Registration Dossier

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:
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

Reference
Name:
Unnamed
Type:
Constituent
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 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 / concentrationsopen allclose all
Dose / conc.:
0 ppm (nominal)
Dose / conc.:
100 ppm (nominal)
Remarks:
approx. 12.75 mg/kg body weight/day (mg/kg bw/d)
Dose / conc.:
300 ppm (nominal)
Remarks:
approx. 37.68 mg/kg bw/d
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
Details on study design:
- Dose selection rationale:
- Rationale for animal assignment (if not random):
- Other:
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 animals)

General toxicity (P0)

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

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

Clinical observations for females during gestation of F1 litters (Tab. IA-003)
There were no test substance-related clinical findings in any F0 parental females of all dose groups during the gestation period for F1 litter.
One sperm positive and one sperm negative female of test group 02 (Nos. 262 and 275 - 300 ppm) and one sperm positive female of test group 03 (No. 291 - 1000 ppm) did not deliver F1 pups and had no implants in the uterus. One sperm negative female of test group 02 (No. 266) did not deliver F1 pups, but had implants in the uterus. These observations were not considered to be associated with the test compound.

Clinical observations for females and offspring during lactation of F1 litters (Tab. IA-004)
There were no test substance-related clinical observations observed in all F0 parental females of all dosed groups during the lactation period.
Two high-dose females (Nos. 292 and 311 - 1000 ppm) had lost their complete litters by PND 4. One female pup in control (dam No. 219, pup No. 7 - 0 ppm) showed chromodacryorrhea (both eyes) during PND 20 - 21 and one female pup in test group 03 (dam No. 306, pup No. 9 - 1000 ppm) showed microphthalmia (both eyes) during PND 18 - 21. These finding were not considered to be treatment-related.
Mortality:
no mortality observed
Description (incidence):
There were no test substance-related or spontaneous mortalities in any of the groups.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
The body weights of the high-dose F0 parental males (1000 ppm) were statistically significantly below the concurrent control from study day 7 onwards (up to 9%), as were the body weights of the mid-dose F0 parental males (300 ppm) from study day 28 onwards (up to 7%), until the
end of the study.
The body weights of the low-dose F0 parental male rats (100 ppm) were comparable to the concurrent control values throughout the entire study. The body weights of the high-dose F0 parental females were statistically significantly below the concurrent control from premating day 7 onwards, throughout gestation and lactation (up to 5%, 15% and 13%, respectively).
The body weights of the mid-dose F0 parental females were statistically significantly below the concurrent control values during PND 4 - 7 (up to 5%). There were no effects on body weights during premating and gestation in this dose group.
The body weights of the low-dose females were comparable to the concurrent control during the entire study. A consistently lower body weight gain was noted in the high-dose F0 parental males (1000 ppm) which became statistically significant during several study sections. The overall average decrease of weight gain was about 25%. The mid-dose F0 parental males (300 ppm) still gained less weight than the controls (overall about 23% less), however the difference to the control was statistically significant in fewer sections than in the high-dose group.
A lower body weight gain was also noted for the low-dose F0 parental males (100 ppm) during the first week of treatment (14% below control). However, this was the only study section where this was seen and there was no effect on body weight during this study section and the
remaining study. Thus, this is considered of no toxicological relevance.
The body weight gain of the high-dose F0 parental females (1000 ppm) was consistently below the concurrent control during gestation (overall about 33%) through early lactation (about 65%). In the other study sections periods of lower weight gain alternated with periods of comparable
or higher weight gains, displaying an inconsistent course. The body weight gain of the low- and mid-dose F0 parental females (100 and 300 ppm) was widely comparable to the concurrent control, with some brief episodes of statistically significantly lower (GD 0-7) or higher (premating days 7-14) weight gains. Those minor changes were considered as spurious findings.
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):
In the F0 generation rats of both sexes in test group 3 (1000 ppm) 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.
Additionally, in the mentioned test group in males mean corpuscular hemoglobin concentration
(MCHC) and in females relative reticulocyte counts were lower compared to controls, but
MCHC was within the historical control range (MCHC 20.36-21.48 mmol/L) and in females
mean values of the reticulocyte counts were not different to the controls. Therefore, both latter
parameter changes were regarded as incidental and not treatment-related.
In rats of both sexes of test group 2 (300 ppm) hemoglobin values were decreased. In males
of this test group MCV and MCH and in females hematocrit values were decreased. MCV was
already decreased in males of test group 1 (100 ppm). In males of test group 2 the measured
parameter hemoglobin was within the historical control range (males: hemoglobin 8.6-9.3
mmol/L). In these individuals also the the calculated red blood cell parameters MCV and MCH
were within or marginally below (MCV in test group 2) historical control ranges (males: MCH
1.00-1.10 fmol; MCV 48.4-51.9 fl). In females of test group 2 (300 ppm) as well as in female
study controls hemoglobin and hematocrit values were not below, but above historical control
ranges (females hemoglobin 8.7-9.3 mmol/L; hematocrit 0.410-0.435 L/L). Measured red blood
cell parameters, hemoglobin in both sexes and hematocrit in females of test group 2 were only
slightly lower compared to study controls (hemoglobin males mean – 6%; females mean -3%,
hematocrit females mean -4% compared to respective control means, Mueller et al., 2006).
Therefore, the alterations of the red blood cell parameters in test group 2 and test group 1 were
regarded as treatment-related, but not adverse.
In rats of both sexes of test groups 2 and 3 (300 and 1000 ppm) prothrombin time
(Hepatoquick’s test HQT) was reduced and in males of both test groups platelet counts were
increased.
Regarding differential blood cell counts in test group 3 (1000 ppm), in rats of both sexes
absolute basophil counts were decreased. Additionally, in males absolute neutrophil counts
and in females absolute and relative monocyte and eosinophil counts and relative basophil
counts were lower compared to controls. All absolute cell counts were within historical control
ranges (males absolute neutrophils 0.70-1.35 Giga/L; absolute basophils 0.00-0.02 Giga/L;
females absolute basophils 0.00-0.02 Giga/L; absolute monocyte 0.06-0.08 Giga/L; absolute
eosinophils 0.08-0.11 Giga/L). Relative monocyte and eosinophil counts in females of test
group 3 were marginally below the historical control ranges (relative monocytes 1.7-2.8 %,
relative eosinophils 2.2-3.7 %). However, because no relevant change above historical control
ranges occurred in the total and absolute differential cell counts all alterations in the differential
blood cell counts in males and females of test group 3 (1000 ppm) were regarded as incidental
and not treatment-related.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
In male F0 generation rats of test group 3 (1000 ppm) aspartate aminotransferase (AST) and
alkaline phosphatase (ALP) activities were increased. ALP activities were already higher in
males of test group 2 (300 ppm), but in this test group it was the only relevantly changed clinical
pathology parameter. ALP means in males of both mentioned test groups was less than 2fold
higher compared to controls. Therefore, the ALP change in males of test group 2 was regarded
as treatment-related, but not adverse (ECETOC Technical Report No. 85, 2002). Alanine
aminotransferase (ALT) activities were lower in males of test group 2 (300 ppm) compared to
controls, but the change was not dose-dependent and therefore it was regarded as incidental
and not treatment-related.
In male and female rats of test group 3 (1000 ppm) urea and albumin levels were increased.
Albumin levels were already higher in females of test group 2 (300 ppm), but in these
individuals, this was the only relevantly changed clinical pathology parameter (total calcium
levels were higher in these individuals as consequence of higher albumin levels). Therefore,
the albumin increase in females of test group 2 (300 ppm) was regarded as treatment-related,
but not adverse (ECETOC Technical Report No. 85, 2002).
Calcium levels were increased in rats of both sexes of test groups 2 and 3 (300 and 1000 ppm).
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). Calcium levels
in males were within, those of females slightly above the historical control range (calcium males
2.45-2.67 mmol/L, females 2.49-2.61 mmol/L).
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 of test groups 2 and 3 (300 and
1000 ppm), increased total protein levels in females of test group 2 and 3, Decreased
cholesterol levels in females of test group 3, increased triglyceride levels in males of test group
2 and 3 and in females of test group 2 (males: sodium 141.1-146.7 mmol/L, chloride 98.8-105.9
mmol/L, triglycerides 0.61-1.29 mmol/L; females: sodium 140.3-145.8 mmol/L, chloride 98.7-
103.2 mmol/L, total protein 60.65-67.01 g/L, cholesterol 0.99-1.70 mmol/L). Triglyceride levels
in females of test group 2 (300 ppm) were higher compared to controls, but the change was
not dose-dependent and therefore, it was regarded as incidental and not treatment-related.
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
Description (incidence and severity):
Please see tables (Tables IC 10 – IC 14) of the attachment.
The decrease in terminal body weight in males and females of test group 02 and 03 (300 and
1000 ppm) was regarded to be treatment related. In test group 02 animals it was still within
historical control values (see PART III).
The kidney and liver weights of males and females of all test groups were increased when
compared to control. The weight changes were regarded to be treatment-related.
The decrease in absolute epididymides and heart weight in males and adrenal glands, heart,
spleen, and thyroid glands in females were thought to be a consequence to the decrease in
terminal body weight. This was assessed to be treatment-related but secondary.
The increase in absolute brain weight of females and relative and absolute uterus weight did
not show a dose response relationship and test group 03 (1000 ppm) weights were not
significantly changed. This was regarded to be incidental.
The relative weight increase in adrenal glands, brain, spleen and testes of male animals as
well as the relative weight increase in brain and uterus of females was thought to be secondary
to the terminal body weight decrease.
The decrease of relative spleen weight of test groups 02 and 03 (300 and 1000 ppm) was still
within historical control values (see PART III) and did not have a histopathological correlate,
and were thus regarded to be not adverse if treatment-related at all.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Gross pathology
Please see tables (Tables IC 10 – IC 14) of the attachment.
The foci in the glandular stomach of females, mainly in test group 03 (1000 ppm) (1/3/3/6) were
regarded to be treatment related.
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.
Fertility:
The female animals (animal Nos. 262, 275), which were not pregnant as well as the male
mating partners (animal Nos. 62, 75) did not show relevant gross lesions.
Neuropathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Please see tables (Tables IC 10 – IC 14) of the attachment.
In the proximal tubules of the kidneys, 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. These findings were
regarded to be treatment-related. Within the papilla, females of test group 02 and 03 (300 and
1000 ppm) showed an increase of multifocal mineral depositions. Nine males of test group 03
(1000 ppm) showed multifocal mineral depositions at the transition between the outer and inner
medulla. The mineralization in males and females was regarded to be treatment-related.

