Ethanol, 2,2'-oxybis-, reaction products with ammonia, morpholine derivs. residues

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2018-07-27 - 2019-11-15

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to other study

Reference

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

2018-02-09 to 2018-04-06

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

no guideline required

Principles of method if other than guideline:

The study was not designed to meet any particular regulatory requirements.

GLP compliance:

no

Remarks:

No claim for compliance with Good Laboratory Practice was made, although the work performed generally followed Good Laboratory Practice principles. No formal study-based Quality Assurance Procedures were performed on this study.

Limit test:

no

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Lot/batch number of test material: 17/19961
- Expiry date 2019-11-22
- Appearance: Brown liquid
- Purity.: Not applicable (UVCB material)


STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At ambient temperature (15 to 25°C); protected from light and moisture.
- Stability and homogeneity of the test material in the vehicle under test conditions (e.g. in the exposure medium) and during storage: The stability of formulations was demonstrated over a period of up to 21 days at 2 to 8°C and 1 day at ambient temperature (15to 25°C) as part of another study, Covance Study Number, XM94DH.
- Solubility and stability of the test material in the vehicle and the exposure medium: not indicated


OTHER SPECIFICS
- Shelf life is 24 months from production release date (22 November 2017).

Species:

rabbit

Strain:

New Zealand White

Details on species / strain selection:

The female New Zealand White (sexually mature, virgin) 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.

Sex:

female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: 14 female New Zealand White rabbits from Envigo RMS.
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation:
*Group 1: approximately 18 to 21 weeks.
*Group 2: approximately 20 to 22 weeks.
*Group 3: approximately 22 to 26 weeks.
- Weight at study initiation:
*Group 1: 2.93 to 3.67 g
*Group 2: 2.55 to 3.09 g
*Group 3: 2.79 to 3.71 g
- Fasting period before study: Restricted [initially 150 g/animal/day during acclimatization up to 200 g/animal/day for all animals five days prior to the onset of treatment (Group 1)].
- Housing: Suspended plastic cages fitted with perforated floor panels and mounted in batteries. Undertrays lined with absorbent paper were changed at least three times a week. Aspen chew block provided to each cage throughout the study and replaced when necessary. Stainless steel key ring attached to the cage.
- Diet: ad libitum, Teklad 2930. 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.
- Water: ad libitum, potable water from the public supply via polycarbonate bottles with sipper tubes. Bottles were changed at appropriate intervals. Water bowls were also provided, where required.
- Acclimation period:
*Group 1: six days before commencement of treatment.
*Group 2: 14 days before commencement of treatment.
*Group 3: 42 days before commencement of treatment.

DETAILS OF FOOD AND WATER QUALITY: The diet contained no added antibiotic or other chemotherapeutic or prophylactic agent. Certificates of analysis were received from the suppliers of the Aspen chew blocks and diet; these were scrutinized and approved before any batch was released for use. Certificates of analysis were also routinely provided by the water supplier. No specific contaminants were known that may have interfered with or prejudiced the outcome of the study and therefore no special assays were performed.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 15-21°C (There were no deviations from these ranges.)
- Humidity (%): 45-70% (There were no deviations from these ranges.)
- Air changes (per hr): Filtered fresh air which was passed to atmosphere and not recirculated. Air changes/hr not indicated.
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 2018-02-09 To: Group 1: 2018-02-23
Group 2: 2018-03-09
Group 3: 2018-04-06

Route of administration:

oral: gavage

Details on route of administration:

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

Vehicle:

water

Remarks:

purified

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
- Individual weighings were diluted to 50% of the final volume and magnetically stirred until uniformly mixed, then made up to final volume with vehicle and magnetically stirred until visually homogenous.
- Formulations were stirred using a magnetic stirrer before and throughout the dosing procedure.
- 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.
- Frequency of preparation: Weekly.
- Storage of formulation: Refrigerated (2 to 8°C).

VEHICLE
- Concentration in vehicle: 200, 150, 100 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg

Analytical verification of doses or concentrations:

no

Remarks:

No formulation analysis was performed on this study.

Duration of treatment / exposure:

Eight days for Group 1 and 14 days for Groups 2 and 3.

Frequency of treatment:

Once daily, at approximately the same time each day.

