2,2'-(octadecylimino)bisethanol

Endpoint summary

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Effects on fertility

Description of key information

The available information is on a related substance 2, 2’-(Octadec-9-enylimino) bisethanol CAS No 25307-17-9, this substance is predominantly C18 unsaturated, while, 2,2’-(Octadecylimino)bisethanol) CAS No 10213-78-2 is predominantly C18 saturated. The unsaturated C18 is more reactive and therefore expected to be more toxic than the equivalent C18 saturated structure constituting for the proposed category a k-nearest neighbor worse-case scenario.

The effects on fertility are based on an OECD 443 on the read-across substance 2,2’-(Octadecylimino)bisethanol) CAS No 10213-78-2.The extent of the local irritancy of the findings in addition to deaths in the high dose group (150 mg/kg/day) resulted in an overall NOAEL being set to 30 mg/kg/day. Litter size, survival indices and sex ratio the number of implantation sites, post-implantation survival, live birth index and the number of offspring on Day 1 (total and live Day 1) were unaffected by parental treatment at dose levels up to and including 150 mg/kg/day. Resulting in the conclusion systemic and reproductive toxicity was determined to have a NOAEL of 150 mg/kg/day.

 

Link to relevant study records

Reference

Endpoint:

extended one-generation reproductive toxicity - basic test design (Cohorts 1A, and 1B without extension)

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

Experimental start date (Animal arrival) 25 March 2020 Experimental completion date (Anatomical Pathology Report ) 23 February 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

See section 13.2 for the read-across justification.

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 443 (Extended One-Generation Reproductive Toxicity Study)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Justification for study design:

Purpose
The purpose of this study was to assess the influence of 2,2' (Octadec 9 enylimino)bisethanol on reproductive performance when administered by oral gavage administration to Han Wistar rats. Cohorts of F1 animals were used to assess the potential for systemic toxicity, and potential effects on sexual maturation and estrous cycles.


Route of Administration
The oral gavage route of administration was chosen to simulate the conditions of possible human exposure.

Rationale for Dose Level Selection
Dose levels of 30, 70 and 150 mg/kg/day were selected in conjunction with the Sponsor based on the findings from a preliminary reproductive study conducted at this laboratory (Covance study no XG05HV).

In the preliminary study oral administration of 2,2'-(Octadec-9-enylimino)bisethanol at dose levels of 30, 70 or 150 mg/kg/day had no adverse effect on general clinical condition (F0/F1), mating performance and fertility (F0), organ weights (F0) or macropathology (F0/F1). However at 150 mg/kg/day significant effects were apparent on both F0 and F1 body weight performance and two out of the eight litters showed high mortality.

Based on the findings in the preliminary study there was nothing to preclude the use of 150 mg/kg/day as the high dose for use on this OECD 443 study, with low and intermediate dose levels of 30 and 70 mg/kg/day, to provide approximately 2-fold dose intervals.

Species:

rat

Strain:

Wistar

Details on species / strain selection:

Animal Model
The rat was chosen as the test species because of the requirement for a rodent species by regulatory agencies. The HanWistar (RccHan™;WIST) strain was used because of the historical control data available at this laboratory.

Sex:

male/female

Details on test animals or test system and environmental conditions:

Strain/Species RccHan™;WIST rat.

Supplier Envigo RMS Limited.

Number of animals ordered 106 males and 106 females; males unrelated to females.
Spare animals were removed from the study room after treatment commenced.

Duration of acclimatization Six days before commencement of treatment.

Age of the F0 animals at the start of the treatment 28 to 34 days old.

Weight range of the F0 animals at the start of the treatment Males 72 to 102 g.
Females 65 to 95 g.

Animal Care and Husbandry
Environmental Control
Animal facility Limited access - to minimize entry of external biological and chemical agents and to minimize the transference of such agents between rooms.

Air supply Filtered fresh air which was passed to atmosphere and not recirculated.

Temperature and relative humidity Monitored and maintained within the range of 20-24°C and 40-70%.

There were no deviations from these ranges.

Lighting Artificial lighting, 12 hours light: 12 hours dark.

Electricity supply Public supply with automatic stand-by generators.

Animal Accommodation
Cages Cages comprised of a polycarbonate body with a stainless steel mesh lid; changed at appropriate intervals.

Solid (polycarbonate) bottom cages were used throughout the study except during pairing.

Grid bottomed cages were used during pairing. These were suspended above absorbent paper which was changed daily.

Cage distribution The cages were distributed on the racking to equalize, as far as possible, environmental influences amongst the groups.

Bedding Solid bottom cages contained softwood based bark-free fiber bedding, which was changed at appropriate intervals each week.

Environmental Enrichment
Aspen wood based product A soft white untreated wood based products; provided to each cage throughout the study (except for F0 females during lactation and except when F1 Cohort 1A animals were separated into single housing overnight during urine collection) and replaced when necessary.

Plastic shelter Provided to each cage throughout the study (except during pairing and during lactation and except when F1 Cohort 1A animals were separated into single housing overnight during urine collection) and replaced at the same time as the cages.

Paper shavings From Day 20 after mating and throughout lactation, approximately two handfuls of paper shavings were provided to each cage as nesting material; this nesting material was changed at the same frequency as the cage bedding.

Diet Supply
Diet SDS VRF1 Certified pelleted diet.

The diet contained no added antibiotic or other chemotherapeutic or prophylactic agent.

Availability Non-restricted (except when diet was removed overnight before blood sampling for hematology or blood chemistry and during urine collection).

Water Supply
Supply Potable water from the public supply via polycarbonate bottles with sipper tubes. Bottles were changed at appropriate intervals.

Availability Non-restricted (except during urine collection).

Supplier Certificates of Analysis
Certificates of analysis for the diet were scrutinized and approved before any batch of diet was released for use. Certificates of analysis were routinely provided by the water supplier.

Certificates of analysis were also received from the suppliers of the softwood based bark-free fiber bedding and Aspen wood based product.

No specific contaminants were known that may have interfered with or prejudiced the outcome of the study and therefore no special assays were performed.

Route of administration:

oral: gavage

Vehicle:

arachis oil

Details on exposure:

Test Item Preparation and Analysis
Method of preparation The required amount of test item was weighed and approximately 50% of the required volume of vehicle was added. It was magnetically stirred until uniformly mixed and then made up to the required volume with vehicle and mixed again with a magnetic stirrer until homogenous.

A series of formulations at the required concentrations were prepared by dilution of individual weighings of the test item.

Frequency of preparation Weekly.

Storage of formulation Refrigerated (2 to 8°C).

Test item accounting
Detailed records of compound usage were maintained. The amount of test item necessary to prepare the formulations and the amount actually used were determined on each occasion. The difference between these amounts was checked before the formulations were dispensed.

Formulation Analysis
Stability and homogeneity Before commencement of treatment, the suitability of the proposed mixing procedures was determined and specimen formulations at 0.5 to 50 mg/mL were analyzed to assess the stability and homogeneity of the test item in the liquid matrix (Covance Study Number NQ85KJ).

• Ambient temperature (15 to 25℃) for 24 hours.
• Refrigerated temperature (2 to 8℃) for eight days.

Achieved concentration Samples of each dose preparation l for Week 1 (F0 and F1 generation) and last week (F1 generation) were analyzed for achieved concentration of the test item.


Formulation A daily record of the usage of formulation was maintained based on weights. This balance was compared with the expected usage as a check of correct administration. No significant discrepancy was found.
Formulations were stirred using a magnetic stirrer before and throughout the dosing procedure.

Details on mating procedure:

F0 Generation
F0 pairing commenced After ten weeks of treatment.

Male/female ratio 1:1 from within the same treatment groups (sibling pairing was not permitted).

Duration of pairing Up to two weeks.

Daily checks for evidence of mating Ejected copulation plugs in cage tray and sperm in the vaginal smear.

Day 0 of gestation When positive evidence of mating was detected.

Male/female separation Day when mating evidence was detected.
Pre-coital interval Calculated for each female as the time between first pairing and evidence of mating.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The analytical method involved extraction in acetone and dilution in acetonitrile/water/formic
acid 50/50/0.1 v/v/v followed by liquid chromatographic analysis with mass spectrometric
detection (LC-MS/MS). Sample concentrations were determined with reference to single
bracketing standards. Procedural recovery samples were prepared concurrently with samples
and results were corrected for the mean recovery value at each level at analysis.

Duration of treatment / exposure:

F0 animals For ten weeks before pairing until termination after litters were weaned.

F1 animals From weaning until termination of respective cohort; although direct exposure starts at weaning on Day 21 of age, all offspring had potential indirect exposure in utero and through the milk during lactation.

Cohort 1A : General toxicity and pathology of the tissues of the male and female reproductive systems - treated from weaning to 13 weeks of age.

Cohort 1B : Spare Cohort - treated from weaning to 14 weeks of age.

Frequency of treatment:

Once daily at approximately the same time each day. Animals were not dosed if parturition was in progress at the scheduled time of administration.

Details on study schedule:

Selection of Offspring to Form F1 Generation
Selection
Nominally Day 21 of age (direct dose administration from Day 21 of age).

Formal start of F1 generation Nominally Day 28 of age

Method Where possible, two male and two female were selected from each selected litter and were allocated to each of the two cohorts. If more were required, up to three males and three females were selected from each selected litter.

Selected animals were microchipped on Day 18 to 21 of age and separated from littermates on Day 21 of age.

Up to two male and two female offspring per group were retained as spares, to provide potential replacement in the event of any mortality. These spares had body weights and clinical signs recorded weekly and were terminated after commencement of the F1 generation.

Dose / conc.:

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

Remarks:

Group 1 (control): F0 generation, F1 generation cohorts 1A and 1B

Dose / conc.:

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

Remarks:

Group 2: F0 generation, F1 generation cohorts 1A and 1B

Dose / conc.:

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

Remarks:

Group 3: F0 generation, F1 generation cohorts 1A and 1B

Dose / conc.:

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

Remarks:

Group 4: F0 generation, F1 generation cohorts 1A and 1B

No. of animals per sex per dose:

F0 generation: three groups of 25 male and 25 female rats

F1 generation
Cohorts 1A and 1B: 20 male and 20 female progeny

Control animals:

yes, concurrent vehicle

Details on study design:

Rationale for Dose Level Selection
Dose levels of 30, 70 and 150 mg/kg/day were selected in conjunction with the Sponsor based on the findings from a preliminary reproductive study conducted at this laboratory (Covance study no XG05HV).

In the preliminary study oral administration of 2,2'-(Octadec-9-enylimino)bisethanol at dose levels of 30, 70 or 150 mg/kg/day had no adverse effect on general clinical condition (F0/F1), mating performance and fertility (F0), organ weights (F0) or macropathology (F0/F1). However at 150 mg/kg/day significant effects were apparent on both F0 and F1 body weight performance and two out of the eight litters showed high mortality.

Based on the findings in the preliminary study there was nothing to preclude the use of 150 mg/kg/day as the high dose for use on this OECD 443 study, with low and intermediate dose levels of 30 and 70 mg/kg/day, to provide approximately 2-fold dose intervals.

Parental animals: Observations and examinations:

Clinical Observations - F0 generation
Animals were inspected visually at least twice daily for evidence of ill-health or reaction to treatment. Cages were inspected daily for evidence of animal ill-health amongst the occupant(s). Any deviation from normal was recorded at the time in respect of nature and severity, date and time of onset, duration and progress of the observed condition, as appropriate.

During the acclimatization period, observations of the animals and their cages were recorded at least once per day.

Signs Associated with Dosing
Detailed observations were performed to establish and confirm a pattern of signs in association with dosing according to the following schedule:

F0 generation Week 1 - Daily.
Weeks 2 to 4 - twice weekly (middle and end of the week)
Week 5 onward - once each week (Days 0, 7, 14 and 20 after mating and Days 1, 7, 14 and 20 of lactation for F0 females).

Detailed observations were recorded at the following times in relation to dose administration:
• Prior to dosing.
• One to two hours after completion of dosing.
• As late as possible in the working day.

Clinical Signs
A detailed physical examination was performed on each animal to monitor general health according to the following schedule:

Physical examination Once each week

After mating of F0 females: Days 0, 5, 12, 18 and 20 after mating and Days 1, 7, 14 and 21 of lactation.

Particular attention was paid to possible signs of neurotoxicity such as convulsions, tremor and abnormalities of gait or behavior.

Mortality
A viability check was performed near the start and end of each working day. Animals were killed for reasons of animal welfare where necessary.

A complete necropsy was performed in all cases

Body weight
F0 males Day that treatment commenced.
Each week.
Before necropsy.
F0 females Day that treatment commenced.
Each week until mating detected.
Days 0, 2, 4, 6, 8, 10, 12, 14, 16, 18 and 20 after mating.
Days 1, 4, 7, 14, 21 and 28 of lactation.
Before necropsy

Food Consumption
The weight of food supplied to each cage, that remaining and an estimate of any spilled was recorded as follows:

F0 males and females Weekly, until paired for mating.

For females after mating food consumption was performed to match the body weight recording:
Days 0-1, 2-3, 4-5, 6-7, 8-9, 10-11, 12-13, 14-15, 16-17 and 18 19 after mating
Days 1-3, 4-6, 7-13, and 14-20 of lactation.

From these records the mean weekly or daily consumption per animal (g/animal/week or g/animal/day) was calculated for each relevant phase.

Parturition Observations and Gestation Length - F0 Generation
Duration of gestation Time that elapsed between mating and commencement of parturition.

Parturition observations From Day 20 after mating animals were checked three times daily for evidence of parturition. The progress and completion of parturition was monitored; numbers of live and dead offspring were recorded and any difficulties observed were noted.

Records Made During Littering Phase - F0 Generation
The records maintained were as follows:

Clinical observations Observed approximately 24 hours after birth (Day 1 of age) and then daily for evidence of ill-health or reaction to treatment.

On Day 1 of age, all offspring received a qualitative assessment of body temperature, state of activity and reaction to handling.

Hematology, Peripheral Blood - F0 Generation
Blood samples were collected after overnight withdrawal of food. Sampling for Cohort 1A was performed on the morning after overnight collection of urine. These animals were, therefore, also deprived of water overnight but had access to water for a minimum period of one hour prior to the commencement of blood sampling procedures. Samples were collected at the following occasions:

Occasion Generation Animals
Termination F0 Adults Ten male and ten female animals per group

Animals were held under light general anesthesia induced by isoflurane. Blood samples (nominally 0.5 mL) were withdrawn from the sublingual vein, collected into tubes containing EDTA anticoagulant and examined for the following characteristics using a Bayer Advia 120 analyzer:
• Hematocrit (Hct)*
• Hemoglobin concentration (Hb)
• Erythrocyte count (RBC)
• Absolute reticulocyte count (Retic)
• Mean cell hemoglobin (MCH)*
• Mean cell hemoglobin concentration (MCHC)*
• Mean cell volume (MCV)
• Total leucocyte count (WBC)
• Differential leucocyte count:
• Neutrophils (N)
• Lymphocytes (L)
• Eosinophils (E)
• Basophils (B)
• Monocytes (M)
• Large unstained cells (LUC)
• Platelet count (Plt)

* Derived values calculated in ClinAxys

Blood film (prepared for all samples) - Romanowsky stain, examined for abnormalities by light microscopy, in the case of flags from the Advia 120 analyzer. Confirmation or a written description from the blood film was made where appropriate. A manual count of the differential white blood cell parameters was performed where necessary.
Additional blood samples (nominally 0.5 mL) were taken into tubes containing citrate anticoagulant and examined using a Stago STA Compact Max analyzer and appropriate reagent in respect of:
• Prothrombin time (PT) - using IL PT Fibrinogen reagent.
• Activated partial thromboplastin time (APTT) - using IL APTT reagent.

Blood Chemistry - F0 Generation
Blood samples were collected after overnight withdrawal of food. Sampling for Cohort 1A was performed on the morning after overnight collection of urine. These animals were, therefore, also deprived of water overnight but had access to water for a minimum period of one hour prior to the commencement of blood sampling procedures. Samples were collected at the following occasions:
Occasion Generation Animals
Termination F0 Adults Ten male and ten female animals per group
F1 Cohort 1A Ten male and ten female animals per group

Animals were held under light general anesthesia induced by isoflurane. Blood samples (nominally 0.7 mL) were withdrawn from the sublingual vein and collected into tubes containing lithium heparin as anticoagulant. After separation, the plasma was examined using a Roche Cobas 6000 Analyzer in respect of:
• Alkaline phosphatase (ALP)
• Alanine aminotransferase (ALT)
• Aspartate aminotransferase (AST)
• Gamma-glutamyl transpeptidase (gGT)
• Total bilirubin (Bili)
• Bile acid (Bi Ac)
• Urea
• Creatinine (Creat)
• Glucose (Gluc)
• Total cholesterol (Chol)
• Sodium (Na)
• Potassium (K)
• Chloride (Cl)
• Calcium (Ca)
• Inorganic phosphorus (Phos)
• Total protein (Total Prot)
• Albumin (Alb)

Albumin/globulin ratio (A/G Ratio) was calculated from total protein concentration and analyzed albumin concentration.


Thyroid Hormone Analysis - TSH and T4

Blood samples were collected at the following occasions:
Occasion Generation Animals
Termination F0 Adults Ten male and ten female animals per group

Ten male and ten female animals per group


Conditions Adults: Following overnight deprivation of food.
Blood sample site Adults: Sublingual vein.
Anesthetic Adults and Offspring on Day 22 of age: Isoflurane
Anticoagulant None.
Tubes Greiner Minicollect - with clot activator.
Blood volume 1 mL
Treatment of samples Samples were kept at ambient temperature (15 to 25°C) for a minimum of 30 minutes prior to centrifugation.
Centrifugation conditions At 2000g for ten minutes at 4°C.
Number of aliquots Adults and offspring on Day 22 of age: Two per animal. Aliquot 1: 0.2 mL serum for T4
Aliquot 2: residual serum for TSH
Final storage conditions Deep frozen (approximately -60°C to -90°C).
Fate of samples Aliquot 1 (T4): dispatched to the Department of Bioanalysis, Covance.
Aliquot 2 (TSH): dispatched to the Department of Immunology & Immunotoxicology, Covance.
T4 Performed by the Department of LC-MS/MS Bioanalysis, Covance.
TSH Performed by the Department of Immunology & Immunotoxicology, Covance.

Oestrous cyclicity (parental animals):

Estrous Cycle Monitoring - F0 Generation
Dry and wet smears were taken as follows:

Dry smears For 15 days before pairing, using cotton swabs.
Wet smears After pairing until evidence of mating confirmed.

For four days before scheduled termination (nominally Days 25 to 28 post partum). Females that failed to litter or mate were retained and smeared for four days starting on the day on which the first batch of ‘true’ Day 25 post partum females started smearing, and were then killed with that first batch of females.

Sperm parameters (parental animals):

Sperm Analysis - F0 Generation
Immediately after scheduled sacrifice of each F0 male and the left vas deferens, epididymis and testis were removed and the epididymis and testis were weighed.

The following tests were performed:
Sperm motility: all groups
A sample of sperm was expressed from the left vas deferens into prewarmed (target 37°C) medium M199, which contained 0.5% w/v bovine serum albumin (BSA Fraction V). A sample for assessment was taken into a 100 µm depth cannula by capillary action and, at least 200 sperm per animal analyzed using the Hamilton Thorne IVOS II Computer Assisted Sperm Analyzer (CASA).

F0 Group 1 Males 9 & 19 had insufficient sperm to assess 200.

Sperm morphology
Groups 1 and 4 A 200 µL aliquot of the sperm/medium mixture (described above) was diluted with 800 µL of 10% neutral buffered formalin. After staining with nigrosine and eosin an air-dried smear was prepared. Slides were examined by light microscopy for the assessment of sperm morphology. At least 200 sperm were assessed for each male, where possible.

F0 Group 1 Males 9 & 19 and Group 4 Male 91 & 98 had insufficient sperm to assess 200.

Sperm morphology: Groups 2 and 3 Fixed samples retained for possible future assessment.

Sperm count: Groups 1 and 4 The left cauda epididymis of each male was weighed and then the tunica was removed, then homogenized for at least 30 seconds in 10 mL of a mixture of 0.9% saline and 0.01% merthiolate (SM). An aliquot of this mixture was added to a pre-prepared IDENT stain tube before being assessed for sperm count using CASA.

