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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:
2015-08-25 to 2016-03
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

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

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Deviations:
no
Principles of method if other than guideline:
The study was also designed to meet the requirements of the following guideline: Japanese Ministry of Agriculture, Forestry and Fisheries, Test Data for Registration of Agricultural Chemicals, 12 Nohsan No. 8147, Agricultural Production Bureau, November 24, 2000
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
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%
- Purity test date: no data
- Lot/batch No.: DR66700414
- Expiration date of the lot/batch: 2016-12-05
- Stability under test conditions: no data
- 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
- correction factor: none
- purity/weighing factor: none

Test animals

Species:
rabbit
Strain:
New Zealand White
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: 96 female (sexually mature, virgin) New Zealand White Rabbits, supplied by Charles River (France)
- Age at start of study (day 0 of gestation): 21-22 weeks
- Weight at day 0 (after mating): 3.17 - 4.62 kg
- Fasting period before study: no data
- Housing: One animal per cage (acclimatization and gestation) one stock male and one female per cage 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. The cages constituting each group were blocked by group and mounted in batteries.
- 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), Teklad diet. The diet contained no added antibiotic or other chemotherapeutic or prophylactic agent. Should an individual show a significant non-treatment related reduced food consumption, moistened diet (50 g pelleted diet moistened with 50 mL of water) was offered, the consumption was recorded. In addition to this diet, a small supplement of autoclaved hay was given on a daily basis to promote gastric motility and a small amount of chopped fresh vegetables were given twice weekly.
- 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: at least 19 days. Any individuals rejected during the acclimatization period were replaced with spare animals of suitable weight from the same batch.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 16-20°C
- Humidity (%): 40-70%
- Air changes (per hr): filtered fresh air which was passed to atmosphere and not recirculated
- Photoperiod (hrs dark / hrs light): 10 hours dark/14 hours light, artificial lighting

IN-LIFE DATES: From: 2016-01-30 To: 2016-02-22 to 2016-02-26

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 groups were formulated by serial dilution with further quantities of vehicle.
- Individual dose volume was calculated from the most recently recorded scheduled body weight.
- Frequency of preparation: weekly
- 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.


VEHICLE
- Concentration in vehicle: no data
- Amount of vehicle (if gavage): 5ml/kg body weight
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples of each formulation prepared for administration in the first and last weeks of treatment were analyzed for achieved concentration of the test item.

The analytical method will involve the dilution of the test formulations with a suitable solvent followed by chromatographic assay.
The formulated samples will be analysed using a method transferred from the sponsor with respect to the determination of the specificity of analysis, limit of
detection, linearity of detector response, repeatability, method accuracy and precision.
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: no data
- Proof of pregnancy: natural mating observed referred to as day 0 of pregnancy
- After mating each female was injected intravenously with 25 i.u. luteinising hormone.
- 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:
days 6 to 28 after mating (22 days)
Frequency of treatment:
once daily, at approximately the same time each day
Duration of test:
30 days
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Dose / conc.:
15 mg/kg bw/day (nominal)
Dose / conc.:
50 mg/kg bw/day (nominal)
Dose / conc.:
115 mg/kg bw/day (nominal)
No. of animals per sex per dose:
22 females per dose
Control animals:
yes
Details on study design:
- Dose selection rationale: the dose levels were selected by the Sponsor based on a preliminary embryo-fetal study in the rabbit (Envigo Study No. TMR0110), in which the high dose of 150 mg/kg/day elicited extended periods of low food intake in some animals and resulted in one animal being killed for reasons of animal welfare, whereas the intermediate dose of 115 mg/kg/day elicited extended periods of low food consumption in some animals but did not result in premature death. A dose level of 150 mg/kg/day was therefore considered too high for subsequent investigation on a main embryo-fetal study, and therefore the dose level of 115 mg/kg/day was selected as the high dose for the current main embryo-fetal development study.
Intermediate and low dose levels of 50 and 15 mg/kg/day, respectively, were selected to evaluate the response at lower dose levels to identify any dose response. The lowest dose level of 15 mg/kg/day was expected to provide a clear No Observed Effect Level for extended periods of low maternal food intake.

