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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:
21 Nov 2013 - 16 Jul 2014 (date of final histopathology report)
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report Date:
2014

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
- Name of test material (as cited in study report): Amides, C16-18 (even), N,N'-ethylenebis
- Substance type: organic, UVCB
- Physical state: off white powder
- Analytical purity: not supplied
- Impurities (identity and concentrations): not supplied
- Composition of test material, percentage of components: N,N'-ethylenedi(stearamide) (10-72%); N,N'-ethane-1,2-diylbishexadecan-1-amide (5-45%); N-(2-Octadecanoylaminoethyl) hexadecanamide (25-50%); unidentified N,N'-ethylenebis fatty alkylamides
- Lot/batch No.: 302243
- Expiration date of the lot/batch: 07 June 2014
- Stability under test conditions: determination not possible due to chemical characteristics of test substance; therefore, formulations for application prepared daily prior to application
- Storage condition of test material: Room temperature in the dark

Test animals

Species:
rat
Strain:
other: Wistar Han:RccHan:WIST
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source:Harlan Laboratories U.K. Ltd., Oxon, UK
- Age at study initiation: 6-8 weeks
- Weight at study initiation: males 215-243 g, females 152-200 g
- Fasting period before study: no
- Housing: groups of 3 or 4 by sex, solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding (Datesand Ltd., Cheshire, UK). Environmental enrichment provided in form of wooden chew blocks and cardboard fun tunnels (Datesand Ltd., Cheshire, UK).
- Diet (e.g. ad libitum): Pelleted diet (Rodent 2014C Teklad Global Certified Diet, Harlan Laboratories U.K. Ltd., Oxon, UK): ad libitum.
- Water (e.g. ad libitum): Mains drinking water from polycarbonate bottles: ad libitum.
- Acclimation period: 8 days

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

IN-LIFE DATES: From: 21 Nov 2013 (first day of treatment) To: 19 Feb 2014 (final day of necropsy)

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
arachis oil
Remarks:
BP
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
As the method employed to measure formulation concentrations is not stability indicating, test item formulations were prepared freshly each day at the appropriate concentrations as a suspension in Arachis oil BP and used within two hours of preparation (stored under ambient condition between preparation and use).

VEHICLE
- Justification for use and choice of vehicle (if other than water): test substance insoluble in water
- Concentration in vehicle: 0, 25, 75, 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:
Due to the technical characteristics of the test item, a chromatographic method of analysis could not be developed, and a gravimetric method was instead employed to measure formulation concentrations. This involved washing the vehicle through a sintered crucible and weighing the residue of the test item.
Samples of dosing formulation were taken once weekly during the first month of treatment and thereafter once monthly and analysed for concentration of the test substance at Harlan Laboratories Ltd., Shardlow, UK, Analytical Services. The results indicate that the prepared test item formulations were within ± 8% of the nominal concentration.
Duration of treatment / exposure:
90 days
Frequency of treatment:
daily
Doses / concentrations
Remarks:
Doses / Concentrations:
100, 300, 1000 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Based on the results of a dose range finder study performed in advance to the main study.
- Rationale for animal assignment: random
- Rationale for selecting satellite groups: no satellite groups included
- Post-exposure recovery period in satellite groups: not applicable
- Section schedule rationale (if not random): no information provided
Positive control:
none

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: No

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: immediately before dosing, up to thirty minutes post dosing and one hour after dosing
- Clinical observations included: overt signs of toxicity, ill-health or behavioural change

BODY WEIGHT: Yes
- Time schedule for examinations: on Day 1 (prior to dosing), at weekly intervals thereafter and at terminal sacrifice

FOOD CONSUMPTION: Yes
- Food consumption for each cage group determined at weekly intervals throughout the study and mean daily diet consumption calculated as g food/animal/day: Yes

