3,7-dimethyloctan-3-ol

Toxicity to reproduction

Administrative data

Endpoint:

extended one-generation reproductive toxicity - basic test design (Cohorts 1A, and 1B without extension)

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2022

Report date:

2022

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 443 (Extended One-Generation Reproductive Toxicity Study)

Version / remarks:

25-Jun-2018

GLP compliance:

yes (incl. QA statement)

Remarks:

GLP CERTIFICATE (FROM THE COMPETENT AUTHORITY): Landesamt für Umwelt, Rheinland Pfalz, 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: (minimum) 10 weeks
- Basis for dose level selection:
Selection of the dose levels listed above was based on the results of an OECD 421 type range-finding study. The test item was administered to groups of 10 male and 10 female healthy young Wistar rats (F0 animals) as a constant homogeneous addition to the food in different concentrations, i.e. 0, 1500, 5000 and 15000 ppm. During lactation period the test item concentrations in the diet of the F0 females were reduced to 50%. The duration of treatment covered a 4 weeks in-life period in males including mating (mating pairs were from the same test group) as well as a 2-weeks in-life period, mating period and the entire gestation and lactation period in females.
Signs of general systemic toxicity were observed in parental animals at 15000 ppm taking reduced body weight development into account. There were no effects on estrous cyclicity, mating, pregnancy including implantation and intrauterine mortality as well as parturition and postnatal development of offspring. Clinical pathology and pathology revealed no changes.
The top concentration of 15000 ppm in this range-finding study targeted for a substance intake of about 1000 mg/kg bw, i.e. the limit dose. However, this target dose level was considerably exceeded, particularly in the F1 rearing animals (up to 1875 mg/kg bw/d). Therefore, a top concentration of 12500 ppm was chosen for the present study to meet a target dose level of about 1000 mg/kg bw/d.
A standard spacing factor between dose groups was applied.

- Exclusion of extension of Cohort 1B
- Exclusion of developmental neurotoxicity Cohorts 2A and 2B
- Exclusion of developmental immunotoxicity Cohort 3
- Route of administration: diet/feed

Test material

Test material information

Constituent 1

Reference substance name:

3,7-dimethyloctan-3-ol

EC Number:

201-133-9

EC Name:

3,7-dimethyloctan-3-ol

Cas Number:

78-69-3

Molecular formula:

C10H22O

IUPAC Name:

3,7-dimethyloctan-3-ol

Details on test material:

- Name of test material (as cited in study report): Tetrahydrolinalool

Test animals

Species:

rat

Strain:

Wistar

Remarks:

Crl:WI(Han)

Details on species / strain selection:

Reason for species selection:
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.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS (used as F0 generation parental animals):
- Source: supplied by Charles River Laboratories, Germany, Sulzfeld
- According to a written statement from the breeder, male and female animals were derived from different litters. This was necessary to rule out the possibility of sibling mating.
- Females: nulliparous and non-pregnant at the beginning of the study
- Age at study initiation: 33 +/1 days
- Fasting period before study: no
- Housing: housed together (up to 5 animals per sex and cage) in Polysulfonate cages Typ 2000P (H-Temp) supplied by TECHNIPLAST, Hohenpeißenberg, Germany with the following exceptions:
Male and female mating partners were housed together during overnight mating.
Females were housed individually during gestation and lactation; dams and their litters were housed together until PND 22 in Polycarbonate cages type III. Pregnant females were provided with nesting material (cellulose wadding) towards the end of gestation.
- Diet: ad libitum
- Water ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24°C
- Humidity (%): 45-65%
- Air changes (per hr): 15 times per hour
- Photoperiod (hrs dark / hrs light): 12 hours (12 hours light from 06.00 h to 18.00 h and 12 hours darkness from 18.00 h to 06.00 h)

IN-LIFE DATES:
F0 generation parental animals and progeny:
Arrival: 13 Oct 2020
Sacrifice of litters after weaning: 17 - 20 Feb, 24 Feb and 01 – 02 Mar 2021,
Sacrifice of parental/ rearing animals: M: 05, 06 and 07 Feb 2021 / F: 01, 02 and 03 Mar 2021
F1 rearing animals (cohorts 1A and 1B):
Sacrifice of parental/ rearing animals: 1A: 26 - 29 Apr 2021 / 1B: 19 - 23 Apr 2021

Administration / exposure

Route of administration:

oral: feed

Details on exposure:

DIET PREPARATION:
The food used was Mouse and rat maintenance diet “GLP”, supplied by Garanovit AG, Kaiseraugst, Switzerland.
The required quantity of test substance was weighed in a beaker depending on the dose group and thoroughly mixed with a small amount of food. Then further amounts of food were added to this premix and thoroughly mixed for 3 minutes. Afterwards, further amounts of food, depending on the dose group, were added to this premix in order to obtain the desired concentrations. Mixing of this final mix was carried out for about 10 minutes in a laboratory mixer. The test-substance preparations were split in aliquots and stored at -18°C in order to offer new test substance preparations. The food in the food hoppers were changed at least every three days.
Analytical verifications of the stability of the test substance in the diet over a period of 19 days under freezer conditions, followed by 3 days at room temperature was verified before the start of the administration period.

DOSE ADJUSTMENT
During the lactation period the test item concentrations in the diet of the F0 females were reduced to 50%. This dietary adjustment derived from historical body weight and food consumption data maintained the dams at the desired target doses of the test item during this period of increased food intake.

Details on mating procedure:

Mating pairs were from the same dose group. Throughout the mating period, each female animal was paired with a predetermined male animal from the same dose group.

- M/F ratio per cage: 1:1
- Length of cohabitation: from about 16.00 h until 06.30 - 09.00 h of the following morning for a maximum of 2 weeks
- Proof of pregnancy: sperm in vaginal smear referred to as gestation day (GD) 0
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged: housed individually during gestation and lactation; dams and their litters were housed together until PND 22 in Polycarbonate cages type III. Pregnant females were provided with nesting material (cellulose wadding) towards the end of gestation.

