1,2-Cyclohexanedicarboxylic Acid, 1-(phenylmethyl) ester, ester with 2,2,4-trimethyl, 1,3-petanediol mono(2-methyl propanoate)

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

03/10/2018 - 21/12/2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

The justification for read across is provided as an attachment in IUCLID Section 13.

Cross-reference

Reason / purpose for cross-reference:

read-across: supporting information

Reference

Endpoint:

developmental toxicity

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

03/10/2018 - 21/12/2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

The justification for read across is provided as an attachment in IUCLID Section 13.

Reason / purpose for cross-reference:

read-across source

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

One female administered 100 mg/kg/day (Animal B0114) died after dosing on GD 27, following clinical observations of gasping respiration. One control female (Animal B0021) was prematurely sacrificed on GD 25 following clinical observations of red discharge from the nose and laboured respiration. At necropsy, the animal was observed with dark lobes of the lung and gelatinous connective tissue surrounding the
oesophagus. The thoracic cavity had red fluid. The demise of these animals was considered inappropriate dosing technique. One female administered 1000 mg/kg/day (Animal B0301) was found dead on GD 23 with dark foci noted on all lobes of the lungs. The lungs inflated at necropsy (hence no rupture). Another female administered 1000 mg/kg/day (Animal B0319) was sacrificed prematurely on GD 22 due to clinical observations of gasping respiration, hunched posture and pale eyes. At necropsy, the lungs were dark, and dark/red frothy contents were noted along the length of the trachea. Given the macroscopic findings in the lungs noted at necropsy, and the deaths
observed at the lower dose levels, these deaths were most probably attributable to inappropriate dosing technique, unrelated to test article toxicity.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

There were no adverse effects on body weights or body weight changes by the test article at any dose level. However, there were slight increases in body weight changes noted toward the end of gestation following 1000 mg/kg/day administration. Although mean values did not attain statistical significance, and as such, were considered not to represent an adverse effect of the test article.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

An increase in mean food consumption was noted for females administered 1000 mg/kg/day, beginning on GD 15, which achieved statistical significance, later in gestation; GD 22-23 (P < 0.05), GD 26-27 (P < 0.01), GD 27-28 (P < 0.001) and GD 28 - 29 (P < 0.05), compared with controls. Mean food consumption towards the end of gestation was higher for females administered 300 mg/kg/day, with a statistically significant increase noted on GD 27 -28 (P < 0.05) compared with controls. These increases were, however, considered not to represent an adverse health effect.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

no effects observed

Description (incidence and severity):

Water consumption was monitored on regular basis throughout the study by visual inspection of the water bottles/containers.

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:

not examined

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Number of abortions:

effects observed, non-treatment-related

Description (incidence and severity):

One female administered 300 mg/kg/day (Animal B0205) exhibited signs of abortion on GD 25 and was sent to necropsy. All foetuses were late deaths at necropsy. No other macroscopic abnormalities were observed. Abortions are occasionally observed in rabbit studies of this type, and in isolation, this finding was considered to have arisen incidentally.

Pre- and post-implantation loss:

effects observed, non-treatment-related

Description (incidence and severity):

After exclusion of the data for the female with no viable foetuses, a dose-related increase in mean percentage post-implantation losses was observed, compared with controls (1.8%, 4.9%, 5.4% or 5.6% for animals administered 0, 100, 300 or
1000 mg/kg/day, respectively). Statistical analysis of these data did not achieve any statistical significance, and values were within the historical control ranges (mean 4.9% (SD 11.45); range 0 -31%); therefore, these intergroup differences were
considered to have arisen incidentally.

Total litter losses by resorption:

effects observed, non-treatment-related

Description (incidence and severity):

One female administered 300 mg/kg/day (Animal B0205) exhibited signs of abortion on GD 25 and was sent to necropsy. All foetuses were late deaths at necropsy. No other macroscopic abnormalities were observed. Abortions are occasionally observed in rabbit studies of this type, and in isolation, this finding was considered to have arisen incidentally.

