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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
reproductive toxicity, other
Remarks:
Range finding study for EOGRTS
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2017

Materials and methods

GLP compliance:
yes
Remarks:
But no inspection by Quality Assurance Unit
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Lysmeral (encapsulated)

Test animals

Species:
rat
Strain:
Wistar
Remarks:
Crl:Wl(Han)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: male animals: about 10 weeks; female animals: about 9 weeks
- Fasting period before study: no
- Housing: 1 animal, Polycarbonate cages type 111, Wooden gnawing blocks, Cellulose wadding toward the end of gestation; Dust-free wooden bedding.
Exceptions:
During mating: 1 male/1 female per cage
During rearing until weaning: 1 dam with her litter
- Diet: ad libitum
- Water: ad libitum)
- Acclimation period: 6 days

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

Administration / exposure

Route of administration:
oral: feed
Vehicle:
other: Test subsance in sunflower oil, encapsulated in aginate capsules
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: max 14 days
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy.
Duration of treatment / exposure:
Continuously via diet during study period (2 weeks premating, 2 weeks mating, gestation and lactation (PND 21)).
Frequency of treatment:
Continuously via diet
Doses / concentrationsopen allclose all
Dose / conc.:
230 ppm (nominal)
Remarks:
2.3-3.6 mg/kg bw/d
Dose / conc.:
750 ppm (nominal)
Remarks:
7.4-11.9 mg/kg bw/d
Dose / conc.:
2 300 ppm (nominal)
Remarks:
25.1 -34.7 mg/kg bw/d
No. of animals per sex per dose:
10
Control animals:
other: control groups fed plain diet (negative control) or placebo alginate capsules with sunflower oil only (placebo control)
Positive control:
not applicable

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes (incl. parturition and lactation behavior)
- Time schedule: at least once daily

BODY WEIGHT: Yes
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).
The following exceptions apply to the female parental animals:
• During the mating period, 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, 14 and 21 .
Body weight was not determined in the females without positive evidence of sperm during mating and gestation periods and in the females without litter during lactation period.


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
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

Generally food consumption was determined once a week for the male and female F0 parental animals, with the following exceptions:
• Food consumption was not be determined during the mating period (male and female F0 animals).
• Food consumption of the F0 females with evidence of sperm was determined for GD 0-7, 7-14 and 14-20.
• Food consumption of the F0 females, which gave birth to a litter was determined for PND 1-4, 4-7, 7-14 and 14-21 .
Food consumption was not determined in the females without positive evidence of sperm during the mating and gestation periods and in females without litter during lactation period.

CLINICAL PATHOLOGY:
Blood samples were withdrawn from F0 animals by puncturing the retrobulbar venous plexus following isoflurane anesthesia.
- Hematology:
1. Leukocytes
2. Erythrocytes
3. Hemoglobin
4. Hematocrit
5. Mean corpuscular volume (MCV)
6. Mean corpuscular hemoglobin (MCH)
7. Mean corpuscular hemoglobin concentration (MCHC)
8. Platelets
9. Differential blood count
10. Reticulocytes
11. Blood smear (only evaluated preparations will be archived)
12. Prothrombin time

- Clinical chemistry
1. Alanine aminotransferase
2. Aspartate aminotransferase
3. Alkaline phosphatase
4. Serum y-glutamyl transferase
5. Sodium
6. Potassium
7. Chloride
8. lnorg. phosphate
9. Calcium
10. Urea
11. Creatinine
12. Glucose
13. Total bilirubin
14. Totalprotein
15. Albumin
16. Globulins
17. Triglycerides
18. Cholesterol
Sperm parameters (parental animals):
Performed in the right testis or right epididymis of all male F0 parental animals sacrificed on schedule:
• Cauda epididymis sperm motility
• Sperm morphology
• Spermatid head count in the testis
• Sperm head count in the cauda epididymis
Litter observations:
Clinical examinations:
- Pup status and litter size after birth: Status (sex, liveborn or stillborn) and number of all pups delivered were determined as soon as possible after birth. At the same time, the pups were examined for gross-morphological changes.
- Pup viability/mortality: checked twice daily on workdays (once in the morning and once in the afternoon) or as a rule, only in the morning on Saturdays, Sundays or public holidays. Pups, which died before the first determination of their status on the day of birth, were defined as stillborn pups.
Clinical signs: All live pups were examined daily for clinical symptoms (including gross-morphological findings) during the clinical inspection of the dams.
- Body weights: Pups were weighed on the day after birth (PND 1) and PND 4,7,14 and 21.
Postmortem examinations (parental animals):
SACRIFICE
All F0 parental animals were sacrificed by decapitation under isoflurane anesthesia.


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

ORGAN WEIGHTS
1. Anesthetized animals
2. Adrenal glands
3. Cauda epididymis
4. Epididymides
5. Prostate
6. Liver
7. Seminal vesicles including coagulating glands
8. Testes

HISTOPATHOLOGY
Testis
Epididymis
Organs were fixed in modified Davidson's solution, whereas the other testis and epididymis was used for sperm parameters.
Postmortem examinations (offspring):
SACRIFICE
On PND 4, as a result of standardization, some of the surplus pups were sacrificed under isoflurane anesthesia with CO2.