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

Group 1

Dose / conc.:

750 mg/kg bw/day (nominal)

Remarks:

Group 2

Dose / conc.:

500 mg/kg bw/day (nominal)

Remarks:

Group 3

No. of animals per sex per dose:

3 females/dose. Spare animals were removed from the study room after treatment commenced.

Control animals:

no

Details on study design:

- Dose selection rationale: In a fourteen day repeat dose study in the Han Wistar rat at dose levels of 250, 500 and 1000 mg/kg/day and a 28 day repeat dose study (Calvert study no: 0436RH11.001) in the Han Wistar rat at dose levels of 100, 500 and 1000 mg/kg/day were well tolerated with no adverse effects on clinical condition, body weight and food consumption and there were no macroscopic abnormalities detected at necropsy. An initial dose of 1000 mg/kg/day was therefore selected in conjunction with the Sponsor for Group 1 animals on this rabbit pilot study.
- Fasting period before blood sampling for clinical biochemistry: not applicable.
- Rationale for selecting satellite groups: Allocation On day of dosing. Random, avoiding allocation of more than one sibling per group.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: weekly during acclimatization and then daily from three days prior to the start of treatment (Day -3), on the day that treatment commenced and daily throughout the treatment period. A viability check was performed near the start and end of each working day.

DETAILED CLINICAL OBSERVATIONS: Not specified

BODY WEIGHT: Yes
- Time schedule for examinations: The weight of each animal was recorded weekly during acclimatization and then daily for three days prior to the start of treatment, on the day that treatment commenced (Day 1), daily throughout the study and before necropsy.

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- The weight of food supplied to each animal, that remaining and an estimate of any spilled was recorded daily throughout the study from three days before treatment started until scheduled termination.

FOOD EFFICIENCY: No

WATER CONSUMPTION AND COMPOUND INTAKE: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: No

CLINICAL CHEMISTRY: No

PLASMA/SERUM HORMONES/LIPIDS: No

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
- Method of Euthanasia: Intravenous injection of sodium pentobarbitone
- All 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.

HISTOPATHOLOGY: Yes
- 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.

Statistics:

No statistical analysis was performed on the study.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

At 1000mg/kg bw/d on Days 7/8 signs were observed in association with dose administration with all three animals observed as underactive and one female had piloerection. Terminal signs included inappetance, reduced water consumption, reduced faecal output and body weight loss. At 500 or 750 mg/kg/day no signs were observed in association with dose administration and
signs at routine physical examination were limited to one female at 750 mg/kg/day which had
low hay consumption on Day 14.

Mortality:

mortality observed, treatment-related

Description (incidence):

Animals receiving 1000 mg/kg/day were euthanised for reasons of animal welfare after
8 days of treatment. On Days 7/8 signs were observed in association with dose
administration with all three animals observed as underactive and one female had
piloerection. Terminal signs included inappetance, reduced water consumption, reduced
faecal output and body weight loss.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

All three females receiving 750 mg/kg/day showed marginal body weight loss over the
treatment period (between 70g and 110g), whilst at 500 mg/kg/day only one female showed
an overall loss in body weight (-100g) with the other two showing an overall gain
(+100/110g).

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Following the onset of treatment females receiving 750 mg/kg/day showed a persistent and
moderate reduction in food consumption when compared with pre-treatment values. At
500 mg/kg/day females also showed a reduction in food consumption following the onset of
treatment however it was not as low as that observed at 750 mg/kg/day and when compared
with pre-treatment values it only persisted in one of the three females.

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

Endocrine 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:

effects observed, treatment-related

Description (incidence and severity):

Macroscopic examination revealed dark abnormal content in the gall bladder for all three animals dosed with 1000 mg/kg bw, depressions in the corpus mucosa of the stomach for two animals and an accentuated liver lobular pattern in one animal. After 14 days of treatment no macroscopic abnormalities were apparent for females that received 500 or 750 mg/kg/day.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

not specified

Conclusions:

It was concluded based on the findings in this study, that a high dose of 750 mg/kg/day would be suitable for use on the preliminary embryo-fetal development study.

Executive summary:

The purpose of this study was to establish doses for a preliminary embryo-fetal toxicity study in the rabbit. Initially three non-pregnant rabbits received the test item at a dose of 1000 mg/kg/day, due to adverse results the animals were sacrificed for welfare reasons on Day 9 of treatment. Based on the results obtained, a further three non-pregnant rabbits/ group were assigned to the study and received a doses of 750 or 500 mg/kg/day for 14 days. 

