures will depend on the results obtained in the study.

COHORT 1B
All cohort 1B animals will be sacrificed by decapitation under isoflurane anesthesia. The exsanguinated animals will be necropsied and assessed by gross pathology, special attention being given to the reproductive organs. Animals which die intercurrently or are sacrificed in a moribund state will be necropsied as soon as possible after their death and assessed by gross pathology.
Organ weights
The following weights will be determined in all animals sacrificed on schedule:
1. Anesthetized animals
2. Cauda epididymis
3. Epididymides
4. Liver
5. Ovaries
6. Pituitary gland
7. Prostate
8. Target organs
9. Testes
10. Seminal vesicles including coagulating gland
11. Uterus (with cervix)
Organ/Tissue fixation
The following organs or tissues will be fixed in 4% formaldehyde solution or in modified Davidson’s solution:
1. All gross lesions
2. Cervix uteri
3. Coagulating glands
4. Epididymis, left (fixed in modified Davidson ´s solution)
5. Liver
6. Ovaries (fixed in modified Davidson´s solution)
7. Pituitary gland
8. Prostate
9. Seminal vesicles
10. Target organs
11. Testis, left (fixed in modified Davidson ´s solution)
12. Uterus
13. Vagina

The testes, epididymides and ovaries of animals that die or will have to be sacrificed intercurrently will be fixed in 4% buffered formaldehyde solution.

Tissue sampling for choline determination
Tissue portions of the liver of all cohort 1 B males and females per group will be sampled.
As soon as possible after dissection of the animals, the livers will be weighed in toto. Immediately thereafter, the lobus sinister medialis of the liver will be separated from the remaining parts of the liver and weighed. The weight of the lobus sinister medialis will be recorded online in the pathology data system but will not be reported within the pathology report. Immediately after weighing, the liver lobe samples will be frozen in liquid nitrogen. Until analysis, the samples will be stored at -80°C.
For the remaining parts of the liver the standard procedures as described above will be followed.

Histopathology
All organs listed above will be preserved in adequate fixative. Histopathological processing and examination by light microscopy will only be conducted if results from cohort 1A are equivocal or in case the test compound is, from the results obtained so far, suspected to be a reproductive or endocrine toxicant.

Specifically, fixation of reproductive organs will be followed by histotechnical processing and examination by light microscopy and assessment of findings.

COHORT 2A
On postnatal day 77, cohort 2A animals will be weighed, counterbalanced, subjected to deep anesthesia (pentobarbital) and sacrificed by perfusion fixation.
SOERENSEN phosphate buffer will be used as the rinsing solution, and a fixation solution according to KARNOVSKY will be used as a fixative.
The perfusion fixed animals will be necropsied with regard to the question of neuropathology, and the visible organs will be assessed by gross pathology as accurately as is possible after a perfusion fixation. The cranial vault and the spinal cord will be opened and the skin will be removed from both hind extremities. In this state, the perfused animals will be stored in a fixation solution according to KARNOVSKY for at least 48 hours.
Animals which die intercurrently or are sacrificed in a moribund state will be necropsied as soon as possible after their death and assessed by gross pathology. These animals will be sacrificed under isoflurane anesthesia with C02.

Organ weights
The following weights will be determined (the brain will be weighed after its removal but before further preparation):
1. Brain (including olfactory bulb)

The terminal body weights will be recorded to calculate the relative organ weights.

Length and width of brain
The length and maximum width of the brain will be 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 will be carefully removed, processed histotechnically in accordance with the data given in the sections of this part of the study plan 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-C5)
9. Spinal cord, thoracic part (Th5-8)
10. Spinal cord, lumbar part (L1-L4)
11. Spinal ganglia (C1-C5 [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 (s. Gasserian)
16. Root fibers, dorsal (C1-C6 and L1-L4)
17. Root fibers, ventral (C1-C6 and L1-L4)

The remaining animal body after trimming will be stored in neutrally buffered, 4% formaldehyde solution.

Neurohistopathology
The histotechnical processing, examination by light microscopy and assessment of findings is performed on organs/tissues mentioned above.
Tissues demonstrating potential treatment–related changes will also be examined in the lower dose groups.
Further examination procedures (e.g. special stains) will depend on the results of the study, especially on the presence of neurofunctional or neuropathological changes.

Morphometry
Thickness measurements of major brain layers (neocortex: frontal and parietal cortices, caudate nucleus/putamen, hippocampus, corpus callosum, cerebellum) will be performed. Measurements will be 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 will be carried out in a cross section which approximates the plane of section on page 88 in Sherwood and Timiras (1970).
• Measurements for the thickness of the hippocampus will be carried out in a cross section which approximates the plane of section on page 110 in Sherwood and Timiras (1970).
• Measurements for the thickness of select folia of the cerebellum will be 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).
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) will be measured vertically to a tangent over a region of the frontal and parietal cortices determined beforehand.
• Caudate nucleus/putamen: The largest lateral extension will be measured.
• Corpus callosum: The width will be measured at the middle line of the cross section.
• Hippocampus: The largest dorsoventral extension will be measured.
• Cerebellum: The width of a select folium (e.g. folium pyramis) will be measured in the middle of a line which runs vertically to a tangent from the tip to the base of the folium.

