Registration Dossier

Toxicological information

Toxicity to reproduction

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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:
2021
Report date:
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)
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: 10 weeks; according to ECHA Compliance Check Decision CCH-D-2114448631-50-01/F
- Basis for dose level selection:
In a previous OECD No. 414 study (BASF, project No. 30R0333/03R011, 2016), Citronellylnitril was administered as an oily solution to groups of 25 time-mated female rats by gavage at doses of 50, 150 and 450 mg/kg body weight/day (mg/kg bw/d) on gestation days (GD) 6 through 19. Maternal toxicity was observed at the highest dose level of 450 mg/kg bw/d, namely increased absolute and relative liver weights (19 and 21% above control) and corresponding clinical-pathological changes (increased triglyceride and cholesterol values). Therefore, the no observed adverse effect level (NOAEL) for maternal toxicity was set to 150 mg/kg bw/d.
In a previous OECD No. 408 study (SPL, project No. 2153-0004, 2008), Citronellylnitril was administered by oral gavage at dose levels of 300,100, 30, 10 and 0 mg/kg bw/d for a period of ninety consecutive days. No adverse effects were observed in clinical examinations. In (histo)pathology, centrilobular, hepatocyte enlargement and increased absolute and relative liver weights at 300 (and partly at 100) mg/kg bw/d were observed. These changes were discussed as adaptive and the NOAEL was set to 300 mg/kg bw/d.
In an OECD No. 415 one-generation reproduction study in rats with an evaluation through sexual maturity in the F1 Generation (CLR, study No. TIF00044, 2011), Citronellylnitril was administered by oral gavage to 25 rats per sex and group at dose levels of 500, 200, 75 and 0 mg/kg bw/d. The purpose of this study was to provide information concerning the effects of Citronellylnitrile on gonadal function, estrous cycles, mating behavior, conception, parturition, lactation and the growth and development of offspring up to day 60 postpartum.
In this study, the no-observable-adverse-effect-level (NOAEL) for systemic toxicity was 200 mg/kg/day based on the below mentioned effects. At 500 mg/kg/day, reductions in body weight gains occurred in F0 male rats prior to cohabitation, followed by persistent reductions in weight gain through the end of the study (10% below control). In pathology, increased absolute and relative brain (males: 4 and 11% above control), spleen (males: 17 and 25% above control, respectively), liver (males: 35 and 44%; females: 15 and 9% above control) and kidney weights (males: 5 and 14% above control) occurred in F0 animals at 500 mg/kg/d. Microscopic findings that could be correlated with the changes in organ weights were not observed. The NOAELs for reproductive performance in the F0 and for viability and growth of the F1 generation offspring was greater than 500 mg/kg/d.
Based on the above-mentioned study results, the following dose levels were selected: 25 mg/kg bw/d as low dose, 100 mg/kg bw/d as intermediate dose, 400 mg/kg bw/d as high dose.
- Exclusion of extension of Cohort 1B: according to ECHA Compliance Check Decision CCH-D-2114448631-50-01/F
- Exclusion of developmental neurotoxicity Cohorts 2A and 2B: according to ECHA Compliance Check Decision CCH-D-2114448631-50-01/F
- Exclusion of developmental immunotoxicity Cohort 3: according to ECHA Compliance Check Decision CCH-D-2114448631-50-01/F
- Route of administration: oral (gavage): according to ECHA Compliance Check Decision CCH-D-2114448631-50-01/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:
3,7-dimethyloct-6-enenitrile
EC Number:
257-288-8
EC Name:
3,7-dimethyloct-6-enenitrile
Cas Number:
51566-62-2
Molecular formula:
C10H17N
IUPAC Name:
3,7-dimethyloct-6-enenitrile
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and batch number of test material: BASF SE, 19000329U0
- Purity: 95.6 area-%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature
- Stability of the test material during storage: Expiry date: 01 May 2021
The stability of the test substance under storage conditions over the test period was guaranteed by the sponsor, and the sponsor holds this responsibility.
- Homogeneity of the test material: given

OTHER
- Chemical name: 3,7-dimethyloct-6-enenitrile
- Physical state/appearance: Liquid/colorless, clear

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 GmbH, Sulzfeld, Germany
- Females nulliparous and non-pregnant: yes
- Age at start of the administration period: 35 ± 1 days
- 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: 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:
• During overnight matings (male and female mating partners were housed together), gestation, lactation and females after weaning the animals were housed individually in Polycarbonate cages type III (supplied by TECHNIPLAST, Hohenpeißenberg, Germany and Becker & Co., Castrop-Rauxel, Germany).
• Dams and their litters were housed together until PND 22 in Polycarbonate cages type III.
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: about 7 days

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

IN-LIFE DATES: From: 05 Nov 2019 (TS administration) To: 08 May 2020 (sacrifice of F1 cohort 1B animals)

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
- The test substance preparations were prepared in intervals, which took into account the analytical results of the stability verification.
- For the preparation of the administration suspensions the test substance was weighed in a graduated flask depending on the dose group, topped up with corn oil and intensely mixed with a magnetic stirrer until it was completely homogenized.
- Before and during administration, the preparations were kept homogeneous with a magnetic stirrer.

