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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2011-11-07 to 2012-04-24
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report date:
2012

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Qualifier:
according to guideline
Guideline:
EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.3100 (90-Day Oral Toxicity in Rodents)
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Specific details on test material used for the study:
Batch No.: 20091203
Purity: 99.42%

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories Ltd, Blackthorn, Bicester, Oxfordshire, United Kingdom
- Age at study initiation:
- Weight at study initiation: males 173 g to 209 g; females 147 g to 175 g.
- Fasting period before study:
- Housing: Groups of 3 or 4 by sex in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding
- Diet: Ad libitum - Rodent 2014C Teklad Global Certified Diet, Harlan Laboratories Ltd
- Water: Ad libitum - mains drinking water
- Acclimation period: 7 d

ENVIRONMENTAL CONDITIONS
- Temperature: Target of 21 ± 2 °C (with possible short term deviations that did not affect integrity of study).
- Humidity 55 % ± 15% (with possible short term deviations that did not affect integrity of study).
- Air changes: ≥ 15 per hr
- Photoperiod: 12 hrs dark / 12 hrs light

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
arachis oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The test item was prepared at the appropriate concentrations as a solution in Arachis oil BP. Results from a previous study showed the formulations to be stable for at least 21 days. Formulations were therefore prepared fortnightly and stored at approximately 4 °C in the dark.

VEHICLE
- Concentrations in vehicle: 2.5 mg/mL, 25 mg/mL, 250 mg/mL
- Amount of vehicle (if gavage): 4 mL/kg bw
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
- The concentration of DEHCH in the test item formulations was determined by gas chromatography (GC) using an external standard technique.
- The results indicated that the prepared formulations were within acceptable limits.
Duration of treatment / exposure:
90 d
Frequency of treatment:
Daily
Doses / concentrationsopen allclose all
Dose / conc.:
10 mg/kg bw/day (actual dose received)
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
10 animals per sex per dose
Control animals:
yes, concurrent vehicle
yes, historical
Details on study design:
- Dose selection rationale: Based on previous studies (Harlan study No. 3058-004)
- Rationale for animal assignment: Stratified body weight randomisation procedure; group mean body weights were then determined to ensure similarity between groups.
Positive control:
None

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS:
- Time schedule:
All animals were examined for overt signs of toxicity, ill-health or behavioural change immediately before dosing, up to 30 mins post-dosing and 1 and 5 hrs after dosing during the working week. Animals were observed immediately before and after dosing and 1 hr after dosing at weekends and public holidays. All observations were recorded.

FUNCTIONAL OBSERVATIONS:
Prior to the start of treatment and at weekly intervals thereafter, all animals were observed for signs of functional/behavioural toxicity. During Week 21, functional performance tests were also performed on all animals together with an assessment of sensory reactivity to different stimuli.
BEHAVIOURAL ASSESSMENTS:
Detailed individual clinical observations were performed for each animal using a purpose built arena. The following parameters were observed:
Gait, tremors, twitching, convulsions, bizarre/abnormal/stereotypic behavior, salivation, pilo-erection, exophthalmia, lachrymation, hyper/hypothermia, skin colour, respiration, palpebral closure, urination, defecation, transfer arousal and tail elevation.

FUNCTIONAL PERFORMANCE TESTS:
-Motor activity:
Twenty purpose-built 44 infra-red beam automated activity monitors were used to assess motor activity. Animals of one sex were tested at each occasion and were randomly allocated to the activity monitors. The tests were performed at approximately the same time each day, under similar laboratory conditions. The evaluation period was 1 h for each animal. The time in seconds each animal was active and mobile was recorded for the overall one hour period and also during the final 20 % of the period (considered to be the asymptotic period).
-Forelimb/hind-limb grip strength:
An automated grip-strength meter was used. Each animal was allowed to grip the proximal metal bar of the meter with its forepaws. The animal was pulled by the base of the tail until its grip was broken. The animal was drawn along the trough of the meter by its tail until its hind paws gripped the distal metal bar. The animal was pulled by the base of the tail until its grip was broken. A record of the force required to break the grip for each animal was made. Three consecutive trials were performed for each animal.