COHORT 2B
On postnatal day 22, cohort 2B animals will be weighed, counterbalanced, subjected to deep anesthesia (isoflurane) and sacrificed by perfusion fixation.
SOERENSEN phosphate buffer will be used as the rinsing solution, and neutrally buffered, 4% formaldehyde solution will be used as a fixative.
The perfusion fixed animals will be necropsied with regard to the question of neuropathology, and the visible organs will be assessed by gross pathology as accurately as is possible after a perfusion fixation. The cranial vault and the spinal cord will be opened and the skin will be removed from both hind extremities. In this state, the perfused animals will be stored in neutrally buffered, 4% formaldehyde solution for at least 48 hours.
Animals which die intercurrently or are sacrificed in a moribund state will be necropsied as soon as possible after their death and assessed by gross pathology. These animals will be sacrificed under isoflurane anesthesia with C02.

Organ weights
The following weights will be determined (the brain will be weighed after its removal but before further preparation):
1. Brain (including olfactory bulb)

The terminal body weights will be recorded to calculate the relative organ weights.

Length and width of brain
The length and maximum width of the brain will be 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 will be carefully removed, processed histotechnically in accordance with the data given in the sections of this part of the study plan and examined:

The following organs or tissues will be fixed in 4% formaldehyde solution:
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 (s. Gasserian)

The animals and the tissue or organ material remaining after trimming will be stored in neutrally buffered, 4% formaldehyde solution.

Neurohistopathology
The histotechnical processing, examination by light microscopy and assessment of findings is performed on organs/tissues mentioned above.

COHORT 3
All Cohort 3 animals will be sacrificed by decapitation under isoflurane anesthesia. The exsanguinated animals will be necropsied and assessed by gross pathology. Animals which die intercurrently or are sacrificed in a moribund state will be necropsied as soon as possible after their death and assessed by gross pathology.

Organ weights
The following weights will be determined in all animals sacrificed on schedule:
1. Anesthetized animals
2. Spleen
3. Thymus

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

Histopathology
Histotechnical processing and examination will be performed at the request of the sponsor only.

SURPLUS F1 GENERATION PUPS
All surplus F1 generation pups will be sacrificed by decapitation under isoflurane anesthesia with CO2. The exsanguinated animals will be necropsied and assessed by gross pathology with special emphasis on the reproductive organs.

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

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

Histopathology
Histotechnical processing and examination will be performed at the request of the sponsor only.

Liver sampling for choline determination
Livers of the blood-sampled surplus PND 22 offspring (10 male and 10 female pups per group) will be sampled.
As soon as possible after dissection of the animals, the livers will be weighed in toto. The weight of the livers will be recorded but will not be reported. Immediately after weighing, the liver samples will be frozen in liquid nitrogen. Until analysis, the samples will be stored at -80°C.

Statistics:

Means and standard deviations will be calculated. In addition, the following statistical analyses will be carried out:
- Water consumption, food consumption, body weight and body weight change (parental animals, rearing animals and pups); estrous cycle length; mating days; duration of gestation; number of delivered pups per litter; developmental landmarks (days up to preputial separation or opening of the vagina), anogenital distance and index; implantation sites; postimplantation loss: DUNNETT test (two-sided)
- Number of live and dead pups and different indices (e.g. mating index, fertility index and gestation index) and number of litters with necropsy findings in pups; developmental landmarks (preputial separation or opening of the vagina): FISHER's exact test
- Absolute and relative pup organ weights; feces, rearing, grip strength forelimbs, grip strength hind-limbs, landing foot-splay test, motor activity, startle response: KRUSKAL-WALLIS and WILCOXON test (two-sided)
- Proportion of pups with necropsy findings per litter, presence of areolas/nipples: WILCOXON test (one-sided)
- Clinical pathology and sperm parameters: KRUSKAL-WALLIS and WILCOXON test
- Weight of the anesthetized animals and absolute and relative organ weights: KRUSKAL-WALLIS and WILCOXON test
- DOFC: WILCOXON test (one-sided)
- Brain weights: KRUSKAL-WALLIS and WILCOXON test
- Brain morphometry: linear measurements of selected brain regions: WILCOXON test

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Description (incidence and severity):

Clinical observations
No clinical signs or changes of general behavior attributable to the test substance were detected in any group.

Detailed clinical observations (DCO)
Male and female animals of all dose groups (100, 300 and 1000 ppm) did not show any abnormalities.

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):

Body weight
1000ppm: reduced b.w. in males and females starting on day 7 (-9% males, up to -13% females)
300ppm: reduced b.w. in males (-7%) starting on day 28. Transient reduction in females during PND4-7 (up to -5%)

Body weight gain
1000ppm: lower b.w. gain, which became statistically significant during several study sections. The overall decrease was app. 25% for males and 33% for females (65% during early lactation).
300ppm: in males: lower b.w. gain of overall 23%, but the difference was significant compared to the control during fewer section as for high dose animals.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Food consumption of the high-dose F0 parental males (1000 ppm) was statistically significantly below the concurrent control values during premating (up to 9%), while food consumption of all low- (100 ppm) and mid-dose (300 ppm) males was comparable to the concurrent control values.