Sperm count: Groups 2 and 3 Samples frozen for possible future assessment.

Homogenization-resistant spermatid counts: Groups 1 and 4 After removal of the tunica, the left testis of each male then homogenized for at least 30 seconds in 25 mL of SM. An aliquot of this mixture was added to a pre-prepared IDENT stain tube before being assessed for homogenization resistant spermatid count using CASA.

Homogenization-resistant spermatid counts: Groups 2 and 3 Samples frozen for possible future assessment.


Light Microscopy
Tissues preserved for examination were examined as follows:
Right testis (except F1 Group 4 male 480, the left tissues were used) A detailed qualitative examination was made, 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, multinucleate or apoptotic germ cells and sloughing of spermatogenic cells into the lumen. Any cell- or stage-specificity of testicular findings was noted.

Vagina The stage of vaginal estrus was evaluated based on vaginal epithelial morphology (and appearance of the uterus and endometrial glands).
All other findings were either reported as "present" or assigned a severity grade. In the latter case one of the following five grades was used - minimal, slight, moderate, marked or severe. A reviewing pathologist undertook a peer review of the microscopic findings.

Litter observations:

Litter size
Daily records were maintained of mortality and consequent changes in litter size from Days 1-21 of age.
On Day 4 of age, litters containing more than eight offspring were reduced to eight by random culling, leaving, whenever possible, four male and four female offspring in each litter.

Sex ratio of each litter
Recorded on Days 1, 4 (before and after culling) and on Day 21 of age.

Individual offspring body weights
Recorded on Days 1, 4 (before culling), 7, 14 and 21 of age.
Selected F1 generation: Days 23, 25, 27* and 29* of age.
* - Only applicable before formal commencement of the F1 generation at nominal four weeks of age (Day 28 of age ± 2 days).

Unselected F1 offspring: Day 22 of age

Weaning of offspring The dam was removed from the litter cage and offspring were weaned on Day 21 of age.

Ano-genital distance Day 1 of age - all offspring.

Nipple/areolae count Day 13 of age - male offspring.


Sexual Maturation - F1 Generation
Males Sexual maturation was assessed by daily examination from Day 38 of age until balano-preputial separation occurred. Body weight was recorded on the day of completion of separation.
Females Sexual maturation was assessed by daily examination from Day 25 of age until vaginal opening occurred. Body weight was recorded on the day of vaginal opening.
For Cohort 1A: a wet smear was taken daily from the day of vaginal opening until first estrus was detected.

Mortality - F1 Generation
A viability check was performed near the start and end of each working day. Animals were killed for reasons of animal welfare where necessary.

A complete necropsy was performed in all cases

Body Weight - F1 Generation
The weight of animals was recorded as follows:
F1 selected animals From nominal Week 4 of age, twice during Week 1 of the F1 generation and weekly thereafter.

Before necropsy.

Food consumption - F1 Generation
F1 selected animals From nominal Week 4 of age, twice during Week 1 of the F1 generation and weekly thereafter.
From these records the mean weekly or daily consumption per animal (g/animal/week or g/animal/day) was calculated for each relevant phase.

Haematology, Peripheral Blood - F1 Cohort 1A Generation
Blood samples were collected after overnight withdrawal of food. Sampling for Cohort 1A was performed on the morning after overnight collection of urine. These animals were, therefore, also deprived of water overnight but had access to water for a minimum period of one hour prior to the commencement of blood sampling procedures. Samples were collected at the following occasions:
Occasion Generation Animals
Termination F1 Cohort 1A Ten male and ten female animals per group

Animals were held under light general anesthesia induced by isoflurane. Blood samples (nominally 0.5 mL) were withdrawn from the sublingual vein, collected into tubes containing EDTA anticoagulant and examined for the following characteristics using a Bayer Advia 120 analyzer:
• Hematocrit (Hct)*
• Hemoglobin concentration (Hb)
• Erythrocyte count (RBC)
• Absolute reticulocyte count (Retic)
• Mean cell hemoglobin (MCH)*
• Mean cell hemoglobin concentration (MCHC)*
• Mean cell volume (MCV)
• Total leucocyte count (WBC)
• Differential leucocyte count:
• Neutrophils (N)
• Lymphocytes (L)
• Eosinophils (E)
• Basophils (B)
• Monocytes (M)
• Large unstained cells (LUC)
• Platelet count (Plt)

* Derived values calculated in ClinAxys

Blood film (prepared for all samples) - Romanowsky stain, examined for abnormalities by light microscopy, in the case of flags from the Advia 120 analyzer. Confirmation or a written description from the blood film was made where appropriate. A manual count of the differential white blood cell parameters was performed where necessary.
Additional blood samples (nominally 0.5 mL) were taken into tubes containing citrate anticoagulant and examined using a Stago STA Compact Max analyzer and appropriate reagent in respect of:
• Prothrombin time (PT) - using IL PT Fibrinogen reagent.
• Activated partial thromboplastin time (APTT) - using IL APTT reagent.


Blood Chemistry - F1 Cohort 1A Generation
Blood samples were collected after overnight withdrawal of food. Sampling for Cohort 1A was performed on the morning after overnight collection of urine. These animals were, therefore, also deprived of water overnight but had access to water for a minimum period of one hour prior to the commencement of blood sampling procedures. Samples were collected at the following occasions:
Occasion Generation Animals
Termination F0 Adults Ten male and ten female animals per group
F1 Cohort 1A Ten male and ten female animals per group

Animals were held under light general anesthesia induced by isoflurane. Blood samples (nominally 0.7 mL) were withdrawn from the sublingual vein and collected into tubes containing lithium heparin as anticoagulant. After separation, the plasma was examined using a Roche Cobas 6000 Analyzer in respect of:
• Alkaline phosphatase (ALP)
• Alanine aminotransferase (ALT)
• Aspartate aminotransferase (AST)
• Gamma-glutamyl transpeptidase (gGT)
• Total bilirubin (Bili)
• Bile acid (Bi Ac)
• Urea
• Creatinine (Creat)
• Glucose (Gluc)
• Total cholesterol (Chol)
• Sodium (Na)
• Potassium (K)
• Chloride (Cl)
• Calcium (Ca)
• Inorganic phosphorus (Phos)
• Total protein (Total Prot)
• Albumin (Alb)

Albumin/globulin ratio (A/G Ratio) was calculated from total protein concentration and analyzed albumin concentration.

Urinalysis - F1 Cohort 1A Generation
Urine samples were collected after overnight withdrawal of food and water at the following occasion:
Occasion Generation Animals
Termination F1 Cohort 1A Ten males and ten females animals per group

The individual samples were examined for the following characteristics:
• Using manual methods:
• Clarity and Color (App) - by visual assessment
• Volume (Vol) - using a measuring cylinder
• pH - using a pH meter
• Specific gravity (SG) - by direct refractometry using a SG meter

• Using Multistix reagent strips interpreted using the Clinitek®500 instrument:
• Glucose (Gluc)
• Ketones (Keto)
• Bile pigments (Bili)
• Blood pigments (UBld)

• Using a Cobas 6000 Analyzer:
• Protein - total (T-Prot) and concentration (Prot)
• Sodium - total (T-Na) and concentration (U-Na)
• Potassium - total (T-K) and concentration (U-K)
• Chloride - total (T-Cl) and concentration (U-Cl)

A microscopic examination of the urine sediment was performed. An aliquot of the urine sample was centrifuged, stained with Kova stain and the resulting deposit spread on a microscope slide. The number of elements seen in nine high or low power fields (HPF or LPF) was recorded in the raw data and entered onto the database and the number seen /HPF or /LPF was derived from these data as described below.
• Epithelial cells (Epi)
• Leucocytes (WBC)
• Erythrocytes (RBC)
• Casts
• Other abnormal components (A)

The slide was also examined for abnormalities in spermatozoa and crystals


Thyroid Hormone Analysis - TSH and T4
Blood samples were collected at the following occasions:
Occasion Generation Animals
Termination F1 Offspring Ten litters per group on Day 4 of age
(pooled litter sample for T4 only and retained pending possible analysis)
Ten male and ten female animals per group on Day 22 of age
(from as many litters as possible)

F1 Adults - Cohort 1A Ten male and ten female animals per group

Animal numbers are documented in Attachment 13.3 and Attachment 13.4.

Conditions Adults: Following overnight deprivation of food.

Offspring: No overnight deprivation of food.
Offspring Day 4 of age: Decapitation
Offspring Day 22 of age: Orbital sinus
Anesthetic Offspring on Day 22 of age: Isoflurane
Offspring Day 4 of age: Not required
Anticoagulant None.
Tubes Greiner Minicollect - with clot activator.
Blood volume Offspring Day 22 of age: 1 mL
Offspring Day 4 of age: Maximum possible.
Treatment of samples Samples were kept at ambient temperature (15 to 25°C) for a minimum of 30 minutes prior to centrifugation.
Centrifugation conditions At 2000g for ten minutes at 4°C.
Number of aliquots Offspring on Day 22 of age: Two per animal. Aliquot 1: 0.2 mL serum for T4
Aliquot 2: residual serum for TSH
Offspring on Day 4 of age: single aliquot for T4 analysis, all available collected.
Final storage conditions Deep frozen (approximately -60°C to -90C).
Fate of samples Aliquot 1 (T4): dispatched to the Department of Bioanalysis, Covance.
Aliquot 2 (TSH): dispatched to the Department of Immunology & Immunotoxicology, Covance.
T4 Performed by the Department of LC-MS/MS Bioanalysis, Covance.
TSH Performed by the Department of Immunology & Immunotoxicology, Covance.

Cohort Specific in Life Investigations - F1 Generation
Estrous Cycle Monitoring - Cohort 1A
Dry and wet smears were taken as follows:

Wet smears (using pipette lavage) Following onset of vaginal patency until first cornified (estrus) smear was recorded.

For at least three days prior to the start of the necropsy phase and on the day of termination.
Dry smears (using cotton swabs) For two weeks from approximately Day 75 of age.

Estrous Cycle Monitoring - Cohort 1B
Wet smears were taken as follows:

Wet smears (using pipette lavage) For at least three days prior to the start of the necropsy phase and on the day of termination.

Postmortem examinations (parental animals):

Any abnormality in the appearance or size of any organ and tissue was recorded and the required tissue samples preserved in appropriate fixative.

The organs weighed, tissue samples fixed and sections examined microscopically (if applicable) are detailed as follows:

Tissue and regions examined

Abnormalities
Adrenals
Brain (cerebellum, cerebrum, midbrain)
Cecum
Colon
Duodenum
Epididymides
Esophagus
Eyes
Femurs - (longitudinal section through joint)
Heart (including auricular and ventricular regions)
Ileum
Jejunum
Kidneys
Liver (section from two lobes)
Lungs (section from two major lobes including bronchi)
Optic nerves
Ovaries with oviduct
Pancreas
Pituitary
Prostate - dorsolateral and ventral combined
Rectum
Sciatic nerves
Seminal vesicles (with coagulating gland)
Skeletal muscle
Skin with mammary glands (inguinal area)
Spinal cord (transverse and longitudinal sections at the cervical, thoracic and lumbar levels)
Spleen
Sternum - bone marrow
Stomach
Testes
Thymus
Thyroid with parathyroids
Trachea
Urinary bladder
Uterus with cervix
Vagina
Vas Deferens


Histology
F0 animals and Cohort 1A
Wet tissues Wet tissues were dispatched to the Test Site (Covance Harrogate, UK) for processing.

Tissue samples were dehydrated, embedded in paraffin wax and sectioned at a nominal four to five micron thickness. For bilateral organs, sections of both organs were prepared. A single section was prepared from each of the remaining tissues required.

Full List All adult animals killed or dying prematurely.

All terminal adult animals of Groups 1 and 4 killed at a scheduled interval.

Processing - reproductive organs and pituitary The reproductive organs were examined from F0 animals in Groups 2 and 3 that showed reduced fertility. This included males that failed to sire a pregnancy and females that were not pregnant, failed to litter or females with a litter death and abnormal estrous cycles.

Processing - stomach and jejunum All F0 and Cohort 1A males and females from Groups 2 and 3.
Processing - duodenum F0: All males from Groups 2 and 3.
Cohort 1A: All males and females from Groups 2 and 3.

Processing - ileum Cohort 1A: All males and females from Groups 2 and 3.

Processing - abnormalities only All F0/Cohort 1A terminal adult animals of Groups 2 and 3 killed at a scheduled interval.

Routine staining Sections were stained with hematoxylin and eosin.

Postmortem examinations (offspring):

Any abnormality in the appearance or size of any organ and tissue was recorded and the required tissue samples preserved in appropriate fixative.

For females of Cohort 1A, counts were performed for the number of ovarian follicles and corpora lutea.

Pathology procedures - Cohort 1A
Tissue and regions examined

Abnormalities
Adrenals
Brain (cerebellum, cerebrum, midbrain)
Cecum
Colon
Duodenum
Epididymides
Esophagus
Eyes
Femurs - (longitudinal section through joint)
Heart (including auricular and ventricular regions)
Ileum
Jejunum
Kidneys
Liver (section from two lobes)
Lungs (section from two major lobes including bronchi)
Lymph nodes - mesenteric
- left axillary
Optic nerves
Ovaries with oviduct
Pancreas
Pituitary
Prostate - dorsolateral and ventral combined
Rectum
Sciatic nerves
Seminal vesicles (with coagulating gland)
Skeletal muscle
Skin with mammary glands (inguinal area)
Spinal cord (transverse and longitudinal sections at the cervical, thoracic and lumbar levels)
Spleen
Sternum- bone marrow
Stomach
Testes
Thymus
Thyroid with parathyroids
Trachea
Urinary bladder
Uterus with cervix
Vagina
Vas Deferens


Pathology procedures - Cohort 1B
Tissue and regions examined

Abnormalities
Adrenals
Brain (cerebellum, cerebrum, midbrain)
Cecum
Colon
Duodenum
Epididymides
Esophagus
Eyes
Femurs - (longitudinal section through joint)
Heart (including auricular and ventricular regions)
Ileum
Jejunum
Kidneys
Liver (section from two lobes)
Lungs (section from two major lobes including bronchi)
Lymph nodes - mesenteric
- left axillary
Optic nerves
Ovaries with oviduct
Pancreas
Pituitary
Prostate - dorsolateral and ventral combined
Rectum
Sciatic nerves
Seminal vesicles (with coagulation gland)
Skeletal muscle
Skin with mammary glands (inguinal area)
Spinal cord (transverse and longitudinal sections at the cervical, thoracic and lumbar levels)
Spleen
Sternum - bone marrow
Stomach
Testes
Thymus
Thyroid with parathyroids
Trachea
Urinary bladder
Uterus with cervix
Vagina
Vas Deferens

Pathology procedures - Unselected F1 offspring on Day 22 of age
Ten male and ten females per group; one male or one female from each litter to ensure that all litters are represented.
Tissue and regions examined

Abnormalities
Brain (cerebellum, cerebrum, midbrain)
Epididymides
Ovaries
Pituitary
Prostate
Seminal vesicles
Skin with mammary glands (inguinal area)
Spleen
Testes
Thymus
Uterus with cervix and oviducts
Vagina


Sperm Analysis - F1 Cohort 1A Generation
Immediately after scheduled sacrifice of each F1 Cohort 1A male and the left vas deferens, epididymis and testis were removed and the epididymis and testis (except F1 Group 4 male 480, the right tissues were used) were weighed.

The following tests were performed:
Sperm motility: all groups A sample of sperm was expressed from the left vas deferens into prewarmed (target 37°C) medium M199, which contained 0.5% w/v bovine serum albumin (BSA Fraction V). A sample for assessment was taken into a 100 µm depth cannula by capillary action and, at least 200 sperm per animal analyzed using the Hamilton Thorne IVOS II ComputerAssisted Sperm Analyzer (CASA).


F1 Group 1 male 412 unable to assess 200 sperm. 20 samples were incorrectly analyzed using the wrong volume, reanalysis of the saved images resulted in 200 sperm not being analyzed for six samples).

Sperm morphology: Groups 1 and 4 A 200 µL aliquot of the sperm/medium mixture (described above) was diluted with 800 µL of 10% neutral buffered formalin. After staining with nigrosine and eosin an air-dried smear was prepared. Slides were examined by light microscopy for the assessment of sperm morphology. At least 200 sperm were assessed for each male, where possible.


F1 Group 1 male 413 unable to assess 200 sperm and Group 4 male 472 less than 200 sperm were analyzed in error.

Sperm morphology: Groups 2 and 3 Fixed samples retained for possible future assessment.

Sperm count: Groups 1 and 4 The left cauda epididymis of each male was weighed and then the tunica was removed, then homogenized for at least 30 seconds in 10 mL of a mixture of 0.9% saline and 0.01% merthiolate (SM). An aliquot of this mixture was added to a pre-prepared IDENT stain tube before being assessed for sperm count using CASA.

Sperm count: Groups 2 and 3 Samples frozen for possible future assessment.

Homogenization-resistant spermatid counts: Groups 1 and 4 After removal of the tunica, the left testis of each male then homogenized for at least 30 seconds in 25 mL of SM. An aliquot of this mixture was added to a pre-prepared IDENT stain tube before being assessed for homogenization resistant spermatid count using CASA.

Homogenization-resistant spermatid counts: Groups 2 and 3 Samples frozen for possible future assessment.


Histology
F0 animals and Cohort 1A
Wet tissues Wet tissues were dispatched to the Test Site (Covance Harrogate, UK) for processing.

Processing Tissue samples were dehydrated, embedded in paraffin wax and sectioned at a nominal four to five micron thickness. For bilateral organs, sections of both organs were prepared. A single section was prepared from each of the remaining tissues required.

Full List All adult animals killed or dying prematurely.

All terminal adult animals of Groups 1 and 4 killed at a scheduled interval.

Processing - reproductive organs and pituitary The reproductive organs were examined from F0 animals in Groups 2 and 3 that showed reduced fertility. This included males that failed to sire a pregnancy and females that were not pregnant, failed to litter or females with a litter death and abnormal estrous cycles.

Processing - stomach and jejunum All F0 and Cohort 1A males and females from Groups 2 and 3.

Processing - duodenum F0: All males from Groups 2 and 3.

Cohort 1A: All males and females from Groups 2 and 3.

Processing - ileum Cohort 1A: All males and females from Groups 2 and 3.

Processing - abnormalities only All F0/Cohort 1A terminal adult animals of Groups 2 and 3 killed at a scheduled interval.

Routine staining Sections were stained with hematoxylin and eosin.
Cohort 1B

Processing Tissue samples were dehydrated and embedded in paraffin wax.
Full List All adult animals killed or dying prematurely.
Processing - abnormalities only All animals.
Processing to block - reproductive organs Al animals (see Section 4).

On completion of the study phase the Histology slides were transferred back to the Test Facility with any necessary supporting documentation. The wet tissues, blocks and raw paper data were returned to the Test Facility.


Immunophenotyping of Spleen Leucocytes - Cohort 1A
Ten males and ten females per group from Cohort 1A were selected for immunophenotyping.
Where possible, one male or one female was assigned from each selected litter.

The whole spleen was weighed. After weighing, a 3-5 mm mid transverse section was removed and retained for histopathological evaluation. The remaining portions of the spleen was then weighed, placed in to a vial of chilled Hank’s Balanced Salt Solution (HBSS) and held in wet ice until processing for analysis.


Light Microscopy
Tissues preserved for examination were examined as follows:
Right testis (except F1 Group 4 male 480, the left tissues were used) A detailed qualitative examination was made, 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, multinucleate or apoptotic germ cells and sloughing of spermatogenic cells into the lumen. Any cell- or stage-specificity of testicular findings was noted.
Ovaries F0 and Cohort 1A - Qualitative evaluation of one section from each ovary
Cohort 1A - Quantitative evaluation of five sections from the middle third from each ovary for the assessment of primordial follicle and small growing follicle population as well as evaluation of corpora lutea numbers in one section from each ovary.
Vagina The stage of vaginal estrus was evaluated based on vaginal epithelial morphology (and appearance of the uterus and endometrial glands).