Examinations

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

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily during the treatment period at the following times during the day: 1 to 2 hours after completion of dosing, as late as possible in the working day. Detailed physical examination on days 0, 6, 12, 18, 24 and 29 after mating
- Detailed physical examination was performed on each animal to monitor general health.

BODY WEIGHT: Yes
- Time schedule for examinations: weekly during acclimatization, on days 0, 3, 6 to 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 supplied to each animal, that remaining and an estimate of any spilled was recorded
- Consumption of hay and vegetables were monitored qualitatively but not quantitatively. No entry was made in the raw data for the provision of vegetables during the period of 26 Jan 16 – 01 February 2016 and thus during this period only, a twice weekly schedule cannot be verified from the raw data.

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

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day #: day 29 after mating
- Females that exhibit pregnancy loss were killed on the day of detection.
- All adult animals were killed by intravenous injection of sodium pentobarbitone
- Macroscopic pathology: a full macroscopic examination of the tissues was performed for each animal. 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:
MORTALITY:
- Time schedule: a viability check was performed near the start and end of each working day.
- Animals were killed for reasons of animal welfare where necessary or if they exhibited pregnancy loss.
Ovaries and uterine content:
For females surviving to term, the following was recorded:
- uterus: gravid uterine weight (including cervix and ovaries).

The following were recorded for all animals (including those prematurely sacrificed, where possible):
- For each ovary/uterine horn: number of corpora lutea, implantation sites, resorption sites (classified as early or late), fetuses (live and dead).
- Apparently non pregnant animals and for apparently empty uterine horns: the absence or number of uterine implantation sites was confirmed.
- Females exhibiting pregnancy loss: expelled uterine contents were identified and examined, as appropriate.
Fetal examinations:
- External examinations: Yes: [all viable fetuses per litter ]
- Soft tissue examinations: No
- Skeletal examinations: Yes [one third per litter ], fixed in Industrial Methylated Spirit and stained with Alizarin Red
- Head examinations: Yes: [appr. 50% of eviscerated fetuses ], fixed in Bouin's fluid
- Remaining fetuses will be eviscerated and fixed in Industrial Methylated Spirit.
- All live fetuses: Internal examination of neck and thoracic and abdominal cavities, sex recorded and individual identification within litter.
- Fetuses were killed by subcutaneous injection of sodium pentobarbitone.
Statistics:
Body weight, gravid uterus weight, food consumption, corpora lutea, implantations, live young, fetal, placental and litter weight data:
- A parametric analysis if Bartlett's test for variance homogeneity (Bartlett 1937) was not significant at the 1% level. For pretreatment 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 comparisons the F1 approximate test was applied. If the F1 approximate test for monotonicity of dose-response was not significant at the 1% level, Williams' test for a monotonic trend was applied. If the F1 approximate test was significant, Dunnett's test (Dunnett 1955, 1964) was performed instead.
- A non-parametric analysis if Bartlett's test was still significant at the 1% level following both logarithmic and square-root transformations. For pretreatment 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 (Wilcoxon 1945) were made. For all other comparisons the H1 approximate test, the non-parametric equivalent of the F1 test described above, was applied.If the H1 approximate test for monotonicity of dose-response was not significant at the 1% level, Shirley's test for a monotonic trend was applied. If the H1 approximate test was significant, Steel's test (Steel 1959) was performed instead.

Litter size and survival indices and fetal, placental and litter weight and gravid uterine weight data, if 75% of the data (across all groups) were the same value:
- Fisher’s exact tests (Fisher 1973).

Pre/post implantation loss and sex ratio: generalized mixed linear model with binomial errors, a logit link function and litter as a random effect (Lipsitz 1991).