Oral administration of test item to New Zealand white rabbits was not tolerated at 1000 mg/kg/day and animals were euthanised for reasons of animal welfare on Day 9. Terminal signs included body weight loss, reduced food/water consumption and reduced faecal output. Macroscopic examination revealed abnormal content of the gall bladder, stomach abnormality and liver abnormality.
Dose levels of 500 and 750 mg/kg/day were tolerated for the14 day treatment period with no adverse effect on clinical condition or body weight and animals were macroscopically normal at necropsy. There was evidence of a moderate but dose related reduction in food consumption at 500 and 750 mg/kg/day with the effect at 750 mg/kg/day persisting throughout the treatment period in all three of the animals compared to only one animal at 500 mg/kg/day.
It was therefore concluded based on the findings in this study that a high dose of 750 mg/kg/day would be suitable for use on the preliminary embryo-fetal development study.

Cross-reference 2

Reason / purpose for cross-reference:

reference to other study

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

02/05/2018- 12/08/2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

not applicable

Remarks:

dose range finding study

GLP compliance:

no

Remarks:

No claim for compliance with Good Laboratory Practice was made, although the work performed generally followed Good Laboratory Practice principles.

Limit test:

no

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Envigo RMS
- Age at study initiation: 18 to 22 weeks old
- Weight at study initiation: 2.50 to 3.67 kg
- Fasting period before study: no
- Housing: 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.
- Diet (e.g. ad libitum): Restricted (initially 150 g/animal/day during acclimatization
up to one week prior to the onset of mating and
200 g/animal/day thereafter).
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 14 days before commencement of mating

ENVIRONMENTAL CONDITIONS
- Temperature (°C): Monitored and maintained within the range of 15-21°C
- Humidity (%): Monitored and maintained within the range of 45-70%.
- Air changes (per hr): /
- Photoperiod (hrs dark / hrs light): 14 hours light : 10 hours dark

IN-LIFE DATES: From: To: 02/05/2019- 15/06/2018

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:

VEHICLE
- Justification for use and choice of vehicle (if other than water): water
- Concentration in vehicle: 0-50-100-150 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg body weight.

Analytical verification of doses or concentrations:

no

Remarks:

The stability of formulations was demonstrated over a period of up to 21 days at 2 to 8°C and one day at ambient temperature (15 to 25°C) as part of another study, Covance Study Number, XM94DH.

Details on mating procedure:

- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1:1
- Length of cohabitation: until natural mating observed
- Verification of same strain and source of both sexes: yes
- Proof of pregnancy: visual mating observation referred to as day 0 of pregnancy
- Any other deviations from standard protocol: after mating each female was injected intravenously with 25 i.u. luteinizing hormone

Duration of treatment / exposure:

Day 6 to Day 28 (inclusive) after mating

Frequency of treatment:

daily

Duration of test:

females were sacrificed on GD 29

Dose / conc.:

0 mg/kg bw/day (nominal)

Dose / conc.:

250 mg/kg bw/day (nominal)

Dose / conc.:

500 mg/kg bw/day (nominal)

Dose / conc.:

750 mg/kg bw/day (nominal)

No. of animals per sex per dose:

6

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: doses were selected based on the results of the pilot study in non pregnant females Covance study number HF46WY
- Rationale for animal assignment (if not random): females were allocated 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 treated group and to prevent more than one sibling female in each group, where possible.

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Animals were inspected visually at least twice daily for evidence of ill-health or reaction to treatment.
Detailed observations were recorded daily 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

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: A detailed physical examination was performed on each animal on Days 0, 6, 12, 18, 24 and 29 after mating to monitor general health.

BODY WEIGHT: Yes
- Time schedule for examinations:Days 0, 3 and 6-29 after mating.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
-The weight of food supplied to each animal, that remaining and an estimate of any spilled
was recorded daily from Day 1 after mating.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 29
- Organs examined: 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.

OTHER:

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other:

Fetal examinations:

- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: all per litter
- Skeletal examinations: No
- Head examinations: No

Statistics:

Where appropriate, group mean values, each with standard deviation (SD), were calculated
from individual data.