All other findings were either reported as "present" or assigned a severity grade. In the latter case one of the following five grades was used - minimal, slight, moderate, marked or severe. A reviewing pathologist undertook a peer review of the microscopic findings

Statistics:

Please refer to "Any other information on materials and methods"

Reproductive indices:

Mating Performance and Fertility - F0 Generation
Individual data was tabulated. Group values were calculated for males and females separately for the following:

Percentage mating (%) = (Number of animals mating /Animals paired) x 100

Conception rate (%) = (Number of animals achieving pregnancy / Animals mated) x 100

Fertility index (%) = (Number of animals achieving pregnancy / Animals paired) x 100

Gestation Length and Gestation Index - F0 Generation
Gestation length was calculated as the number of gestation days up to and including the day on which offspring were first observed, with Day 1 = day of mating for calculation purposes. Where parturition had started overnight, this value was adjusted by subtracting half of one day.

Gestation index was calculated for each group as:

Gestation index (%) = (Number of live litters born /Animals paired) x 100

Offspring viability indices:

Sexual Maturation - F1 Generation
Individual values were tabulated for age and body weight at completion. Group mean values were calculated from individual values presented.

Survival Indices - F0 Generation
The following were calculated for each litter:
Post implantation survival index (%) = Total number of offspring born x 100
Total number of uterine implantation sites

Post-implantation survival index was expressed as 100% where the number of offspring exceeded the number of implantation sites recorded.
Live birth index (%) = (Number of offspring on Day 1 after littering / Total number of offspring born) x 100

Viability index (%) = (Number of live offspring on Day 4 before culling / Number of live offspring on Day 1 after littering) x 100

Lactation index (%) = (Number of live offspring on Day 21 after littering / Number of live offspring on Day 4 (after culling) ) x 100

Group mean values were calculated from individual litter values.

Sex Ratio - F0 Generation

The percentage of male offspring in each litter was calculated at Day 1, and for live offspring on Days 1, 4 (before and after culling) and 21 of age.

Percentage males = Number of males in litter x 100
Total number of offspring in litter

Group mean values were calculated from individual litter values.

Offspring Examinations - F0 Generation

Ano-genital distance were presented both as absolute/unadjusted and adjusted for body weight, using the weight recorded on Day 1 of age.

A check was performed to assess for the presence or absence of nipple/areolae for the male offspring on Day 13 of age. As no nipples were present, no data is presented.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

A few animals receiving 150 mg/kg/day were observed with increased salivation in association with dose administration; this sign is often seen in association with oral gavage administration and is attributed to the taste of the formulation rather than an effect of treatment.

No further signs were observed at either routine physical examination or in association with dose administration that were considered to relate to administration of the test item.

Mortality:

mortality observed, treatment-related

Description (incidence):

A total of four animals (one male and three females) were euthanized for welfare reasons.
Group 1(Control) male no. 18 was euthanized on Day 43 of treatment. Terminal signs included abnormal breathing and partially closed eyelids. Macroscopic findings at necropsy were limited to an enlarged mandibular lymph node. However, no histopathological lesions accounting for the poor clinical condition were observed.

Three females (animal nos. 278, 279 and 280 that received 150 mg/kg/day) were sacrificed following the day parturition completed, Day 2 of lactation and Day 21 of gestation, respectively.
• Terminal signs for female no 278 included thin build, labored breathing, piloerection, pallor and a hunched posture. Macroscopic examination revealed a distended stomach with abnormal gastrointestinal tract content, small spleen and thick/dark red fluid in the vagina.
Histopathological evaluation revealed treatment related findings such as slight accumulation of the foamy macrophages within the jejunum and minimal epithelial hyperplasia of the non-glandular gastric region, decreased cellularity of the splenic red pulp, minimal degranulation of the pancreas, moderate involution/atrophy of the thymus, one uterine polyp and moderate intraluminal haemorrhage the vagina and uterus.
• Terminal signs for female no. 279 included thin build, piloerection, pallor and a hunched posture. Macroscopic examination revealed thickened non-glandular mucosa of the stomach, abnormal caecal and rectal content, pale areas on the lungs and generally pale organs.
Histopathological evaluation revealed treatment related findings such as slight accumulation of the foamy macrophages within the jejunum and moderate oedema with slight hyperplasia of the gastric non-glandular region, minimal focal accumulation of the macrophages within the lung, moderate degranulation of the pancreas, moderate atrophy/involution of the thymus, moderate ulceration of the urinary bladder and moderate diffuse hyperplasia of the uterus with slight fibrinoid necrosis of the vascular media.

• Terminal signs for female no. 280 included decreased activity, piloerection, pale eyes and partially closed eyelids. Macroscopic examination revealed thickened non-glandular mucosa of the stomach, abnormal gastrointestinal tract content, abnormal nasal turbinate content and a small spleen. Histopathological evaluation revealed treatment related findings such as slight accumulation of the foamy macrophages within the jejunum and slight oedema, minimal mixed inflammation, moderate ulceration and moderate epithelial hyperplasia of the non-glandular gastric region, decreased cellularity of the splenic white pulp, slight degranulation of the pancreas and moderate diffuse hyperplasia of the uterus.

The premature deaths at 150 mg/kg/day were considered to be related to treatment.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Males receiving 2,2'-(Octadec-9-enylimino)bisethanol had low body weight gain resulting in low mean absolute body weights from Day 8 of study up to termination; a dose response was apparent. The overall bodyweight gain from Day 1 to 120 was significantly low for male receiving 70 or 150 mg/kg/day (p<0.01).

During the 10-week pre-pairing treatment period body weight gain for females during Week 4 of treatment was significantly low at all dose levels; however, the overall gain for females before pairing showed no adverse effects of treatment.

During gestation body weight gain was slightly low during Days 18-20 for females receiving 150 mg/kg/day (p<0.01); however, the overall weight gain GD0-20 showed no adverse effect of treatment.

On Day 1 of lactation mean body weight for females receiving 150 mg/kg/day was low when compared with Controls (p<0.01), however overall the bodyweight gain at 70 or 150 mg/kg/day from Day 1-21 of lactation was high when compared with Control values (p<0.01); these differences were attributed to high weight gain during the first two weeks of lactation.

PLEASE REFER TO THE ATTACHED TABLES; "BODY WEIGHT AND BODY WEIGHT CHANGE - GROP MEAN VALUES FOR MALES AND FEMALES BEFORE PAITING (F0)

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Males receiving 70 or 150 mg/kg/day showed low food consumption from Week 1 of treatment (p<0.05/0.01); a dose response was apparent. Males at 30 mg/kg/day also showed occasions of low consumption during Week 2 (p<0.05), Week 5(p<0.01) and Week 9 (p<0.05) of treatment.
Before pairing females at 150 mg/kg/day showed low food consumption during Weeks 4, 5, 7, 8 and 9 (p<0.05/0.01); food consumption for females receiving 30 or 70 mg/kg/day was unaffected by treatment.

During gestation females receiving 150 g/kg/day showed slightly low food consumption, with statistical significance achieved on Days 2-6 and 12-20 (p<0.05/0.01); food consumption at 30 or 70 mg/kg/day was similar to Controls.

Food consumption during lactation was unaffected by administration of 2,2'-(Octadec-9-enylimino)bisethanol at dose levels up to and including 150 mg/kg/day.

PLEASE REFER TO THE ATTACHED TABLE: "FOOD CONSUMPTION - GROUP MEAN VALUES BEFORE PAIRING (F0)

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Males and females receiving 70 or 150 mg/kg/day and males receiving 30 mg/kg/day showed slightly but statistically significantly shorter prothrombin clotting times when compared with Controls.

At 150 mg/kg/day the mean neutrophil count for males and females was high when compared with Controls, the difference attaining statistical significance for the male animals (p<0.01). Females at 150 mg/kg/day also showed a high mean eosinophil count when compared with Controls (p<0.05).

Males at 150 mg/kg/day also showed:
• low mean haemoglobin (p<0.01)
• low mean cell haemoglobin (p<0.01)
• low mean cell volume (p<0.05)
• high platelet count (p<0.01)

Contrary to male animals, females at all dose levels showed high mean cell haemoglobin (p<0.05) and a longer activated partial thromboplastin time at 150 mg/kg/day (p<0.01) when compared with Controls.

The effects on erythrocyte parameters in males are possibly treatment related but are only of a minor nature. The percentage of reticulocytes is not affected. Females are much less affected, actually showing a non dose related small increase of MCH which in the absence of anemia has no biological meaning.

PLEASE REFER TO THE ATTACHED TABLE: "HAEMATOLOGY - GROUP MEAN VALUES AT SCHEDULED TERMINATION (F0)"

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Alanine transaminase activity for males and females receiving 150 mg/kg/day was elevated when compared with Controls (p<0.05). Males at 150 mg/kg/day also showed low total protein and albumin (p<0.01), with males showing a high A/G ratio at 70 mg/kg/day (p<0.05) and 150 mg/kg/day (p<0.01).

Other statistically significant differences were as follows:
• gamma-glutamyl transpeptidase activity at 150 mg/kg/day increased for male animals only (p<0.01)
• bile acid concentration at 150 mg/kg/day was high for males (p<0.01) but low for females (p<0.05)
• urea concentration at 70 and 150 mg/kg/day were maginally high for males only (p<0.05); however, there was no dose response and most individual values were within the concurrent control range.

• cholesterol levels at all dose levels were slightly low for females only (p<0.05)
• sodium concentrations at 150mg/kg/day were high for males (p<0.05) and low for females (p<0.05).

As observed differences to Control were of relative minor clinical significance, and mostly showed no consistency between males and females, these effects are not considered to represent adverse treatment related effects.

PLEASE REFER TO THE ATTACHED TABLE: "BLOOD CHEMISTRY - GROUP MEAN VALUES AT SCHEDULED TERMINATION (F0)"

Endocrine findings:

effects observed, non-treatment-related

Description (incidence and severity):

Thyroid Hormone Analysis
Thyroid stimulating hormone (TSH)

Administration of 2,2'-(Octadec-9-enylimino)bisethanol by oral gavage led to no significant differences in serum TSH levels in F0 adult male animals, F1 male offspring or F1 adult female animals at dose levels up to and including 150 mg/kg/day when compared with Controls.

However, serum TSH levels were high when compared with Controls for the following:
• F0 adult females at 150 mg/kg/day
• F1 female offspring at 150 mg/kg/day
• F1 adult males at 70 or 150 mg/kg/day

These differences lacked consistency and showed no correlation with thyroxine levels and were therefore considered to be unrelated to administration of the test item.

Thyroxine (T4)

The mean serum T4 concentrations in samples obtained from F1 offspring on Day 22 of age, F0 adult females and F1 adult males were slightly variable across treatment Groups 2 to 4, but within ±20% of the mean T4 values in the respective Control group.

F0 adult male animals and F1 adult female animals that received 150 mg/kg/day showed mean serum T4 levels that were low when compared with their Control group (at 77% and 75% of Controls, respectively). As the observed differences to control are relatively minor, and show no consistent pattern between ages and sexes, they are not considered to represent adverse effects from treatment with the test substance.

PLEASE REFER TO THE TABLES IN ANY OTHER INFORMATION ON RESULTS: "GROUP MEAN SERUM TSH CONCENTRATIONS IN MALE RATS" AND "FEMALE RATS"

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

effects observed, non-treatment-related

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

In unscheduled and terminal sacrifice animals, 2,2’-(Octadec-9-enylimino) bisethanol-related microscopic findings were observed in the stomach (both sexes administered 30, 70 or 150 mg/kg/day), jejunum (both sexes administered 70 or 150 mg/kg/day) and duodenum (males administered 150 mg/kg/day).

The predominant microscopic finding in the stomach was characterized as diffuse minimal to moderate epithelial hyperplasia of the non-glandular region associated with erosion/vesicle or ulceration in a few animals. Edema and mixed inflammation were considered secondary to the erosion/vesicle or ulceration. This finding correlated with the 2,2’-(Octadec-9-enylimino) bisethanol related macroscopic observation of edema and thickening of the non-glandular mucosa.
Foamy cell accumulation observed within the jejunum (both sexes) and duodenum (males only) correlated with the 2,2’-(Octadec-9-enylimino) bisethanol related macroscopic observation of abnormal color and thickening.

PLEASE REFER TO THE TABLE IN ANY OTHER INFORMATION ON RESULTS: "MICROSCOPIC FINDINGS - TERMINAL SACRIFICE F0 GENERATION"

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

With the exception of one female at 150 mg/kg/day all females showed regular 4/5-day estrous cycles.

Stage of Estrous Cycle at Termination
The majority of females showed an estrus smear prior to scheduled termination.

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

No adverse effects were observed following treatment with 2,2'-(Octadec-9-enylimino) bisethanol at dose levels up to and including 150 mg/kg/day.

Reproductive performance:

no effects observed

Description (incidence and severity):

Pre-Coital Interval
Pre-coital interval was unaffected by treatment with the vast majority of animals showing positive evidence for mating within four days of pairing, with the exception of one Control female (13-14 days) and one female at 70 mg/kg/day (5-8 days)

Mating Performance and Fertility
Mating performance and fertility did not show any adverse effects of treatment.

Gestation Length and Gestation Index
The duration of gestation was unaffected by treatment at dose levels up to and including 150 mg/kg/day; however, at 150 mg/kg/day the gestation index was slightly low when compared with Controls (91% vs 100%). This was related to the sacrifice for welfare reasons of one female on GD21 and another following completion of parturition.

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

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

System:

gastrointestinal tract

Organ:

stomach

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

At 150 mg/kg/day there was a slight increase in the number of offspring (litters) with clinical signs; three litters with offspring cold to touch and two litters scattered in the cage. The general condition of offspring at 30 or 70 mg/kg/day was similar to Controls

No signs were observed at either routine physical examination or in association with dose administration that could be associated with administration of the test item.

Mortality / viability:

mortality observed, treatment-related

Description (incidence and severity):

A total of eight animals died prematurely; death for five of these animals is related to accidental trauma rather than administration of the test item. From the remaining three deaths two are also unrelated to administration of the test item: Group 1 female no. 616 (no vagina) and in Group 2 male no. 512 (multiple pathology). The death of Group 4 male no. 466 is probably related to the observed effects in GI-system and is possibly test substance treatment related.

• Group 2 male no. 512 (30 mg/kg/day) was killed for reasons of animal welfare on Day 47 of the F1 generation. Terminal signs included thin build and piloerection. Macroscopic examination dark areas on the liver, small epididymides, small testes, small thymus and a distended urinary bladder. Histopathological examination revealed signs of sexual immaturity with absence of sperm, distension of the urinary bladder, thymic atrophy/involution and depletion of the pancreatic acini.

• Group 4 male no 466 (150 mg/kg/day) was killed for welfare reasons on Day 64 of the F1 generation following significant body weight loss. Macroscopic examination revealed that the stomach had an irregular surface and depressions on the non-glandular mucosa. Histopathological evaluation revealed treatment related findings such as slight accumulation of the foamy macrophages within the jejunum, ileum and duodenum, diffuse hypertrophy of the villi within the ileum and duodenum, and oedema, inflammation, erosion/vesicle and hyperplasia of the gastric non-glandular region and involution/atrophy of the thymus.

• Group 4 male no. 550 (150 mg/kg/day) was killed for welfare reasons on Day 25 of the F1 generation. Terminal signs and macroscopic findings were related to trauma of the left tibia; death was not attributed to treatment. Histopathological findings included a callus formation suggesting the bone trauma on the tibia.

• Group 4 male no. 559 (150 mg/kg/day) was found dead on Day 10 of the F1 generation. No clinical signs were observed up to Day 9. Macroscopic examination revealed findings including a perforated esophagus and adhesions in the thoracic cavity. Histopathological examination revealed minimal to moderate diffuse mixed inflammation within the thoracic cavity including heart, lungs and thymus. This death was attributed to dosing trauma and is not attributed to administration of the test item.

• Group 4 male no. 560 (150 mg/kg/day) was found dead and cannibalised on Day 74 of the F1 generation. Macroscopic examination revealed fluid in the thoracic cavity. Histopathological examination revealed minimal to moderate diffuse mixed inflammation within the thoracic cavity including heart, lungs and thymus. This death was attributed to dosing trauma and is not attributed to administration of the test item.

• Group 1 Control female no. 616 was despatched to necropsy on Day 55 of age having failed to achieve vaginal opening; macroscopic examination revealed that the vulva was imperforate. Histopathological evaluation revealed the absence of vagina and imperforate vulva.

• Group 3 female no 650 (70 mg/kg/day) was killed for welfare reasons on Day 60 of the F1 generation. Terminal signs include decreased activity, noisy breathing and pale ears. Macroscopic examination revealed a perforated esophagus, thick clear fluid in the thoracic cavity and the heart had a thickened pericardium; Histopathological examination revealed minimal diffuse mixed inflammation within the thoracic cavity including oesophagus, heart and lungs and involution/atrophy of the thymus; this death is attributed to doing trauma and is not attributed to administration of the test item.

• Group 3 female no 655 (70 mg/kg/day) was killed for welfare reasons on Day 11 of the F1 generation. Terminal signs included decreased activity, laboured breathing, thin build and piloerection. Macroscopic examination revealed findings of a perforated esophagus with fluid and adhesions in the thoracic cavity. Histopathological examination revealed treatment related findings such as slight accumulation of foamy cells within the sinuses of the mesenteric lymph node, slight to moderate mixed inflammation within the thoracic cavity, lungs and thymus, minimal hepatic necrosis and minimal splenic extramedullary hematopoiesis; this death is attributed to doing trauma and is not attributed to administration of the test item.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

From Day 1 of age mean body weights for both male and female offspring at 150 mg/kg/day were significantly low when compared with Controls (p<0.01). Body weight gain at 150 mg/kg/day was also low from Day 1 up to Day 7 for males (p<0.01) and from Day 1 up to Day 14 for females (p<0.05/0.01); the overall body weight gain for both male and female offspring from Day 1 up to weaning on Day 21 of age was low at 150 mg/kg/day when compared with Controls (p<0.01).

Offspring body weight for offspring at 30 and 70 mg/kg/day showed no adverse effects of parental treatment.


At weaning on Day 21 of age selected F1 males at all dose levels and females at 30 mg/kg/day showed slight differences in mean bodyweight that were not dose related. However, both males and females at 70 or 150 mg/kg/day showed low bodyweight gain up to Day 25 of age (p<0.01) and a dose response was apparent.
At the formal commencement of the F1 generation on nominal Day 28 (±2) of age, the mean bodyweight for selected F1 animals at 150 mg/kg/day was low when compared with Controls (p<0.01). Subsequent body weight gain from Day 1 to Day 64 of the F1 generation was low for males receiving 70 or 150 mg/kg/day (p<0.01) and for females at 150 mg/kg/day (p<0.05).

PLEASE REFER TO THE ATTACHED TABLES: "BODY WEIGHT AND BODY WEIGHT CHANGE - GROUP MEAN VALUES FOR OFFSPRING (F1)"
and
"BODY WEIGHT AND BODY WEIGHT CHANGE - GROUP MEAN VALUES FOR MALES AND FEMALES (F1)"

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

From Day 1 of the F1 generation males at 150 mg/kg/day showed slightly but significantly low food consumption when compared with Controls (p<0.05/0.01) and at 70 mg/kg/day low food consumption was recorded for male animals from Day 15 of the F1 generation low food consumption (p<0.05/0.01).

Females at 150 mg/kg/day showed slightly low food consumption from Day 8 of the F1 generation (p<0.05/0.01).

Food consumption for females at 70 mg/kg/day and for males and females at 30 mg/kg/day was essentially similar to Controls and considered unaffected by treatment.

PLEASE REFER TO THE ATTACHED TABLE: "FOOD CONSUMPTION - GROUP MEAN VALUES FOR MALES AND FEMALES (F1)"

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Males and females that received the test item had statistically significantly shorter prothrombin clotting times when compared with Controls (p<0.05/0.01); similar to the results for F0 animals.
Males at all dose levels showed a low haematocrit (p< 0.05/0.01) and males at 150 mg/kg/day had low haemoglobin (p<0.01) when compared with Controls. The biological relevance of this finding is limited considering the lack of changes in eyrthrocytes, with no shifts in mean cell hemaglobin, mean cell hemaglobin conentrtaion and mean cell volume.