The liver revealed a minimal to mild centrilobular hypertrophy in single males of test group 03
(1000 ppm) and females of test groups 02 and 03 (300 and 1000 ppm). This finding was
regarded to be treatment-related.

The submucosa of the glandular stomach of females was distended by an edema with
infiltration of inflammatory cells. Furthermore, some females of test group 02 and 03 (300 and
1000 ppm) revealed erosion/ulcers in the mucosa of the glandular stomach. These findings in
test groups 02 and 03 (300 and 1000 ppm) were regarded to be treatment-related.
All other findings occurred either individually or were biologically equally distributed over control
and treatment groups. They were considered to be incidental or spontaneous in origin and
without any relation to treatment.
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):
CHOLINE CONCENTRATION IN BLOOD AND LIVER SAMPLES
The analytical results demonstrated the clear presence of choline in all plasma samples from
the animals dosed with the test substance 2,2’-iminodiethanol (100 ppm, 300 ppm and 1000
ppm dosed animals) and in those from control, non-dosed animals.
In general, it can be stated that the presence of the test substance 2,2’-iminodiethanol led to a
reduction in the content of choline in the plasma samples analyzed. This effect appears to be
dose-dependent, in that higher dose levels were associated with greater choline reduction.
This effect is most clearly visible at lower dose levels (100 ppm and 300 ppm), at which
dramatic plasma choline levels could be seen. At higher dosing levels, although further minor
plasma choline content reduction was observed, this was by no means as drastic.
The analytical results demonstrated the clear presence of choline in all liver samples from the
animals dosed with the test substance 2,2’-iminodiethanol (100 ppm,300 ppm and 1000 ppm
dosed animals) and in those from control, non-dosed animals. This was true from all time points
investigated (4-day old pups, 22-day old pups and ~90-day old adolescents).
In general, it can be stated that the presence of the test substance 2,2’-iminodiethanol led to a
reduction in the content of choline in the liver samples analyzed. This effect appears to be dose-dependent, in that higher dose levels were associated with greater choline reduction, but
only up to moderate dosing levels (300 ppm and 100 ppm, depending on the sampling day).
At higher dosing levels, no further dramatic liver choline content reduction was observed.
This effect was however, not observed in 4-day old animals, in which no clearly definable dosedependent
trend is evident. In 22-day old animals this effect could be clearly observed,
although the choline levels of the 100 ppm dosed animals have not yet attained minimal
concentrations. In ~90-day old animals the effect is dramatic in that the liver choline levels of
all non-control animals have reached an approximate minimum. Only a relatively minor further
dose-dependency can be observed at this time point.

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 for the F1 litter, revealed
regular cycles in the females of all test groups including the control. The mean estrous cycle
duration in the different test groups was similar: 3.9 days in control and in the low- and middose
groups and 4.0 days in the high-dose group.

Differential ovarian follicle count - F0 generation parental animals
The results of the differential ovarian follicle count (DOFC) – comprising the numbers of
primordial and growing follicles, as well as the combined incidence of primordial plus growing
follicles – did not reveal significant differences between the control group 00 and animals of
test group 03.
Reproductive function: sperm measures:
effects observed, non-treatment-related
Description (incidence and severity):
Concerning motility of the sperms and sperm head counts in the testis and in the cauda
epididymidis no treatment-related effects were observed. 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
For nearly all F0 parental males, which were placed with females to generate F1 pups, copulation was confirmed. Copulation was not confirmed for test group 02 male No. 75 paired with test group 02 female No. 275. Thus, the male mating index was 100% in the control and
test groups 01 and 03 and 97% in test group 02. Fertility was proven for most of the F0 parental males within the scheduled mating interval for
F1 litter. Two males of test group 02 (Nos. 62 and 75 - 300 ppm) did not generate F1 pups. One male of test group 02 (No. 66) and one male of test group 03 (No. 91) did not generate F1 pups, but implants were found in the uterus of the corresponding females (No. 266 - test
group 02 and No. 291 - test group 03).
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.
Thus, the test substance 2,2’-iminodiethanol did not adversely affect fertility and reproduction
of the F0 generation parental males.

Female reproduction and delivery data
The female mating index calculated after the mating period for F1 litter was 100% in the control and test groups 01 and 03 and 97% in test group 02. 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 following exceptions:
 Test group 02
female No. 262 (mated with male No. 62) did not become pregnant
female No. 275 (mated with male No. 75) did not become pregnant
The fertility index varied between 97% and 100% without showing 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 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 gestation index was 100% in the control and test groups 01, 96% in test group 02 and
97% in test group 03. These values reflect the normal range of biological variation inherent in the strain of rats used for this study.
The mean number of implantation sites was 12.3 / 12.2 / 11.4 and 7.8** (**:p<=0.01) implants/dam in test groups 00 - 03, 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 in test groups 00 - 03, respectively.
The rate of liveborn pups was not affected by the test substance, as indicated by live birth indices of 98% / 99% / 98% and 96% in test groups 00 - 03, respectively. Despite that the number of litters with stillborn pups was higher at the top dose (2 / 2 / 4 and 8** (**:p<=0.01) in
test groups 00 - 03, respectively), the total number of stillborn pups was comparable between the groups (7 / 2 / 5 and 8 in test groups 00 - 03, respectively) and does not indicate an association to treatment.
The number of cannibalized pups was 0 / 2 / 2 and 10** (**:p<=0.01) in test groups 00 - 03, respectively.

Effect levels (P0)

open allclose all
Key result
Dose descriptor:
NOAEL
Remarks:
based on a lower number of implants, prolonged/irregular estrous cycles as well as pathological changes in sexual organs, pituitary and mammary glands of both genders
Effect level:
100 ppm (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
gross pathology
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 function (oestrous cycle)
reproductive performance

Results: P1 (second parental generation)

General toxicity (P1)

Clinical signs:
not examined
Mortality:
mortality observed, treatment-related
Description (incidence):
Pup number and status at delivery
Whereas the litter size of the F1 litters at the top dose (1000 ppm) was lower (11.9 / 11.8 / 11.1 and 7.3** (**:p<=0.01) pups/dam in test groups 00 - 03, respectively) the postimplantation loss as well as the rates of liveborn and stillborn F1 pups were evenly distributed about the groups.
Thus the lower litter size in the high-dose group was a consequence of a lower number of implants and not due to prenatal or perinatal mortality.
Mortality are based on stillborn pups, dead pups, pups sacrificed moribund and cannibalized pups.

Pup viability/mortality
The viability index indicating pup mortality during early lactation (PND 0 - 4) varied between 100% / 99% / 99% and 93% in test groups 00 - 03. The lower index in the high-dose group was slightly below the historical control range. This slightly higher pup mortality at the high dose
came from a 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 (292, 311) none of the pups survived.
The absolute number of pups surviving early lactation (PND 0 - 4) varied between 350 / 349 / 291* (*:p<=0.05) and 191** (**:p<=0.01) in test groups 00 - 03. 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.
Body weight and weight changes:
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
not examined
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not examined

Reproductive function / performance (P1)

Reproductive function: oestrous cycle:
effects observed, treatment-related
Description (incidence and severity):
please compare with results for the F1 generation details
Reproductive function: sperm measures:
effects observed, non-treatment-related
Description (incidence and severity):
Sperm parameters
Concerning motility of the sperms and sperm head counts in the testis and in the cauda
epididymidis no treatment-related effects were observed. In males of test group 13 (1000 ppm)
incidence of abnormal sperms was slightly higher compared to controls, although not
statistically significant. This was due to two individuals (no. 474 and 477) having sperm with
missing heads, abnormal hook of the head, bent heads and combined morphological changes
of head and tail. These morphological changes occurred also in controls. The mean incidence
of the morphological changes on the whole was still in the range of historical controls (abnormal
sperms 6.0-7.5 %). Therefore, this change was regarded as incidental and not treatmentrelated.
Reproductive performance:
not examined
Description (incidence and severity):
No F2 generatio nas been perfomed according to the agreed study protocol.

Results: F1 generation

General toxicity (F1)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
There were no test substance-related adverse clinical signs observed in any of the F1 generation pups of the different test groups.

F1 rearing animals, Cohort 1A (14R0781A)
Clinical observations for males and females
No clinical signs or changes of general behavior, which may be attributed to the test substance,
were detected in any of the low- and mid-dose males and in any female animal of all dose
groups.
Two high-dose (1000 ppm) males (No. 476 and 478) showed high-stepping gait and
piloerection recurring in several study sections. In addition, both animals had small testes which
were detected in study week 5 for the first time.

