Ethanol, 2,2'-iminobis-, N-C12-18-alkyl derivs.

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental start date (Pre study chemistry ) 22 November 2017 Experimental completion date Fetal pathology data 19 February 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other:

Version / remarks:

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

Deviations:

no

Principles of method if other than guideline:

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

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Ethanol, 2,2'-iminobis-, N-C12-18-alkyl derivs.

EC Number:

276-014-8

EC Name:

Ethanol, 2,2'-iminobis-, N-C12-18-alkyl derivs.

Cas Number:

71786-60-2

Molecular formula:

Not applicable

IUPAC Name:

2,2'-(C12-18 evennumbered alkyl imino) diethanol

Test material form:

liquid

Details on test material:

- Chemical name: Ethanol, 2,2'-iminobis-, N-C12-18-alkyl derivs.
- EC number: 276-014-8

To the best of knowledge, the sample used is representative to the boundary composition shared and agreed by each registrant.

Specific details on test material used for the study:

Test item: Ethanol, 2,2’-iminobis-, N-C12-18-alkyl derives.
CAS number: 71786-60-2.
Appearance: Light yellow/ pale orange Liquid.
Storage conditions: At ambient temperature (15 to 25°C), in the dark.

Test animals

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

Strain/Species New Zealand White rabbit.
Supplier Envigo RMS.
Number of animals ordered 96 females.
Duration of acclimatization 19 days before commencement of pairing.
Age of the animals at the start of the study (Day 0 of gestation) 19 to 23 weeks old.
Weight range of the animals at the start of the study (Day 0 of gestation) 2.41 to 4.08 kg


3.3.4 Environmental Control
Rabbit facility Partial barrier with 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 15-21°C and 45-70%.
On one occasion the relative humidity was outside the indicated ranges, this deviation was minor and/or of short duration and was not considered to have influenced the health of the animals and/or the outcome of the study.
Lighting Artificial lighting, 14 hours light : 10 hours dark.
Alarm systems Activated on ventilation failure and when temperature/humidity limits exceeded.
Electricity supply Public supply with automatic stand-by generators.


Cages Suspended cages fitted with perforated floor panels and mounted in batteries. Undertrays lined with absorbent paper were changed at least three times a week. Cages were also fitted with a plastic resting platform.
Cage distribution The cages constituting each group were blocked by group and mounted in batteries.
Number of animals per cage
Acclimatization one female.
During mating one stock male and one female.
Gestation one female.
3.3.6 Environmental Enrichment
Aspen chew product A soft white untreated wood product; provided to each cage throughout the study and replaced when necessary.
Cage paper Provided to each cage from Day 20 after mating and replaced as necessary.
Stainless steel key ring Attached to the cage.
3.3.7 Diet Supply
Diet Teklad 2930 Diet.
The diet contained no added antibiotic or other chemotherapeutic or prophylactic agent.

Should an individual show a significant non-treatment related reduced food consumption, moistened diet (50 g pelleted diet moistened with up to 50 mL of water) was offered, the consumption was recorded.
In addition to this diet, a small supplement of autoclaved hay was given on a daily basis to promote gastric motility and a small amount of chopped fresh vegetables were given twice weekly. Consumption of hay and vegetables were monitored qualitatively but not quantitatively.
3.3.8 Water Supply
Supply Potable water from the public supply via polycarbonate bottles with sipper tubes. Bottles were changed at appropriate intervals. Water bowls were also used in addition to a bottle.
Availability Non-restricted.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: 0.5% methyl cellulose

Details on exposure:

Method of preparation The required amount of test item was weighed and 90% of the final volume of vehicle was added and magnetically stirred. The remaining vehicle was added and the formulation was stirred using a magnetic stirrer until homogenous.
Frequency of preparation Weekly.
Storage of formulation Refrigerated (2-8°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.
3.2.2 Formulation Analysis
Stability and homogeneity Before commencement of treatment, the suitability of the proposed mixing procedures was determined and specimen formulations at 2.5 and 250 mg/mL were analyzed to assess the stability and homogeneity of the test item in the liquid matrix. The stability was confirmed as 15 days at refrigerated temperature (2 to 8°C) and one day ambient temperature (15 to 25°C).
Achieved concentration Samples of each formulation prepared for administration on Day 6 and 28 of treatment were analyzed for achieved concentration of the test item.