Indices:

Pre-implantation loss (%) = (Number of corpora lutea - Number of implantations) x 100
/Number of corpora lutea

Post-implantation loss (%)= (Number of implantations - Number of live fetuses) /Number of implantations x 100

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Signs were observed for the two high dose animals that were euthanized for welfare reasons
and included low diet/hay consumption, reduced faecal/urinary output and thin build.
There were no signs at routine physical examination for females receiving 250 or
500 mg/kg/day that could be related to treatment and there were no signs observed in
association with dose administration at dose levels up to and including 750 mg/kg/day.

Mortality:

mortality observed, treatment-related

Description (incidence):

Two females Nos. 21 and 23 receiving 750 mg/kg/day were euthanized for reasons of animal welfare on GD 25 and Day 20 after mating, respectively. Following the welfare euthanasia of these two animals and
review of the data at 750 mg/kg/day, it was concluded that a high dose of 750 mg/kg/day
would be too high for use on a main study. This dose group was therefore terminated prematurely.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Body weight gain for females at 750 mg/kg/day from GD6 to GD23 was approximately
13% of Controls; this group was terminated prematurely for reasons of animal welfare.
Body weight gain for females receiving 500 mg/kg/day during the treatment period (GD6 to
GD29) was slightly low at approximately 85% of Controls; this difference did not attain
statistical significance.
Body weight gain for females receiving 250 mg/kg/day during the treatment period (GD6 to
GD29) was unaffected by treatment.
The maternal body weight loss following adjustment for the gravid uterine weight was higher
for females that received 500 mg/kg/day when compared with Controls, minus 220g vs 160g.
The maternal body weight change at 250 mg/kg/day was considered to be unaffected by treatment.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

Animals receiving 750 mg/kg/day showed low food consumption following the onset of
treatment on GD6, with the difference attaining statistical significance on GDs 6, 7, 8, and
10. The total food consumption for females receiving 750 mg/kg/day up to premature
termination was low when compared with Controls (approximately 76 % of Controls).
At 500 mg/kg/day food consumption was also slightly low following the onset of treatment;
although the differences did not attain statistical significance. Overall the total food
consumption at this dose level was slightly low, approximately 86% of Controls during GD6
to GD28.
Food consumption at 250 mg/kg/day during the treatment period was considered to be
unaffected by treatment.

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

Mean placental weight at 500 mg/kg/day was marginally low at approximately 90% of
Controls; this difference was not apparent at 250 mg/kg/day.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

For the two animals which were terminated early for welfare reasons macroscopic examination at necropsy revealed congested stomach fundus and lungs with congested/dark areas for animal No. 21,
whilst no abnormalities were observed for No. 23.

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Changes in number of pregnant:

no effects observed

Details on maternal toxic effects:

Following the welfare euthanasia of two animals at 750 mg/kg/day, it was concluded that a high dose of 750 mg/kg/day would be too high for use on a main study. This dose group was therefore terminated prematurely on GD25. The maternal developmental toxicity indices were not calculated for this group.

Fetal body weight changes:

no effects observed

Description (incidence and severity):

Litter and fetal weight was unaffected by maternal treatment at 250 or 500 mg/kg/day.

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

no effects observed

Details on embryotoxic / teratogenic effects:

Following the welfare euthanasia of two animals at 750 mg/kg/day, it was concluded that a high dose of 750 mg/kg/day would be too high for use on a main study. This dose group was therefore terminated prematurely on GD25. The fetal developmental toxicity indices were not calculated for this group.

Conclusions:

The high dose 750mg/kg bw/d was not tolerated and 2 females were terminated for welfare reasons. Based on the results of this study, doses of 125, 250 and 500 mg/kg/day were considered suitable for use on the main embryo-fetal development study.

Executive summary:

Oral administration of test item to New Zealand White rabbits from Day 6 to Day 28 of gestation at dose levels of 250, 500 or 750 mg/kg/day was not tolerated at the high dose. Two out of the six females at 750 mg/kg/day showed inappetance, body weight loss and poor clinical condition and the group was terminated prematurely for reasons of animal welfare. At 500 mg/kg/day there was evidence of maternal toxicity without deterioration in general condition; low body weight gain (high maternal weight loss), low food consumption and
slightly low placental weight. There was no evidence of any effect of treatment at 250 mg/kg/day.
Based on the results of this study, doses of 125, 250 and 500 mg/kg/day were considered suitable for use on the main embryo-fetal development 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:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