VEHICLE
- Concentration in vehicle: 0.63, 2.5, 10.0 g/100 mL in low, mid and high dose, respectively
- Amount of vehicle (if gavage): 4 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
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 corn at room temperature over a period of 7 days had been verified prior to the start of the study.
The applicable form of the test-substance preparation was completely miscible with corn oil and thus a solution. Therefore, the test-substance preparation was considered to be homogenous.
Samples of the test substance preparations were sent to the analytical laboratory during the study period (at the beginning, towards the middle and towards the end) for verification of the concentrations.
Duration of treatment / exposure:
F0 males: 10 weeks (premating) + 2 weeks (mating) + max. 6 weeks (post-mating)
F0 females: 10 weeks (premating) + 2 weeks (mating) + approx. 6 weeks (pregnancy + lactation)
F1 animals (Cohort 1A and 1B): post weaning until an approx. age of 13 weeks
Frequency of treatment:
once daily
Details on study schedule:
F0 generation animals and their progeny:
The male and female animals were about 4 weeks old when they arrived from the breeder. During an acclimatization period of about 7 days, animals with lowest and highest body weights were eliminated from the study and used for other purposes. The 100 male and 100 female animals required for the study were about 5 weeks old at the beginning of treatment and their weight variation did not exceed 20 percent of the mean weight of each sex.
The assignment of the animals to the different test groups was carried out using a randomization program, according to their weight one day before the beginning of the administration period (day -1).
After the acclimatization period, the test substance was administered to the animals orally by gavage, once daily at approximately the same time in the morning. Females in labor were not treated. The treatment lasted up to one day prior to sacrifice. The animals of the control group were treated with the vehicle (corn oil), in the same way. The volume administered each day was 4 mL/kg body weight. The calculation of the administration volume was based on the most recent individual body weight.
After a minimum of 10 weeks after the beginning of treatment, males and females from the same dose group were mated, overnight at a ratio of 1 : 1 (for details see: Pairing of F0 generation parental animals).
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 2 different cohorts which were subjected to specific postweaning examinations.
On PND 4 blood samples were collected from 10 surplus (culled) F1 pups per sex and group. On PND 22 blood samples were collected from 10 surplus F1 pups per sex and group.
Blood samples were taken from 10 animals per test group of the F0 parental animals and cohort 1A animals.
Before 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 (selection see below), to be placed into cohorts according to the scheme presented under 3.7.3. 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.

Standardization of litters (F1 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.

Pups after standardization/weaning:
With the exception of those F1 generation pups, which were chosen as F1 rearing animals, and those F1 pups, which were chosen for blood sampling on PND 4 and 22, all pups were sacrificed under isoflurane anesthesia with CO2 after standardization or weaning. The pups chosen for blood sampling were sacrificed by decapitation under isoflurane anesthesia.
All culled pups, including stillborn pups and those that died during their rearing period, were subjected to a macroscopic (external and visceral) examination.
All pups without any notable findings or abnormalities were discarded after their macroscopic evaluation. Animals with notable findings or abnormalities were further evaluated on a case-by-case basis (e.g., histopathological evaluation or special staining), depending on the findings noted.
Doses / concentrationsopen allclose all
Dose / conc.:
25 mg/kg bw/day (nominal)
Remarks:
test group 01 (parental animals), test group 11 (cohort 1A and 1B animals)
Dose / conc.:
100 mg/kg bw/day (nominal)
Remarks:
test group 02 (parental animals), test group 12 (cohort 1A and 1B animals)
Dose / conc.:
400 mg/kg bw/day (nominal)
Remarks:
test group 03 (parental animals), test group 13 (cohort 1A and 1B animals)
No. of animals per sex per dose:
F0: 25 animals per sex per dose
F1A: 20 animals per sex per dose
F1B: 25 animals per sex per dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
In a previous OECD No. 414 study (BASF, project No. 30R0333/03R011, 2016), Citronellylnitril was administered as an oily solution to groups of 25 time-mated female rats by gavage at doses of 50, 150 and 450 mg/kg body weight/day (mg/kg bw/d) on gestation days (GD) 6 through 19. Maternal toxicity was observed at the highest dose level of 450 mg/kg bw/d, namely increased absolute and relative liver weights (19 and 21% above control) and corresponding clinical-pathological changes (increased triglyceride and cholesterol values). Therefore, the no observed adverse effect level (NOAEL) for maternal toxicity was set to 150 mg/kg bw/d.
In a previous OECD No. 408 study (SPL, project No. 2153-0004, 2008), Citronellylnitril was administered by oral gavage at dose levels of 300,100, 30, 10 and 0 mg/kg bw/d for a period of ninety consecutive days. No adverse effects were observed in clinical examinations. In (histo)pathology, centrilobular, hepatocyte enlargement and increased absolute and relative liver weights at 300 (and partly at 100) mg/kg bw/d were observed. These changes were discussed as adaptive and the NOAEL was set to 300 mg/kg bw/d.
In an OECD No. 415 one-generation reproduction study in rats with an evaluation through sexual maturity in the F1 Generation (CLR, study No. TIF00044, 2011), Citronellylnitril was administered by oral gavage to 25 rats per sex and group at dose levels of 500, 200, 75 and 0 mg/kg bw/d. The purpose of this study was to provide information concerning the effects of Citronellylnitrile on gonadal function, estrous cycles, mating behavior, conception, parturition, lactation and the growth and development of offspring up to day 60 postpartum.
In this study, the no-observable-adverse-effect-level (NOAEL) for systemic toxicity was 200 mg/kg/day based on the below mentioned effects. At 500 mg/kg/day, reductions in body weight gains occurred in F0 male rats prior to cohabitation, followed by persistent reductions in weight gain through the end of the study (10% below control). In pathology, increased absolute and relative brain (males: 4 and 11% above control), spleen (males: 17 and 25% above control, respectively), liver (males: 35 and 44%; females: 15 and 9% above control) and kidney weights (males: 5 and 14% above control) occurred in F0 animals at 500 mg/kg/d. Microscopic findings that could be correlated with the changes in organ weights were not observed. The NOAELs for reproductive performance in the F0 and for viability and growth of the F1 generation offspring was greater than 500 mg/kg/d.
Based on the above-mentioned study results, the following dose levels were selected: 25 mg/kg bw/d as low dose, 100 mg/kg bw/d as intermediate dose, 400 mg/kg bw/d as high dose.
- 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.
• 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 parental animals and F1 rearing animals, with the following exceptions:
• not determined after the 10th premating week (male F0 animals) and during the mating period (male and female F0 animals).
• during pregnancy, food consumption of the F0 females with evidence of sperm was determined weekly for GD 0-7, 7-14 and 14-20.
• during lactation, food consumption of the F0 females, which gave birth to a litter was determined for PND 1-4, 4-7, 7-10, 10-14, 14-18 and 18-21.
Food consumption was not determined in the females without positive evidence of sperm during mating and gestation periods, in the females without litter during lactation period and in the females after weaning.