Females that received 70 or 150 mg/kg/day had low haemoglobin (p<0.01) and mean cell haemoglobin concentration (p<0.05/0.01). Females at all dose levels had low mean cell haemoglobin (p<0.05/0.01) and females at 150 mg/kg/day also had low mean cell volume (p<0.05)and a high platelet count (p<0.01). The majority of these differences were not evident in the male animals with the exception of low haemoglobin for males that received 150 mg/kg/day. The biological relevance of the low hemagolobin is limited in view of no changes in hematocrit, RBC and reticulocytes, and only minor shifts in mean cell hemaglobin, mean cell hemaglobin conentrtaion and mean cell volume.

White blood cell counts were high for males at 150 mg/kg/day and for females at all dose levels.

• Leucocyte counts high for males at 150 mg/kg/day (p<0.05) and for females high at all dose levels(p<0.05)

• Neutrophil and monocyte counts high for males at 70 of 150 mg/kg/day(p<0.01)

• Large unstained cell count high for males at 150 mg/kg/day(p<0.05)

PLEASE REFER TO THE ATATCHED TABLE: "HAEMATOLOGY - GROUP MEAN VALUES AT SCHEDULED TERMINATION (F1 COHORT 1A)"

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Males that received 70 or 150 mg/kg/day showed low Alkaline phosphatase activity (p<0.01), which is of no biological significance, and males at 150 mg/kg/day had slightly high Alanine amino-transferase activity (p<0.01). This was not evident in female animals, with females at 150 mg/kg/day showing high Gamma glutamyl transpeptidase activity (p<0.01).

Both males and females at 150 mg/kg/day had low cholesterol levels when compared with Controls (male p<0.05, females p<0.01)

Glucose and potassium levels were low for males at dose levels up to and including 150 mg/kg/day when compared with Controls (p<0.01).

Creatinine levels for females at all dose levels were low when compared with Controls (p<0.05/0.01); this decrease is oif noi biological/toxicological significance.

When compared with Controls males at 150 mg/kg/day also had marginally high plasma urea (p<0.05), with low total protein (p<0.05) and a high A/G ratio (p<0.01).

PLEASE REFER TO THE ATTACHED TABLE: "BLOOD CHEMISTRY - GROUP MEAN VALUES AT SCHEDULED TERMINATION (F1 COHORT 1A)"

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

Urinary protein levels were significantly high for both male and females that received the test item at dose levels up to and including 150 mg/kg/day (p<0.05/0.01), but total urinary protein levels were unaffected by treatment. Other differences included:

• Low urinary output for males at 70 and 150 mg/kg/day (p<0.05)

• Low total potassium levels for males at 70 and 150 mg/kg/day (p<0.05/0.01)

• Low pH for females at all dose levels (p<0.05/0.01)

PLEASE REFER TO THE ATTACHED TABLE: "URINALYSIS - GROUP MEAN VALUES AT SCHEDULED TERMINATION (F1 COHORT 1A)"

Sexual maturation:

effects observed, non-treatment-related

Description (incidence and severity):

At 150 mg/kg/day completion of sexual maturation for both males and females was approximately three days later than the concurrent Controls; however, as the mean body weight at completion was similar to Controls, these delays are considered to be related to a general delay in physical development rather than a specific effect on sexual maturation.

PLEASE REFER TO THE ATTACHED TABLE: "SEXUAL MATURATION - GROUP MEAN AGE AND BODY WEIGHT AT COMPLETION (F1)"

Anogenital distance (AGD):

effects observed, treatment-related

Description (incidence and severity):

Ano-genital distance of male offspring on Day 1 of age was unaffected by parental treatment at dose levels up to and including 150 mg/kg/day.

The ano-genital distance for female offspring at 150 mg/kg/day was significantly smaller than Controls (p<0.05), but as this is a decrease rather than an increase in distance it is of no biological/toxicological significance; values at 30 or 70 mg/kg/day were similar to Controls

PLEASE REFER TO THE ATTACHED TABLE: "ANO-GENITAL DISTANCE - GROUP MEAN AND ADJUSTED VALUES FOR OFFSPRING (F1)"

Nipple retention in male pups:

no effects observed

Description (incidence and severity):

No nipples were seen on examination of male offspring on Day 13 of age

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Male offspring derived from the group that received 150 mg/kg/day showed slightly but statistically significantly low absolute mean brain and thymus weight when compared with Controls; in the absence of any similar observation in the female offspring at the same dose level, and since body weight relative brain and thymus weights were similar to Controls, these differences were considered to reflect the lower terminal body weights and not a specific effect of treatment on these organs.

* PLEASE REFER TO THE TABLE IN ANY OTHER INFORMATION ON RESULTS: "ORGAN WEIGHT PARAMETERS - UNSELECTED OFFPSRING ON DAY 22 OF AGE (F1)"

F1 Generation (Cohort 1A)
Body weight relative kidney weights were statistically significantly high for both males and females at all dose levels (p<0.05/0.01). Females at all dose levels and males at 70 or 150 mg/kg/day had significantly high body weight relative spleen weight (p<0.05/0.01).

2,2’-(Octadec-9-enylimino) bisethanol-related low absolute and body weight relative thymus weights were noted at the terminal sacrifice of males that received 150 mg/kg/day (p<0.01 and p<0.05 respectively). Males at 70 or 150 mg/kg/day also had high body weight relative heart weight(p<0.05) and males at 150 mg/kg/day had high bodyweight relative adrenal weight. (p<0.01).

High bodyweight relative mesenteric lymph nodes weight and liver weight were recorded for females that received 150 mg/kg/day (p<0.01 and p<0.05 respectively). The high mesenteric lymph node weights may be associated with the test item related microscopic changes identified in this tissue.

No test item related microscopic changes were detected in any of the other organs and thus no definite or adverse effect of treatment is inferred. Differences in relative brain weight and reproductive organ relative weights were considered to be secondary to the effect on body weight.

PLEASE REFER TO THE TABLE IN ANY OTHER INFORMATION ON RESULTS:"ORGAN WEIGHT PARAMETERS - TERMINAL SACRIFICE F1 GENERATION (COHORT 1A)"

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Macroscopic examination of offspring that died or were killed prior to scheduled termination or were culled on Day 4 of age did not reveal any findings that could be attributed to parental treatment.

Macroscopic examination of unselected F1 offspring on Day 22 of age did not reveal any abnormality and as such no data is presented.

F1 Generation (Cohort 1A)
At terminal sacrifice, thickening was noted for the stomach of some animals administered 70 or 150 mg/kg/day. This finding was inconsistently associated with depression, in both sexes, and correlated with the 2,2’-(Octadec-9-enylimino) bisethanol related microscopic observation of diffuse hyperplasia of the non-glandular mucosa, erosion/vesicles, ulceration, edema and mixed inflammation.

PLEASE REFER TO THE TABLE IN ANY OTHER INFORMATION ON RESULTS: "MACROSCOPIC FINDINGS - TERMINAL SACRIFICE F1 GENERATION (COHORT 1A)"

F1 Generation (Cohort 1B)
At terminal sacrifice, thickening was noted for the stomach of occasional animals administered 70 or 150 mg/kg/day. This finding was inconsistently accompanied with depression, in both sexes, and correlated with the 2,2’-(Octadec-9-enylimino) bisethanol related microscopic observation of diffuse hyperplasia of the non-glandular mucosa, erosion/vesicles, ulceration, edema and mixed inflammation.

PLEASE REFER TO THE TABLE IN ANY OTHER INFORMATION ON RESULTS: "MACROPSCOPIC FINSINGS - TERMINAL SACRIFICE F1 GENERATION (COHORT 1B)"

F1 Generation (Cohort 1B)
Low absolute thymus weights were noted at terminal sacrifice in males that received 150 mg/kg/day.

Other variations in organ weights consisted of decreases/increases of absolute or relative weights of pituitary glands (both sexes administered 150 mg/kg/day), testes, epididymides and seminal vesicles and coagulating glands (males administered 70 or 150 mg/kg/day) and ovaries (females administered 150 mg/kg/day).

The effects of 2,2’-(Octadec-9-enylimino) bisethanol administration on organ weights and organ weights relative to body weight are mostly characterised as consequential to changes observed in body weight and not indicative of specific organ toxicity. The lower absolute and relative thymus weight in males at 70 and at 150 mg/kg/day is indicative for a secondary thymus involution.

PLEASE REFER TO THE TABLE IN ANY OTHER INFORMATION ON RESULTS: "ORGAN WEIGHT PARAMETERS - TERMINAL SACRIFICE F1 GENERATION (COHORT 1B)"

Histopathological findings:

effects observed, treatment-related

Description (incidence and severity):

In unscheduled and terminal sacrifice animals, 2,2’-(Octadec-9-enylimino) bisethanol-related microscopic findings were observed in the stomach (males administered 70 or 150 mg/kg/day and females administered 30 or 150 mg/kg/day), jejunum (both sexes administered 30, 70 or 150 mg/kg/day), duodenum (both sexes administered 150 mg/kg/day), ileum (both sexes administered 70 or 150 mg/kg/day), mesenteric lymph nodes (males and females administered 70 or 150 mg/kg/day) and thymus (males administered 70 or 150 mg/kg/day).

Findings in the stomach included diffuse hyperplasia of the non-glandular mucosa, erosion/vesicles, ulceration, edema and mixed inflammation and correlated with macroscopic observations of thickening and/or depression. Findings in the jejunum, duodenum and ileum included diffuse accumulation of foamy cells within the lamina propria and lymphangiectasis accompanied by hypertrophic villi within the duodenum and ileum. Changes observed within the mesenteric lymph nodes consisted of foamy cell accumulation within the sinuses with sinus dilation and reflect the lymph nodes drainage function of the intestinal lesions. Changes observed within the thymus consisted of atrophy/involution and considered as probably due to body weight loss.


F1 Generation (Cohort 1B)
In unscheduled and terminal sacrifice animals, 2,2’-(Octadec-9-enylimino) bisethanol-related microscopic findings were observed in the stomach (males administered 70 or 150 mg/kg/day and females administered 150 mg/kg/day) and jejunum (males administered 150 mg/kg/day); histopathological examination was limited to those with macroscopic abnormality.

Findings in the stomach included diffuse hyperplasia of the non-glandular mucosa, erosion/vesicles, ulceration, edema and mixed inflammation and correlated with macroscopic observations of thickening and/or depression. Findings in the jejunum included diffuse accumulation of foamy cells within the lamina propria.

PLEASE REFER TO THE TABLES IN ANY OTHER INFORMATION ON RESULTS:
a) "MICROSCOPIC FINDINGS - TERMINAL SACRIFICE F1 GENERATION (COHORT 1A)"
b) "MICROSCOPIC FINDINGS - TERMINAL SACRIFICE F1 GENERATION (COHORT 1B)"

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Litter Size, Survival Indices and Sex Ratio
The number of implantation sites, post-implantation survival, live birth index and the number of offspring on Day 1 (total and live Day 1) were unaffected by parental treatment at dose levels up to and including 150 mg/kg/day.

However, at 150 mg/kg/day the viability index on Day 4 of age was slightly low when compared with Controls at 94.6% compared with 100% in the Control group; this was attributed to litter no 289 with a viability index of 18% and no relationship to treatment was inferred.

Litter survival following standardization of the litter size on Day 4 of age up to weaning on Day 21 of age was unaffected by treatment at all dose levels.

Sex ratio showed no effects of treatment.

Estrous cycles following Vaginal Opening - Cohort 1A
The interval between completion of vaginal opening and the first estrus smear showed no adverse effects of treatment.

Estrous Cycles from nominal Day 75 of age - Cohort 1A
Assessment of estrous cycles from nominal Day 75 of age showed a slight but statistically significant shift at 150 mg/kg/day from a 4-day cycle to 4/5 day or 5-day cycles when compared with Controls (p<0.05). At 70 mg/kg/day the incidence of 4/5 day cycles was also high when compared with Controls, but this difference did not attain statistical significance. The biological significance of these findings is minimal since the normal cycle duration is 4-5 days.

Estrous cycles at 30 mg/kg/day were similar to Controls

Stage of Estrous Cycle at Termination - Cohort 1A and 1B
The majority of females showed an estrus smear prior to scheduled termination.

Ovarian Follicle Counts and Corpora Lutea - Cohort 1A
Assessment of the follicle and corpora lutea counts in the Control and high dose did not show any adverse effects of treatment.

Sperm Assessment - Cohort 1A
Following treatment at 150mg/kg/day, there was a slight but statistically significant decrease in cauda epididymis total (million), this was within the historical control range. Motility, motion, testis and morphology parameters were unaffected.

There were positive statistically significant increases in some motion parameters (STR, LIN) and decreases in morphological parameters (tail abnormal and head misshapen).

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

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

System:

gastrointestinal tract

Organ:

stomach

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Key result

Reproductive effects observed:

no

Lowest effective dose / conc.:

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

Treatment related:

no

 

Group Mean Serum TSH Concentration (pg/mL) in Male Rats

Group	Treatment	Dose (mg/kg/day)		F0 Adults Day 28	F1 Offspring Day 22	F1 Adults Cohort 1A Day 91
1	Control	0	Mean	1260	287	542
			SD	591	NA	304
			%CV	46.8	NA	56.1
			N	10	10	9
2	2,2'-(Octadec-9-enylimino) bisethanol	30	Mean	2170	374	799
			SD	1240	145	473
			%CV	56.9	38.8	59.2
			N	10	10	10
3	2,2'-(Octadec-9-enylimino) bisethanol	70	Mean	1280	315	1140
			SD	729	NA	617
			%CV	56.7	NA	54.3
			N	10	10	10
4	2,2'-(Octadec-9-enylimino) bisethanol	150	Mean	1550	409	1090
			SD	984	202	527
			%CV	63.6	49.2	48.3
			N	10	10	10

Where fewer than 66.7% of the individual values were BLQ, the mean was calculated using 0.5 x LLOQ. SD and %CV were not calculated

 

 

Group Mean Serum TSH Concentration (pg/mL) in Female Rats

Group	Treatment	Dose (mg/kg/day)		F0 Adults Day 28	F1 Offspring Day 22	F1 Adults Cohort 1A Day 91
1	Control	0	Mean	441	238	585
			SD	267	NA	226
			%CV	60.7	NA	38.6
			N	10	10	10
2	2,2'-(Octadec-9-enylimino) bisethanol	30	Mean	607	421	738
			SD	324	NA	501
			%CV	53.4	NA	67.9
			N	10	10	10
3	2,2'-(Octadec-9-enylimino) bisethanol	70	Mean	543	305	627
			SD	631	NA	224
			%CV	116.1	NA	35.7
			N	9	10	10
4	2,2'-(Octadec-9-enylimino) bisethanol	150	Mean	904	998	579
			SD	393	876	NA
			%CV	43.5	87.8	NA
			N	10	10	10

Where fewer than 66.7% of the individual values were BLQ, the mean was calculated using 0.5 x LLOQ. SD and %CV were not calculated

 

 

 

 

Organ Weight Parameters – Terminal Sacrifice F0 Generation

Sex	2,2’- (Octadec-9-enylimino) bisethanol
	Males				Females
Dose (mg/kg/day)	0	30	70	150	0	30	70	150
Kidney
Absolute Weight (g)	2.484	97	99	97	1.685	101	113	115
Relative Mean Organ Weight (%)	0.545	101	110**	120**	0.693	103	110**	119**
Liver
Absolute Weight (g)	13.267	98	94	96	9.222	101	107	113
Relative Mean Organ Weight (%)	2.90	102	105	118**	3.77	103	105	117**
Spleen
Absolute Weight (g)	0.670	99	104	100	0.536	99	103	104
Relative Mean Organ Weight (%)	0.147	103	116**	124**	0.221	100	101	107
Adrenals
Absolute Weight (g)	0.059	105	98	100	0.073	104	107	107
Relative Mean Organ Weight (%)	0.0130	110	108	123**	0.0303	104	104	110*
Brain
Absolute Weight (g)	2.121	100	100	99	1.908	100	100	100
Relative Mean Organ Weight (%)	0.466	105	111**	122**	0.790	101	98	102
Epididymides
Absolute Weight (g)	1.462	99	99	96
Relative Mean Organ Weight (%)	0.323	103	110*	118**
Sex	2,2’- (Octadec-9-enylimino) bisethanol
	Males				Females
Dose (mg/kg/day)	0	30	70	150	0	30	70	150
Testes
Absolute Weight (g)	3.929	100	101	102
Relative Mean Organ Weight (%)	0.862	104	113**	126**
Ovaries and oviducts
Absolute Weight (g)					0.133	110	113	113
Relative Mean Organ Weight (%)					0.0548	112*	110*	117**
* = p<0.05; ** = p<0.01 statistically significant difference (absolute or relative) compared with respective control mean value.

 

 

Macroscopic Findings – Terminal Sacrifice F0 generation

Sex	2,2’-(Octadec-9-enylimino) bisethanol
	Males				Females
Dose Level (mg/kg/day)	0	30	70	150	0	30	70	150
Stomach
Number Examined	24	25	25	25	24	24	22	20
Thickened	0	8	9	21	0	3	11	9
Edematous	0	0	0	0	0	2	2	1
Jejunum
Number examined	24	25	25	25	24	24	22	20
Abnormal color	0	0	2	5	0	0	0	3
Thickened	0	0	0	9	0	0	0	1

 

Microscopic Findings – Terminal Sacrifice F0 Generation

Sex	2,2’-(Octadec-9-enylimino) bisethanol
	Males				Females
Dose Level (mg/kg/day)	0	30	70	150	0	30	70	150
Stomach
Number Examined	25	25	25	25	25	25	25	25
Hyperplasia, Epithelial, Non-Glandular Region
Minimal	0	8	6	4	0	6	6	5
Slight	0	3	9	13	0	2	12	13
Moderate	0	0	3	6	0	1	2	7
Erosion/Vesicle, Non-Glandular Region
Minimal	0	0	0	0	0	0	0	0
Slight	0	0	1	0	0	0	0	2
Moderate	0	0	0	0	0	0	2	0
Ulceration, Non-Glandular Region
Minimal	0	0	2	1	0	0	0	0
Slight	0	1	0	0	0	1	0	1
Moderate	0	0	1	0	0	0	0	2
Edema, Non-Glandular Region
Minimal	0	1	1	0	0	3	1	2
Slight	0	1	2	1	0	0	4	5
Moderate	0	0	0	0	0	0	1	5
Inflammation, Non-Glandular Region
Minimal	0	1	2	2	0	4	6	7
Slight	0	0	1	2	0	0	0	2
Jejunum
Number Examined	25	25	25	25	24	25	25	25
Accumulation, Foamy Cells
Minimal	0	0	3	0	0	0	16	2
Slight	0	0	12	3	0	0	3	11
Moderate	0	0	7	22	0	0	0	12
Duodenum
Number Examined	25	25	25	25	25	0	0	25
Accumulation, Foamy Cells
Minimal	0	0	0	3	0	-	-	0
Slight	0	0	0	6	0	-	-	0
Moderate	0	0	0	0	0	-	-	0

 

Formulation Analysis

The mean achieved concentrations of formulation samples taken during the course of the study were within 6% of the nominal concentration, confirming the accuracy of formulation. The difference from mean remained within 2%, confirming precise analysis.

For First Week (F0 generation), procedural recoveries were outside of the range established during the validation. The standard and sample responses remained consistent throughout, and therefore the procedural recoveries were considered to be prepared incorrectly and were not used to correct results. All system suitability criteria for this run was met except standard comparison. At most the standards were made with an error of 10%, and the worse possible outcome is that the results are marginally outside of the acceptance criteria stated in the study plan. The results are reported for information only but considered meaningful.

For Week 1 (F1 generation) Group 1 due to a response at the same retention time as the test item, re-dilution of original samples took place and contingency samples were analyzed. Both the re-dilution of original samples and contingency samples confirmed the original results, however the response is less than the quantifiable range for the method. There are four results reported for this group.

For Last week (F1 generation), Group 2 had a difference from mean outside of specified limits. Re-dilutions of original samples provided variable results and therefore were deemed unreliable. The contingency samples were analyzed, and confirmed the original results, therefore four results are reported for this group and the co-efficient of variation was 10.17%.

For Last week (F1 generation) contingency analysis, procedural recoveries were outside of the range established during the validation. No results were deemed outliers and therefore were used to correct results.