FOOD EFFICIENCY:
- Body weight gain per rat in g/food consumption per rat in g per unit time X 100 calculated as time-weighted averages (per week) from the group mean consumption and group mean body weight gain data: Yes

WATER CONSUMPTION: Yes
- Time schedule for examinations: daily for each cage group by visual inspection of the water bottles for any overt changes


OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: pre-treatment and before termination of treatment (during Week 12).
- Dose groups that were examined: all control and high dose animals. Examinations included observation of the anterior structures of the eye, pupillary and corneal blink reflex. Following pupil dilatation with 0.5% Tropicamide solution (Mydriacyl 0.5%, Alcon Laboratories (UK) Ltd., Hemel Hampstead, UK), detailed examination of the internal structure of the eye using a direct ophthalmoscope was performed.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: obtained from lateral tail vein on Day 90; where necessary, repeat samples were obtained by cardiac puncture prior to necropsy on Day 91 (animals killed by intravenous overdose of suitable barbiturate agent followed by exsanguination)
- Anaesthetic used for blood collection: No
- Animals fasted: No
- How many animals: all surviving animals from each test and control group
- Parameters examined: hemoglobin (Hb), erythrocyte count (RBC), hematocrit (Hct), mean corpuscular hemoglobin (MCH), mean corpuscular volume (MCV), mean corpuscular hemoglobin concentration (MCHC), total leucocyte count (WBC), differential leucocyte count: neutrophils (Neut), lymphocytes (Lymph), monocytes (Mono), basophils (Bas), platelet count (PLT), prothrombin time (CT), activated partial thromboplastin time (APTT)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: obtained from lateral tail vein on Day 90; where necessary, repeat samples were obtained by cardiac puncture prior to necropsy on Day 91 (animals killed by intravenous overdose of suitable barbiturate agent followed by exsanguination)
- Animals fasted: No
- How many animals: all surviving animals from each test and control group
- Parameters examined: urea, glucose, total protein (Tot.Prot.), albumin, albumin/globulin (A/G) ratio (by calculation), sodium (Na+), potassium (K+), chloride (CL-), calcium (Ca++), inorganic phosphorus (P), aspartate aminotransferase (ASAT), alanine aminotransferase (ALAT), alkaline phosphatase (AP), creatinine (Creat), total cholesterol (Chol), total bilirubin (Bili), bile acids

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Functional/behavioural observations were done prior to the start of treatment and at weekly intervals thereafter. During Week 12, approximately at the same time each occasion (at least 2 hours after dosing), functional performance tests were also performed together with an assessment of sensory reactivity to different stimuli. The evaluation period was 1 hour for each animal.
- Dose groups that were examined: all animals
- Battery of functions tested: sensory activity (grasp response, vocalisation, toe pinch, tail pinch, finger approach, touch escape, pupil reflex, blink reflex, startle reflex) / forelimb/hindlimb grip strength / motor activity / other: behavioural assessment (gait, tremors, twitches, convulsions, bizarre/abnormal/stereotypic behaviour, salivation, pilo-erection, exophthalmia, lachrymation, hyper/hypothermia, skin colour, respiration, palpebral closure, urination, defecation, transfer arousal, tail elevation)
Sacrifice and pathology:
GROSS PATHOLOGY: Yes: All animals were subjected to a full external and internal examination (not further specified), and any macroscopic abnormalities were recorded.
HISTOPATHOLOGY: Yes: Samples of the following tissues were removed from all animals and preserved in buffered 10% formalin, except where stated: adrenals, aorta (thoracic), bone & bone marrow (femur incl. stifle joint; retained only and not processed), bone & bone marrow (sternum), brain (incl. cerebrum, cerebellum, pons), caecum, colon, duodenum, epididymides (preserved in Bouin's fluid, then transferred to Industrial Methylated Spirit (IMS) approx. 48 h later; preserved in Modified Davidson's fluid), esophagus, eyes (fixed in Davidson's fluid), gross lesions, heart, ileum (incl. Peyer's patches), jejunum, kidneys, liver, lungs (with bronchi; lungs were inflated to approx. normal inspiratory volume with buffered 10% formalin before immersion in fixative), lymph nodes (mandibular and mesenteric), mammary glands, muscle (skeletal; retained only and not processed), ovaries, pancreas, pituitary, prostate, rectum, salivary glands (submaxillary), sciatic nerve, seminal vesicles, skin (hind limb), spinal cord (cervical, mid-thoracic, lumbar), spleen, stomach, testes (preserved in Bouin's fluid, then transferred to Industrial Methylated Spirit (IMS) approx. 48 h later; preserved in Modified Davidson's fluid), thymus, thyroid/parathyroid, tongue (retained only and not processed), trachea, urinary bladder, uterus (with cervix), vagina.