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 the diet over a period of 19 days under freezer conditions, followed by 3 days at room temperature was verified before the start of the administration period.

Homogeneity and Concentration Control Analysis:
Analyses were carried out 5 times during the study. At the beginning, in the middle and toward the end of the study, as well as for female diets once in during lactation period, according to the following:
Beginning of the study - Concentration control (mid dose) and Homogeneity analyses (low + high dose)
Mid of the study - Concentration control (mid dose) and Homogeneity analyses (low + high dose)
End of the study - Concentration control (mid dose) and Homogeneity analyses (low + high dose)
Two lactation periods - Concentration control (mid dose) and Homogeneity analyses (low + high dose)
Technique: GC-FID
Results:
Considering the low relative standard deviation in the homogeneity analysis, it can be
concluded that the test substance was distributed homogeneously in mouse and rat
maintenance diet „GLP“ meal.
The mean values and single values of the test substance in mouse and rat maintenance diet „GLP“ meal were found to be in the range of 90 % – 110 % of the nominal concentrations.
These results demonstrate the correctness of the concentrations of the test subtance in the diet

Duration of treatment / exposure:

F0 males: 10 weeks pre-mating, 2 weeks mating, post-mating (approx. 16 weeks)
F0 females: 10 weeks pre-mating, 2 weeks mating, during pregnancy and lactation (approx 19 weeks)
F1 Cohort 1A+1B: (in-utero development, pre-weaning), approx. 10 weeks post-weaning

Administration period:
F0 generation parental animals and progeny - M: 20 Oct 2020 - 06 Feb 2021 / F: 20 Oct 2020 -02 Mar 2021
F1 rearing animals (cohorts 1A and 1B) - 1A: 24 Feb - 28 Apr 2021 / 1B: 25 Feb - 22 Apr 2021

Frequency of treatment:

continuously via the diet

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

1 200 ppm

Remarks:

600 ppm (during lactation period)

F0 males (mean): 84 mg/kg bw/d
F1A males (mean): 102 mg/kg bw/d
F1B males (mean): 102 mg/kg bw/d

F0 females (mean): 100, 87, 119 mg/kg bw/d (premating, gestation, lactation) -> 104 mg/kg bw/d (overall mean)
F1A females (mean): 107 mg/kg bw/d
F1B females (mean): 108 mg/kg bw/d

Doses / concentrations 2

Dose / conc.:

4 000 ppm

Remarks:

2000 ppm (during lactation period)

F0 males (mean): 278 mg/kg bw/d
F1A males (mean): 338 mg/kg bw/d
F1B males (mean): 347 mg/kg bw/d

F0 females (mean): 330, 287, 398 mg/kg bw/d (premating, gestation, lactation) -> 345 mg/kg bw/d (overall mean)
F1A females (mean): 362 mg/kg bw/d
F1B females (mean): 361 mg/kg bw/d

Doses / concentrations 3

Dose / conc.:

12 500 ppm

Remarks:

6250 ppm (during lactation period)

F0 males (mean): 887 mg/kg bw/d
F1A males (mean): 1073 mg/kg bw/d
F1B males (mean): 1122 mg/kg bw/d

F0 females (mean): 1024, 887, 1185 mg/kg bw/d (premating, gestation, lactation) -> 1053 mg/kg bw/d (overall mean)
F1A females (mean): 1108 mg/kg bw/d
F1B females (mean): 1148 mg/kg bw/d

No. of animals per sex per dose:

F0 parental generation: 25 animals/sex/dose
F1 rearing animals (cohort 1A): 20 animals/sex/dose (1 male and 1 female pup/litter)
F1 rearing animals (cohort 1B): 25 animals/sex/dose (1 male and 1 female pup/litter)

Control animals:

yes, plain diet

Details on study design:

- Rationale for animal assignment: random
- Fasting period before blood sampling for clinical biochemistry: 16-20 hours

Examinations

Parental animals: Observations and examinations:

MORTALITY
A check for moribund or dead animals was made twice daily on working days or once daily
(Saturday, Sunday or on public holidays).

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least once daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Detailed clinical observations were performed in all F0 parental animals once before the administration and subsequently once per week during the administration period. The examinations started in the morning.
Parameters examined:
1. Abnormal behavior in handling
2. Fur
3. Skin
4. Posture
5. Salivation
6. Respiration
7. Activity/arousal level
8. Tremors
9. Convulsions
10. Abnormal movements
11. Gait abnormalities
12. Lacrimation
13. Palpebral closure
14. Exophthalmos (Protruding eyeball)
15. Assessment of the feces excreted during the examination (appearance/consistency)
16. Assessment of the urine excreted during the examination
17. Pupil size

BODY WEIGHT: Yes
- Time schedule for examinations: In general, the body weight of the male and female F0 parental animals was determined once a week at the same time of the day (in the morning), with the following exceptions:
• During the mating period of the F0 parental animals, the females were weighed on the day of positive evidence of sperm (GD 0) and on GD 7, 14 and 20.
• Females with litter were weighed on the day after parturition (PND 1) and on PND 4, 7, 10, 14, 18 and 21.

FOOD CONSUMPTION AND COMPOUND INTAKE:
- 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 with the following exceptions:
• Food consumption was not determined during the mating period (male and female parental animals).
• Food consumption of the females with evidence of sperm was determined for GD 6-7, 13-14 and 19-20.
• Food consumption of the females which gave birth to a litter was determined for PND 3-4, PND 6-7, PND 9-10 and PND 13-14, 17-18 and 20-21.
• Food consumption of females showing no positive evidence of sperm in the vaginal smear was determined once a week after this mating interval as will be the males
• Food consumption of females without litter and after weaning (PND 21) was determined once a week


- The mean daily intake of test substance (group means) was calculated based upon mean body weight values of each cage (the mean of the calculated average body weights during an interval, e.g. application days 0-7) and the mean food consumption per cage (during the same interval).