For all other occasions, procedural recoveries remained within the range established during the validation, confirming the continued accuracy of the analytical procedure.

 

Organ Weight Parameters – Unselected offspring on Day 22 of age (F1)

Sex	2,2’- (Octadec-9-enylimino) bisethanol
	Males				Females
Dose (mg/kg/day)	0	30	70	150	0	30	70	150
Body weight (g)	49.2	99	100	91*	54.1	107	106	95
Thymus
Absolute Weight (g)	0.196	97	99	88*	0.192	106	109	91
Relative Mean Organ Weight (%)	0.398	98	99	97	0.426	99	103	96
Spleen
Absolute Weight (g)	0.212	109	97	88	0.192	116	118	104
Relative Mean Organ Weight (%)	0.431	110	96	97	0.426	108	111	109
Brain
Absolute Weight (g)	1.461	97	96	94*	1.367	103	102	97
Relative Mean Organ Weight (%)	2.970	98	96	104	3.031	96	97	101
* = p<0.05; ** = p<0.01 statistically significant difference (absolute or relative) compared with respective control mean value.

 

Organ Weight Parameters – Terminal Sacrifice F1 Generation (Cohort 1A)

Sex	2,2’- (Octadec-9-enylimino) bisethanol
	Males				Females
Dose (mg/kg/day)	0	30	70	150	0	30	70	150
Thymus
Absolute Weight (g)	0.411	93.4	81.5**	66.4**	0.323	106.5	97.5	96.9
Relative Mean Organ Weight (%)	0.110	94.5	90.9	84.5*	0.145	108.9	101.3	106.2
Mesenteric lymph nodes
Absolute Weight (g)	0.018	100	100	133.3	0.015	153.3	126.6	173.3*
Relative Mean Organ Weight (%)	0.0049	104	112.2	173.4	0.0070	150	131.4	181.4*
Kidney
Absolute Weight (g)	2.297	107	105	90*	1.448	104	105	105
Relative Mean Organ Weight (%)	0.619	107*	117**	113**	0.648	107**	110**	115**
Heart
Absolute Weight (g)	1.029	101	94	84**	0.744	103	97	92
Relative Mean Organ Weight (%)	0.278	101	105*	107*	0.333	105	101	101
Sex	2,2’- (Octadec-9-enylimino) bisethanol
	Males				Females
Dose (mg/kg/day)	0	30	70	150	0	30	70	150
Liver
Absolute Weight (g)	12.689	103	93	84*	7.520	103	104	109
Relative Mean Organ Weight (%)	3.40	102	104	105	3.36	105	108	120**
Spleen
Absolute Weight (g)	0.634	107	98	93	0.443	110	105	107
Relative Mean Organ Weight (%)	0.171	106	109*	117**	0.198	113*	109*	117**
Adrenals
Absolute Weight (g)	0.067	97	98	92	0.074	100	96	96
Relative Mean Organ Weight (%)	0.0181	97	110	117**	0.330	102	101	106
Brain
Absolute Weight (g)	2.038	102	97	93**	1.887	100	97*	96**
Relative Mean Organ Weight (%)	0.553	102	109*	118**	0.848	103	101	106
Epididymides
Absolute Weight (g)	1.274	100	95	87**
Relative Mean Organ Weight (%)	0.345	100	107	110**
Testes
Absolute Weight (g)	3.637	102	100	99
Relative Mean Organ Weight (%)	0.98	103	113**	125**
Ovaries and oviducts
Absolute Weight (g)					0.121	109	101	110
Relative Mean Organ Weight (%)					0.540	112	106	121**
* = p<0.05; ** = p<0.01 statistically significant difference (absolute or relative) compared with respective control mean value.

 

 

 

Organ Weight Parameters – Terminal Sacrifice F1 Generation (Cohort 1B)

Sex	2,2’- (Octadec-9-enylimino) bisethanol
	Males				Females
Dose (mg/kg/day)	0	30	70	150	0	30	70	150
Pituitary
Absolute Weight (g)	0.009	100	100	89**	0.013	92	100	85**
Relative Mean Organ Weight (g)	0.0023	100	104	113*	0.0057	95	96	88**
Thymus
Absolute Weight (g)	0.372	110	98	73**	0.325	99	102	91
Relative Mean Organ Weight (g)	0.0956	111	103	88	0.141	103	102	98
Sex	2,2’- (Octadec-9-enylimino) bisethanol
	Males				Females
Dose (mg/kg/day)	0	30	70	150	0	30	70	150
Testes
Absolute Weight (g)	3.743	101	103	102
Relative Mean Organ Weight (g)	0.96	101	109**	125**
Seminal vesicles and coagulating glands
Absolute Weight (g)	1.045	105	120*	97
Relative Mean Organ Weight (g)	0.267	105	128**	119**
Epididymides
Absolute Weight (g)	1.280	104	103	94
Relative Mean Organ Weight (g)	0.329	104	109*	115**
Ovaries and oviducts
Absolute Weight (g)	N/A	N/A	N/A	N/A	0.124	104	106	110**
Relative Mean Organ Weight (g)	N/A	N/A	N/A	N/A	0.0540	108	107	119**
* = p<0.05; ** = p<0.01 statistically significant difference (absolute or relative) compared with respective control mean value.

 

 

Macroscopic Findings – Terminal Sacrifice F1 Generation (Cohort 1A)

Sex	2,2’-(Octadec-9-enylimino) bisethanol
	Males				Females
Dose Level (mg/kg/day)	0	30	70	150	0	30	70	150
Stomach
Number Examined	20	20	20	20	20	20	20	20
Thickened	1	1	5	7	0	0	3	4
Depression	0	0	0	2	0	0	1	1

 

Macroscopic Findings – Terminal Sacrifice F1 Generation (Cohort 1B)

Sex	2,2’-(Octadec-9-enylimino) bisethanol
	Males				Females
Dose Level (mg/kg/day)	0	30	70	150	0	30	70	150
Stomach
Number Examined	20	20	20	20	20	20	20	20
Thickened	0	0	3	2	0	0	0	1
Depression	0	0	3	4	0	0	0	2

 

 

 

Microscopic Findings – Terminal Sacrifice F1 Generation (Cohort 1A)

Sex	2,2’-(Octadec-9-enylimino) bisethanol
	Males				Females
Dose Level (mg/kg/day)	0	30	70	150	0	30	70	150
Stomach
Number Examined	20	20	20	20	20	20	20	20
Hyperplasia, Epithelial, Non-Glandular Region
Minimal	0	0	6	7	0	0	0	6
Slight	0	0	1	7	0	0	0	7
Moderate	0	0	0	3	0	0	0	1
Erosion/Vesicle, Non-Glandular Region
Minimal	0	0	0	0	0	1	0	0
Slight	0	0	2	3	0	0	0	3
Moderate	0	0	0	1	0	0	0	1
Ulceration, Non-Glandular Region
Minimal	0	0	0	0	0	0	0	0
Slight	0	0	0	2	0	0	0	0
Moderate	0	0	0	2	0	1	0	1
Edema, Non-Glandular Region
Minimal	0	0	0	4	0	1	0	5
Slight	0	0	4	3	0	0	0	1
Moderate	0	0	0	3	0	0	0	0

 

Sex	2,2’-(Octadec-9-enylimino) bisethanol
	Males				Females
Dose Level (mg/kg/day)	0	30	70	150	0	30	70	150
Stomach
Inflammation, Non-Glandular Region
Minimal	0	0	3	5	0	0	0	3
Slight	0	0	2	4	0	1	0	2
Jejunum
Number Examined	20	20	20	20	20	20	20	20
Accumulation, Foamy Cells
Minimal	0	1	15	1	0	2	8	0
Slight	0	0	0	7	0	0	0	9
Moderate	0	0	0	10	0	0	0	11
Marked	0	0	0	2	0	0	0	0
Lymphangiectasis
Minimal	0	0	0	2	0	0	0	0
Duodenum
Number Examined	20	20	20	20	20	20	20	20
Accumulation, Foamy Cells
Minimal	0	0	0	10	0	0	0	2
Slight	0	0	0	2	0	0	0	1
Hypertrophy, Villous
Minimal	0	0	0	12	0	0	0	1
Slight	0	0	0	2	0	0	0	0
Lymphangiectasis
Minimal	0	0	0	3	0	0	0	0
Ileum
Number Examined	20	20	20	20	19	20	19	20
Accumulation, Foamy Cells
Minimal	0	0	4	12	0	0	3	6
Slight	0	0	0	0	0	0	0	0
Moderate	0	0	0	0	0	0	0	0
Hypertrophy, Villous
Minimal	0	0	0	8	0	0	0	1
Slight	0	0	0	0	0	0	0	0
Lymphangiectasis
Minimal	0	0	0	1	0	0	0	0
Mesenteric Lymph Node
Number Examined	20	20	20	20	19	20	20	20
Accumulation, Foamy Cells
Minimal	0	0	8	0	0	0	7	0
Slight	0	0	11	3	0	0	11	5
Moderate	0	0	1	14	0	0	1	13
Marked	0	0	0	2	0	0	0	2
Dilated/Cystic, Sinuses
Minimal	0	2	3	0	0	1	1	2
Slight	0	0	1	5	0	0	0	11
Moderate	0	0	0	14	0	0	0	6
Thymus
Number Examined	20	18	18	20	20	0	1	20
Involution/Atrophy
Minimal	0	0	2	13	0	0	1	1
Slight	0	1	0	0	0	0	0	0

 

Microscopic Findings – Terminal Sacrifice F1 Generation (Cohort 1B)

Sex	2,2’-(Octadec-9-enylimino) bisethanol
	Males				Females
Dose Level (mg/kg/day)	0	30	70	150	0	30	70	150
Stomach
Number Examined	0	1	5	8	0	0	0	3
Hyperplasia, Epithelial, Non-Glandular Region
Slight	0	0	2	1	0	-	-	2
Moderate	0	0	2	5	0	-	-	1
Erosion/Vesicle, Non-Glandular Region
Minimal	0	0	0	1	0	-	-	0
Slight	0	0	0	1	0	-	-	0
Ulceration, Non-Glandular Region
Slight	0	0	0	0	0	-	-	1
Moderate	0	0	0	1	0	-	-	0
Edema, Non-Glandular Region
Minimal	0	0	2	1	0	-	-	2
Slight	0	0	0	2	0	-	-	0
Inflammation, Non-Glandular Region
Minimal	0	0	2	2	0	-	-	1
Slight	0	0	0	2	0	-	-	1
Jejunum
Number Examined	0	1	0	1	0	-	-	0
Accumulation, Foamy Cells
Minimal	0	0	0	1	0	-	-	0

 

Conclusions:

Based on the results of this study, it is concluded that the No Observed Adverse Effect Level (NOAEL) for both systemic and reproductive toxicity in the Han Wistar rat is 150 mg/kg/day and the NOAEL for the local gastrointestinal irritant effect was not established and lies below 30 mg/kg/day.

Executive summary:

The purpose of this study was to assess the influence of 2,2'‑(Octadec‑9‑enylimino)bisethanol on reproductive performance when administered  by oral gavage administration to Han Wistar rats. Cohorts of F1 animals were used to assess the potential for systemic toxicity, and potential effects on sexual maturation and estrous cycles.

Dose levels for the EOGRTS were selected after evaluation in a RF in which four groups of eight male and eight female rats received 2,2’‑(Octadec-9-enylimino)bisethanol at doses of 0, 30, 70 or 150 mg/kg/day by oral gavage. Males were treated daily for 15 days before pairing and until termination. Females were treated daily for 15 days before pairing, throughout pairing, gestation and lactation. The F1 generation comprised of 10 male and 10 female progeny from each group, and they continued to receive the relevant dose, as per the F0 generation, throughout the study until termination following sexual maturation at approximately eight weeks of age. The oral administration at dose levels of 30, 70 or 150 mg/kg/day had no adverse effect on general clinical condition (F0/F1), mating performance and fertility (F0), organ weights (F0) or macropathology (F0/F1). However, at 150 mg/kg/day clear effects were apparent on both F0 and F1 body weight performance and two out of the eight litters at this dose level were compromised resulting in high mortality/litter death. It was not clear whether this is indicative for a too severe perinatal toxicity or mostly accidental as suggested based on results on similar structures. Based on the findings in this preliminary study it was decided to use of 150 mg/kg/day as the high dose in the subsequent OECD 443 study.

In the F0 generation of the EOGRTS, three groups of 25 male and 25 female rats received 2,2'‑(Octadec‑9‑enylimino)bisethanol at dose levels of 30, 70 or 150 mg/kg/day at a volume dose of 4 mL/kg/day. Males were treated for ten weeks before pairing, up to necropsy after litters were weaned. Females were treated for ten weeks before pairing, throughout pairing up to necropsy on Day 28 of lactation.

For the F0 generation data were recorded on clinical condition, body weight, food consumption, estrous cycles, mating performance and fertility, gestation length and parturition observations and reproductive performance.  Clinical pathology (hematology, blood chemistry and thyroid-related hormones), sperm assessment, organ weight, macroscopic pathology and microscopic pathology investigations were performed. 

For F1 offspring, clinical condition, litter size and survival, sex ratio, body weight, ano‑genital distance, organ weights and macropathology were assessed.  Nipple counts were performed on male offspring on Day 13 of age.  Serum samples that were collected from selected offspring on Day 22 of age were analysed for thyroid-related hormones.

The F1 generation comprised of two cohorts:

Cohort 1A: 20 male and 20 female progeny were selected from each dose group and continued to receive 2,2'‑(Octadec‑9‑enylimino)bisethanol at doses of 0 (Control), 30, 70 or 150 mg/kg/day from weaning until scheduled sacrifice at approximately Week 13 of age. 

Cohort 1B: 20 male and 20 female progeny were selected from each dose group and continued to receive 2,2'‑(Octadec‑9‑enylimino)bisethanol at doses of 0 (Control), 30, 70 or 150 mg/kg/day from weaning until scheduled sacrifice at approximately Week 14 of age. 

For F1 Cohort 1A, data were recorded on clinical condition, body weight, food consumption, sexual maturation and estrous cycles. Clinical pathology (hematology, blood chemistry, urinalysis and biomarkers), sperm assessment, ovarian follicle and corpora lutea counts, organ weight, macroscopic pathology, full microscopic pathology and immunophenotyping investigations were performed. 

For F1 Cohort 1B, data was recorded on clinical condition, body weight, food consumption, sexual maturation, organ weight and macroscopic pathology investigations were performed.

 

Results

Oral administration of 2,2’-(Octadec-9-enylimino) bisethanol to Han Wistar rats at dose levels of 30, 70 and 150 mg/kg/day showed no treatment-related effects on general condition (F0/F1), estrous cycles (F0/F1), thyroid hormones(F0/F1), sperm parameters (F0/F1), mating performance (F0), fertility (F0), gestation length (F0), ano genital distance (F1), spleen cell immunophenotyping (F1) or sexual maturation (F1).

There was a total of four premature deaths at 150 mg/kg/day that were considered to relate to treatment; three F0 females and one F1 male.  Macroscopic and histopathological examination of these animals revealed findings related to the stomach and small intestine. 

Histopathological examination of F0 and F1 animals at scheduled termination revealed findings in the non-glandular portion of the stomach that consisted of epithelial hyperplasia associated with erosion/vesicle or ulceration, edema and mixed inflammation and correlated with macroscopic observations of thickened stomach and depressions. The combination of these findings was considered adverse in this study at all dose levels. The higher neutrophil counts for F0 males and females at 150 mg/kg/day, the higher eosinophil counts in F0 females at 150 mg/kg/day, and the higher white blood cell counts in F1 males at 150 mg/kg/day and F1 females at all dose levels were considered likely to reflect the mixed inflammation in the stomach.

In the jejunum (F0/F1), duodenum (F0/F1) and ileum (F1) showed histopathological changes at scheduled termination.  

• In the F0 adults the jejunum and duodenum showed foamy cell accumulation within the lamina propria, probably related to the absorption of the test item and/or metabolites and correlated with the macroscopic observations of thickened and abnormal color. The jejunum was more severely affected than the duodenum. At the severity observed this is likely to compromise function and indicates an adverse effect for the males and females administered 70 or 150 mg/kg/day.
• In the F1 adults the intestine showed diffuse accumulation of the foamy cells within the lamina propria and lymphangiectasis (duodenum, jejunum and ileum) accompanied by villous hypertrophy (duodenum and ileum), with the jejunum being  most severely affected. At the severity noted is likely to compromise function and indicates an adverse effect for the males and females administered 70 or 150 mg/kg/day.  The mesenteric lymph nodes showed accumulation of the foamy cells and dilation of the sinuses: this change is secondary to the intestinal findings, reflecting the drainage function of these lymph nodes.

For both the F0 and F1 adult animals the reductions observed inlife on both body weight gain and food consumption are therefore considered to be secondary to these histopathological findings.  As a consequence of these effects on the F0 adult animals the body weight and body weight gain of F1 offspring was low at 150 mg/kg/day and the F1 adults showed atrophy/involution of the thymus.

Body weight relative kidney weights for both F0 and F1 adult animals were marginally high when compared with Controls but absolute kidney weights were generally unaffected.  F0 males at 70 and 150 mg/kg/day and F1 males at 150 mg/kg/day had marginally high plasma urea levels.  There was no histopathological correlate  for these findings and no adverse or conclusive effect on renal function was inferred.

There were a number of minor changes in haematology, blood chemistry and urinalysis and organ weight parameters, but none were supported by any test item related microscopic changes in the full range of tissues examined and are of uncertain significance and limited biological relevance and may be incidental or minor consequences to the primary pathology observed and likely not direct effects of the test substance.

The stomach changes are considered to represent a local effect of the irritancy of the test item rather than any systemic effect of toxicity however in view of the mortality at 150 mg/kg/day and the extent of the histopathological changes in the stomach at all doses and small intestine at 70 and 150 mg/kg/day these findings are considered as adverse.  Therefore the No Observed Adverse Effect Level (NOAEL) for the local irritant effect was not established and the NOAEL for both systemic and reproductive toxicity is considered to be 150 mg/kg/day.

 

Conclusion

Based on the results of this study, it is concluded that the No Observed Adverse Effect Level (NOAEL) for both systemic and reproductive toxicity in the Han Wistar rat is 150 mg/kg/day and the NOAEL for the local gastrointestinal irritant effect was not established and lies below 30 mg/kg/day.

 

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

125 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

High quality study but it is read across, from an extended one generation reproduction study on 2, 2’-(Octadec-9-enylimino) bisethanol CAS No 25307-17-9 which is an appropriate read across to for 2,2’-(Octadecylimino)bisethanol CAS No 10213-78-2.

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

We have no specific data on toxicity to reproduction for 2, 2’-(Octadecylimino)bisethanol CAS No 10213-78-2 or for 2, 2’-(Octadec-9-enylimino) bisethanol CAS No 25307-17-9 which is considered to be an appropriate read across substance. The available information is on a related substance 2, 2’-(Octadec-9-enylimino) bisethanol CAS No 25307-17-9, this substance is predominantly C18 unsaturated, while, 2,2’-(Octadecylimino)bisethanol) CAS No 10213-78-2 is predominantly C18 saturated. The unsaturated C18 is more reactive and therefore expected to be more toxic than the equivalent C18 saturated structure constituting for the proposed category a k-nearest neighbor worse-case scenario.

 

The category approach was undertaken to identify an appropriate read-across substance for 2, 2’-(Octadecylimino)bisethanol CAS No 10213-78-2 based on sufficient data density and k-nearest neighbor structural identity that could be considered a reasonably conservative approach. 2, 2’-(Octadec-9-enylimino) bisethanol CAS No 25307-17-9 is the output of this approach. Lower alkyl chain-lengths are available in the category such as: 2,2’-[C16-18( evennumbered, C18 unsaturated) alkyl imino] diethanol, (PFAEO-T), CAS No 1218787-32-6; 2,2’-(C16-18 evennumbered alkyl imino) diethanol, (PFAEO-HT), CAS No 1218787-30-4; and 2,2’-(C12-18 evennumbered alkyl imino) diethanol, (PFAEO-C), 71786-60-2. It is theorized that decreasing alkyl chain length on this family of tertiary amines leads to increased toxicity among several endpoints.

