1,2,4-Oxadiazole, 3-phenyl-5-(2-thienyl)-

Repeated dose toxicity: dermal

Administrative data

Endpoint:

short-term repeated dose toxicity: dermal

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From September 4, 2012 to January 11, 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study was conducted according to OECD Guideline 410 and OPPTS Guideline 870.3200, in compliance with GLP.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 410 (Repeated Dose Dermal Toxicity: 21/28-Day Study)

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3200 (Repeated Dose Dermal Toxicity -21/28 Days)

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

3-phenyl-5-(thiophen-2-yl)-1,2,4-oxadiazole

EC Number:

810-533-8

Cas Number:

330459-31-9

Molecular formula:

C12H8N2OS

IUPAC Name:

3-phenyl-5-(thiophen-2-yl)-1,2,4-oxadiazole

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

- Name of test material (as cited in study report): MON 102100

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Inc., Raleigh
- Age at study initiation: 44 d old
- Housing: Individually in clean, stainless steel, wire-mesh cages suspended above cage-board. Racks were rotated within the animal room at least once every two weeks during the study to change the physical location from one area of the room to another to ensure similarly varied environmental exposure for all animals. Animals were maintained in accordance with the Guide for the Care and Use of Laboratory Animals (National Research Council, 1996).
- Diet: PMI Nutrition International, LLC, Certified Rodent LabDiet® #5002. Basal diet were provided ad libitum throughout the study, except during the period of fasting prior to clinicalpathology blood collection.
- Water: Reverse osmosis-treated (on-site) drinking water, delivered by an automatic watering system, provided ad libitum
- Acclimation period: 14 d. During acclimation period, each animal was observed twice daily for mortality and changes in general appearance or behavior.

The results of the diet and water analyses are maintained at WIL Research. No contaminants were present in animal feed or water at concentrations sufficient to interfere with the objectives of this study.

ENVIRONMENTAL CONDITIONS
- Temperature: 21.2°C - 22.0°C
- Humidity:34.1% - 48.3%
- Air changes (per h): 10
- Photoperiod: 12 h light/12 h dark cycle

Administration / exposure

Type of coverage:

other: Skin was covered with a wrap consisting of gauze secured in place with non-irritating tape

Vehicle:

water

Details on exposure:

On the day prior to dosing, and during the study as needed (after completion of the daily exposure), the hair was clipped from the back (down each side to the ventral surface) and flanks of each animal using an electric clipper; a different set of clippers was used for the control and test substance-treated groups to avoid potential cross contamination.

Test substance moistened with the vehicle, deionized water (0.6 or 0.7 mL), was administered by topical application to the shaved intact dorsal surface (covering approximately 10% of the body surface area) of three groups of rats once daily for up to 28 consecutive days. The test substance was allowed to remain in contact with the skin for 6 h (±30 min), during which time the treated skin was covered with a wrap consisting of gauze secured in place with non-irritating tape, and the rats wore Elizabethan collars. At the end of the 6 h (±30 min) exposure period, the collars and
dressings were removed and the test sites were wiped and cleaned using gauze soaked in deionized water to remove any remaining test substance from the skin. A concurrent control group received the vehicle on a comparable regimen.

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

28 d

Frequency of treatment:

Daily

Doses / concentrations

Remarks:

Doses / Concentrations:
100, 300 and 1,000 mg/kg bw/day
Basis:

No. of animals per sex per dose:

10 males and 10 females per dose group

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Dose levels were selected based on using the recommended limit test exposure level of 1,000 mg/kg/day as the Group 4 target exposure concentration (references in the OPPTS 870.3200 testing).

- Four days prior to the initiation of dose administration, all available rats were weighed and examined in detail for physical abnormalities. The animals judged suitable for assignment to the study were selected for use in a computerized randomization procedure based on body weight stratification in a block design. Individual body weights at randomization were within ± 20% of the mean for each sex.

Examinations

Observations and examinations performed and frequency:

SURVIVAL
All animals were observed twice daily, once in the morning and once in the afternoon, for mortality and moribundity.