F1 rearing animals, Cohort 1B (14R0781B)
Clinical observations for males and females
No clinical signs or changes of general behavior, which may be attributed to the test substance,
were detected in any of the low- and mid-dose male and female animals.
Several high-dose male and female animals (Nos. 693, 694, 697, 785, 797, 798) showed highstepping
gait and piloerection recurring during several study sections. Affected male (No. 697)
had small testes in addition, which were detected for the first time in test week 5. These findings
were considered as treatment-related and adverse.
Female No. 693 had also a malocclusion, which is considered as a spurious finding.

Detailed clinical observations (DCO)
No clinical signs or changes of general behavior, which may be attributed to the test substance,
were detected in any of the low- and mid-dose male and female animals (100 and 300 ppm).
A number of high-dose male and female animals (1000 ppm) exhibited high-stepping gait and
piloerection on seveal occasions during the study, as follows:
- Male animal No. 693: high-stepping gait during study weeks 6 - 8 and piloerection on study
week 2 and during weeks 6 - 8
- Male animal No. 697: high-stepping gait during study weeks 5 - 6 and piloerection on week
6
- Female animal No. 785: high-stepping gait and piloerection during study weeks 6 - 8
- Female animal No. 797: high-stepping gait on study week 8
- Female animal No. 798: high-stepping gait on study week 1 and during study weeks 5 - 8
as well as piloerection during study weeks 1 - 2 and study weeks 5 – 8.
These clinical observations were considered to be treatment-related and adverse.


F1 rearing animals, Cohort 2A (14R0782A)
Clinical observations for males and females
No clinical signs or changes of general behavior, which may be attributed to the test substance,
were detected in any of the test substance-treated male as well as in low- and mid-dose female
animals.
One high-dose (1000 ppm) female (No. 937) showed high-stepping gait during study weeks 6
– 7 and piloerection during study weeks 5 - 7. These findings were considered as treatmentrelated
and adverse.
A malocclusion observed in high-dose male animal No. 833 is considered as a spurious finding.


F1 rearing animals, Cohort 2A (14R0782A)
Detailed clinical observations (DCO)
No clinical signs or changes of general behavior, which may be attributed to the test substance,
were detected in any of the test substance treated male and in any low- and mid-dose female
animal.
One high-dose (1000 ppm) female (No. 937) showed high-stepping gait and piloerection during
study week 6.

F1 rearing animals, Cohort 3 (14R0783)
Clinical observations for males and females
One high-dose (1000 ppm) male (No. 1240) showed high-stepping gait and piloerection
recurring in several study sections. In addition, the animal had small testes which were
detected in study week 4 for the first time. A malocclusion observed in this male was considered
as a spurious finding. High-dose female animal No. 1336 showed high-stepping gait on study
week 4 and piloerection during study weeks 3 – 4.

Detailed clinical observations (DCO)
No clinical signs or changes of general behavior, which may be attributed to the test substance,
were detected in any of the low- and mid- dose male and female animals.
One high-dose (1000 ppm) male (No. 1240) showed high-stepping gait on study week 2 and
piloerection during study weeks 1 - 2. High-dose female animal No. 1336 showed high-stepping
gait and piloerection on study week 4. These findings were considered as treatment-related
and adverse.
Mortality / viability:
mortality observed, treatment-related
Description (incidence and severity):
Pup number and status at delivery
Whereas the litter size of the F1 litters at the top dose (1000 ppm) was lower (11.9 / 11.8 / 11.1 and 7.3** (**:p<=0.01) pups/dam in test groups 00 - 03, respectively) the postimplantation loss as well as the rates of liveborn and stillborn F1 pups were evenly distributed about the groups.
Thus the lower litter size in the high-dose group was a consequence of a lower number of implants and not due to prenatal or perinatal mortality.
Mortality are based on stillborn pups, dead pups, pups sacrificed moribund and cannibalized pups.

Pup viability/mortality
The viability index indicating pup mortality during early lactation (PND 0 - 4) varied between 100% / 99% / 99% and 93% in test groups 00 - 03. The lower index in the high-dose group was slightly below the historical control range. This slightly higher pup mortality at the high dose
came from a 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 (292, 311) none of the pups survived.
The absolute number of pups surviving early lactation (PND 0 - 4) varied between 350 / 349 / 291* (*:p<=0.05) and 191** (**:p<=0.01) in test groups 00 - 03. 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 (14R0781A)
Mortality
There were no test substance-related mortalities in any of the groups up to a dose of 300 ppm.
One high-dose male (No. 462, 1000 ppm) was sacrificed moribund in study week 0, after
showing lateral position, apathy, hypothermia and diarrhea.

F1 rearing animals, Cohort 1B (14R0781B)
There were no test substance-related mortalities in any of the groups up to 300 ppm. One high-dose male (No. 687) and one high-dose female (No. 781) were found dead on study week 8, respectively.


F1 rearing animals, Cohort 2A (14R0782A)
Mortality
There were no test substance-related or spontaneous mortalities in any of the groups.

F1 rearing animals, Cohort 3 (14R0783)
Mortality
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 (14R0781A)
The body weights of the high-dose males were statistically significantly below the concurrent
control values during the entire study period, the final weight was about 24% below control.
The body weights of the mid-dose males were statistically significantly below the concurrent
control during the first week after weaning, and stayed below control until the end of the study
(final weight about 6% below control) albeit the difference was not statistically significant.
The body weights of the low-dose male rats were comparable to the concurrent control values
throughout the study.
The body weights of the high-dose females were statistically significantly below the concurrent
control values during the entire study period, the final weight was about 15% below control.
The body weights of the mid-dose females were below control throughout the study (final
weight about 5% below control), although the difference was small and not statistically
significant.
The body weights of the low-dose females were comparable to the concurrent control during
the entire study.
The body weight gain of the high-dose males was statistically significantly below the concurrent
control throughout the study except of the last study week, the average reduction of weight gain was about 25%. Mid-dose males gained about 5% less weight, the reduction was
statistically significantly below the concurrent control during study days 35 - 49 (up to 28%).
The body weight change of the low-dose males was comparable to the concurrent control
during the entire study.
The high-dose females gained statistically significantly less weight than the concurrent control
during the study (14% on days 0 – 63), further their weight gain was statistically significantly
below control on days 0 – 7 and 42 - 49 (about 13% and 42%, respectively).
The body weight change of the low and mid-dose females was comparable to the concurrent
control during the study. The statistically significantly increased body weight change in the lowdose
females during study days 21 - 28 was considered to be spontaneous in nature.


F1 rearing animals, Cohort 2A (14R0782A)
Body weight data
The body weights of the high-dose males were statistically significantly below the concurrent
control values during the entire study period, the final weight was about 13% below control.
The body weights of the low- and mid-dose male rats were comparable to the concurrent
control values throughout the study.
The body weights of the high-dose females were statistically significantly below the concurrent
control values during the entire study period, the final weight was about 16% below control.
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.
The body weights of the low-dose females were comparable to the concurrent control during
the entire study.
The body weight gain of the high-dose males was statistically significantly below the concurrent
control during several sections of the study, the average reduction of weight gain was about
11%.
The body weight change of the low- and mid-dose males was comparable to the concurrent
control during the entire study.
There was no consistent effect on the body weight gain of the high-dose females, although
they gained overall less weight than the concurrent control during the study (about 12%).
The body weight change of the low and mid-dose females was comparable to the concurrent
control during the study. The statistically significantly decreased body weight change in the
low-dose females during study days 28 - 35 was considered to be spontaneous in nature.

F1 rearing animals, Cohort 3 (14R0783)
Body weight data
The body weights of the high-dose males were statistically significantly below the concurrent
control values during the entire study period, the final weight was about 19% below control; as
were the body weights of the high-dose females, their final weight was about 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.
The body weight gain of the high-dose males was statistically significantly below the concurrent
control during several sections of the study, the average reduction of weight gain was about
19%.
The body weight change of the low- and mid-dose males was comparable to the concurrent
control during the entire study.
There was no consistent effect on the body weight gain of the high-dose females, although
they gained overall less weight than the concurrent control during the study (about 8%).
The body weight change of the low and mid-dose females was comparable to the concurrent
control during the study. The statistically significantly increased body weight change in the middose
females during study days 0 - 7 was considered to be spontaneous in nature.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
F1 rearing animals, Cohort 1A (14R0781A)
Food consumption
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
17%. Food consumption of the mid-dose males 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 low-dose males was comparable to the concurrent control values
throughout the study.
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
the study was comparable to control.
Food consumption of the low- and mid-dose females was comparable to the concurrent control
throughout the study.

F1 rearing animals, Cohort 1B (14R0781B)
Food consumption of the high-dose males was statistically significantly below the concurrent
control during the entire study (up to 18%), the average decrease was about 14%.
There was no significant effect on food consumption of the low- and mid-dose males throughout
the entire study.
Food consumption of the high-dose females was statistically significantly below the concurrent
control during major parts of the study period (up to 17%), the average dcrease was about 9%.
There was no significant effect on food consumption of the low- and mid-dose females
throughout the entire study.


F1 rearing animals, Cohort 2A (14R0782A)
Food consumption
Food consumption of the high-dose males was below the concurrent control during the entire
study period (up to 12%), the average reduction was about 11%.
Food consumption of the high-dose females was statistically significantly below the concurrent
control during study days 0 - 7 and 14 - 21 (about 15% and 10%, respectively), the average
reduction was about 8%.
Food consumption of the low- and mid-dose males and females was comparable to the
concurrent control throughout the study.


