2-ethylhexyl 12-(acetoxy)octadecanoate

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

May 2015 - November 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

repeated dose toxicity: oral, other

Remarks:

OECD 422

Type of information:

experimental study

Adequacy of study:

key study

Study period:

May 2015 - November 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

Identification : SE7B
Physical State/Appearance : Clear colorless liquid
Chemical Name : Fatty Acids, C8-C18 and C18 unsaturated, reaction product
with isomerized oleic acid homopolymer 2- ethylhexyl ester,
hydrogenated
Chemical Formula : C44H86O4
Purity : >95%
Batch Number : 0000140602
Label : SE7B Lot#0000140602
Date Received : 20 February 2015
Storage Conditions : In the dark, at room temperature
Expiry Date : 18 February 2017

Species:

rat

Strain:

Wistar

Details on species / strain selection:

Wistar Han™:RccHan™:WIST

Sex:

male/female

Details on test animals or test system and environmental conditions:

A sufficient number of male and female Wistar Han™:RccHan™:WIST strain rats were obtained from Harlan Laboratories U.K. Ltd., Blackthorn, Bicester, Oxon, UK. On receipt the animals were examined for signs of ill-health or injury. The animals were acclimatized for eight days during which time their health status was assessed. A total of one hundred and sixteen animals (fifty eight males and fifty eight females) were accepted into the study. At the start of treatment the males weighed 317 to 360g, the females weighed 188 to 232g, and were approximately twelve weeks old.

Initially, all animals were housed in groups of three or five in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding (Datesand Ltd., Cheshire, UK). During the pairing phase,the non-recovery animals were transferred to polypropylene grid floor cages suspended over trays lined with absorbent paper on a one male: one female basis within each dose group. Following evidence of successful mating, the males were returned to their original cages. Mated females were housed individually during gestation and lactation in solid floor polypropylene cages with stainless steel mesh lids and softwood flakes. Recovery group animals were housed in groups of five in solid floor polypropylene cages with stainless steel mesh lids and furnished with softwood flake bedding.

The animals were allowed free access to food and water. A pelleted diet (Rodent 2018C Teklad Global Certified Diet, Harlan Laboratories U.K. Ltd., Oxon, UK.) was used. Mains drinking water was supplied from polycarbonate bottles attached to the cage. Environmental enrichment was provided in the form of wooden chew blocks and cardboard fun tunnels (Datesand Ltd., Cheshire, UK) except for paired animals and mated females during gestation and lactation. Mated females were also given softwood flakes, as bedding, throughout gestation and lactation. The diet, drinking water, bedding and environmental enrichment was considered not to contain any contaminant at a level that might have affected the purpose or integrity of the study.

The animals were housed in a single air-conditioned room within the Envigo Research Limited, Shardlow, UK Barrier Maintained Rodent Facility. The rate of air exchange was at least fifteen air changes per hour and the low intensity fluorescent lighting was controlled to give twelve hours continuous light and twelve hours darkness. Environmental conditions were continuously monitored by a computerized system, and print-outs of hourly temperatures and humidities are included in the study records. The Study Plan target ranges for temperature and relative humidity were 22 ± 3 °C and 50 ± 20% respectively; there were short term deviations from these targets were considered not to have affected the purpose or integrity of the study; see deviations from Study Plan.

The animals were randomly allocated to treatment groups using a stratified body weight randomization procedure and the group mean body weights were then determined to ensure similarity between the treatment groups. The cage distribution within the holding rack was also randomized. The animals were uniquely identified within the study by an ear punching system routinely used in these laboratories.

Route of administration:

oral: gavage

Vehicle:

arachis oil

Details on analytical verification of doses or concentrations:

Test Item Preparation
For the purpose of this study the test item was prepared at the appropriate concentrations as a solution in Arachis oil BP. The stability and homogeneity of the test item formulations were determined by Envigo Research Limited, Shardlow, UK, Analytical Services. Results show the formulations to be stable for at least 21 days. Formulations were therefore prepared fortnightly and stored at approximately 4°C in the dark.

Samples of the test item formulation were taken and analyzed for concentration of SE7B at Envigo Research Limited, Shardlow, UK, Analytical Services. The results indicate that the prepared formulations were within ± 10% of the nominal concentration.

The test item was administered daily by gavage using a stainless steel cannula attached to a disposable plastic syringe. Control animals were treated in an identical manner with 4 mL/kg of Arachis oil BP.

Animals were allocated to treatment groups as follows:

Control
Dose Level(mg/kg bw/day) - 0
Treatment Volume (mL/kg) - 4
Concentration (mg/mL) - 0
Animal Numbers - Male - 12(1-12)
Animal Numbers - Female - 12(13-24)

Recovery Control
Dose Level(mg/kg bw/day) - 0
Treatment Volume (mL/kg) - 4
Concentration (mg/mL) - 0
Animal Numbers - Male - 5(97-101)
Animal Numbers - Female - 5(102-106)

Low
Dose Level(mg/kg bw/day) - 100
Treatment Volume (mL/kg) - 4
Concentration (mg/mL) - 25
Animal Numbers - Male - 12(25-36)
Animal Numbers - Female - 12(37-48)

Intermediate
Dose Level(mg/kg bw/day) - 300
Treatment Volume (mL/kg) - 4
Concentration (mg/mL) - 75
Animal Numbers - Male - 12(49-60)
Animal Numbers - Female - 12(61-72)