SENSORY REACTIVITY:
Each animal was individually assessed for sensory reactivity to auditory, visual and proprioceptive stimuli. The following parameters were observed;
Grasp response, vocalization, toe pinch, tail pinch, finger approach, touch escape, pupil reflex, blink reflex, startle reflex.

BODY WEIGHT (INDIVIDUAL):
- Time schedule for examinations: Day 1 and weekly thereafter

FOOD CONSUMPTION (PER GROUP)
- Recorded weekly for each group

WATER CONSUMPTION (PER GROUP)
- Recorded daily for each group

OPHTHALMOSCOPIC EXAMINATION:
- Time schedule for examinations: Pre-treatment, wk 12 (before termination of treatment)
- Dose groups that were examined: Control, 1000 mg/kg bw
- Examination included observation of the anterior structures of the eye, pupillary and corneal blink reflex.Following pupil dilation with 0.5 % Tropicamide solution, detailed examination of the internal structure of the eye using a direct ophthalmoscope was performed.

HAEMATOLOGY
- Time schedule for collection of blood: Day 90 (repeat samples on day 91 if necessary)
- Anaesthetic used for blood collection: No data
- Animals were not fasted prior to sampling.
- All animals examined.
- Parameters examined: Haemoglobin (Hb), erythrocyte count (RBC), haematocrit (Hct), erythrocyte indices (mean corpuscular haemoglobin (MCH), mean corpuscular volume (MCV), mean corpuscular haemoglobin concentration (MCHC)), total leucocyte count (WBC), Differential leucocyte counts (neutrophils (Neut), lymphocytes (Lymph), monocytes (Mono), eosinophils (Eos), basophils (Bas)), platelet count (PLT), Reticulocyte count (Retic) (methylene blue stained slides were prepared but reticulocytes were not assessed). Prothrombin time (CT) was assessed by 'Innovin' and Activated partial thromboplastin time (APTT) was assessed by 'Actin FS' using samples collected into sodium citrate solution (0.11 mol/L).

CLINICAL CHEMISTRY
- Time schedule for collection of blood: Day 90 (repeat samples on day 91 if necessary)
- Animals not fasted
- All animals examined
- Parameters examined: The following parameters were measured on plasma from blood collected into tubes containing lithium heparin anti-coagulant: Urea, glucose, total protein (Tot.Prot.), albumin, albumin/Globulin (A/G) ratio (by calculation), sodium (Na⁺), potassium (K⁺), chloride (Cl⁻), calcium (Ca²⁺), inorganic phosphorus (P), aspartate aminotransferase (ASAT), alanine aminotransferase (ALAT), alkaline phosphatase (AP), creatinine (Creat), total cholesterol (Chol), total bilirubin (Bili), bile acids (Bile),

URINALYSIS: Not performed
Sacrifice and pathology:
GROSS PATHOLOGY:
- On completion of the dosing period all animals were killed by intravenous overdose of sodium pentobarbitone followed by exsanguination.
- All animals were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.

ORGAN WEIGHTS:
The following organs, removed from animals that were killed at the end of the study, were dissected free from fat and weighed before fixation:
Adrenals, brain, epididymides, heart, kidneys, liver, ovaries, spleen, tested, thymus and uterus.