On Day 0 (first dose), one male and one female from 300 mg/kg bw/day dose group were found dead prior to completion of the 6 h (±30 min) dosing period and were replaced with other male and female replacement animals, prior to dosing on the following day; therefore, these animals were only dosed for 27 consecutive days. Also on Day 0, one female (300 mg/kg bw/day) died during dose application and was replaced with other replacement female. These early deaths were considered to be procedure-related and not considered test substance-related.

CLINICAL OBSERVATIONS
Clinical examinations were performed twice daily, prior to dose administration and 1 to 2 h following bandage removal. The absence or presence of findings was recorded for individual animals at the scheduled intervals. Detailed physical examinations were conducted on all animals within four days of receipt, on the day of randomization, and weekly (± 1 d) during the dosing period, and on the day of the scheduled necropsy. Any observations noted outside of the above specified intervals were also recorded.

DERMAL OBSERVATIONS:
Dermal observations of the dosing site were conducted during the week prior to randomization (± two days), on the day of randomization, prior to dosing on study day 0, weekly (± 2 d; prior to dosing) during the dosing period, and on the day of the scheduled necropsy for erythema and oedema in accordance with a four step grading system, of very slight, slight, moderate, and severe. Other remarkable dermal findings, if present, were recorded. The presence or absence of findings was recorded for individual animals.

BODY WEIGHTS
Individual body weights were recorded within four days of receipt, on the day of randomization, on study Day -1, weekly (± one day) throughout the study, and on the day prior to the scheduled necropsy (non-fasted). Mean body weights and mean body weight changes were calculated for the corresponding intervals. Final body weights (fasted) were recorded on the day of the scheduled necropsy.

FOOD CONSUMPTION
Individual food consumption was calculated beginning one week prior to randomization (± two days), on the day of randomization, and weekly (± one day) throughout the study. Food intake was calculated as g/animal/day and g/kg/day for the corresponding body weight intervals. In addition, food efficiency (body weight gained as percent of feed consumed) was calculated.

CLINICAL PATHOLOGY:
Blood and urine samples for clinical pathology evaluations (haematology, coagulation, serum chemistry, and urinalysis) were collected from all animals at the scheduled necropsy (study Day 28). The animals were fasted overnight prior to blood collection while in metabolism cages for urine collection. Blood was collected for haematology and serum chemistry evaluation via the retro-orbital sinus of animals anesthetized by inhalation of isoflurane. The site of collection, including left or right, was documented for each animal. Blood was collected for coagulation parameters at the time of euthanasia via the vena cava of animals euthanized by inhalation of carbon dioxide. Blood was collected into tubes containing potassium EDTA (haematology), sodium citrate (coagulation), or no anticoagulant (serum chemistry).

HEMATOLOGY AND COAGULATION PARAMETERS: Total leukocyte count (WBC), erythrocyte count (RBC), haemoglobin (HGB), haematocrit (HCT), mean corpuscular volume (MCV), mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration (MCHC), platelet count (PLATELET), prothrombin time (PT), activated partial thromboplastin time, reticulocyte count percent (RETIC), absolute (RETIC ABSOLUTE), differential leukocyte count, percent a and absolute, haemoglobin distribution width, platelet estimate, and red cell morphology (RBC Morphology).

SERUM CHEMISTRY: Total protein, globulin albumin/globulin ratio (A/G ratio), total bilirubin (total Bili), urea nitrogen, creatinine, alkaline phosphatase (ALP), alanine aminotransferase (ALT), aspartate aminotransferase (AST), gamma glutamyltransferase (GGT), glucose, total cholesterol, calcium, chloride, phosphorus, potassium, sodium, sorbitol dehydrogenase (SDH), triglycerides (Triglyceride), haemolysis, lipemia and Icterus.