WATER CONSUMPTION: No

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):
Estrous cycle length was evaluated by daily analysis of vaginal smear for all F0 female 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 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.

- 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 (before standardization), 7, 14 and 21.

- 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.

- Vaginal opening
All female F1 pups selected to become the F1 rearing animals (cohort 1A and 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 the F1 rearing animals (cohort 1A and 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.

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)
The concentrations of TSH were determined by radioimmunoassay (RIA), using commercially available RIA test kits. T4 was examined via ELISA.

OESTROUS CYCLICITY (F1 ANIMALS)
In all cohort 1A females, vaginal smears were collected after vaginal opening until the first cornified smear (estrous) was recorded. The estrous cycle was also evaluated in cohort 1A and 1B females for 2 weeks around PND 75.
An internal peer review of the estrous cycle data prepared around PND 75 including all vaginal smears taken from cohorts 1A and 1B female animals of test group 0 (0 mg/kg bw/d) was performed because the estrous cycle data of the control group (e.g. cycle length) was outside the range of the historical control data of the test facility.
In general, the original and peer reviewed estrous cycle data was comparable.
At necropsy, an additional vaginal smear was examined to determine the stage of estrous cycle for each cohort 1A and 1B female with scheduled sacrifice.

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. Pituitary gland
26. Prostate
27. Rectum
28. Sciatic nerve
29. Seminal vesicles
30. Skeletal muscle
31. Spinal cord (cervical, thoracic, lumbar)
32. Spleen
33. Stomach (forestomach and glandular stomach)
34. Testis, left
35. Thymus
36. Thyroid glands
37. Trachea
38. Urinary bladder
39. Uterus
40. Vagina
41. Vas deferens

Animals that died or were sacrificed in a moribund state were processed histotechnically and assessed like control animals.

The ovaries and eyes with optic nerve of animals that died or were sacrificed intercurrently were fixed in 4% neutral buffered formaldehyde solution.

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

For technical reasons, the left testis, left epididymis, and the eyes with optic nerves of all animals of the F0 parental control and high dose groups sacrificed at scheduled dates were embedded in paraplast after fixation. For the same reason, the ovaries of all F0 females in all test groups were embedded in paraplast.

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

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).


Subcontraction of histotechnical processing
With the exception of intermediate test groups, the histotechnical processing (cutting and HE staining) of all organs of all F0 parental animals and the histotechnical processing (paraplast embedding, cutting, HE staining) of all organs of all cohort 1A animals (according to study plan, all amendments and raw data) was accomplished by the test site TPL Path Labs GmbH (TPL) Sasbacher Straße 10, 79111 Freiburg; Germany. The ovaries of both the F0 generation animals, were processed at the test facility and were not sent to TPL.
The study phase histotechnical processing was performed under the test site code 1701/20.
Raw data of the study phase, as well as wet tissues, paraplast blocks and HE-stained slides were sent to the test facility for archiving for at least the period of time specified in the GLP principles.

Intermediate test groups:
The thyroid and liver of test group 01 and 02 male animals (F0) were processed histotechnically at BASF SE.

Histopathological evaluation of the HE-stained slides was performed by the study pathologist.

Peer Review
After completion of the histopathological assessment by the study pathologist an internal peer review including liver and thyroid glands of all examined male animals of the F0 parental generation and the spleen of all male animals of the F1, rearing animals, cohort 1A was performed (BASF SE, Ludwigshafen). Results presented in the pathology report reflect the consensus opinion of the study pathologist and the peer review pathologist.
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 test groups 10 and 13 (cohort 1A females) 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)
All surplus F1 generation pups that were not used for the following organ weight determinations were sacrificed under isoflurane anesthesia with CO2. The selected pups for organ weight determination were sacrificed by decapitation under isoflurane anesthesia. All animals were necropsied and assessed by gross pathology with special emphasis on the reproductive organs.

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

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


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.

- 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. 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 in modified Davidson´s solution)
35. Thymus
36. Thyroid glands (with parathyroid glands)
37. Trachea
38. Urinary bladder
39. Uterus
40. Vagina
41. Vas (ductus) deferens

Animals that died or were sacrificed in a moribund state were processed histotechnically and assessed like control animals.

The ovaries and eyes with optic nerve of animals that died or were sacrificed intercurrently were fixed in 4% neutral buffered formaldehyde solution.

For technical reasons, the ovaries of all cohort 1A females in all test groups were embedded in paraplast.

The left testis and left epididymis of all Cohort 1A animals sacrificed at scheduled dates were fixed in modified Davidson’s solution, whereas the right testis and epididymis were used for sperm parameters analysis.

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


Subcontraction of histotechnical processing
With the exception of intermediate test groups as listed below, the histotechnical processing (cutting and HE staining) of all organs of all F0 parental animals and the histotechnical processing (paraplast embedding, cutting, HE staining) of all organs of all cohort 1A animals (according to study plan, all amendments and BASF raw data) was accomplished by the test site TPL Path Labs GmbH (TPL) Sasbacher Straße 10, 79111 Freiburg; Germany. The ovaries of both the F0 generation animals and the Cohort 1A animals, were processed at the test facility and were not sent to TPL.
The study phase histotechnical processing was performed under the test site code 1701/20.
Raw data of the study phase, as well as wet tissues, paraplast blocks and HE-stained slides were sent to the test facility for archiving for at least the period of time specified in the GLP principles.

Intermediate test groups:
The spleen of test group 11 and 12 male animals (F1, cohort 1A) were processed histotechnically at BASF SE.

Histopathological evaluation of the HE-stained slides was performed by the study pathologist.


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. Testes
10. Seminal vesicles including coagulating gland (fixed)
11. 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. All gross lesions
2. Adrenal glands
3. Cervix uteri
4. Coagulating glands
5. Epididymis, left
6. Liver
7. Ovaries
8. Pituitary gland
9. Prostate
10. Seminal vesicles including coagulating glands
11. Testis
12. Uterus
13. Vagina

Histotechnical processing and examination by light microscopy was not performed

For technical reasons, the ovaries of all cohort 1B females of all test groups were embedded in paraplast.


