Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

An extended one-generation reproductive toxicity study (EOGRTS) using the study design outlined in EU method B.56/OECD 443 - cohorts 1A and 1B with extension to include the F2 generation and cohorts 2A and 2B (developmental neurotoxicity) for Santicizer S278 is planned and was approved based on ECHA communication/decision number TPE-D-2114516050-69-01/F. Therefore, this endpoint will be updated on availability of data upon study completion.

Effects on developmental toxicity

Description of key information

A OECD 414 study (Harlan, 2014) conducted on 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 is available. The test material Santicizer 278 was administrated to pregnant female rats from day 6 post coitum by continuous dietary exposure at concentration of 100, 500 and 1000 mg/kg/day. No mortality was recorded at any of the dose tested. Treatment caused slightly lower body weight in all dose groups but did not deviate more than 10% from the Control group. A dose-dependent differences was recorded in relative liver weights. This may be related to hypertrophy commonly observed with phthalate esters as a class of peroxisome proliferators. In the observations deriving from the hysterectomies, no test-item related embryofetal toxicity was observed at any of the administered doses. No effects on mean foetal weight was per litter basis was observed. At the external examination, one litter from 1000 mg/kg/day showed one foetus with a supernumerary digit on the left hind paw (variation). Furthermore, one litter from the Control group had one foetus with head abnormalities, including no recognizable nose, naris, eyes, mouth or mandible; two protuberances from the forehead and edema in the hind paw (abnormalities). There was no evidence of a compound-related increase in the occurrence of these findings. The skeletal examination of the foetuses did not reveal any toxicologically relevant alterations. The slight delay in ossification (skull bones and sternebrae) recorded in some litters from 1000 mg/kg/day compared to the Control group should be considered a variation without pathological meaning. The visceral examination revealed some abnormalities, however there was no evidence of a test-related increase in their occurrence. Furthermore, common variations mainly involving urogenital morphology and other findings were recorded in all the groups including Control. These alterations should be regarded as spontaneous variations with limited pathological relevance. Based on the results of this study, the dose of 500 mg/kg/day is considered the No Observed Adverse Effect Level (NOAEL) for pregnant females due to the slightly lower body-weight gain recorded at 1000 mg/kg/day compared to the Control group. With respect to the effects on embryofetal development, 1000 mg/kg/day is considered to be the No Observed Adverse Effect Level (NOAEL). Regarding the potential for teratogenic effects, 1000 mg/kg/day is considered to be the No Observed Adverse Effect Level (NOAEL).

A recent OECD 414 study (Covance, 2019) conducted on 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 is available. 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.

 

Link to relevant study records

Referenceopen allclose all

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
24 March 2014 - 15 July 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
yes
Remarks:
Deviations had no impact on the study results
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
- Identification: Santicizer® 278
- Batch: 130521
- Purity: 98%
- CAS no.: 16883-83-3
- Arrival Date: 4 November 2013
- Production Date: 28 October 2013
- Expiry / Retest Date: 10 years after the manufacturing date
- Storage Conditions: At room temperature (20 ± 5 ºC), protected from humidity and in the dark
- Safety Precautions: Routine hygienic procedures (gloves, goggles, face mask)
No correction for purity was made.
Species:
rat
Strain:
Wistar
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories Models, S.L.,Harlan Laboratories, B.V., Postbus 553, 5800AN Venray, Netherlands
- Age at study initiation: 11 weeks minimum
- Weight at study initiation: 185-293 g
- Housing: Cages with standard, granulated, softwood Lignocel S8/15 bedding (supplied by Harlan Laboratories Models, S.L.).
- Diet (e.g. ad libitum): Powder standard rat/mouse maintenance diet ad libitum V1530 (supplied by Ssniff)
- Water (e.g. ad libitum): Tap water ad libitum in bottles.
- Acclimation period: At least five days minimum prior to mating under test conditions.
- Sex: Males and females (nulliparous and nonpregnant)

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18-24 ºC
- Humidity (%): 40-60%
- Air changes (per hr): 15-20 air changes per hour
- Photoperiod (hrs dark / hrs light): 12-hours light and 12 hours dark

IN-LIFE DATES: From: To: 19 March 2014 - 15 July 2014
Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on exposure:
DIET PREPARATION
- Dose formulation concentrations (ppm): calculation based on an estimation of the daily food consumption and body weight in order to achieve the target dose levels.
- Mixing appropriate amounts with (Type of food): dietary admixture
- Storage temperature of food: Feed preparations were stored at room temperature (20 ± 5 ºC) in the dark up to 8 days.


Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples were analyzed and results were available before formulations were administered to the animals.
The formulations were quantified following the analytical method developed in Study S46126 (Dose Range Finder). The homogeneity and accuracy of the formulations as well as their stability were verified in the present study.The acceptance range was 100 ± 20 % of nominal content.

Details on mating procedure:
- Mating details: Females and males were housed in a cage when sexually mature for approximately 16 hours. At least 22 different males participated in each dose group.
- Vaginal smears were taken daily until mating was confirmed.
- M/F ratio per cage: 1/1
- Length of cohabitation: 16 hr
- Proof of pregnancy: 1. vaginal smear was sperm-positive 2. Copulation plug was observed.
- The day of mating was designated day 0 post coitum.
Duration of treatment / exposure:
From Day 6 post coitum (first exposure) to cesarean section - on Day 20 post coitum (last exposure)
Frequency of treatment:
Ad Libitum
Duration of test:
From Day 6 post coitum (first exposure) to cesarean section - on Day 20 post coitum (last exposure)
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Control
Dose / conc.:
100 mg/kg bw/day (nominal)
Remarks:
Low dose
Dose / conc.:
500 mg/kg bw/day (nominal)
Remarks:
Medium dose
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Remarks:
High dose
No. of animals per sex per dose:
Females
Group 1: 1-22
Control: 1-22
Group 2: 23-44
Group 3: 45-66
Group 4: 67-98
Control animals:
yes, concurrent no treatment
Details on study design:
Rational for the dose level selection: The dose levels were selected based on a Range-Finding study in Wistar rats: Study No. S46104.
Maternal examinations:
Morbidity / Mortality: Twice daily
Clinical Signs: Detailed clinical signs for signs of reaction to treatment and/or symptoms of ill health during days 6-19 post coitum. Daily cage signs in remaining days.
Food Consumption: Periods: days 0-3, 3-6, 6-9, 9-12, 12-15, 15-18 and 18-20
Body Weights: Daily
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Postmortem examination, including gross macroscopic examination of all internal organs with emphasis on the uterus, uterine contents, position of fetuses in the uterus and the number of corpora lutea was performed and the data recorded. The uteri (and contents) of all females was weighed during necropsy on day 20 post coitum to enable the calculation of the corrected bodyweight gain.


Fetal examinations:
Fetuses were removed from the uterus, sexed, weighed individually, examined for gross external abnormalities They were serially sectioned and examined (evaluation of the internal structures of the heads, thoracic and abdominal organs). The tissue was preserved in a solution of 96% ethyl alcohol in
individual containers. Descriptions of any abnormalities and variations were recorded. 2. The remaining fetuses were eviscerated and fixed in 70% alcohol. Then they were placed in a solution of potassium hydroxide with Alizarin red S (for clearing and staining ossified bone) and a solution of glycerin/alcohol for preservation and storage (Dawson, 1926). The skeletons were examined and all abnormal findings and variations were recorded. The specimens were individually preserved.
Statistics:
Statistical methods used:
• The Dunnett-test (Dunnett, 1955)
• The Steel-test (Miller, 1981)
• Fisher's exact-test (Fisher, 1950)
Indices:
Pre- and post-implantation losses, embryonic and fetal deaths, live and dead fetuses, abnormal fetuses, fetal sex ratios and fetal body weights were calculated from the on-line recorded reproduction data.

For reproduction data, group mean values were calculated both on a litter basis and on a percentage per group basis. Mean fetal weights were calculated from the individual weights per group and per litter.



Clinical signs:
no effects observed
Description (incidence and severity):
No treatment- related clinical signs were recorded.

Mortality:
no mortality observed
Description (incidence):
No mortality was recorded among the females treated with the test item.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Body-weight gain (%) was dose dependently lower in all test item groups than in the control group. These differences were statistically significant from day 7 post coitum onwards at 1000 mg/kg/day on days 7, 10 to 12, 14 to 17 and 19 to 20 at 500 mg/kg/day and on day 14 post coitum at 100 mg/kg/day.
There were no significant differences in the weight of the gravid uterus; however the adjusted body-weight gain was statistically lower in females belonging to the high-dose group.

Treatment caused slightly lower body weight in all dose groups but did not deviate more than 10% from the Control group. In addition, the dose-dependent differences recorded in relative liver weights may be related to hypertrophy commonly observed with phthalate esters as a class of peroxisome proliferators.



Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
Higher food consumption was occasionally observed at 1000 mg/kg/day with respect to the Control group. Statistically significant differences were recorded from days 6-9 and 18-20 post coitum in absolute and relative values and from days 12-15 in relative values.
No differences with respect to the control group were recorded in the remaining groups.

Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
Significantly higher relative liver weights were observed at 500 and 1000 mg/kg/day with respect to Control females. No differences were recorded at 100 mg/kg/day in relative liver weight, and no differences in absolute liver weight were recorded at any dose level.
Significantly lower kidney weights were recorded at 1000 mg/kg/day compared to the control group. No differences were recorded in relative mean values.
At 100 and 500 mg/kg/day, the kidney weights were comparable to the control group.

Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
0 and 100 mg/kg/day: no findings were recorded.
500 mg/kg/day: female no. 48 had right kidney with enlarged, pale and misshapen cranial region, dilated pelvis and pale medulla. In addition, female no. 63 had enlarged right kidney and small and pale left kidney.
1000 mg/kg/day: female no. 72 had yellowish stomach contents.
These findings could be considered of no toxicological relevance and within the range of normal background lesions that may be seen in rats of this strain.




Neuropathological findings:
not specified
Number of abortions:
no effects observed
Pre- and post-implantation loss:
effects observed, non-treatment-related
Description (incidence and severity):
One female (no. 10) at control group and one (female no. 86) at 1000 mg/kg/day had 100% postimplantation losses. No relevant test-item-related differences were recorded in postimplantation losses or total fetuses.
Total litter losses by resorption:
no effects observed
Description (incidence and severity):
Female no. 88 at 1000 mg/kg/day showed remains of blood in vagina on day 13 post coitum. This female had three embryonic resorptions at cesarean section. Remaining implantation sites were live fetuses.
Early or late resorptions:
no effects observed
Description (incidence and severity):
Female no. 88 at 1000 mg/kg/day showed remains of blood in vagina on day 13 post coitum. This female had three embryonic resorptions at cesarean section. Remaining implantation sites were live fetuses.
Dead fetuses:
no effects observed
Changes in pregnancy duration:
no effects observed
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): no effects observed
Changes in number of pregnant:
effects observed, non-treatment-related
Description (incidence and severity):
The following females were not pregnant:

Group 1 Control: 11
Group 2 Low dose: 25, 29, 30, 38, 92
Group 3 Mid dose: 53, 60, 61, 62, 66
Group 4 High dose: 67, 76, 97

Other effects:
no effects observed
Details on maternal toxic effects:
-Viability / Mortality
No mortality was recorded.

-Signs of Reaction to Treatment
Female no. 88 at 1000 mg/kg/day showed remains of blood in vagina on day 13 post coitum. This female had three embryonic resorptions at cesarean section. Remaining implantation sites were live fetuses. In addition, some findings in skin/fur were recorded in two females from Control, one from 100 and three from 500 mg/kg/day groups.
These findings (scabs and/or nodules) were considered incidental.

-Food Consumption
Higher food consumption was occasionally observed at 1000 mg/kg/day with respect to the Control group. Statistically significant differences were recorded from days 6-9 and 18-20 post coitum in absolute and relative values and from days 12-15 in relative values.
No differences with respect to the control group were recorded in the remaining groups.

-Body Weights
Body-weight gain (%) was dose dependently lower in all test item groups than in the control group. These differences were statistically significant from day 7 post coitum onwards at 1000 mg/kg/day on days 7, 10 to 12, 14 to 17 and 19 to 20 at 500 mg/kg/day and on day 14 post coitum at 100 mg/kg/day.
There were no significant differences in the weight of the gravid uterus; however the adjusted body-weight gain was statistically lower in females belonging to the high-dose group.

-Necropsy Findings
No findings were recorded in the Control group or at 100 mg/kg/day. At 500 mg/kg/day, female no. 48 had right kidney with enlarged, pale and misshapen cranial region, dilated pelvis and pale medulla. In addition, female no. 63 had enlarged right kidney and small and pale left kidney. At 1000 mg/kg/day, female no. 72 had yellowish stomach contents. These findings could be considered of no toxicological relevance and within the range of normal background lesions that may be seen in rats of this strain.

-Organ Weights
Significantly higher relative liver weights were observed at 500 and 1000 mg/kg/day with respect to Control females. No differences were recorded at 100 mg/kg/day in relative liver weight, and no differences in absolute liver weight were recorded at any dose level.
Significantly lower kidney weights were recorded at 1000 mg/kg/day compared to the control group. No differences were recorded in relative mean values.
At 100 and 500 mg/kg/day, the kidney weights were comparable to the control group.