3.4.2 Administration
Route Oral gavage using a suitably graduated syringe and a rubber catheter inserted via the mouth.
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 28 (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:

Preparation of Calibration Standards
A primary standard solution (500 μg/mL) was prepared by dissolving an accurately weighed quantity (ca. 50 mg) of Ethanol, 2,2’-Iminobis-, N-C12-18-alkyl derives in diluent (100 mL). A secondary standard solution (10 μg/mL) was prepared by appropriate dilution of the primary standard using diluent. A tertiary standard solution (1 μg/mL) was
prepared by appropriate dilution of the secondary standard using diluent.

Solutions for instrument calibration were prepared by appropriate dilution of the tertiary standard using diluent and contained Ethanol, 2,2’-Iminobis-, N-C12-18-alkyl derives at nominal concentrations of 10 ng/mL, 20 ng/mL, 40 ng/mL, 50 ng/mL, 60 ng/mL, 80 ng/mL and 100 ng/mL.

Calibration solutions were injected onto the LC-MS/MS, at the beginning of each sample analysis sequence, using the conditions detailed in the instrumentation parameters section. The standard prepared at 50 ng/mL was injected in duplicate to bracket every three samples.

Preparation of Test Samples
A representative sample of test formulation (1 mL, accurately weighed) was dissolved using ultrasonic vibration in a suitable volume of diluent. The extract was then diluted further with diluent, to provide a solution containing Ethanol, 2,2’-Iminobis-, N-C12-18-alkyl derives at an expected concentration of 50 ng/mL or as close as practical.
The concentration of Ethanol, 2,2’-Iminobis-, N-C12-18-alkyl derives in the final solution was quantified by LCMS/MS as detailed in the instrumentation parameters section.

Preparation of Recovery Samples
Procedural recoveries were prepared by fortifying samples (1 mL) of control matrix (0.5% methylcellulose) with known amounts of Ethanol, 2,2’-Iminobis-, N-C12-18-alkyl
derives. The prepared procedural recoveries were analyzed in accordance with the analytical procedure.

Instrumentation Parameters
High performance liquid Agilent 1100 pump, Perkin Elmer PE200 Autosampler and Sciex API 3000 equivalent mass spectrometer
chromatograph and mass spectrometer
(LC-MS/MS):

Column: Agilent Poroshell SB C18, 2.7 μm, 100 x 4.6 mm

Column temperature: 45ºC

Sample temperature: Ambient

Mobile Phase A: 0.1M ammonium acetate (aq)/acetic acid 100/0.1 v/v

Mobile Phase B: MeOH/acetic Acid 100/0.1 v/v

Linear Gradient:
Time (minutes) %A %B
0.0 60 40
4.0 30 70
6.0 5.0 95
8.0 5.0 95
8.1 70 30
12.0 70 30

Flow rate: 0.5 mL/minute

Ionisation: Turboionspray positive ion mode

Source temperature: +500°C

Collision gas: Nitrogen
Collision energy: 35 eV
Dwell time: 100 ms
Pause time: 5 ms
Ions to be monitored: m/z 274.5  m/z 106.1 (used for quantitate)
Run time: 12 minutes
Approximate retention time: 8.1 minutes

Method Validation
The analytical procedure was successfully validated for Ethanol, 2,2’-Iminobis-, N-C12-18-alkyl derives in 0.5% methylcellulose with respect to the specificity of chromatographic analysis, limit of quantification, the linearity of detector response, repeatability and method accuracy and precision. Results are summarized below:
* The specificity of the LCMS/MS assay was demonstrated by any peak at the characteristic retention time for Ethanol, 2,2’-Iminobis-, N-C12-18-alkyl derives in
the control sample chromatogram being less than 20% of the response of standard 1

The limit of quantification was deemed to be the lowest standard, 10 ng/mL.