Food consumption of the high-dose F0 females was statistically significantly below the concurrent control values throughout premating, gestation and lactation periods (up to 17%, 13% and 29%, respectively). For the mid-dose females (300 ppm) food consumption was statistically significantly below the concurrent control values during PND 4 - 7 (about 7%). There were no effects on food consumption during premating and gestation in this dose group. Food consumption of the low-dose females was comparable to the concurrent control during the entire study.

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

effects observed, treatment-related

Description (incidence and severity):

Water consumption of all F0 parental male rats of all test groups was comparable to the concurrent control throughout the entire study. The statistically significantly increased water consumption in the low-dose males during study days 18 - 21 and in the mid- and high-dose males during study days 42 - 46 was considered as spontaneous in nature.

In the high-dose F0 females (1000 ppm) water consumption was statistically significantly below concurrent control during GD 14 – 18 (up to 18%) and during the entire lactation period (up to 45%). The overall mean water consumption during lactation was about 35% below control. There was no effect on water consumption during premating in this dose group.
For the mid-dose females (300 ppm) water consumption was statistically significantly below the concurrent control values during PND 1 - 2, 7 - 8 and 20 - 21 (about 11%, 11% and 15%, respectively). The overall mean water consumption during lactation was about 10% below control. There was no effect on water consumption during premating and gestation in this dose group.
Water consumption of the low-dose females (100 ppm) was comparable to the concurrent control during the entire study.

Ophthalmological findings:

no effects observed

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

1000ppm:
Red blood cell (RBC) counts, hemoglobin and hematocrit values as well as the calculated red blood cell indices mean corpuscular volume (MCV) and mean corpuscular hemoglobin content (MCH) were decreased.
Reduced prothrombin time and increased platelet count in males
Incidental changes:
Reduced mean corpuscular hemoglobin concentration (MCHC) in males and relative lower reticulocyte counts in females: MCHC values stayed within the historical control range, and absolute reticulocyte counts did not differ from controls.
Decreased absolute number of basophils, but within the historical control range
Decreased absolute neutrophil counts in males, but within the historical control range
Decreased absolute and relative monociye and eosinophil counts and decreased relative basophil counts in females: all absolute values stayed within the historical control range, relative counts were only marginally decreased.

300ppm:
Reduced prothrombin time and increased platelet count in males
Incidental changes:
slightly decreased hemoglobin, MCV and MCH in males: within or marginally below the historical control range
slightly decreases hemoglobin, hematocrit values in females: both values (as well as the control values) are already above the historical control range

100ppm
slightly decreased MCV in males, but within the historical control range

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

1000ppm:
increased AST, ALP activities
Increased urea and albumin

300ppm
increased ALP activity in males, but since this was the only altered parameter, and the differences was less than two fold, the change was regarded as treatment-related, but not adverse (ECETOC Technical Report No. 85, 2002)
Lower ALT activity in males. But since there was no relation to dosing, this change was considered incidental.
Higher albumin levels in females, but since this was the only changed clinical pathology parameter (total calcium levels were higher in these individuals as consequence of higher albumin levels), it was considered as treatment related, but not advserse (ECETOC Technical Report No. 85, 2002)

The following clinical chemistry values were within historical control ranges and therefore, the alterations were regarded as incidental and not treatment-related:
increased sodium and chloride values in rats of both sexes (300 and 1000ppm)
increased total protein levels in females (300 and 1000ppm)
decreased cholesterol levels in females (1000ppm)
increased triglyceride levels in males (300 and 1000ppm) and females (300ppm only)

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

No treatment-related changes among urinalysis parameters were observed.

In males of test groups 2 and 3 (300 and 1000 ppm) urine specific gravity was lower and urine volume was higher (not statistically significantly) compared to controls. In conjunction with histopathological alterations in the kidneys, this change was regarded as treatment-related and adverse.

Behaviour (functional findings):

no effects observed

Immunological findings:

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Please see tables (Tables IC 10 – IC 14) of the attachment for details.

In the proximal tubules of the kidneys in the mid and high dose males and high dose females, there were signs of degeneration and regeneration characterized by apoptotic cell death, debris in the tubular lumen, increased numbers of large vesicular nuclei, increased basophilic staining and nuclear crowding. Within the papilla, females exposed to 300 and 1000 ppm showed an increase of multifocal mineral depositions. Nine high dose males showed multifocal mineral depositions at the transition between the outer and inner medulla.

The liver revealed a minimal to mild centrilobular hypertrophy in single high dose males and mid and high dose females.

The submucosa of the glandular stomach of females in all dose gropus was distended by edema with infiltration of inflammatory cells. Furthermore, some mid and high dose females revealed erosion/ulcers in the mucosa of the glandular stomach.

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.

Fertility
The female animals (Nos. 262, 275), which were not pregnant as well as their male mating partners (Nos. 62, 75) did not show relevant histopathological findings consistent with impaired fertility.

Histopathological findings: neoplastic:

no effects observed

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Platelet activating factor (PAF)
The median concentration of the platelet activating factor (PAF) concentration in serum of high dose females was decreased by 31% compared to controls. This decrease was statistically significant when the two-sided Jonckheere-Terpstra trend test was applied. No such difference was observed in males.

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

Estrous cycle data, generated during the last 2 weeks prior to mating in F0 animals, revealed regular cycles in the females of all groups. The mean estrous cycle duration in the different test groups varied between 3.9 and 4.0 days.

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 and treated groups.