GROSS NECROPSY
After sacrifice, pups were examined externally, eviscerated and their organs were assessed macroscopically.

OTHER: Generation of samples to establish and validate acetylcholine esterase activity test used for the main study (EOGRTS; BASF 03R0179/14R092):
PND 4: Erythrocytes; Serum; Cortex; Cerebellum; Medulla oblongata with Pons and cervical spinal cord; Remaining brain parts (brain stem); Heart; Diaphragma; Musculus gastrocnemius; whole brain.
PND 22: Erythrocytes; Serum; Cortex; Cerebellum; Striatum; Hippocampus; Medulla oblongata with Pons, Cervical spinal cord; Heart; Diaphragma; Musculus gastrocnemius.
Statistics:
DUNNETT test (two-sided): Food consumption, body weight and body weight change (parental animals/F1 rearing animals and pups); mating days; duration of gestation; number of delivered pups per litter; implantation sites; postimplantation loss

FISHER's exact test:Number of live and dead pups and different indices (e.g. mating index, fertility index and gestation index) and
number of litters with necropsy findings in pups.

WILCOXON test (one-sided): Proportion of pups with necropsy findings per litter.

KRUSKAL-WALLIS and WILCOXON test: Clinical pathology and sperm parameters. Weight of the anesthetized animals and absolute and relative organ weights.
Reproductive indices:
Male mating index (%) = number of males with confirmed mating / number of males placed with females * 100
Male fertility index (%) = number of males proving their fertility / number of males placed with females * 100
Female mating index (%) = number of females mated / number of females placed with males * 100
Female fertility index (%) = number of females pregnant / number of females mated * 100
Gestation index (%) = number of females with live pups on the day of birth / number of females pregnant * 100
Live birth index (%) = number of liveborn pups at birth / total number of pups born * 100
Postimplantation loss (%) = number of implantations – number of pups delivered / number of implantations * 100
Offspring viability indices:
Viability index (%) = number of live pups on day 4* after birth / number of live pups on the day of birth * 100
Lactation index (%) = number of live pups on day 21 after birth / number of live pups on day 4* after birth * 100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
High dose group (30 mg/kg bw/d):
Males: BW decreased week 2-7 (5%-11% below placebo control) ; BWC decreased during the different periods (53-84% below placebo control); week 0-7 (45% below placebo control).
Females - Premating: BW decreased week 2 (5% below placebo control); Body weight loss in week 0-1; BWC decreased week 0-2 (59% below placebo control).
Females - Gestation: BW decreased day 14, 20 (10-16% below placebo control). BWC decreased during the different periods (32-43% below placebo control); day 0-20 (40% below placebo control).
Females - Lactation: BW decreased day 1-21 (6-11% below placebo control); BWC decreased day 1-21 (26% below placebo control).

Mid dose group (10 mg/kg bw/d):
Females - Premating: BWC decreased week 0-1 (36% below placebo control); week 0-2 (12% below placebo control).
Females - Gestation: BW decreased day 14, 20 (6% below placebo control); BWC decreased day 0-7 (30% below placebo control).
Females - Lactation: BW decreased day 1-14 (6-9% below placebo control). BWC increased day 1-21 (64% above placebo control).


No evident test substance related effects were observed on other parameters and in the low dose group (3 mg/kg bw/d).
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
High dose group (30 mg/kg bw/d):
Males: decreased week 0-1 (9% below placebo control).
Females - Premating: decreased week 0-1 (14% below placebo control); decreased week 0-2 (8% below placebo control).
Females - Lactation: decreased during the different periods (44-48% below placebo control); decreased day 1-21 (46% below placebo control).

Mid dose group (10 mg/kg bw/d):
Males: decreased week 0-1 (7% below placebo control).
Females - Lactation: decreased during the different periods (8-20% below placebo control); decreased day 1-21 (14% below placebo control).

No evident test substance related effects were observed on other parameters and in the low dose group (3 mg/kg bw/d).
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
High dose group (30 mg/kg bw/d):
Males:
Decrease in platelet counts, neutrophil counts.
Increase in prothrombin time.

Females:
Decrease in hemoglobin, hematocrit, mean corrpuscular volume, platelet counts, eosinophil counts.
Increase in reticulocyte counts, prothrombin time.


Mid dose group (10 mg/kg bw/d):
Males:
Decrease in neutrophil counts.

Females:
Decrease in hematocrit, platelet counts.
Increase in prothrombin time.

No evident test substance related effects were observed on other parameters and in the low dose group (3 mg/kg bw/d).
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
High dose group (30 mg/kg bw/d):
Males:
Increase in aspartate aminotransferase (AST) (26% above placebo control);
Decrease in total protein, albumin, globulin, cholesterol, triglycerides, sodium, calcium

Females:
Increase in aspartate aminotransferase (AST) (47% above placebo control); and gamma glutamyltransferase (GGT) (24 fold); creatinine, total bilirubin, chloride, inorganic phosphate
Decrease in total protein, albumin, globulin, cholesterol, triglycerides, calcium.

