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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:
2020
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2020
Report Date:
2020

Materials and methods

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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Specific details on test material used for the study:
Test item: Pentyl propionate.
Test item identity (including alternative names): Amyl propionate, 1-Pentyl propionate.
CAS Number: 624-54-4
Action of test item: Industrial chemical.
Batch number: D229I9D725.
Storage conditions: at ambient temperature (15 to 25°C).
Flammable liquid and vapor, therefore containers should be kept tightly sealed and away from heat, sparks, open flames and hot surfaces.
Certificate of analysis (or other appropriate documentation): Composition and/or appropriate characteristics.
Purity: 99.96%.
Specific gravity: 0.87.
Appearance: Colorless, clear liquid.
Expiry/Retest date: 17 April 2021.

Test animals

Species:
rat
Strain:
other: Crl:CD(SD)
Details on test animals and environmental conditions:
Animals:
The rat was chosen as the test species because of the requirement for a rodent species by regulatory agencies. The Sprague Dawley [Crl:CD(SD)] strain was used because of the historical control data available at this laboratory.

Strain/Species: Sprague Dawley, Crl:CD(SD) rat.
Supplier: Charles River (UK) Ltd
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): 71 days old.
Weight range of the animals at the start of the study (Day 0 of gestation): 209 to 284 g

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.

Environmental Control:
Animal 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 20-24C 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.
Bedding: Solid bottom cages contained softwood based bark-free fiber bedding, 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

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.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:

Vehicle: Corn oil.
Method of preparation: The required amount of test item was weighed out and 50% of the final volume of vehicle was added to the test item. The formulation was magnetically stirred until all the test item was uniformly mixed. Vehicle was added to achieve the final required volume and the formulation was
magnetically stirred until homogenous. Magnetic stirring was continued during the transfer of the formulations, via syringe, to the final containers.
Frequency of preparation: Weekly.
Storage of formulation: Refrigerated temperature (2 to 8°C).
Test item accounting: Detailed records of compound usage were maintained. The amount of test item necessary to prepare the formulations and the amount actually used were determined on each occasion. The difference between these amounts was checked before the formulations were dispensed.

Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Formulation Analysis:
Stability and homogeneity: Before commencement of treatment, the suitability of the proposed mixing procedures was determined and specimen formulations at 1 and 250 mg/mL were analyzed to assess the stability and homogeneity of the test item in the liquid matrix (Covance Study Number FC62JS).
Formulations were shown to be stable when stored as follows:
Ambient temperature (15 to 25°C) for 24 hours.
Refrigerated temperature (2 to 8°C) for 15 days.
Achieved concentration: Samples of each of the first and last preparation formulations were analyzed for achieved concentration of the test item.
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:
Females were treated from Day 6 to Day 19 (inclusive) after mating, once daily at approximately the same time each day.
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:
Three groups of 20 females received pentyl propionate at doses of 100, 330 or 1000 mg/kg/day by oral gavage administration, from Day 6 to 19 after mating. A similarly constituted control group received the vehicle, corn oil, at the same volume dose as treated groups. Animals were killed on Day 20 after mating for reproductive assessment and fetal examination.
Doses / concentrationsopen allclose all
Dose / conc.:
100 mg/kg bw/day
Dose / conc.:
330 mg/kg bw/day
Dose / conc.:
1 000 mg/kg bw/day
No. of animals per sex per dose:
20 females per dose group
Control animals:
yes, concurrent vehicle
Details on study design:
Dose levels were selected in conjunction with the Sponsor based on findings from the preliminary embryo-fetal development study in the Sprague Dawley rat.
In the preliminary study pregnant Sprague Dawley rats were given 250, 500 or 750 mg/kg/day pentyl propionate from Days 6 to 19 of gestation. No conclusive reaction to treatment was observed, in contrast to the results of a 14 day toxicity study in female Sprague Dawley rats where treatment at both 750 and 1000 mg/kg/day was associated with an initial 8 g loss of body weight, with all females in both groups showing weight loss. By the end of treatment, the 750 and 1000 mg/kg/day groups had gained weight, but overall body weight gains were lower compared to controls. It was considered that pregnant females were not more sensitive than non-pregnant females to treatment with pentyl propionate. Therefore, the high dose was set at 1000 mg/kg/day with a three-fold interval in doses.

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 during the treatment period at the following times in relation to dose administration:
 Pre-dose observation
 One to two hours 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.

Thyroid Hormone Analysis
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: The number of uterine implantation sites were checked after staining with ammonium sulphide [modification of the Salewski staining technique (Salewski, 1964)].
Fetal examinations:
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 and ano-genital distance 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.
Bouin’s fixed fetuses: Serial sections were examined for visceral abnormalities.
Alizarin Red stained fetuses: Assessed for skeletal development and abnormalities.
Statistics:
The analyses were carried out using the individual animal as the basic experimental unit. For litter/fetal findings the litter was taken as the treated unit and the basis for statistical analysis and biological significance was assessed with relevance to the severity of the anomaly and the incidence of the finding within the background control population.
The following data types were analyzed at each timepoint separately:
Body weight, using absolute weights 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)
Placental, litter and fetal weights
Ano-genital distance, average for each litter adjusted for litter average fetal body weight
Organ weights, absolute and adjusted for terminal body weight
The following comparisons were performed:
Group 1 vs 2, 3 and 4
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. All group values and SD were calculated from the individual litter values.