Linearity was confirmed over the nominal concentration range 10 ng/mL to 100 ng/mL with a coefficient of determination >0.99.

The repeatability was <10% for six replicate injections of standard solutions containing Ethanol, 2,2’-Iminobis-, N-C12-18-alkyl derives at nominal concentrations of 10 ng/mL and 100 ng/mL.

A mean procedural recovery value of 85.3% (CV=2.29%, n=5) was obtained for 2.5 mg/mL and 91.5% (CV=6.82%, n=5) was obtained for 250 mg/mL. A repeat validation at 250 mg/mL formulation confirmed these results. Therefore, the recovery limit was set at 80 to 110% recovery due to the nature of the test item.

Homogeneity and Stability of Dose Formulations
The homogeneity and stability of Ethanol, 2,2’-Iminobis-, N-C12-18-alkyl derives in 0.5% methylcellulose formulations was assessed with respect to the level of concentration at nominal concentrations of 2.5 mg/mL and 250 mg/mL.

Homogeneity was confirmed during distribution between the bottles, during magnetic stirring for 4 hours, and on re-suspension following storage at ambient temperature for 1 day and refrigeration for up to 15 days. At each time-point, the mean analyzed concentration for the three samples remained within 8% of the initial time zero value and the coefficient of variation was less than 6%. This is with the exception of 250 mg/mL day 15 which was –10.6% from the initial time zero value. As result is just outside of acceptance criteria of ±10% from initial time zero value and it is within 6% of nominal value the formulation was considered to be stable for 15 days.

For the discrete samples the mean analyzed concentration for the two samples remained within 3% of the initial time zero value at each time point for each concentrations.

Recovery results during the trial remained within the acceptable range of 80% to 110% showing the continued accuracy of the method.

Concentration of Dose Formulations
The mean concentrations were within the applied limits of +10/-15% confirming the accuracy of formulation. The difference between the samples remained within 4%, confirming precise analysis.

Recovery results during Day 6 and Day 28 remained within the acceptable range of 80% to 110% showing the continued accuracy of the method. Samples were corrected for the mean procedural recovery. This is with the exception of day 28 43.77 mg/mL recovery was out of the acceptance limits of 80 to 110% recovery and therefore has been excluded as per SOP.

CONCLUSION
The analytical procedure was successfully validated with respect to specificity of chromatographic analysis, limit of quantification, linearity of detector response, repeatability, method accuracy and precision.

The homogeneity and stability was confirmed for Ethanol, 2,2’-Iminobis-, N-C12-18-alkyl derives in 0.5% methylcellulose formulations at nominal concentrations of 2.5 mg/mL and 250 mg/mL during distribution between the bottles, during magnetic stirring for 4 hours, ambient temperature storage (15 to 25ºC) for 1 day and refrigerated storage (2 to 8ºC) for up to 15 days.

The mean concentrations of Ethanol, 2,2’-Iminobis-, N-C12-18-alkyl derives in test formulations analyzed for the study were within ±10% of nominal concentrations, confirming accurate formulation. The difference from mean remained within 4%, confirming precise analysis.

Recovery results during the trial and on Day 6 and Day 28 remained within the acceptable range of 80 to 110%. Samples were corrected for the mean procedural recovery.

Details on mating procedure:

Mating
Male/female ratio 1:1 using identified stock New Zealand White bucks.
Checks Natural mating observed.
After mating Each female was injected intravenously with 25 i.u. luteinizing hormone.
Day 0 of gestation On the day of mating.
A colony of stud males was maintained specifically for the purpose of mating; these animals were not part of the study and were maintained as stock animals.