F1 rearing animals, Cohort 3 (14R0783)
Food consumption
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 (14R0781A)
Water consumption of the high-dose males was below the concurrent control values throughout the study (average about 13% lower), the difference became statistically significant on study days 0 – 21, 24 - 28 and 49 - 56 (up to 24%, 14% and 19%, respectively). A statistically
significantly reduced water consumption was also noted for the mid-dose males during study days 7 - 10 (about 10%).
Water consumption of the high-dose females was below the concurrent control values throughout the study (average about 14% lower), the difference became statistically significant during several parts of the study period (up to 25%). 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 1B (14R0781B)
Water consumption of the high-dose males was below the concurrent control values throughout
the study (average about 13% lower), the difference became statistically significant during
major parts of the study (up to 23%). A statistically significantly reduced water consumption
was also noted for the mid-dose males towards the end of treatment (study days 52 – 59; about
10 - 17%).
Water consumption of the low-dose males was consistently comparable to the concurrent
control during the entire study, except of a decrease on days 52 – 56 which is considered of
no toxicological relevance.
Water consumption of the high-dose females was below the concurrent control values
throughout the study (average about 16% lower), the difference became statistically significant
during major parts of the study period (up to 29%). Mid-dose females also had a consistenly
lower water consumption than control (average about 10%), the difference becoming
statistically significant during study days 24 – 28, 31 – 38 and 49 - 56 (up to 18% below control).
Water consumption of the low-dose females was comparable to the concurrent control during
the entire study.


F1 rearing animals, Cohort 2A (14R0782A)
Water consumption
Water consumption of the high-dose males was below the concurrent control values during
major parts of the study (average about 7% lower), the difference became statistically
significant on study days 0 - 11 (up to 22%).
Water consumption of the low and mid-dose males was comparable to the concurrent control
during the entire study.
Water consumption of the high-dose females was below the concurrent control values
throughout the study (average about 13% lower), the difference became statistically significant
during several parts of the study period (up to 18%). Mid-dose females occasionally had
statistically significantly lower water consumption during the study period (up to 23% below
control).
Water consumption of the low-dose female animals was comparable to the concurrent control
values during the entire study.

F1 rearing animals, Cohort 3 (14R0783)
Water consumption
Water consumption of the high-dose males was below the concurrent control values during
major parts of the study (average about 9% lower), the difference became statistically
significant on study days 0 - 7 (up to 16%).
Water consumption of the low and mid-dose males was comparable to the concurrent control
during the entire study.
Water consumption of the high-dose females was below the concurrent control values
throughout the study (average about 11% lower), the difference became statistically significant
during several parts of the study period (up to 18%).
Water consumption of the low- and mid-dose female animals was comparable to the concurrent
control values during the entire study.
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
In rats of both sexes of the F1 generation (PND92) of test group 13 (1000 ppm) 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) were decreased.
In rats of both sexes of test group 12 (300 ppm) hemoglobin and hematocrit values were
decreased and additionally in females RBC counts were decreased and in males of the
mentioned test group MCV and MCH were lower compared to controls. In females of this test
group hemoglobin values and RBC counts were within and hematocrit values were marginally
below historical control ranges (females: hemoglobin 8.1-8.9 mmol/L, RBC 7.49-7.93 Tera/L,
hematocrit 0.383-0.421 L/L). However, hematocrit mean in females of test group 12 was only
9% lower compared to the controls and this was the only relevantly changed red blood cell
parameter. In males of test group 12, the measured red blood cell parameters hemoglobin and
hematocrit were within historical control range (males hemoglobin 8.4-9.5 mmol/L, hematocrit
0.400-0.446 L/L) and only the calculated red blood cell indices MCV and MCH were below
historical control ranges (males MCV 50.1-54.7 fL, MCH 1.05-1.18 fmol). Therefore, the red
blood cell parameter changes in rats of both sexes of test group 12 (300 were regarded as
treatment-related but not adverse.
In females of test group 11 (100 ppm) RBC counts, hemoglobin and hematocrit values and in
males of the same test group MCH were still lower compared to study controls, but all values
apart from MCH in males were within historical control ranges (females: hemoglobin 8.1-8.9
mmol/L, RBC 7.49-7.93 Tera/L, hematocrit 0.383-0.421 L/L). In males of test group 11 MCH
was still lower compared to historical controls, but the measured red blood cell parameters (i.e.
RBC counts hemoglobin and hematocrit values) were not changed. Therefore, the mentioned
red blood cell alterations in both sexes of test group 11 (100 ppm) were regarded as incidental
and not treatment-related.
In females of test group 13 (1000 ppm) prothrombin time (Hepatoquick’s test, HQT) was
reduced. In males of test groups 12 and 13 (300 and 1000 ppm) platelet counts were increased.
In females of test group 12 (300 ppm) platelet counts were increased, but they were not dosedependently
changed. Therefore, these platelet count alterations were regarded as incidental
and not treatment-related.
Regarding the differential blood cell counts, in females of test group 13 (1000 ppm) absolute
and relative monocyte counts were decreased and this is regarded as adverse finding.
The following changes in the differential blood cell counts were within the historical control
ranges. Therefore, they were regarded as incidental and not treatment-related: decreased
absolute and relative monocyte counts in females of test groups 11 and 12 (100 and 300 ppm),
decreased relative basophil counts in females of test groups 11, 12 and 13, increased relative
lymphocyte counts in males of test group 13, decreased relative monocyte and eosinophil
counts in males of test groups 12 and 13, decreased absolute monocyte counts in males of
test group 12 (females: relative monocytes 1.2-1.9 %, absolute monocytes 0.05-0.08 Giga/L,
relative basophils 0.1-0.5 %; males: relative lymphocytes 77.7-87.0 %, relative monocytes 0.9-
2.3 %, relative eosinophils 0.8-2.0 %, absolute monocytes 0.07-0.14 Giga/L).
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
In rats of both sexes of the F1 generation (PND92) of test group 13 (1000 ppm), aspartate
aminotransferase (AST) activities were increased. Additionally, in males of test groups 12 and
13 (300 and 1000 ppm) alkaline phosphatase (ALP) activities were higher compared to
controls. However, the ALP activity mean in test group 2 was within the historical control range
(ALP 1.64-2.80 μkat/L) and therefore, this change was regarded as incidental and not
treatment-related.
In male and female rats of test group 13 (1000 ppm) urea levels were increased.
In females of test groups 11, 12 and 13 (100, 300 and 1000 ppm) creatinine levels were
decreased and glucose levels were increased. However, both parameters were not dosedependently
changed regarding means as well as medians. Therefore these alterations were
regarded as incidental and not treatment-related.
In females of test groups 12 and 13 (300 and 1000 ppm) total protein, albumin and calcium
levels were higher compared to controls. Albumin levels were within historical control ranges
and total protein levels of test group 12 were within and those of test group 13 marginally above
the historical control range (albumin 38.12-42.64 g/L, total protein 58.83-65.33 g/L). Calcium
values were slightly above the historical control range (calcium 2.38-2.63 mmol/L). 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.
The following parameter changes were within historical control ranges and therefore they were
regarded as incidental and not treatment-related: increased sodium in both sexes of test groups
12 and 13 (300 and 1000 ppm), increased chloride levels in both sexes of test group 13,
increased calcium levels in males of test group 13, decreased levels of cholesterol in females
of test groups 12 and 13 (in test group 13 not statistically significant) (males: sodium 140.7-
145.4 mmol/L, chloride 97.9-103.4 mmol/L, calcium 2.42-2.71 mmol/L; females: sodium 140.5-
144.1 mmol/L, chloride 100.1-104.9 mmol/L, cholesterol 1.04-1.65 mmol/L). Inorganic
phosphate levels were decreased and triglyceride levels were increased in females of test
group 12 (300 ppm), but the changes were not dose-dependent and therefore they were regarded as incidental and not treatment-related. Calcium levels were already increased in
females of test group 11 (100 ppm), but this value was at the upper border of the historical
controls (females, calcium 2.38-2.63 mmol/L) and this was the only changed clinical
chemistry parameter among these individuals. Therefore, calcium level increase in females of
test group 11 was regarded as maybe treatment-related, but not adverse (ECETOC Technical
Report No. 85, 2002).
Urinalysis findings:
effects observed, 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 test groups 11, 12 and 13 (100, 300 and 1000 ppm) 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):
Vaginal opening
Each female F1 pup, which was selected to be raised as F1 female (cohorts 1A, 1B, 2A, 3),
was evaluated for commencement of sexual maturity. 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 test groups 00-03.
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
Each male F1 pup, which was selected to be raised as F1 male (cohorts 1A, 1B, 2A, 3), was
evaluated for commencement of sexual maturity. 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 test groups 00-03.
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.

F1 rearing animals, Cohort 1A (14R0781A)
Estrous cycle data
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 (4.1 / 4.0 / 4.2 and 5.5*
(*:p<=0.05) days in test groups 10-13). There was no particular cycle phase prolonged, some
high-dose F1A females with a longer cycle (beyond 5 days) had a prolonged diestrous, others
had equally longer estrous and/or metestrous phases. One female (No. 562) stayed
exceptionally long in estrous (11 days in 2 cycles). One female (No. 597) was in diestrous for
14 days which is usually considered as a sign of pseudopregnancy, without this animal the
average cycle duration would have been 4.9 days.