HISTOPATHOLOGY:
- Samples of the following tissues were removed from all animals and preserved in buffered 10 % formalin: Adrenals, aorta (thoracic), bone & bone marrow (femur including stifle joint), bone & bone marrow (sternum), brain (including cerebrum, cerebellum and pons), caecum, colon, duodenum, gross lesions, heart, ileum (including Peyer’s patches), jejunum, kidneys, pancreas, pituitary, rectum, salivary glands (submaxillary), sciatic nerve, skin (hind limb), spinal cord (cervical, mid-thoracic and lumbar), spleen, stomach, liver, lungs (with bronchi)#, lymph nodes (cervical and mesenteric), mammary glands, muscle (skeletal) oesophagus, thymus, thyroid/parathyroid, tongue, trachea, urinary bladder and uterus.
# Lungs were inflated to approximately normal respiratory volume with bufffered 10 % formalin before immersion in fixative.
All tissues from control and 1000 mg/kg bw/day dose group animals were prepared as parafin blocks, sectioned at a nominal thickness of 5 µm and stained with haematoxylin and eosin for subsequent microscopic examination. Any macroscopically observed lesions were also processed.
- Eyes were removed from all animals and fixed in Davidson’s fluid.
- Ovaries were removed from all female animals and preserved in buffered 10 % formalin.
- Prostate and seminal vesicles (including, coagulating gland) were removed from all male animals and preserved in buffered 10 % formalin.
- Epididymides and testes were removed from all male animals, preserved in Bouin's fluid and then transferred to Industrial Methylated Spirits (IMS) approx. 48 hrs later.
Statistics:
Data were processed to give group mean values and standard deviations where appropriate.

Where appropriate, quantitative data were analysed by the Provantis Tables and Statistics Module. For each variable, the most suitable transformation of the data was found, the use of possible covariates checked and the homogeneity of means assessed using ANOVA or ANCOVA and Bartlett’s test. The transformed data were analysed to find the lowest treatment level that showed a significant effect, using Williams' Test for parametric data or Shirley's Test for non-parametric data. If no dose response was found, but the data showed non-homogeneity of means, the data were analysed by a stepwise Dunnett (parametric) or Steel (non-parametric) test to determine significant differences from the control group. Finally, if required, pair-wise tests were performed using Student's t-test (parametric) or the Mann-Whitney U test (non-parametric).

In addition, histopathological findings were analysed using Fisher's exact test.

Results and discussion

Results of examinations

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
- There were no toxicologically significant effects detected throughout the study period.
-Animals of either sex treated with 1000 mg/kg bw/day showed episodes of increased salivation from Day 26 (males) and Day 53 (females) onwards and one female treated with 100 mg/kg bw/day also showed an isolated incident of increased salivation on Day 53.
- One male treated with 1000 mg/kg bw/day showed an isolated incident of red/brown staining around the mouth on Day 37. Observations of this nature are commonly observed following the oral administration of an unpalatable or slightly irritant test item formulation, and in the absence of any associated changes are considered not to be of toxicological importance.
- One male treated with 1000 mg/kg bw/day had an abnormal gait between Days 87 and 90. At necropsy this animal had a thickened/enlarged femur and this was considered to be the reason for the abnormal gait. In isolation this was considered not to be of toxicological importance.
- One male treated with 100 mg/kg bw/day had exophthalmia in the left eye between Days 68 and 90. In the absence of a true dose related response this finding was considered not to be of toxicological importance.
- One female reated with 100 mg/kg bw/day had a damaged tail tip from Day 3 onwards. Observations of this nature are commonly observed in group housed animals and are considered not to be related to treatment.
- Scab formation was evident in one control male between Days 45 and 56. In the absence of treatment, this was considered to be a low incidence finding occasionally observed in laboratory maintained rats.
Mortality:
no mortality observed
Description (incidence):
There were no unscheduled deaths during the study.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
-There were no toxicologically significant effects detected in body weight development.
- Males treated with 1000 and 100 mg/kg bw/day showed a statistically significant reduction (P < 0.05) in body weight gain during Week 11. No adverse effect was evident in the overall body weight gain for these males and in isolation was considered not to be of toxicological importance.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
- No adverse effect in overall food consumption was detected in treated animals when compared to controls.
Food efficiency:
no effects observed
Description (incidence and severity):
- No adverse effect in food efficiency was detected in treated animals when compared to controls.
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
- There were no treatment related effects detected in water consumption.
Ophthalmological findings:
no effects observed
Description (incidence and severity):
- There were no treatment related ocular effects detected.
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
- There were no toxicologically significant effects detected in the haematological parameters examined.
- Males treated with 100 mg/kg bw/day showed a statistically significant increase (P < 0.05) in mean corpuscular haemoglobin concentration whilst females from this treatment group showed a statistically significant increase (P < 0.05) in neutrophil count. In the absence of a true dose related response the intergroup differences were considered not to be of toxicological significance.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
- There were no toxicologically significant effects detected in the blood chemical parameters examined.
- Males treated with 1000 mg/kg bw/day showed statistically significant increases (P < 0.01) in sodium and phosphorus concentrations and a statistically significant reduction (P < 0.05) in alkaline phosphatase. Males treated with 100 mg/kg bw/day also showed an increase in phosphorus. Males from all treatment groups and females treated with 1000 mg/kg bw/day showed a statistically significant increase (P < 0.05 - 0.01) in potassium concentration. Females from all treatment groups also showed a statistically significant increase (P < 0.01) in calcium concentration. The majority of individual values were within normal ranges for rats of the strain and age used and in the absence of any associated histology correlates the intergroup differences were considered not to be of toxicological importance.
- Females from all treatment groups showed statistically significant increases (P < 0.05) in total protein and albumin levels. The majority of individual values were within normal ranges for rats of the strain and age used and in the absence of true dose related responses the intergroup differences were considered not to be of toxicological importance.
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
- Weekly open field arena observations did not reveal any treatment-related effects for treated animals when compared to controls. All inter- and intragroup differences in urination, defecation and transfer arousal scores were considered to be a result of normal variation for rats of the strain and age used, and the differences were of no toxicological importance.