High
Dose Level(mg/kg bw/day) - 1000
Treatment Volume (mL/kg) - 4
Concentration (mg/mL) - 250
Animal Numbers - Male - 12(73-84)
Animal Numbers - Female - 12(85-96)

Recovery High
Dose Level(mg/kg bw/day) - 1000
Treatment Volume (mL/kg) - 4
Concentration (mg/mL) - 250
Animal Numbers - Male - 12(107-111)
Animal Numbers - Female - 12(112-116)

Duration of treatment / exposure:

43-57 days

Frequency of treatment:

Daily

Dose / conc.:

0 mg/kg bw/day (actual dose received)

Remarks:

Control

Dose / conc.:

0 mg/kg bw/day (actual dose received)

Remarks:

Recovery Control

Dose / conc.:

100 mg/kg bw/day (actual dose received)

Remarks:

Low

Dose / conc.:

400 mg/kg bw/day (actual dose received)

Remarks:

Intermediate

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

Remarks:

High

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

Remarks:

Recovery High

No. of animals per sex per dose:

Control - 12M/12F
Recovery Control - 5M/5F
Low - 12M/12F
Intermediate - 12M/12F
High - 12M/12F
Recovery High - 5M/5F

Control animals:

yes, concurrent vehicle

Details on study design:

Chronological Sequence of Study
Non-Recovery Dose Groups
i. Groups of twelve male and twelve female animals were treated daily at the appropriate dose level throughout the study (except for females during parturition where applicable).The first day of dosing was designated as Day 1 of the study.
ii. Prior to the start of treatment and once weekly thereafter, all animals were observed for signs of functional/behavioral toxicity.
iii. On Day 15, animals were paired on a 1 male: 1 female basis within each dose group for a maximum of fourteen days.
iv. Following evidence of mating (designated as Day 0 post coitum) the males were returned to their original cages and females were transferred to individual cages.
v. On completion of the pairing phase (during Week 6), five selected males per dose group were evaluated for functional/sensory responses to various stimuli.
vi. Pregnant females were allowed to give birth and maintain their offspring until Day 5 post partum. Litter size, offspring weight and sex, surface righting and clinical signs were also recorded during this period.
vii. At Day 4 post partum, five selected females per dose group were evaluated for functional/sensory responses to various stimuli.
viii. Blood samples were taken from five males from each dose group for hematological and blood chemical assessments on Day 42. The male dose groups were killed and examined macroscopically on Day 43 or 44.
ix. Blood samples were taken from five randomly selected females from each dose group for hematological and blood chemical assessment on Day 4 post partum. At Day 5 post partum, all females and offspring were killed and examined macroscopically. Any female which did not produce a pregnancy was also killed and examined macroscopically.

Recovery Dose Groups
i. Groups of five male and five female rats were dosed according to dose group continuously up to the point of sacrifice of non-recovery males at which time treatment was discontinued.
ii. The males and females were maintained without treatment for a further fourteen days.
iii. Urinalysis was performed for all males during the final week of recovery.
iv. Blood samples were taken for hematological and blood chemical assessment on Day 56.
v. After fourteen days of recovery, males and females were killed and examined macroscopically.

Observations and examinations performed and frequency:

Clinical Observations
All animals were examined for overt signs of toxicity, ill-health and behavioral change immediately before dosing, up to thirty minutes after dosing, and one hour after dosing, during the working week (except for females during parturition where applicable). During the treatment-free period, recovery animals were observed daily. All observations were recorded.

Functional Observations
Prior to the start of treatment and at weekly intervals thereafter, all non-recovery animals were observed for signs of functional/behavioral toxicity. Functional performance tests were also performed on five selected males and females from each non-recovery dose level, prior to termination, together with an assessment of sensory reactivity to various stimuli.

Behavioral Assessments
Detailed individual clinical observations were performed for each animal using a purpose built arena. The following parameters were observed:
Gait
Hyper/Hypothermia
Tremors
Skin color
Twitches
Respiration
Convulsions
Palpebral closure
Bizarre/Abnormal/Stereotypic behavior
Urination
Salivation
Defecation
Pilo-erection
Transfer arousal
Exophthalmia
Tail elevation
Lachrymation

This test was developed from the methods used by Irwin (1968) and Moser et al (1988). The scoring system used is outlined in The Key to Scoring System and Explanation for Behavioral Assessments and Sensory Reactivity Tests.


Functional Performance Tests
Motor Activity. Purpose-built 44 infra-red beam automated activity monitors were used to assess motor activity. Animals were randomly allocated to the activity monitors. The tests were performed at approximately the same time on each occasion (at least two hours after dosing), under similar laboratory conditions. The evaluation period was thirty minutes for each animal.

The percentage of time each animal was active and mobile was recorded for the overall thirty minute period and also during the final 20% of the period (considered to be the asymptotic period, Reiter and Macphail, 1979).

Forelimb/Hindlimb Grip Strength. An automated 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 gripwas broken. The animal was drawn along the trough of the meter by the 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. The assessment was developed from the method employed by Meyer et al (1979).

