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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:
2015-10-13 to 2016-01-05
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report Date:
2016

Materials and methods

Principles of method if other than guideline:
The study design was based on OECD guideline 407.
GLP compliance:
no
Remarks:
the work performed generally followed GLP principles
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Details on test material:
- Physical state: liquid
- Storage condition of test material: Room temperature
Specific details on test material used for the study:
- Name of test material (as cited in study report): POPDA
- Substance type: yellow liquid
- Physical state: liquid
- Analytical purity: 100%
- Impurities (identity and concentrations): no data
- Purity test date: no data
- Lot/batch No.: DR66700414
- Expiration date of the lot/batch:2016-12-05
- Stability under test conditions: refrigerated (nominally 4 – 8°C) or ambient (nominally 21°C) storage for 15 days
- Storage condition of test material: at ambient temperature in the dark
- Action of test substance: reaction products of propane-1,2-diol, propoxylated by amination of the terminal hydroxyl groups
- purity/weighing factor: none
- total correction factor: none

Test animals

Species:
rabbit
Strain:
New Zealand White
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: 36 female (sexually mature, virgin) New Zealand White rabbits, supplied by Charles River (France)
- Age at start of the study: 18-19 weeks (pilot phase); 24 to 25 weeks (preliminary phase)
- Weight range at start of the study: 3.15 - 4.26 kg (pilot phase); 3.87 - 4.54 kg (preliminary phase)
- Fasting period before study: no data
- Housing: individual housing during acclimatization and gestation, one stock male and one female during mating; suspended cages fitted with perforated floor panels and mounted in batteries. Undertrays lined with absorbent paper were changed at least 3 times a week. Cages were also fitted with a plastic resting platform. The cages constituting each group were blocked by group and mounted in batteries.
- Diet (e.g. ad libitum): restricted, initially 150 g/rabbit/day during acclimatisation up to one week prior to the onset of mating and 200 g/animal/day thereafter; Harlan Teklad 2930, pelleted 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 (50g pelleted diet moistened with 50 ml of water) was offered, the consumption was recorded. In addition, a small supplement of autoclaved hay was given on a daily basis to promote gastric mobility and a small amount of chopped fresh vegetables were given twice weekly.
- 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 also provided.
- Acclimation period: 15 days (pilot phase), 55 days (preliminary phase)

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 16-20 °C
- Humidity (%): 40-70%
- Although conditions were occasionally outside the indicated ranges, these deviations were minor and/or of short duration and were considered not to have influenced the health of the animals and/or the outcome of the study.
- Air changes (per hr): filtered fresh air which was passed to atmosphere and not recirculated
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES:
- Pilot phase: from 2015-11-03 to: 2015-11-26
- Preliminary phase: from 2015-12-13 to 2016-01-05

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Remarks:
purified
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
- The required amount of test item for the highest concentration was mixed with sufficient vehicle to form a solution and mixed with a magnetic stirrer. The solution was made up to the required volume with vehicle and stirred again with a magnetic stirrer. The remaining group was formulated by serial dilution with further quantities of vehicle.
- Frequency of preparation: weekly, batches may be prepared in advance but will be used within documented stability limits.
- Storage of formulations: at ambient temperature (21°C)
- 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.
- Formulations were stirred using a magnetic stirrer before and throughout the dosing procedure.