URINALYSIS: Parameters evaluated were specific gravity (SG), pH, urobilinogen (URO), total volume (TVOL), colour (COL), clarity (CLA), protein (PRO), glucose (GLU), ketones (KET), bilirubin (BIL), occult blood (BLD), leukocytes (LEU), nitrites (NIT) and microscopy of sediment.

OPHTHALMIC EXAMINATIONS
Ocular examinations were conducted on all animals prior to randomization (study Day -8) and near the end of the dosing period (study Day 27). All ocular examinations were conducted using an indirect ophthalmoscope and slit lamp biomicroscope preceded by pupillary dilation with an appropriate mydriatic agent.

Sacrifice and pathology:

MACROSCOPIC EXAMINATION:
A complete necropsy was conducted on all animals. Animals were euthanized by carbon dioxide inhalation followed by exsanguination. The necropsies included, but were not limited to, examination of the external surface, all orifices, and the cranial, thoracic, abdominal, and pelvic cavities, including viscera. The tissues and organs were collected for analysis were adrenals, aorta, bone with marrow, femur, sternum, bone marrow smear, brain, cerebrum level, cerebrum level, cerebellum with medulla/pons, cervix, epididymides, eyes with optic nerve, gastrointestinal tract, oesophagus, stomach, duodenum, jejunum, ileum, cecum, colon, rectum, heart, kidneys, larynx, liver (sections of 2 lobes), lungs (including bronchi, fixed by inflation with fixative), lymph nodes, nasal cavity, ovaries with oviducts, pancreas, peripheral nerve (sciatic), peyer’s patches, pituitary, pharynx, prostate, salivary glands, seminal vesicles, skeletal muscle (rectus femoris), skin (with mammary gland), spinal cord (cervical, thoracic, lumbar), spleen, testes, thymus, thyroid (with parathyroid, if present), trachea, urinary bladder, uterus, vagina, gross lesions.

ORGAN WEIGHTS:
The organs weighed from all animals at the scheduled necropsy were adrenals, brain, epididymides, heart, kidneys, liver, ovaries with oviducts, spleen, testes, thymus, thyroid with parathyroids and uterus.

SLIDE PREPARATION AND MICROSCOPIC EXAMINATION:
Microscopic examination was performed on all tissues listed under macroscopic examination from all animals in the control and 1,000 mg/kg bw/day groups at the scheduled necropsy. In addition, gross lesions were examined from all animals and correlated to microscopic findings, if possible. Adrenal glands, liver, and skin (treated and untreated) were examined from the 100 and 300 mg/kg bw/day groups. Missing tissues were identified as not found at necropsy, lost at necropsy, lost during processing, or other designations as appropriate. Tissues may appear on the report tables as not examined due to the tissue not being in the plane of section, not present at trimming, etc.

Statistics:

Each mean was presented with the standard deviation (S.D.) and the number of animals (N) used to calculate the mean. All statistical tests were performed using the WIL Toxicology Data Management System (WTDMS™). Analyses were conducted using two-tailed tests (except as noted otherwise) for minimum significance levels of 1% and 5%, comparing each test substance treated group to the control group by sex.
Microscopic findings were compared using a one-tailed Fisher’s exact test for significance levels of 5% and 1%.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Dermal irritation:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

(300 and 1,000 mg/kg bw/day group males and females)

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):

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Details on results:

SURVIVAL:
There were no test substance related deaths; however, on study day 0, one 300 mg/kg bw/day group female died during dose application and one male and one female were found dead prior to completion of the 6 h (± 30 min) dosing period. These deaths were not considered test substance-related, but instead a result of the dose application and wrapping procedures. Due to the timing of the early deaths (all occurring on study Day 0), these animals were replaced.

CLINICAL OBSERVATIONS:
Test substance-related clinical observations of yellow material around the urogenital area was noted in the 1,000 mg/kg bw/day group males and 300 and 1,000 mg/kg bw/day group females and yellow material around the anogenital area was noted in the 1,000 mg/kg bw/day group females. The effects were noted mainly during the first week of test substance administration, in a variable frequency, and in no more than three of the 10 animals in a group. Only in one 1,000 mg/kg bw/day group female were these effects observed after study Day 15. All other clinical findings in the test substance treated groups were noted with similar incidence in the control group, were limited to single animals, were not noted in a dose related manner, and were common findings for laboratory rats of this age and strain.

