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Developmental toxicity / teratogenicity

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Administrative data

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

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report date:
2014

Materials and methods

Test guideline
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)

Test material

Constituent 1
Chemical structure
Reference substance name:
Benzyl 3-isobutyryloxy-1-isopropyl-2,2-dimethylpropyl phthalate
EC Number:
240-920-1
EC Name:
Benzyl 3-isobutyryloxy-1-isopropyl-2,2-dimethylpropyl phthalate
Cas Number:
16883-83-3
Molecular formula:
C27H34O6
IUPAC Name:
Benzyl 2,2,4-trimethyl-1-[(2-methylpropanoyl)oxy]pentan-3-yl benzene-1,2-dicarboxylate
Constituent 2
Reference substance name:
Reaction mass of benzyl 1-(isobutyryloxy)-2,2,4-trimethylpentan-3-yl phthalate and benzyl 3-(isobutyryloxy)-2,2,4-trimethylpentyl phthalate
EC Number:
701-008-3
Molecular formula:
C27H34O6
IUPAC Name:
Reaction mass of benzyl 1-(isobutyryloxy)-2,2,4-trimethylpentan-3-yl phthalate and benzyl 3-(isobutyryloxy)-2,2,4-trimethylpentyl phthalate
Details on test material:
Identification: Santicizer® 278
Batch Number: 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: (provided by the Sponsor)
At room temperature (20 ± 5 ºC), protected from humidity and in the dark
Safety Precautions: Routine hygienic procedures (gloves, goggles, face mask)
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.

Test animals

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

Administration / exposure

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)
Doses / concentrationsopen allclose all
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.

Examinations

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.



Results and discussion

Results: maternal animals

General toxicity (maternal animals)

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

Maternal developmental toxicity

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.














Effect levels (maternal animals)

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

Results (fetuses)

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

Effect levels (fetuses)

open allclose all
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

Fetal abnormalities

Key result
Abnormalities:
no effects observed

Overall developmental toxicity

Key result
Developmental effects observed:
no

Applicant's summary and conclusion

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).