Fetal, Litter and Placental Weights:
Mean fetal weights were calculated for each litter. Values were presented for male, female and overall fetal weight. Litter weight was calculated as the sum of all fetal weights. Mean placental weight was also calculated for each litter.
Group mean values and SD were calculated using individual litter mean values.

Ano-Genital Distance:
Ano-genital distance were presented both as absolute/unadjusted and adjusted for fetal body weight, using the weight recorded at necropsy.

Detailed Fetal Examination:
Findings from external, visceral and skeletal examination of fetuses are tabulated on an individual basis for affected litters and fetuses, linking the results of initial external examinations with subsequent visceral and/or skeletal examinations to fetal weight.
Group incidences of observations on fetuses and litters are summarized in terms of major or minor abnormalities or as skeletal variants. The incidence of structural changes are presented as numeric fetal and litter incidences.
Findings observed were classified, according to severity and incidence, as:
Major abnormalities: normally rare, definitely detrimental to normal subsequent development, possibly lethal, e.g. ventricular septal defect
Minor abnormalities: minor differences from normal that are detected relatively frequently considered to have little detrimental effect and may be a transient stage in development e.g. bipartite centrum, dilated ureter.
Variants: alternative structures or stages of development occurring regularly in the control population, e.g. number of ribs, incomplete ossification of 5th and 6th sternebrae.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
no effects observed
Description (incidence and severity):
No signs were observed in association with dose administration and there were no signs at routine physical examination that could be attributed to administration of pentyl propionate at all dose levels.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Description (incidence and severity):
The body weight performance and gain during gestation Days 6 to 20 of females that received 100, 330 or 1000 mg/kg bw/day pentyl propionate was comparable to controls.
The mean gravid uterine weight, adjusted terminal body weight and adjusted body weight gain (Days 6 to 20) for females treated with pentyl propionate was comparable to controls.
Food consumption and compound intake (if feeding study):
no effects observed
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:
no effects observed
Description (incidence and severity):
The analysis of serum thyroxine (T4) and triiodothyronine (T3) concentrations at scheduled termination on Day 20 of gestation revealed that mean serum T3 concentrations at 330 or 1000 mg/kg bw/day were found to be slightly but statistically significantly lower when compared with the control group. Mean serum T4 concentrations were found to be slightly lower at 330 or 1000 mg/kg bw/day, but did not attain statistical significance.
The analysis of serum TSH concentrations performed at scheduled termination on Day 20 of gestation revealed that the TSH level was similar at all dose levels when compared with the control group and no statistically significant differences were observed.
It was concluded that there was no clear effect of treatment on serum T3, T4 and TSH concentrations in pregnant rats at a dose level of 100, 330 or 1000 mg/kg/day, when administered via oral gavage.
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
The thyroids and parathyroids weights (both absolute and adjusted for terminal body weight) from pentyl propionate treated females were comparable to controls.
Gross pathological findings:
no effects observed
Description (incidence and severity):
There were no adverse findings at macroscopic examination of females that were treated with pentyl propionate.
One female (animal No. 63) that received 1000 mg/kg bw/day pentyl propionate had pale rims on the placenta and abnormal dark fluid in the uterus but all fetuses survived to Day 20 of gestation. Low food consumption (11 g /day) was recorded for this female during Days 18 to 20 of gestation.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
No findings related to treatment with pentyl propionate given by oral gavage were seen in the thyroid glands of Sprague-Dawley rats during pregnancy.
Histopathological findings: neoplastic:
not examined

Maternal developmental toxicity

Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
On Day 20 of gestation, mean numbers of corpora lutea, implantations, resorptions, the number of live young and sex ratio were considered to be unaffected by treatment with pentyl propionate.
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed
Changes in pregnancy duration:
no effects observed
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
One female (animal No. 61) that received 1000 mg/kg/day pentyl propionate was found to be not pregnant at necropsy.

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: There was no effect of treatment on maternal clinical condition, body weight, food consumption, thyroid weight, macropathology or histopathology of the maternal thyroid.

Maternal abnormalities

Abnormalities:
no effects observed

Results (fetuses)

Fetal body weight changes:
no effects observed
Description (incidence and severity):
Placental, total litter and fetal weights were unaffected by treatment with pentyl propionate.
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
no effects observed
Changes in postnatal survival:
no effects observed
External malformations:
no effects observed
Description (incidence and severity):
The incidence of major and minor abnormalities and skeletal variants show no relationship to treatment.
Skeletal malformations:
no effects observed
Visceral malformations:
no effects observed
Other effects:
no effects observed
Description (incidence and severity):
The ano-genital distance of male and female fetuses were considered to be unaffected by maternal treatment with pentyl propionate.

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Sex:
not specified
Basis for effect level:
other: Embryo-fetal survival, fetal weight and ano-genital distance and development were unaffected by maternal treatment.