Reproductive function: sperm measures:

effects observed, non-treatment-related

Description (incidence and severity):

In males of test group 3 (1000 ppm) incidence of abnormal sperms was slightly higher compared to controls. This was mainly due to missing heads, shortened heads, an abnormal hook of the head and in some samples combined morphology changes of head and tail. However, at least the first three mentioned morphological changes occurred also in the controls and the incidences were still in the range of historical controls (abnormal sperms 6.0-7.5 %). Therefore, these changes were regarded as incidental and not treatment-related.

Reproductive performance:

effects observed, treatment-related

Description (incidence and severity):

Male reproduction data
The male mating index was 100% in the control, low and high dose group, and 97% in the mid dose due to one male for whigh copulation was not confirmed.
Two mid dose males did not generate F1 pups. One additional male of this group and one high dose male did not generate F1 pups, but implants were found in the uterus of the corresponding females. Thus, the male fertility index ranged between 93% and 100% without showing any relation to dosing. This reflects the normal range of biological variation inherent in the strain of rats used for this study. The apparently infertile male rats did not show relevant gross or microscopic lesions.

Female reproduction and delivery data
The female mating index was 100% in control, low and high dose test groups, and 97% in the mid dose. The mean duration until sperm was detected (GD 0) varied between 2.2 and 3.1 days without any relation to the dose level.
All female rats delivered pups or had implants in utero with the exception of two mid dose females that did not become pregnant. The fertility index thus varied between 97% and 100% without any relation to the dose level. This reflects the normal range of biological variation inherent in the strain of rats used for this study. The non-pregnant females had no relevant gross lesions or microscopic findings.
The gestation index varied between 97% and 100%, which reflects the normal range of biological variation inherent in the strain of rats used for this study. The mean duration of gestation was 22.0 / 22.0 / 22.1 and 22.5** (**:p<=0.01) days in the control, low-, mid- and high-dose groups (0, 100, 300 and 1000 ppm), respectively.
The mean number of implantation sites was 12.3 / 12.2 / 11.4 and 7.8** (**:p<=0.01) implants/dam in the control, 100, 300, and 1000ppm groups, respectively. The post-implantation loss did not show any statistically significant differences between the groups and all values were well within the historical control range. Corresponding to the lower number of implants the mean number of F1 pups delivered per dam was lower in the high-dose group as well, the numbers were 11.9 / 11.8 / 11.1 and 7.3** (**:p<=0.01) pups/dam.
The rate of liveborn pups was not affected by the test substance. Despite a higher number of litters with stillborn pups at the top dose (2 / 2 / 4 and 8** (**:p<=0.01) in control, 100, 300, and 1000ppm groups, respectively), the total number of stillborn pups was comparable between the groups (7 / 2 / 5 and 8 from control to high dose) and does not indicate an association to treatment. The number of cannibalized pups was higher in the high dose group (0 / 2 / 2 and 10** (**:p<=0.01).

Effect levels (P0)

1

Key result

Dose descriptor:

NOAEL

Remarks:

general toxicity

Effect level:

100 ppm (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: evidence for distinct kidney and liver toxicity and effects on heamatotolgy parameters, as well as corresponding effects on water consumption, food consumption, body weights and clinical-pathological parameters

2

Key result

Dose descriptor:

NOAEL

Remarks:

fertility and reproductive performance

Effect level:

300 ppm (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reproductive performance

Results: F1 generation

General toxicity (F1)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

F1 rearing animals, Cohort 1A / 1B / 2A / 3
Several high-dose (1000 ppm) animals showed high-stepping gait and piloerection recurring in several study sections, which were considered treatment related and adverse.

Mortality / viability:

mortality observed, treatment-related

Description (incidence and severity):

Pub mortality
The viability index indicating pup mortality during early lactation (PND 0 - 4) was 99% or 100% in the control, low and mid dose group. The lower value of 93% in the high-dose group was slightly below the historical control range and was due to higher number of dead (4 vs. 1 in control) and cannibalized (10** (**:p<=0.01) vs. 0 in control) pups which were distributed across 8 litters. In 2 of those 8 litters none of the pups survived. The absolute numbers of pups surviving early lactation (PND 0 - 4) from control to high dose were 350 / 349 / 291* (*:p<=0.05) and 191** (**:p<=0.01). The significantly lower number in the mid-dose group reflects the incidentally lower number of liveborn litters in this group (27 vs. 30 in the control) and not an effect on pup survival. The lactation index indicating pup mortality on PND 4 - 21 was 100% in all test groups.

F1 rearing animals, Cohort 1A
There were no test substance-related mortalities in any of the groups up to a dose of 300 ppm. One high-dose male was sacrificed moribund in study week 0, after showing lateral position, apathy, hypothermia and diarrhea.

F1 rearing animals, Cohort 1B
There were no test substance-related mortalities in any of the groups up to 300 ppm. One high-dose male and one high-dose female were found dead on study week 8.


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 or spontaneous mortalities in any of the groups.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Mean birth weights of the pups were comparable across all test groups including control. Mean body weights of the high-dose pups (1000 ppm) were statistically significantly below the concurrent control values from PND 4 until weaning, the difference was about 13% at maximum. A slight decrease of pup weights was still noted in the mid-dose group beginning on PND 14 (male pups only) and definitive on PND 21 (about 6% below control). Mean body weight gain of the high-dose pups was statistically significantly below the concurrent control values during the entire lactation period (about 21 - 25% below control). A similar decrease though less pronounced (7 – 12%) was still noted for the mid-dose pups (300 ppm); the difference to the control became statistically significant during PND 4 - 7, 14 - 21 and 4 - 21. No test compound-related influence on F1 pup body weights/body weight gain were noted in the low-dose group (100 ppm).