DERMAL OBSERVATIONS:
There was no dermal irritation observed for any animal in the control or test substance treated groups.

BODY WEIGHTS:
Test substance related lower body weights were noted in the 300 and 1,000 mg/kg bw/day group males and females. Lower mean body weight gains were generally noted in the 300 and 1,000 mg/kg bw/day group males and females; these values were statistically significantly lower for intervals 13 to 20 (300 and 1,000 mg/kg bw/day group males) and 20 to 27 (1,000 mg/kg bw/day group males) when compared to the control group. As a result of the lower mean body weight gains, lower mean cumulative body weight gains (occasionally statistically significant) were noted. On study Day 27, mean body weights in the 300 and 1,000 mg/kg bw/day group males were 5.1% and 8.5% lower than the control group, respectively, and mean body weights in the 300 and 1,000 mg/kg bw/day group females were 5.5% and 4.3% lower than the control group, respectively. The mean cumulative body weight gains from study Days -1 to 27 were lower than control by 15% and 28% in the males and by 21% and 19% in the females for the 300 and 1,000 mg/kg bw/day groups, respectively. There were no test substance related effects on body weight in the 100 mg/kg bw/day group males or females. Lower mean body weight gains were noted in the 100 mg/kg bw/day group females from study Days 13 to 20; however, the difference was not considered test substance related due to the transient or inconsistent pattern throughout the study.

FOOD CONSUMPTION:
Test substance-related lower food consumption and food efficiency was noted in the
1000 mg/kg/day group males. Statistically lower mean food consumption values were noted in the 1,000 mg/kg bw/day group males from study Day -1 to 6 (g/animal/day and g/kg/day) and 13 to 20 (g/animal/day) when compared to the control group. Additionally, a lower mean food efficiency value was noted in the 1,000 mg/kg bw/day group males from study Day 20 to 27 when compared to the control group. Although the food consumption and food efficiency differences were minimal, the slightly lower values likely contributed to the lower cumulative body weight gains and lower mean body weight values in the 1,000 mg/kg bw/day group males. There were no other test substance related effects on food consumption or food efficiency. Some statistically significant food consumption and/or food efficiency differences were noted in the 100 and 300 mg/kg bw/day group males; however, these differences were not considered treatment-related due to the small magnitude of the changes, changes occurring in the opposite direction from that generally associated with an adverse effect, and/or the presence of a transient or inconsistent pattern throughout the study. There were no effects noted in the females.

HEMATOLOGY AND COAGULATION:
There were no test substance-related alterations in haematology and coagulation parameters. However, a statistically significant difference was observed when the control and test substance treated groups were compared. A lower mean corpuscular haemoglobin (MCH) value was noted in the 300 mg/kg bw/day group females; however, it was not considered test substance-related due to the lack of a dose response.

SERUM CHEMISTRY:
Test substance related alterations in serum chemistry parameters include higher bilirubin in the 1,000 mg/kg bw/day group males, higher cholesterol in the 300 and 1,000 mg/kg bw/day group females, and lower alanine aminotransferase (ALT) in the 1,000 mg/kg bw/day group females. Statistically significantly higher mean bilirubin value was noted in the 1, 000 mg/kg bw/day group males when compared to the control group. The elevated mean total bilirubin value in the 1,000 mg/kg bw/day group males was associated with minimal changes in the urine.
Higher mean cholesterol values were noted in the 300 and 1,000 mg/kg bw/day group females when compared to the control group and occurred in a dose responsive manner. A lower mean ALT value in the 1,000 mg/kg bw/day group females was considered test substance related; however, this change was not toxicologically relevant. There were no other test substance related effects on serum chemistry parameters. However, some statistically significant differences were observed when the control and test substance treated groups were compared. Higher mean total protein was noted in the 100 mg/kg bw/day group males but was not considered test substance related due to the lack of a dose response. Higher mean albumin values in the 300 and 1,000 mg/kg bw/day group females were not test substance related, as the change was minimal and due to biologic variation.

