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Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
27th March 2018 -12th June 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report date:
2018

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Chemical structure
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
Specific details on test material used for the study:
Test item: Ethanol, 2,2’-iminobis-, N-C12-18-alkyl derives
Test item identity (including alternative names):
Ethomeen C/12
2,2’-(C12-18 even numbered alkyl imino)diethanol.
Bis(2-hydroxyethyl)cocoalkylamine.
CAS number: 71786-60-2 and 61791-31-9
Intended use: Substance used in industry.
Appearance: Light yellow liquid.
Storage conditions: At ambient temperature (15 to 25C), in the dark.
Supplier: Sponsor
Batch number: 1373142
Stability/expiry date: 28 November 2018
Purity: 98%

Test animals

Species:
rat
Strain:
Wistar
Details on test animals or test system and environmental conditions:
Strain/Species RccHan™:WIST rat.
Supplier Envigo (RMS) UK Limited
Number of animals ordered 90 females.
Spare animals were removed from the study room after treatment commenced.
Duration of acclimatization Five days before commencement of pairing.
Age of the animals at the start of the study (Day 0 of gestation) 77 to 86 days old.
Weight range of the animals at the start of the study (Day 0 of gestation) 176-221 g

Environmental Control
Rodent 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 20-24ºC 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 during the acclimatization and gestation periods. Grid bottomed cages were used during pairing. Cages were suspended above absorbent paper which was changed daily during pairing.
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 (sterilized by autoclaving), which was changed at appropriate intervals each week.
Number of animals per cage Acclimatization up to four animals, During pairing one (stock) male and one female. Gestation one female

Environmental Enrichment
Aspen chew block A soft white untreated wood block; provided to each cage throughout the study (except during pairing) and replaced when necessary.
Plastic shelter Provided to each cage throughout the study (except during pairing) and replaced at the same time as the cages.

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

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

Availability: Non-restricted.

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 chew blocks.

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

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
arachis oil
Details on exposure:
1 Control 0 mg/kg/day 0 mg/ml dosed at 4ml/kg
2 Ethanol, 2,2’-iminobis-, N-C12-18-alkyl derives 10 mg/kg/day, 2.5 mg/ml, dose at 4 ml/kg
3 Ethanol, 2,2’-iminobis-, N-C12-18-alkyl derives 30 mg/kg/day, 7.5 mg/ml, dosed at 4 ml/kg
4 Ethanol, 2,2’-iminobis-, N-C12-18-alkyl derives 125 mg/kg/day, 31.25 mg/ml, dosed at 4 ml/kg
# Expressed in terms of material as supplied.

Correction factor: None.
Vehicle: Arachis oil.
Method of preparation:
The test item was prepared for administration as a series of graded concentrations in the vehicle. Starting with the low concentration (2.5 mg/mL), the formulation was prepared by weighing out the
required amount of test item and adding approximately 50% of the final volume of vehicle. It was then magnetically stirred. The solution was made up to the required volume with the vehicle and stirred
using a magnetic stirrer until homogenous.

The procedure was repeated for the mid and high dose (7.5 and 31.25 mg/mL).
Frequency of preparation Weekly, and prepared in advance of the first day of dosing.
Storage of formulation: Refrigerated (2 to 8 °C) for up to 20 days.
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.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Formulation Analysis
Stability and homogeneity The homogeneity and stability of formulations during storage was confirmed and supplied by the Sponsor (Harlan Project Nos. 0142-0416 and 0142-0417).

The mean concentrations were within 5% of the nominal concentration, confirming the accuracy of formulation. The difference between the samples remained within 3%, confirming precise analysis.

Achieved concentration: Samples of each formulation prepared for administration Days 6 and 19 after mating were analyzed for achieved concentration of the test item.

Achieved procedural recovery results for both Day 6 and Day 19 were significantly greater than the validated range for this method. A different system was used for these occasions and is consid
ered to be the reason for this, as all results are corrected for the mean procedural recovery value at analysis there is no impact on the results.
Details on mating procedure:
Mating
Male/female ratio 1:1 with identified stock males.

Daily checks for evidence of mating.

Ejected copulation plugs in cage tray and vaginal smears were checked for the presence of sperm. Day 0 of gestation When positive evidence of mating was detected.

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:
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 19 (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. No significant discrepancy was found.

Formulations were stirred using a magnetic stirrer before and throughout the dosing procedure.
Frequency of treatment:
Females were treated from Day 6 to Day 19 (inclusive) after mating, once daily at approximately the same time each day.
Duration of test:
Termination is on Day 20 after mating.
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
Control - Vehicle arachis oil
Dose / conc.:
10 mg/kg bw/day (actual dose received)
Dose / conc.:
30 mg/kg bw/day (actual dose received)
Dose / conc.:
125 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
20 femlaes per dose
1 Control No 1-20
2 10mg/kg/day No 21-40
3 30 mg/kg/day No 41-60
4 125 mg/kg/day No 61-80
Control animals:
yes, concurrent vehicle
Details on study design:
The study consisted of one control and three treated groups.

