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Administrative data

Description of key information

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.
Based on the absence of treatment related findings it is concluded that the NOAEL 1000 mg/kg bw.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: oral
Remarks:
combined repeated dose and reproduction / developmental screening
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
Qualifier:
equivalent or similar 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
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

Route of administration:
oral: gavage
Vehicle:
arachis oil
Details on oral 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
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 period 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 period, gestation and lactation and at least up to and including the day before sacrifice (day 4 post partum).
Frequency of treatment:
daily
Remarks:
Doses / Concentrations:
0, 100, 300 and 1000 mg/kg bw
Basis:
actual ingested
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.
Observations and examinations performed and frequency:
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
- Time schedule: 5 males/dose week 6 of treatment (post-pairing), 5 females/dose day 4 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.
Sacrifice and pathology:
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

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
Other examinations:
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.
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
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
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
not examined
Details on results:
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
Key result
Dose descriptor:
NOAEL
Remarks:
systemic
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
Critical effects observed:
not specified
Conclusions:
The NOAEL for systemic effects in this study 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.

Based on the absence of treatment related findings it is concluded that the NOAEL 1000 mg/kg bw.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
Klimisch 1

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Mode of Action Analysis / Human Relevance Framework

No effects that could be related to effects of the substance were observed.

Additional information

Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
Guideline study under GLP

Justification for classification or non-classification

Based on the absence of toxicity, the substance does not need to be classified.