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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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: This study is rated a "1" because it applied GLP, used appropriate testing procedures, and followed the test guideline that was in place at the time the study was performed
Cross-reference
Reason / purpose for cross-reference:
reference to other study
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Remarks:
Dose range finding study
Adequacy of study:
other information
Study period:
8-March-1994 to 1-April-1994
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Reason / purpose for cross-reference:
reference to other study
Qualifier:
equivalent or similar to guideline
Guideline:
other: U.S. EPA TSCA 40 CFR, Part 798
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
12-May-1981
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Specific details on test material used for the study:
Expiration Date: February 1999
Description: Colorless liquid
Storage Condition: Room temperature
The test material was assumed 100% pure for the purpose of dosing
Species:
rat
Strain:
Sprague-Dawley
Remarks:
Crl:CDBR (SD) VAF/Plus
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Inc., Kingston Facility, Stone Ridge, New York
- Age at study initiation: Females - Approximately 10 weeks
- Weight at study initiation: Females - 228 to 275 grams
- Housing: Single housed during the study period, except during mating.
- Diet (e.g. ad libitum): Purina Certified Rodent Chow (5002 meal), ad libitum
- Water (e.g. ad libitum): Automatic watering system, ad libitum
- Acclimation period: 14 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24
- Humidity (%): 40 - 70
- Photoperiod (hrs dark / hrs light): 12 hours dark/light

Route of administration:
oral: gavage
Vehicle:
corn oil
Remarks:
The stability and homogeneity of DIDP in com oil was evaluated at 0.8% and 20% w/v. Stability data indicated the test material was stable in com oil at room temperature for at least eight days.
Details on exposure:
The undiluted test material was thoroughly mixed in carrier prior to dispensing.
The test material/carrier mixtures were prepared weekly. Dosing solutions were prepared at the following doses found under Doses/Concentrations.

Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The samples were diluted using ethyl acetate and were analyzed for homogeneity, stability and concentration verification using High Pressure Liquid Chromatography (HPLC).
Concentration verification analysis indicated that all solutions were within 1% of the nominal concentrations for Week 1 samples.
Details on mating procedure:
On the initial scheduled mating day, females were placed in males' cages in a 1:1 (male: female) ratio. Males and females were paired based on sequential Physical Identification Number. A suitable number of animals were co-housed in an attempt to produce a suitable number of mated animals to accommodate lab scheduling. Mating was confirmed on the following morning by observation of a copulatory plug (vaginal) and/or by the presence of sperm in a vaginal rinse. The day on which mating was confirmed was the female's Day 0 of gestation (GD 0).