 

Bis (2-hydroxyethyl) coco alkylamine (CAS Number 61791-31-9) has shown to affect reproductive parameters in rats. The oral administration of PFAEO-C to rats by gavage, at dose levels of 125, 30 and 10 mg/kg/day, resulted in treatment related effects at 125 and 30 mg/kg/day. These effects consisted of a localized irritant effect, with an almost complete regression observed following the treatment-free period. Due to the inevitable variability in the counting of corpora lutea in females four days after littering, ranges observed inside that of historical controls, and the lack of a dose response, these differences are not considered to be evidence of a toxic effect on reproduction. Lower litter sizes due to lower numbers of corpora lutea and implantation sites, and higher post implantation losses were evident at 125 mg/kg/day. A NOEL was therefore considered to be 30 mg/kg/day for reproductive toxicity. There is currently a EOGRTS being performed on PFAEO-C that will help to better define the nature of this toxicity.

 

A OECD 422 has also been completed on the substance 2,2’-[C16-18( evennumbered, C18 unsaturated) alkyl imino] diethanol CAS No 1218787-32-6. The OECD 422 on PFAEO-T showed substance related effects in the F0 generation. These effects included irritation/corrosion at the site of local exposure of the GI tract, foamy macrophage accumulation in the mesenteric lymph nodes, and mortality. The NOAEL is based on a just too severe toxicity at 175 mg/kg bw/day leading to significant mortality and early termination of all males before mating, and several females at end pregnancy/early lactation, although this seemed to be well tolerated during the RF. Despite the serious toxicity, affects on fertility seem limited with only post-implantation index being slightly lower than control which was an effect of the maternal animals sacrificed in extremis in late gestation. It is likely that this is a secondary effect as it was only observed at the high dose which consisted of excessive maternal toxicity, though direct effects of the test substance cannot necessarily be ruled out. At 75 mg/kg/day there were no effects to reproduction thus resulting in this being the established NOAEL.

 

The available study on 2, 2’-(Octadec-9-enylimino) bisethanol is an  TG OECD 443 extended one generation reproductive toxicity study. There were observed reductions in body weight gain and food consumption in both the F0 and F1 generations as well as increases in some hematological parameters. These effects were determined to be secondary to the gastrointestinal inflammation and irritancy observed at all doses. The extent of the local irritancy of the findings in addition to deaths in the high dose group (150 mg/kg/day) resulted in an overall NOAEL being set to 30 mg/kg/day. Litter size, survival indices and sex ratio the number of implantation sites, post-implantation survival, live birth index and the number of offspring on Day 1 (total and live Day 1) were unaffected by parental treatment at dose levels up to and including 150 mg/kg/day. Resulting in the conclusion systemic and reproductive toxicity was determined to have a NOAEL of 150 mg/kg/day.

 

Justification for selection of Effect on fertility via oral route:

Oral administration of 2, 2’-(Octadec-9-enylimino) bisethanol CAS No 25307-17-9 at dose levels of 30, 70 and 150 mg/kg/day showed no treatment-related effects on general condition (F0/F1), estrous cycles (F0/F1), thyroid hormones(F0/F1), sperm parameters (F0/F1), mating performance (F0), fertility (F0), gestation length (F0), ano genital distance (F1), spleen cell immunophenotyping (F1) or sexual maturation (F1).

 

There was a total of four premature deaths at 150 mg/kg/day that were considered to relate to treatment; three F0 females and one F1 male.  Macroscopic and histopathological examination of these animals revealed findings related to the stomach and small intestine. For both the F0 and F1 adult animals the reductions observed inlife on both body weight gain and food consumption were considered to be secondary to these histopathological findings. As a consequence of these effects on the F0 adult animals the body weight and body weight gain of F1 offspring was low at 150 mg/kg/day and the F1 adults showed atrophy/involution of the thymus.

 

The stomach changes are considered to represent a local effect of the irritancy of the test item rather than any systemic effect of toxicity however in view of the mortality at 150 mg/kg/day and the extent of the histopathological changes in the stomach at all doses and small intestine at 70 and 150 mg/kg/day these findings are considered as adverse. Therefore the No Observed Adverse Effect Level (NOAEL) for the local irritant effect was not established and the NOAEL for both systemic and reproductive toxicity is considered to be 150 mg/kg/day.

 

Justification for selection of Effect on fertility via inhalation route:

Due to the low vapour pressure and the physical form of the substance, 2,2’-(Octadecylimino)bisethanol) CAS No 10213-78-2 being a waxy solid a room temperature inhalation is not an expected route of exposure in the workplace.  In addition the irritant properties to the skin would make such testing very technically difficult.

 

Justification for selection of Effect on fertility via dermal route:

Due to the irritant properties to the skin such testing is not scientifically justified due to animal welfare considerations.  It would be expected to be absorbed less through the skin than orally, therefore data from the oral dosing studies will be sufficient to calculate dermal DNELs based on the available oral repeat dose NOAEL data, following ECHA guidance.

 

Effects on developmental toxicity

Description of key information

There is a break in the category approach for PFAEOs for developmental toxcity that lies somewhere between category members 2,2’-(C12-18 evennumbered alkyl imino) diethanol CAS No 71786-60-2 (primarily C12 alkyl chain) and 2,2’-[C16-18( evennumbered, C18 unsaturated) alkyl imino] diethanol CAS No 1218787-32-6 (primarily C16-18 alkyl chain). Results from OECD 414 on 2,2’-[C16-18( evennumbered, C18 unsaturated) alkyl imino] diethanol CAS No 1218787-32-6 do not show the same foetal malformations observed in the OECD 414  on 2,2’-(C12-18 evennumbered alkyl imino) diethanol CAS No 71786-60-2 at comparable dose levels, high dose 125 and 150 mg/kg bw/day respectively. 

There is an OECD414 pre-natal developmental toxicity study on 2, 2’-(Octadec-9-enylimino) bisethanol CAS No 25307-17-9 an appropriate read across substance for  2,2’-(Octadecylimino)bisethanol) CAS No 10213-78-2. This study showed no indication of developmental toxicity in the foetuses, the NOEL being 150mg/kg (the top dose).  There was also no indication of toxicity in the pregnant females.  This study does not include dosing during the implantation period, information concerning this is read across to the OECD443 study on the same substance.  The available information is on a related substance 2, 2’-(Octadec-9-enylimino) bisethanol CAS No 25307-17-9, this substance is predominantly C18 unsaturated, while, 2,2’-(Octadecylimino)bisethanol) CAS No 10213-78-2 is predominantly C18 saturated. The unsaturated C18 is more reactive and therefore expected to be more toxic than the equivalent C18 saturated structure constituting a worse-case scenario. There was no indication of changes to litter size, survival indices, sex ratio, the number of implantation sites, post-implantation survival, live birth index and the number of offspring on Day 1 (total and live Day 1) were unaffected by parental treatment at dose levels up to and including 150 mg/kg/day.

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental toxicity

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

19 August 2021 - 03 December 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

See read-across documentation in section 13.1

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

other: • Japanese Ministry of Agriculture, Forestry and Fisheries, Test Data for Registration of Agricultural Chemicals, 12 Nohsan No. 8147, Agricultural Production Bureau, November 24, 2000.

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Wistar

Remarks:

RccHan®:WIST. (Han Wistar) rat

Details on test animals or test system and environmental conditions:

Strain/Species RccHan®:WIST. (Han Wistar) rat.
Supplier Envigo RMS Ltd. (UK)
Number of animals ordered 88 time-mated females.
Duration of acclimatization Four days from arrival on Day 2 after mating to commencement of treatment on Day 6 after mating.
Age of the animals at the start of the study (Gestation Day 2) 11 weeks old.
Weight range of the animals at the start of the study (Gestation Day 3) 171 to 238 g.

3.3.2 Mating
Method Natural mating with Han Wistar males at the supplier’s facility. Males and females were not related.
Day 0 of gestation When positive evidence of mating was detected.
Delivery to Labcorp On Day 2 after mating

3.3.3 Allocation and Identification
Allocation Randomly to each group, on day of arrival.
Method Allocation was controlled to prevent time mated females mated with the same male partner being allocated to the same treatment group.
Identification of animals Each animal was assigned a number and identified uniquely within the study by a microchip inserted subcutaneously in the dorsal cervical region.
Identification of cages Each cage label was color-coded according to group and was numbered uniquely with cage and study number, as well as the identity of the occupant.

3.4 Animal Care and Husbandry

3.4.1 Environmental Control
Animal facility Limited access - to minimize entry of external biological and chemical agents and to minimize the transference of such agents between rooms.
Air supply Filtered fresh air which was passed to atmosphere and not recirculated.
Temperature and relative humidity Monitored and maintained within the range of 20-24C and 40-70%.
There were no deviations from these ranges.
Lighting Artificial lighting, 12 hours light: 12 hours dark.
Electricity supply Public supply with automatic stand-by generators.

3.4.2 Animal Accommodation
Cages Cages comprised of a polycarbonate body with a stainless steel mesh lid; changed at appropriate intervals.
Solid (polycarbonate) bottom cages were used during the acclimatization and gestation periods.
Cage distribution The cages constituting each group were blocked by group and mounted in batteries.
Bedding Solid bottom cages contained softwood based bark-free fiber bedding, which was changed at appropriate intervals each week.
Number of animals per cage One.

3.4.3 Environmental Enrichment
Aspen wood based products A soft white untreated wood block: provided to each cage throughout the study and replaced when necessary.
Plastic shelter Provided to each cage throughout the study and replaced at the same time as the cages.

3.4.4 Diet Supply
Diet SDS VRF1 Certified, pelleted diet.
The diet contained no added antibiotic or other chemotherapeutic or prophylactic agent.
Availability Non-restricted.

3.4.5 Water Supply
Supply Potable water from the public supply via polycarbonate bottles with sipper tubes. Bottles were changed at appropriate intervals.
Availability Non-restricted.

3.4.6 Supplier Certificates of Analysis
Certificates of analysis for the diet are scrutinized and approved before any batch of diet was released for use. Certificates of analysis were routinely provided by the water supplier.
Certificates of analysis were also received from the suppliers of the softwood based bark-free fiber bedding and Aspen wood based products.
No specific contaminants were known that may have interfered with or prejudiced the outcome of the study and therefore no special assays were performed.

Route of administration:

oral: gavage

Vehicle:

arachis oil

Details on exposure:

Route Oral gavage using a suitably graduated syringe and a flexible cannula inserted via the mouth.

Method For each group, the required amount of dose was drawn up into the syringe and cannula. The outside of the cannula was wiped clean of any formulation residue with a clean disposable tissue and the end of the flexible cannula was lightly tapped onto clean tissue to remove any remaining droplets. The cannula was then dipped into a container filled with 5% glucose solution to aid intubation.

Treated at Constant doses in mg/kg/day.

Volume dose 4 mL/kg body weight.

Individual dose volume Calculated from the most recently recorded scheduled body weight.

Control (Group 1) Vehicle at the same volume dose as treated groups.

Frequency Females were treated from Day 6 to Day 20 (inclusive) after mating, once daily at approximately the same time each day.

Formulation A daily record of the usage of formulation was maintained based on weights. This balance was compared with the expected usage as a check of correct administration.
Formulations were stirred using a magnetic stirrer before and throughout the dosing procedure.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Correction factor None.

Vehicle Arachis oil

Method of preparation The test material was warmed until melted and gently inverted until homogenous. The required amount of test item was weighed. Approximately 50% of the final volume of vehicle was added and heated to around 60°C in a water bath whilst magnetically stirring until uniformly mixed. The remaining vehicle was added to achieve the required volume and the formulation was mixed using a magnetic stirrer and heated in water bath to around 60°C until homogenous. The formulation was transferred to the final containers, via syringe, whilst magnetically stirring.

Frequency of preparation Twice weekly.

Storage of formulation Refrigerated (2 to 8C).

Test item accounting Detailed records of compound usage were maintained. The amount of test item necessary to prepare the formulations and the amount actually used were determined on each occasion. The difference between these amounts was checked before the formulations were dispensed.


Stability and homogeneity The homogeneity and stability of formulations during storage were determined as part Labcorp Study Number MH28JS. In that study, formulations in the range 1 to 100 mg/mL were determined to be stable for:
• One day at ambient temperature (15 to 25C)
• Four days when stored refrigerated (2 to 8C)

Achieved concentration Samples of each formulation prepared for administration in the first and last weeks of treatment were analyzed for achieved concentration of the test item.

Analysis The method of analysis and results are presented in Attachment 14.2.

Details on mating procedure:

Method Natural mating with Han Wistar males at the supplier’s facility. Males and females were not related.

Day 0 of gestation When positive evidence of mating was detected.

Delivery to Labcorp On Day 2 after mating

Duration of treatment / exposure:

Females were treated from Day 6 to Day 20 (inclusive) after mating, once daily at approximately the same time each day.

Volume dose 4 mL/kg body weight.

Individual dose volume Calculated from the most recently recorded scheduled body weight.

Frequency of treatment:

Once daily

Duration of test:

14 days Day 6 to Day 20 post-coitum

Dose / conc.:

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

Remarks:

Control

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

No. of animals per sex per dose:

22 Females

Control animals:

yes, concurrent vehicle

Maternal examinations:

3.6.1 Clinical Observations
Animals were inspected visually at least twice daily for evidence of ill-health or reaction to treatment. Cages were inspected daily for evidence of animal ill-health. Any deviation from normal was recorded at the time in respect of nature and severity, date and time of onset, duration and progress of the observed condition, as appropriate.

During the acclimatization period, observations of the animals and their cages were recorded at least once per day.

Signs Associated with Dosing
Detailed observations were recorded daily during the treatment period at the following times in relation to dose administration:
• Pre-dose observation
• One to two hours after completion of dosing
• As late as possible in the working day.

Clinical Signs
A detailed physical examination was performed on each animal on Days 3, 5, 12, 18 and 21 after mating to monitor general health.

3.6.2 Body Weight
The weight of each adult was recorded on Days 3 and 6-21 after mating.

3.6.3 Food Consumption
The weight of food supplied to each adult, that remaining and an estimate of any spilled was recorded for the periods Days 3-5, 6-8, 9-11, 12-14, 15-17 and 18-20 after mating inclusive.

All adult animals were subject to a detailed necropsy. After a review of the history of each animal, a full macroscopic examination of the tissues was performed. All external features and orifices were examined visually. Any abnormality in the appearance or size of any organ and tissue (external and cut surface) was recorded and the required tissue samples preserved in appropriate fixative.
Schedule Animals were killed on Day 21 after mating.

Organ Weights
For bilateral organs, left and right organs were weighed together. Requisite organs were weighed for animals killed at scheduled intervals.

Fixation
Tissues were routinely preserved in 10% Neutral Buffered Formalin

3.7.3 Histology
Processing Tissue samples were dehydrated, embedded in paraffin wax and sectioned at a nominal four to five micron thickness. For bilateral organs, sections of both organs were prepared. A single section was prepared from each of the remaining tissues required.

Thyroid All adult females.

Routine staining Sections were stained with hematoxylin and eosin.

3.7.4 Light Microscopy

Ovaries and uterine content:

The following were recorded for all animals:
Uterus Gravid uterine weight (including cervix and ovaries).

For each ovary/uterine horn Number of:
Corpora lutea.
Implantation sites.
Resorption sites (classified as early or late).
Fetuses (live and dead).

Apparently non pregnant animals and for apparently empty uterine horns
The number of uterine implantation sites were checked after staining with ammonium sulphide (modification of the Salewski staining technique (Salewski, 1964)).

Blood sampling:

3.6.4 Thyroid Hormone Analysis
Blood samples were collected at the following occasion GD 21 at necropsy

Parameters
Triiodothyronine (T3)
Thyroxine (T4)
Thyroid stimulating hormone (TSH)

Sequence of blood sampling on each occasion To minimize any potential confounding effect of the time of day of blood sampling, the time of blood sampling was controlled to allow satisfactory inter-group comparisons. Animals were sampled in a random order, within a two-hour time period, where possible

Conditions No overnight deprivation of food.

Blood sample site Sublingual vein.

Anesthetic Isoflurane.

Anticoagulant None.

Blood tubes Greiner Minicollect tubes with clotting activator.

Blood volume 1.0 mL.

Treatment of samples Samples were kept at ambient temperature (15 to 25°C) for a minimum of 30 minutes prior to centrifugation.

Centrifugation conditions At 2000g for ten minutes at 4°C.
Serum was transferred to appropriately labelled polypropylene “cryo” tubes using plastic disposable pipettes and then mixed by gentle ten-fold inversion. Following mixing, each serum sample was divided in two aliquots.

Number of aliquots Two per animal.
Aliquot 1: 0.2 mL serum for T3/T4
Aliquot 2: residual serum for TSH

Final storage conditions Deep frozen (approximately -60°C to -90C) pending analysis.

Fate of samples Aliquot 1 (T3 and T4): dispatched to the department of LC MS/MS Bioanalysis, Labcorp.
Aliquot 2: dispatched to the Department of Immunology and Immunotoxicology, Labcorp.

T3 and T4 Performed by the department of LC-MS/MS Bioanalysis, Labcorp.
The method of analysis and results are presented in Attachment 14.3.

TSH Performed by the Department of Immunology and Immunotoxicology, Labcorp.
The method of analysis and results are presented in Attachment 14.4.

Fetal examinations:

3.7.6 Fetal Examination and Processing
Examination of all viable fetuses and placentae Dissected from the uterus, individually weighed and identified within the litter using a coding system based on their position in the uterus. Fetuses examined externally with abnormalities recorded. Particular attention was paid to the external genital organs of male fetuses. The sex and ano-genital distance of each fetus was recorded.

50% of fetuses in each litter Sexed internally, eviscerated and fixed in Industrial Methylated Spirit (IMS).

50% of fetuses in each litter Sexed internally and examined for visceral abnormalities by fresh microdissection (Modified Staples technique) and subsequently fixed in Bouin’s solution.

Processing Bouin’s fixed fetuses had heads removed post-fixation and heads were processed by Wilsons free-hand serial sectioning. Torsos were retained in Bouin’s solution.
IMS fixed fetuses were processed and stained with Alizarin Red.

3.7.7 Fetal Pathology Examination
Bouin’s fixed heads Serial sections were examined for visceral abnormalities.

Alizarin Red stained fetuses Assessed for skeletal development and abnormalities.

Statistics:

This report only contains serial observations pertaining to the gestation period (from Day 3 after mating).
Where appropriate, group mean values, each with standard deviation (SD), were calculated from individual data. Summary tabulated data was normally restricted to data derived from females/litters with live young at Day 21 after mating.

For all adult parameters, the analyses were carried out using the individual animal as the basic experimental unit. For litter/fetal findings the litter was taken as the treated unit and the basis for statistical analysis and biological significance was assessed with relevance to the severity of the anomaly and the incidence of the finding within the background control population.
The following data types were analyzed at each timepoint separately:
Body weight, using absolute weights and gains over appropriate study periods
Gravid uterine weight and adjusted body weight
Food consumption, over appropriate study periods
C-section litter data (corpora lutea, implantations, pre/post implantation loss, live young and sex ratio - percentage male)
Placental, litter and fetal weights
Ano-genital distance, average for each litter adjusted for litter average fetal body weight
Organ weights, absolute

The following comparisons were performed:
Group 1 vs 2, 3 and 4

The following sequence of statistical tests was used for body weight, gravid uterus weight, food consumption, corpora lutea, implantations, pre/post implantation loss, live young, sex ratio - percentage male, placental, litter and fetal weights, ano-genital distance and organ weight data

For litter average ano genital distance, analysis of covariance was performed using average fetal weight for each litter as covariate (Angervall and Carlstrom, 1963), unless non parametric methods were applied. The treatment comparisons were made on adjusted group means in order to allow for differences in fetal weight which might influence the ano genital distance.

Indices:

Prenatal losses are separated into pre- and post-implantation phases. Pre-implantation loss was considered to reflect losses due to non-fertilization of ova and failure to implant. It was calculated from the formula:
Pre-implantation loss (%) = (Number of corpora lutea - Number of implantations) / Number of corpora lutea x 100

Where the number of implantations exceeded the number of corpora lutea observed, pre implantation loss was assumed to be zero (i.e. no pre-implantation loss was considered to have occurred).
Post-implantation loss was calculated from the formula:
Post-implantation loss (%) = (Number of implantations - Number of live fetuses) / Number of implantations x 100
All group values and SD were calculated from the individual litter values.