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

Route of Administration
The oral (gavage) route of administration was chosen to simulate the conditions of potential human exposure during manufacture, handling or use of the test item.

Rationale for Dose Level Selection
The doses used in this study (0, 10, 30 and 125 mg/kg/day) were selected in conjunction with the Sponsor.

The dose levels were chosen based on the results of a combined repeat dose toxicity study with a reproduction/developmental toxicity screening test repeated OECD 422 study (Harlan Project No.0142 0417). In that study there were no adverse clinical signs and no effects on body weight or food consumption, however slightly increased liver and spleen weights were observed at macroscopic examination. At microscopic examination, acanthosis was seen in the forestomach of males and females treated at 125 mg/kg/day. Since the forestomach present in the rodent is not present in the human stomach and the functionality of the stomach was not compromised, these findings were not considered to be indicative of a risk to human health. Low litter size (due to low corpora lutea and low implantation counts) was evident at 125 mg/kg/day.
Therefore, 125 mg/kg/day was selected as the high dose level in this study. The intermediate and low dose levels of 30 mg/kg/day and 10 mg/kg/day, respectively, were selected to allow evaluation of any dose related trends.

Allocation and Identification

Allocation: On the day of positive evidence of mating (Day 0). Only females showing at least two copulation plugs were allocated.

Method: To group and cage position in the sequence of mating. Females mating on any one day were evenly distributed amongst the groups.

Allocation was controlled to prevent any stock male from providing more than one mated female in each 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(s).

Examinations

Maternal examinations:
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 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 recorded daily at the following times in relation to dose administration:
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, 5, 12, 18 and 20 after mating to monitor general health.

Body Weight
The weight of each adult was recorded on Days 0, 3 and 6-20 after mating.

Food Consumption
The weight of food supplied to each adult, that remaining and an estimate of any spilled was recorded for the periods Days 0-2, 3-5, 6-9, 10-13, 14-17 and 18-19 after mating inclusive.

Terminal Investigations
Method of Kill
Method of kill for all adult animals was Carbon dioxide asphyxiation.

Method of kill for fetuses Chilling on a cool plate (approximately 0C).

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 were killed on Day 20 after mating. Sequence To allow satisfactory inter-group comparison.
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.
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. The sex of each fetus was recorded.

Examination of nominally 50% of fetuses in each litter Sexed internally and eviscerated.

Fixation Fetuses eviscerated were fixed in Industrial Methylated Spirit (IMS).

Remaining fetuses were fixed whole in Bouin’s fluid. Processing Bouin’s fixed fetuses were subject to free-hand serial sectioning.

IMS fixed fetuses were processed and stained with Alizarin Red.

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

Alizarin Red stained fetuses Assessed for skeletal development and abnormalities.
Statistics:
Statistical Analysis
The following data types were analyzed at each timepoint separately:
Body weight, using absolute values 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)
Fetal, placental and litter weight
 
No statistical analysis were performed on the results of the fetal examinations only a comparison of incidence with the concurrent and historical controls.
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)/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:
See attached Annex Fetal examination - Major Historical control data

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
One female receiving 125 mg/kg/day was observed with rales on Day 20 of gestation. There were no clinical signs that were considered to be associated to treatment with Ethanol,
2,2’-Iminobis-, N-C12-18-Alkyl Derives.

There were no dosing signs recorded, hence no data has been presented.
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Body weight performance of females treated with Ethanol, 2,2’-Iminobis-, N-C12-18-Alkyl Derives was generally similar to the Controls throughout gestation. See Table 1 and 2 attached.

Body weight gain was slightly lower for females receiving 125 mg/kg/day when compared with Controls.

The mean gravid uterine weight for females treated with Ethanol, 2,2’-Iminobis-, N-C12-18-Alkyl Derives was comparable to the mean gravid uterine weight from the Control females.

Adjusted body weight change (0-20) of females treated with 125 mg/kg/day of Ethanol, 2,2’-Iminobis-, N-C12-18-Alkyl Derives was statistically significantly low when compared to the Controls
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Food consumption for females treated at 125 mg/kg/day of Ethanol, 2,2’-Iminobis-, N-C12-18-Alkyl Derives was slightly but statistically significant lower during Days 6-14 of gestation, resulting in a statistically significantly low overall food intake during the treatment period (Days 6-18), when compared with the Control group. See Table 3 attached.