Sensory Reactivity
Each animal was individually assessed for sensory reactivity to auditory, visual and proprioceptive stimuli.
This assessment was developed from the methods employed by Irwin (1968) and Moser et al (1988).
The following parameters were observed:
Grasp response
Touch escape
Vocalization
Pupil reflex
Toe pinch
Blink reflex
Tail pinch
Startle reflex
Finger approach

Body Weight
Individual body weights were recorded on Day 1 (prior to dosing) and then weekly for males until termination and weekly for females until pairing. During pairing phase females were weighed daily until mating was confirmed. Body weights were then recorded for females on Days 0, 7, 14 and 20 post coitum, and on Days 1 and 4 post partum. Recovery animals were weighed on Day 1 (prior to dosing) and then weekly until termination.

Food Consumption
During the pre-pairing period, weekly food consumption was recorded for each cage of adults. This was continued for males after the mating phase. For females showing evidence of mating, food consumption was recorded for the periods covering post coitum Days 0-7, 7-14 and 14-20. For females with live litters, food consumption was recorded on Days 1 and 4 post partum.
Weekly food consumptions were performed for each cage of recovery group animals throughout the study period.
Food efficiency (the ratio of body weight change/dietary intake) was calculated retrospectively for nonrecovery males (except during the mating phase) and recovery group animals throughout the study period and for non-recovery females during the pre-pairing phase. Due to offspring growth and milk production,food efficiency could not be accurately calculated during gestation and lactation.

Water Consumption
Water intake was measured daily during the pre-pairing phase of the study.
Intergroup differences did not indicate any need for more formal gravimetric measurements.

Reproductive Performance
Mating
Non-recovery animals were paired on a 1 male: 1 female basis within each dose group, for a period of up to fourteen days. Cage tray-liners were checked each morning for the presence of ejected copulation plugs and each female was examined for the presence of a copulation plug in the vagina. A vaginal smear was prepared for each female and the stage of estrus or the presence of sperm was recorded. The presence of sperm within the vaginal smear and/or vaginal plug in situ was taken as positive evidence of mating (Day 0 of gestation) and the males were subsequently returned to their original holding cages. Mated females were housed individually during the period of gestation and lactation.

Pregnancy and Parturition
Each pregnant female was observed at least three times a day (early morning, mid-day and as late as possible during the normal working day) around the period of expected parturition. Observations were carried out early morning and as late as possible at weekends and public holidays. The following was recorded for each female:
i. Date of pairing
ii. Date of mating
iii. Date and time of observed start of parturition
iv. Date and time of observed completion of parturition

Litter Data
On completion of parturition (Day 0 post partum), the number of live and dead offspring was recorded.
Offspring were individually identified within each litter by tattoo on Day 1 post partum.
For each litter the following was recorded:
i. Number of offspring born
ii. Number of offspring alive recorded daily and reported on Days 1 and 4 post partum
iii. Sex of offspring on Days 1 and 4 post partum
iv. Clinical condition of offspring from birth to Day 5 post partum
v. Individual offspring weights on Days 1 and 4 post partum (litter weights were calculated retrospectively from this data)

Physical Development
All live offspring were assessed for surface righting reflex on Day 1 post partum.

Laboratory Investigations
Hematological and blood chemical investigations were performed on five males and five females selected from each non-recovery test and control group prior to termination (Day 42 for males and Day 4 post partum for females). In addition hematological and blood chemical investigations were performed on all recovery group animals after the fourteen day treatmentfree period at termination (Day 56). Blood samples were obtained from the lateral tail vein. Where necessary repeat samples were taken by cardiac puncture at termination. Animals were not fasted prior to sampling.
Urinalytical investigations were performed on five non-recovery males from the control and each test group during the final week of treatment and on all recovery males during the final week of the recovery period.
Urine samples were collected overnight by housing the rats in metabolisms cages. Animals were maintained under conditions of normal hydration during collection but without access to food.

Hematology
The following parameters were measured on blood collected into tubes containing potassium EDTA anticoagulant:
Hemoglobin (Hb)
Erythrocyte count (RBC)
Hematocrit (Hct)
Erythrocyte indices - mean corpuscular hemoglobin (MCH)
- mean corpuscular volume (MCV)
- mean corpuscular hemoglobin concentration (MCHC)
Total leukocyte count (WBC)
Differential leukocyte count - 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).

Blood Chemistry
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 (Ca++)
Inorganic phosphorus (P)
Aspartate aminotransferase (ASAT)
Alanine aminotransferase (ALAT)
Alkaline phosphatase (AP)
Creatinine (Creat)
Total cholesterol (Chol)
Total bilirubin (Bili)
Bile acids
Gamma glutamyltranspeptidase
Triglycerides (Trigs)

Urinalysis
The following parameters were measured on collected urine:
Volume
Ketones
Specific
Gravity Bilirubin
pH
Urobilinogen
Protein
Blood
Glucose
Appearance

Sacrifice and pathology:

Pathology
Necropsy
Adult non-recovery males were killed by intravenous overdose of a suitable barbiturate agent followed by exsanguination on Day 43 or 44. Adult non-recovery females were killed by intravenous overdose of a suitable barbiturate agent followed by exsanguination on Day 5 post partum. Surviving offspring were terminated via intracardiac overdose of a suitable barbiturate agent. Any females which failed to achieve pregnancy or produce a litter were killed on or after Day 25 post coitum.
For all non-recovery females, the uterus was examined for signs of implantation and the number of uterine implantations in each horn was recorded. This procedure was enhanced; as necessary, by staining the uteri with a 0.5% ammonium polysulphide solution (Salewski 1964). The corpora lutea were also counted.
Recovery group animals were killed by intravenous overdose of a suitable barbiturate agent followed by exsanguination on Day 57.
All adult animals and offspring, including those dying during the study, were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.