Early or late resorptions:

no effects observed

Dead fetuses:

effects observed, non-treatment-related

Description (incidence and severity):

One female administered 300 mg/kg/day (Animal B0205) exhibited signs of abortion on GD 25 and was sent to necropsy. All foetuses were late deaths at necropsy. No other macroscopic abnormalities were observed. Abortions are occasionally observed in rabbit studies of this type, and in isolation, this finding was considered to have arisen incidentally.

Changes in pregnancy duration:

not examined

Changes in number of pregnant:

effects observed, non-treatment-related

Description (incidence and severity):

On GD 29, there were 20 pregnant females (with live foetuses) in each of the dose groups, including controls. One female administered 100 mg/kg/day was pregnant, but had no viable foetuses (in utero) at the end of the study. In isolation, and as this was also observed in the low dose, hence a lack of dose response, this finding was considered a low-incidence finding occasionally observed in studies of this type and was considered incidental. One female administered 300 mg/kg/day and one control female were found not pregnant at the end of the study. Animals are supplied time-mated from the breeders prior to any dosing; therefore, these non-pregnancies were unrelated to the test article.

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

Test article-related related weight changes were evident; although not statistically different, mean carcass weight, corrected body weight change, and total weight change were higher for dams administered 1000 mg/kg/day compared with controls at
caesarean section. These intergroup differences were considered not to represent an adverse effect test article administration.

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: no maternal effects observed

Key result

Abnormalities:

no effects observed

Fetal body weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Mean foetal weights were marginally elevated for foetuses from litters of dams administered 1000 mg/kg/day compared with controls (adjusted for litter size - males, 10%; females, 6%), although statistical significance was not achieved. Nevertheless, these findings were considered not to represent an adverse effect of maternal test article administration.

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:

not examined

External malformations:

effects observed, non-treatment-related

Skeletal malformations:

effects observed, non-treatment-related

Visceral malformations:

effects observed, non-treatment-related

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: teratogenic

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: embryofetal development

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

no

Dose Formulation Analyses

The mean % nominal concentration should be between 85% to 115% and with a relative standard deviation (RSD) 10.0%. Results were within these criteria. The test article was not detected in the control samples.

Conclusions:

The test material Santicizer 278 adminstered at 100, 300, or 1000 mg/kg/day, was well tolerated in the pregnant rabbit, with no clinical signs and no adverse effect on overall body weight, body weight gain, food consumption or on pregnancy or foetal parameters. Therefore, based on the above findings, the No Observed Adverse Effect Level (NOAEL) for maternal and embryo-foetal development was considered to be 1000 mg/kg/day.

Executive summary:

This data is being read across from the source study that tested Santicizer S278 (benzyl 3-(isobutyryloxy)-1-isopropyl-2,2-dimethylpropyl phthalate) based on analogue read across.

The objective of this study was to determine the effects of prenatal exposure of the test article, Benzyl 3-isobutyryloxy-1-isopropyl-2,2-dimethylpropyl phthalate, a Reaction Mass of Benzyl (1R,1S) 2,2,24-trimethyl-1-[(2‑methylpropanoyl)oxy]pentan-3-yl benzene-1,2-dicarboxylate and Benzyl (3R,3S) 2,2,4-trimethyl-3-[(2-methylpropanoyl)oxy]pentyl benzene-1,2-dicarboxylate, to the pregnant animal and on embryonic and foetal development. Four groups of 22 time-mated female New Zealand White: Hra:(NZW)SPF rabbits were administered 0 (Corn oil, vehicle), 100, 300, or 1000 mg/kg/day of the test article by oral gavage from Gestation Day (GD) 6 to 28, inclusive, at a dose volume of 1 mL/kg.

Assessment of toxicity was based on clinical observations (including postdose observations), body weights, and food consumption. Complete necropsies were performed on all animals, and any macroscopic abnormalities were recorded. The progress and outcome of pregnancy were evaluated, and foetuses were evaluated for malformations and variations.

