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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
extended one-generation reproductive toxicity - with F2 generation (Cohorts 1A, and 1B with extension)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Study initiation date 19.Nov.2019; study not finalized yet
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2021

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 443 (Extended One-Generation Reproductive Toxicity Study)
Version / remarks:
25 June 2018
GLP compliance:
yes (incl. QA statement)
Remarks:
BASF SE Experimental Toxicology and Ecology 67056 Ludwigshafen, Germany
Limit test:
no
Justification for study design:
SPECIFICATION OF STUDY DESIGN FOR EXTENDED ONE-GENERATION REPRODUCTION TOXICITY STUDY WITH JUSTIFICATIONS

- Premating exposure duration for parental (P0) animals : 2 weeks; according to ECHA TP decision TPE-D-2114460918-36-01/F
F1 animals of Cohort 1B are mated to produce the F2 generation and, thus, the premating exposure duration will be ten weeks for these animals. Consequently the fertility parameters will be covered allowing an evaluation of the full spectrum of effects on fertility in these animals. Thus, shorter premating exposure duration for parental (P) animals may be considered.
- Basis for dose level selection : Based on a range finding study
- Inclusion of extension of Cohort 1B: yes; according to ECHA TP decision TPE-D-2114460918-36-01/F
- Termination time for F2 : terminal sacrifice of the F2 weanlings.
- Exclusion of developmental neurotoxicity Cohorts 2A and 2B : according to ECHA TP decision TPE-D-2114460918-36-01/F
- Exclusion of developmental immunotoxicity Cohort 3 : according to ECHA TP decision TPE-D-2114460918-36-01/F
- Route of administration : oral (gavage): according to ECHA TP decision TPE-D-2114460918-36-01/F
- Choice of species, strain: The rat is the preferred animal species for reproduction studies according to test guidelines. This strain was selected since extensive historical control data were available for Wistar rats.

Test material

Constituent 1
Chemical structure
Reference substance name:
Citral
EC Number:
226-394-6
EC Name:
Citral
Cas Number:
5392-40-5
Molecular formula:
C10H16O
IUPAC Name:
3,7-dimethylocta-2,6-dienal

Test animals

Species:
rat
Strain:
Wistar
Remarks:
Crl:WI(Han)
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH,
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 14 weeks old at the beginning of treatment (P)
- Weight at study initiation: weight variation did not exceed 20 percent of the mean weight of each sex (P)
- Fasting period before study: no
- Housing: up to 5 animals per sex and cage
From delivery to randomization, during overnight matings (male and female mating partners were housed together), gestation, lactation and females after weaning the animals were housed individually. Dams and their litters were housed together until PND 21.
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: approx. 2 weeks
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 45-65
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 02 Dec. 2019 (TS administration) To: 09 Jun. 2020 (sacrifice of F1 cohort 1B animals)

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
CMC (carboxymethyl cellulose)
Remarks:
0.5% CMC suspension in drinking water (with 5 mg/ 100 mL Tween 80)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:

VEHICLE
- Concentration in vehicle: 0.25, 0.8, 2.5 g/ml in low, mid and high dose, respectively
- Amount of vehicle (if gavage): 10 ml/kg bw/d
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: max. 2 weeks
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged: individually
- Any other deviations from standard protocol:
Throughout the mating period, each female animal was paired with a predetermined male animal from the same dose group except for one case in the high dose test group F1 (Co1B), which was paired with 2 females.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analytical verifications of the stability of the test substance in 0.5% CMC suspension in drinking water (with 0.5 mg/ 100 mL Tween 80) in a refrigerator over a period of 7 days at room temperature were verified prior to the start of the study in a similar batch.

At the beginning (during premating) of the administration 3 samples each were taken from the lowest and highest concentration for potential homogeneity analyses. The 3 samples were withdrawn from the top, middle and bottom of the preparation vessel. These samples were used as a concentration control at the same time. At the above mentioned time point additionally one sample from the mid concentration was taken for concentration control analysis. Once during the middle and once during the end of the administration each 1 sample was taken from the low, mid and high concentration for a concentration control analysis.
Duration of treatment / exposure:
F0 males: 2 weeks (premating) + 2 weeks (mating) + max. 6 weeks (post-mating)
F0 females: 2 weeks (premating) + 2 weeks (mating) + approx. 6 weeks (pregnancy + lactation)
F1 males (Co 1B): 10 weeks (post-weaning/premating) + 2 weeks (mating) + 4 weeks (post-mating)
F1 males (Co 1B): 10 weeks (post-weaning/premating) + 2 weeks (mating) + approx. 6 weeks (pregnancy + lactation)
F1 animals (Co 1A): post weaning until an approx. age of 13 weeks
Frequency of treatment:
once daily
Females in labor were not treated.
Details on study schedule:
F0 parental animals:
After a minimum of 2 weeks after the beginning of treatment, males and females from the same dose group were mated. The females were allowed to deliver and rear their pups (F1 generation pups) until PND 4 (standardization) or PND 21 or 22 (depending on the cohort). Pups of the F1 litter were selected (F1 rearing animals) and assigned to Cohorts 1A and 1B. During weaning of the F1 pups the F0 generation parental male animals were sacrificed. After weaning of F1 pups the F0 generation parental female animals were sacrificed.