Deviations:

no

Test guideline 2

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

Test material information

Constituent 1

Reference substance name:

Ethanol, 2,2'-oxybis-, reaction products with ammonia, morpholine derivs. residues

EC Number:

272-712-1

EC Name:

Ethanol, 2,2'-oxybis-, reaction products with ammonia, morpholine derivs. residues

Cas Number:

68909-77-3

Molecular formula:

Not applicable (UVCB)

IUPAC Name:

2-(2-hydroxyethoxy)ethan-1-ol; 2-[1-(morpholin-4-yl)ethoxy]ethan-1-amine; 2-{2-[bis(2-hydroxyethyl)amino]ethoxy}ethan-1-ol; 4-{2-[2-(morpholin-4-yl)ethoxy]ethyl}morpholine; morpholin-3-one

Test material form:

liquid

Details on test material:

Name: Ethanol, 2,2'-oxybis-, reaction products with ammonia, morpholine derivs. residues
Physical state: liquid
Appearance: brown liquid

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch number of test material: Huntsman Europe BVBA and 17/19961
- Expiration date of the lot/batch: 2019-11-22
- Purity test date: Not applicable (UVCB)

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At ambient temperature (15 to 25°C); protected from light and moisture.
- Stability under storage conditions: Not specified
- Stability under test conditions: Before commencement of treatment, the suitability of the proposed mixing procedures was determined and specimen formulations at 2and 200mg/mL were analyzed to assess the stability of the test item in the liquid matrix.
- Solubility and stability of the test substance in the solvent/dispersant/vehicle/test medium: Not specified
- Reactivity of the test substance with the solvent/vehicle /test medium (if applicable): Not specified

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Mixed to vehicle (purified water)
- Final preparation: The required amount of test item was weighed. Approximately 50% of the final volume of vehicle was added and magnetically stirred until the test material was uniformly mixed. The remaining vehicle was added to achieve the required volume and the formulation was mixed using a magnetic stirrer until homogeneous.
A series of formulations at the required concentrations were prepared in ascending order.
- Frequency of preparation: Weekly, in advance of dose administration.
- Storage of formulation: Refrigerated at 2 to 8°C

OTHER SPECIFICS
- Physical State/appearance: Brown liquid
- other information: No correction factor

Test animals

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Envigo RMS.
- Age at study initiation (day 0 of gestation): 19 to 23 weeks old
- Weight at study initiation: 2.82 to 4.61 kg
- Fasting period before study: No
- Housing: 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.
Number of animals per cage: acclimatization one female, during mating one stock male and one female, during gestation one female.
- Diet (e.g. ad libitum): Teklad 2930 Diet, the diet contained no added antibiotic or other chemotherapeutic or prophylactic agent. Availability was restricted (initially 150g/animal/day during acclimatization up to one week prior to the onset of mating and 200g/animal/day thereafter). Should an individual show a significant non-treatment related reduced food consumption, moistened diet (50 g pelleted diet moistened with 20 to 50 mL of water) was offered and 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.

- Water (e.g. ad libitum): Ad libitum. Potable water from the public supply via polycarbonate bottles with sipper tubes. Bottles were changed at appropriate intervals. Water bowls were offered to individuals during acclimatization.
- Acclimation period: 19 days before commencement of mating.


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 15 - 21°C
- Humidity (%): 45 - 70%.
- Air changes (per hr): Filtered fresh air which was passed to atmosphere and not recirculated.
- Photoperiod (hrs dark / hrs light): 10/14

IN-LIFE DATES: From: 2018-07-04 To: 2018-08-24

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
-The test item was prepared at the appropriate concentrations as a solution in Distilled Water.
- The stability of formulations was demonstrated over a period of up to 21 days at 2 to 8°C and one day at ambient temperature (15 to 25°C). Formulations were therefore prepared weekly and stored at approximately 2 to 8 ºC.
VEHICLE
- Justification for use and choice of vehicle (if other than water): not applicable (vehicle is water)
- Concentration in vehicle: 0, 25, 50,100 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg body weight
- Purity: not specified

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The homogeneity and stability was confirmed for test item in Distilled Water formulations at nominal concentrations of 2 mg/mL and 200 mg/mL when stored refrigerated for 21 days and at room temperature in the light for 24 hours.
Samples of test item formulations were taken on two occasions and analyzed for concentration and the results indicate that the prepared formulations were within 100-112% of the nominal concentration.