Duration of treatment / exposure:

Females Day 6 to 28 after mating

Frequency of treatment:

Daily

Duration of test:

Day 0-6: Mating
Day 6-28: Treatment
Day 29: Necorpsy

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Doses / concentrations 2

Dose / conc.:

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

Doses / concentrations 3

Dose / conc.:

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

Doses / concentrations 4

Dose / conc.:

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

No. of animals per sex per dose:

22 females

Control animals:

yes, concurrent vehicle

Details on study design:

Animal Model
The rabbit was chosen as the test species because of the requirement for a non-rodent species by regulatory agencies. The New Zealand White strain was used because of the historical control data available in this laboratory.

Rationale for Dose Level Selection
The doses used in this study (0, 20, 60 and 175 mg/kg/day) were selected in conjunction with the Sponsor.

Dose levels were selected following the completion of the pilot and preliminary studies (Envigo Study numbers: LP82XG and LT98XB). In the Pilot study (LP82XG) oral administration at 125, 175 or 200 mg/kg/day to New Zealand White rabbits was well tolerated, and did not elicit any marked signs of maternal toxicity. A dose level of 200 mg/kg/day, resulted in a number of clinical signs (predominantly little hay eaten, little water drunk, few faeces, which were of reduced size or abnormal colour and all females were of thin build). There were no deaths or post dose signs observed and no treatment related macroscopic findings, but there were adverse effects on body weight gain and food consumption. No clinical or significant post dose signs were observed at the second dose level of 175 mg/kg/day, and there were no overall adverse effects on body weight or food consumption and no treatment related macroscopic findings.

In the Preliminary study (LT98XB) oral administration at 20, 60 or 175 mg/kg/day to New Zealand White rabbits was well tolerated, and did not elicit any marked clinical signs of maternal toxicity. There were no deaths or post dose signs observed and no treatment related macroscopic findings, but there were slight effects on body weight gain and food consumption at 175 mg/kg/day, and overall group mean body weight gain was slightly low in females treated at 60 mg/kg/day when compared with the Controls.

Therefore dose levels of 20, 60 and 175 mg/kg/day were considered suitable for use on this main study.

Examinations

Maternal examinations:

Serial Observations
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 or if they exhibited pregnancy loss.
A complete necropsy was performed in all cases.

Clinical Observations
Animals were inspected visually at least twice daily for evidence of ill-health or reaction to treatment. Cages and cage-trays were inspected daily for evidence of animal ill-health amongst the occupant. 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 0, 6, 12, 18, 23 and 29 after mating to monitor general health.

Body Weight
The weight of each adult was recorded weekly during acclimatization, on the day of mating and on Days 0, 3 and 6-29 after mating.

Food Consumption
The weight of food supplied to each animal, that remaining and an estimate of any spilled was recorded daily from Day 1 after mating.

Terminal Investigations
Method of Kill
Method of kill for all adult animals Intravenous injection of sodium pentobarbitone.
Method of kill for fetuses Subcutaneous injection of sodium pentobarbitone.

Necropsy
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 surviving until the end of the scheduled study period were killed on Day 29 after mating.
Sequence To allow satisfactory inter-group comparison.

Ovaries and uterine content:

For females surviving to term, the following was recorded:
Uterus Gravid uterine weight (including cervix and ovaries).

The following were recorded for all animals (including those prematurely sacrificed, where possible):
For each ovary/uterine horn
Number of: Corpora lutea.
Implantation sites.
Intrauterine deaths (classified as early or late resorptions).
Fetuses (live and dead).

Non pregnant animals The absence or number of uterine implantation sites was confirmed.

Fetal examinations:

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. Examined externally with abnormalities recorded. All fetuses were subject to a gross internal examination of the viscera of the neck, thorax and abdominal cavities and the sex of each fetus was also recorded.

Fixation
Nominally one half of eviscerated fetuses were decapitated; heads were initially stored in Bouin’s fluid.
Remaining eviscerated fetuses and torsos were fixed in Industrial Methylated Spirit.

