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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
16 Oct 2014 to
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study according to OECD 422 under GLP

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Type:
Constituent
Test material form:
other: viscous liquid
Details on test material:
Identification : Fats and Glyceridic oils, vegetable, winterized, reaction
products with ammonia-ethanolamine reaction products
Physical State/Appearance : Brown extremely viscous liquid
Storage Conditions : Ambient, in the dark

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories U.K. Ltd., Blackthorn, Bicester, Oxon, UK
- Strain: Wistar Han™:RccHan™
- Age at study initiation: ca.12 weeks
- Weight at study initiation: Males: 322-378 g; Females: 197-222 g
- Fasting period before study: none
- Housing: Pretest and premating period: 4 animals/cage; Mating period: one male and one female/cage; Postmating: males 4/cage, females individually
- Diet: Rodent 2018C Teklad Global Certified Diet (Harlan Laboratories U.K. Ltd., Oxon, UK) ad libitum
- Water: Tap water in bottles ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 50 ± 20%
- Air changes (per hr): ≥15/hr
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
arachis oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: every 14 days stored at ca. 4 °C in the dark

VEHICLE: Arachis oil BP
- Justification for use and choice of vehicle (if other than water): not indicated
- Concentration in vehicle: 0, 25, 75 and 250 mg/mL
- Dose volume: 4 mL/kg
Details on mating procedure:
- M/F ratio per cage: 1/1
- Length of cohabitation: 1-4 days
- Proof of pregnancy: vaginal plug or 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:
Actual concentrations in the test substance formulations were 105-112% of nominal

System: HPLC-MS
HPLC System: HPLC Agilent 1200 MSD
Column: Symmetry C18: 50 mm*3.0 mm: 3.5 um
Column temperature: 30 °C
Detection method: MS/SIM
Injection volume: 10 µL
Mobile Phase: 0.05% ammonium formate in water with 0.1% formic acid methanol (30/70 v/v)
Column flow rate: 0.5 mL/min
Retention time: Approx. 2.5-15 min

Calibration solution: 0.05 g of the test substance was weighed in a 100-mL flask and brought to volume with methanol (0.5 mg/mL). Aliquots were further diluted with methanol to a concentration of 0.01 mg/mL

HPLC-MS gave several peaks with one considered to be representative (area changes with known concentration). Linearity of the system demonstrated over 0-0.0177 mg/mL (R2 of fit 0.997).

Analysis for accuracy of preparation, homogenicity and stability:
accuracy of preparation: 98-104 % of nominal (3.68-3.92 mg/g and 250-253 mg/g fortification)
homogenicity: 95.6 ± 1.2% (at 3.75 mg/mL) and 102 ± 0.4% (at 250 mg/mL)
stability : mean recovery 97-98% over 17 days
Duration of treatment / exposure:
Males: two weeks prior to mating, during mating and at least up to and including the day before sacrifice (at least a total of 42 days).
Females: two weeks prior to mating, during mating and at least up to and including the day before sacrifice (day 4 post partum).
Frequency of treatment:
daily
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 100, 300 and 1000 mg/kg bw
Basis:
nominal conc.
No. of animals per sex per dose:
12/sex
Control animals:
yes, concurrent vehicle
Details on study design:
Dose selection rationale: based on a 14-day dose-range-finding toxicity study in rats (Harlan Laboratories Study 41403024, Fourteen Day Repeated Dose Oral (Gavage) Range-Finding Toxicity Study in the Rat), using dose levels of 0, 500, 750 and 1000 mg/kg/day.

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

DETAILED CLINICAL OBSERVATIONS: Yes (Gait, Hyper/Hypothermia, Tremors, Skin color, Twitches, Respiration, Convulsions, Palpebral closure, Bizarre/Abnormal/Stereotypic behavior, Urination, Salivation, Defecation, Pilo-erection, Transfer arousal, Exophthalmia, Tail elevation, Lachrymation)
- Time schedule: once weekly in a standard arena

BODY WEIGHT: Yes
- Time schedule for examinations:
P males: at pretest and weekly during the pre-pairing, pairing period and post-pairing period.
P females: at pretest and weekly during the pre-pairing, daily during the pairing period and on day 0, 7, 14 and 20 post-coitum and day 1 and 4 post-partum.

FOOD CONSUMPTION : yes
- Time schedule for examinations:
P males: weekly during the pre-pairing and post-pairing period.
P females: weekly during the pre-pairing period and days 0-7, 7-14, 14-21 post coitum and days 1-4 post partum.