No test article-related deaths occurred, however, there were 5 incidental mortalities on this study,one female administered 1000 mg/kg/day was sacrificed prematurely on GD 22, another female administered 1000 mg/kg/day was found dead on GD 23. One female administered 100 mg/kg/day died after dosing on GD 27. One control female was sacrificed on GD 25. These deaths were considered attributable to inappropriate dose administration and unrelated to test article toxicity. One female administered 300 mg/kg/day exhibited signs of abortion on GD 25 and so was sacrificed prior to scheduled termination. At termination, 20 females from each of the dose groups, including controls, were pregnant with live foetuses.

There were no test article-related clinical observations or postdose observations evident. No adverse effects on body weights or body weight change were noted. An increase in mean food consumption for females administered 1000 mg/kg/day, compared with controls was noted, but this was considered to be non-adverse. At the scheduled sacrifice, no test article-related macroscopic findings were noted. Marginally higher carcass weight, corrected body weight change, and total weight change were evident for females administered 1000 mg/kg/day, compared with controls, however, this was considered not to represent an adverse effect of test article administration. No test article-related effects were noted on pre- or post-implantation losses, litter size, sex ratio. Higher foetal weights were considered not to represent an adverse effect of test article administration, and no test article-related foetal malformations or variations were noted.

In conclusion, once daily oral gavage administration of 0, 100, 300, or 1000 mg/kg/day Benzyl 3‑isobutyryloxy-1-isopropyl-2,2-dimethylpropyl phthalate, a Reaction Mass of Benzyl (1R,1S) 2,2,24-trimethyl-1-[(2-methylpropanoyl)oxy]pentan 3-yl benzene-1,2-dicarboxylate and Benzyl (3R,3S) 2,2,4-trimethyl-3-[(2‑methylpropanoyl)oxy]pentyl benzene-1,2-dicarboxylate, was well tolerated in the pregnant rabbit, with no clinical signs and no adverse effect on overall body weight, body weight gain, food consumption or on pregnancy or foetal parameters.

Therefore, based on the above findings, the No Observed Adverse Effect Level (NOAEL) for maternal and embryo-foetal development was considered to be 1000 mg/kg/day.

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:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

not applicable

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

Deviations:

not applicable

Test guideline 3

Qualifier:

according to guideline

Guideline:

EU Method B.31 (Prenatal Developmental Toxicity Study)

Deviations:

not applicable

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Reaction Mass of Benzyl (1R,1S) 2,2,4-trimethyl-1-[(2-methylpropanoyl)oxy]pentan-3-yl benzene-1,2-dicarboxylate and Benzyl (3R,3S) 2,2,4-trimethyl-3-[(2-methylpropanoyl)oxy]pentyl benzene-1,2-dicarboxylate

EC Number:

701-008-3

Molecular formula:

C27H34O6

IUPAC Name:

Reaction Mass of Benzyl (1R,1S) 2,2,4-trimethyl-1-[(2-methylpropanoyl)oxy]pentan-3-yl benzene-1,2-dicarboxylate and Benzyl (3R,3S) 2,2,4-trimethyl-3-[(2-methylpropanoyl)oxy]pentyl benzene-1,2-dicarboxylate

Specific details on test material used for the study:

TEST MATERIAL NAME: Benzyl 3-isobutyryloxy-1-isopropyl-2,2-dimethylpropyl phthalate, a Reaction Mass of Benzyl (1R,1S) 2,2,4-trimethyl-1-[(2-methylpropanoyl)oxy]pentan-3-yl benzene-1,2-dicarboxylate and Benzyl (3R,3S) 2,2,4-trimethyl-3-[(2-methylpropanoyl)oxy]pentyl benzene-1,2-dicarboxylate
CAS Number: 16883-83-3
EC Number: 701-008-3
Molecular weight: 454.4
Trading name: Santicizer® 278
Batch number: 2984
Appearance: clear oily liquid
Molecular weight: 454.4 g/mol
Received on: 02 March 2018
Storage Temperature: 15 25°C protected from light
Purity: 98.51%
Retest date: 10 April 2019


The test article information and certificate of analysis provided by the Sponsor are considered an adequate description of the characterisation, purity and stability of the test article. Determinations of stability and characteristics of the test article were the responsibility of the Sponsor.