Processing
Bouin’s fixed fetal heads were subject to free-hand serial sectioning.
Industrial Methylated Spirit fixed fetuses and torsos were processed and stained with Alizarin Red.

Fetal Pathology Examination
Bouin’s fixed heads Serial sections were examined for soft tissue abnormalities.
Alizarin Red stained fetuses and torsos Assessed for skeletal development and abnormalities.

Statistics:

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

Indices:

Reproductive Assessment
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) / x 100
Number of corpora lutea

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) / x 100
Number of implantations
All group values and SD (as appropriate) were calculated from the individual litter values.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

One female receiving 175 mg/kg/day (4F 73) was killed for welfare reasons on Day 21 of gestation following a sustained period of low/negligible food consumption (pellets), and little hay eaten and decreased number of faecal pellets which were of reduced size and abnormal pale colour. This female also had reduced urine output and was of thin build. Macroscopic examination revealed the cecum to have abnormal fluid contents. This female was pregnant, and all fetuses appeared grossly normal.

A second female receiving 175 mg/kg/day (4F 83) was killed for welfare reasons on Day 26 of gestation due to marked breathing impairment which consisted of wet rales, irregular breathing, decreased activity, and excessive salivation. There were no macroscopic abnormalities. This female was pregnant, and all fetuses appeared grossly normal.

Signs observed at routine examination following the start of treatment on Day 6 of gestation consisted of decreased number of faecal pellets which were of reduced size and/or of abnormal pale colour for up to 15 females receiving 175 mg/kg/day. Reduced urine output was observed for up to 6 females and little hay eaten, little water drunk and little diet eaten was also recorded for up to 4 females receiving 175 mg/kg/day; in addition, 4 females had an ulceration on the muzzle and 4 females had red/orange staining on the cage undertray.

Following dose administration one female receiving 20 mg/kg/day was observed with dry rales on Days 14 and 17 after mating. There were no other signs observed following dose administration.


PLEASE REFER TO ATTACHED TABLE: Clinical signs - group distribution of observations

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Following the start of treatment (Days 6-8 of gestation) group mean body weight loss was evident in all treated groups, it was minor at 20 and 60 mg/kg/day, but was greater and statistical significance was attained at 175 mg/kg/day.

Group mean statistically significant body weight loss was still evident until Day 11 of gestation. From Day 8 of gestation group mean body weight gain was similar to Controls for females receiving 20 or 60 mg/kg/day. However, statistically significantly reduced overall body weight gain was evident at 175 mg/kg/day and a minor decrease in body weight gain was evident at 20 and 60 mg/kg/day.

The mean gravid uterine weight for females treated with Ethanol, 2,2' iminobis , N C12 18 alkyl derives was slightly low 175 mg/kg/day when compared with the Control females. Adjusted body weight loss was higher for females receiving 175 mg/kg/day when compared to the Controls.

PLEASE REFER TO ATTACHED TABLE - Body weight and body weight change - group mean vakues (kg) during gestation

PLEASE REFER TO ATTACHED TABLE - Gravid uterine weight, adjusted bodyweight and adjusted body weight change - group mean values (kg) on Day 29 of gestation

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Following the start of treatment group mean food intake was statistically significantly reduced for females receiving 175 mg/kg/day (Day 6) which was statistically significantly reduced during Days 6-20 of gestation when compared with Controls. Food intake was comparable with Controls from Day 21 of gestation.

Group mean food intake for females receiving 20 or 60 mg/kg/day was similar to Controls. However, individual responses were very varied with some individual having periods of inappetance, but the number of individuals was greater for females receiving 175 mg/kg/day.

PLEASE REFER TO ATTACHED TABLE - Food consumption - group mean values (g/animal/day) during gestation

Food efficiency:

not examined

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

not examined

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

Description (incidence and severity):

Macroscopic examination on Day 29 after mating did not reveal any adverse findings, that were considered related to treatment.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Maternal developmental toxicity

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

Litter data as assessed by the mean numbers of implantations, early, late and total resorptions, the total number of live young and the sex ratio, were considered to be unaffected by treatment when compared with Controls.