Organ Weights
The following organs were dissected free from fat and weighed before fixation from five selected males and
five selected females from each non-recovery dose group and from all recovery group animals. Tissues
shown with a * were weighed from all remaining animals:
Adrenals
Prostate*
Brain
Seminal vesicles*
Epididymides*
Spleen
Heart
Testes*
Kidneys
Thymus
Liver
Thyroid (weighed post-fixation with Parathyroid)
Ovaries*
Uterus* (weighed with Cervix)
Pituitary* (post fixation)

Histopathology
Samples of the following tissues were removed from five selected males and five selected females from each dose group and preserved in buffered 10% formalin, except for Epididymides and Testes which were preserved in Modified Davidsons fluid and eyes fixed in Davidson’s fluid. Lungs were inflated to approximately normal inspiratory volume with buffered 10% formalin before immersion in fixative.
Tissues shown with a * were preserved from all remaining animals:
Adrenals
Muscle (skeletal)
Aorta (thoracic)
Ovaries*
Bone & bone marrow (femur including stifle joint)
Pancreas
Bone & bone marrow (sternum)
Pituitary*
Brain (including cerebrum, cerebellum and pons)
Prostate*
Caecum
Rectum
Coagulating gland*
Salivary glands (submaxillary)
Colon
Sciatic nerve
Duodenum
Seminal vesicles*
Epididymides*
Skin (hind limb)
Esophagus
Spinal cord (cervical, mid-thoracic and lumbar)
Eyes
Gross lesions*
Spleen
Heart
Stomach
Ileum (including peyer’s patches)
Thyroid/parathyroid
Jejunum
Trachea
Kidneys
Testes*
Liver
Thymus
Lungs (with bronchi)
Urinary bladder
Lymph nodes (mandibular and mesenteric)
Uterus/Cervix*
Mammary gland*
Vagina*

Tissues were dispatched to the Test Site (Envigo CRS Limited, Occold, Eye Research Center, Eye, Suffolk, IP23 7PX) for processing (Principal Investigator: D Roberts). The tissues from five selected non-recovery control and 1000 mg/kg bw/day dose group animals, any animals dying during the study, and any animals which failed to mate or did not achieve a pregnancy were prepared as paraffin blocks, sectioned at a nominal thickness of 5 μm and stained with hematoxylin and eosin for subsequent microscopic examination. The tissues shown in bold from the remaining control and 1000 mg/kg bw/day animals and animals which did not achieve a pregnancy were also processed. In addition, sections of testes from all control and 1000 mg/kg bw/day males were also stained with Periodic Acid-Schiff (PAS) stain and examined.
Detailed qualitative examination of the testes was undertaken, taking into account the tubular stages of the spermatogenic cycle. The examination was conducted in order to identify treatment-related effects such as missing germ cell layers or types, retained spermatids, multinucleated or apoptotic germ cells and sloughing of spermatogenic cells into the lumen.
Any cell-or stage-specificity of testicular findings was noted.Microscopic examination was conducted by the Study Pathologist (P Millar at Peter Millar Associates Ltd. 3 Queen Charlotte Lane, Edinburgh EH6 6AY).

Clinical signs:

no effects observed

Description (incidence and severity):

There were no clinical signs of toxicity related to the test item.

Mortality:

no mortality observed

Description (incidence):

There were no unscheduled deaths during the study.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There were no detrimental effects detected for overall body weight development in treated animals of either sex.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

No adverse effects were detected in food consumption or food efficiency for treated males throughout the treatment period or in treated females during maturation, gestation or lactation.

Food efficiency:

no effects observed

Description (incidence and severity):

No adverse effects were detected in food consumption or food efficiency for treated males throughout the treatment period or in treated females during maturation, gestation or lactation.

Water consumption and compound intake (if drinking water study):

no effects observed

Description (incidence and severity):

Daily gravimetric assessment of the water bottles throughout the pre-pairing phase showed no effect on water intake when compared to controls. Water bottles were assessed by visual inspection for the remainder of the study.

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

There were no toxicologically significant changes in the haematological parameters measured.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

No toxicologically significant changes were detected in blood chemical parameters.

Urinalysis findings:

no effects observed

Description (incidence and severity):

There were no treatment-related urinalytical effects detected for any treated male dose groups.

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

There were no treatment-related effects detected in animals of either sex treated with 1000, 300 or 100 mg/kg bw/day.
There were no treatment-related changes in functional performance for animals of either sex treated with 1000, 300 or 100 mg/kg bw/day
There were no treatment-related changes in sensory reactivity for animals of either sex treated with 1000, 300 or 100 mg/kg bw/day.

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

No toxicologically significant changes were evident in the organ weights measured.

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no treatment-related macroscopic abnormalities detected.

Neuropathological findings:

not specified

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

There was no evidence of test item-related histopathological findings.

Histopathological findings: neoplastic:

no effects observed

Description (incidence and severity):

There was no evidence of test item-related histopathological findings.