Peer Review
After completion of the histopathological assessment by the study pathologist an internal peer review including liver and thyroid glands of all examined male animals of the F0 parental generation and the spleen of all male animals of the F1, rearing animals, cohort 1A was performed (BASF SE, Ludwigshafen). Results presented in the pathology report reflect the consensus opinion of the study pathologist and the peer review pathologist.

Statistics:
- DUNNETT test (two-sided): Food consumption (parental and rearing animals), body weight and body weight change (parental animals, pups and rearing animals; for the pup weights, the litter means were used), estrous cycle duration, number of mating days, duration of gestation, number of implantation sites, postimplantation loss and % postimplantation loss, number of pups delivered per litter, 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 with liveborn pups, females with stillborn pups, females with all stillborn pups, live birth index, pups stillborn, pups died, pups cannibalized, pups sacrificed moribund, viability index, lactation index, number of litters with affected pups at necropsy, sexual maturation data (vaginal opening, preputial separation)
- WILCOXON-test (one-sided): Presence of areolae/nipples, proportions of affected pups per litter with necropsy observations, 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-): DOFC
- KRUSKAL-WALLIS test (two-sided) + WILCOXON-test (two-sided): 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
* 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:
effects observed, non-treatment-related
Description (incidence and severity):
No treatment-related, adverse findings were observed.

Clinical observations for males and females (except gestation and lactation period)
Transient salivation during a short time period after gavage dosing (up to 2 hours) was noted for all male and female animals of test group 03 (400 mg/kg bw/d) and six males of test group 02 (100 mg/kg bw/d) during the entire study period. It is likely, that this temporary finding was induced by a bad taste of the test substance or local affection of the upper digestive tract. It is, however, not considered to be an adverse finding indicating systemic toxicity.
Male animal No. 33 of test group 01 (25 mg/kg bw/d) showed an unsteady gait from study day 35 onwards, starting between 2 -5 h after application on study day 35. One female animal No. 175 of test group 02 (100 mg/kg bw/d) showed respiration sounds from study day 74 onwards as well as during the mating period. These individual observations were not related to dose
and, therefore, not assessed as treatment-related.
Note: On pre-mating day 38, the clinical examinations between 2-5 hours after treatment were not performed for all animals of test groups 00-03. Thus, no documentation exists in the electronic system and are not shown in tables. These missing records do not have an influence on the validity of the present study.

Clinical observations for females during gestation of F1 litters
Transient salivation during a short time period after gavage dosing (up to 2 hours) was noted in female animals of test group 03 (400 mg/kg bw/d) during the entire gestation period. It is likely, that this temporary finding was induced by a bad taste of the test substance or local affection of the upper digestive tract. It is, however, not considered to be an adverse finding indicating systemic toxicity.

Clinical observations for females and offspring during lactation of F1 litters
Transient salivation (please see above) in female animals of test group 03 (400 mg/kg bw/d) was still present during lactation.
Female animal No. 141 of test group 01 (25 mg/kg bw/d) showed a swelling on the right side at the mammary line during PND 14 and 24. Female animal No. 125 of test group 00 (control group, 0 mg/kg bw/d) as well as female animal No. 158 of test group 02 (100 mg/kg bw/d) had no more pups alive on PND 0 (complete litter loss). These observations were not considered to be associated with the test compound since there were not related to dose.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, non-treatment-related
Description (incidence):
There were no test substance-related mortalities in any of the groups.
One female animal (No. 176) of test group 03 (400 mg/kg bw/d) was found dead on PND 3. The death of this animal was not preceded by any specific preterminal clinical signs. This isolated death of one single animal was assessed as spontaneous and not related to treatment.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Mean body weights as well as mean body weight change values of all test substance-treated male and female F0 animals were comparable to the concurrent control values throughout the entire study.
In male animals of test group 03 (400 mg/kg bw/d) the mean body weight change value was significantly decreased during premating days 56 – 63. The mean body weight change values in female animals of test group 03 (400 mg/kg bw) were also significantly decreased during premating days 49 – 56 and 63 - 70. However, during premating days 56 – 63 the mean body weight change value in female animals of test group 03 (400 mg/kg bw/d) was significantly increased. The body weight change values were decreased/increased during a short time period and are within the normal range of biological variation. Therefore, these findings were considered to be spontaneous in nature and not treatment related.
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. In test group 03 (400 mg/kg bw/d), food consumption was significantly increased in male animals on study day 35 (20% above control) and in female animals on PND 21 (14 % above control). Since all other intervals were comparable to control, these slight temporary increases were assessed as spontaneous and not related to treatment.
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Description (incidence and severity):
No treatment-related, adverse changes among hematological parameters were observed.
At the end of the administration period, in parental females of test groups 02 and 03 (100 and 400 mg/kg bw/d) absolute monocyte counts were significantly increased. However, this was the only altered hematology parameter among these individuals. Therefore, it was regarded as treatment related but non-adverse (ECETOC Technical Report No. 85, 2002).
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
At the end of the administration period, in parental females of test group 03 (400 mg/kg bw/d) triglyceride levels were significantly increased. This alteration in combination with increased liver weights was regarded as a treatment-related and adverse effect (ECETOC Technical Report No. 85, 2002).
Endocrine findings:
effects observed, non-treatment-related
Description (incidence and severity):
In parental males (test groups 01, 02 and 03; 25, 100, 400 mg/kg bw/d), no treatment-related alterations of T4 and TSH levels were observed.
In F0 females of test group 03 (400 mg/kg bw/d), T4 values were significantly decreased. However, the T4 mean was within the historical control range, whereas that one of the study controls was above this range (F0 females, T4 24.28-43.26 nmol/L). Therefore, this alteration was regarded as incidental and not treatment related.
Urinalysis findings:
effects observed, non-treatment-related
Description (incidence and severity):
No treatment-related, adverse changes among urinalysis parameters were observed.
At the end of the administration period, the urine pH value in males and females of test group 03 (400 mg/kg bw/d) as well as in females of test group 02 (100 mg/kg bw/d) was significantly decreased. Additionally, in males of test group 03, urine volume was significantly decreased, and urine specific gravity was significantly increased. Among these individuals the levels of ketone bodies in the urine und the numbers of granular and epithelial cell casts in the urine sediment were significantly increased. However, all mentioned changes were observed without relevant changes in other clinical pathology parameters and without any histopathological change in the kidneys. Therefore, these changes were regarded as maybe treatment related but non-adverse.
In parental females of test groups 02 and 03 (100 and 400 mg/kg bw/d), urine specific gravity was significantly increased and in females of test group 02 urine volume was significantly decreased. However, these changes were not dose dependent. Therefore, they were regarded as incidental and not treatment related.
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Treatment-related findings were noted in liver and thyroid in male animals of test group 03.