Resorptions: exact Wilcoxon rank sum test (Wilcoxon 1945)
Indices:
- Pre-implantation loss (%) = (Number of corpora lutea – Number of implantations) / Number of corpora lutea x 100
Where the total number of implantations exceeds corpora lutea observed, pre-implantation loss was assumed to be 0.

- Post implantation loss (%) = (Number of implantations – Number of live fetuses) / Number of implantations x 100
Historical control data:
Historical control data will be routinely supplied for selected observations where this information is considered to assist interpretation of study data, and will normally include both fetal and litter incidences.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
- Female number 82 receiving 115 mg/kg/day was despatched to necropsy for reason of animal welfare on day 23 of gestation following a prolonged period of marked inappetance from day 9 of gestation and subsequent body weight loss of 590 g from day 6 of gestation. At necropsy this female showed 8 normal implantations, and there were no remarkable macroscopic observations. This prolonged period of reduced food intake is likely to be related to treatment.
- Female number 83 receiving 115 mg/kg/day was despatched to necropsy following evidence of abortion. This female had shown body weight loss of 310 g from day 10 of gestation, and food consumption had been very low from day 11 of gestation. Fetal material was observed in the cage under tray on day 20 of gestation. At necropsy this female showed a pale liver, and the animal was confirmed as pregnant with 12 implantations, 11 of which were live young and one was an aborted implant. Due to its isolated nature, a relationship between treatment and abortion is considered unproven.
- Female number 58 receiving 50 mg/kg/day was despatched to necropsy for reason of animal welfare following the observation of a small amount of clotted blood, and subsequently further blood, in the cage under tray, and signs of piloerection and dark eyes on day 15 of gestation. This female had progressively lost bodyweight (to a total of 260 g) with the most marked loss occurring from day 13 of gestation, and food consumption was very low during days 13 and 14. At necropsy there were no macroscopic changes, but the animal was confirmed as pregnant with 11 implantations, which were all noted to be early resorptions. This rabbit therefore showed total litter resorption. Our preliminary evaluation is that this is not related to treatment.
- Female number 65 receiving 50 mg/kg/day was despatched to necropsy for reason of animal welfare on day 23 of gestation following the observation of a swelling on the ventral abdomen (suspected hernia) on days 22-23 of gestation. This female also showed signs of little hay/water consumed, underactive behaviour, reduced body temperature (assessed by touch), deep and irregular breathing, thin build, few faeces, reduced urine output and dark eyes on day 23 of gestation. Body weight loss of 330 g was evident from day 15 of gestation, and food consumption was markedly low from day 14 of gestation. At necropsy the hernia was confirmed (jejunum protruded through the abdomen wall), stomach contents were dark and liquefied, the stomach was distended with dark areas on the glandular mucosa, the jejunum and duodenum were distended and the caecum had firm contents. This female was pregnant with 10 implantations, all of which were late resorptions. This rabbit therefore also showed total litter resorption. Our preliminary evaluation is that this is not related to treatment.
- In animals surviving to scheduled termination there was an increased incidence of females showing clinical signs indicative of inappetance at 115 mg/kg/day. These signs included thin build, little hay eaten, reduced urine output and few, small and pale faeces. Three females receiving 50 mg/kg/day were also noted to have thin build.
- There were no other test article related clinical signs, nor any dosing signs, observed.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Description (incidence):
- Two females receiving 115 mg/kg/day, and two females receiving 50 mg/kg/day, were killed for reasons of animal welfare.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
- At 115 mg/kg/day mean body weight gain during days 6-14 of gestation was similar to Controls, however, during days 14-28 of gestation mean bodyweight loss of approximately 120 g was evident, compared to body weight gain (ca. 90 g) in the Control group. 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 greater than Controls at 115 mg/kg/day, confirming that the maternal portion of gestational body weight gain was affected by treatment at this high dose level.
- Body weight performance at 15 or 50 mg/kg/day was unaffected by treatment with mean body weight gain during days 6-14 and 14-28 being similar to Control values. When adjusted for the contribution of the gravid uterus, adjusted body weight change values at 50 or 15 mg/kg/day were similar to Controls indicating that maternal weight gain was unaffected by treatment at these dose levels.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
- Mean values of food consumption at 115 mg/kg/day were lower than Controls from day 15 of gestation to termination on day 29 of gestation. Resulting overall mean food consumption values for the treatment period (days 6-28) were 79 % of Controls.
- Food consumption at 50 or 15 mg/kg/day was similar to Controls throughout gestation and unaffected by treatment.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
- There were no maternal findings observed at macroscopic examination that were considered to relate to treatment.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not specified