HAEMATOLOGY: yes
- blood samples were taken at necropsy from 10 F0 parental per group by puncturing the retrobulbar venous plexus following isoflurane anesthesia.
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 / URINALYSIS: yes
- blood samples were taken at necropsy from 10 F0 parental per group 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.

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):

Estrous cycle length was evaluated by daily analysis of vaginal smear for all F0 parental animals for a minimum of 3 weeks prior to mating. Determination was continued throughout the pairing period until the female exhibited evidence of copulation.

At necropsy, an additional vaginal smear was examined to determine the stage of estrous cycle for each F0 female with scheduled sacrifice.

Sperm parameters (parental animals):

After the organ weight determination, the following parameters were determined in the right testis or right epididymis of all male F0 parental animals and cohort 1A males 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 male pups on PND 13 and on PND 20.

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

- Vaginal opening
All female F1 pups selected to become F1 rearing animals (cohort 1A + 1B) 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 F1 rearing animals (cohort 1A + 1B) 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.


DETAILED CLINICAL OBSERVATIONS (Cohorts 1A, 1B): Yes
- Time schedule: Detailed clinical observations were performed at weekly intervals during the administration period. The examinations started in the morning.
Parameters examined:
1. Abnormal behavior in handling
2. Fur
3. Skin
4. Posture
5. Salivation
6. Respiration
7. Activity/arousal level
8. Tremors
9. Convulsions
10. Abnormal movements
11. Gait abnormalities
12. Lacrimation
13. Palpebral closure
14. Exophthalmos (Protruding eyeball)
15. Assessment of the feces excreted during the examination (appearance/consistency)
16. Assessment of the urine excreted during the examination
17. Pupil size

BODY WEIGHT F1 (rearing animals): Yes
- Time schedule for examinations: Ionce a week at the same time of the day (in the morning)

FOOD CONSUMPTION AND COMPOUND INTAKE F1:
- 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 F1B parental animals and F1 rearing animals.

ESTROUS CYCLING (F1A + F1B)
In all cohort 1A females, vaginal smears were collected after vaginal opening until the first cornified smear (estrous) was recorded. The estrous cycle also was evaluated in cohort 1A and cohort 1B females for 2 weeks around PND 75. At necropsy, an additional vaginal smear was examined to determine the stage of estrous cycle for each F1B female and cohort 1A female with scheduled sacrifice.

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
- Male animals: Before weaning of the F1 pups the F0 generation parental male animals were sacrificed (max. 6 weeks post-mating).
- Maternal animals: After weaning of F1 pups the F0 generation parental female animals were sacrificed.

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

ORGAN WEIGHTS
The following weights were determined in all animals sacrificed on schedule:
1. Anesthetized animals (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). Fixation was followed by histotechnical processing, examination by light microscopy and assessment of findings according to the list below:
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 (fixed)
11. Esophagus
12. Eyes with optic nerve (fixed)
13. Heart
14. Ileum
15. Jejunum (with Peyer’s patches)
16. Kidneys
17. Liver
18. Lungs
19. Lymph nodes, (axillary, mesenteric)
20. Mammary gland (male and female)
21. Ovaries (fixed)
22. Oviducts
23. Pancreas
24. Parathyroid glands
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. Testis, left (fixed)
35. Thymus
36. Thyroid glands
37. Trachea
38. Urinary bladder
39. Uterus
40. Vagina
41. Vas deferens

For a better visualization of the droplets in the kidneys of male animals, a CAB stain was performed in all males. In addition, immunohistochemistry was performed in single control and high dose groups males (F0 + F1A) with Rat alpha 2u-Globulin (AUG) MAb (Clone 129736), Mouse IgG1.

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 about 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, selected F1 pups were sacrificed by decapitation under isoflurane anesthesia and blood was sampled for determination of serum thyroid hormone concentrations.

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. Parathyroid 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

For a better visualization of the droplets in the kidneys of male animals, a CAB stain was performed in all males. In addition, immunohistochemistry was performed in single control and high dose groups males (F0 + F1A) with Rat alpha 2u-Globulin (AUG) MAb (Clone 129736), Mouse IgG1.

- Splenic lymphocytes subpopulation analysis
10 animals/ sex of cohort 1A were used to perform a splenic lymphocyte subpopulation analysis using one half of the spleen by immunophenotyping with a FACSLyric flow cytometer. Parameters assessed:
B lymphocytes
T lymphocytes
CD4+ lymphocytes
CD8+ lymphocytes
Natural killer cells


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. Testes
11. Uterus (with cervix)

- Histopathology (Cohort 1B): not performed

Statistics:

- DUNNETT test (two-sided): 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, % live female day x

- KRUSKAL-WALLIS test (two-sided) + WILCOXON-test (two-sided): Number of cycles and Cycle Length, Organ weight parameters

- KRUSKAL-WALLIS test + WILCOXON-test (one or two-sided): Urine pH, volume and specific gravity, Blood parameters and splenic lymphocytes subpopulations (uni- or bidirectional changes)

Reproductive indices:

Male reproduction data:

Male mating index (%) = number of males with confirmed mating* / number of males placed with females x 100
* defined by a female with vaginal sperm or with implants in utero

Male fertility index (%) = number of males proving their fertility* / number of males placed with females x 100
* defined by a female with implants in utero


Female reproduction and delivery data:

Female mating index (%) = number of females mated* / number of females placed with males x 100
* defined as the number of females with vaginal sperm or with implants in utero

Female fertility index (%) = number of females pregnant* / number of females mated** x 100
* defined as the number of females with implants in utero
** defined as the number of females with vaginal sperm or with implants in utero

Gestation index (%) = number of females with live pups on the day of birth / number of females pregnant* x 100
* defined as the number of females with implants in utero

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

Offspring viability indices:

Viability index (%) = number of live pups on the day 4* after birth / number of live pups on day 21 after birth x 100
* before standardization of litters (i.e. before culling)

Lactation index (%) = number of live pups on day 21 after birth / number of live pups on day 4* after birth x 100
* after standardization of litters (i.e. after culling)

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Body weights (test substance related/adverse):
- High dose males: mean body weight was slightly decreased (statistical significant from study day 56 -70 and day 91-105; Maximum by -6.6 % on study day 105).