Other effects:

not specified

Details on results:

JUSTIFICATION OF NO OBSERVED EFFECT LEVEL
Oral administration of the test item SE7B to rats for a period of up to eight weeks at dose levels of up to 1000 mg/kg bw/day did not result in any treatment-related changes. The ‘No Observed Adverse Effect Level’ (NOAEL) was, therefore considered to be 1000 mg/kg bw/day.
The following differences between treated and control animals were considered not to be indicative of test item toxicity:

Clinical Observations
- There were isolated incidents of one male and one female treated with 1000 mg/kg bw/day showing signs of generalised fur loss. Two control males and two males treated with 1000 mg/kg bw/day had scab formation. This was considered likely to be due to a physical injury. Both of these observations were considered to be of no toxicological importance.

Funtional Observations
Functional Performance Tests
- Males treated with 1000 and 300 mg/kg bw/day attained a statistically significant reduction in the fore limb Test 1 and all treated female groups attained a statistically significant increase in the fore limb Test 3. All individual values were within the background control ranges and without the presence of doserelated response. Therefore, these findings were considered to be incidental and to be of no toxicological importance.
- Overall activity for non recovery males treated with 1000 mg/kg bw/day was lower when compared to controls, subsequently attaining statistical significance but without any dose relationship. The corresponding value in high non-recovery females was comparable with control and as there were no apparent signs of neurotoxicity for any of these animals. The intergroup difference was considered to be incidental.

Body Weight
- Intergroup differences were observed, in particular for the recovery male achieving statistical significance in some instances, however, overall body weight gain was comparable to controls. Overall weight gains for all non-recovery male dose groups were slightly higher than controls and there was no effect of treatment on female body weight development during pre-pairing, gestation or lactation.

Haematology
- Non-recovery males treated with 1000 mg/kg bw/day showed a statistically significant increase (p<0.05) in hematocrit levels when compared to controls. Individual values were all within the normal background ranges for animals of the age, sex and strain and in the absence of any supporting histopathological correlates the intergroup difference was considered to be incidental and of no toxicological importance.

Blood Chemistry
- Non-recovery males treated with 1000 mg/kg bw/day showed a statistically increase in chloride concentration. Non-recovery females from all treatment groups showed a statistically significant reduction in alkaline phosphates; which there were no dose relationships. Recovery high dose males showed a statistical significant reduction in Albumin/globulin ratio values and an increase in bile acid when compared to controls. All individual values were all within the normal background ranges for animals of the age, sex and strain and in the absence of any supporting histopathological correlates the intergroup differences were considered to be incidental and of no toxicological importance.

Organ Weights
- Incidental findings were confined to all treated non-recovery males showing a statistically significant increase (p<0.05) in absolute and relative brain weights when compared to controls. Recovery high dose males revealed a statistically significant reduction (p<0.05) in absolute and relative thyroid/parathyroid weights. The majority of the individual values were within the anticipated background ranges and in the absence of any histopathological correlates these changes were considered to be of no toxicological importance.

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:

behaviour (functional findings)

body weight and weight gain

clinical biochemistry

clinical signs

food consumption and compound intake

food efficiency

gross pathology

haematology

histopathology: neoplastic

histopathology: non-neoplastic

mortality

organ weights and organ / body weight ratios

urinalysis

water consumption and compound intake

Key result

Critical effects observed:

no

Results tables attached ( Results Tables - Repeated Dose Oral OECD 422 - SE7B.pdf)

Conclusions:

The oral administration of SE7B to rats by gavage, at dose levels of 100, 300 and 1000 mg/kg bw/day, was tolerated well. Based on these results the ‘No Observed Adverse Effect Level’ (NOAEL) for systemic toxicity was therefore considered to be 1000 mg/kg bw/day.
The ‘No Observed Effect Level’ (NOEL) for reproductive toxicity was considered to be 1000 mg/kg bw/day.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

hydrogenation products of (esterification products of 2-ethylhexan-1-ol with (Estolide formation products of oleic acid and Fatty acids, C8-18 and C18-unsatd. (branched or linear))

IUPAC Name:

hydrogenation products of (esterification products of 2-ethylhexan-1-ol with (Estolide formation products of oleic acid and Fatty acids, C8-18 and C18-unsatd. (branched or linear))

Test material form:

liquid

Specific details on test material used for the study:

Identification : SE7B
Physical State/Appearance : Clear colorless liquid
Chemical Name : Fatty Acids, C8-C18 and C18 unsaturated, reaction product
with isomerized oleic acid homopolymer 2- ethylhexyl ester,
hydrogenated
Chemical Formula : C44H86O4
Purity : >95%
Batch Number : 0000140602
Label : SE7B Lot#0000140602
Date Received : 20 February 2015
Storage Conditions : In the dark, at room temperature
Expiry Date : 18 February 2017

Test animals

Species:

rat

Strain:

Wistar

Remarks:

Wistar Han™:RccHan™:WIST

Sex:

male/female

Details on test animals or test system and environmental conditions:

A sufficient number of male and female Wistar Han™:RccHan™:WIST strain rats were obtained from Harlan Laboratories U.K. Ltd., Blackthorn, Bicester, Oxon, UK. On receipt the animals were examined for signs of ill-health or injury. The animals were acclimatized for eight days during which time their health status was assessed. A total of one hundred and sixteen animals (fifty eight males and fifty eight females) were accepted into the study. At the start of treatment the males weighed 317 to 360g, the females weighed 188 to 232g, and were approximately twelve weeks old.