Test animals

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Covance Research Products, Denver, Pennsylvania, USA
- Age at study initiation: Not speciefied, however, at the time of mating, females weighed at least 2.7 kg.
- Weight at study initiation: Females weighed between 2.81 kg and 3.69 kg at the start of dosing
- Fasting period before study: Not specified
- Housing: Animals were individually housed in cages, suitable tray liners were provided and changed at least weekly.
- Diet (e.g. ad libitum): Animals had ad libitum access to LabDiet 5322 (PMI Nutrition International). Each batch of diet was analysed for specific constituents and contaminants. Upon arrival and on some occasions when low food consumption was observed, animals were provided with moist hay or approximately 50 g of moistened diet. This was not included in any food consumption calculations.No contaminants were present in the diet at levels which might have interfered with achieving the objective of the study.
- Water (e.g. ad libitum): Water from the main tap supply was provided ad libitum via water bottles. The water is periodically analysed for specific contaminants. No contaminants were present in the water at levels which might have interfered with achieving the objective of the study.
- Acclimation period: Animals were delivered to Covance by GD 3, upto 3 days acclimatisation.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 15 °C to 21°C
- Humidity (%): >45%
- Air changes (per hr): Rooms were air-conditioned to provide a minimum of 15 air changes/hour.
- Photoperiod (hrs dark / hrs light): 12 hour light/12 hour dark cycle

IN-LIFE DATES: From: 03/10/2018 to 21/12/2018 (Completion of in-life phase)

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Formulations were prepared weekly and were frozen (<10°C) until required. Formulations were thawed prior to dosing and assigned a 6-hour expiry from time of thawing.

The test article was formulated as a suspension in corn oil following Dispensary SOPs and the formulation method (Method 8381237_O_01D), as maintained in the study data.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Based on trial preparations performed at the Test Facility to select a suitable vehicle and to establish a suitable formulation procedure.
- Concentration in vehicle: Group 1 (Control): 0 mg/mL; Group 2: 100 mg/mL; Group 3: 300 mg/mL; Group 4: 1000 mg/mL.
- Amount of vehicle (if gavage): 1 mL/Kg
- Lot/batch no. (if required): Not specified

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analyses were performed by using a validated analytical procedure.

Concentration Analysis:
Formulations prepared for use on the first and last day of dosing were analysed to determine the achieved concentration. Triplicate samples were removed from the middle of the test article formulations and analysed. A single sample was taken from the middle of vehicle formulations and was analysed.

Stability and Homogeneity Analysis:

Test article formulations at 1 mg/mL concentration have been shown to be stable for 6 hours at room temperature (15 to 25°C) and at least 12 days in frozen storage (<-10°C) and were homogenous (Covance Study 8381238).

Test article formulations at a concentration of 1000 mg/mL have been shown to be stable for up to 12 days at room temperature (15 to 25°C) and in frozen storage (<-10°C) and were homogenous (Covance Study 8381238).

Details on mating procedure:

Eighty-eight time-mated female New Zealand White: Hra:(NZW)SPF rabbits were obtained from Covance Research Products, Denver, Pennsylvania, United States of America, in order to provide sufficient animals for study selection.

At the time of mating, females weighed at least 2.7 kg. Each female was mated with one proven male at the supplier’s laboratory. The day on which mating was observed was designated as Gestation Day (GD) 0. Animals were delivered to Covance by GD 3. Females weighed between 2.81 kg and 3.69 kg at the start of dosing.