VEHICLE
- Concentration in vehicle:
- pilot phase: 15 mg/ml (75 mg/kg) and 30 mg/ml (150 mg/kg);
- preliminary phase: 23 mg/ml (115 mg/kg) and 30 mg/ml (150 mg/kg)
- Amount of vehicle (if gavage): 5 ml/kg
Analytical verification of doses or concentrations:
no
Details on mating procedure:
- Impregnation procedure: cohoused
- If cohoused: no data
- M/F ratio per cage: 1:1 using identified stock New Zealand White bucks
- Length of cohabitation: no data
- Further matings after two unsuccessful attempts: no data
- Verification of same strain and source of both sexes: yes, a colony of stock males of the same strain was maintained specifically for the purpose of mating; these animals are not part of the study and were maintained as stock animals.
- Proof of pregnancy: natural mating observed referred to as day 0 of pregnancy
- checks: natural mating observed
- after mating: each female was injected intravenously with 25 i.u. luteinising hormone
- day 0 of gestation = day of mating
Duration of treatment / exposure:
days 6 to 28, after mating
Frequency of treatment:
once daily, at approximately the same time each day
Duration of test:
22 days (day 6 to day 28 after mating)
No. of animals per sex per dose:
6 females per dose (pilot and preliminary phase), except control group (3 in pilot phase and 3 in preliminary phase)
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
Pilot phase: the dose levels for the Pilot phase were selected in conjunction with the Sponsor based on a previous Preliminary Toxicity study in the New Zealand White Rabbit (Study number TMR0109). In that study, a single dose of 600 mg/kg/day was not tolerated, and was associated with marked macroscopic findings relating to depressions or perforation on the stomach, and other gastro-intestinal findings including dark areas on the jejunum and thickened omental adipose tissue.
Repeat daily dosing at a lower dose of 300 mg/kg/day was initially tolerated for a period of 11 days, but did elicit a marked reduction in food consumption for two out of three females. After receiving 11 doses one female was found dead and at macroscopic examination showed multiple depressions on the stomach. Repeat daily dosing at 300 mg/kg/day was subsequently suspended due to the similarity of the stomach findings in the decedent to those observed at the higher dose of 600 mg/kg/day. At necropsy a total of two out of three females receiving a daily dose of 300 mg/kg/day showed a marked reduction in food intake and macroscopic findings in the stomach and GI tract whilst the third animal was not similarly affected.
It is considered that daily dosing at 300 mg/kg/day for a period of 11 days or more is clearly unsuitable for any further investigation. Due to the marked nature of the findings observed in the stomach and GI tract the starting high dose level for the pilot phase of this preliminary embryo-fetal study in the New Zealand White Rabbit is set at 150 mg/kg/day, with the low dose set at 75 mg/kg/day.
Preliminary phase: Results from the pilot phase of the current study suggested potential effects in individual animals in terms of food consumption, and the possibility of marginally low fetal weights, at 150 mg/kg/day. The high dose level of 150 mg/kg/day was therefore repeated on the preliminary phase in order to substantiate these findings. A lower dose level of 115 mg/kg/day was also implemented on the preliminary phase to investigate the response between 75 and 150 mg/kg/day.

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least twice daily (at least once per day during the acclimatisation period)
- Animals visually inspected for evidence of reaction to treatment or ill-health. Cages and cage-trays were inspected daily for evidence of animal ill-health amongst the occupant.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily during the treatment period at the following times in relation to dose administration: pre-dose observation, 1 to 2 hours after dosing, as late as possible in the working day; detailed physical examination was performed on days 0, 6, 12, 18, 23 and 29 after mating to monitor general health.

BODY WEIGHT: Yes
- Time schedule for examinations: weekly during acclimatisation, on day of mating (day 0) and days 3 and 6-29 after mating
- all adult animals

FOOD CONSUMPTION: Yes
- Time schedule: daily from day 1 after mating
- all adult animals
- The weight of food supplied to each animal, that remaining and an estimate of any spilled was recorded.

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

POST-MORTEM EXAMINATIONS:
- Necropsy: all adult animals were subject to a detailed necropsy. Animals surviving until the end of the scheduled study period were killed at day 29 after mating; All adult animals were killed by intravenous injection of sodium pentobarbitone. Fetuses were killed by subcutaneous injection of sodium pentobarbitone.
- Macroscopic pathology: 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.