Mean body weights of the low-and mid-dose male animals were comparable to the concurrent control values throughout the entire study. Mean body weights of females were comparable across all groups during pre-pairing, gestation and lactation.

Body weight changes (test substance related):
- High dose males: slightly decreased (statistical significant from study day 0-7 (-12%), day 49 – 56 (-19%) and whole period (-9%) vs ctrl.).
- High dose females: slightly decreased during gestation (statistical significant on GD 0-7 (- 17%) and on GD 0-20 ( -10 %) vs ctrl.).

Further findings:
- Mid dose males: significantly decreased between study day 77-84 (-31%.)
- Low dose males: significantly decreased between study day 56-63 (-22%) and day 77-84 (-34%).
Spontaneous, non-test substance related effects were observed in mid dose females (increase at day 7-14 premating, non-dose related), and mid/high dose females at lactation (decrease btw. LD18-21, non-dose related, overall comparable during full lactation period).

No other deviations between the test groups and control group could be observed in body weight and body weight change values.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

In general, mean food consumption of all male and female animals of all test groups was comparable to the concurrent control values throughout the entire study.

Slight temporary differences (assessed as spontaneous and not related to treatment):
High dose males: -14 % below control (study day 97 - 98)
Mid dose males: -10 % below control (study day 91)

High dose females: -11 % below control (PND 21)
Mid dose females: -8 % below control (study day 21)

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related, adverse changes observed.

Non-adverse or incidental/ non-treatment related findings:

High dose females:
- absolute neutrophil count decreased (-> treatment-related, but not adverse as the decrease was the only changed differential blood cell count, and total white blood cell counts were also not altered (ECETOC Technical Report No. 85, 2002).

Mid dose females:
- mean corpuscular volume (MCV) slightly decreased (within the historical control range)

Low dose females:
- absolute neutrophil count decreased (within the historical control range)

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Treatment-related and adverse effects observed:
High dose males:
- Urea levels were significantly increased (+19% related to ctrl.)
- Glucose values were significantly decreased (-16% related to ctrl.)

Treatment-related and non-adverse findings:
High dose males:
- Total bilirubin values were significantly decreased. This change without any change in the red blood cell parameters were most probably due to an increased conjugation of bilirubin followed by an accelerated excretion via the bile which is caused by a liver enzyme induction. This mechanism is regarded as treatment-related, but adaptive

Incidental/ non-treatment related findings:
Mid dose males:
- urea value increased (within historical control range)
High dose females:
- Creatinine levels decreased (within historical control range)
- Inorganic phosphate value decreased (within historical control range)
- Albumin levels increased (within historical control range)
- Triglyceride levels increased (within historical control range)
Mid dose females:
- Creatinine levels decreased (within historical control range)
Low dose females:
- Creatinine levels decreased (within historical control range)

Endocrine findings:

no effects observed

Description (incidence and severity):

Thyroid hormones:

No treatment related alterations of T4 and TSH levels were observed in F0 males and females.

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

Treatment-related and adverse effects observed:
High dose males:
- pH value of the urine was significantly decreased (5.2 vs. 6.4 in ctrls.)
- numbers of transitional epithelial cells and of granular and epithelial casts in urine sediment were significantly increased
These changes were not observed in F0 females. In combination with histopathologic findings these alterations were most probably due to an α2u-Globulinuria which is regarded as species-specific, treatment related and adverse finding in rats which is not relevant for humans (Hard et al., 2018).
No other treatment-related changes among urinalysis parameters were observed.

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Treatment-related and adverse findings:

Kidney
- minimal to slight casts with a granular appearance were present within tubules (high dose males).
- eosinophilic globules within the cytoplasm of tubular epithelium and/or within the tubular lumen (a dose-dependent increase in number of affected animals and severity).

For visualization and grading, a CAB stain was performed in the kidneys of all males.

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: neoplastic:

no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

- Mean estrous cycle duration was 4.6, 4.4, 4.4 and 4.2 days in control, low, mid and high dose groups, respectively.
Estrous cycle data, generated during the last 3 weeks prior to mating to produce the F1 litter, revealed regular cycles in the females of all test groups including the control and the mean estrous cycle duration was similar.

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

Concerning motility of the sperms and the incidence of abnormal sperms in the cauda epididymidis as well as sperm head counts in the testis and in the cauda epididymidis, no treatment-related effects were observed.

Reproductive performance:

no effects observed

Description (incidence and severity):

Males:
- Male mating index was 100% in all test groups. For all F0 parental males, which were placed with females to generate F1 pups, copulation was confirmed.
- Male fertility index was 92%, 96%, 92%, 88% in control, low mid and high dose groups, respectively, reflecting the normal range of biological variation inherent in the strain of rats used for this study
- Mean pre-coital interval was 2.8, 3.0, 2.8 and 2.7 days in control, low mid and high dose groups, respectively

Females:
- Female mating index was 100% in all test groups.
- All female rats delivered pups/had implants in utero, except:
High dose group: 3 females did not become pregnant.
Mid dose group: 2 females did not become pregnant.
Low dose group: 1 female did not become pregnant.
Ctrl group: 2 females did not become pregnant.
- Female fertility index was 92%, 96%, 92%, 88% in control, low mid and high dose groups without any relation to the dose.
- Mean duration of gestation was similar in all test groups (i.e. between 22.0 and 22.2 days).
- Gestation index was 100% in all test groups.
- Implantation was not affected by the treatment. Mean number of implantation sites was comparable between all test substance-treated groups and the control, taking normal biological variation into account (12.8 / 12.1 / 12.6 and 11.4 implants/dam in control, low mid and high dose groups, respectively)
- Postimplantation loss did not show any statistically significant differences between the groups (2.2 / 2.8 / 3.6 and 0.7 mean% in control, low mid and high dose groups, respectively). Thus, no indications for test substance-induced intrauterine embryo-/fetolethality.
- Mean number of F1 pups delivered per dam remained unaffected (12.5 / 11.8 / 12.1 and 11.3. pups/dam in control, low mid and high dose groups, respectively).
- Live birth indices were 99.3% / 98.2% / 99.6% and 98.8% in control, low mid and high dose groups, respectively
- Number of stillborn pups (2, 5, 1, 3 in control, low mid and high dose groups, respectively) was not significantly different between the test groups.