Initially, all animals were housed in groups of three or five in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding (Datesand Ltd., Cheshire, UK). During the pairing phase, the non-recovery animals were transferred to polypropylene grid floor cages suspended over trays lined with absorbent paper on a one male: one female basis within each dose group. Following evidence of successful mating, the males were returned to their original cages. Mated females were housed individually during gestation and lactation in solid floor polypropylene cages with stainless steel mesh lids and softwood flakes. Recovery group animals were housed in groups of five in solid floor polypropylene cages with stainless steel mesh lids and furnished with softwood flake bedding.

The animals were allowed free access to food and water. A pelleted diet (Rodent 2018C Teklad Global Certified Diet, Harlan Laboratories U.K. Ltd., Oxon, UK.) was used. Mains drinking water was supplied from polycarbonate bottles attached to the cage. Environmental enrichment was provided in the form of wooden chew blocks and cardboard fun tunnels (Datesand Ltd., Cheshire, UK) except for paired animals and mated females during gestation and lactation. Mated females were also given softwood flakes, as bedding, throughout gestation and lactation. The diet, drinking water, bedding and environmental enrichment was considered not to contain any contaminant at a level that might have affected the purpose or integrity of the study.

The animals were housed in a single air-conditioned room within the Envigo Research Limited, Shardlow, UK Barrier Maintained Rodent Facility. The rate of air exchange was at least fifteen air changes per hour and the low intensity fluorescent lighting was controlled to give twelve hours continuous light and twelve hours darkness. Environmental conditions were continuously monitored by a computerized system, and print-outs of hourly temperatures and humidities are included in the study records. The Study Plan target ranges for temperature and relative humidity were 22 ± 3 °C and 50 ± 20% respectively; there were short term deviations from these targets were considered not to have affected the purpose or integrity of the study; see deviations from Study Plan.

The animals were randomly allocated to treatment groups using a stratified body weight randomization procedure and the group mean body weights were then determined to ensure similarity between the treatment groups. The cage distribution within the holding rack was also randomized. The animals were uniquely identified within the study by an ear punching system routinely used in these laboratories.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

arachis oil

Details on exposure:

Test Item Preparation
For the purpose of this study the test item was prepared at the appropriate concentrations as a solution in Arachis oil BP. The stability and homogeneity of the test item formulations were determined by Envigo Research Limited, Shardlow, UK, Analytical Services. Results show the formulations to be stable for at least 21 days. Formulations were therefore prepared fortnightly and stored at approximately 4°C in the dark.
Samples of the test item formulation were taken and analyzed for concentration of SE7B at Envigo Research Limited, Shardlow, UK, Analytical Services. The results indicate that the prepared formulations were within ± 10% of the nominal concentration.

The test item was administered daily by gavage using a stainless steel cannula attached to a disposable plastic syringe. Control animals were treated in an identical manner with 4 mL/kg of Arachis oil BP.
Animals were allocated to treatment groups as follows:
Control
Dose Level(mg/kg bw/day) - 0
Treatment Volume (mL/kg) - 4
Concentration (mg/mL) - 0
Animal Numbers - Male - 12(1-12)
Animal Numbers - Female - 12(13-24)

Recovery Control
Dose Level(mg/kg bw/day) - 0
Treatment Volume (mL/kg) - 4
Concentration (mg/mL) - 0
Animal Numbers - Male - 5(97-101)
Animal Numbers - Female - 5(102-106)

Low
Dose Level(mg/kg bw/day) - 100
Treatment Volume (mL/kg) - 4
Concentration (mg/mL) - 25
Animal Numbers - Male - 12(25-36)
Animal Numbers - Female - 12(37-48)

Intermediate
Dose Level(mg/kg bw/day) - 300
Treatment Volume (mL/kg) - 4
Concentration (mg/mL) - 75
Animal Numbers - Male - 12(49-60)
Animal Numbers - Female - 12(61-72)

High
Dose Level(mg/kg bw/day) - 1000
Treatment Volume (mL/kg) - 4
Concentration (mg/mL) - 250
Animal Numbers - Male - 12(73-84)
Animal Numbers - Female - 12(85-96)

Recovery High
Dose Level(mg/kg bw/day) - 1000
Treatment Volume (mL/kg) - 4
Concentration (mg/mL) - 250
Animal Numbers - Male - 12(107-111)
Animal Numbers - Female - 12(112-116)

Details on mating procedure:

Non-recovery animals were paired on a 1 male: 1 female basis within each dose group, for a period of up to fourteen days. Cage tray-liners were checked each morning for the presence of ejected copulation plugs and each female was examined for the presence of a copulation plug in the vagina. A vaginal smear was prepared for each female and the stage of estrus or the presence of sperm was recorded. The presence of sperm within the vaginal smear and/or vaginal plug in situ was taken as positive evidence of mating (Day 0 of gestation) and the males were subsequently returned to their original holding cages. Mated females were housed individually during the period of gestation and lactation.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Concentrations determined by GC using an external standard technique.
Formulations investigated comprise test itemin the range o 90% to 109%, thus within acceptable limits of ±10%
Detection system was found to have acceptable linearity, and the proceedure had acceptable recoveries of test item in the vehicle.

