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Description of key information

Twelve rats/sex were treated with the test substance (oral gavage) at 0, 100, 300 and 1000 mg/kg bw during 14 days (pre-mating), during mating and 14 days thereafter (males). Females were further treated during gestation and until 5 days post-partum. No adverse effects on mortality, clinical and functional observations, body weight, food and water consumption, haematology and clinical chemistry, macroscopy, organ weights and histopatology were found. Incidental findings reported were within historical control values and without a dose-response relationship. Therefore the NOAEL is considered 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:
12-03-2013 to
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Stusy according to the guidelines under GLP
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
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Strain: Wistar Han™:RccHan™:WIST strain
- Source: Harlan Laboratories U.K. Ltd., Blackthorn, Bicester, Oxon, UK
- Age at study initiation: ca. 12 weeks
- Weight at study initiation: males 313 - 387g; females 192 - 236g,
- Fasting period before study:
- Housing: pre-mating (males post mating) 4/cage in solid floor polypropylene cages; during mating 1M/1F in polypropylene grid floor cages; gestation/lactation: individually in solid floor polypropylene cages with stainless steel mesh lids and softwood flakes
- Diet: Rodent 2018C Teklad Global Certified Diet, Harlan Laboratories U.K. Ltd., Oxon, UK ad libitum
- Water: ad libitum
- Acclimation period: 14 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 2 °C
- Humidity (%): 55 ± 15%
- Air changes (per hr): ≥ 15/hour
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From 09 May 2013 (first day of treatment) to 23 June 2013 (final day of necropsy).
Route of administration:
oral: gavage
Vehicle:
arachis oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:weekly (based on outcome of stability test)

VEHICLE: Arachis oil BP (total volume 4 mL/kg)
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analyses By HPLC-MS (one test-item related peak)

Analytical standard: standard solution of 0.01 mg/L in methanol
Linearity: proven over 0-0.01638 mg/L
recovery: 98-102% of nominal (fortified samples)
accuracy of preparation : 93-109% of nominal (4 weekly samples)
stability over 21 days:93-97% of initial
Duration of treatment / exposure:
Males: 14 days prior to mating; upto 14 days during mating and 14 days post-mating
Females: 14 days prior to mating; upto 14 days during mating; average 21 days of gestation; 4 days of lactation
Frequency of treatment:
once daily
Remarks:
Doses / Concentrations:
0, 30, 300 and 1000 mg/kg bw
Basis:
actual ingested
No. of animals per sex per dose:
12/sex/dose
Control animals:
yes, concurrent vehicle
Details on study design:
Dose selection rationale: based on outcome of dose range finding study where no signs of toxicity were observed at 1000 mg/kg bw
Positive control:
NA
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: week days : before dosing, soon after dosing, and one hour and five hours after dosing
weekend and holidays: before dosing, soon after dosing, and one hour after dosing
- Cage side observations checked were included.in tables in the report

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly on all animals individually
- Check for Gait, Hyper/Hypothermia, Tremors, Skin colour, Twitches, Respiration. Convulsions, Palpebral closure, Bizarre/Abnormal/Stereotypic behaviour, Urination, Salivation, Defecation, Pilo-erection, Transfer arousal, Exophthalmia, Tail elevation, Lachrymation

BODY WEIGHT: Yes
- Time schedule for examinations: weekly, for females after mating on day 0, 7, 14 and 20 of gestation and day 1 and 4 of lactation

FOOD CONSUMPTION: yes
- Time schedule for examinations: weekly (cage basis), for females after mating on day 0, 7, 14 and 20 of gestation and day 1 and 4 of lactation

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Yes based on the cage side measurements of food consumption

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes / No / No data
- Time schedule for examinations: daily during pre-mating period

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: males on day 42, females on day 4 of lactation
- Anaesthetic used for blood collection: No, withdrawal from the lateral tail vein
- Animals fasted: No
- How many animals: 5/sex of control and 1000 mg/kg bw groups
- Parameters checked: Haemoglobin (Hb), Erythrocyte count (RBC), Haematocrit (Hct), mean corpuscular haemoglobin (MCH), mean corpuscular volume (MCV), mean corpuscular haemoglobin concentration (MCHC), Total leucocyte count (WBC), neutrophils (Neut), lymphocytes (Lymph), monocytes (Mono), eosinophils (Eos), basophils (Bas), platelet count (PLT), Reticulocyte count (Retic), PTT and APTT

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: males on day 42, females on day 4 of lactation
- Anaesthetic used for blood collection: No, withdrawal from the lateral tail vein
- Animals fasted: No
- How many animals: 5/sex of control and 1000 mg/kg bw groups
- Parameters checked. Urea, Calcium (Ca++),,Glucose, Inorganic phosphorus (P),Total protein (Tot.Prot.),Aspartate aminotransferase (ASAT),Albumin Alanine aminotransferase (ALAT), Albumin/Globulin (A/G) ratio (by calculation) ,Alkaline phosphatase (AP), Sodium (Na+), Creatinine (Creat),,Potassium (K+), total cholesterol (Chol), Chloride (Cl-), Total bilirubin (Bili), Bile acids

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations:
- Dose groups that were examined: 5/sex of control and 1000 mg/kg bw groups
- Battery of functions tested: motor activity, hind/forelimb grip strength, sensory reactivitys (Grasp response, Touch escape, Vocalisation, Pupil reflex, Toe pinch, Blink reflex, Tail pinch, Startle reflex, Finger approach)

Sacrifice and pathology:
Sacrifice: males on day 43, non-pregant females on day 25 after mating, females and offspring on day 5 post-partum by overdose of pentobarbitone