Effect levels (P0)

1

Dose descriptor:

NOAEL

Remarks:

general systemic toxicity F0

Effect level:

4 000 ppm

Based on:

test mat.

Remarks:

Males: 278 mg/kg bw/d Females: 345 mg/kg bw/d

Sex:

male/female

Basis for effect level:

body weight and weight gain

clinical biochemistry

urinalysis

histopathology: non-neoplastic

2

Dose descriptor:

NOAEL

Remarks:

fertility / reproductive performance

Effect level:

12 500 ppm

Based on:

test mat.

Remarks:

Males: 887 mg/kg bw/d Females: 1024 mg/kg bw/d in females during the pre-pairing period, 887 mg/kg bw/ during gestation and 1185 mg/kg bw/d during lactation

Sex:

male/female

Remarks on result:

other: highest dose tested

Target system / organ toxicity (P0)

Critical effects observed:

not specified

Results: F1 generation

General toxicity (F1)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Pups:
No test substance-related adverse clinical signs observed in any of the different test groups.
Incidental/ non-treatment related findings:
- Absent tail in 1 low-dose male pup end from PND 10 onwards (-> spontaneous finding in rats and no dose-dependency).

Cohort 1A:
No clinical signs or changes of general behavior, which may be attributed to the test substance, were detected in any of the male and female animals in any of the groups.
Incidental/ non-treatment related findings:
- Injury (neck region, bilateral) in 1 high dose male during study days 28 – 48.

Detailed clinical observations:
No clinical signs or changes of general behavior, which may be attributed to the test substance, were detected in any of the male and female animals in any of the groups.

Cohort 1B:
No clinical signs or changes of general behavior, which may be attributed to the test substance, were detected in any of the male and female F1B parental animals in any of the groups.

Incidental/ non-treatment related findings:
- non palpable testes (both) of 1 high dose male from study day 0 to 22 + piloerection from study day 0 to 32 and a high-stepping gait from study day 7 to 26. This animal also had a lower body weight / body weight gain compared to the other males of this group.
- piloerection of 1 high dose female from study day 10 onwards and a high-stepping gait from study day 24 onwards.
Due to their isolated occurrence these findings were considered not to be associated with the test compound.

Detailed clinical observations:
No clinical signs or changes of general behavior, which may be attributed to the test substance, were detected in any of the male and female animals in any of the groups.

Incidental/ non-treatment related findings:
- non palpable testes (both) of 1 high dose male on study days 0, 7, 14 and 21 + piloerection on study days 0, 7, 14, 21 and 28 as well as a high-stepping gait on study days 7, 14 and 21, when DCO was performed.
- piloerection of 1 high dose female from study day 14 onwards and a high-stepping gait from study day 28 onwards on the specific days when DCO performed.
These findings were considered not to be associated with the test compound.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

no mortality observed

Description (incidence and severity):

Pups:
Viability index (pup survival during early lactation PND 0 - 4): 98.2 % / 97.1 % / 98.6 % and 98.8 % in control, low mid and high dose groups, respectively.

Lactation index (pup survival on PND 4 - 21): 100 % / 100 % / 99.6 % and 100% in control, low mid and high dose groups, respectively.

Cohort 1A:
There were no test substance-related or spontaneous mortalities in any of the groups.

Cohort 1B:
There were no test substance-related or spontaneous mortalities in any of the groups.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Pups:
Body weights:
High dose (male + female pups + both sexes combined): statistically significantly decrease on PND 21 (- 9% for both sexes combined).

No test compound-related influence on pup body weights/pup body weight change were noted in all pups of low and mid dose test groups.

Body weight change:
High dose (male + female pups + both sexes combined): significantly below the concurrent control values during PND 4 - 7, PND 14 – 21 and PND 1 - 21 (up to 17 %).


Cohort 1A:
Body weights (test substance related/adverse):
- significantly decreased in high dose males during the entire study with a maximum of -9.2 % vs ctrl. on study day 0.
- significantly lower in high dose females on study day 0 (-8.1% vs ctrl) and 7 (-6.2 % vs ctrl.).

Mean body weights in low and mid dose animals were comparable to the concurrent control values throughout the entire study.

Body weight changes (test substance related/adverse):
- significantly decreased in high dose males from study day 35 - 42 (-18% vs ctrl.) and from study day 0-56 (-9% vs ctrl.).

Other findings:
- significantly decreased in mid dose males from study day 28 to 35 (-14% vs ctrl.).
- significantly decreased in low dose males from study day 42 to 49 (-21% vs ctrl.).
Body weight change values of all female test groups were comparable to the concurrent control values throughout the entire study.

Cohort 1B:
Body weights (test substance related/adverse):
- statistically significantly below the concurrent control values in high-dose males from study day 7 onwards, with a maximum of -9 % vs ctrl. on study day 14.
- significantly decreased in high-dose females on study days 7, 35 and 42 up to -6 % vs ctrl on study day 7.

Mean body weights of the low-and mid-dose F1B males and females were comparable to the concurrent control values throughout the entire study.

Body weight changes (test substance related/adverse):
- significantly decreased in male animals of the high dose group during study days 0-7, 7-14 and 0-49 (up to -11 % vs ctrl) as well as in female animals during study day 21-28 (-16 % vs ctrl).