Liver
In the liver, an increased incidence of minimal centrilobular hepatocellular hypertrophy was observed, characterized by minimally increased size of the cytoplasm.
- Hypertrophy, centrilobular, grade 1 (males): 1 / 1 / 0 / 11 in control, low, mid and high dose groups, respectively

Thyroid glands
In the thyroid glands, there was an increased incidence of minimal follicular
hypertrophy/hyperplasia characterized by a high prismatic shape of cells and smaller size of follicular lumina.
- Hypertrophy/hyperplasia, follicular cell, grade 1 (males): 2 / 2 / 2 / 10 in control, low, mid and high dose groups, respectively

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

Fertility
The female animals (No. 108, 144), which were not pregnant as well as one of the male mating
partners (No. 44) did not show relevant histopathological findings consistent with impaired
fertility.
Male animal No. 8 showed marked tubular degeneration in the left testis and severe
oligospermia bilateral in the epididymides, which was considered the cause for the lack of
offspring of the mating pair of animals 8/108. This was considered a spontaneous occurrence
in the control group.
Decedents
The female animal No. 176 was found dead 104 days after start of exposure. No macroscopic
or microscopic findings were observed that could explain the death of this animal.
Histopathological findings: neoplastic:
effects observed, treatment-related

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
Estrous cycle data, generated during the last 3 weeks prior to mating to produce the F1 litter, revealed regular cycles in the females of all test groups including the control. The mean estrous cycle duration was comparable between the groups: 4.0 / 4.1 / 4.1 and 4.0 days in test groups 00 - 03, respectively.
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
Concerning motility of the sperms and the incidence of abnormal sperms in the cauda epididymidis as well as sperm head counts in the testis and in the cauda epididymidis of parental males, no treatment-related effects were observed.
Reproductive performance:
effects observed, non-treatment-related
Description (incidence and severity):
For all F0 parental males, which were placed with females to generate F1 pups, copulation was confirmed. Thus, the male mating index was 100% in all test groups (00 - 03).
Fertility was proven for most of the F0 parental males within the scheduled mating interval for F1 litter.
Male animal No. 8 of test group 00 (control; 0 mg/kg bw/d) as well as male animal No. 44 of test group 01 (25 mg/kg bw/d) did not generate F1 pups.
Thus, the male fertility index ranged between 96% and 100%, reflecting the normal range of biological variation inherent in the strain of rats used for this study.

The female mating index calculated after the mating period for F1 litter was 100% in all test groups.
The mean duration until copulation was detected (GD 0) varied between 2 and 3 days without any relation to dosing.
All female rats delivered pups or had implants in utero with the following exception:
• Test group 00 (control, 0 mg/kg bw/d): Female No. 108 (mated with male No. 8) did not become pregnant
• Test group 01 (25 mg/kg bw/d): Female No. 144 (mated with male No. 44) did not become pregnant
These animals did not show relevant gross or histopathological findings consistent with impaired fertility, except of control animal No. 8.
The fertility index ranged between 96% and 100% without showing any relation to dosing.
The mean duration of gestation was comparable in all test groups (i.e. between 22.3 and 22.6 days).
The gestation index ranged between 95.8% and 100% without showing any relation to dosing.
Implantation was not affected by the treatment since the mean number of implantation sites was comparable between all test substance-treated groups and the control, taking normal biological variation into account (13.7 / 12.7 / 13.7 and 12.3 implants/dam in test groups 00 - 03, respectively). Furthermore, there were no indications for test substance-induced intrauterine embryo-/fetolethality since the postimplantation loss did not show any statistically significant differences between the groups (11.6 / 10.2 / 10.5 and 4.1 mean% in test groups 00 - 03, respectively), and the mean number of F1 pups delivered per dam remained unaffected (12.2 / 11.4 / 12.2 and 12.1 pups/dam, respectively in test groups 00 - 03).
The rate of liveborn pups was also not affected by the test substance, as indicated by live birth indices of 97% / 97% / 97% and 99% in test groups 00 - 03. Moreover, the number of stillborn pups was not significantly different between the test groups.

Effect levels (P0)

open allclose all
Key result
Dose descriptor:
NOAEL
Remarks:
fertility and reproductive performance
Effect level:
400 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Dose descriptor:
NOAEL
Effect level:
400 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: Increased triglyceride values in combination with increased liver weights

Target system / organ toxicity (P0)

Key result
Critical effects observed:
no

Results: F1 generation

General toxicity (F1)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
F1 generation pups/litters
There were no test substance-related adverse clinical signs observed in any of the F1 generation pups of the different test groups.
Male animal No. 134-02 of test group 01 (25 mg/kg bw/d) showed a poor general condition as well as a reduced nutritional condition on PND 8. Therefore, this animal was sacrificed moribund on PND 8. Further, reduced nutritional condition was observed in male animal No. 135-09 of test group 01 (25 mg/kg bw/d) on PND 1. Closed eyelids was detected in male animal 183-03 of test group 03 (400 mg/kg bw/d) from PND 19 onwards. Since all these findings occurred in individual pups mostly without relation to dose, they were not considered to be treatment-related.


F1 rearing animals, Cohort 1A
Transient salivation during a short time period after gavage dosing (up to two hours) was noted for all male and female animals of test group 13 (400 mg/kg bw/d) during the entire study. It is likely, that this temporary finding was induced by a bad taste of the test substance or local affection of the upper digestive tract. It is, however, not considered to be an adverse finding indicating systemic toxicity.
No further clinical signs or changes of general behavior, which may be attributed to the test substance, were detected in any treated male and female animals of all test groups 11, 12 and 13 (25, 100 and 400 mg/kg bw/d).
The findings in control group were assessed as spontaneous in nature, i.e. male animal. No. 216 showed a small testis on the right side from study day 14 to 34 and female animal No. 303 showed a kinked tail from study day 14 onwards.