F1 rearing animals, Cohort 1A
1000ppm: statistically significantly reduced body weight and body weight gain during the entire study period, the final weight was about 24% and 15% below control for males and females, respectively.
300ppm: The body weights of males and females were reduced by 6% and 5%, albeit the difference was not statistically significant. Body weight gain in males was significantly reduced during study days 35-49 (up to 28%), while there was no significant difference for females.
100ppm: There was no effect on body weight or body weight gain.

F1 rearing animals, Cohort 2A
The body weights and body weight gain of the high-dose males and females were statistically significantly below the concurrent control values during the entire study period, the final weight was about 13% and 16% below control, respectively. The body weights of the mid-dose females were below control throughout the study (final weight about 6% below control), although the difference was small and not statistically significant. There was no difference for mid dose males and low dose males and females.

F1 rearing animals, Cohort 3
The body weights of the high-dose males and females and body weight change in males were statistically significantly below the concurrent control values during the entire study period, the final weight was about 19% and 14% below control. The body weights of the low- and mid-dose male and female rats were comparable to the concurrent control values throughout the study.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

F1 rearing animals, Cohorts 1A / 1B / 2
Food consumption of the high-dose males was statistically significantly below the concurrent control values during the entire study period (up to 22%), the average reduction was about 11-17%, depending on which cohort was evaluated.
Food consumption of the high-dose females was statistically significantly below the concurrent control occasionally during the study (up to 11%), however, the average food intake throughout was only significantly different in cohorts 1B and 2 (reduction by app. 8-9%).
Food consumption of the mid-dose males (cohort 1A only) was below the concurrent control values throughout the study (average 6%), although the difference was statistically significant only during study days 0 – 7.
Food consumption of all most mid dose males as well as all mid dose females and low-dose males and females was comparable to the concurrent control values throughout the study.

F1 rearing animals, Cohort 3
Food consumption of the high-dose males was below the concurrent control during the entire study period although the difference was not statistically significant, the average reduction was about 11%. Food consumption of the high-dose females was also below the concurrent control throughout the study, although the difference was slight (about 5%) and not statistically significant. Food consumption of the low- and mid-dose males and females was comparable to the concurrent control throughout the study.

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

effects observed, treatment-related

Description (incidence and severity):

F1 rearing animals, Cohort 1A and 1B
Water consumption of the high-dose animals was below the concurrent control values throughout the study (average about 13%-16% lower). A statistically significantly reduced water consumption was also noted for the mid-dose males during parts of the study. Mid-dose females occasionally had statistically significantly lower water consumption during the study period (up to 18% below control). Water consumption of the low-dose male and female animals was comparable to the concurrent control values during the entire study.

F1 rearing animals, Cohort 2A
Water consumption of the high-dose males and females was below the concurrent control values during major parts of the study (average about 7% -13% lower). Mid-dose females occasionally had statistically significantly lower water consumption during the study period (up to 23% below control). Water consumption of the low and mid-dose males and low dose females was comparable to the concurrent control during the entire study.

F1 rearing animals, Cohort 3
Water consumption of the high-dose animals was below the concurrent control values during major parts of the study (average about 9% -11% lower). Water consumption in the low and mid-dose was comparable to the concurrent control during the entire study.

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

1000ppm:
decreased red blood cell (RBC) counts, hemoglobin and hematocrit values as well as red blood cell indices mean corpuscular volume (MCV and mean corpuscular hemoglobin content (MCH) in both sexes
Reduced prothrombine time in females
increased platelet counts in males
decreased absolute and relative monocyte counts in females

300ppm:
decreased hemoglobin and hematocrit values in both sexes. Additionally in females RBC counts were decreased and in males of the mentioned test group MCV and MCH were lower compared to controls. However, almost all values were within the historical control range (with the exception of a slight reduction in hematocrit in females and the calculated values in males). Therefore, the red blood cell parameter changes in rats of both sexes of this group were regarded as treatment-related but not adverse.
increased platelet counts in males

100ppm: In females, RBC counts, hemoglobin and hematocrit values and in males MCH were still lower compared to study controls, but all values apart from MCH in males were within historical control ranges. Since all measured red blood cell parameters in males were unaltered, the mentioned change in MCH is regarded as incidental and not treatment-related.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

1000ppm:
increased aspartate aminotransferase (AST) activities in both sexes
increased alkaline phophatase (ALP) activities in males
increased blood urea in both sexes
increased total protein (slightly outside HC), albumin (within HC) and calcium levels (slightly outside HC) in females. As already mentioned in the corresponding paragraph in the F0 generation, unbound and protein-bound calcium levels were measured. In order to maintain a constant free calcium level, total calcium level has to increase with higher albumin levels. Therefore, calcium changes were secondary to the albumin level increases and were regarded as treatment-related, but not adverse (ECETOC Technical Report No. 85, 2002). Albumin and total protein alterations were regarded as incidental and not treatment-related.