After confirmation of mating, each mated female was returned to its own cage. New females then were placed in the males' cages until the required number of mated females was obtained by continuous cohabitation in consideration of lab scheduling. Mated females were assigned to dose groups in the order of mating. Accordingly, the first confirmed mated female was assigned to Group 1, the next to Group 2, and so on until all mated animals for a given day were assigned to dose groups. On subsequent days, the next group in sequence was filled by the first confirmed mated female on that day, and so on. Assignments were made until all groups were filled with confirmed mated females.
Duration of treatment / exposure:
Mated females were dosed once daily by oral gavage from GD 6 through GD 15. Dosing volumes were 5 ml/kg for all groups and were based on the most recent body weights. Animals were dosed at approximately the same time each day (+1.0 hour).
Frequency of treatment:
Once daily by oral gavage from GD 6 through GD 15.
Duration of test:
Surviving animals were terminated on Gestation Day 21.
Dose / conc.:
0 mg/kg bw/day
Remarks:
Carrier control
Dose / conc.:
40 mg/kg bw/day
Remarks:
Low
Dose / conc.:
200 mg/kg bw/day
Remarks:
Intermediate
Dose / conc.:
500 mg/kg bw/day
Remarks:
Intermediate
Dose / conc.:
1 000 mg/kg bw/day
Remarks:
High
No. of animals per sex per dose:
7 females/ dose group
Control animals:
yes
Details on study design:
The test material was administered by oral gavage to 5 groups of female rats at doses of 40, 200, 500, and 1000 mg/kg. Group 1 served as a control and received the carrier (com oil) only. Mated females were dosed once daily from Gestation Day (GD) 6 through GD 15. Dosing volumes were 5 ml/kg for all groups and were based on the animals' most recent body weight.
Maternal examinations:
Animals were examined for viability at least twice daily during the treatment period and at least once daily at other times during the study.
Body weights were taken prior to selection, and on GD 0, 6,9, 12, 15,18, and 21.
Food consumption was measured for mated females concurrently with body weights.
A clinical examination was given to each female prior to selection, and daily during gestation.
Ovaries and uterine content:
Mated females were euthanized on GD 21. Euthanasia was by C02 asphyxiation and exsanguination. All females assigned to groups were examined by gross necropsies.
Uterine weight with ovaries attached was recorded at the time of necropsy. Body weight was recorded on the day of necropsy. Uterine contents were examined and the numbers and locations of implantation sites, early and late resorptions, live, and dead were counted. Corpora lutea also were counted.
Blood sampling:
Not examined.
Fetal examinations:
Each live fetus was weighed and examined externally for gross malformations.
Fetal sex was determined by external examination. All live fetuses subsequently were euthanized by intramuscular injection into the tongue with sodium pentobarbital and discarded without further examination.
Statistics:
Please refer to Any other information on materials and methods incl. tables for the full text.
Clinical signs:
no effects observed
Description (incidence and severity):
There were no clinical signs which were judged to be related to treatment with DIDP.
The majority of dams were free of observable abnormalities during the entire gestation period. Clinical signs were limited to one 500 mg/kg dam which had a broken incisor from GD 12 to GD 17. In addition, one control dam was observed with red vaginal discharge and delivered two live pups just prior to her scheduled cesarean-section on GD 21. All clinical findings were considered incidental and unrelated to treatment with DIDP.
Mortality:
no mortality observed
Description (incidence):
All animals survived to scheduled study termination on Gestation Day 21.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
All animals displayed increases in body weight over their initial values. There were no statistically significant differences in mean gestation body weight, mean gestation body weight change, mean uterine weight, or mean corrected body weight between treated and control animals.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
There was a statistically significant decrease in mean food consumption of the 40 mg/kg dose group females compared with controls during the GD 12-15 interval.
In the absence of a clear dose response or other statistically significant differences in mean food consumption between the low dose and control animals, this finding was considered spurious.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Endocrine findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were no postmortem findings which were judged to be the result of treatment with DIDP. The majority of animals had no adverse findings at necropsy. Postmortem findings were limited to one 500 mg/kg dose group female with a distended/thickened area on the esophagus and white/yellow caseous material in the thoracic cavity. These observations probably were related to the dosing procedure and were not considered treatment-related.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed
Changes in pregnancy duration:
no effects observed
Changes in number of pregnant:
no effects observed
Key result
Dose descriptor:
NOAEL
Effect level:
>= 1 000 mg/kg diet
Based on:
test mat.
Remarks on result:
not determinable due to absence of adverse toxic effects
Fetal body weight changes:
no effects observed
Description (incidence and severity):
There were no statistically significant differences in mean fetal body weight between the treated and control fetuses of either sex.
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
not examined
Changes in litter size and weights:
no effects observed
Anogenital distance of all rodent fetuses:
not examined
Changes in postnatal survival:
not examined
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
All fetuses in all groups were free of observable abnormalities at the external examination.
One control female, one 40 mg/kg dose female, three 200 mg/kg dose females, one 200 mg/kg dose male, and one 500 mg/kg dose male were stunted, but otherwise within normal limits. In the absence of a clear dose response or differences in the mean fetal body weight of the control and treated animals, this finding was considered incidental and not biologically important.
Skeletal malformations:
not examined
Visceral malformations:
not examined
Key result
Dose descriptor:
NOAEL
Effect level:
>= 1 000 mg/kg diet
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects
Conclusions:
In a developmental toxicity range finding study, 1,2-Benzenedicarboxylic acid, di-C9-11-branched alkyl esters, C10-rich did not induce any apparent toxicity at the tested doses. Therefore, the maternal and fetal NOAELs (No Observable Adverse Effect Level) were established at 1000 mg/kg under the conditions of this study. Thus, the high dose level for a subsequent definitive developmental toxicity study in rats is recommended to be 1000 mg/kg/day (the limit dose).
Executive summary:

This study was conducted to provide general information in order to select appropriate dose levels for a subsequent developmental toxicity study with di-isodecylphthalate (DIDP), designated MRD-94-775, in the rat.



The test material was administered by oral gavage to 5 groups of Crl:CDBR female rats at doses of 40, 200, 500, and 1000 mg/kg. Group 1 served as a control and received the carrier (com oil) only. Mated females were dosed once daily from Gestation Day (GD) 6 through GD 15. Dosing volumes were 5 ml/kg for all groups and were based on the animals' most recent body weight.