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.
Ovaries and uterine content:
- The uterine content was examined after termination only for females exhibiting pregnancy loss.
- For females surviving to full term (day 29 after mating), the following was recorded: gravid uterine weight (including cervix and ovaries)
- The following was recorded for all animals (including those prematurely sacrificed, where possible): for each ovary/uterine horn: number of: corpora lutea, implantation sites, intrauterine deaths (assessed as early or late resorptions), fetuses (live and dead); apparently non-pregnant animals and for apparently empty uterine horns: the absence or number of uterine implantation sites was confirmed.
- Any photographs of unusual findings were taken at the discretion of the necropsy supervisor.
- Fetuses and placentae were dissected from the uterus and weighed individually.
Fetal examinations:
- External examinations: Yes, each placenta and fetus was externally examined.
- Soft tissue examinations: No
- Skeletal examinations: No
- Head examinations: No
- Internal examination of neck and thoracic and abdominal cavities was performed and any abnormalities were recorded, sampled as appropriate and retained in appropriate fixative.
- The sex of each fetus was recorded.
- Grossly normal fetuses were discarded.
Statistics:
No statistical analysis performed.
Indices:
- Prenatal losses are seperated into pre- and post-implantation phases. Pre-implantation loss was considered to reflect losses due to non-fertilization of ova and failure to implant.
Pre-implantation loss (%) = (Number corpora lutea – Number implantations)/Number corpora lutea x 100
Where the total number of implantations exceeds corpora lutea observed, preimplantation loss was assumed to be zero (i.e. no pre-implantation loss was considered to have occurred).
- Post implantation loss (%) = (Number implantations – Number live fetuses)/Number implantations x 100
- All group values and SD (as appropriate) were calculated from the individual litter values.
Historical control data:
no data

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
Mortality and clinical signs:
One female (number 15) receiving 150 mg/kg/day in the pilot phase was despatched to necropsy for reasons of animal welfare following an extended period of inappetance and clinical signs indicative of inappetance (little diet/water consumed, small and reduced faeces) and underactive behaviour. Upon veterinary examination it was deemed necessary to despatch this female to necropsy. Macroscopic examination revealed no findings, in particular no findings related to abnormalities of the gastro-intestinal tract. This mortality was considered to be related to inappetance, which is likely to be caused by the test material.There were no premature deaths at 150 mg/kg/day in the prelim phase.
There were no premature mortalities at 115 or 75 mg/kg/day.
In animals surviving to scheduled termination there were no consistent clinical signs, nor were there any dosing signs, observed that were considered to be related to treatment at any of the dose levels investigated.
Signs considered to be related to treatment related effects upon food consumption included two animals at 115 mg/kg/day, and one at 150 mg/kg/day, on the prelim phase that were observed to be thin, and pale and few faeces, which were observed in 4 animals at 115 mg/kg/day and few faeces in two animals receiving 150 mg/kg/day.

Body weight and gravid uterine weight:
Body weight was not clearly affected by treatment.
On the pilot phase, group mean overall body weight gain during Days 6-29 of gestation was lower than controls amongst females receiving 75 or 150 mg/kg/day; however, when mean values of body weight and body weight gain were adjusted for the contribution of the gravid uterus, overall maternal mean body weight loss during Days 6-29 of gestation was similar to controls at both dose levels investigated. This suggested that the reduced gravid uterine weight observed was the influencing factor in treated animals not gaining as much weight as controls and that the maternal portion of gestational body weight gain was unaffected by treatment.
On the prelim phase, slight group mean overall bodyweight loss was evident at 115 mg/kg/day and overall bodyweight gain lower than that of Controls was evident at 150 mg/kg/day. When mean values of body weight and body weight gain were adjusted for the contribution of the gravid uterus, overall maternal mean body weight loss during Days 6-29 of gestation was slightly greater than controls at both dose levels.
At 115 mg/kg/day overall body weight change was heavily influenced by two females (numbers 23 and 24) showing marked weight loss of 0.40 – 0.41 kg, however, all other females at this dose level showed lower weight gain than in controls.
At 150 mg/kg/day on the prelim phase overall body weight change was heavily influenced by a single female (number 28) that showed weight loss of 0.20 kg, however, all other females at this dose level (i.e. 5/6 females) showed overall weight gain that was similar to controls.
Individual values of weight change following adjustment for the contribution of the gravid uterus indicated 4 / 6 females at 115 mg/kg/day, and two females at 150 mg/kg/day, with greater adjusted body weight loss than controls. Control values were also influenced by one female (number 6) showing overall body weight gain after adjustment for the contribution of the gravid uterus, due to a very low litter size (3 implantations).