Upon arrival, all animals were given a clinical inspection for ill health. Acclimation was limited by mated status, and an inspection was performed by the Named Animal Care and Welfare Officer (NACWO) before the start of dosing to ensure suitability for the study.

Duration of treatment / exposure:

From Day 6 to Day 28 post-coitum, inclusive. A dose volume of 1 mL/kg was used, and dose volumes were calculated from the most recently recorded body weight for each animal.

Frequency of treatment:

Once daily, 7 days a week

Duration of test:

Once daily oral gavage 7 days a week from Day 6 to Day 28 post-coitum, inclusive.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Control

Doses / concentrations 2

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

Low Dose

Doses / concentrations 3

Dose / conc.:

300 mg/kg bw/day (nominal)

Remarks:

Medium Dose

Doses / concentrations 4

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

High Dose

No. of animals per sex per dose:

22 females/dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The oral route of exposure was selected because this is a possible route of human exposure during manufacture, handling or use of the test material.

The dose level selection was based on a previous dose range-finding study (Covance Study 8381236) where the test article was administered to the pregnant New Zealand White rabbit at dose levels of 350, 750, and 1000 mg/kg/day. Body weight, body weight change, or food consumption were unaffected by test article administration at any dose level. Implantation sites, litter sizes, or foetal weights were unaffected, and no test article-related foetal malformations or variations were noted. Therefore, the high dose level of 1000 mg/kg/day was selected and is also the limit dose for this study type. The intermediate dose level of 300 mg/kg/day represents a dose level approximately 3-fold lower than the high dose. The low dose level of 100 mg/kg/day represents a dose level approximately 3-fold lower than the intermediate dose, and anticipated to be the no observed adverse effect level.

- Rationale for animal assignment (if not random): Animals were assigned to dose groups based on GD 0 body weight data obtained from the supplier (i.e., all animals confirmed as mated on a specific day were randomised to groups based on body weight). Animals were individually identified by electronic implant. Cages were placed in dose group order across the batteries.

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily, in the morning and at the end of the working day (animals were observed for general health/mortality and moribundity)

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at least once daily, beginning on Day 6 post-coitum and lasting up to the day prior to necropsy.

BODY WEIGHT: Yes
- Time schedule for examinations: Animals were individually weighed on Days 3, 6, 8, 9, 12, 15, 17, 19, 22, 25, 28, and 29 post-coitum.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Food consumption for each animal determined and mean daily diet consumptiony: Yes (Food consumption was quantitatively measured for once daily from 4 post-coitum until necropsy. Food consumption was calculated as g/animal/day).
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No data

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: Water consumption was monitored on regular basis throughout the study by visual inspection of the water bottles/containers.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 29 post-coitum

Unscheduled Deaths: Terminal procedures were also followed for animals that died or were sacrificed at an unscheduled interval.

Scheduled Euthanasia:
Females were sacrificed in replicate order. Food was not removed prior to scheduled necropsies. Females were sacrificed on GD 29 by an intravenous injection (overdose) of sodium pentobarbitone.

Scheduled necropsy was conducted on the following days:
Females surviving to planned necropsy: Day 29 post-coitum

Necropsy: Females were sacrificed on GD 29 by an intravenous injection (overdose) of sodium pentobarbitone. Immediately subsequent to this, and once death had been confirmed, the major blood vessels were severed to exsanguinate the animal. All lesions were recorded and retained in the relevant fixative. The ovaries and uteri were removed and examined.

Ovaries and uterine content:

Females were sacrificed on GD 29 by an intravenous injection (overdose) of sodium pentobarbitone. Immediately subsequent to this, and once death had been confirmed, the major blood vessels were severed to exsanguinate the animal. All lesions were
recorded and retained in the relevant fixative. The ovaries and uteri were removed and examined, and the following data were recorded.