Maternal developmental toxicity

Number of abortions:
not examined
Pre- and post-implantation loss:
not examined
Total litter losses by resorption:
not examined
Early or late resorptions:
not examined
Dead fetuses:
not examined
Changes in pregnancy duration:
not examined
Changes in number of pregnant:
not examined
Other effects:
not specified

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Effect level:
50 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity

Maternal abnormalities

Key result
Abnormalities:
no effects observed

Results (fetuses)

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

Details on embryotoxic / teratogenic effects:
Litter data:
- In addition to the premature deaths observed, there was also one female in each treatment group that was observed to be not pregnant at necropsy on day 29 after mating. The following assessment of litter data is therefore based on a total of 22, 21, 19 and 19 pregnant females reaching full term in Groups 1-4, respectively.
- Mean post-implantation loss at 115 mg/kg/day was marginally higher than Control values, resulting in a slight increase in the numbers of early and late, and therefore total, resorptions; and subsequently slightly fewer live young when compared with Control values.
- Embryo-fetal survival was unaffected by treatment at 15 or 50 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.

Placental, litter and fetal weight
- Mean placental, litter and fetal weights at 115 mg/kg/day were marginally lower than Controls.
- Mean placental, litter, and male, female and overall fetal weights at 15 or 50 mg/kg/day were similar to Controls and unaffected by treatment.

Macropathology:
- At 115 mg/kg/day there was an increased fetal and litter incidence of the minor abnormalities of large anterior fontanelle, delayed/incomplete ossification/ unossified sternebrae, pubes, epiphyses, talus, metacarpals/metatarsals/phalanges compared to the concurrent Control group, and which were outside of Historical Control Data (HCD) ranges (See Annex 3). This delay in ossification is a transient stage in fetal development that may be attributed to the slight decrease in mean fetal weight observed and as such this is not considered to be adverse.
- In addition, at 115 mg/kg/day there was also a slightly increased incidence of the minor abnormality of fused/partially fused/misaligned sternebral hemicentres, with associated misaligned costal cartilage when compared to concurrent Control, and these were also slightly outside of HCD ranges. As these incidences were small (max 4 fetuses from 3 litters) a relationship to treatment is considered not proven.
- At 15 and 50 mg/kg/day there was a high incidence of the minor abnormality of delayed/incomplete ossification/ unossified sternebrae compared to concurrent control and outside of HCD ranges. As at 115 mg/kg/day this delay in ossification is a transient stage in fetal development and as such this is not considered to be adverse.

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
115 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: fetotoxicity

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Key result
Developmental effects observed:
no

Any other information on results incl. tables

Formulation analysis:

The mean concentrations of the test substance in test formulations were within applied limits +/- 10%, confirming the accuracy of formulation. Difference from mean values were within 2% confirming precise analysis.

Applicant's summary and conclusion

Conclusions:
Based on the results of this prenatal developmental toxicity study, it was concluded that the No-Adverse-Effect-Level (NOAEL) of the test substance for maternal toxicity was 50 mg/kg/day as the higher dose of 115 mg/kg/day elicited low food consumption that directly resulted in a single mortality, and elicited clinical signs relating to inappetance and overall body weight loss during gestation compared with body weight gain in all other groups.
The NOAEL for embryo-fetal survival, development and growth is set at 115 mg/kg/day when the test substance is administered during organogenesis in the rabbit.