WATER CONSUMPTION: yes
- Time schedule for examinations:
P-males and females daily during pre-pairing

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood:
5 selected males: on day 42 of treatment (on day of sacrifice)
5 selected females: on day 5 post partum (on day of sacrifice)
- Anaesthetic used for blood collection: No
- Animals fasted: No
- Parameters checked: Erythrocyte count, Hemoglobin, Hematocrit, Mean corpuscular volume, Mean corpuscular hemoglobin, Mean corpuscular hemoglobin concentration, Platelet (thrombocyte) count, Reticulocyte count, Total leukocyte count, Differential leukocyte count (Neutrophils, Lymphocytes, Monocytes, Eosinophils, Basophils), prothrombine time, APTT.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood:
5 selected males: on day 42 of treatment (on day of sacrifice)
5 selected females: on day 5 post partum (on day of sacrifice)
- Anaesthetic used for blood collection: No
- Animals fasted: No
- Parameters checked: Urea, Calcium, Glucose, Inorganic phosphorus, Total protein, Aspartate aminotransferase,Albumin Alanine aminotransferase, Albumin/Globulin ratio, Potassium, Total cholesterol,Chloride, Total bilirubin, Bile acids

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes post-partum
- Battery of functions tested: Motor activity, Grip strength (hind and fore limb), Grasp response, Touch escape, Vocalization, Pupil reflex, Toe pinch, Blink reflex, Tail pinch, Startle reflex, Finger approach.

Date of pairing, Date of mating, Date and time of observed start of parturition, Date and time of observed completion of parturition
Sperm parameters (parental animals):
Detailed qualitative examination of the testes was undertaken, taking into account the tubular stages of the spermatogenic cycle. The examination was conducted in order to identify treatment-related effects such as missing germ cell layers or types, retained spermatids, multinucleated or apoptotic germ cells and sloughing of spermatogenic cells into the lumen.
Litter observations:
PARAMETERS EXAMINED
The following parameters were examined in offspring:
number and sex of pups, live births, postnatal mortality, weight (day 1 and 4), clinical signs, presence of gross anomalies, rightening reflex

Postmortem examinations (parental animals):
GROSS PATHOLOGY: Yes : on all animals

HISTOPATHOLOGY: Yes : on 5 animals/sex of the control and high dose group
Adrenals Muscle (skeletal) , Aorta (thoracic), Ovaries, Bone & bone marrow (femur including stifle joint), Bone & bone marrow (sternum), Brain (including cerebrum, cerebellum and pons), Prostate, Pancreas, Pituitary, Caecum, Rectum, Jejunum, Colon, Duodenum, Ileum (including Peyer’s patches) Coagulating gland, Salivary glands (submaxillary), Seminal vesicles, Epididymides, Skin (hind limb), Esophagus, Sciatic nerve, Spinal cord (cervical, mid-thoracic and lumbar), Eyes, Spleen, Heart, Stomach, Thyroid/parathyroid, Trachea, Kidneys, Testes , Liver, Thymus, Lungs (with bronchi), Urinary bladder, Lymph nodes (mandibular and mesenteric), Uterus/Cervix, Mammary gland, Vagina, Gross lesions
on all animals/sex from the control and the high dose group: reproductive organs, mammary gland and pituitary ; Uterus examined for implantation sites

Organ weights:
on 5 animals/sex of the control and high dose group: Adrenals, Kidneys, Brain, Liver, Thymus, Thyroid and parathyroids, Heart, Spleen
on all animals: Testes, Prostate, Seminal vesicle, Epididymides, Ovaries, Uterus(with Cervix), Pituitary
Postmortem examinations (offspring):
GROSS NECROPSY
Statistics:
Provantis Tables and Statistics Module including
•ANOVA
•Bartlett test
•Williams test or Shirley test
•Dunnett's or Steel test
•Student's t-test or Mann Whitney U-test
plus additional tests when the above mentioned methods were not analyzed by the Provantis data capture system
Reproductive indices:
Percentage Mating (%) = (Number of females mated/Number of females paired) x 100
Pregnancy rate (%) = (Number of females achieving pregnancy/Number of females mated) x 100
Gestation Index (%) = (Number of females with live pups/Number of females pregnant) x 100



Offspring viability indices:
Viability index (%) = (Number of alive pups on Day 4/Number of offspring alive on Day 1)x 100
Live birth index (%) = (Number of offspring alive on Day 1/ Number of offspring delivered)x 100




Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Salivation in males and females at 1000 mg/kg bw (related to test substance palatability)
Mortality:
mortality observed, non-treatment-related
Description (incidence):
2 Males (1 at 300 and 1 at 1000 mg/kg bw) died due to intubation errors
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Males: 300 and 1000 mg/kg bw: Hb significantly decreased: MCHC (significantly) decreased; 1000 mg/kg bw: increased WBC/leukocytes
Females: 1000 mg/kg bw: increased WBC/significantly increased neutrophiles
All effects were within historical background ranges
:
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
Males: 300 and 1000 mg/kg bw: significantly decreased inorganic phosphorus
Females: 300 and 1000 mg/kg bw: (significantly) decreased inorganic phosphorus; at 1000 mg/kg: significantly increased TP and cholesterol
All effects were within historical background ranges
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed
Description (incidence and severity):
MATING INDEX: 100% at all doses

PREGNANCY RATE: 100% at all doses

PARTURATION INDEX: 100% at all doses

Details on results (P0)