Pregnancy status
Gravid uterus weight
Terminal body weight (recorded for adjusted gravid uterus weight calculations
only and have not been reported)
Number of corpora lutea
Number and intrauterine position of implantations, subdivided into:
Live foetuses
Early intrauterine deaths
Late intrauterine deaths
Dead foetuses

Early intrauterine deaths were classified as those which showed decidual or placental tissue only. Late intrauterine deaths showed embryonic or foetal tissue in addition to placental tissue. Dead foetuses were classified as those that appeared to have died
shortly before necropsy. Implantations and foetues were sequentially allocated numbers commencing with the site nearest to the left ovary.

The uterus of any apparently non-pregnant female was immersed in a 10% ammonium sulphide solution to reveal any evidence of implantation.

Fetal examinations:

Live foetuses were sacrificed by an intraperitoneal injection of sodium pentobarbitone (overdose), followed by confirmation of cessation of circulation. Individual foetal and placental weights were recorded, and foetuses were examined externally and sexed internally. All foetuses in each litter were examined for visceral and heart abnormalities by micro-dissection; foetuses were eviscerated, and carcasses were processed for skeletal examination. The foetal carcasses for skeletal preparations were processed to clear the soft tissue and stain the ossified bone with Alizarin Red S, examined in 50% glycerol and retained in glycerol/propylene glycol. Intact heads of approximately one-half of the foetuses/litter were removed (after foetuses were sacrificed) and processed in Bouin’s fluid. Heads were then examined by the Wilson sectioning method. Foetal head sections were retained in 10% neutral-buffered formalin.

Statistics:

Please see 'Any other information on materials and methods incl. tables' for information on Statistics.

Indices:

A number of indices were used, where appropriate, to evaluate reproductive function:

% Pre-Implantation Loss = Number of corpora lutea - number of implantations / Number of corpora lutea X 100
% Post-Implantation Loss = Number of implantations - number of live embryos / Number of implantations X 100
Corrected Body Weight (Carcass Weight) = Terminal body weight - uterine weight
Corrected Weight Change = Carcass weight - GD 3 body weight
Total Weight Change = GD 29 body weight - GD 3 body weight
% Male Fetuses = Number of male fetuses / Number of fetuses of determined sex X 100

Historical control data:

Historical Control Data of Caesarian section data presented in Annex in Study Report (Please see 'Attached background material').

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

One female administered 100 mg/kg/day (Animal B0114) died after dosing on GD 27, following clinical observations of gasping respiration. One control female (Animal B0021) was prematurely sacrificed on GD 25 following clinical observations of red discharge from the nose and laboured respiration. At necropsy, the animal was observed with dark lobes of the lung and gelatinous connective tissue surrounding the
oesophagus. The thoracic cavity had red fluid. The demise of these animals was considered inappropriate dosing technique. One female administered 1000 mg/kg/day (Animal B0301) was found dead on GD 23 with dark foci noted on all lobes of the lungs. The lungs inflated at necropsy (hence no rupture). Another female administered 1000 mg/kg/day (Animal B0319) was sacrificed prematurely on GD 22 due to clinical observations of gasping respiration, hunched posture and pale eyes. At necropsy, the lungs were dark, and dark/red frothy contents were noted along the length of the trachea. Given the macroscopic findings in the lungs noted at necropsy, and the deaths
observed at the lower dose levels, these deaths were most probably attributable to inappropriate dosing technique, unrelated to test article toxicity.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

There were no adverse effects on body weights or body weight changes by the test article at any dose level. However, there were slight increases in body weight changes noted toward the end of gestation following 1000 mg/kg/day administration. Although mean values did not attain statistical significance, and as such, were considered not to represent an adverse effect of the test article.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

An increase in mean food consumption was noted for females administered 1000 mg/kg/day, beginning on GD 15, which achieved statistical significance, later in gestation; GD 22-23 (P < 0.05), GD 26-27 (P < 0.01), GD 27-28 (P < 0.001) and GD 28 - 29 (P < 0.05), compared with controls. Mean food consumption towards the end of gestation was higher for females administered 300 mg/kg/day, with a statistically significant increase noted on GD 27 -28 (P < 0.05) compared with controls. These increases were, however, considered not to represent an adverse health effect.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

no effects observed

Description (incidence and severity):

Water consumption was monitored on regular basis throughout the study by visual inspection of the water bottles/containers.