Duration of treatment / exposure:

57 days

Frequency of treatment:

Daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Control

Doses / concentrations 2

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

Low

Doses / concentrations 3

Dose / conc.:

300 mg/kg bw/day (nominal)

Remarks:

Intermediate

Doses / concentrations 4

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

High

No. of animals per sex per dose:

Control: 12 M, 12 F
Recovery Control: 5 M, 5F
Low: 12 M, 12 F
Intermediate: 12 M, 12 F
High: 12 M, 12 F
Recovery High: 5 M, 5 F

Control animals:

yes, concurrent vehicle

Details on study design:

Chronological Sequence of Study
Non-Recovery Dose Groups
i. Groups of twelve male and twelve female animals were treated daily at the appropriate dose level throughout the study (except for females during parturition where applicable).The first day of dosing was designated as Day 1 of the study.
ii. Prior to the start of treatment and once weekly thereafter, all animals were observed for signs of functional/behavioral toxicity.
iii. On Day 15, animals were paired on a 1 male: 1 female basis within each dose group for a maximum of fourteen days.
iv. Following evidence of mating (designated as Day 0 post coitum) the males were returned to their original cages and females were transferred to individual cages.
v. On completion of the pairing phase (during Week 6), five selected males per dose group were evaluated for functional/sensory responses to various stimuli.
vi. Pregnant females were allowed to give birth and maintain their offspring until Day 5 post partum. Litter size, offspring weight and sex, surface righting and clinical signs were also recorded during this period.
vii. At Day 4 post partum, five selected females per dose group were evaluated for functional/sensory responses to various stimuli.
viii. Blood samples were taken from five males from each dose group for hematological and blood chemical assessments on Day 42. The male dose groups were killed and examined macroscopically on Day 43 or 44.
ix. Blood samples were taken from five randomly selected females from each dose group for hematological and blood chemical assessment on Day 4 post partum. At Day 5 post partum, all females and offspring were killed and examined macroscopically. Any female which did not produce a pregnancy was also killed and examined macroscopically.

Recovery Dose Groups
i. Groups of five male and five female rats were dosed according to dose group continuously up to the point of sacrifice of non-recovery males at which time treatment was discontinued.
ii. The males and females were maintained without treatment for a further fourteen days.
iii. Urinalysis was performed for all males during the final week of recovery.
iv. Blood samples were taken for hematological and blood chemical assessment on Day 56.
v. After fourteen days of recovery, males and females were killed and examined macroscopically.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Description (incidence and severity):

There were no clinical signs of toxicity related to the test item.

Mortality:

no mortality observed

Description (incidence):

There were no unscheduled deaths during the study.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There were no detrimental effects detected for overall body weight development in treated animals of either sex.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

No adverse effects were detected in food consumption or food efficiency for treated males throughout the treatment period or in treated females during maturation, gestation or lactation.

Food efficiency:

no effects observed

Description (incidence and severity):

No adverse effects were detected in food consumption or food efficiency for treated males throughout the treatment period or in treated females during maturation, gestation or lactation.

Water consumption and compound intake (if drinking water study):

no effects observed

Description (incidence and severity):

Daily gravimetric assessment of the water bottles throughout the pre-pairing phase showed no effect on water intake when compared to controls. Water bottles were assessed by visual inspection for the remainder of the study.

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

There were no toxicologically significant changes in the haematological parameters measured.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

No toxicologically significant changes were detected in blood chemical parameters.

Urinalysis findings:

no effects observed

Description (incidence and severity):

There were no treatment-related urinalytical effects detected for any treated male dose groups.

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

There were no treatment-related effects detected in animals of either sex treated with 1000, 300 or 100 mg/kg bw/day.
There were no treatment-related changes in functional performance for animals of either sex treated with 1000, 300 or 100 mg/kg bw/day
There were no treatment-related changes in sensory reactivity for animals of either sex treated with 1000, 300 or 100 mg/kg bw/day.

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

There was no evidence of test item-related histopathological findings.

Histopathological findings: neoplastic:

no effects observed

Description (incidence and severity):

There was no evidence of test item-related histopathological findings.

Other effects:

not specified

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

not specified

Description (incidence and severity):

No treatment-related effects were detected in mating performance.

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

No treatment-related effects were detected in mating performance.

Reproductive performance:

no effects observed

Description (incidence and severity):

There were no treatment-related differences in fertility. One control female and one female treated with 100 mg/kg bw/day were non-pregnant following positive evidence of mating. No histopathological abnormalities were detected in either the male or females to explain the failure of the animals to breed successfully. Two females treated with 100 mg/kg bw/day showed positive evidence of mating however failed to give birth to any live offspring.
There were no differences in gestation lengths. Gestation lengths were between 22 and 23½ days.

Effect levels (P0)

Key result

Dose descriptor:

NOEL

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

mortality

body weight and weight gain

food consumption and compound intake

food efficiency

water consumption and compound intake

haematology

clinical biochemistry

urinalysis

organ weights and organ / body weight ratios

gross pathology

histopathology: non-neoplastic

histopathology: neoplastic

reproductive function (oestrous cycle)

reproductive function (sperm measures)

reproductive performance

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Description (incidence and severity):

There were no clinical signs of toxicity observed in offspring, or any macroscopic abnormalities detected at terminal kill, litter response is based on all litters reared to termination on Day 5 of lactation/age. The incidental clinical signs detected throughout the control and treatment groups consisting of small, no milk in stomach, ear damage, hind limb malformation, missing and found dead were considered to be low incidence findings observed in offspring in studies of this type and were considered unrelated to test item toxicity.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

no mortality observed

Description (incidence and severity):

There was no effect of treatment on the mean number of corpora lutea, implantation sites, and pre- or post implantation losses. Of the litters born, litter size at birth and subsequently on Day 1 and 4 post partum was comparable to controls. Sex ratio (percentage male offspring) for the offspring was similar in all groups.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Offspring body weights and litter weights on Days 1 and 4 post partum were comparable to controls. Body weight changes for the pups across all treatment groups were similar to controls.
Statistical analysis of surface righting reflex did not reveal any significant intergroup differences.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

There were no toxicologically significant changes in the haematological parameters measured

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

No toxicologically significant changes were detected in blood chemical parameters

Urinalysis findings:

no effects observed

Description (incidence and severity):

No treatment related effects were detected for any treated male dose groups.