300ppm:
increased alkaline phophatase (ALP) activities in males, but within the historical control range. It was thus considered incidental.
increased total protein, albumin, and calcium levels in females, but only calcium levels were marginally outside the historical control range and considered as potentially treatement related, but not adverse (s. above)

Urinalysis findings:

effects observed, non-treatment-related

Description (incidence and severity):

In F1 rats of both sexes (PND 92) no treatment-related, adverse changes among urinalysis parameters were observed.
In females of all test groups urine volume was lower and urine specific gravity was higher compared to controls. However, both parameters were not dose-dependently changed. Statistically significance in this case occurred because of a high urine volume and a low specific gravity in the controls. Therefore, the changes were regarded as incidental and not treatment-related.

Sexual maturation:

effects observed, treatment-related

Description (incidence and severity):

Nipple/ Aerola anlagen
The apparent number and percentage of male pups having areolae was not influenced by the test substance when examined on PND 12. During re-examination on PND 20, no areolae were detected at all in male pups of all test groups.

Anogenital distance: No test substance-related findings.

Vaginal opening
The first day when vaginal opening was observed was PND 27, the last was PND 38. The mean number of days to reach the criterion in the control and 100, 300 and 1000 ppm test groups amounted to 29.7 / 30.2 / 30.6* (*:p<=0.05) and 32.0** (**:p<=0.01) days. The mean body weight on the day, when vaginal opening was recorded, amounted to 85.1 g / 87.4 g / 84.8 g and 80.4 g in the control to high dose test groups. The values for pubertal age and weight of control females were both at the lower end of the historical control range; thus the apparent statistical increase in age at attainment in the middose group is considered to be due to the very low concurrent control values in this study and not treatment-related. In the high-dose group the pubertal age is just above the upper limit of the historical range while the weight at puberty is below the historical control range. This clearly indicates that the later onset of puberty is a consequence of a general developmental delay and not a specific effect on the timing of puberty.

Preputial separation
The first day when preputial separation was observed was PND 38, the last was PND 66. The mean number of days to reach the criterion in the control and 100, 300 and 1000 ppm test groups amounted to 41.1 / 41.2 / 41.8 and 43.3** (**:p<=0.01) days. The mean body weight on the day, when preputial separation was recorded, amounted to 168.5 g / 168.6 g / 165.7 g and 145.2 g** (**:p<=0.01) in the control to high dose groups. In the high-dose group the pubertal age is well within of the historical range while the weight at puberty is distinctly below the historical control range. This indicates that the apparent later onset of puberty may be a spurious finding, and if at all, is a consequence of a general developmental delay and not a specific effect on the timing of puberty.

Estrous cycle
F1 rearing animals, Cohort 1A / 1B
Estrous cycle data, generated during a maximum of 3 weeks, revealed regular cycles in the females of control as well as of the low- and mid-dose groups. The mean estrous cycle duration was prolonged in the high-dose group (app. 5 days* vs. 4.1/4.2 days in the control (*:p<=0.05)). There was no particular cycle phase prolonged.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

F1 rearing animals, Cohort 1A / 1B
Absolute kidney weights were significantly increased in all dose groups, but without clear relation to dosing, while the relative weight was dose-dependently altered. Absolute liver weights were dose dependently increased in females of all dose groups, as well was in mid and high dose males of cohort 1B. The relative weight was increased in males and females of all groups. Changes in liver and kidney weights are partly caused by the terminal body weight decrease, but as all groups are affected (the body weight was mainly decreased in high dose animals) and the values are outside the historical control range, the changes were assessed as treatment-related.

Absolute weights of the seminal vesicles, cauda epididymides, epididymides, and heart were significantly decreased in high dose males. The relative weight of the pituitary gland was increased in high dose males of cohort 1B. These changes are considered to be caused by the decrease in terminal body weight, but the changes in reproductive organs would have been less pronounced without the three male animals which revealed histopathologic findings in these organs. Additionally, in cohort 1B, absolute prostate and testes weights were reduced. But as there was no change in relative testes weight, and as the change in prostate weight was less than for cohort 1A, these changes were regarded as secondary to the body weight decrease.

The increased relative adrenal gland weight in high dose males was higher than what could be expected due to the terminal body weight decrease. But as there were no histopathologic findings corresponding to the weight increase this finding might be treatment-related but was assessed as non-adverse.

The significantly increased relative uterus weights in mid and high dose females of cohort 1B were also thought to be caused by the terminal body weight reduction.

Please see tables (Tables IC 16 – IC 36) of the attachment for details.



F1 rearing animals, Cohort 3 (Immunotoxicity cohort)

Please see tables (Tables IC 40 – IC 47) of the attachment.
The increase in relative spleen weight in males of this test group was thought to be a consequence to the body weight reduction and therefore secondary.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Cohort 1A / 1B
1000ppm:
The light-brown discoloration in the liver of twelve males and two females corresponded in many cases to the diagnosis “fatty change, peripheral” and were regarded to be treatment-related.
The size reduction of prostate, seminal vesicle, epididymides and testes in three males corresponded to histopathological findings.
The foci in the glandular stomach were partly diagnosed as erosion/ulcer, but histopathologically there was not a significantly higher incidence compared to control. Therefore, it was regarded to be incidental.