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:

not examined

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Maternal developmental toxicity

Number of abortions:

effects observed, non-treatment-related

Description (incidence and severity):

One female administered 300 mg/kg/day (Animal B0205) exhibited signs of abortion on GD 25 and was sent to necropsy. All foetuses were late deaths at necropsy. No other macroscopic abnormalities were observed. Abortions are occasionally observed in rabbit studies of this type, and in isolation, this finding was considered to have arisen incidentally.

Pre- and post-implantation loss:

effects observed, non-treatment-related

Description (incidence and severity):

After exclusion of the data for the female with no viable foetuses, a dose-related increase in mean percentage post-implantation losses was observed, compared with controls (1.8%, 4.9%, 5.4% or 5.6% for animals administered 0, 100, 300 or
1000 mg/kg/day, respectively). Statistical analysis of these data did not achieve any statistical significance, and values were within the historical control ranges (mean 4.9% (SD 11.45); range 0 -31%); therefore, these intergroup differences were
considered to have arisen incidentally.

Total litter losses by resorption:

effects observed, non-treatment-related

Description (incidence and severity):

One female administered 300 mg/kg/day (Animal B0205) exhibited signs of abortion on GD 25 and was sent to necropsy. All foetuses were late deaths at necropsy. No other macroscopic abnormalities were observed. Abortions are occasionally observed in rabbit studies of this type, and in isolation, this finding was considered to have arisen incidentally.

Early or late resorptions:

no effects observed

Dead fetuses:

effects observed, non-treatment-related

Description (incidence and severity):

One female administered 300 mg/kg/day (Animal B0205) exhibited signs of abortion on GD 25 and was sent to necropsy. All foetuses were late deaths at necropsy. No other macroscopic abnormalities were observed. Abortions are occasionally observed in rabbit studies of this type, and in isolation, this finding was considered to have arisen incidentally.

Changes in pregnancy duration:

not examined

Changes in number of pregnant:

effects observed, non-treatment-related

Description (incidence and severity):

On GD 29, there were 20 pregnant females (with live foetuses) in each of the dose groups, including controls. One female administered 100 mg/kg/day was pregnant, but had no viable foetuses (in utero) at the end of the study. In isolation, and as this was also observed in the low dose, hence a lack of dose response, this finding was considered a low-incidence finding occasionally observed in studies of this type and was considered incidental. One female administered 300 mg/kg/day and one control female were found not pregnant at the end of the study. Animals are supplied time-mated from the breeders prior to any dosing; therefore, these non-pregnancies were unrelated to the test article.

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

Test article-related related weight changes were evident; although not statistically different, mean carcass weight, corrected body weight change, and total weight change were higher for dams administered 1000 mg/kg/day compared with controls at
caesarean section. These intergroup differences were considered not to represent an adverse effect test article administration.

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: no maternal effects observed

Maternal abnormalities

Key result

Abnormalities:

no effects observed

Results (fetuses)

Fetal body weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Mean foetal weights were marginally elevated for foetuses from litters of dams administered 1000 mg/kg/day compared with controls (adjusted for litter size - males, 10%; females, 6%), although statistical significance was not achieved. Nevertheless, these findings were considered not to represent an adverse effect of maternal test article administration.

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:

not examined

External malformations:

effects observed, non-treatment-related

Skeletal malformations:

effects observed, non-treatment-related

Visceral malformations:

effects observed, non-treatment-related

Effect levels (fetuses)

1

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: teratogenic

2

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: embryofetal development

Fetal abnormalities

Key result

Abnormalities:

no effects observed

Overall developmental toxicity

Key result

Developmental effects observed:

no

Any other information on results incl. tables

Dose Formulation Analyses

The mean % nominal concentration should be between 85% to 115% and with a relative standard deviation (RSD) 10.0%. Results were within these criteria. The test article was not detected in the control samples.