All tissues from control and 1000 mg/kg bw/day dose group animals were prepared as paraffin blocks, sectioned at a nominal thickness of 5 µm and stained with Hematoxylin and Eosin for subsequent microscopic examination. Any macroscopically observed lesions were also processed.
Other examinations:
ORGAN WEIGHTS: from all animals killed at the end of the study.
Adrenals, brain, epididymides, heart, kidneys, liver, ovaries, spleen,testes, thymus, uterus.
Statistics:
Data were processed to give summary incidence or group mean and standard deviation values where appropriate.
Where considered appropriate, quantitative data was subjected to statistical analysis to detect the significance of intergroup differences from control; statistical significane was achieved at a level of p<0.05. Statistical analysis was performed on the following parameters:
Grip strength, motor activity, body weight change, hematology, blood chemistry, absolute organ weights, body weight-relative organ weights.

Data were analysed using the decision tree from the Provantis Tables and Statistics Module as follows:
Where appropriate, data transformations were performed using the most suitable method. The homogeneity of variance from mean values was analsed using Bartlett's test. Intergroup variances were assessed using suitable ANOVA, or if required, ANCOVA with appropriate covariances. 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 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'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 were performed using the Student t-test (parametric) or the Mann-Whitney U test (non-parametric).

Probability values (p) are presented as follows:
p<0.01 **
p<0.05 *
p≥0.05 (not significant)

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Mortality:
no mortality observed
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:
no effects observed
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:
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
One 1000 mg/kg bw/day female (No. 76) was terminated on humane grounds on Study Day 29 due to a rapid decline in clinical condition. Clinical signs observed prior to termination involved abnormal respiration, prostration, dehydration and hypothermia. Macroscopic examination revealed reddening of the lungs and submandibular lymph nodes supported by histopathological findings of slight pulmonary congestion. Because of the isolated nature of this mortality and in view of the inferential evidence this death is assumed to be the result of a mal-dose and not associated with test item toxicity. There were no other deaths in the study.

There were no treatment-related clinical findings observed in the test or control animals killed at study termination. Incidental findings were confined to one 300 mg/kg bw/day male (No. 43) observed to have a scab on the head between Days 39 and 48.

BODY WEIGHT AND WEIGHT GAIN
No adverse effects on body weight and body weight gain were detected in either sex of test animals or controls throughout the treatment period.

Females treated at 300 and 1000 mg/kg bw/day showed a slight but statistically significant increase (p<0.05) in body weight gain during Week 6 of the study. However, an isolated increase in body weight gain is unlikely to represent an adverse effect on health and was therefore considered to be of no toxicological consequence.

FOOD CONSUMPTION
Dietary intake in test animals of either sex remained similar to that of the respective controls throughout the treatment period

FOOD EFFICIENCY
Food Efficiency (the ratio of body weight gain to dietary intake) in test animals of either sex remained similar to that of the respective controls throughout the treatment period.

WATER CONSUMPTION
Daily visual inspection of water bottles did not reveal any significant intergroup differences between control and treated animals.