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

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

Standardization of litters (F1 and F2 generation pups)
On PND 4, the individual litters were standardized in such a way that, where possible, each litter contained 5 male and 5 female pups (always the first 5 pups/sex and litter were taken for further rearing). If individual litters did not have 5 pups/sex, the litters were processed in such a way that the most evenly distributed 10 pups per litter were present for further rearing (e.g. 6 male and 4 female pups). Surplus animals were sacrificed. Standardization of litters was not performed in litters with < 10 pups.
Doses / concentrationsopen allclose all
Dose / conc.:
25 mg/kg bw/day (nominal)
Dose / conc.:
80 mg/kg bw/day (nominal)
Dose / conc.:
250 mg/kg bw/day (nominal)
No. of animals per sex per dose:
F0: 25 animals pers sex per dose
F1A: 20 animals pers sex per dose
F1B: 25 animals pers sex per dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: based on range-finding study
- Fasting period before blood sampling for clinical biochemistry: yes, for about 16-20 hours
Positive control:
not applicable

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule:
Mortality: twice daily on working days or once daily (Saturday, Sunday or on public holidays).
Cage side observations: at least daily for any signs of morbidity, pertinent behavioral changes and signs of overt toxicity before the administration as well as within 2 hours and within 5 hours after the administration. The parturition and lactation behavior of the dams was generally evaluated in the mornings in combination with the daily clinical inspection of the dams. On weekdays (except Saturday, Sunday and public holidays) the parturition behavior of the dams was inspected in the afternoons in addition to the evaluations in the mornings.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule:
F0: animals once before the administration and subsequently once per week
F1 (Cohort 1A/1B): at weekly intervals during the administration period.

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

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
Generally, food consumption was determined once a week (over a period of 7 days) for male and female F0 and F1 parental animals and F1 rearing animals, with the following exceptions:
• not determined after the 2nd premating week (male F0 animals) and after the 10th premating week (male F1 animals).
• determined with evidence of sperm of the F0 and F1 females for GD 0-7, 7-14 and 14-20.
• detemined for the F0 and F1 females, which gave birth to a litter for PND 1-4, 4-7, 7-10, 10-14, 14-18 and 18-21.
Food consumption was not determined in the females without positive evidence of sperm during mating and gestation periods, in the females without litter during lactation period and in the females after weaning.

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


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

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

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

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

Urinalysis
Parameters examined:
- pH
- Protein (PRO)
- Glucose (GLU)
- Ketones (KET)
- Urobilinogen (UBG)
- Bilirubin (BIL)
- Blood
- Specific gravity
- Sediment
- Color, turbidity (COL, TURB)
- Volume (VOL)
Oestrous cyclicity (parental animals):
evaluated by daily analysis of vaginal smear for all F0 female parental rats for a minimum of 2 weeks and for all F1 female parental rats (= cohort 1B) for a minimum of 3 weeks prior to mating. Determination was continued throughout the pairing period until the female exhibited evidence of copulation.
In all cohort 1A females, vaginal smears were collected after vaginal opening until the first cornified smear (estrous) was recorded. The estrous cycle also was evaluated in cohort 1A females for 2 weeks around PND 75.
At necropsy, an additional vaginal smear was examined to determine the stage of estrous cycle for each F0 female and cohort 1A and 1B female with scheduled sacrifice.
Sperm parameters (parental animals):
After the organ weight determination, the following parameters were determined in the right testis or right epididymis of all male F0 parental animals sacrificed on schedule:
- Sperm motility
- Sperm morphology
- Sperm head count (cauda epididymis)
- Sperm head count (testis)

Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes

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

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

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

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

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

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

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

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


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

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

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

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

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


ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: no

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: no
Postmortem examinations (parental animals):
SACRIFICE
All F0 generation parental animals were sacrificed by decapitation under isoflurane anesthesia.

GROSS NECROPSY
The exsanguinated animals were necropsied and assessed by gross pathology; special attention being given to the reproductive organs.)

ORGAN WEIGHTS
The following weights were determined in all animals sacrificed on schedule:
1. Anesthetized animals (final body weight)
2. Adrenal glands (fixed)
3. Brain
4. Cauda epididymis
5. Epididymides
6. Heart
7. Kidneys
8. Liver
9. Ovaries
10. Pituitary gland (fixed)
11. Prostate (ventral and dorsolateral part together, fixed)
12. Testes
13. Seminal vesicles including coagulating glands (fixed)
14. Spleen
15. Thymus (fixed)
16. Thyroid glands (with parathyroid glands) (fixed)
17. Uterus with cervix
All paired organs were weighed together (left and right).