Applicant's summary and conclusion

Conclusions:

The test material Santicizer 278 adminstered at 100, 300, or 1000 mg/kg/day, was well tolerated in the pregnant rabbit, with no clinical signs and no adverse effect on overall body weight, body weight gain, food consumption or on pregnancy or foetal parameters. Therefore, based on the above findings, the No Observed Adverse Effect Level (NOAEL) for maternal and embryo-foetal development was considered to be 1000 mg/kg/day.

Executive summary:

The objective of this study was to determine the effects of prenatal exposure of the test article, Benzyl 3-isobutyryloxy-1-isopropyl-2,2-dimethylpropyl phthalate, a Reaction Mass of Benzyl (1R,1S) 2,2,24-trimethyl-1-[(2‑methylpropanoyl)oxy]pentan-3-yl benzene-1,2-dicarboxylate and Benzyl (3R,3S) 2,2,4-trimethyl-3-[(2-methylpropanoyl)oxy]pentyl benzene-1,2-dicarboxylate, to the pregnant animal and on embryonic and foetal development. Four groups of 22 time-mated female New Zealand White: Hra:(NZW)SPF rabbits were administered 0 (Corn oil, vehicle), 100, 300, or 1000 mg/kg/day of the test article by oral gavage from Gestation Day (GD) 6 to 28, inclusive, at a dose volume of 1 mL/kg.

Assessment of toxicity was based on clinical observations (including postdose observations), body weights, and food consumption. Complete necropsies were performed on all animals, and any macroscopic abnormalities were recorded. The progress and outcome of pregnancy were evaluated, and foetuses were evaluated for malformations and variations.

No test article-related deaths occurred, however, there were 5 incidental mortalities on this study,one female administered 1000 mg/kg/day was sacrificed prematurely on GD 22, another female administered 1000 mg/kg/day was found dead on GD 23. One female administered 100 mg/kg/day died after dosing on GD 27. One control female was sacrificed on GD 25. These deaths were considered attributable to inappropriate dose administration and unrelated to test article toxicity. One female administered 300 mg/kg/day exhibited signs of abortion on GD 25 and so was sacrificed prior to scheduled termination. At termination, 20 females from each of the dose groups, including controls, were pregnant with live foetuses.

There were no test article-related clinical observations or postdose observations evident. No adverse effects on body weights or body weight change were noted. An increase in mean food consumption for females administered 1000 mg/kg/day, compared with controls was noted, but this was considered to be non-adverse. At the scheduled sacrifice, no test article-related macroscopic findings were noted. Marginally higher carcass weight, corrected body weight change, and total weight change were evident for females administered 1000 mg/kg/day, compared with controls, however, this was considered not to represent an adverse effect of test article administration. No test article-related effects were noted on pre- or post-implantation losses, litter size, sex ratio. Higher foetal weights were considered not to represent an adverse effect of test article administration, and no test article-related foetal malformations or variations were noted.

In conclusion, once daily oral gavage administration of 0, 100, 300, or 1000 mg/kg/day Benzyl 3‑isobutyryloxy-1-isopropyl-2,2-dimethylpropyl phthalate, a Reaction Mass of Benzyl (1R,1S) 2,2,24-trimethyl-1-[(2-methylpropanoyl)oxy]pentan 3-yl benzene-1,2-dicarboxylate and Benzyl (3R,3S) 2,2,4-trimethyl-3-[(2‑methylpropanoyl)oxy]pentyl benzene-1,2-dicarboxylate, was well tolerated in the pregnant rabbit, with no clinical signs and no adverse effect on overall body weight, body weight gain, food consumption or on pregnancy or foetal parameters.

Therefore, based on the above findings, the No Observed Adverse Effect Level (NOAEL) for maternal and embryo-foetal development was considered to be 1000 mg/kg/day.