FUNCTIONAL PERFORMANCE TESTS
- There were no toxicologically significant changes in functional performance.
- Males from all treatment groups showed a statistically significant reduction in overall activity. In the absence of a true dose related response or any supporting clinical observations to suggest an effect of neurotoxicity, the intergroup differences were considered not to be of toxicological significance.
- Females treated with 1000 mg/kg bw/day showed a statistically significant increase in mean fore limb grip strength. The statistical significance was minimal (P < 0.05) and in the absence of any supporting clinical observations to suggest an effect of neurotoxicity, the finding was considered to be of no toxicological significance.

SENSORY REACTIVITY ASSESSMENTS
There were no treatment-related changes in sensory reactivity.
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
- There were no toxicologically significant effects detected in the organ weights measured.
- Males treated with 1000 mg/kg bw/day showed a statistically significant (P < 0.05) increase in spleen weight both absolute and relative to terminal body weight. In the absence of any associated histology correlates the intergroup difference was considered not to be of toxicological importance.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
- There were no toxicologically significant macroscopic abnormalities detected.
- One male treated with 1000 mg/kg bw/day had increased renal pelvic space in the right kidney. This effect is considered to be a congenital abnormality and unrelated to treatment.
- One male treated with 1000 mg/kg bw/day had a thickened/enlarged femur. Microscopic examination revealed a bone fraction reaction and therefore in isolation this was considered unrelated to treatment.
- One male treated with 100 mg/kg bw/day had an enlarged left eye. In the absence of a true dose related response the intergroup difference was considered not to be of toxicological importance.
- One control female and one female treated with 10 mg/kg bw/day had reddened lungs at necropsy. In the absence of a dose related response or the administration of test item in the case of the control female the intergroup differences were considered to incidental.
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
- There were no treatment related microscopic abnormalities detected.
- The findings recorded were within the range of normal background lesions which may be recorded in rats of the strain and age used.

Effect levels

Key result
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: overall effects

Target system / organ toxicity

Key result
Critical effects observed:
no

Applicant's summary and conclusion

Conclusions:
In a study to OECD Guideline 408 and to GLP, the authors determined that the 90 d oral NOAEL of the test substance in rats was ≥ 1000 mg/kg bw/d.