PLEASE REFER TO ATTACHED APPENDIX - Litter data - individual values

Total litter losses by resorption:

no effects observed

Description (incidence and severity):

Litter data as assessed by the mean numbers of implantations, early, late and total resorptions, the total number of live young and the sex ratio, were considered to be unaffected by treatment when compared with Controls.

PLEASE REFER TO ATTACHED APPENDIX - Litter data - individual values

Early or late resorptions:

no effects observed

Description (incidence and severity):

Litter data as assessed by the mean numbers of implantations, early, late and total resorptions, the total number of live young and the sex ratio, were considered to be unaffected by treatment when compared with Controls.

PLEASE REFER TO ATTACHED APPENDIX - Litter data - individual values

Dead fetuses:

no effects observed

Description (incidence and severity):

Litter data as assessed by the mean numbers of implantations, early, late and total resorptions, the total number of live young and the sex ratio, were considered to be unaffected by treatment when compared with Controls.

PLEASE REFER TO ATTACHED APPENDIX - Litter data - individual values

Changes in pregnancy duration:

no effects observed

Changes in number of pregnant:

effects observed, non-treatment-related

Description (incidence and severity):

Two females receiving 20 mg/kg/day (2F No. 26 and 29), three females receiving 60 mg/kg/day (3F No. 51, 59 and 62) and two females receiving 175 mg/kg/day (4F No. 77 and 79) were not pregnant. The following assessment is based on the 22, 20, 19 and 18 females with live young at termination on Day 29 of gestation in the Control group and at 20, 60 and 175 mg/kg/day, respectively.

Effect levels (maternal animals)

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Basis for effect level:

body weight and weight gain

food consumption and compound intake

Maternal abnormalities

Abnormalities:

no effects observed

Results (fetuses)

Fetal body weight changes:

effects observed, treatment-related

Description (incidence and severity):

Group mean placental, total litter weights, and male or female and overall fetal weights were slightly reduced for females that received 175 mg/kg/day when compared with the Controls. Statistical significance was attained for placental, male and overall fetal weights.

Group mean weights for females receiving 20 or 60 mg/kg/day were similar to Controls.

PLEASE REFER TO ATTACHED TABLE - Placental, litter and fetal weights - group mean values (g) on Day 29 of gestattion

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

not examined

Changes in litter size and weights:

effects observed, treatment-related

Description (incidence and severity):

Group mean placental, total litter weights, and male or female and overall fetal weights were slightly reduced for females that received 175 mg/kg/day when compared with the Controls. Statistical significance was attained for placental, male and overall fetal weights.

Group mean weights for females receiving 20 or 60 mg/kg/day were similar to Controls.

PLEASE REFER TO ATTACHED TABLE - Placental, litter and fetal weights - group mean values (g) on Day 29 of gestattion

Changes in postnatal survival:

not examined

External malformations:

no effects observed

Skeletal malformations:

effects observed, treatment-related

Description (incidence and severity):

At 175 mg/kg/day there was an increased incidence of fetuses/litters with incompletely ossified cervical vertebrae and metacarpals/phalanges forepaws when compared to concurrent control and this incidence was outside of Historical Control Data range. Incomplete ossification is a transient stage in fetal development, not considered adverse and is likely associated with the slight decrease in mean fetal weight seen at this dose level likely due to the reduced maternal food consumption (Cappon et al 2005).

PLEASE REFER TO ATATCHED TABLE - Fetal examinations - minor skeletal abnormality findings - group incidences

Visceral malformations:

effects observed, treatment-related

Description (incidence and severity):

At 175 mg/kg/day there was an increased incidence of fetuses/litters with incompletely ossified cervical vertebrae and metacarpals/phalanges forepaws when compared to concurrent control and this incidence was outside of Historical Control Data range. Incomplete ossification is a transient stage in fetal development, not considered adverse and is likely associated with the slight decrease in mean fetal weight seen at this dose level likely due to the reduced maternal food consumption (Cappon et al 2005).

