Reaction mass of N,N-ethanediylbis- Hexadecanamide, N-[2-[(1-oxohexadecyl)amino]ethyl]-Octadecanamide, Hexadecanoic acid, 12-hydroxystearic acid, reaction products with ethylenediamine, N,N-ethanediylbis-Octadecanamide and Octadecanoic acid, 12-hydroxystearic acid, reaction products with ethylenediamine

Endpoint summary

Description of key information

NOEL (subchronic, oral, rat; structural analogue) ≥ 1000 mg/kg bw/day (male/female)

NOEL (14 days DRF, oral, rat) ≥ 1000 mg/kg bw/day (male/female)

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

weight of evidence

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.

Qualifier:

according to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

other: Wistar Han:RccHan:WIST

Sex:

male/female

Details on test animals or test system 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)

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

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

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)

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.

Key result

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

Key result

Critical effects observed:

no

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.

Conclusions:

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.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

The available information comprises adequate, reliable (Klimisch score 1 and 2, respectively) and consistent studies, and is thus sufficient to fulfil the standard information requirements set out in Annex VIII-X, 8.6., of Regulation (EC) No 1907/2006.

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

Additional information

Oral

Subacute

A 14 day dose range finder with Licolub FA 6 resulted in no effects up to the limit dose of 1000 mg/kg bw/d.

Subchronic

A subchronic oral toxicity study performed with the structural analogue, Licowax C, according to OECD guideline 408 in male and female Wistar rats is available (McRae, 2014). The test substance was administered by gavage to three groups, each of 10 male and female Wistar strain rats, for up to 90 consecutive days at dose levels of 100, 300 and 1000 mg/kg bw/day. A control group of 10 males and females was dosed with vehicle alone. Clinical signs, functional observations, body weight change, dietary intake and water consumption were monitored during the study. Hematology and blood chemistry were evaluated for all animals at the end of the study. An ophthalmoscospic examination was also performed on control group and high dose animals. All animals were subjected to gross necropsy examination, and histopathological examination of selected tissues including reproductive organs and tissues from the high dose and control animals was performed at the end of the study.

There were no treatment-related effects observed in any of the investigated parameters. The oral administration of Amides, C16 -C18 (even), N,N'-ethylenebis to rats by oral gavage at dose levels of 100, 300 and 1000 mg/kg bw/day did not result in any test substance-related systemic toxicity.

Based on the results of the study the subchronic NOEL for systemic toxicity of the test substance administered to male and female Wistar rats by oral gavage was determined to be ≥ 1000 mg/kg bw/day. There had been no test substance-related findings up to and including the highest administered dose of 1000 mg/kg bw/day.

Chronic

In a 2-year feeding study, rats were exposed to the structural analogue, Licowax C, at dose levels of 5000, 20000 and 50000 ppm via the feed, which were equivalent to 250, 1000 and 2500 mg/kg bw/day (Weigand, 1963). The doses were converted assuming an animal weight of 400 g and a daily food consumption of 20 g/day, according to the recommendations of the WHO or the Toxicologist's pocket Handbook, CRC press, 2000. In the study, mortality, clinical signs, body weights, haematology and urinalysis parameters were assessed, and all survivors of the 104-week exposure period and all animals deceased before scheduled sacrifice were subjected to macroscopic and histopathological examination of heart, lung, liver, kidney and spleen. Despite the unusual high dose for chronic exposure, no adverse effects related to treatment could be detected after 104 weeks of exposure. Frequency and characteristics of findings, including the sporadic occurrence of tumours in the experimental groups, did not differ significantly from those observed in the control group.

Based on the results of that study the chronic NOAEL for Amides, C16-C18 (even), N,N'-ethylenebis for rats was considered to be ≥ 2500 mg/kg bw/day. This assumption is rather conservative, since the actual NOAEL might range between 5000-7500 mg/kg bw/d, based on the authors information that daily food consumption equalled about 10-15% of the animal’s body weight.

Although giving useful information on repeated dose toxicity in rodents the study does not have the potential for a key study according to today's standards. It was conducted according to the standards of 1960, and many details required for an appropriate study nowadays are missing, e.g. information on the effects on reproductive organs. Therefore, the study as such does not fulfil the requirements for a key study, it can only be of supporting character.

Therefore, especially in the light of the extremely high NOAEL ≥ 2500 mg/kg bw/day observed in the chronic study with Amides, C16-C18 (even), N,N'-ethylenebis, the use of a NOEL ≥ 1000 mg/kg bw/day from the subchronic toxicity study is considered as justified and sufficiently conservative for risk assessment.

Justification for classification or non-classification

The available data on the repeated dose toxicity of Licolub FA 6 and its structural analogue via the oral route do not meet the criteria for classification according to Regulation (EC) 1272/2008 and are therefore conclusive but not sufficient for classification.