Food consumption:
At 150 mg/kg/day, four out of a total of 12 animals at this dose level showed periods of low food intake. Females 15 (pilot phase) and 28 (prelim phase) showed low food from day 15 after mating, female 16 showed low food during days 15-20 after mating and female 18 showed low food intake from day 22 after mating.
At 115 mg/kg/day three out of six females showed periods of low food intake. Female 23 showed low food from day 13 after mating, female 24 showed low food intake from day 14 after mating and female 19 showed low food from day 24 after mating.
These incidences were compared to no similar cases in the control or 75 mg/kg/day groups.

Macropathology
There were no maternal findings observed at macroscopic examination of adult females receiving 75, 115 or 150 mg/kg/day that were considered to relate to treatment.

Effect levels (maternal animals)

Key result
Dose descriptor:
dose level: other
Remarks:
no remarks
Effect level:
ca. 115 mg/kg bw/day (nominal)
Based on:
test mat.
Remarks:
no remarks
Basis for effect level:
mortality
Remarks on result:
other: 115 mg/kg considered appropriate high dose in main OECD414 study due to mortality observed at 150 mg/kg as a direct result of sustained period of very low food intake.

Maternal abnormalities

Abnormalities:
not specified

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
Litter data:
All females on the pilot phase were pregnant. One Group 3 female receiving 150 mg/kg/day was killed for welfare reasons therefore the assessment of litter data is based on a total of 6, 6 and 5 pregnant females reaching full term in Groups 1-3, respectively.
One female on the preliminary phase was found to be not pregnant at necropsy, female 27 receiving 150 mg/kg/day. The assessment of litter data on the preliminary phase is therefore based on a total of 6, 6 and 5 pregnant females reaching full term in Groups 1, 4 and 5, respectively.
Embryo-fetal survival was unaffected by treatment at 75, 115 or 150 mg/kg/day with mean numbers of implantations, resorptions, live young and percentages of sex ratio and pre- and post-implantation loss being similar to control values.

Placenta, litter and fetal weight:
Mean placental weight at 150 mg/kg/day was marginally lower than controls in the pilot phase but similar to Controls in the preliminary phase, therefore considered unrelated to treatment.
On the pilot phase mean litter weight was lower than controls in both treated groups, despite mean litter size being considered essentially similar across all groups. In the preliminary phase, mean litter weight was similar to Controls at 150 mg/kg/day but marginally lower than controls at 115 mg/kg/day.
Mean male, female and overall fetal weights at 75, 115 or 150 mg/kg/day were marginally lower than controls, with evidence of a slight dose response apparent. This was replicated on both the pilot and prelim phases at 150 mg/kg/day.

Macropathology:
There were no consistent fetal abnormalities observed at macroscopic examination of adult females receiving 75, 115 or 150 mg/kg/day.
There was one fetus in a litter of an adult female receiving 75 mg/kg/day that showed cardiovascular abnormalities including dilated aortic arch, narrow pulmonary trunk and ventricular septal defect.
At 115 mg/kg/day there were three fetuses from a litter of an adult female receiving 115 mg/kg/day with dark thyroids, two of these fetuses also showed enlarged thyroids.
There was one fetus in a litter of an adult female receiving 150 mg/kg/day on the pilot phase that showed the cardiovascular abnormality of retroesophageal subclavian artery, and further findings of small lungs and bilateral forepaw flexure, and a single fetus at 150 mg/kg/day on the preliminary phase that showed unilateral forepaw extension and flexure.
In isolation these findings are currently of uncertain relationship to treatment.

Effect levels (fetuses)

Key result
Dose descriptor:
dose level:
Effect level:
150 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
not specified
Basis for effect level:
fetal/pup body weight changes
changes in litter size and weights
Remarks on result:
not determinable

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
Based on the results of this study it is concluded that a dose level of 150 mg/kg/day would be too high for investigation on a main embryo-fetal study in the pregnant New Zealand White Rabbit, due to the mortality observed as a direct result of a sustained period of very low food intake.
A dose level of 115 mg/kg/day would therefore be considered appropriate for use as the high dose on a main embryo-fetal study in the pregnant New Zealand White Rabbit. This dose level could be expected to elicit slight maternal toxicity in terms of prolonged periods of lower food intake in some females, but did not result in any premature mortality on this preliminary study.