Historical control data:

Available and used for comparison where necessary and to help validate controls.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

The appearance and behavior of all animals were unaffected by treatment.

Isolated findings of decreased activity, salivation and piloerection were reported in one animal receiving 150 mg/kg/day, but in view of the low incidence, this was considered unrelated to treatment.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Overall body weight gain (Day 6-21 after mating) was statistically significantly lower than control in females receiving 150 mg/kg/day (overall reduction of 50%). The extent of the reduction from control was greatest from Day 13-21 after mating (55% reduction, compared to a 41% reduction from Day 6-13 after mating).

Body weight gain was unaffected in females receiving 15 or 50 mg/kg/day.

On Day 21 of gestation, gravid uterine weight was statistically significantly lower than control in females receiving 150 mg/kg/day (overall reduction 28%). When adjusted for the contribution of the gravid uterus, there was a mean loss of body weight (-6 g) in females receiving 150 mg/kg/day.

Gravid uterus weight and adjusted body weight gain was unaffected in females receiving 15 or 50 mg/kg/day.

Food efficiency:

effects observed, treatment-related

Description (incidence and severity):

Overall mean food intake during the dosing period (Day 6-21 after mating) was 24% lower than controls in females receiving 150 mg/kg/day. There was no similar finding at 15 or 50 mg/kg/day.

Endocrine findings:

effects observed, non-treatment-related

Description (incidence and severity):

The analysis of thyroid hormone concentrations on Day 21 of gestation revealed statistically significantly low triiodothyronine concentration in females receiving 150 mg/kg/day, compared with control.
Thyroxine concentration was slightly lower than control in females receiving 50 or 150 mg/kg/day and thyroid stimulating concentration was slightly high in females receiving 150 mg/kg/day but in each case, there was no dose response and statistical significance was not attained and, as such, this was not attributable to treatment.

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

There were no organ weight differences considered to be related to treatment with 2,2’-(C16-18 (even numbered, C18 unsaturated) alkyl imino) diethanol in the thyroid and parathyroid glands.
Any changes in the absolute thyroid and parathyroid weights of treated females were considered to be incidental and not related to treatment, as there was no microscopic correlate. It is possible that any changes in the absolute thyroid and parathyroid weights of females receiving 150 mg/kg/day were secondary to lower than control bodyweight gain and lower than control food intake seen in females receiving 150 mg/kg/day, and not related to treatment.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no macroscopic findings considered to be related to treatment with 2,2’-(C16-18 (even numbered, C18 unsaturated) alkyl imino) diethanol.
The thyroids of one female treated with 150 mg/kg/day were macroscopically small, and this finding was considered to represent normal biological variation as there was no microscopic correlate.

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

No test item related microscopic findings were seen in the thyroid gland.

Histopathological findings: neoplastic:

no effects observed

Number of abortions:

no effects observed

Pre- and post-implantation loss:

effects observed, non-treatment-related

Description (incidence and severity):

The number of live offspring was slightly low and pre-and post-implantation loss were slightly high at 150 mg/kg/day, with the mean number of live offspring and pre- and post-implantation losses being outside of the historical control range. However, the pre-implantation loss, although outside the range of the historical control data, was only marginally higher than that at 50 mg/kg/day, did not attained statistical significance and represents a period prior to the start of dosing. The number of live offspring and post-implantation losses did not attain statistical significance and individual values were similar to control. These variations were therefore not attributable to treatment

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Changes in pregnancy duration:

no effects observed

Changes in number of pregnant:

effects observed, non-treatment-related

Description (incidence and severity):

At scheduled termination on Day 21 after mating, two control females (Nos. 14 and 17), three females receiving 15 mg/kg/day (Nos. 34, 35 and 41), one female receiving 50 mg/kg/day (No. 57) and four females receiving 150 mg/kg/day (Nos. 71, 72, 80 and 82) were not pregnant. In addition, one female receiving 15 mg/kg/day (No. 25), one female receiving 50 mg/kg/day (No. 45) and one female receiving 150 mg/kg/day (No. 79) had a total litter resorption

Other effects:

no effects observed

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Basis for effect level:

body weight and weight gain

Fetal body weight changes:

effects observed, treatment-related

Description (incidence and severity):

Total litter weight was 29% lower than control at 150 mg/kg/day, the difference attaining statistical significance. There was no effect on total litter weight for females receiving 15 or 50 mg/kg/day.

Changes in litter size and weights:

effects observed, treatment-related

Description (incidence and severity):

Total litter weight was 29% lower than control at 150 mg/kg/day, the difference attaining statistical significance. There was no effect on total litter weight for females receiving 15 or 50 mg/kg/day.

Anogenital distance of all rodent fetuses:

no effects observed

Changes in postnatal survival:

no effects observed

External malformations:

no effects observed

Skeletal malformations:

no effects observed

Visceral malformations:

no effects observed

Other effects:

not specified

Details on embryotoxic / teratogenic effects:

Placental weight was slightly higher than control in females receiving 150 mg/kg/day. There was no effect of maternal treatment on male, female or overall fetal weight at any dose level investigated

Key result

Dose descriptor:

NOAEL

Effect level:

> 150 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Developmental effects observed:

no

Lowest effective dose / conc.:

150 mg/kg bw/day (nominal)

Treatment related:

no

Summary of Thyroid Hormone Levels after 13 Weeks of Treatment

Group/sex	1F	2F	3F	4F
Dose level (mg/kg/day)	0	15	50	150
Thyroxine (pg/mL)	19500	21300	17700	16000
Triiodothyronine (pg/mL)	493	515	494	354**
Thyroid stimulating hormone (pg/mL)	580	725	596	741

** p≤0.01

Conclusions:

It is concluded that that oral administration of 2,2’-(C16-18 (even numbered, C18 unsaturated) alkyl imino) diethanol to Han Wistar rats during the organogenesis and fetal growth phases of pregnancy at doses of 0, 15, 50 or 150 mg/kg/day was generally tolerated but cause an adverse reduction of weight gain, associated with low food intake, in females receiving 150 mg/kg/day. The number of implantations, resorptions and live young, the pre- and post implantation loss and sex ratio were considered unaffected by treatment. The No-Observed-Adverse-Effect Level (NOAEL) for maternal toxicity was therefore considered to be 50 mg/kg/day, based on the effects on body weight gain and body weight loss when corrected for gravid uterus. The NOAEL for embryo-fetal survival and development was considered to be 150 mg/kg/day.

Executive summary:

The purpose of this study was to assess the influence of 2,2’-(C16-18 (even numbered, C18 unsaturated) alkyl imino) diethanol on embryo-fetal survival and development when administered during the organogenesis and fetal growth phases of pregnancy in the Han Wistar rat. Dose levels of 0, 15, 50, 150 mg/kg bw/day. The dose levels for this study were selected based on the results of Labcorp Study Number 8466317. In that study, a dose of 150 mg/kg/day was tolerated by pregnant Han Wistar rats, but resulted in a reduction of weight gain (associated with low food intake), which, when adjusted for gravid uterus weight, resulted in an overall body weight loss. There was no effect on embryo-fetal survival or development. The lower body weight gain was considered not to preclude the use of the 150 mg/kg/day in this main study, which was, therefore, selected as the highest dose.

The purpose of this study was to assess the influence of 2,2’-(C16-18 (even numbered, C18 unsaturated) alkyl imino) diethanol on embryo-fetal survival and development when administered during the organogenesis and fetal growth phases of pregnancy in the Han Wistar rat. This study demonstrated that doses up to 150 mg/kg/day were generally tolerated with no treatment-related clinical signs and no premature deaths, though there was evidence of a toxic response in pregnant females which was manifest as an adverse reduction of body weight gain (50% reduction from control), adjusted weight loss and a sustained reduction in food consumption. There was no treatment-related effect on the number of implantations, resorptions and live young, the pre- and post implantation loss and sex ratio and there were no treatment-related findings at the fetal pathology investigations, indicating that there was no effect on embryo-fetal development. Although there was a statistically significant decrease of triiodothyronine concentration in females receiving 150 mg/kg/day, there was considered to be no effect of treatment on the hormonal control of the thyroid gland, since thyroxine and thyroid stimulating hormone concentrations were unaffected and there was no effect on thyroid gland weight and pathology.

Fetal showed bodyweight reductions of up to 29% in the high dose group when compared with that of control. This effect was not considered adverse. Addtionally, secondary effects of maternal toxicity at 150 mg/kg bw/day  resulting in an average body weight loss of 50% when corrected for gracid uterine weight as this may have contributed to reduced fetal bodyweight. 

The reproductive assessment revealed high pre- and post-implantation loss in females receiving 150 mg/kg/day, resulting in a lower number of implantations and live young, which was adverse, with all values being outside of the background control range. The reduction in live young correlated with lower total litter weights and higher placental weights at this dose level. There was, however, no effect on individual fetal weights and the increased placental weights were likely due to the lower numbers of live young in each litter.

Maternal NOAEL was set to 50 mg/kg bw/day due to severe effects on body weight gain.

Fetal NOAEL is 150 mg/kg/day due to lack of adverse effects seen.

Reference 2

Endpoint:

developmental toxicity

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

19th June 2013 to 17 the December 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Read across to CAS 25307-17-9, 2,2’-(Octadecylimino)bisethanol, is seen as a worst case as this is predominantly C18 unsaturated compared to CAS no 10213-78-2 Octadecyl bis(2-hydroxyethyl)amine which is predominantly C18 saturated. The unsaturated C18 is more reactive and therefore expected to be more toxic than the equivalent C18 saturated structure.

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

OECD Guidelines for Testing of Chemicals No. 422 “Combined Repeated Dose Toxicity Study with the Reproduction/ Developmental Toxicity Screening Test”

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Japanese Ministry of Agriculture, Forestry and Fisheries Testing guidelines for Toxicology Studies, 12 NohSan No 8147 (24 November 2000)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.31 (Prenatal Developmental Toxicity Study)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

A total of ninety-six time-mated female Sprague-Dawley Crl:CD (SD) IGS BR strain rats were obtained from Charles River (UK) Limited, Margate, Kent. Animals were delivered in two batches containing females prior to Day 3 of gestation. The day that positive evidence of mating was observed was designated Day 0 of gestation. On arrival the females weighed 190g to 269g.

The animals were housed individually in solid-floor polypropylene cages with stainless steel lids furnished with softwood flakes (Datesand Ltd., Cheshire, UK). The animals were allowed free access to food and water. A pelleted diet (Rodent 2018C Teklad Global Certified Diet, Harlan UK, Oxon, UK) was used. Certificates of analysis of the batches of diet used are given in Appendix 14. 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). 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 Harlan Laboratories Ltd., 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 mean temperatures and humidity were included in the study records. The Study Plan target ranges for temperature and relative humidity were 22 ± 3 ºC and 50 ± 20% respectively; there were no deviations from these targets.

The animals were randomly allocated to treatment groups using a randomization procedure based on stratified body weight to ensure similarity between the treatment groups. 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 exposure:

As Arachis Oil was successfully used on both the twenty-eight day and ninety toxicity studies, the same vehicle and dosage (4 mL/kg body weight) was employed in this study. The stability and homogeneity of the test item formulations were previously determined by Harlan Laboratories Ltd., Shardlow, UK Analytical Services and showed the formulations to be stable for at least twenty one days at 4 °C. Formulations were therefore prepared in two separate bulk formulations (covering up to 9 days) and divided into daily aliquots and stored at approximately +4 °C in the dark.

Samples were taken of each test item formulation and were analyzed for concentration of 2,2'-(octadec-9-enylimino)bisethanol CAS No 25307-17-9 at Harlan Analytical Laboratory, Shardlow. The results indicate that the prepared formulations were within 94% to 104% of the nominal concentration and within acceptable limits of the nominal concentration.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The test item concentration in the test samples was determined by gas chromatography (GC) using an external standard technique. The test item gave a chromatographic profile consisting of a single peak.

Test Item
The test item described in the main part of this study was also used as the analytical standard.

Preparation of standard solutions
Stock solutins of test item in methanol were prepared for external standard calibration. An aliquot, 100 mg of test item was exactly weighed into a 100 mL volumetric flask and brought to volume with methanol to yield a solution with a concentration of 1 mg/mL. Aliquots of this stock standard solutins were used to prepare working standard solutions in methanol with a concentration of 0.1 mg/mL.

Analysis of samples
The formulations recieved were extracted with methanol. An aliquot of test item formulation was accurately weighed into a volumetric flask and brought to volume with methanol. This was then ultra-sonicated for 15 minutes and centrifuged to 4500 rpm for 10 minutes. Where necessary, sample solutions were further diluted with methanol to achieve the working concentration.

Preparation of accuracy samples
Samples of Arachis Oil BP were accurately fortified with known amounts of test item equivalent to the lowest and highest anticipated dose concentrations. These sample were then prepared for analysis.

Instrumental Setip

HC system: Agilent Technologues 5890, incorporating autosampler and workstation
Column: DB-1 (15 m x 0.53 mm id x 1.5 micro-m film)
Oven temperature program: Oven: 200°C for 0 minute, with 10°C/minute to 300°C, for 12 minutes
Injection temperature: 300°C
Flame ionisation detector temperature: 300°C
Injection volume: 1 micro-litre
Retention time: ~ 4.5 mins

Details on mating procedure:

Not described in the study

Duration of treatment / exposure:

Between Days 5 and 19 of gestation, inclusive.

Frequency of treatment:

Daily

Duration of test:

20 days

Dose / conc.:

0 mg/kg bw/day

Remarks:

Vehicle control

Dose / conc.:

15 mg/kg bw/day

Dose / conc.:

50 mg/kg bw/day

Dose / conc.:

150 mg/kg bw/day

No. of animals per sex per dose:

24 females

Control animals:

yes, concurrent vehicle

Details on study design:

The animals were randomly allocated to treatment groups using a randomization procedure based on stratified body weight to ensure similarity between the treatment groups. The animals were uniquely identified within the study by an ear punching system routinely used in these laboratories.

Justification
The rat was selected for this study as it is a readily available rodent species historically used in safety evaluation studies and is acceptable to appropriate regulatory authorities.

Maternal examinations:

Following arrival, all animals were examined for overt signs of toxicity, ill-health or behavioral changes once daily during the gestation period. Additionally, during the dosing period, observations were recorded immediately before and soon after dosing and one hour post dosing. An additional observation was also performed five hours after dosing during the normal working week. All observations were recorded.

Body Weight
Individual body weights were recorded on Day 3 (before the start of treatment) and on Days 5, 6, 7, 8, 11, 14 and 17 of gestation. Body weights were also recorded for animals at terminal kill (Day 20).

Food Consumption
Food consumption was recorded for each individual animal at Day 3, 5, 8, 11, 14, 17 and 20 of gestation.

Water Consumption
Water intake was observed daily by visual inspection of the water bottles for any overt changes.

Ovaries and uterine content:

Post Mortem
All animals were killed by carbon dioxide asphyxiation followed by cervical dislocation on Day 20 of gestation. All animals were subjected to a full external and internal examination and any macroscopic abnormalities were recorded. The ovaries and uteri of pregnant females were removed, examined and the following data recorded:

i) Number of corpora lutea
ii) Number, position and type of intrauterine implantation
iii) Fetal sex
iv) External fetal appearance
v) Fetal weight
vi) Placental weight
vii) Gravid uterus weight

Implantation types were divided into:

Early Death: No visible distinction between placental/decidual tissue and embryonic tissue

Late Death: Separate embryonic/fetal and placental tissue visible

Dead Fetus: A fetus that had died shortly before necropsy. These were included as late deaths for reporting purposes

All implantations and viable fetuses were numbered according to their intrauterine position as follows (as an example):


Left Horn Cervix Right Horn

L1 L2 L3 L4 L5 L6 L7 L8 R1 R2 R3 R4 R5 R6 R7 R8
V1 V2 V3 V4 V5 V6 V7 V8 V9 V10 V11 V12 V13 V14 V15 V16

V = viable fetus

Fetal examinations:

The fetuses were killed by subcutaneous injection of sodium pentobarbitone. Fetuses from each litter were divided into two groups and examined for skeletal alterations and soft tissue alterations. Alternate fetuses were identified using an indelible marker and placed in Bouin’s fixative. Fetuses were subsequently transferred to distilled water and examined for visceral anomalies under a low power binocular microscope and then stored in 10% Buffered Formalin. The remaining fetuses were identified using cardboard tags marked with chinagraph pencil and placed 70% IMS in distilled water. The fetuses were subsequently eviscerated, processed and the skeletons stained with alizarin red S before being transferred to 50% glycerol for examination of skeletal development and anomalies and storage.

Statistics:

The following parameters were analyzed statistically, where appropriate, using the test methods outlined below:

Body weight and body weight change (including adjustment for the contribution of the gravid uterus), food consumption, gravid uterus weight, litter data and fetal litter and placental weights: Bartlett’s test for homogeneity of variance. Where the data were shown to be homogeneous one way analysis of variance and, if significant, Dunnett’s multiple comparison test was employed, where the data were found to non homogeneous Kruskal-Wallis and, if significant, pairwise analysis of control values against treated values using the Mann-Whitney ‘U’ test was employed. Fetal evaluation parameters, including skeletal or visceral findings were analyzed by Kruskal-Wallis and, if significant, Mann-Whitney ‘U’ test.

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

Description (incidence and severity):

There was no effect of treatment on body weight or body weight gain, including when adjusted for the contribution of the gravid uterus, throughout the treatment period at 15, 50 or 150 mg/kg bw/day.

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Number of abortions:

no effects observed

Pre- and post-implantation loss:

effects observed, non-treatment-related

Description (incidence and severity):

At 150 mg/kg bw/day, mean pre-implantation loss was lower than control with differences attaining statistical significance. As animals were not dosed until implantation had occurred, these differences were incidental and unrelated to treatment.

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Changes in pregnancy duration:

not examined

Description (incidence and severity):

Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): no effects observed

Changes in number of pregnant:

no effects observed

Details on maternal toxic effects:

Maternal toxic effects:no effects

Details on maternal toxic effects:
No effects of any toxicological significance

Key result

Dose descriptor:

NOEL

Effect level:

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

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Key result

Dose descriptor:

NOEL

Effect level:

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

Based on:

test mat.

Basis for effect level:

other: developmental toxicity

Fetal body weight changes:

no effects observed

Description (incidence and severity):

Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): not examined

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

no effects observed

Changes in postnatal survival:

not examined

External malformations:

no effects observed

Skeletal malformations:

no effects observed

Visceral malformations:

no effects observed

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
Litter Responses
Litter Data and Litter Placental and Fetal Weights
There was no obvious effect of maternal treatment on the number of implantations, subsequent embryofetal survival and litter size, sex ratio and mean fetal, litter and placental weights on Day 20 of gestation at 15, 50 or 150 mg/kg bw/day.

At 150 mg/kg bw/day, mean pre-implantation loss was lower than control with differences attaining statistical significance. As animals were not dosed until implantation had occurred, these differences were incidental and unrelated to treatment.

At 15 and 50 mg/kg bw/day, higher mean female fetal weight and mean fetal weight attained statistical significance compared to control. In the absence of any similar increase in fetal weight at 150 mg/kg bw/day, this finding was considered to reflect normal biological variation and was unrelated to treatment.

Fetal Examination
Neither the type, incidence or distribution of findings observed externally at necropsy examination and subsequently during detailed visceral and skeletal assessment of the fetuses indicated any effect of treatment on fetal development.