Details on mating procedure:

- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1:1
- Length of cohabitation: 6 days
- Further matings after two unsuccessful attempts: not specified
- Verification of same strain and source of both sexes: not specified
- Proof of pregnancy: Expelled uterine contents were identified and examined, as appropriate
- Any other deviations from standard protocol: No applicable

Duration of treatment / exposure:

Day 6 to 28 after mating

Frequency of treatment:

Once daily at approximately the same time each day.

Duration of test:

29 days

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Control

Doses / concentrations 2

Dose / conc.:

125 mg/kg bw/day (nominal)

Remarks:

Group 2

Doses / concentrations 3

Dose / conc.:

250 mg/kg bw/day (nominal)

Remarks:

Group 3

Doses / concentrations 4

Dose / conc.:

500 mg/kg bw/day (nominal)

Remarks:

Group 4

No. of animals per sex per dose:

22 females per dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: the doses were selected based on the outcome of the tolerability study in non-pregnant rabbits ( Renaut, 2019) and the dose range finding study in pregnant rabbits (Renaut, 2019). The conclusion of the pilot study in non-pregnant rabbits was that the maximum tolerated dose was 750mg/kg bw/d due to mortality at the high dose of 1000mg/kg bw/d. The dose range finding study in pregnant rabbits established that the maximum tolerated dose was 500mg/kg bw/d due to the poor clinical condition and euthanasia for animal welfare considerations of the animals treated with the high dose of 750mg/kg bw/d.
for this reason the doses selected for the main study were 500-250-125-0 mg/kg bw/d.
- Rationale for animal assignment (if not random): Where possible only females mating at least twice 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 treated group and to prevent more than one sibling female in each group, where possible.

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily
- Cage side observations checked in table were included.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Animals were inspected visually at least twice daily for evidence of ill-health or reaction to treatment. A detailed physical examination was performed on each animal on Days 0, 6, 12, 18, 24 and 29 after mating to monitor general health.

BODY WEIGHT: Yes
- Time schedule for examinations: The weight of each adult was recorded weekly during acclimatization, on Days 0, 3 and 6-29 after mating

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- The weight of food supplied to each animal, that remaining and an estimate of any spilled was recorded daily from Day 1 after mating.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

POST-MORTEM EXAMINATIONS: Yes
- 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.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other: Fetuses (live and dead).

Fetal examinations:

- External examinations: Yes [half per litter]
- Soft tissue examinations: Yes [ half per litter]
- Skeletal examinations: Yes [half per litter]
- Head examinations: Yes: [ half per litter ]

Statistics:

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:

A parametric analysis was performed if Bartlett's test for variance homogeneity (Bartlett 1937) was not significant at the 1% level. For pre-treatment data, analysis of variance was used to test for any group differences. Where this was significant (p<0.05) inter group comparisons using t-tests, with the error mean square from the one-way analysis of variance, were made. For all other analyses the F1 approximate test was applied.

A non-parametric analysis was performed if Bartlett's test was still significant at the 1% level following both logarithmic and square-root transformations. For pre-treatment data, Kruskal-Wallis’ test (Kruskal and Wallis 1952, 1953) was used to test for any group differences. Where this was significant (p<0.05) inter group comparisons using Wilcoxon rank sum tests (Wilcoxon1945) were made. For all other analyses the/The H1 approximate test, the non-parametric equivalent of the F1 test described above, was applied.

Significant differences between the groups compared were expressed at the 5% (p<0.05) or 1% (p<0.01) level.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

no effects observed

Description (incidence and severity):

There were no signs were observed at either routine physical examination or in association with dose administration that could be related to treatment.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

One female dosed at 125 mg/kg/day was euthanized prematurely with evidence of abortion. Uterine examination revealed a single implantation that was resorbing. This was therefore considered to be a resorption rather than abortion and as there was a solely a single implant it is considered to relate to biological efficiency rather than an effect of treatment.