Group mean food intake during Days 10-14 of gestation was slightly but statistically significantly lower for females receiving 30 mg/kg/day when compared with Controls.
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
Description (incidence and severity):
There were no macroscopic findings in dams on Day 20 of gestation that were attributable to treatment
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed

Maternal developmental toxicity

Number of abortions:
no effects observed
Pre- and post-implantation loss:
effects observed, non-treatment-related
Description (incidence and severity):
Post implantation loss (%) was statistically significantly high and total resorptions were higher in females treated at 125 mg/kg/day when compared to the Control group. However, as the number of live fetuses was comparable to the Controls, no definite effect of treatment is inferred.
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Description (incidence and severity):
On Day 20 of gestation, mean numbers of corpora lutea, implantations, the number of live young and sex ratio were comparable to the Controls, and were considered to be unaffected by treatment with Ethanol, 2,2’-Iminobis-, N-C12-18-Alkyl Derives. See Table 4 attached
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):
One female (No. 38) treated at 10 mg/kg/day with Ethanol, 2,2’-Iminobis-, N-C12-18-Alkyl Derives was found not to be pregnant at macroscopic examination, all other females were pregnant.
Other effects:
no effects observed

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Effect level:
125 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:
no effects observed
Description (incidence and severity):
Group mean placental weights, litter weights, and male or female fetal weights were all unaffected by treatment with Ethanol, 2,2’-Iminobis-, N-C12-18-Alkyl Derives. See Table 5 attached.
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Description (incidence and severity):
On Day 20 of gestation, the sex ratio was comparable to the Controls, and were considered to be unaffected by treatment with Ethanol, 2,2’-Iminobis-, N-C12-18-Alkyl Derives.
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
Group mean placental weights, litter weights, and male or female fetal weights were all unaffected by treatment with Ethanol, 2,2’-Iminobis-, N-C12-18-Alkyl Derives.
Changes in postnatal survival:
not examined
External malformations:
effects observed, treatment-related
Description (incidence and severity):
At 125 mg/kg/day there were 8 litters with similar major abnormalities, the majority affecting the head, eye and vertebral column. These abnormalities include, but are not limited to, Exencephaly/Me
ningoencephalocele/Acephalostomia and Cleft lip; Anophthalmia and Microphthalmia; Spina Bifida/Holorachischisis. Major abnormalities of this combination and severity are rare in rats and the majority are outside of Historical Control Data range.

At 30 mg/kg/day there was an incidence of Microphthalmia (not associated with severe cranial abnormalities). An incidence of 1 fetus in 1 litter is extremely low and is seen at this incidence within the control population as documented within the Historical Control Data range. However, as there were also 2 fetuses in 2 litters with Microphthalmia at 125 mg/kg/day as well as Anophthalmia, a
relationship to treatment cannot be ruled out.

See Tables 6, 7 and 8. and APPENDIX Fetal Examination for individual data.
Skeletal malformations:
effects observed, treatment-related
Description (incidence and severity):
At 125 mg/kg/day there were 8 litters with similar major abnormalities, the majority affecting the head, eye and vertebral column. These abnormalities include, but are not limited to, Exencephaly/Meningoencephalocele/Acephalostomia and Cleft lip; Anophthalmia and Microphthalmia; Spina Bifida/Holorachischisis. Major abnormalities of this combination and severity are rare in rats and the majority are outside of Historical Control Data range.

Also, at 125 mg/kg/day there was an increased incidence of medially thickened/kinked ribs, short supernumerary cervical ribs, delayed ossification of 5th/6th sternebrae andthoracic/sacrocaudal vertebral elements and partially undescended lobe(s) of thymus compared to concurrent control.

See Tables 6, 7 and 8. and APPENDIX Fetal Examination for individual data.

The incidences of delayed ossification were within Historical Control Data range. This is an indication of fetal immaturity and as a transient stage in fetal development, not thought to be adverse.
Visceral malformations:
effects observed, treatment-related
Description (incidence and severity):
At 125 mg/kg/day there were 8 litters with similar major abnormalities, the majority affecting the head, eye and vertebral column. These abnormalities include, but are not limited to, Exencephaly/Me
ningoencephalocele/Acephalostomia and Cleft lip; Anophthalmia and Microphthalmia; Spina Bifida/Holorachischisis. Major abnormalities of this combination and severity are rare in rats and the majority are outside of Historical Control Data range.
Other effects:
not examined
Details on embryotoxic / teratogenic effects:
At 125 mg/kg/day there were 8 litters with similar major abnormalities, the majority affecting the head, eye and vertebral column. These abnormalities include, but are not limited to, Exencephaly/Meningoencephalocele/Acephalostomia and Cleft lip; Anophthalmia and Microphthalmia; Spina Bifida/Holorachischisis. Major abnormalities of this combination and severity are rare in rats and the majority are outside of Historical Control Data range.