CLINICAL SIGNS AND MORTALITY:
2 Males (1 at 300 and 1 at 1000 mg/kg bw) died due to intubation errors
Salivation in males and females at 1000 mg/kg bw (related to test substance palatability)

BODY WEIGHT AND WEIGHT GAIN: No treatment related effects

FOOD CONSUMPTION : No treatment related effects

WATER CONSUMPTION : No treatment related effects

HAEMATOLOGY:
Males: 300 and 1000 mg/kg bw: Hb significantly decreased: MCHC (significantly) decreased; 1000 mg/kg bw: increased WBC/leukocytes
Females: 1000 mg/kg bw: increased WBC/significantly increased neutrophiles
All effects were within historical background ranges

CLINICAL CHEMISTRY:
Males: 300 and 1000 mg/kg bw: significantly decreased inorganic phosphorus
Females: 300 and 1000 mg/kg bw: (significantly) decreased inorganic phosphorus; at 1000 mg/kg: significantly increased TP and cholesterol
All effects were within historical background ranges

NEUROBEHAVIOUR: No treatment related effects

ORGAN WEIGHTS: No treatment related effects

GROSS PATHOLOGY: No treatment related effects

HISTOPATHOLOGY: No treatment related effects

SPERMATOGENESIS: No treatment related effects

IMPLEMENTATION SITES/CORPORA LUTEA: No treament related effects

MATING INDEX: 100% at all doses

PREGNANCY RATE: 100% at all doses

PARTURATION INDEX: 100% at all doses

Effect levels (P0)

Dose descriptor:
NOAEL
Effect level:
>= 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no effects

Results: F1 generation

General toxicity (F1)

Clinical signs:
not specified
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
VIABILITY INDEX (OFFSPRING): 100%, 99.3%, 99.3%, 98.6% at 0, 100, 300 and 1000 mg/kg bw

LIVE BIRTH INDEX (OFFSPRING): 100%, 97.6%, 98.5%, 100% at 0, 100, 300 and 1000 mg/kg bw

Body weight and weight changes:
no effects observed
Sexual maturation:
not specified
Organ weight findings including organ / body weight ratios:
not specified
Gross pathological findings:
no effects observed
Histopathological findings:
not specified

Developmental neurotoxicity (F1)

Behaviour (functional findings):
no effects observed
Description (incidence and severity):
no treatment related effects in rightening reflex

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Details on results (F1)

VIABILITY INDEX (OFFSPRING): 100%, 99.3%, 99.3%, 98.6% at 0, 100, 300 and 1000 mg/kg bw

LIVE BIRTH INDEX (OFFSPRING): 100%, 97.6%, 98.5%, 100% at 0, 100, 300 and 1000 mg/kg bw


BEHAVIOURAL EFFECTS (OFFSPRING): no treatment related effects in rightening reflex

BODY WEIGHT (OFFSPRING): no treatment related effects

SEX RATIO (OFFSPRING): no treatment related effects

MACROSCOPY (OFFSPRING): no treatment related effects

Effect levels (F1)

Dose descriptor:
NOAEL
Generation:
F1
Effect level:
>= 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no effects

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

Averages maternal animals

pregnancy: 12,12,12, 12 at 0, 100, 300 and 1000 mg/kg bw

corpora lutea: 13.3, 13.4, 12.3, 13.7 at 0, 100, 300 and 1000 mg/kg bw

implants: 12.8, 12.9, 12.0, 13.0 at 0, 100, 300 and 1000 mg/kg bw

pre-coital time: 1 -4 days, no treatment related effect

gestation length: 22 -23.5 days no treatment related effects

females with live offspring (day 1-4): 12, 12, 12,12 at 0, 100, 300 and 1000 mg/kg bw

Percentage Mating: 100% at all dose levels

Pregnancy rate: 100% at all dose levels

Gestation Index: 100% at all doselevels,

Applicant's summary and conclusion

Conclusions:
The NOAEL on reproductive and developmental effects is 1000 mg/kg bw.
The NOAEL for maternal toxicity is 1000 mg/kg bw
Executive summary:

Rats (12/sex/dose) received the test substance by gavage at 0, 100, 300 and 1000 mg/kg bw daily for at least 6 weeks (including two weeks prior to mating, through mating, pregnancy and early lactation for females). The test design followed was according to OECD 422.

Two males died due to intubation errors. No treatment related effects were found on clinical signs, body weight (gain), food and water consumption, haematology, clinical chemistry, behavioural assessments, organ weights macroscopic investigations and histology.

No effects were reported related to mating, fertility and reproduction (sperm quality, number corpora lutea, number implantation sites, pre- and postimplantion losses or sex ratio). No effects were found on pregnancy rate, gestation length, viability of the offspring, offspring weight or any other parameters observed.

Based on the above mentioned the NOAEL for systemic effects in parental animals is 1000 mg/kg bw. The NOAEL for effects on reproduction and development is 1000 mg/kg bw.