F1 rearing animals, Cohort 1B (14R0781B)
Estrous cycle data
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 (4.2 / 4.0 / 4.1 and 5.0*
(*:p<=0.05) days in test groups 10-13). A number of females displayed irregular cycles,
however they did not show a unique pattern of change. Specifically, there was no particular
cycle phase prolonged in all females, some females had a prolonged diestrous, others had
equally longer estrous and/or metestrous phases. Three females (Nos. 777, 785, 797) had no
complete cycle at all (estrous missing), one of them (No. 797) was in diestrous during almost
the entire observation period. Another female (No. 798) stayed in diestrous for 10 days, had
then one regular cycle and afterwards remained in estrous for the remaining observation
period.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
F1 rearing animals, Cohort 1A
Please see tables (Tables IC 16 – IC 23) of the attachment.
The reduction in terminal body weight in males of test group 12 and 13 (300 and 1000 ppm)
and females of test group 13 (1000 ppm) was regarded to be treatment-related. The increase
in kidney and liver weight (relative) was partly caused by the terminal body weight decrease
but as most of the weights were outside the historical control values (see PART III) they were
assessed to be treatment-related.
The increased relative adrenal gland weight in test group 13 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
not assessed to be adverse. The same comes true for the increase in adrenal weight in test
group 12 (300 ppm) females.
The male reproductive organs in test group 13 (1000 ppm) were mainly decreased by the
reduction in terminal body weight but this change would be less pronounced without the three
male animals which revealed histopathologic findings in these organs.
All other changed weight parameters were regarded to have been caused by the decrease in
terminal body weight.

F1 rearing animals, Cohort 1B
Please see tables (Tables IC 33 – IC 36) of the attachment.
The significant reduction in terminal body weight of males and females of test group 12 and 13
(300 and 1000 ppm) was regarded to be treatment-related. The terminal body weight of males
and females of test group 12 (300 ppm) was within the range of historical controls (see PART
III). The liver weight increase in males and females of all treated groups was regarded to be
treatment-related.The significant decrease in absolute weight of male sex organs in test group 13 (1000 ppm)
was regarded to have been mainly caused by the reduced terminal body weight but it would
have been less pronounced without the three male animals which revealed histopathologic
findings in these organs. The same comes true for the significant increase of relative weights
of pituitary gland in males of test group 13 (1000 ppm) and testes weight in test group 12 and
13 (300 and 1000 ppm). Whereas, for the significantly decreased prostate weight in test group
13 (1000 ppm) a treatment-related effect cannot be excluded, as the relative weight was still
decreased. But as it was decreased less than in F1 generation, cohort 1A animals, it was still
regarded to be secondary to the body weight reduction.
The significantly changed ovary weights in test group 12 and 13 (300 and 1000 ppm) were
regarded to be incidental due to the to a missing dose response relationship and because it
cannot be explained by histologic findings. The significantly increased relative uterus weights
in test group 12 and 13 (300 and 1000 ppm) were also thought to be caused by the terminal
body weight reduction.

F1 rearing animals, Cohort 3 (Immunotoxicity cohort)
Please see tables (Tables IC 40 – IC 47) of the attachment.
Male and female animals of test group 13 (1000 ppm) revealed a significant decrease in
terminal body weight which was regarded to be treatment-related. 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.

Cyclophosphamide monohydrate
Absolut and relative organ weights
When compared to the control group 10 (set to 100%), the mean absolute and relative weight
parameters of test group 14 (positive control) were significantly decreased A significant decrease in absolute and relative weights of the spleen and thymus occurred in the positive control male and female animals. This result was expected.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
The light-brown discoloration in the liver of twelve males and two females of test group 13
(1000 ppm) 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 (animals No. 476, 478, and 479) of test group 13 (1000 ppm)
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 1B (14R0781B)
Gross pathology
(Table IC 37)
In test group 13 (1000 ppm) three males (animal Nos. 693, 694, 697) revealed reduced size of
the testes, epididymides, prostate and seminal vesicle. Four females (animal Nos. 777, 785,
797, 798) of test group 13 (1000 ppm) had reduced size of the ovaries. All these male and
female animals revealed a severely lower body weight when compared to the average of this
group or the control group. The weight decrease was therefore regarded to be treatmentrelated
but secondary to the reduced terminal body weight.
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.

F1 rearing animals, Cohort 3 (Immunotoxicity cohort)
Gross pathology
(Table IC 48 – IC 49)
One male of test group 13 (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, Cohort 1A
Please see tables (Tables IC 26 – IC 31) of the attachment.
Similar to the findings described for the F0 generation animals, in the proximal tubules of the
kidneys signs of degeneration and regeneration (apoptotic cell death, debris in the tubular
lumen, increased numbers of large, vesicular nuclei, increased basophilic staining, nuclear
crowding) were seen. These findings were regarded to be treatment-related. Within the papilla,
females of test group 13 (1000 ppm) showed an increase of multifocal mineral deposition.
Males of test group 12 and 13 (300 and 1000 ppm) showed multifocal mineral depositions at
the transition between the outer and inner medulla. The mineralization in males and females
was regarded to be treatment-related.

Males and females of test group 12 and 13 (300 and 1000 ppm) showed a centrilobular
hypertrophy in the liver. Three males of test group 13 (1000 ppm) revealed a peripheral
hypertrophy. Furthermore, males of test group 12 and 13 (300 and 1000 ppm) and females of
test group 13 (1000 ppm) showed clear, round vacuoles in the peripheral area. Via the ORO
stain it could be demonstrated that they representing lipid vacuoles. These findings were
regarded to be treatment-related.

In the male mammary gland of test group 13 (1000 ppm) 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 in test group 13 (1000 ppm) revealed a higher incidence of eosinophilic amorphous
material which was secreted into the ducts. These findings were regarded to be treatmentrelated.
In males a similar secretion was seen, but there was no significant difference between
control and treated animals.

Three males (animal Nos. 476, 478, 479) of test group 13 (1000 ppm) 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 three animals did not have sperm within
the epididymides (aspermia) and reduced size of the secondary sexual organs (prostate,
seminal vesicle, and coagulating gland). One male (animal No. 479) showed multifocal
degeneration of the testicular tubules in addition. The immaturity in these animals was regarded
to be a consequence to the reduced body weight. The tubular degeneration in animal No. 479
was assumed to be treatment-related.
Only in one male (animal No. 462), which was sacrificed in a moribund state, the reduced size
of the ductus deferens did not match a finding in the testes. In this animal, the premature
sacrifice was regarded to be responsible for the smaller size.
The vacuolation in the ductus deferens in four males of test group 12 (300 ppm) might still be
treatment-related but due to the missing findings in all other sexual organs it was not assumed
to be adverse.

In test group 13 (1000 ppm) one female revealed a diffuse atrophy of the ovary. Six females
revealed luteal cysts. These findings were regarded to be treatment related. The one cyst in a
test group 12 female (300 ppm) 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. These cysts differed from the cysts that occur sporadically as background
lesion in the pituitary gland, also in this study. Spontaneous cysts which were observed in
control and treated animals had a ciliated epithelium and a mucinous content. The treatmentrelated
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.
In the thyroid glands of males of test group 11 and 12 (100 and 300 ppm) there was a slight
increase in hypertrophy of the follicular epithelium. As in test group 13 (3000 ppm) no such
finding was observed it was regarded to be incidental. Branchiogenic cysts occurred in control
and treated animals and are regarded to be incidental findings. In these study, there was a
slight increase in test group 13 animals (1000 ppm) compared to control animals. Nevertheless,
it was regarded to be incidental and not related to treatment.
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)
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 – revealed significant differences between the control group 10 and animals of test
group 13

F1 rearing animals, Cohort 1B (14R0781B)
Please see tables (Tables IC 38 – IC 39) of the attachment.
Three males (animal Nos. 693, 694, 697) of test group 13 (1000 ppm) showed the histological
picture of juvenile and immature testes which were characterized by lower numbers or even
none fully developed and differentiated germ cell rows. In addition, there were no or only very
single sperm present within testicular tubules. Consequently, these three animals did not have
sperm within the epididymides (aspermia) and reduced size of the secondary sexual organs
(prostate, seminal vesicle, and coagulating gland).
Two males (animal No. 694 and 697) showed a slight to moderate multifocal degeneration of
the testicular tubules in addition. Two males revealed a minimal degeneration in the testicle,
only. These findings were regarded to be treatment-related.
In test group 1 (100 ppm) one male also had an immature testis and tubular degeneration.

Four females (animal Nos. 777, 785, 797, 798) of test group 13 (1000 ppm) revealed a diffuse
atrophy of the ovaries, represented by lower numbers of functional corpora and reduction in
size. Two of these females did not have any corpora lutea present. Furthermore, in the ovaries
of six females of this test group and one female of test group 12 (300 ppm) luteal cysts were
observed. These were large cystic cavities surrounded by luteinized and non-luteinized
granulosa cells. These findings were regarded to be treatment-related.

NEUROPATHOLOGY
Summary tables of the results are to be found in Part D of PART I; individual tables are to be
found in Part D of PART II. Abbreviations and histopathological grading used in pathology
report and tables can be found in the appendix.

F1 rearing animals, Cohort 2A (Developmental Neurotoxicity Cohort, adults)
Weight parameters
(Tables ID 1 – ID 4)
Length and width of brain
(Table ID 5)
All length and width measurements were without any findings. Only the length parameter in
females of test group 13 was minimally decreased. As the width of the brain in this test group
was comparable with the control animals and the absolute brain weight was unchanged, a
treatment-related effect seems unlikely.
Gross lesions
(Table ID 6)
Only the moribund sacrificed male animal No 837 revealed a dilation of jejunum, ileum, cecum
and colon. These findings were assessed as incidental and not related to treatment.
No other gross findings were recorded.
Histopathology
(Table ID 7 – ID 9)
The medulla oblongata and the spinal cord of treated 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.

In the pars distalis of the pituitary gland, multifocally distributed very small eosinophilic cysts
with a non-ciliated, irregular border and eosinophilic homogenous content were seen.