Dose descriptor:
NOAEL
Remarks:
pregnant female
Effect level:
500 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
body weight and weight gain
Fetal body weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
The body weight of fetuses was significantly lower in the control group than at 100 and 1000 mg/kg/day when analyzed on an individual basis. These differences were no longer found when body weight was evaluated on a per litter basis.
Litter no. 85 from 1000 mg/kg/day showed all fetuses with lower weight compared to those in the other litters of the group but this was considered to be a chance finding that was unrelated to treatment.
No differences were recorded in fetal body weight at 500 mg/kg/day either on an individual or per litter basis.
Changes in sex ratio:
no effects observed
Description (incidence and severity):
No treatment-related differences were recorded in sex ratio or distribution of males and femin the uterus content compared to the Control group. The differences recorded at 100 mg/kg/day were considered incidental.
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
No treatment-related findings were recorded.

- Group 1: Control group
Litter no. 3 had two fetuses (no. 211 and 212) with fused placenta (variation). Litter no. 15 had one fetus (no. 667) with head abnormalities including no recognizable nose, naris, eyes, mouth or mandible; two protuberances from the forehead and edema in the hind paw (abnormalities).

Group 2: 100 mg/kg/day
No findings were recorded.

Group 3: 500 mg/kg/day
No findings were recorded.

Group 4: 1000 mg/kg/day
Litter no. 69 had one fetus (no. 151) with hematoma in neck (variation).
Litter no. 78 had one fetus (no. 285) with a supernumerary digit between toes 1 and 2 of left hind paw (abnormality).
No further findings were recorded in the remaining fetuses examined.

Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
No toxicologically relevant alterations were observed in the skeletal examination.

There was a slightly higher percentage of litters with lower ossification of some skull bones and sternebrae at 1000 mg/kg/day compared to the Control group. Although some differences recorded were statistically significant, the findings observed were not considered of pathological relevance.
The skeletal examination revealed a range of variations in all groups. There was no indication of a test-item-related trend in the type or incidence of these findings.
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
No test–item-related findings were observed in the visceral examination.
The incidence of common variations such as dilated renal pelvis, malpositioned kidney, dilated and/or convoluted ureter, dilated stomach, left-side umbilical artery, long cranial thymus, malpositioned testis and kidney, additional small lobe in the liver was similar in all groups including control group.
Details on embryotoxic / teratogenic effects:
No treatment - related effects
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
Conclusions:
The test material Santicizer 278 administrated to female rats at 100, 500, 1000 mg/kg/day did not show embryofetal development nor teratogenic effects at any of the selected doses.
Executive summary:

The test material Santicizer 278 was administrated to pregnant female rats from day 6 post coitum by continuous dietary exposure at concentration of 100, 500 and 1000 mg/kg/day. No mortality was recorded at any of the dose tested. Treatment caused slightly lower body weight in all dose groups but did not deviate more than 10% from the Control group. A dose-dependent differences was recorded in relative liver weights. This may be related to hypertrophy commonly observed with phthalate esters as a class of peroxisome proliferators. In the observations deriving from the hysterectomies, no test-item related embryofetal toxicity was observed at any of the administered doses. No effects on mean foetal weight was per litter basis was observed. At the external examination, one litter from 1000 mg/kg/day showed one foetus with a supernumerary digit on the left hind paw (variation). Furthermore, one litter from the Control group had one foetus with head abnormalities, including no recognizable nose, naris, eyes, mouth or mandible; two protuberances from the forehead and edema in the hind paw (abnormalities). There was no evidence of a compound-related increase in the occurrence of these findings. The skeletal examination of the foetuses did not reveal any toxicologically relevant alterations. The slight delay in ossification (skull bones and sternebrae) recorded in some litters from 1000 mg/kg/day compared to the Control group should be considered a variation without pathological meaning.

The visceral examination revealed some abnormalities, however there was no evidence of a test-related increase in their occurrence. Furthermore, common variations mainly involving urogenital morphology and other findings were recorded in all the groups including Control. These alterations should be regarded as spontaneous variations with limited pathological relevance.

Based on the results of this study, the dose of 500 mg/kg/day is considered the No Observed Adverse Effect Level (NOAEL) for pregnant females due to the slightly lower body-weight gain recorded at 1000 mg/kg/day compared to the Control group. With respect to the effects on embryofetal development, 1000 mg/kg/day is considered to be the No Observed Adverse Effect Level (NOAEL). Regarding the potential for teratogenic effects, 1000 mg/kg/day is considered to be the No Observed Adverse Effect Level (NOAEL).

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
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
not applicable
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Deviations:
not applicable
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
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.
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)
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.
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Control
Dose / conc.:
100 mg/kg bw/day (nominal)
Remarks:
Low Dose
Dose / conc.:
300 mg/kg bw/day (nominal)
Remarks:
Medium Dose
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.
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').
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
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:

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.

Justification for classification or non-classification

Additional information