Fetal abnormalities

Abnormalities:
no effects observed

Overall developmental toxicity

Developmental effects observed:
no

Any other information on results incl. tables

The mean concentrations of pentyl propionate in formulation samples taken from the first and last preparation were within 8% of the nominal concentration, confirming the accuracy of formulation. The difference from mean remained within 2%, confirming precise analysis. Procedural recoveries remained within the validated range, confirming the continued accuracy of the analytical methodology.

Additional details of the study and historical control information can be found in the attachments. See "attached background information".

Applicant's summary and conclusion

Conclusions:
Oral administration of pentyl propionate at dose levels of 100, 330 or 1000 mg/kg bw/day to pregnant Sprague Dawley rats throughout organogenesis and the fetal growth phases was well tolerated. There was no effect of treatment on maternal clinical condition, body weight, food consumption, thyroid weight, macropathology or histopathology of the maternal thyroid. Embryo-fetal survival, fetal weight and ano-genital distance and development were unaffected by maternal treatment. There was no statistically significant effect on mean serum T4 or TSH concentrations, however, mean serum T3 levels at 330 or 1000 mg/kg bw/day were slightly but statistically significantly lower than controls, with no dose response apparent. In isolation, this difference was considered of uncertain relationship to treatment, and non adverse and of doubtful biological significance.
It was therefore concluded that the no observed adverse effect level (NOAEL) for maternal toxicity and embryo-fetal toxicity is 1000 mg/kg bw/day.
Executive summary:

The purpose of this study is to assess the influence of pentyl propionate, on embryo-fetal survival and development when administered during the organogenesis and fetal growth phases of pregnancy in the Sprague Dawley rat according to OECD 414.

Three groups of 20 females received pentyl propionate at doses of 100, 330 or 1000 mg/kg/day by oral gavage administration, from Day 6 to 19 after mating. A similarly constituted control group received the vehicle, corn oil, at the same volume dose as treated groups. Animals were killed on Day 20 after mating for reproductive assessment and fetal examination.

Clinical observations, body weights and food consumption were recorded. Adult females were examined macroscopically at necropsy on Day 20 after mating, blood samples were taken for thyroid hormone analysis and the gravid uterus weight and thyroid weight were recorded. Microscopic pathology investigations of the thyroids were also undertaken. Ano‑genital distance was measured for fetuses and all fetuses were examined macroscopically at necropsy and subsequently by detailed internal visceral examination or skeletal examination.

The mean concentrations of formulation samples taken from the first and last preparation were within 8% of the nominal concentration, confirming the accuracy of formulation. The difference from mean remained within 2%, confirming precise analysis.

The clinical condition of pregnant females receiving 100, 330 or 1000 mg/kg/day pentyl propionate were unaffected by treatment.

The overall group mean body weight performance and gain (Days 6-20) of females treated with pentyl propionate was comparable to controls. The mean gravid uterine weight for females treated with pentyl propionate was comparable to the mean gravid uterine weight from the control females, as was adjusted body weight gain.

Overall group mean food consumption of females that received 100, 330 or 1000 mg/kg/day pentyl propionate was comparable to controls throughout Days 6 to 20 of gestation.

The thyroids and parathyroids weights from pentyl propionate treated females were comparable to controls.

There were no adverse findings at macroscopic examination of females that were treated with pentyl propionate. No test item related microscopic findings were observed in the thyroid glands.

At scheduled termination, serum samples taken for thyroxine (T4), triiodothyronine (T3) and thyroid stimulating hormone (TSH) revealed thatmean serum T3 concentrations at 330 or 1000 mg/kg bw/day were found to be slightly but statistically significantly lower when compared with the control group. Mean serum T4 concentrations were found to be slightly lower at 330 or 1000 mg/kg bw/day, but did not attain statistical significance. The analysis of serum TSH concentrations performed at scheduled termination on Day 20 of gestation

revealed that the TSH level was similar at all dose levels when compared with the control group and no statistically significant differences were observed.

One female (animal No. 61) that received 1000 mg/kg/day pentyl propionate was found to be not pregnant at necropsy.

On Day 20 of gestation, mean numbers of corpora lutea, implantations, resorptions, the number of live young and sex ratio were considered to be unaffected by treatment with pentyl propionate.

Placental, total litter and fetal weights were unaffected by treatment with pentyl propionate.

The ano-genital distance of male and female fetuses were considered to be unaffected by maternal treatment with pentyl propionate.

The incidence of major and minor abnormalities and skeletal variants show no relationship to treatment.

There were no histopathological findings seen in the thyroid glands related to treatment with pentyl propionate.

Oral administration of pentyl propionate at dose levels of 100, 330 or 1000 mg/kg bw/day to pregnant Sprague Dawley rats throughout organogenesis and the fetal growth phases was well tolerated. There was no effect of treatment on maternal clinical condition, body weight, food consumption, thyroid weight, macropathology or histopathology of the maternal thyroid. Embryo-fetal survival, fetal weight and ano-genital distance and development were unaffected by maternal treatment.

It was therefore concluded that the no observed adverse effect level (NOAEL) for maternal toxicity and embryo-fetal toxicity is 1000 mg/kg bw/day.