OPHTHALMOSCOPIC EXAMINATION
No ocular changes were detected in any control or high dose animal prior to start of treatment or prior to termination.

HAEMATOLOGY
There were no adverse treatment-related effects detected in the hematological parameters examined.

Males treated at 1000 mg/kg bw/day were observed to have a statistically significant reduction (p<0.05) for partial thromboplastin time in comparison with the controls. However, given the absence of any supporting adverse changes, this isolated finding was considered incidental and of no toxicological importance.

CLINICAL CHEMISTRY
There were no adverse treatment-related effects detected in the blood chemistry parameters examined.

Incidental findings were characterised by a slight but statistically significant reduction (p<0.05) in total protein and plasma albumin levels in 1000 mg/kg bw/day with a similar trend for albumin also identified in 300 mg/kg bw/day males in comparison with controls. In addition, all dose groups displayed a slight reduction (p<0.01) in plasma potassium levels in comparison with controls. However, individual values were almost all within the anticipated historical ranges, and as there was no convincing dose-dependent response nor supporting evidence of liver or kidney impairment these intergroup differences were considered incidental and of no toxicological importance.

NEUROBEHAVIOUR
There were no treatment-related changes in the behavioural assessments.
There were no treatment-related changes in the functional performance tests performed.

Females treated at 100 and 300 mg/kg bw/day showed a statistically significant reduction (p<0.05) in one of three forelimb grip strength measurements in comparison with the female control. In the absence of a dose dependent trend or supporting evidence to suggest neurotoxicity, this finding was considered incidental and of no toxicological importance.

There were no treatment-related changes in the sensory reactivity assessments. All inter and intra group differences in sensory scores were considered to be a result of normal variation for rats of the species and strain used and, therefore, were considered to be of no toxicological significance.

ORGAN WEIGHTS
There were no treatment-related changes in organ weight detected.

Females treated at 300 mg/kg bw/day showed a slight but statistically significant increase (p<0.05) in both absolute and relative (to terminal body weight) ovary weight in comparison with controls. However, no such organ weight changes were detected among females treated at 1000 mg/kg bw/day, and there were no adverse histopathological findings in the ovaries; accordingly this intergroup difference was considered incidental and not to be related to treatment. Additionally, the observed value was well within the historical ranges of ovary weights for female rats of this strain and age.

GROSS PATHOLOGY
There were no treatment-related macroscopic abnormalities detected in animals examined at study termination.

Incidental changes were limited to one 300 mg/kg bw/day male (No. 47) noted to have an enlarged spleen; with no corroborating evidence to indicate a relationship to treatment this finding was attributed to biological variation. The remainig exception involved four instances of reddened lungs at necropsy, a finding occasionally seen at necropsy and consiedered to be associated with the terminal exsanguination procedure.

HISTOPATHOLOGY: NON-NEOPLASTIC
There were no treatment-related microscopic findings.

HISTORICAL CONTROL DATA
Normal ranges for hematological, blood chemical, absolute organ weight and body weight-relative organ weight values for rats of the strain and age used in the study have been provided, which had last been updated in October 2012.

Observed group means for the mentioned parameters were usually within the historical ranges; outliers can be explained by deviating values of single animals within a group as indicated by a high group-specific standard deviation. However, these were considered as typical biological variations occurring in rats of this strain and age and were, due to the absence of supporting histopathological or functional observations, considered as not related to treatment with the test substance.

Effect levels

Dose descriptor:
NOEL
Effect level:
>= 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No test item associated systemic toxicity up to the highest applied dose level

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

CONCLUSION

The oral administration of Amides, C16-C18 (even), N,N'-ethylenebis to rats by gavage, at dose levels of 100, 300, and 1000 mg/kg bw/day for ninety consequtive days did not result in any test item related systemic toxicity, and on this basis the 'No Observed Effect Level' (NOEL) for systemic toxicity was considered to be greater than 1000 mg/kg bw/day.

Applicant's summary and conclusion