GROSS PATHOLOGY: Yes
full necropsy on all animals. For females examination of the uterus for implementation sites and corpora lutea (if necessary Salewski staining)

ORGAN WEIGHTS:Yes of 5/sex of the control and high dose group
Adrenals, Prostate*, Brain, Seminal vesicles*, Epididymides*, Spleen, Heart, Testes*, Kidneys, Thymus, Liver, Thyroid (weighed post-fixation with Parathyroid), Ovaries*, Uterus (weighed with Cervix)*, Pituitary (post fixation)*
* weighed from all remaining animals

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

The tissues shown with * from the remaining control and 1000 mg/kg bw/day animals and animals which did not achieve a pregnancy were also processed. In addition, sections of testes and epididymides from all control and 1000 mg/kg bw/day males were also stained with Periodic Acid-Schiff (PAS) stain and examined.

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.


Other examinations:
i. Pre-coital Interval

ii. Fertility Indices
-Mating Index (%)
-Pregnancy Index (%)

iii Gestation Length

iv. Parturition Index (%)

v Implantation Losses (%)

vi Live Birth and Viability Indices

vii Sex Ratio (% males)
Statistics:
Data were analysed using the decision tree from the Provantis TM Tables and Statistics Module as detailed as follows:

Where appropriate, data transformations were performed using the most suitable method. The homogeneity of variance from mean values was analysed using Bartlett’s test. Intergroup variance were assessed using suitable ANOVA, or if required, ANCOVA with appropriate covarities. Any transformed data were analysed to find the lowest treatment level that showed a significant effect using the Williams Test for parametric data or the Shirley Test for nonparametric data. If no dose response was found but the data shows non-homogeneity of means, the data were analysed by a stepwise Dunnett’s (parametric) or Steel (non-parametric) test to
determine significant difference from the control group. Where the data were unsuitable for these analyses, pair-wise tests was performed using the Student t-test (parametric) or the MannWhitney U test (non-parametric).
Data not analysed by the Provantis data capture system were assessed separately using the SPSS statistical package. Initially, the homogeneity of the data was assessed using Levene’s test. Where Levene’s test was shown to be non-significant (p≥0.05), parametric analysis of the data was applied, incorporating analysis of variance (ANOVA). If this data was shown to be significant, this analysis was followed by pair-wise comparisons using Dunnett’s test. Where
Levene’s test was significant, non-parametric analysis of the data was analysed incorporating the Kruskal-Wallis test which if significant, was followed by the Mann-Whitney U test. Dose response relationship was also be investigated by linear regression. Where the data was unsuitable for these analyses, then pair-wise tests were performed using the Student t-test (parametric) or the Mann-Whitney U test (non-parametric).

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
increased in females at 1000 mg/kg bw during gestation
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
increased in females at 1000 mg/kg bw during gestation
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Description (incidence and severity):
increased in females at 300 and 1000 mg/kg bw during gestation
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
see below
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: no treatment related effects

BODY WEIGHT AND WEIGHT GAIN: at 1000 mg/kg bw increased in females during gestation

FOOD CONSUMPTION: at 1000 mg/kg bw increased in females during gestation

FOOD EFFICIENCY: no treatment related effects

WATER CONSUMPTION: at 300 and 1000 mg/kg bw increased in females

HAEMATOLOGY: decreased haematocrit in males at all dose levels (no dose-response relationship and within historical data)

CLINICAL CHEMISTRY: decreased creatinine in males at all dose levels (no dose-response relationship and within historical data)

NEUROBEHAVIOUR: no treatment related effects

ORGAN WEIGHTS: decreased thyroid weight in males at all dose levels (no dose-response relationship and within historical data), Increased weights of pituitary and ovaries in females at 1000 mg/kg bw

GROSS PATHOLOGY: no treatment related effects

HISTOPATHOLOGY: no treatment related effects

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 adverse effects were noted that could be related to treatment with the test substance.
Critical effects observed:
not specified
Conclusions:
The NOAEL for parental animals in a repeated dose-reproduction test according to OECD 422 is 1000 mg/kg bw
Executive summary:

Twelve rats/sex were treated with the test substance (oral gavage) at 0, 100, 300 and 1000 mg/kg bw during 14 days (pre-mating), during mating and 14 days thereafter (males). Females were further treated during gestation and until 5 days post partum. No adverse effects on mortality, clinical and functional observations, body weight, food and water consumption, haematology and clinical chemistry, macroscopy, organ weights and histopatology were found. Incidental findings reported were within historical control values and without a dose-response relationship. Therfore the NOAEL is considered 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

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
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
The oral toxicity test did not show systemic effects at the highest dose tested. The NOAEL of 1000 mg/kg bw is considered suitable to be the starting point for risk assessment. The outcome of the test can be extrapolated to a dermal NOAEL based on route-to-route extrapolation. The dermal absorption is set at 10%, which is similar to that for oral absorption.

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available
Quality of whole database:
No local effects were seen in the irritation tests and in the mouse lymphoma test up to a concentration of 5%

Additional information

Justification for selection of repeated dose toxicity inhalation - systemic effects endpoint:
Based on the vapour pressure (2.3E-03 Pa at 25 °C) and the extreme viscosity of the test substance, exposure via the inhalation route is considered negligible.

Justification for selection of repeated dose toxicity inhalation - local effects endpoint:
Based on the vapour pressure (2.3E-03 Pa at 25 °C) and the extreme viscosity of the test substance, exposure via the inhalation route is considered negligible.

Justification for classification or non-classification

Based on the outcome of the repeated dose reproduction study no classification for toxicity after repeated exposure is considered necessary.