The body weight change values in low and mid dose males and females were comparable to the concurrent control values throughout the entire study.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Cohort 1A:
Treatment-related and adverse effects observed:
High dose males: significantly decreased on study days 13-14, 27-28 and 55-56 (approximately -10% vs ctrl.)
High dose females: significantly decreased on study day 41-42 (-19% vs ctrl).

Slight temporary differences (assessed as spontaneous and not related to treatment):
- Mid dose males: -8% vs ctrl on study day 27-28
- Low dose males: -10% vs ctrl on study day 55-56
- Low dose females: -14% vs ctrl study day 41-42
Overall food consumption was comparable to controls at low + mid dose animals and in high dose females. Therefore, these statistically significant differences are considered to be incidental and not treatment-related.

Cohort 1B:
Food consumption of all test substance treated animals were comparable to the concurrent control values throughout the entire study.

Slight temporary differences (assessed as spontaneous and not related to treatment):
- High-dose females: -14 % vs ctrl. on study day 34-35

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Cohort F1A

No treatment-related, adverse changes among hematological parameters were observed.

Treatment-related and non-adverse findings:
High dose males:
- Absolute + relative monocyte counts were significantly decreased at the end of the administration period
High dose females:
- Absolute + relative neutrophil cell counts were significantly decreased
- Relative lymphocyte counts were compensatory, significantly increased
Mid dose males:
- Absolute + relative monocyte counts were significantly decreased at the end of the administration period
Mid dose females:
- Relative neutrophil cell counts were significantly decreased
- Relative lymphocyte counts were compensatory, significantly increased

Changes were regarded as treatment-related but non-adverse, because the absolute cell counts were altered isolated without any other changed differential cell count portion (ECETOC Technical Report No. 85, 2002).

Incidental/ non-treatment related findings:
High dose females:
- significant increase in relative monocyte counts (within the historical control range)
Mid dose males:
- Total red blood cell (RBC) counts + hemoglobin (HGB) + hematocrit (HTC) values were significantly increased (within the historical control range).
Mid dose females:
- significant increase in relative monocyte counts (within the historical control range)

Low dose females:
- absolute neutrophil counts were significantly decreased (within the historical control range)

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Cohort 1A:

Treatment-related and adverse effects observed:
High dose males:
- Urea levels were significantly increased (+18% related to ctrl.)
- Creatinine levels were significantly increased (+9% related to ctrl.)
High dose females:
- Urea levels were significantly increased (+15% related to ctrl.)
- Glucose levels were significantly increased (+13% related to ctrl.)

Treatment-related and non-adverse findings:
High dose males:
- Total bilirubin values were significantly decreased.
High dose females:
- Total bilirubin values were significantly decreased.
Mid dose females:
- Total bilirubin values were significantly decreased.
This change without any change in the red blood cell parameters were most probably due to an increased conjugation of bilirubin followed by an accelerated excretion via the bile which is caused by a liver enzyme induction. This mechanism is regarded as treatment related but adaptive rather than adverse.

Incidental/ non-treatment related findings:
High dose males:
- Chloride level decreased ((within historical control range)
Mid dose males:
- Chloride level decreased ((within historical control range)
Low dose females:
- Urea levels decreased (not dose dependent)
- Creatinine levels decreased (not dose dependent)

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

Cohort 1A:

Treatment-related and adverse effects observed:
High dose males:
- pH value of the urine was significantly decreased (5.2 vs. 6.4 in ctrls.)
- the specific gravity was increased
- amount of blood (erythrocytes), transitional epithelial cells and of granular and epithelial casts were significantly increased
- urine volume was decreased (not statistically significantly)
In combination with histopathologic findings these alterations were most probably due to an α2u-Globulinuria which is regarded as species specific, treatment-related, adverse finding in rats which is not relevant for humans (Hard et al., 2018).
The higher amount of blood (erythrocytes) in the urine sediment is the only change which cannot attributed to the α2u-Globulinuria and therefore it may be regarded as treatment-related and adverse.

No other treatment-related changes among urinalysis parameters were observed.

Sexual maturation:

effects observed, treatment-related

Description (incidence and severity):

F1 generation pups/litters

Sex ratio
No substantial differences between the control and the test substance-treated groups on the day of birth and on PND 21; slight differences were regarded to be spontaneous in nature.

Vaginal opening:
- First day of vaginal opening was PND 29
- Last day of vaginal opening was PND 42.
- Mean number of days to reach the criterion was 35.0; 36.2; 35.7 and 36.8** (** p<=0.01) days in the control, low mid and high dose groups, respectively.
- The mean body weight on the day of vaginal opening was 112.4 g, 118.4 g, 114.0 g and 113.1 g in the control, low mid and high dose groups, respectively.
The apparent delay in high dose animals is small, the timing well within the historical control range (HCD: 29.5 - 38.8 days) and related to lower postnatal body weight development of the affected offspring. Thus, the observed statistical difference is considered not to be toxicologically relevant.

Preputial seperation:
- First day of peputial separation was PND 41
- Last day of peputial separation was PND 51
- Mean number of days to reach the criterion was 46.0, 45.6, 45.7 and 47.3* (* p<=0.05) days in the control, low mid and high dose groups, respectively.
- The mean body weight on the day of preputial separation was 202.1 g, 200.4 g, 198.9 g and 196.5 g in the control, low mid and high dose groups, respectively.
The mean number of days in the high dose group was outside the historical range of the rat strain used (40.1 to 45.2 days). However, the animals of this group reached sexual maturity even at a slightly lower body weight when compared to controls. Therefore, the difference in
timing of preputial separation is considered not to be a delay in commencement of sexual maturity, but due to lower postnatal body weight development.

Anogenital distance (AGD):

effects observed, non-treatment-related

Description (incidence and severity):

The anogenital distance and anogenital index of all test substance treated male and female pups was comparable to the concurrent control values.