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.
Male animal. No. 216 of the control group (Test group 10, 0 mg/kg bw/d) showed a small testis on the right side from study day 14 to 34. This finding was also seen on the respective days of detailed clinical observation, i.e. study days 14, 21 and 28. Further, female animal No. 303 of the control group (Test group 10, 0 mg/kg bw/d) showed a kinked tail from study day 14 onwards, which was also seen on the respective days of detailed clinical observation from study day 14 onwards. These findings in the control group were assessed as spontaneous in
nature.


F1 rearing animals, Cohort 1B
No treatment-related, adverse clinical signs were observed.
Transient salivation during a short time period after gavage dosing (up to 2 hours) was noted for all male and female animals of test group 13 (400 mg/kg bw/d) during the entire study period starting on study day 2. It is likely, that this temporary finding was induced by a bad taste of the test substance or local affection of the upper digestive tract. It is, however, not considered to be an adverse finding indicating systemic toxicity.
An anomaly of teeth was observed in male animal No. 447 of test group 11 (25 mg/kg bw/d) from study day 35 to 57. This finding was considered to be spontaneous in nature and not related to test substance treatment.
Note: On study day 7, the clinical examinations between 2-5 hours after treatment were not performed for all animals of test groups 10 – 13 in cohort 1B. Thus, no documentation exists in the electronic system and are not shown in tables. These missing records do not have an influence on the validity of the present study.

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.
An anomaly of teeth was observed in male animal No. 447 of test group 11 from study day 35 to 57 as well as on the respective days of detailed clinical observation, i.e. study days 35, 42, 49 and 56. This finding was considered to be spontaneous in nature.
Dermal irritation (if dermal study):
not examined
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
F1 generation pups/litters
The viability index indicating pup survival during early lactation (PND 0 - 4) varied between 99% / 98% / 98% and 93% in test groups 00 – 03 (0, 25, 100 and 400 mg/kg bw/d).
Female animal No. 176 of test group 03 was found dead on PND 3. Therefore, all remaining pups of that animal were sacrificed on PND 3. This led to a viability index of 93% in test group 03. However, it was not assessed as related to treatment.
The lactation index indicating pup survival on PND 4 - 21 was 100% / 99% / 100% and 100% in test groups 00 - 03.


F1 rearing animals, Cohort 1A
There were no test substance-related mortalities in any of the groups.
Female animal No. 380 of test group 13 (400 mg/kg bw/d) was found dead on study day 26. The death of this animal was not preceded by any specific preterminal clinical signs. Therefore, the death was assessed as spontaneous and not related to treatment.


F1 rearing animals, Cohort 1B (03S032_1B)
There were no test substance-related mortalities in any of the groups.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
F1 generation pups/litters
In general, the mean body weights and body weight change values of all male and female pups in all test substance-treated groups were comparable to the concurrent control values throughout the entire study.
On PND 1, mean body weights of female animals as well as female and male animals combined of test group 01 (25 mg/kg bw/d) were significantly increased. In addition, the mean body weight of female animals of test group 01 (25 mg/kg bw/d) was significantly increased on PND 4. Since no dose-response relationship occurred, these isolated findings were considered to be spontaneous in nature and not related to treatment.


F1 rearing animals, Cohort 1A
In general, no treatment-related, adverse findings in body weight (change) were observed. In males of test group 13, mean body weight was slightly decreased towards the end of the treatment period (6% below control, on study day 63). Consistently, body weight change was significantly decreased during study days 0 – 63. This was assessed as treatment-related but not adverse since the level of decrease was slight. Females of test group 13 showed a mean body weight comparable to control values.
The following findings were not related to dose and, therefore, not assessed as treatment related.
Female animals of test group 12 showed a significant increase in body weight on study days 0 and 7.
In males of test group 13 and 12, mean body weight change values were significantly decreased during study days 42 – 49, without a dose-response relationship.


F1 rearing animals, Cohort 1B
In general, mean body weight and mean body weight change values of all male and female animals of test groups 11, 12 and 13 (25, 100, 400 mg/kg bw/d) were comparable to the concurrent test group 10 (control group; 0 mg/kg bw/d).
In male animals of test group 13, body weight change was significantly decreased during study days 42 – 49. Since test group 13 males gained weight comparable to control during the entire study phase, this was assessed as not related to treatment.
All other findings observed in the test groups, e.g. increased body weight of test group 12 females on study day 21, increased body weight change of test groups 12 and 13 females during study days 14 – 21 and decreased body weight change of test group 11 males during 14 – 21, showed no relation to dose and were assessed to be spontaneous.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
F1 rearing animals, Cohort 1A
In general, mean food consumption was comparable to the concurrent control values in all test groups over the entire study period.
In test group 11, males and females showed a significant decrease in mean food consumption during study days 49 - 56 and 42 - 56, respectively. Since this was not related to dose, it was not assessed as treatment-related.