One female dosed at 500 mg/kg/day prematurely delivered six live offspring, one dead offspring and one late resorption within the animal facility. The live offspring were euthanized within the animal facility and dispatched to necropsy for subsequent examinations. Macroscopic examination of female no 72 and offspring did not reveal any findings that could be attributed to treatment. As this was an isolated incidence it was considered to be unrelated to administration of Amine C8.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

As treatment progressed weight gain for females receiving 500 mg/kg/day was low when compared with Controls and overall the bodyweight gain from GD6 to GD29 bodyweight gain was 17% of Controls (p<0.01). The overall body weight gain for females at 125 or 250 mg/kg/day were similar to the Controls and showed no adverse effect of treatment.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

The mean food consumption for females receiving 500 mg/kg/day was slightly but consistently low from Day 6 to Day 27 of gestation when compared with Controls, with the total consumption over the treatment period at approximately 79% of the Control value.
At 250 mg/kg/day there were occasions of slightly low consumption however overall the food consumed was similar to Control values.
Food consumption at 125 mg/kg/day was unaffected by treatment with Amine C8.

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:

effects observed, non-treatment-related

Description (incidence and severity):

The maternal body weight loss following adjustment for the gravid uterine weight was slightly greater than Controls for females that received 500 mg/kg/day; however this difference did not attain statistical significance. The maternal weight loss for females at 125 or 250 mg/kg/day were similar to the Controls and showed no adverse effect of treatment.

Gross pathological findings:

no effects observed

Description (incidence and severity):

Macroscopic examination of females did not reveal any findings that could be attributedto treatment with Amine C8.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Maternal developmental toxicity

Number of abortions:

no effects observed

Pre- and post-implantation loss:

effects observed, non-treatment-related

Description (incidence and severity):

The post-implantation loss (%) for females at 500mg/kg/day was slightly high when compared with Controls and the resultant mean litter size was low when compared with Controls (p<0.05). However, the live litter size and percentage of post implantation loss were within the historical control data range so no effect of treatment is inferred.

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

Changes in number of pregnant:

effects observed, non-treatment-related

Description (incidence and severity):

One control female and 3 females treated at 250 mg/kg/day were found to be not pregnant at macroscopic examination. There was no clear effect of treatment on offspring survival and litter size at 125 or 250 mg/kg/day.

Effect levels (maternal animals)

Key result

Dose descriptor:

NOEL

Effect level:

250 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

body weight and weight gain

food consumption and compound intake

Maternal abnormalities

Key result

Abnormalities:

no effects observed

Results (fetuses)

Fetal body weight changes:

no effects observed

Description (incidence and severity):

Group mean placental weights, male or fetal weight and overall fetal weight were essentially similar to Controls at dose levels up to and including 500 mg/kg/day.

Reduction in number of live offspring:

not examined

Changes in sex ratio:

not specified

Changes in litter size and weights:

effects observed, non-treatment-related

Description (incidence and severity):

The mean litter weight for females that received 500 mg/kg/day was slightly but significantly low when compared with Controls; this was attributed to the low live litter size rather than the mean fetal weight at this dose level.

Changes in postnatal survival:

not examined

External malformations:

not specified

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

The range and incidence of major abnormalities at 125 and 500 mg/kg/day showed no relationship to treatment. At 500 mg/kg/day there was a slight increased incidence of full supernumerary 13th ribs and unilateral caudal shift of pelvic girdle when compared with both the Concurrent Controls and the historical control data range. The combination of these skeletal variants at 500 mg/kg/day indicates a slight shift in rib/vertebral configuration which is not considered adverse.

Visceral malformations:

not examined

Effect levels (fetuses)

Key result

Dose descriptor:

NOEL

Effect level:

250 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

skeletal malformations

Fetal abnormalities

Key result

Abnormalities:

effects observed, non-treatment-related

Overall developmental toxicity

Key result

Developmental effects observed:

yes

Lowest effective dose / conc.:

500 mg/kg bw/day (nominal)

Treatment related:

no

Relation to maternal toxicity:

not specified

Relevant for humans:

not specified

Applicant's summary and conclusion

Conclusions:

Oral administration of the test substance to New Zealand White rabbits from Day 6 to Day 28 of gestation at dose levels of 125, 250 or 500 mg/kg/day was associated with low maternal body weight gain and food consumption at 500 mg/kg/day.
Embryo-fetal survival, fetal and placental weight were unaffected by treatment and the fetal pathology findings at 500 mg/kg/day were minor skeletal variants which were not considered adverse.
It was therefore concluded that 250 mg/kg/day was the no observed effect level (NOEL) for both maternal toxicity and embryo-fetal development.