The anogenital index in female low dose animals was slightly, but significantly lower (-4 %) when compared to controls. Because no dose-response and other endocrine-related findings occurred, this finding was considered to be spontaneous in nature.

Nipple retention in male pups:

no effects observed

Description (incidence and severity):

The percentage of male pups having nipples/areolae was not influenced by the test substance when examined on PND 13 and on PND 20.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

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

Absolute organ weights
- Spleen (males): 106% / 92% / 82%* versus ctrl in low, mid and high dose groups, respectively
* p <= 0.05
The reduced mean absolute weight of the spleen was considered potentially treatment-related as a dose-response relationship could be observed.
All other mean absolute weight parameters and all mean relative weight parameters did not show significant differences when compared to the control group.

Cohort 1A:
Terminal body weights:
Males: 98% / 97% / 90%** versus ctrl in low, mid and high dose groups, respectively

Absolute organ weights
- Adrenal glands (females): 97% / 96% / 87%** versus ctrl in low, mid and high dose groups, respectively
- Brain (males): 99% / 100% / 93%** versus ctrl in low, mid and high dose groups, respectively
- Heart (males): 101% / 100% / 92%* versus ctrl in low, mid and high dose groups, respectively
- Heart (females): 98% / 98% / 93%** versus ctrl in low, mid and high dose groups, respectively
- Prostate (males): 106% / 98% / 86%* versus ctrl in low, mid and high dose groups, respectively
*: p <= 0.05, **: p <= 0.01
High dose males showed decreased mean absolute weights of brain (1.928 g), heart (0.954 g), and prostate (0.697 g) which were minimally below the historical control range (brain: 2.056 – 2.084 g; heart: 1.027 – 1.059 g; prostate: 0.775 – 0.811 g).
In high dose females, the mean absolute weights of the adrenal glands (71.050 mg) and the heart (0.673 g) were minimally lower than the control range (adrenal glands: 78.250 – 92.950 mg; heart: 0.687 – 0.709 g). These changes were interpreted as possibly treatment-related, but not adverse as they lacked correlating histopathological findings.

Relative organ weights
- Adrenal glands (males): 101% / 102% / 111%** versus ctrl in low, mid and high dose groups, respectively
- Adrenal glands (females): 98% / 95% / 88%** versus ctrl in low, mid and high dose groups, respectively
- Brain (males): 101% / 102% / 102%* versus ctrl in low, mid and high dose groups, respectively
- Heart (females): 99% / 97%* / 95%** versus ctrl in low, mid and high dose groups, respectively
- Kidneys (males): 107%** / 110%** / 117%** versus ctrl in low, mid and high dose groups, respectively
- Liver (males): 99% / 100% / 109%** versus ctrl in low, mid and high dose groups, respectively
- Testes (males): 102% / 103% / 109%* versus ctrl in low, mid and high dose groups, respectively
*: p <= 0.05, **: p <= 0.01
In males of all test groups, the mean relative weight of the kidneys was increased (0.667%, 0.683%, and 0.727% in low, mid and high dose groups, respectively) and minimally exceeded the his-
torical control range (0.652 – 0.663%). These changes were considered treatment-related and probably a consequence of the histopathological findings.


A significant decrease in terminal body weight was observed in high dose males, leading to an increased mean relative organ weight of the adrenal glands, the brain, and the testes.

In high dose females, the mean relative weights of the adrenal glands (0.036%) and the heart (0.339%) were minimally lower than the control range (adrenal glands: 0.038 – 0.046%; heart: 0.341 – 0.349%). These changes were interpreted as possibly treatment-related, but not adverse as they lacked correlating histopathological findings.
The mean relative liver weight (2.687%) in high males as well as the mean relative heart weight (0.346%) in mid dose females were within the historical control range (liver males: 2.537 – 2.716%; heart females: 0.341 – 0.349%) and hence considered incidental.

All other mean absolute and relative weight parameters did not show significant differences when compared to the control group.


Cohort 1B:

Terminal body weights:
Males: 101% / 98% / 93%** versus ctrl in low, mid and high dose groups, respectively
Females: 100% / 99% / 95%* versus ctrl in low, mid and high dose groups, respectively

Absolute organ weights
- Adrenal glands (females): 97% / 95% / 86%** versus ctrl in low, mid and high dose groups, respectively
- Prostate (males): 103% / 102% / 86%** versus ctrl in low, mid and high dose groups, respectively

Similarly to cohort 1A, the mean absolute weight of the prostate (0.694 g) in high dose males was minimally below the historical control range (0.754 – 0.758 g).
In high dose females, the mean absolute weight of the adrenal glands (68,84 mg) was also below the historical control range (78.560 – 79.720 mg). These changes were interpreted as possibly treatment-related, but not adverse as no correlating histopathological findings were observed in cohort 1A


Relative organ weights
- Adrenal glands (males): 102% / 104% / 116%** versus ctrl in low, mid and high dose groups, respectively
- Adrenal glands (females): 97% / 96% / 90%** versus ctrl in low, mid and high dose groups, respectively
- Liver (males): 96%* / 98% / 108%** versus ctrl in low, mid and high dose groups, respectively
- Liver (females): 95%* / 98% / 99% versus ctrl in low, mid and high dose groups, respectively
- Testes (males): 101% / 104% / 106%** versus ctrl in low, mid and high dose groups, respectively

*: p <= 0.05, **: p <= 0.01

A significant decrease of terminal body weight was observed in high dose males and females leading to an increased mean relative organ weight of the adrenal glands and the testes.
Similarly to cohort 1A, the mean relative liver weight (2.806%) minimally exceeded the historical range (2.608 - 2.641%).
In high dose females, the mean relative weight of the adrenal glands (0.036%) was also below the historical control range (0.038 - 0.040%). These changes were interpreted as possibly treatment-related, but not adverse as no correlating histopathological findings were observed in cohort 1A.
The mean relative liver weight of low dose males (2.498%) and females (2.458%) was below the historical control range (males: 2.608 - 2.641%; females: 2.587 – 2.608%), but this was considered as not treatment-related because of a missing dose-response relationship.
All other mean absolute and relative weight parameters did not show significant differences when compared to the control group.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Pups:
No treatment-related findings were observed.