F1 rearing animals, Cohort 3 (Immunotoxicity cohort)
One male (1000 ppm) revealed reduced size of testes, epididymides, seminal vesicle and prostate. This was regarded to be caused by the severely reduced terminal body weight and therefore regarded to be treatment-related but secondary.


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

Histopathological findings:

effects observed, treatment-related

Description (incidence and severity):

F1 rearing animals, Cohorts 1A and 1B
Please see tables (Tables IC 26 – IC 31, IC 38 - IC 39) of the attachment for details.

In mid and high dose animals, signs of degeneration and regeneration were observed in the proximal tubules of the kidneys (apoptotic cell death, debris in the tubular lumen, increased numbers of large, vesicular nuclei, increased basophilic staining, nuclear crowding). Within the papilla, high dose females showed an increase of multifocal mineral deposition. Mid and high dose males showed multifocal mineral depositions at the transition between the outer and inner medulla. These findings were regarded as treatment-related.

Mid and high dose animals showed a centrilobular hypertrophy in the liver. Additionally, three high dose males revealed a peripheral hypertrophy. Mid and high dose males and high dose females showed clear, round vacuoles in the peripheral area, which represent lipid vacuoles as determined via the ORO stain. These findings were regarded to be treatment-related.

In the mammary gland of high dose males, there was an increase of female-like mammary gland structures (tubule-alveolar structure, less amount of cytoplasm, increase in basophilia). One male revealed a moderate diffuse hyperplasia of the mammary gland tissue in addition. Females of this group revealed a higher incidence of eosinophilic amorphous material which was secreted into the ducts. These findings were regarded to be treatmentrelated.

Six high dose males (three per cohort) showed the histological picture of juvenile and immature testes, which were characterized by lower numbers or not fully developed and differentiated germ cell rows. In addition, there were no or only very few sperm present within testicular tubules. Consequently, these animals did not have sperm within the epididymides (aspermia) and reduced size of the secondary sexual organs (prostate, seminal vesicle, and coagulating gland). Three of these males additionally showed multifocal degeneration of the testicular tubules. Two males only revealed a minimal degeneration in the testicle. The immaturity in these animals was regarded to be a consequence to the reduced body weight. The tubular degeneration was assumed to be treatment-related. Vacuolation in the ductus deference occured in 12 high dose males of cohort 1A.

The vacuolation in the ductus deferens in four mid dose males might still be treatment-related, but due to the missing findings in all other sexual organs it was regarded as non-adverse.

Five high dose female revealed a diffuse atrophy of the ovary. Two of these females did not have any corpora lutea present. Twelve females of this group revealed luteal cysts. These findings were regarded to be treatment related. The one cyst in a mid dose female might be treatment-related, but as luteal cysts sometimes also occur in control animals it might also be an incidental finding (McInnes 2012).

In the pars distalis of the pituitary gland of males and females of all test groups eosinophilic cysts were observed. (This finding was also observed in cohorts 2A and 2B) These cysts differed from the cysts that occur sporadically as background,lesion in the pituitary gland. Spontaneous cysts, which were observed in control and treated animals, had a ciliated epithelium and a mucinous content. The treatment-related, very small eosinophilic cysts revealed a non-ciliated, irregular border with an eosinophilic homogenous content and were multifocally distributed within the pars distalis occasionally revealing clear vacuoles at the border.

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


Differential ovarian follicle count – F1 rearing animals, Cohorts 1A and 1B
(Table IC 32)
A dose dependent decrease in primordial and growing ovarian follicles was observed, which became statistically significant in high dose females.

Other effects:

effects observed, treatment-related

Description (incidence and severity):

CHOLINE CONCENTRATION IN BLOOD SAMPLES
Blood was withdrawn from cohort 1B animals shortly before sacrifice (week 13).
Choline was detected in all plasma samples. In males, there was a reduction in choline concentration from 1600ng/ml to 1400ng/ml after treatment with 100ppm of the test substance. Treatment with higher doses led to a further dose-dependent reduction in choline levels down to app. 1300ng/ml. In females, choline levels were dose dependently reduced at 100 and 300ppm (app. 1400ng/mL: control; 1300ng/mL: 100ppm, 1080ng/ml: 300ppm). Increasing the dose to 1000ppm led only a minimal further reduction. All in all, a dose-dependent reduction in choline levels was observed. This effect is more pronounced at lower concentrations, as a minimal levels seem to be approached at higher doses.

CHOLINE CONCENTRATION IN LIVER SAMPLES
Liver samples were analyzed in the F1 generation on PND4, 22, and 90.
There was no clear difference between treated and control animals on PND 4.
In 22-days old animals, choline content was dose-dependently reduced. Minimum concentration of app. 1/2 that of the control animals was already reached after treatment with 300ppm.
In 90-days old animals, all doses led to a drastic reduction of choline content in the liver. The minimum was already reached in the 100ppm group. Higher doses did not result in a further reduction of choline levels.

Sex ratio
The sex distribution and sex ratios of live F1 pups on the day of birth and on PND 21 did not show substantial differences between the control and the test substance-treated groups; slight differences were regarded to be spontaneous in nature.

Sperm parameters
No treatment related effects concerning motility of the sperms and sperm head counts in the testis and in the cauda epididymidis. In high dose males, the incidence of abnormal sperm was slightly higher due to two individuals having sperm with missing heads, abnormal hook of the head, bent heads and combined morphological changes of head and tail. These changes occured also in the control group, and incidences were within the historical control range. It was therefore regarded as incidental.