Morphometry
(Table ID 10)
All morphometric brain measurements were without any findings. Only some single parameters
in male (hippocampus right, base of lobus vermis cerebrelli no 8) or female (parietal cortex left)
animals of test group 13 showed a statistical significant increase. As no other values were
changed, this minimal width increase is assumed as incidental and not related to treatment.

F1 animals, Cohort 2B (Developmental Neurotoxicity Cohort, weanlings)
Weight parameters
(Tables ID 11– ID 14)
The statistically significant decrease of terminal body weight in male and female animals of test
group 12 (females only) and 13 is regarded as treatment-related. The decrease of brain weight
in test group 13 males is assumed to be secondary to the delayed development of the animals,
nevertheless the length and the width of brains are comparable to control animals.
The increased relative brain weights of test group 12 (females only) and 13 (both sexes)
animals are secondarily linked to the decreased terminal body weight. The increase in absolute
brain weight in test group 11 females is regarded as incidental.

Length and width of brain
(Table ID 15)
All length and width measurements were without any findings.
Gross lesions
(Table ID 16)
No gross findings were recorded.
Histopathology
(Table ID 17 - ID 18)
No treatment-related neurohistopathological findings were recorded.
In the pars distalis of the pituitary gland, multifocally distributed very small eosinophilic cysts
with a non-ciliated, irregular border and an eosinophilic homogenous content were seen in four
males and five females (each out of ten) of test group 13 (1000 ppm). This finding is regarded
as treatment-related.

Other effects:
effects observed, treatment-related
Description (incidence and severity):









Thyroid hormones
In F1 generation females of test group 11 and 12 (100 and 300 ppm) at PND 4 and in females
at PND 22 of test group 13 (1000 ppm) T4 was higher compared to controls. In test group 13
(1000 ppm) only two males had a sufficient sample volume for the measurement of the thyroid
hormones at PND 4, but no female pup was available.
The T4 means in F1 generation males and females of test group 13 at PND 92 were 19%/18%
higher compared to controls (medians 18%37% higher), although the values were not
statistically significantly changed.

Platelet activating factor (PAF) determination in serum of F0 parents
The platelet activating factor (PAF) concentration in serum of the F0 females is reduced dosedependently
when regarding medians with a decrease of 31% in test group 3 (1000 ppm)
compared to controls. This decrease in females of test group 3 was statistically significant when
the two-sided Jonckheere-Terpstra trend test was applied. The PAF levels decrease could not
been observed among the F0 males.

CHOLINE CONCENTRATION IN BLOOD AND LIVER SAMPLES
The analytical results demonstrated the clear presence of choline in all plasma samples from
the animals dosed with the test substance 2,2’-iminodiethanol (100 ppm, 300 ppm and 1000
ppm dosed animals) and in those from control, non-dosed animals.
In general, it can be stated that the presence of the test substance 2,2’-iminodiethanol led to a
reduction in the content of choline in the plasma samples analyzed. This effect appears to be
dose-dependent, in that higher dose levels were associated with greater choline reduction.
This effect is most clearly visible at lower dose levels (100 ppm and 300 ppm), at which
dramatic plasma choline levels could be seen. At higher dosing levels, although further minor
plasma choline content reduction was observed, this was by no means as drastic.
The analytical results demonstrated the clear presence of choline in all liver samples from the
animals dosed with the test substance 2,2’-iminodiethanol (100 ppm,300 ppm and 1000 ppm
dosed animals) and in those from control, non-dosed animals. This was true from all time points
investigated (4-day old pups, 22-day old pups and ~90-day old adolescents).
In general, it can be stated that the presence of the test substance 2,2’-iminodiethanol led to a
reduction in the content of choline in the liver samples analyzed. This effect appears to be dose-dependent, in that higher dose levels were associated with greater choline reduction, but
only up to moderate dosing levels (300 ppm and 100 ppm, depending on the sampling day).
At higher dosing levels, no further dramatic liver choline content reduction was observed.
This effect was however, not observed in 4-day old animals, in which no clearly definable dosedependent
trend is evident. In 22-day old animals this effect could be clearly observed,
although the choline levels of the 100 ppm dosed animals have not yet attained minimal
concentrations. In ~90-day old animals the effect is dramatic in that the liver choline levels of
all non-control animals have reached an approximate minimum. Only a relatively minor further
dose-dependency can be observed at this time point.

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 of the F1 generation (PND 60) dosed with the test substance. SRBC titers were statistically
significantly lower in rats of the positive control group (dosed with Cyclophosphamide).

Splenic lymphocyte subpopulations
In females of the F1 generation (PND 92) of test group 13 (1000 ppm) 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 compared to controls.
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).

Details on results (F1)

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 during the
measurement. Amplitude and latency of reaction to a startle stimulus and habituation to the
test environment corresponded to the age of these animals at PND 24, when normal biological
variation inherent in the strain of rats used for this experiment was considered.

Functional observational battery (FOB)
Home cage observations:
No test substance-related or spontaneous findings were observed in male and female animals
of all test groups during the home cage observation.
Open field observations:
The open field observations did not reveal any test substance-related findings in male and
female animals of all test groups.
Sensorimotor tests/reflexes:
There were no test substance-related findings in male and female animals of all test groups.
Quantitative Parameters:
No test substance-related impaired parameters (number of rearings, grip strength of fore- and
hindlimbs and landing foot splay test) were observed in male and female animals of all test
groups. This includes a statistically significantly lower grip strength for forelimbs of the highdose
females, which is considedered to be an isolated, and thus incidental, finding.

Motor activity measurement (MA)
Motor activity (number of beam interrupts) of male and female animals was not influenced by
the test compound at all dose levels (100, 300 and 1000 ppm). Overall activity levels and
habituation to the test environment corresponded to the age of these animals at PND 70. Any
occasional differences between treated animals of these dose groups and their concurrent
control (like lower numbers of beam interrupts in the low- and high-dose females during interval
3), whether statistically significant or not, were regarded as incidental findings and not related
to the test compound.

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.

Anogenital distance/anogenital index
Anogenital distance and index of all test substance treated pups (100; 300 and 1000 ppm)
were comparable to the concurrent control values.

Nipple/ areola anlagen
The apparent number and percentage of male pups having areolae was not influenced by the
test substance when examined on PND 12. However, owing to the high background rate in
control animals on this day, we habitually recheck all animals for nipples/areolae on PND 20,
one day prior to weaning. During this re-examination no areolae were detected at all in male
pups of all test groups.

Effect levels (F1)

open allclose all
Key result
Dose descriptor:
NOAEL
Remarks:
reproductive performance
Generation:
F1
Effect level:
300 ppm (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: reproductive performance
Key result
Dose descriptor:
NOAEL
Remarks:
developmental toxicity in the F1 progeny
Generation:
F1
Effect level:
100 ppm (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: developmental toxicity in the F1 progeny
Key result
Dose descriptor:
NOAEL
Remarks:
developmental neurotoxicity
Generation:
F1
Effect level:
300 ppm (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: developmental neurotoxicity
Key result
Dose descriptor:
NOAEL
Remarks:
developmental immunotoxicity
Effect level:
300 ppm (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: developmental immunotoxicity

Overall reproductive toxicity

Reproductive effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
Under the conditions of the present modified extended 1-generation reproduction toxicity study
the NOAEL (no observed adverse effect level) for general toxicity is 100 ppm for the F0
parental animals, based on evidence for distinct kidney toxicity and stomach irritation, as well
as corresponding effects on water consumption, food consumption, body weights and clinicalpathological
parameters, which were observed at the LOAEL (Lowest Observed Adverse Effect
Level) of 300 ppm. Similar toxicity was noted in the adolescent F1 animals, which had no
stomach irritation but liver toxicity in addition.

The NOAEL for fertility and reproductive performance for the F0 and F1 rats is 300 ppm,
based on a lower number of implants, prolonged/irregular estrous cycles as well as pathological
changes in sexual organs, pituitary and mammary glands of both genders at the LOAEL
(Lowest Observed Adverse Effect Level) of 1000 ppm. However, eosinophilic cysts in the
pituitary gland were present in the F1 animals of cohort 1A down to the 100 ppm dose level,
but no assessment on adversity of this finding is possible at present. Therefore, no NOEL can
be established for this particular effect.

The NOAEL for developmental toxicity in the F1 progeny is 100 ppm, based on impaired
pup survival at 1000 ppm as well as reduced pup body weights in the F1 offspring, which were
observed at the LOAEL (Lowest Observed Adverse Effect Level) of 300 ppm. As these weight
reductions were only observed in the presence of maternal toxicity, including lower weight gain
during pregnancy, they are not regarded as independent effect of the treatment.

The NOAEL for developmental neurotoxicity for the F1 progeny is 300 ppm, based on
adverse clinical observations, impaired auditory startle response and corresponding
neuropathological findings at the LOAEL (Lowest Observed Adverse Effect Level) of 1000
ppm. In addition, increased T4 values were noted in adult and adolescent males at 1000 ppm
as well as newborn and weanling females at 100 and 300 ppm.

The NOAEL for developmental immunotoxicity for the F1 progeny is 300 ppm, based on
effects on the T-helper cells and cytotoxic T-cells in the spleen in the F1 females at the LOAEL
(Lowest Observed Adverse Effect Level) of 1000 ppm. Lower mean and median anti-SRBC
IgM antibody titers of the positive control group (4.5 mg/kg bw/d cyclophosphamide, oral)
demonstrated that the test system worked properly.
Executive summary:

2,2’-iminodiethanol was administered to groups of 30 male and 30 female healthy young

Wistar rats as addition to the drinking water in concentrations of 100, 300 and 1000 ppm. The

vehicle control group was given plain drinking water. Analyses confirmed the correct concentration and the stability of the test substance in drinking water.