A few pups showed spontaneous findings at gross necropsy, such as postmortem autolysis (without any relation to dosing)

Cohort 1A:
All findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.

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

Histopathological findings:

effects observed, treatment-related

Description (incidence and severity):

Cohort 1A:
Treatment-related and adverse findings:

Kidney
- minimal to slight (moderate in 1 animal) casts with a granular appearance were present within tubules (low, mid, high dose males).
- eosinophilic globules within the cytoplasm of tubular epithelium and/or within the tubular lumen (a dose-dependent increase in severity).
- minimal increase in the presence of basophilic tubules. The severity remained minimal with single tubules presenting with basophilic cytoplasm, but otherwise normal morphology.

For visualization and grading, a CAB stain was performed in the kidneys of all males.

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.

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

Estrous cycle of Cohort 1A:
- Mean estrous cycle duration was 4.0 / 3.9 / 3.9 and 4.0 days in control, low, mid and high dose groups, respectively.
Estrous cycle data, generated during 2 weeks, revealed regular cycles in the females of all test groups including the control and the mean estrous cycle duration was comparable.


Estrous cycle of Cohort 1B:
- Mean estrous cycle duration was 3.9 / 3.9 / 4.0 and 4.4** (**:p<=0.01) days in control, low, mid and high dose groups, respectively.
Estrous cycle data, generated during 2 weeks, revealed regular cycles in the females of all test groups. The slightly prolonged average in the high dose group is due to a longer diestrous in 3 females, which is still normal in the tested rat strain. The average cycle length is within the historical control range (HCD = 3.9 - 4.8 days) and a comparable increase was not observed in the corresponding cohort 1A females. Thus, the apparent prolongation is considered as an incidental finding.


Spermanalysis
Cohort 1A:
Concerning motility of the sperms and the incidence of abnormal sperms in the cauda epididymidis as well as sperm head counts in the testis and in the cauda epididymidis, no treatment related effects were observed.

Differential ovarian follicle count
Cohort 1A:
DOFC – comprising the numbers of primordial and growing follicles, as well as the combined incidence of primordial plus growing follicles – did not reveal significant differences between the control and the high dose group.


Thyroid hormones
PND 4 surplus pups:
No treatment-related alterations of T4 and TSH levels were observed.

PND 22 pups:
T4 males: 42.79 / 41.12 / 49.04 / 59.37** nmol/L in ctrl, low, mid and high dose groups, respectively
TSH males: 2.46 / 2.57 / 2.38 / 2.67 µg/L in ctrl, low, mid and high dose groups, respectively
T4 females: 46.19 / 48.41 / 53.55 / 58.23** nmol/L in ctrl, low, mid and high dose groups, respectively
TSH females: 2.39 / 2.22 / 2.58 / 2.73 µg/L in ctrl, low, mid and high dose groups, respectively
** p <=0.01

In high dose male and females T4 levels were significantly increased but TSH values were not altered. T4 values were within historical control ranges (T4 males 50.57-67.08 nmol/L, T4 females, 44.85-73.70 nmol/L),whereas the T4 levels in the control group were at the low border (females) or below these ranges (males). Since T4 levels in PND4 pups were not altered, this isolated T4 increase was regarded as incidental and not treatment-related.

Cohort F1A (d90):
No treatment-related alterations of T4 and TSH levels were observed.


Lymphocyte subpopulations in spleen
Cohort F1A:
No alterations in the absolute and relative lymphocyte subpopulation cell counts in the spleen tissue (B-, T-lymphocytes, CD4-, CD8-T-lymphocytes and natural killer (NK) cells) were ob-
served at PND90 in both sexes.

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Effect levels (F1)

1

Dose descriptor:

NOAEL

Remarks:

general systemic toxicity F1 adults

Generation:

F1

Effect level:

4 000 ppm

Based on:

test mat.

Remarks:

Males: 338 to 347 mg/kg bw/d Females: 361 to 362 mg/kg bw/d

Sex:

male/female

Basis for effect level:

body weight and weight gain

food consumption and compound intake

clinical biochemistry

urinalysis

histopathology: non-neoplastic

2

Dose descriptor:

NOAEL

Remarks:

developmental toxicity F1

Generation:

F1

Effect level:

4 000 ppm

Based on:

test mat.

Remarks:

431 mg/kg bw/d during lactation

Sex:

male/female

Basis for effect level:

body weight and weight gain

Target system / organ toxicity (F1)

Critical effects observed:

no

Overall reproductive toxicity

Reproductive effects observed:

no

Applicant's summary and conclusion

Conclusions:

Under the conditions of the present extended one-generation reproduction toxicity study the NOAEL for general, systemic toxicity is at 4000 ppm (about 278 mg/kg bw/d in males and 345 mg/kg bw/d in females) in the F0 parental rat, based on slightly reduced body weights, mainly in males.

The NOAEL in the F1 adult rats is 4000 ppm (about 338 to 347 mg/kg bw/d in males and 361 to 362 mg/kg bw/d in females).

The NOAEL for fertility and reproductive performance for the F0 and F1 parental rats is 12500 ppm (about 887 mg/kg bw/d and 1024 mg/kg bw/d in females during the pre-pairing period, 887 mg/kg bw/ during gestation and 1185 mg/kg bw/d during lactation).

The NOAEL for developmental toxicity in the F1 progeny is 4000 ppm (about 431 mg/kg bw/d during lactation), based on slightly reduced preweaning body weight gain, which was observed at the LOAEL (Lowest Observed Adverse Effect Level) of 12500 ppm (about 1185 mg/kg bw/d during lactation). However, these effects are considered to be mainly due to compound uptake of the offspring via food and not a direct developmental toxic effect.