F1 rearing animals, Cohort 1B
In general, food consumption of all male and female animals of all test groups was comparable to the concurrent control values throughout the entire study.
In test group 13, food consumption was significantly increased in males during study days 21 - 28 and 35 – 42 (each 8% above control) and in females during study days 21 - 28 (8% above control). However, this minor increase was only temporary during one or two intervals. Therefore, the findings were not assessed as treatment-related.
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):
At PND90 in F1 males of test group 13 (400 mg/kg bw/d) absolute reticulocyte counts were significantly increased. This change in combination with increased spleen weights and increased gradings of extramedullary hematopoiesis was regarded as treatment related and adverse (ECETOC Technical Report No. 85, 2002).
Additionally, in males of this test group absolute and relative eosinophil counts were significantly decreased. Eosinophil count decreases were isolated, without any change of other differential blood cell parameters. Therefore, also this change was regarded as treatment related but non-adverse (ECETOC Technical Report No. 85, 2002).
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
No treatment-related, adverse changes among clinical chemistry parameters were observed.
At PND90 in males of test group 13 (400 mg/kg bw/d), cholesterol values were significantly decreased. However, this was an isolated change without any other clinical chemistry alterations. Therefore, this change was regarded as treatment related but non-adverse.
In F1A females of test group 11 (25 mg/kg bw/d), alkaline phosphatase (ALP) activities were significantly increased but this change was not dose dependent. Therefore, it was regarded as incidental and not treatment related.
Urinalysis findings:
effects observed, non-treatment-related
Description (incidence and severity):
No treatment-related, adverse changes among urinalysis parameters were observed.
Urine pH values in males and females of test group 13 (400 mg/kg bw/d) were significantly decreased. Additionally, in females of this test group urine volume was decreased (not statistically significantly). These changes reflect the adaptation of the kidneys towards less fluid income as well as the physiological regulation of urine pH value keeping the homeostasis of the blood pH value. Therefore, these changes were regarded as treatment related but adaptive rather than adverse.
In F1A males of test group 11 (25 mg/kg bw/d) urine pH values were also decreased, but the change was not dose dependent and therefore, it was regarded as incidental and not treatment related.
Sexual maturation:
effects observed, non-treatment-related
Description (incidence and severity):
Vaginal opening
Each female F1 pup, which was selected to become a rearing female, was evaluated for commencement of sexual maturity. The first day when vaginal opening was observed was PND 27, the last was PND 36. The mean number of days to reach the criterion in the control and 25, 100 and 400 mg/kg bw/d test groups was 31.9; 31.6; 31.6 and 32.6 days, respectively. The mean body weight on the day, when vaginal opening was recorded, was 97.4, 96.8, 98.4 and
101.8 g in test groups 00 – 03, respectively. Neither a statistically significant nor a toxicologically relevant effect was noted in any of the treatment groups.

Preputial separation
Each male F1 pup, which was selected to become a rearing male, was evaluated for commencement of sexual maturity. The first day when preputial separation was observed was PND 38, the last was PND 44. The mean number of days to reach the criterion in the control and 25, 100 and 400 mg/kg bw/d test groups was 39.6, 40.0, 40.3 and 40.4 days, respectively. The mean body weight on the day, when preputial separation was recorded, was 162.5, 160.6, 163.5 and 162.5 g in test groups 00 – 03, respectively. Statistical significance was observed in
test groups 03 and 02. However, their mean values are close to the mean value of the historical control data (HCD, preputial separation: mean of means: 41.9 days). Therefore, this was not assessed as treatment-related, adverse finding.
Anogenital distance (AGD):
no effects observed
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.
Nipple retention in male pups:
no effects observed
Description (incidence and severity):
The apparent number and percentage of male pups having nipples/areolae was not influenced by the test substance when examined on PND 13 as well as on PND 20.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Rearing animals, cohort 1A
Absolute organ weights
When compared with the control group 10 (=100%), the following mean absolute weights were significantly changed:
- Kidneys (females): 101% / 104% / 110%** versus ctrl in low, mid and high dose groups, respectively
- Liver (females): 97% / 107% / 1113%* versus ctrl in low, mid and high dose groups, respectively
*: p <= 0.05, **: p <= 0.01

All other mean absolute weight parameters did not show significant differences when compared to the control group 10.

Relative organ weights
When compared with control group 10 (=100%), the following mean relative weights were significantly changed:
- Kidneys (males): 101% / 101% / 110%** versus ctrl in low, mid and high dose groups, respectively
- Liver (males): 98% / 99% / 111%* versus ctrl in low, mid and high dose groups, respectively
- Spleen (males): 99% / 100% / 107%* versus ctrl in low, mid and high dose groups, respectively
- Kidneys (females): 104% / 103% / 112%** versus ctrl in low, mid and high dose groups, respectively
- Liver (females): 100% / 105% / 114%** versus ctrl in low, mid and high dose groups, respectively
- Spleen (females): 98% / 100% / 107%* versus ctrl in low, mid and high dose groups, respectively
*: p <= 0.05, **: p <= 0.01

All other mean relative weight parameters did not show significant differences when compared to the control group 10.

The increased mean absolute and relative weights of the kidneys and liver of test group 13 male and female animals and the increased mean relative weights of the spleen of male test group 13 animals were regarded to be treatment-related.
The increased mean relative weight of the spleen of female test group 13 animals was regarded as incidental as there were no changes in histopathology or hematology parameters.
As described in amendment No. 3 to the study plan, five animals of each test group were sacrificed without prior fasting period. In males of test groups 10 and 13, vacuolation consistent with glycogen storage was noted in these animals. The weights of the livers of these five animals per group were approximately 1-5g higher than other animals of the respective groups which were fasted. In females, livers of the non-fasted animals were also heavier than other animals of the respective groups, albeit to a smaller degree than in males. Histopathologically,
these animals also showed vacuolation consistent with glycogen storage. As the magnitude of change was comparable in each group, this did not interfere with the interpretation of this study.


Rearing animals, cohort 1B
Absolute organ weights
When compared with the control group 10 (=100%), the following mean absolute weights were significantly changed:
- Liver (males): 96% / 105% / 108%* versus ctrl in low, mid and high dose groups, respectively
- Prostate (males): 91%* / 96% / 84%** versus ctrl in low, mid and high dose groups, respectively
- Testes (males): 98% / 99% / 92%** versus ctrl in low, mid and high dose groups, respectively
- Liver (females): 99% / 103% / 109%** versus ctrl in low, mid and high dose groups, respectively
*: p <= 0.05, **: p <= 0.01

All other mean absolute weight parameters did not show significant differences when compared to the control group 10.

Relative organ weights
When compared with control group 10 (=100%), the following mean relative weights were significantly changed:
- Liver (males): 98% / 103% / 113%** versus ctrl in low, mid and high dose groups, respectively
- Prostate (males): 93% / 94% / 88%** versus ctrl in low, mid and high dose groups, respectively
- Liver (females): 98% / 102% / 108%** versus ctrl in low, mid and high dose groups, respectively
*: p <= 0.05, **: p <= 0.01

All other mean relative weight parameters did not show significant differences when compared to the control group 10.