Effect levels (fetuses)

open allclose all
Dose descriptor:
LOAEL
Effect level:
30 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
visceral malformations
Remarks on result:
other: microphthalmia
Key result
Dose descriptor:
NOAEL
Effect level:
10 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
external malformations
skeletal malformations

Fetal abnormalities

Key result
Abnormalities:
effects observed, treatment-related
Localisation:
external: eye
external: face
skeletal: skull
skeletal: vertebra
visceral/soft tissue: eye
Description (incidence and severity):
At 125 mg/kg/day there were 8 litters with similar major abnormalities, the majority affecting the head, eye and vertebral column. These abnormalities include, but are not limited to, Exencephaly/Meningoencephalocele/Acephalostomia and Cleft lip; Anophthalmia and Microphthalmia; Spina Bifida/Holorachischisis. Major abnormalities of this combination and severity are rare in rats and the
majority are outside of Historical Control Data range.
Also, at 125 mg/kg/day there was an increased incidence of medially thickened/kinked ribs, short
supernumerary cervical ribs, delayed ossification of 5th/6th sternebrae and thoracic/sacrocaudal vertebral elements and partially undescended lobe(s) of thymus compared to concurrent control.
The incidences of delayed ossification were within Historical Control Data range. This is an indication of fetal immaturity and as a transient stage in fetal development, not thought to be adverse.
At 30 mg/kg/day there was an incidence of Microphthalmia (not associated with severe cranial abnormalities). An incidence of 1 fetus in 1 litter is extremely low and is seen at this incidence within the control population as documented within the Historical Control Data range. However, as there were also 2 fetuses in 2 litters with Microphthalmia at 125 mg/kg/day as well as Anophthalmia, a relationship to treatment cannot be ruled out.

Overall developmental toxicity

Key result
Developmental effects observed:
yes
Lowest effective dose / conc.:
30 mg/kg bw/day (actual dose received)
Treatment related:
yes
Relation to maternal toxicity:
developmental effects in the absence of maternal toxicity effects
Dose response relationship:
yes
Relevant for humans:
not specified

Applicant's summary and conclusion

Conclusions:
The results from the OECD414 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.
Executive summary:

The purpose of this study was to assess the influence of Ethanol, 2,2’-iminobis-, N-C12-18-alkyl derives, substance used in industry, on embryo-fetal survival and development when administered during the organogenesis and fetal growth phases of pregnancy in the Han Wistar rat.

 

Three groups of 20 females received Ethanol, 2,2’-iminobis-, N-C12-18-alkyl derives at doses of 10, 30 or 125 mg/kg/day by oral gavage administration, from Day 6 to 19 after mating. A similarly constituted Control group received the vehicle, arachis oil, at the same volume dose as the treated groups. Animals were killed on Day 20 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 20 after mating and the gravid uterus weight recorded. All fetuses were examined macroscopically at necropsy and subsequently by detailed internal visceral examination or skeletal examination.

 

Results

The mean concentrations were within 5% of the nominal concentration, confirming the accuracy of formulation. The difference between the samples remained within 3%, confirming precise analysis.

 

Dose levels of 0, 10, 30 and 125 mg/kg/day were well tolerated with no signs observed following dose administration and no adverse clinical signs. At 125 mg/kg/day, minor maternal toxicity was manifest as a slight reduction in mean body weight gain during Days 6-19 and 19-20 of gestation and a statistically significant reduction in adjusted body weight gain (69% of Controls for Day 6-20), and statistically significantly lower group mean food consumption during Days 6-14 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 also all considered to be unaffected by treatment.

 

Fetal development was severely compromised at 125 mg/kg/day, there were 8 litters with similar major abnormalities, the majority affecting the head, eye and vertebral column. At 30 mg/kg/day there was an incidence of Microphthalmia (not associated with severe cranial abnormalities). An incidence of 1 fetus in 1 litter is extremely low and is seen at this incidence within the control population as documented within the Historical Control Data range, which was also observed for 2 fetuses in 2 litters at 125 mg/kg/day, therefore a relationship to treatment at 30 mg/kg/day is uncertain but cannot be ruled out. There were no major abnormalities considered to be related to treatment at 10 mg/kg/day.

 

Conclusion

It was concluded from this study that the dosage of 125 mg/kg/day was the maternal no-observed-adverse-effect-level (NOAEL). Due to the extent and nature of the major abnormalities seen at 125mg/kg/day and the uncertainty of the fetal pathology findings at 30 mg/kg/day, the no-observed-adverse-effect-level (NOAEL) for embryo-fetal survival and development is 10 mg/kg/day.