URINALYSIS:
Test substance related alterations in urinalysis parameters were limited to lower pH values in the 300 and 1,000 mg/kg bw/day group males, a higher mean urobilinogen value in the 1,000 mg/kg bw/day group males, and red urinary discoloration in one 100 mg/kg bw/day male and in the 300 and 1,000 mg/kg bw/day group males and females. Lower mean pH values were noted in the 300 and 1,000 mg/kg bw/day group males (statistically significant in the 1,000 mg/kg bw/day group). The lower mean pH values were not associated with microscopic kidney lesions. A statistically significantly higher mean urobilinogen value was noted in the 1,000 mg/kg bw/day group males when compared to the control group and was associated with a higher mean total bilirubin value. Red discoloration of the urine was noted at 100, 300, and 1,000 mg/kg bw/day dose levels in the males and at 300 and 1,000 mg/kg bw/day dose levels in the females. While the cause of this red discoloration of the urine is not confirmed, it is most likely a urinary excretion of the test substance or a metabolite and not indicative of renal or systemic toxicity. Occasionally the urines from these animals were also noted positive for nitrite.

OPHTHALMIC EXAMINATIONS:
No ophthalmic lesions indicative of toxicity were observed in any of the test substance-treated groups. All findings observed were typical in prevalence and appearance for laboratory rats of this age and strain.

MACROSCOPIC EXAMINATION:
Review of the gross necropsy observations revealed no observations that were considered to be associated with administration of the test substance.

ORGAN WEIGHTS:
Test substance related changes in organ weights in the 1,000 mg/kg bw/day group included higher heart weights and lower thymus weights in males; higher kidney, liver, and thyroid/parathyroid weights in females; and higher lung weights in males and females. In the 300 mg/kg bw/day group, test substance related organ weight changes were limited to lower thymus weights in males. Statistically significantly higher mean lung weights (absolute and relative to final body weight) were noted in the 1,000 mg/kg bw/day group males and females when compared to the control group. The higher lung weights were not associated with microscopic lung changes. A statistically significantly higher mean heart weight (relative to final body weight) was noted in the 1,000 mg/kg bw/day group males when compared to the control group; however, this was not associated with microscopic heart findings. Statistically significantly lower mean thymus weights (absolute and relative to final body weight) were noted in the 300 and 1,000 mg/kg bw/day group males but were not associated with microscopic changes. Although not associated with microscopic findings, statistically significantly higher mean kidney weight (relative to final body weight) and thyroid/parathyroid weights (absolute [not statistically significant] and relative to final body weight) were noted in the 1,000 mg/kg bw/day group females. Higher mean liver weights (absolute [not statistically significant] and relative to final body weight [statistically significant]) were noted in the 1,000 mg/kg bw/day group females and correlated to microscopic findings but were not associated with any liver enzyme elevations suggestive of toxicity. The relative liver weight in the 1,000 mg/kg bw/day females was 12% higher than that in the control group. All test substance related organ weight changes were minimal and were not considered adverse. A statistically significantly lower mean absolute adrenal gland weight was noted in the 1,000 mg/kg bw/day group females when compared to the control group; however, this change was considered to be a result of an effect on final body weight. There were no other test substance-related effects on organ weights.