Key result

Dose descriptor:

NOEL

Effect level:

>= 150 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Abnormalities:

no effects observed

Developmental effects observed:

not specified

Mortality There were no unscheduled deaths during the study. Clinical Observations The low incidence of clinical sign observed during the study did not indicate any effect of treatment at 15, 50 or 150 mg/kg bw/day. Body Weight There was no effect of treatment on body weight or body weight gain, including when adjusted for the contribution of the gravid uterus, throughout the treatment period at 15, 50 or 150 mg/kg bw/day. Food Consumption There was no effect of treatment on food consumption throughout the treatment period at 15, 50 or 150 mg/kg bw/day. Water Consumption Daily visual inspection of water bottles did not reveal any overt intergroup differences. Post Mortem Studies No macroscopic abnormalities were detected for parental females at scheduled termination on Day 20 of gestation. Litter Responses Litter Data and Litter Placental and Fetal Weights There was no obvious effect of maternal treatment on the number of implantations, subsequent embryofetal survival and litter size, sex ratio and mean fetal, litter and placental weights on Day 20 of gestation at 15, 50 or 150 mg/kg bw/day. At 150 mg/kg bw/day, mean pre-implantation loss was lower than control with differences attaining statistical significance. As animals were not dosed until implantation had occurred, these differences were incidental and unrelated to treatment. At 15 and 50 mg/kg bw/day, higher mean female fetal weight and mean fetal weight attained statistical significance compared to control. In the absence of any similar increase in fetal weight at 150 mg/kg bw/day, this finding was considered to reflect normal biological variation and was unrelated to treatment. Fetal Examination Neither the type, incidence or distribution of findings observed externally at necropsy examination and subsequently during detailed visceral and skeletal assessment of the fetuses indicated any effect of treatment on fetal development.

Conclusions:

The No Observed Effect Level (NOEL) for the pregnant females and the survival, growth and embryofetal development of the offspring was considered to be 150 mg/kg bw/day.

Executive summary:

Introduction

The study was designed to investigate the effects of the test item on embryonic and fetal development following repeated administration by gavage to the pregnant female during gestation including the period of organogenesis.

 

The study was designed to comply with the following guidelines:

 

·        US EPA Health Effects Test Guideline OPPTS 870.3700, ‘Prenatal Developmental Toxicity Study’ (August 1998)

·        Japanese Ministry of Agriculture, Forestry and Fisheries Testing guidelines for Toxicology studies, 12 NohSan No 8147, (24 November 2000)

·        OECD Guidelines for Testing of Chemicals, No 414, ‘Prenatal Developmental Toxicity Study’ (adopted 22 January 2001)

·        Commission Regulation (EC) No 440/2008 of 30 May 2008 test methods pursuant to Regulations (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH)

  

Methods…….

The test item was administered by gavage to three groups each of twenty-four time mated Sprague-Dawley Crl:CD^®(SD) IGS BR strain rats, between Days 5 and 19 of gestation inclusive at dose levels 15, 50, and 150 mg/kg bw/day. A further group of twenty-four time mated females was exposed to the vehicle only (Arachis oil) over the same treatment period to serve as a control.

Clinical signs, body weight change, food and water consumptions were monitored during the study. 

 

All females were terminated on Day 20 of gestation and subjected to gross necropsy including examination of the uterine contents. The number of corpora lutea, number, position and type of implantation, placental weights, fetal weight, sex and external and internal macroscopic appearance were recorded. Half of each litter were examined for detailed skeletal development and the remaining half were subjected to detailed visceral examination.

 

Results…….

Adult Responses

Mortality

There were no unscheduled deaths during the study.

 

 Clinical Observations

Clinical sign did not indicate any effect of treatment at 15, 50 or 150 mg/kg bw/day.

  

Body Weight

Body weight and body weight gain, including adjustment for the contribution of the gravid uterus, was unaffected by treatment at 15, 50 or 150 mg/kg bw/day.

 

 Food Consumption

Food consumption was unaffected by treatment at 15, 50 or 150 mg/kg bw/day.

 

 Water Consumption

Daily visual inspection of water bottles did not reveal any overt intergroup differences.

 

 Post Mortem Studies

No macroscopic abnormalities were detected for parental females at 15, 50 or 150 mg/kg bw/day.

 

 Litter Responses

Litter Data and Litter Placental and Fetal Weights

The number of implantations, subsequent embryofetal survival and litter size, sex ratio and mean fetal, litter and placental weights on Day 20 of gestation were unaffected by maternal treatment at 15, 50 or 150 mg/kg bw/day.

 

 Fetal Examination

There was no effect of maternal treatment on morphological development of the fetuses at 15, 50 or 150 mg/kg bw/day.

 

 Conclusion

The No Observed Effect Level (NOEL) for the pregnant females and the survival, growth and embryofetal development of the offspring was considered to be 150 mg/kg bw/day.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

150 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

There is a high quality OECD 414 study on 2, 2’-(Octadec-9-enylimino) bisethanol CAS No 25307-17-9 which shows no adverse effects and affirms the OECD443 conducted on the same substance for developmental toxicity which additionally covers the period immediately after implantation.

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

General reasoning

The category approach was undertaken to identify an appropriate read-across substance for 2, 2’-(Octadecylimino)bisethanol CAS No 10213-78-2 based on sufficient data density and k-nearest neighbor structural identity that could be considered a reasonably conservative approach. 2, 2’-(Octadec-9-enylimino) bisethanol CAS No 25307-17-9 is the output of this approach. Lower alkyl chain-lengths are available in the category such as: 2,2’-[C16-18( evennumbered, C18 unsaturated) alkyl imino] diethanol, (PFAEO-T), CAS No 1218787-32-6; 2,2’-(C16-18 evennumbered alkyl imino) diethanol, (PFAEO-HT), CAS No 1218787-30-4; and 2,2’-(C12-18 evennumbered alkyl imino) diethanol, (PFAEO-C), 71786-60-2. It is theorized that decreasing alkyl chain length on this family of tertiary amines leads to increased toxicity among several endpoints.

 

Read-across to PFAEO O:

The OECD414 pre-natal development study included dose levels of 15, 50 and 150mg/kg bodyweight of 2, 2’-(Octadec-9-enylimino) bisethanol CAS No 25307-17-9, administered by gavage for days 5 to 19 of pregnancy as a solution in arachis oil. This substance is the appropriate read across substance for the appropriate read across substance for 2,2’-(Octadecylimino)bisethanol) CAS No 10213-78-2. The dose levels were selected based on those used in the 28 day and 90 day repeat dose studies. The adult females did not show any signs of toxicity in this study such as on bodyweights, or food or water consumption, but there was no histopathological examination of their stomachs, making it not possible to see local adverse (irritant/corrosive) effects due to the irritant nature of the test material as seen in the other repeat dose studies.

The absence of toxic effects in the adult females is not considered to be of concern as we have good evidence from the 28 day repeat dose study that higher doses could result in mortality or severe toxicity due to local effects in the stomach.

The number of implantations, subsequent embryo foetal survival and litter size, sex ratio and mean foetal, litter and placental weights on Day 20 of gestation were unaffected by maternal treatment at 15, 50 or 150 mg/kg bw/day.

There was no effect of maternal treatment on morphological development of the foetuses at 15, 50 or 150 mg/kg bw/day.

As the OECD414 study is in line with the results observed in the OECD 443 study with the same substance.

 

Read-across to PFAEO T

An OECD 414 was also conducted on 2,2’-(C16-18 (even numbered, C18 unsaturated) alkyl imino) diethanol CAS No 1218787-32-6, which has an average shorter alkyl chain-length (C16-18). It is concluded that that oral administration of 2,2’-(C16-18 (even numbered, C18 unsaturated) alkyl imino) diethanol to Han Wistar rats during the organogenesis and fetal growth phases of pregnancy at doses of 0, 15, 50 or 150 mg/kg/day was generally tolerated but cause an adverse reduction of weight gain, associated with low food intake, in maternal females receiving 150 mg/kg/day. There was an adverse increase of pre- and post-implantation loss and a lower number of live young and smaller litter size at 150 mg/kg/day. This could possibly be a secondary effect of maternal local toxicity and bodyweight lose, though a direct test item related effect cannot be ruled out. The No-Observed-Adverse-Effect Level (NOAEL) for maternal toxicity and embryo-fetal survival and development was therefore considered to be 50 mg/kg/day. 

An OECD 422 on 2,2’-(C16-18 (even numbered, C18 unsaturated) alkyl imino) diethanol did have lower live birth indexes though this is thought to be a factor of the reduced post-implatation indexes. Further lower foetal bodyweights, PND 4 survival and PND 13 surival were all decreased at 150 mg/kg bw/day compared to that of controls. This factors are reduced as three maternal females were sacrificed in late gestation /early lactation. This leads the authors of the study to the conclusion that 2,2’-(C16-18 (even numbered, C18 unsaturated) alkyl imino) had no effect on implantation count and live birth index. Reduced offspring survival (low Day 4 and Day 13 lactation survival indices) resulted in a conservative NOAEL of 75 mg/kg bw/day. Mortalities in maternal animals resulted in a NOAEL of 75 mg/kg bw/day for repeat-dose toxicity.

 

Addtional category information:

2,2’-(C12-18 evennumbered alkyl imino) diethanol CAS No 71786-60-2
has shown in particular to affect developmental and reproductive parameters in both rats and some developmental parameters in rabbits. 

The results from the OECD 414 pre-natal development study in rats indicate that Ethanol, 2,2’-Iminobis-, N-C12-18-Alkyl Derives produces major skeletal abnormalities in the skull and spinal cord including effects in the eyes of microphthalmia and anophthalmia. The incidence of one fetus in one litter at 30 mg/kg/day is within the historical control range so 30mg/kg/day could be the true NOAEL, however a conservative NOAEL of 10mg/kg/day was selected as it was not possible to be sure that this effect was not treatment related.

The results from the OECD 414 pre-natal development study in rabbits indicate that Ethanol, 2,2’-Iminobis-, N-C12-18-Alkyl Derives showed treatment related toxicity at the high dose of 175 mg/kg/day causing overt maternal toxicity which manifested as body weight loss and low food consumption (one female was killed following a sustained period of low/negligible food intake and associated clinical signs), however, embryo fetal survival was unaffected but mean fetal weights were slightly low and there was an increase in incomplete ossification, a transient, non-adverse minor fetal finding. It was concluded from this study that the dosage of 60 mg/kg/day is the maternal no observed-adverse-effect-level (NOAEL), and the no observed adverse-effect-level (NOAEL) for embryo-fetal survival, growth and development is 60 mg/kg/day.

There is currently a EOGRTS being performed on PFAEO-C that will help to better define the nature of this toxicity.

 

On-going work

An addtional OECD 414 is being conducted currently on 2,2’-(C16-18 evennumbered alkyl imino) diethanol CAS No 1218787-30-4. While no conclusions can yet be made on the study itself, the conclusion of the study will help to solidify conclusions and trends in the category. 

 

Conclusion

There is a clear break in the trends of developmental toxicity for the category of PFAEOs. This break occurs somewhere between C12 and C16 alkyl chain lengths. The avialable information on the category points to increasing chain lengths and increasing saturation resulting in decreased toxicity. 2, 2’-(Octadecylimino)bisethanol CAS No 10213-78-2 is at the upper edge of category in terms of both chain-length and saturation. As there is no estabished trend for the endpoint of developmental toxcity in terms of degrees of severity it is prudent to examine category members with the closest structural relationship in order to determine risk and hazard while still ensuring a conservative approach. The substance 2, 2’-(Octadec-9-enylimino) bisethanol CAS No 25307-17-9 provides the best canidate for this approach.

The No Observed Effect Level (NOEL) for the pregnant females and the survival, growth and embryo-foetal development of the offspring was considered to be 150 mg/kg bw/day. 2, 2’-(Octadec-9-enylimino) bisethanol CAS No 25307-17-9, even at this dose level it did not show any indications of potential for developmental toxicity in this study. The EOGRT study demonstrated no adverse effects on endpoints relating to fertility in regards to 2, 2’-(Octadec-9 -enylimino) bisethanol CAS No 25307-17-9 and by read across also to 2,2’-(Octadecylimino)bisethanol) CAS No 10213-78-2. A study on 2,2’-[C16-18( evennumbered, C18 unsaturated) alkyl imino] diethanol which has a shorter chain-length than that of the target substance. This helps to solidify a trend for the category as the OECD 414 on PFAEO-T did not show the severe malformations seen with 2,2’-(C12-18 evennumbered alkyl imino) diethanol which has an even shorter average chain-length. 2, 2’-(Octadec-9 -enylimino) bisethanol is the most similar category member to 2,2’-(Octadecylimino)bisethanol) CAS No 10213-78-2 that would result in a conservative estimate on account of its unsaturation.

This approach satisfies sufficient category data density, establishing structural similarity, and providing a conservative approach for the purposes of read-across.

 

Justification for selection of Effect on developmental toxicity: via oral route:

We have a Klimisch 1 full GLP compliant OECD414 study on 2, 2’-(Octadec-9-enylimino) bisethanol CAS No 25307-17-9 an appropriate read across substance for  2,2’-(Octadecylimino)bisethanol CAS No 10213-78-2. However this OECD414 study does not cover the implantation period, which is covered by the OECD443 on the same substance.

 

Justification for selection of Effect on developmental toxicity: via inhalation route:

Due to the low vapour pressure and the physical form of the substance, it being a waxy solid a room temperature inhalation is not an expected route of exposure in the workplace.  In addition the irritant properties to the skin would make such testing very technically difficult.

 

Justification for selection of Effect on developmental toxicity: via dermal route:

The skin irritant properties of this substance mean the repeated dose dermal studies are not scientifically justified due to concerns for animal welfare.  Also t would be expected to be absorbed less through the skin than orally, therefore it is possible to calculate dermal DNELs based on the available oral repeat dose NOAEL data, based on ECHA guidance.

 

Justification for classification or non-classification

General reasoning

There is no specific test data on potential reproductive toxic effect of for 2,2’-(Octadecylimino)bisethanol) CAS No 10213-78-2, an appropriate read across substance 2’-(Octadec-9-enylimino) bisethanol CAS No 25307-17-9 was determined appropriate to read-across for developmental toxicity endpoints based of comparable structures. 2, 2’-(Octadec-9-enylimino) bisethanol CAS No 25307-17-9, is predominantly C18 unsaturated, while, 2,2’-(Octadecylimino)bisethanol) CAS No 10213-78-2 is predominantly C18 saturated. The unsaturated C18 is more reactive and therefore expected to be more toxic than the equivalent C18 saturated structure constituting a conservative trend approach worse-case scenario.

 

Sufficent data density and structural similarity exist on the proposed category for the given endpoints of reproductive and developmental toxicity that would allow reasonable conclusions to be drawn. Available trends within the data of the category allow for assertion that lower alkyl-chain lengths led to increasing severity of adversity of foetal toxic effects. 2’-(Octadecylimino)bisethanol) CAS No 10213-78-2 is on the upper boundary of alkyl carbon chain-lengths for the cateogry and is most structurally similar to 2’-(Octadec-9-enylimino) bisethanol CAS No 25307-17-9 by compostion and affirmed by k-nearest neighbor analysis. Addtionally, these compounds share high degrees of similarity via the tanimoto index. As stated above, within the trends established in this category approach 2’-(Octadec-9-enylimino) bisethanol CAS No 25307-17-9 a worse-case than in regard to reproductive and developmental toxicity.

 

Specific reasoning to Reproductive classification

The OECD443 study on 2’-(Octadec-9-enylimino) bisethanol CAS No 25307-17-9 showed litter size, survival indices and sex ratio the number of implantation sites, post-implantation survival, live birth index and the number of offspring on Day 1 (total and live Day 1) were unaffected by parental treatment at dose levels up to and including 150 mg/kg/day (highest dose tested). With observable effects on reduced food consumption and body weight gain being attributed to secondary effects as the result of gastrointestinal tract irritancy. 

The OECD 422 that is available for 2,2’-[C16-18( evennumbered, C18 unsaturated) alkyl imino] diethanol CAS No 1218787-32-6 demonstrated that treatment at 175 mg/kg bw/day exceeded the tolerable intake level of the animals and resulted in the mortality of four male rats and an addtional three females that were in late gestation/early lactation.  One female at 175 mg/kg/day and one female at 30 mg/kg/day failed to litter and were confirmed to not be pregnant at necropsy. Implantation count and live birth index were unaffected by maternal treatment with 2,2’-[C16-18( evennumbered, C18 unsaturated) alkyl imino] diethanol CAS No 1218787-32-6. As a result of a low post-implantation survival index at 175 mg/kg/day, total litter size was slightly lower than Control. At 175 mg/kg/day the viability index on Day 4 of lactation and lactation index on Day 13 were lower than Control as a result of the maternal mortalites. At 75 or 30 mg/kg/day, litter size and survival indices were unaffected by maternal treatment.

These results indicate that there is little concern for reproductive toxicity for 2,2’-(Octadecylimino)bisethanol) CAS No 10213-78-2 as it has a increased level of saturation and increased chain length which both show trends of decreasing concerns toxicity among reproductive endpoints.

 

Specific reasoning to Developmental classification

There is a break in the category approach for PFAEOs for developmental toxcity that lies somewhere between category members 2,2’-(C12-18 evennumbered alkyl imino) diethanol CAS No 71786-60-2 (primarily C12 alkyl chain) and 2,2’-[C16-18( evennumbered, C18 unsaturated) alkyl imino] diethanol CAS No 1218787-32-6 (primarily C16-18 alkyl chain). Results from OECD 414 on 2,2’-[C16-18( evennumbered, C18 unsaturated) alkyl imino] diethanol CAS No 1218787-32-6 do not show the same foetal malformations observed in the OECD 414  on 2,2’-(C12-18 evennumbered alkyl imino) diethanol CAS No 71786-60-2 at comparable dose levels, high dose 125 and 150 mg/kg bw/day respectively. 

This information presents with two distinct posibilities either a.) there is a break in developmental toxicity between C12 and C16 alkyl chain lengths for this endpoint, or b.) overt maternal toxicity at the highest dose level tested (150 mg/kg bw/day) for 2,2’-[C16-18( evennumbered, C18 unsaturated) alkyl imino] diethanol CAS No 1218787-32-6 thus any higher dosing structure to elicit similar responses would be too detrimental to the welfare of maternal animals to evaluate the response. Regardless, it is clear that 2,2’-(C12-18 evennumbered alkyl imino) diethanol CAS No 71786-60-2 is not a clear indicator of developmental toxicity for other category members with alkyl chains that exceed C16. Severe maternal toxicity from corrosive effects driven by the lipophilic nature of the molecule seems to take precedent in this category at chain lengths of C16-18 and does not result in developmental malformations observed with their C12 counterparts at the tolerable doses tested.

 "In principle, adverse effects on reproduction seen only at very high
dose levels in animal studies (for example doses that induce prostration, severe inappetence, excessive mortality) would not normally
lead to classification,..." Section 3.7.2.5.8. EC 1272/2008

Obsereved trends in the data among available OECD 414s in the category suggest that maternal toxicity decreases with increasing chain length. The OECD 414 on 2,2’-[C16-18( evennumbered, C18 unsaturated) alkyl imino] diethanol CAS No 1218787-32-6 did show high pre- and post-implantation loss in females receiving 150 mg/kg/day, resulting in a lower number of implantations and live young. The reduction in live young correlated with lower total litter weights and higher placental weights at this dose level. There was, however, no effect on individual fetal weights and the increased placental weights were likely due to the lower numbers of live young in each litter.  Addtionally, secondary effects of maternal toxicity at 150 mg/kg bw/day  resulting in an average body weight loss of of maternal animals by 50% when corrected for gravid uterine weight as this may have contributed to reduced fetal bodyweight and acted a as a possible precursour for implantation loss due to the poor health of the maternal animals. These effects on reproductive and developmental parameters were not observed at lower dose levels that did not induce maternal bodyweight loss.

 

Conclusions

The OECD414 pre-natal development study in rats, showed no evidence of developmental toxicity even at the top dose of 150mg/kg bodyweight, therefore while this study does not include dosing during the pre-mating period until day 4, it does not indicate any potential for developmental toxicity (adverse effects on the morphological development of the foetus). The combine evidence from the OECD414 and OECD433 studies indicates that 2,2’-(Octadecylimino)bisethanol can be considered not to induce adverse effects on the morphological development of the foetus in rats or exhibit direct effects on fertility in either sex, therefore it is not necessary to classify this substance for reproductive or developmental toxicity based on current data from read across to 2, 2’-(Octadec-9-enylimino) bisethanol CAS No 25307-17-9.

Based on available informaiton on the category, where the substance 2,2’-(Octadecylimino)bisethanol) CAS No 10213-78-2 lies in the category with respect to chain length, the breaks in toxicity, and reproductive/developmental effects only occuring at doses that induce maternally toxic effects resulting in bodyweight loss it is not considered necessary to categorize 2,2’-(Octadecylimino)bisethanol) CAS No 10213-78-2 based on available infromation for developmental or reproductive toxicity.