Sexual maturation:

not examined

Anogenital distance (AGD):

not examined

Nipple retention in male pups:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

No toxicologically significant changes were evident in the organ weights measured

Gross pathological findings:

no effects observed

Description (incidence and severity):

No treatment-related macroscopic abnormalities were detected in interim death or terminal kill offspring. The incidental findings observed were those occasionally observed in reproductive studies of this type and were considered to be unrelated to toxicity of the test item.

Histopathological findings:

no effects observed

Description (incidence and severity):

There was no evidence of test item-related histopathological findings. All of the histopathological findings encountered were considered to have occurred spontaneously or post mortem.

Other effects:

not specified

Developmental neurotoxicity (F1)

Behaviour (functional findings):

no effects observed

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

no effects observed

Details on results (F1)

JUSTIFICATION OF NO OBSERVED EFFECT LEVEL
Oral administration of the test item SE7B to rats for a period of up to eight weeks at dose levels of up to 1000 mg/kg bw/day did not result in any treatment-related changes. The ‘No Observed Adverse Effect Level’ (NOAEL) was, therefore considered to be 1000 mg/kg bw/day.
The following differences between treated and control animals were considered not to be indicative of test item toxicity:

Clinical Observations
- There were isolated incidents of one male and one female treated with 1000 mg/kg bw/day showing signs of generalised fur loss. Two control males and two males treated with 1000 mg/kg bw/day had scab formation. This was considered likely to be due to a physical injury. Both of these observations were considered to be of no toxicological importance.

Funtional Observations
Functional Performance Tests
- Males treated with 1000 and 300 mg/kg bw/day attained a statistically significant reduction in the forelimb Test 1 and all treated female groups attained a statistically significant increase in the fore limb Test 3. All individual values were within the background control ranges and without the presence of dose-related response. Therefore, these findings were considered to be incidental and to be of no toxicological importance.
- Overall activity for non recovery males treated with 1000 mg/kg bw/day was lower when compared to controls, subsequently attaining statistical significance but without any dose relationship. The corresponding value in high non-recovery females was comparable with control and as there were no apparent signs of neurotoxicity for any of these animals. The intergroup difference was considered to be incidental.

Body Weight
- Intergroup differences were observed, in particular for the recovery male achieving statistical significance in some instances, however, overall body weight gain was comparable to controls. Overall weight gains for all non-recovery male dose groups were slightly higher than controls and there was no effect of treatment on female body weight development during pre-pairing, gestation or lactation.

Haematology
- Non-recovery males treated with 1000 mg/kg bw/day showed a statistically significant increase (p<0.05) in hematocrit levels when compared to controls. Individual values were all within the normal background ranges for animals of the age, sex and strain and in the absence of any supporting histopathological correlates the intergroup difference was considered to be incidental and of no toxicological importance.

Blood Chemistry
- Non-recovery males treated with 1000 mg/kg bw/day showed a statistically increase in chloride concentration. Non-recovery females from all treatment groups showed a statistically significant reduction in alkaline phosphates; which there were no dose relationships. Recovery high dose males showed a statistical significant reduction in Albumin/globulin ratio values and an increase in bile acid when compared to controls. All individual values were all within the normal background ranges for animals of the age, sex and strain and in the absence of any supporting histopathological correlates the intergroup differences were considered to be incidental and of no toxicological importance.

Organ Weights
- Incidental findings were confined to all treated non-recovery males showing a statistically significant increase (p<0.05) in absolute and relative brain weights when compared to controls. Recovery high dose males revealed a statistically significant reduction (p<0.05) in absolute and relative thyroid/parathyroid weights. The majority of the individual values were within the anticipated background ranges and in the absence of any histopathological correlates these changes were considered to be of no toxicological importance.

Effect levels (F1)

Key result

Dose descriptor:

NOEL

Generation:

F1

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

viability

clinical signs

mortality

body weight and weight gain

gross pathology

histopathology: non-neoplastic

histopathology: neoplastic

Overall reproductive toxicity

Key result

Reproductive effects observed:

no

Any other information on results incl. tables

Results tables attached ( Results Tables - OECD 422 - SE7B.pdf)

Applicant's summary and conclusion

Conclusions:

The oral administration of SE7B to rats by gavage, at dose levels of 100, 300 and 1000 mg/kg bw/day, was tolerated well. Based on these results the ‘No Observed Adverse Effect Level’ (NOAEL) for systemic toxicity was therefore considered to be 1000 mg/kg bw/day. The ‘No Observed Effect Level’ (NOEL) for reproductive toxicity was considered to be 1000 mg/kg bw/day.