The overall mean doses of 2,2’-iminodiethanol throughout all study sections and across all

cohorts were 12.75 mg/kg body weight/day (mg/kg bw/d) in the 100 ppm group, 37.68 mg/kg

bw/d in the 300 ppm group and 128.35 mg/kg bw/d in the 1000 ppm group.

There were no test substance-related mortalities or adverse clinical observations noted

in the F0 generation parental animals at any dose level as well as in the F1 adolescents at the

low- and mid-dose level (100 and 300 ppm).

 

The high-dose of the test substance (1000 ppm) produced mortalities or adverse clinical

observations in the F1 adolescents. Three high-dose offspring (2 males, 1 female) in two

rearing cohorts (1A, 1B) were either sacrificed moribund or found dead at different timepoints

during the study. These casualties went along with adverse clinical observations such as highstepping gait and piloerection recurring across all F1 cohorts in several study sections.

Altogether 11 high-dose animals of both sexes in cohorts 1A, 1B, 2A and 3 were affected.

Some of these affected high-dose male F1 offspring had, in addition, small testes for which a

histopathological correlate was found. As these animals had a severe reduction in terminal

body weight, this size reduction was regarded to be secondary to the body weight decrease.

Treatment with 2,2’-iminodiethanol produced no effect on water consumption in the F0

males at all dose levels, while F0 females at 300 and 1000 ppm had decreased water

consumption beginning during gestation and distinct to severe during lactation. The F1 adolescents

showed this effect consistently and distinctly across all cohorts at the 1000 ppm dose level and

less pronounced and consistent, but still noticeable, at the 300 ppm dose level. No such effect

was noted at 100 ppm. In the affected groups reduced water consumption was associated with

reduced urine volume and histopathological findings in the kidneys.

 

In the 1000 ppm F0 parental males and females food consumption was consistently reduced,

at the 300 ppm level reduced food consumption was only noted in females during lactation. In

the 1000 ppm F1 males and females food consumption was consistently reduced postweaning,

although there was some variability in the extent of the reduction across the 1A, 1B,

2A and 3 cohorts. At the 300 ppm level slightly and non-statistically significant reduced food

consumption was only noted in the cohort 1A males. In contrast to this, food consumption of all

males and females at the 100 ppm level remained unchanged.

In the 1000 ppm F0 parental males and females body weights were consistently reduced

throughout all study sections beginning on study day 7. At the 300 ppm level reduced body

weights were noted in F0 parental males from study day 28 and in females during lactation

only. This was caused by a similarly affected body weight gain, though the course of body

weight changes was variable in the different study sections, generally for the females being more severe during the gestation/lactation period.

 

Body weights/body weight gain of the high-dose (1000 ppm) Cohort 1A, Cohort 1B, Cohort 2A

and Cohort 3 animals were similarly reduced as of the F0 parental animals, however showing

some variability across cohorts. At 300 ppm changes of body weights/body weight gain look

rather nonuniform and mild, however, there appears to be evidence that this dose group was

affected as well.

 

Regarding clinical pathology in F0 generation male and female rats of test group 3 (1000

ppm) as well as in F1 generation male and female rats at PND92 of test group 13 (1000 ppm)

a microcytic anemia was present indicated by decreased red blood cell (RBC) counts

hematocrit and hemoglobin values as well as decreased mean corpuscular volume (MCV).

Increased urea values in male and female rats of the same test groups in the F0 and F1

generation at PND 92 were due to an increased protein metabolism. This is confirmed by higher

albumin levels in the rats of both sexes of test group 3 (1000 ppm) in the F0 generation and

most probably also by a reduced prothrombin time in females of test group 13 (1000 ppm) of

the F1 generation at PND 92; indicating higher synthesis of coagulation factors.

Higher activities of alkaline phosphatase in males of the F0 generation of test group 3 and of

the F1 generation in test group 13 may have been caused by a reduced food consumption of

these animals. Higher aspartate aminotransferase (AST) activities in F0 males of test group 3

and in male and female rats of the F1 generation of test group 13 may be due to a liver cell

effect, although other tissues could also have been involved because AST is not a liver-specific

enzyme in rats.

 

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.

 

The increase of platelet counts in F0 males of test groups 2 and 3 (300 and 1000 ppm) as well

as males of the F1 generation in test groups 12 and 13 (300 and 1000 ppm) as well as the

shortened prothrombin time (Hepatoquick’s test) in males and females of test groups 2 and 3

of the F0 generation and in F1 females of test group 13, indicated a dysregulation of the

coagulation homeostasis. This correlates with decreased serum platelet activating factor (PAF)

values in F0 females of test group 3 (1000 ppm).

 

Regarding pathology, target organs were the kidneys, liver and glandular stomach.

F0 generation parental animals

 

The terminal body weight in the F0 generation parental males and females of test group 03

(1000 ppm) was decreased to below historical control values and for test group 02 animals

(300 ppm) it was within historical control values (see PART III). This reflects the different extent

of body weight effects at 300 and 1000 ppm in-life.

In the kidneys of males of test group 02 and test group 03 and in the kidneys of females of test

group 03, degeneration/regeneration of the proximal tubules was observed. This was also regarded as treatment related

 

 

The decrease in terminal body weight in males of test group 12 and 13 (300 and 1000 ppm)

and females of test group 13 (1000 ppm) reflects the body weight effect at 300 and 1000 ppm

in life.

 

Similar to the findings in the kidneys of the F0 generation, males and females of test group 12

and 13 (300 and 1000 ppm) revealed tubular degeneration and regeneration. This was also

reflected by the increased kidney weight. In test group 11 (100 ppm) animals the kidney weight

was still increased, but was regarded to be non-adverse due to a missing histopathologic

correlate. Comparable to the F0 generation, animals of the F1 generation revealed also

mineralization: male animals at the transition between inner and outer medulla and females in

the papilla. As for the F0 generation this finding was regarded to be treatment-related but not

as adverse.

 

Males and females of test group 13 (1000 ppm) revealed centrilobular hypertrophy in the liver.

Male animals also had a peripheral hypertrophy. This correlated with the increased liver

weights. Furthermore, in both sexes an increase of fatty change was observed in the peripheral

area. These findings in combination with clinical pathology findings were regarded to be

adverse. In males of test group 12 (300 ppm), the relative liver weight was increased, three

animals showed centrilobular hypertrophy and fatty change. Due to the additional fatty change

in combination with the hypertrophy, it was regarded as adverse. Females of the same test

group revealed increased liver weights and one animal a centrilobular hypertrophy, but no

increase in fatty change when compared to control and no relevant findings in clinical

pathology. It was therefore regarded to be treatment-related but not as adverse. The same

comes true for the increased liver weight in test group 11 males and females (100 ppm), in

which no relevant histopathologic findings were observed.

 

The mammary gland of males of test group 13 (1000 ppm) revealed a female phenotype

(feminization) in four out of 16 animals. One male animal showed in addition a diffuse

hyperplasia of the mammary gland. Females of this test group showed an increase in secretion.

These findings were regarded to be adverse.

 

In the left testis, three males of test group 13 (1000 ppm) revealed immature testicular tubules

which corresponded to the macroscopic finding “size reduced”. In one male, there was focal

degeneration in addition. As a consequence, the secondary sexual glands showed also a

reduction in size. In the corresponding epididymis of the affected animals aspermia was found.

In the ductus deferens size reduction and an increase in macrovesicular vacuolation was

observed. The vacuolation in the ductus deferens of test group 12 animals (300 ppm) might be

still treatment-related but due to the absence of findings in all other sexual organs this was

regarded to be non-adverse. These three males revealed a severe decrease in terminal body

weight. Therefore, this size decrease in the above-mentioned organs was most likely due to

the body weight reduction and was assessed to be treatment-related but a secondary effect.

A delayed maturation of the testicular epithelium is known to occur in animals which have

reduced body weights (McInnes, 2012). But as in cohort 1A animals (and in cohort 1B animals)

degeneration of the testicular epithelium was observed in addition to the delay in maturation

as well as in animals without a delay in maturation in these organs, the degeneration of tubular

epithelium in the testis was assumed to be an adverse but secondary effect.

 

Six out of 20 females of test group 13 (1000 ppm) showed an increased incidence of luteal

cysts in the ovary. One female revealed a diffuse atrophy of the ovaries. Luteal cysts might

develop in case the follicle fails to ovulate. These findings were regarded to be treatment-related

and adverse.

 

Males and females of all treated groups revealed cysts in the pars distalis of the pituitary gland

which were filled with a homogenous eosinophilic material and differed from the spontaneously

observed cysts (e.g. remnants of the Rathke’s pouch). It could not be determined what material

was present in these cysts and whether they were functionally active or not. Thus this finding

could not be assessed with regard to potential adversity.

 

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 and growing

follicles – showed significant differences between the control group 10 and animals of test

group 13. The DOFC was performed in cohort 1A and cohort 1B animals together in a fully

blinded manner. This statistically significant decrease in primordial and growing follicles was

regarded to be adverse.

 

F1 rearing animals, cohort 1B

 

Target organs were the ovaries and testes. No other organs were examined

histopathologically.

 

The decrease in terminal body weight in males and females of test group 13 (1000 ppm)

reflects the body weight effect at 1000 ppm in life.

 

In males and females of all treated groups the liver weight was significantly increased which

was regarded to be treatment-related. Although a conclusive interpretation of adversity cannot

be made without a histopathological or clinical pathological examination, similar effects as have

been detected in cohort 1A livers are the most likely explanation for this finding.