The statistically significantly increased mean absolute and relative weights of the liver in test group 13 male and female animals were regarded to be treatment – related.
The mean absolute weights of the prostate were statistically significantly decreased in test groups 11 and 13. The weights of control (0.847g), test groups 01 (0.774g) and 12 (0.815g) were above the historical control range of 0.686 – 0.748g. The mean absolute weight of test group 13 (0.708g) was within the historical control range. The statistically significantly decreased mean relative weight of the prostate of test group 13 (0.228%) was within the historical control range of 0.213 – 0.231%, while all other test groups including controls lay above this range (test group 10: 0.26%, test group 11: 0.241%, test group 12: 0.246%). As there was no clear dose-response, and the weights of the prostate in cohort 1A were not significantly changed, these weight changes were regarded to be incidental.
The statistically significantly decreased mean absolute weight of the testes in test group 13 males (3.259g) was below the historical control range of 3.338 – 3.51g, while the control test group 10 (3.532g) was above this range. As there were no statistically significant changes in cohort 1A or the parental generation nor any treatment-related findings in spermanalysis or histopathology in cohort 1A and the parental generation, these changes were regarded to be
incidental.


Surplus F1 generation pups on PND 22 (F1 weanlings not selected for cohorts)
Absolute and relative organ weights
None of the mean absolute and relative weight parameters showed significant differences when compared to the control group 00.
Gross pathological findings:
no effects observed
Description (incidence and severity):
Rearing animals, cohort 1A
All findings occurred either individually or were well-described spontaneous lesions (focal constriction/herniation of the liver in two test group 11 females (McInnes 2012). They were considered to be incidental or spontaneous in origin and without any relation to treatment.


Rearing animals, cohort 1B
All findings occurred either individually or were well-described spontaneous lesions (focal constriction/herniation of the liver in two test group 11 females (McInnes 2012). They were considered to be incidental or spontaneous in origin and without any relation to treatment.


Surplus F1 generation pups on PND 22 (F1 weanlings not selected for cohorts)
No gross lesions were observed.
Histopathological findings:
effects observed, treatment-related
Description (incidence and severity):
Rearing animals, cohort 1A
Increased hematopoiesis (reticulocytes) was noted in the spleen of male animals of test group 13.
- Hematopoiesis, extramedullary, grade 1 (males): 8 / 8 / 10 / 6 in control, low, mid and high dose groups, respectively
- Hematopoiesis, extramedullary, grade 2 (males): 6 / 3 / 3 / 6 in control, low, mid and high dose groups, respectively
- Hematopoiesis, extramedullary, grade 3 (males): 0 / 0 / 0 / 8 in control, low, mid and high dose groups, respectively

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


Decedents
The female animal No. 380 was found dead 26 days after start of exposure. No macroscopic or microscopic findings were observed that could explain the death of this animal.
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
Thyroid hormones
In F1 PND4 and PND22 pups (test groups 01, 02 and 03; 25, 100, 400 mg/kg bw/d), no treatment-related alterations of T4 and TSH levels were observed.
At PND90 in F1A males of test group 13 (400 mg/kg bw/d), T4 values were significantly decreased. However, in this test group T4 as well as TSH mean values were within historical control ranges (F1A males, T4 49.46-88.73 nmol/L, TSH 2.61-9.90 μg/L). Therefore, T4 change in males of test group 13 was regarded as incidental and not treatment related.
AT PND90 in F1A females of test groups 11, 12, and 13 (25, 100 and 400 mg/kg bw/d) no changes of T4 and TSH values were observed.

Spermanalysis:
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 of F1A males, no treatment-related effects were observed.

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Details on results (F1)

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

Differential ovarian follicle count
The results of the differential ovarian follicle count (DOFC) – comprising the numbers of primordial and growing follicles, as well as the combined incidence of primordial plus growing follicles – did not reveal significant differences between the control group 10 and animals of test group 13

F1 rearing animals, Cohort 1A
Estrous cycle data generated for 2 weeks revealed regular cycles in the females of all test groups 10, 11, 12 and 13 (0, 25, 100 and 400 mg/kg bw/d). The mean estrous cycle duration was comparable between the groups: 5.1 / 4.4 / 5.3 and 4.3 in test groups 10 - 13, respectively.
The slightly higher mean value in the control and mid-dose group was assessed to be spontaneous and without relation to dose.

F1 rearing animals, Cohort 1B
Estrous cycle data generated for 2 weeks revealed regular cycles in females of all test groups 10 - 13. The mean estrous cycle duration in the different test groups was comparable: 5.4 / 4.5 / 4.4 and 4.8 days in test groups 10, 11, 12 and 13 (0, 25, 100 and 400 mg/kg bw/d), respectively.

Spermanalysis of F1 generation
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 of F1A males, no treatment-related effects were observed.

Thyroid hormones of F1 generation
In F1 PND4 and PND22 pups (test groups 01, 02 and 03; 25, 100, 400 mg/kg bw/d), no treatment-related alterations of T4 and TSH levels were observed.
At PND90 in F1A males of test group 13 (400 mg/kg bw/d), T4 values were significantly decreased. However, in this test group T4 as well as TSH mean values were within historical control ranges (F1A males, T4 49.46-88.73 nmol/L, TSH 2.61-9.90 μg/L). Therefore, T4 change in males of test group 13 was regarded as incidental and not treatment related.
AT PND90 in F1A females of test groups 11, 12, and 13 (25, 100 and 400 mg/kg bw/d) no changes of T4 and TSH values were observed.

Effect levels (F1)

open allclose all
Key result
Dose descriptor:
NOAEL
Remarks:
developmental toxicity
Generation:
F1
Effect level:
400 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
400 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
100 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: increased absolute reticulocyte counts in combination with increased spleen weights and higher grades of extramedullary hematopoiesis (ncreased red blood cell metabolism)

Target system / organ toxicity (F1)

Key result
Critical effects observed:
no

Overall reproductive toxicity

Key result
Reproductive effects observed:
no

Applicant's summary and conclusion