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

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

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

DOFC (Cohort 1A)
A differential ovarian follicle count (DOFC) was conducted in control and high dose test groups according to Plowchalk et.al. (1993). In general, sections were prepared with 2 - 3 µm thickness and serial sections were taken every 100 µm to complete up to 15 – 20 cut levels across the whole ovarian tissue. For the counting of primordial and growing follicles, H&E stained slides were prepared from all cut levels.


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

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

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


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

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

- Organ weights (Cohort 1A)
The following weights were determined in all animals sacrificed on schedule:
1. Anesthetized animals (final body weight)
2. Adrenal glands (fixed)
3. Brain
4. Cauda epididymis
5. Epididymides
6. Heart
7. Kidneys
8. Liver
9. Lymph nodes, axillary (10 animals per sex per group, cohort 1A animals only)
10. Lymph nodes, mesenteric (10 animals per sex per group, cohort 1A animals only)
11. Ovaries
12. Pituitary gland (fixed)
13. Prostate (ventral and dorsolateral part together, fixed)
14. Testes
15. Seminal vesicles including coagulating glands (fixed)
16. Spleen
17. Thymus (fixed)
18. Thyroid glands (with parathyroid glands) (fixed)
19. Uterus with cervix
All paired organs were weighed together (left and right).

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


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

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

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

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

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


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

- Pup organ weights (F2 pups)
After the scheduled sacrifice, the brain, spleen and thymus of 1 pup/sex per F2 litter were weighed. Normally, the first male and the first female pups/litter were taken for these determinations. The corresponding in-life pup weights determined on PND 21 were used to calculate the relative organ weights.
Statistics:
- DUNNETT test (two-sided): Water consumption (parental and rearing animals), food consumption (parental and rearing animals), body weight and body weight change (parental animals, rearing animals and pups; for the pup weights, the litter means were used), gestation days, duration of sexual maturation (days to vaginal opening, days to preputial separation), anogenital distance, anogenital index
- FISHER'S EXACT test (one-sided): Male and female mating indices, male and female fertility indices, gestation index, females mated, females pregnant, females delivering, females with liveborn pups, females with stillborn pups, females with all stillborn pups
- WILCOXON-test (one-sided): Urinalysis parameters (apart from pH, urine volume, specific gravity, color and turbidity)
- WILCOXON-test (one-sided) with BONFERRONI-HOLM adjustment: Spermanalysis parameters
- WILCOXON test (one-sided+) with BONFERRONI-HOLM adjustment: Mating days until day 0 pc, %postimplantation loss, pups stillborn, %perinatal Loss, nipple development
- WILCOXON test (one-sided-) with BONFERRONI-HOLM adjustment: Implantation sites, pups delivered, pups liveborn, live pups day x, viability index, lactation index
- WILCOXON-test (one-sided-): DOFC
- WILCOXON test (two-sided): % live male day x, % live female day x
- KRUSKAL-WALLIS test (two-sided) + WILCOXON-test (two-sided): Number of cycles and Cycle Length, pup organ weights (absolute and relative), Organ weight parameters
- KRUSKAL-WALLIS test + WILCOXON-test (one or two-sided): Blood parameters, Urine pH, volume and specific gravity
Reproductive indices:
Male mating index (%) = number of males with confirmed mating / number of males placed with females x 100
Male fertility index (%) = number of males proving their fertility / number of males placed with females x 100
Female mating index (%) = number of females mated / number of females placed with males x 100
Female fertility index (%) = number of females pregnant / number of females mated x 100
Gestation index (%) = number of females with live pups on the day of birth / number of females pregnant x 100
Offspring viability indices:
Live birth index (%) = number of liveborn pups at birth / total number of pups born x 100
Postimplantation loss (%) = number of implantations – number of pups delivered / number of implantations x 100
Viability index (%) = number of live pups on day 4* after birth / number of live pups on the day of birth x 100
Lactation index (%) = number of live pups on day 21 after birth / number of live pups on day 4* after birth x 100

Results and discussion

Results: P0 (first parental generation)

Effect levels (P0)

Remarks on result:
other: The study is still ongoing.

Results: P1 (second parental generation)

Effect levels (P1)

Remarks on result:
other: The study is still ongoing.

Results: F1 generation

Effect levels (F1)

Remarks on result:
other: The study is still ongoing.

Results: F2 generation

Effect levels (F2)

Remarks on result:
other: The study is still ongoing.

Overall reproductive toxicity

Reproductive effects observed:
not specified

Applicant's summary and conclusion