PLEASE REFER TO ATTACHED TABLE - Fetal examinations - minor visceral abnormality and necropsy findings - group incidences

Effect levels (fetuses)

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

not specified

Basis for effect level:

changes in litter size and weights

skeletal malformations

Overall developmental toxicity

Key result

Developmental effects observed:

yes

Lowest effective dose / conc.:

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

Treatment related:

yes

Relation to maternal toxicity:

developmental effects occurring together with maternal toxicity effects, but not as a secondary non-specific consequence of maternal toxicity effects

Dose response relationship:

yes

Relevant for humans:

not specified

Applicant's summary and conclusion

Conclusions:

Treatment at the high dose of 175 mg/kg/day caused 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.

Executive summary:

The purpose of this study was to assessthe influence of Ethanol, 2,2’-iminobis-, N-C12-18-alkyl derives(a substance used in industry) on embryo-fetal survival and development when administered during the organogenesis and fetal growth phase of pregnancy in the New Zealand White Rabbit.

Three groups of 22 females receivedEthanol, 2,2’-iminobis-, N-C12-18-alkyl derives at doses of 20, 60 or 175 mg/kg/day by oral (gavage) administration, from Day 6 to 28 after mating. A similarly constituted Control group received the vehicle, 0.5% methylcellulose at the same volume dose (4ml/kg/day) as treated groups. Animals were killed on Day 29 after mating for reproductive assessment and fetal examination.

Clinical observations, body weight and food consumption were recorded. Adult females were examined macroscopically at necropsy on Day 29 after mating and the gravid uterus weight recorded. All fetuses were examined macroscopically at necropsy and subsequently by detailed internal visceral examination of the head or skeletal examination.

Results

The mean concentrations of Ethanol, 2,2’-Iminobis-, N-C12-18-alkyl derives in test formulations analyzed for the study were within ±10% of nominal concentrations, confirming accurate formulation. The difference from mean remained within 4%, confirming precise analysis.

Oral administration at 20, 60 mg/kg/day to New Zealand White rabbits was well tolerated, and did not elicit any marked clinical signs of maternal toxicity. There were no deaths or post dose signs observed and no treatment related macroscopic findings at these levels.

Two animals receiving 175 mg/kg/day were killed for welfare reasons in late gestation. One female was killed on Day 21 of gestation following a prolonged period of low/negligible food consumption and clinical signs related to low food intake, macroscopic examination revealed the cecum to have abnormal fluid contents. The second female was killed for welfare reasons on Day 26 of gestation due to marked breathing impairment which consisted of wet rales, irregular breathing, decreased activity, and excessive salivation. There were no macroscopic abnormalities. Both females were pregnant, and all fetuses appeared grossly normal.

At 175 mg/kg/day, maternal toxicity was manifest asstatistically significant bodyweight loss which persisted until Day 11 of gestation and overall reduced body weight gain,and statistically significantly lower group mean food consumption during Days 6-20 of gestation. 

There was considered to be no adverse effect of maternal treatment upon numbers of implantations, early, late and total resorptions, number of live young and sex ratio and the extent of pre and post-implantation loss. Placental, litter and fetal weights were slightly low at 175 mg/kg/day and macroscopic assessment on Day 29 of gestation did not reveal any adverse findings that were considered related to treatment withEthanol, 2,2’-iminobis-, N-C12-18 alkyl derives.

At 175 mg/kg/day there was an increased incidence of fetuses/litters with incompletely ossified cervical vertebrae and metacarpals/phalanges forepaws when compared to concurrent control and this incidence was outside of Historical Control Data range. Incomplete ossification is a transient stage in fetal development, not considered adverse and is likely associated with the slight decrease in mean fetal weight seen at this dose level likely due to the reduced maternal food consumption(Cappon et al 2005).

Conclusion

Treatment at the high dose of 175 mg/kg/day caused 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.