MICROSCOPIC EXAMINATION:
Test substance related microscopic findings included epidermal hyperplasia in the treated skin (100, 300, and 1,000 mg/kg bw/day group males and females), cytoplasmic alteration of hepatocytes (300 and 1,000 mg/kg bw/day group males and 100, 300, and 1,000 mg/kg bw/day group females), and vacuolation of the adrenal cortex (100, 300, and 1,000 mg/kg bw/day group males and 1,000 mg/kg bw/day group females).
Treated skin: Minimal to mild epidermal hyperplasia, characterized by thickening of the stratum spinosum and granulosum and hypertrophy of the basal cell layer, was noted in the 100, 300, and 1,000 mg/kg bw/day group males and females. A similar change was noted in a low number (4/10) of control group males but was not observed in control group females. The incidence of epidermal hyperplasia was statistically significant in the 100, 300, and 1,000 mg/kg bw/day group males and females when compared to the control group.
Liver: Minimal to mild cytoplasmic alteration of the hepatocytes was noted in the 300 and 1,000 mg/kg bw/day group males and mild cytoplasmic alteration of the hepatocytes was noted in the 100, 300, and 1,000 mg/kg bw/day group females. Cytoplasmic alteration was characterized by tinctoral variations in staining characteristics of centrilobular hepatocytes (pale pink) when compared to periportal and midzonal hepatocytes. Periportal and midzonal hepatocytes were slightly smaller and more eosinophilic than centrilobular hepatocytes; however, hypertrophy of centrilobular hepatocytes was not detected. The incidence of cytoplasmic alteration was statistically significant in the 1,000 mg/kg bw/day group females when compared to the control group. The higher liver weights and corresponding histologic correlate are considered to be likely a compensatory response possibly related to liver enzyme induction and a nonadverse effect of test substance administration. Adrenal cortex: Minimal to moderate vacuolation, characterized by multiple fine vacuoles and/or larger distinct vacuoles in the zona fasciculata and/or zona reticularis, was noted in the 100, 300, and 1,000 mg/kg bw/day group males and 1,000 mg/kg bw/day group females. Two females in the 1,000 mg/kg bw/day group had multifocal, minimal brown pigment in cortical cells of the zona fasciculata. Adrenal cortical vacuolation was also observed in control group males (5/10) and females (3/10). The incidence of adrenal cortical cytoplasmic vacuolation in the 300 and 1,000 mg/kg bw/day group males and 1,000 mg/kg bw/day group females was statistically significant when compared to the control group. There were no other test substance-related histologic changes. One male in the 1,000 mg/kg bw/day group had a focal area of tubular necrosis in the cortex of the right kidney. This was not considered to be test substance-related given the focal nature of the lesion. One female in the 1,000 mg/kg bw/day group had minimal acute pyelonephritis of the right kidney. This was not considered test substance related given the background nature of the lesion. Remaining histologic changes were considered to be incidental findings or related to some aspect other than administration of the test substance. There was no test substance-related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.

Effect levels

Dose descriptor:

NOAEL

Effect level:

100 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Based on lower mean cumulative body weight gains in the 300 and 1,000 mg/kg bw/day group males and females, and adrenal cortical cytoplasmic vacuolation in the 300 mg/kg bw/day group males and 1,000 mg/kg bw/day group males and females

Target system / organ toxicity

Critical effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

In a four week repeated dose dermal toxicity study, NOAEL was considered to be 100 mg/kg bw/day.

Executive summary:

A four week dermal toxicity study in Sprague-Dawley rats was conducted at concentrations of 0, 100, 300, and 1,000 mg/kg bw/day test substance according to OECD Guideline 410 and OPPTS 870.3200, in compliance with GLP. Ten males and ten females per dose group were used. Test substance moistened with the vehicle (deionized water) was applied topically under occlusive conditions to the shaved intact dorsal surface of three groups of rats once daily for 6 h for up to 28 consecutive days. A concurrent control group received the vehicle on a comparable regimen. There were no test substance-related effects on survival, dermal observations, haematology, coagulation, ophthalmic examinations and macroscopic observations. Treatment-related lower mean cumulative body weight gains were observed in the 300 and 1,000 mg/kg bw/day group males and females. Also adrenal cortical cytoplasmic vacuolation were observed in the 300 mg/kg bw/day group males and 1,000 mg/kg bw/day group males and females. Hence, the NOAEL was considered to be 100 mg/kg bw/day (Mertens JJWM, 2014).