Mid dose group (10 mg/kg bw/d):
Males:
Decrease in total protein

Females:
Increase in aspartate aminotransferase (AST) (23% above placebo control); and gamma glutamyltransferase (GGT) (9 fold).
Decrease in total protein, albumin, globulin, triglycerides, calcium.

No evident test substance related effects were observed on other parameters and in the low dose group (3 mg/kg bw/d).
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
High dose group (30 mg/kg bw/d):
Epididymis:
• ductal atrophy: 8 /10 males (minimal to moderate), not observed in placebo control.
• oligospermia: 6 /10 males (slight to moderate), not observed in placebo control.
• cellular debris: 2 out of 10 males (slight to moderate), not observed in placebo control.

Testis:
• tubular degeneration: 3 / 10 males (minimal to moderate) vs. 1 / 10 males in placebo control


The ductal atrophy of the epididymides was characterized by a segmental or generalized narrowing the ductal lumina, accompanied by increasing height and sometimes folding of the epithelium. The affected segments were at the junction of distal corpus and cauda. With increasing severity the ductal atrophy was generalized. The ductal narrowing was consistent with reduced sperm content (oligospermia).

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

Reproductive function / performance (P0)

Reproductive function: sperm measures:
effects observed, treatment-related
Description (incidence and severity):
Reduction in mean fraction of motile sperm in the cauda epididymis in the high dose (25%) versus placebo control and low, mid dose group (85%, 88%, 86% at ctrl, 3, 10 mg/kg bw/d)
Increase in mean fraction of abnormal sperm in the cauda epididymis in the high dose (72.3%) versus placebo control and low, mid dose group (6.2%, 6.6%, 7.0% at ctrl, 3, 10 mg/kg bw/d)
Reduction in mean sperm head count in the cauda epididymis in the high dose (469 mio/g) versus placebo control and low, mid dose group (674, 760, 640 mio/g at ctrl, 3, 10 mg/kg bw/d)
No effect on mean spermatid head counts in the testis (124, 107, 127, 115 mio/g at ctrl, 3, 10, 30 mg/kg bw/d).
Reproductive performance:
effects observed, treatment-related
Description (incidence and severity):
Reduction in male fertility index in the high dose (40%) versus placebo control and low, mid dose group (100%, 90%, 100% at ctrl, 3, 10 mg/kg bw/d)
Reduction in female fertility index in the high dose (44%) versus placebo control and low, mid dose group (100%, 90%, 100% at ctrl, 3, 10 mg/kg bw/d)
Increase in the duration of gestation in the high dose group (23 days) versus placebo control and low, mid dose group (22.2, 22.3, 22.0 days at ctrl, 3, 10 mg/kg bw/d)
Decrease of mean implantation sites in the high dose group (4.5) versus placebo control and low, mid dose group (11.5, 11.8, 10.1 at ctrl, 3, 10 mg/kg bw/d)
Increase in mean % postimplantation loss in the high dose group (16.7%) versus placebo control and low, mid dose group (3.8, 3.9, 3.7% at ctrl, 3, 10 mg/kg bw/d), but increase was not observed for the endpoints mean or total postimplantation loss.
Decrease of the total pups delivered in the high dose group (16) versus placebo control and low, mid dose group (111, 102, 97 at ctrl, 3, 10 mg/kg bw/d)
Decrease of mean pups delivered/ litter in the high dose group (4.0) versus placebo control and low, mid dose group (11.1, 11.3, 9.7 at ctrl, 3, 10 mg/kg bw/d)
Decrease of females with liveborn in the high dose group (4) versus placebo control and low, mid dose group (10, 9, 10 at ctrl, 3, 10 mg/kg bw/d), but no increase in females with stillborn. No effects on the gestation index respectively, i.e. 100% in all dose groups.


Mating indices, mating days live birth index and number of stillborns were not affected by test substance treatment.

Results: F1 generation

Details on results (F1)

Decrease of the viability index (survival day 1-4) in the high (75%) and mid dose group (86%) versus placebo control and low dose group (95%, 99% at ctrl, 3 mg/kg bw/d). The lactation index (survival day 4-21) was not affected by test substance treatment.

Higher % of live females compared to live males in the high dose group (69% / 31% at day 0 and 67% / 33% at day 21) versus placebo control (53% / 47% at day 0 and day 21). Finding is a potential chance finding due to the low number of pups in the high dose group compared to the placebo control group.


Pup body weights (male + female pups): Decreased on day 1, 4, 7, 14, 21 in the mid (13-21% below placebo control) and high dose group (18-32% below placebo control).
Pup body weight change:
Decreased during the different periods in the mid (9-28% below placebo control) and high dose group (24-51% below placebo control).
Day 1-21 (male+ female pups): Decreased in the mid (13% below placebo control) and high dose group (33% below placebo control).

No test substance related pup necropsy findings were observed.

Applicant's summary and conclusion