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not specified

Description (incidence and severity):

F1 rearing animals, Cohort 2A (Developmental Neurotoxicity Cohort, adults)
AUDITORY STARTLE RESPONSE
The maximum amplitude of the high-dose males and females (1000 ppm) was below the concurrent control during the entire measurement, while latency of reaction to a startle stimulus corresponded to the age of these animals. There was also no habituation to the test environment seen in these animals, males slightly more affected by this than females. The difference in the amplitude was statistically significant in measurement block 1 and 1 - 5 (males) as well as block 5 (females).
No influence of the test substance on auditory startle habituation (maximum amplitude and latency) was observed in the low- and mid-dose male and female animals.

FOB
Home cage observations: No test substance-related findings.
Open field observations: No test substance-related findings.
Sensorimotor tests/reflexes: No test substance-related findings.
Number of rearings, grip strength of fore- and hindlimbs and landing foot splay test: No test substance-related findings.
Motor activity measurement (number of beam interrupts): No test substance-related findings. Overall activity levels and habituation to the test environment corresponded to the age of these animals at PND 70.

NEUROPATHOLOGY
There was no treatement-related effect on brain size (width and length measurements).
All morphometric brain measurements were without any findings. Minimal changes in some single parameters in high dose male (hippocampus right, base of lobus vermis cerebrelli no 8) or female (parietal cortex left) animals were assessed as incidental and not related to treatment.
The medulla oblongata and the spinal cord of high dose animals revealed a minimal to marked, multifocal degeneration of nerve fibers. The finding was characterized by disintegrated myelin sheaths, vacuolation of myelin sheaths, pyknotic nuclei of oligodendroglia and spheroids as well as gitter cells within the lesions. The lesions were especially visible in longitudinal sections and less visible in cross sections of the spinal cord.

THYROID HORMONES (measured in cohort 1A)
Low and mid dose females on PND 4 as well as high dose females on PND 22 showed an increase in T4 compared to the control. On PND 4, only 2 high dose males and no females were available for hormone measurement due to insufficient blood sample volume.
On PND 92, T4 means in high dose males and females of test group 13 at PND 92 were 19%/18% higher compared to controls (medians 18% and 37% higher), although the values were not statistically significantly changed.

F1 animals, Cohort 2B (Developmental Neurotoxicity Cohort, weanlings)
There was no treatement-related effect on brain size (width and length measurements).
No treatment-related neurohistopathological findings were recorded.

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

effects observed, non-treatment-related

Description (incidence and severity):

T-cell dependent antibody response (Anti SRBC IgM antibodies)
Six days after immunization, no changes in the SRBC IgM titers were found in male and female rats (PND 60) of all test groups. SRBC titers were statistically significantly lower in rats of the positive control group (dosed with Cyclophosphamide).

Splenic lymphocyte subpopulations
relative T-helper cell (CD4+/CD3+ lymphocytes in spleen tissue) counts were decreased and relative cytotoxic Tcell (CD8+/CD3+ lymphocytes in spleen tissue) counts were increased in high dose females. This led to a decrease of the CD4/CD8 ratio (not statistically significantly). Absolute T-helper cell counts were not altered in the mentioned test group, but absolute cytotoxic T-cells were also increased (although not statistically significantly).

Effect levels (F1)

1

Key result

Dose descriptor:

NOAEL

Remarks:

general toxicity

Generation:

F1

Effect level:

100 ppm (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: evidence for distinct kidney and liver toxicity and effects on heamatotolgy parameters, as well as corresponding effects on water consumption

2

Key result

Dose descriptor:

NOAEL

Remarks:

developmental toxicity in the F1 progeny

Generation:

F1

Effect level:

300 ppm (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

viability

other: female phenotype of male mammary glands as well as effects on testes and ovaries

3

Key result

Dose descriptor:

NOAEL

Remarks:

developmental neurotoxicity

Generation:

F1 (cohort 2A)

Effect level:

300 ppm (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

developmental neurotoxicity

Remarks on result:

other: Demyelination already observed in 90-day study

4

Key result

Dose descriptor:

NOAEL

Remarks:

developmental immunotoxicity

Generation:

F1 (cohort 3)

Effect level:

ca. 1 000 ppm (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no effects on DIT

Target system / organ toxicity (F1)

1

Critical effects observed:

yes

Lowest effective dose / conc.:

300 ppm

System:

haematopoietic

Organ:

blood

Treatment related:

yes

Dose response relationship:

yes

2

Critical effects observed:

yes

Lowest effective dose / conc.:

300 ppm

System:

hepatobiliary

Organ:

liver

Treatment related:

yes

Dose response relationship:

yes

3

Critical effects observed:

yes

Lowest effective dose / conc.:

300 ppm

System:

urinary

Organ:

kidney

Treatment related:

yes

Dose response relationship:

yes

Overall reproductive toxicity

Reproductive effects observed:

yes

Lowest effective dose / conc.:

1 000 ppm (nominal)

Treatment related:

yes

Relation to other toxic effects:

reproductive effects occurring together with other toxic effects, but not as a secondary non-specific consequence of other toxic effects

Dose response relationship:

yes