The reduced absolute and relative prostate weight in test group 13 (1000 ppm) males could be

related to treatment. But as the terminal body weight was also significantly decreased and the

cohort 1B animals were treated the same way as the cohort 1A animals and there were no

histopathologic findings in the prostate that could explain the weight decrease beside the three

affected animals, it was regarded to be secondary to the body weight reduction.

 

Three males of test group 13 (1000 ppm) had reduced testes sizes and corresponding reduced

size of the prostate and seminal vesicle that was thought to be secondary to the testes findings.

Microscopically, the gross lesion was related to immaturity as described for cohort 1A. Two of

these males had in addition tubular degeneration and two males showed minimal tubular

degeneration without other findings. In test group 11 (100 ppm), one male also revealed

immature testicular epithelium and degeneration. As there was no male affected in test group

12 (300 ppm) this finding was regarded to be incidental. The immaturity and degeneration in

test group 13 animals (1000 ppm) was regarded to be treatment-related and adverse, however,

as in the cohort 1A animals are most likely a secondary effect of the body weight reduction, a hypothesis supported when the individual animal data is taken into account.

 

Four females of test group 13 (1000 ppm) had macroscopically reduced ovaries that could be

related microscopically to a diffuse atrophy of the ovary. As described for cohort 1A animals

here were six females showing luteal cysts in the same test group and in two females of test

group 13 (1000 ppm) no corpora lutea could be observed. These findings were regarded

treatment-related and adverse. The single animal in test group 12 (300 ppm) showing luteal

cysts was regarded to be incidental.

 

F1 rearing animals, cohort 3 (Immunotoxicity)

 

There was a reduction in terminal body weight in test group 13 (1000 ppm) in males and

females which reflects the body weight effect at 1000 ppm in life.

Macroscopically there was one male showing reduced size of testes, epididymides, prostate

and seminal vesicle. As no microscopic investigation was performed a detailed diagnosis could

not be made. But considering the testis effects in the other cohorts it is most likely the same

finding here.

 

There were no indications from clinical examinations as well as gross and histopathology, that

2,2’-iminodiethanol adversely affected the fertility or reproductive performance of the F0

parental animals up to and including the administered dose of 300 ppm. Estrous cycle data,

mating behaviour, conception, gestation, parturition, lactation and weaning as well as sexual

organ weights and gross and histopathological findings of these organs (specifically the

differential ovarian follicle count) were comparable between the rats of these groups including

control and ranged within the historical control data of the test facility.

 

The high dose of the test item (1000 ppm) exerted effects on a number of parameters such as

number of implants and duration of gestation in the F0 parental animals as well as estrous

cyclicity and morphology of pituitary, ovaries, testes (subsequently accessory sexual glands)

and mammary glands in the F1 offspring.

 

The high-dose F0 generation animals were successfully paired, fertility and gestation indices

were comparable to the concurrent control. There was, however, a significantly lower number

of implants and subsequently lower litter size noted along with a small but significant increase

in duration of gestation. There were no morphological changes detected in tissues related to

reproduction which could have explained these effects.

 

The high dose F1 generation females had a prolonged estrous cycle and the cycle was irregular

in a higher number of females. There was no obvious pattern of change in the affected females,

the irregularities consisted likewise of prolonged diestrous, estrous or metestrous. These

irregularities corresponded with ovarian atrophy, pituitary and luteal cysts as well as a decrease

in primordial and growing ovarian follicles, as described in the pathology/neuropathology

sections.

 

Some high-dose F1 males had smaller and lightweight testes which were immature and, in

some cases, showed degeneration of tubular epithelia. Cysts in the pars distalis of pituitary

were also present. Most of the affected males exhibited distinct clinical symptoms of systemic

toxicity as well. In addition, some high-dose F1 males had feminized and/or hyperplastic

mammary glands, while high-dose females showed an increase in secretion, as described in

the pathology section.

 

For all liveborn male and female pups of the F0 parents, no test substance-induced signs of

developmental toxicity were noted at dose levels as high as 100 ppm. Postnatal survival, pup

body weight gain as well as post-weaning development of the offspring of this test group until

puberty remained unaffected by the test substance. Furthermore, clinical and/or gross

necropsy examinations of the F1 pups revealed no adverse findings.

The high dose of the test substance (1000 ppm) caused effects on pup survival during early

lactation. While the lower litter size in this dose group was a consequence of a lower number

of implants and not due to prenatal or perinatal mortality, the slightly lower viability index came

from a higher number of dead and cannibalized pups which were distributed across 8 litters. In

2 of those 8 litters none of the pups survived. Altogether, the lower viability index in this group

was only slightly below the historical control range. Postnatal survival after PND 4 of the

offspring of all test groups until weaning remained unaffected by the test substance.

Furthermore, clinical and/or gross necropsy examinations of the weaned F1 pups revealed no

adverse findings.

 

Pup body weight development of the mid and high-dose F1 offspring (300 and 1000 ppm)

was affected by the treatment. These offspring had similar weights as the control right after

birth but gained significantly less weight than control offspring from PND 4 (high-dose) or PND

14 (mid-dose) onwards. The decrease in terminal body weight in the 300 and 1000 ppm male

and 1000 ppm female weanlings not selected for cohorts reflects the body weight effect in life.

The impairment of body weight gain in the F1 offspring continued after weaning in the offspring

selected for cohorts.

 

Measurement of thyroid hormones revealed higher T4 values in F0 males of test group 3

(1000 ppm) as well as in F1 rats of both sexes in test group 13 (1000 ppm) at PND 92 as well

as in F1 females of the same test group at PND 22 and in females of test groups 11 and 12 at

PND 4. The increased values were within historical control data and thus not biologically significant. In addition, there were no significant changes of TSH observed in any dose groups.

 

Anogenital distance of all test substance treated F1 pups (100, 300 and 1000 ppm) was

comparable to the concurrent control values, as were anogenital indices.

In addition, the check for the presence of nipples/areolas, also a very sensitive marker of

potential endocrine-mediated imbalances, revealed no test substance-related effects at all.

Vaginal opening and preputial separation are commonly used developmental markers for

onset of puberty in laboratory rats. A statistically significant delay in vaginal opening of about

1-2 days beyond the control was observed in the female F1 offspring of the mid- and high-dose

groups (300 and 1000 ppm). The values for pubertal age and weight of mid-dose females were

both at the lower end of the historical control range; thus the apparent statistical increase 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. A statistically significant delay in preputial separation

of about 2 days beyond the control was observed in the male F1 offspring of the high-dose

group (1000 ppm). 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.

No clinical signs of developmental neurotoxicity were evident in male and female F1

offspring at dose levels as high as 300 ppm. There were no compound related effects on motor

activity, auditory startle habituation, and in the field observation battery following exposure to

the test compound in these animals.

 

Some findings which might be related to an affection of the nervous system were observed in

the F1 offspring at 1000 ppm. Clinically, high-stepping gait and piloerection were observed

recurring across all F1 cohorts in several study sections. The only notable finding in

neurobehavioral testing, however, were lower maximum amplitudes in the auditory startle

response test of the high-dose F1 males and females in Cohort 2A, while FOB and motor

activity testing remained without findings. In the auditory startle test there was also no

habituation to the test environment seen in these animals, males slightly more affected by this

than females. Notably, no corresponding effects were recorded for startle response latency.

In addition, regarding neuropathology, treatment-related findings were seen in the medulla

oblongata, spinal cord and pituitary gland of Cohort 2A animals (adults, PND 77) as well as in

the pituitary gland of Cohort 2B animals (weanlings, PND 22).

 

F1 rearing animals, cohort 2A (adults)

The terminal body weight was decreased in male and female test group 13 animals, reflects

the body weight effect at 1000 ppm in life.

 

The medulla oblongata and the spinal cord of male and female animals of test group 13 (1000

ppm) revealed a minimal to marked, multifocal degeneration of nerve fibers. This 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

visible especially in longitudinal sections and less in cross sections of the spinal cord. As also

in several control animals minimal spontaneous degeneration was seen, only degeneration

with a higher severity grade was assessed as treatment-related and adverse.

In the pars distalis of the pituitary gland of male and female animals (all test groups),

multifocally distributed very small eosinophilic cysts with a non-ciliated, irregular border and an

eosinophilic homogenous content were seen. As no functional or mechanistic data are

available, this finding could not be assessed with regard to potential adversity.

The brain weight determination, brain length and width measurements as well as brain

morphometry and neuropathological examination by light microscopy of all other tissues did

not reveal further treatment-related findings.

 

F1 rearing animals, cohort 2B (weanlings)

The terminal body weight was decreased in male and female test group 13 animals and in

females of test group 12, which reflects the body weight effect at 300 and 1000 ppm in life.

In the pars distalis of the pituitary gland of four male and five female animals of test group 13

(1000 ppm), multifocally distributed, very small eosinophilic cysts with a non-ciliated, irregular

 

border and an eosinophilic homogenous content were seen. As no functional or mechanistic

data are available, this finding could not be assessed with regard to potential adversity.

There was no evidence that the test substance produced any developmental immunotoxicity

up to and including a dose of 300 ppm in both sexes and 1000 ppm in males. Neither T-cell

dependent anti-SRBC IgM antibody response, nor absolute and relative lymphocyte

subpopulation cell counts in the spleen tissue (B-, T-lymphocytes, CD4-, CD8-T-lymphocytes

and natural killer (NK) cells) displayed any treatment-related changes.

However, a test compound-related effect on the T-helper cells and cytotoxic T-cells in the

spleen in the high-dose F1 females (1000 ppm) cannot be excluded.