Males and females were paired and housed overnight until confirmation of mating (sperm/plug = GD 0). Each mated female then was returned to its own cage and new females were placed in the males' cages until the required number of mated females was obtained. Mated females were assigned to dose groups in the order of mating. Clinical observations were made daily during gestation. Body weight and food consumption measurements were made on GD 0, 6, 9, 12, 15, 18, and 21. On GD 21, animals were euthanized and cesarean sections were performed. Gross necropsies were performed, uterine weights with ovaries attached were measured, uterine contents were examined, and the required uterine implantation data were recorded. All live fetuses were weighed, sexed externally, examined externally for gross malformations, and subsequently euthanized and discarded without further examination.



All maternal animals survived to scheduled study termination on GD 21. There were no treatment-related clinical signs observed during gestation and the majority of dams were free of observable abnormalities. There were no statistically significant differences in mean gestation body weight, mean gestation body weight change, mean uterine weight, or mean corrected body weight between treated and control females. Similarly, there were no biologically significant differences in mean gestation food consumption between treated and control animals. There were no postmortem findings which were judged to be the result of treatment with DIDP. Additionally, there were no statistically significant differences in any of the mean uterine implantation parameters between the treated and control females.



There were no statistically significant differences in mean fetal body weight between the treated and control fetuses of either sex. All fetuses in all groups were free of observable abnormalities at the external examination. One control female, one 40 mg/kg dose female, three 200 mg/kg dose females, one 200 mg/kg dose male, and one 500 mg/kg dose male were stunted, but otherwise within normal limits.



In conclusion, overt signs of maternal toxicity were not apparent at any dose level tested, as indicated by the absence of adverse clinical or postmortem findings or adverse effects on body weight, food consumption, and/or uterine implantation data. Similarly, there were no treatment-related adverse effects on fetal development or growth observed at any dose level tested. Therefore, the maternal and fetal NOAELs (No Observable Adverse Effect Level) were established at 1000 mg/kg under the conditions of this study.

Data source

Referenceopen allclose all

Reference Type:
publication
Title:
Unnamed
Year:
1995
Report date:
1995
Reference Type:
publication
Title:
Unnamed
Year:
1999
Reference Type:
study report
Title:
Unnamed
Year:
1995
Report date:
1995

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
other: Official Journal of European Communities L 133 Methods for Determination of Toxicity, Teratogenicity (Annex V, Adopted November 18, 1987)
Deviations:
no
GLP compliance:
yes

Test material

Constituent 1
Chemical structure
Reference substance name:
1,2-Benzenedicarboxylic acid, di-C9-11-branched alkyl esters, C10-rich
EC Number:
271-091-4
EC Name:
1,2-Benzenedicarboxylic acid, di-C9-11-branched alkyl esters, C10-rich
Cas Number:
68515-49-1
Molecular formula:
C28 H46 O4
IUPAC Name:
1,2-Benzenedicarboxylic acid, di-C9-11-branched alkyl esters, C10 rich
Constituent 2
Reference substance name:
1,2-benzenedicarboxylic acid, di-C9,C10 and C11 branched alkyl ester, C10 Rich
IUPAC Name:
1,2-benzenedicarboxylic acid, di-C9,C10 and C11 branched alkyl ester, C10 Rich
Details on test material:
- Name of test material (as cited in study report): di-isodecyl phthalate
- CAS RN 68515-49-1

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Inc
- Age at study initiation: 9 weeks
- Weight at study initiation: 210 to 270 g
- Fasting period before study:
- Housing: Single house during the study period, except during mating
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 13 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 to 24.4
- Humidity (%): 40 to 70
- Photoperiod: 12 hrs dark / 12 hrs light

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on mating procedure:
- Impregnation procedure: cohoused
- If cohoused:
- 1:1 M/F ratio per cage:
- Length of cohabitation: until observation of proof of pregnancy.
Mated females returned to individual cages. New females placed in males'cages until required number of mated females obtained for study.
- Proof of pregnancy: vaginal plug / sperm in vaginal smear, referred to as day 0 of pregnancy
Duration of treatment / exposure:
Gd 6 through 15
Frequency of treatment:
daily
Duration of test:
All females euthanised on GD21
Doses / concentrations
Remarks:
Doses / Concentrations:
100, 500, 1000 mg/kg
Basis:
actual ingested
No. of animals per sex per dose:
no males; up to 25 mated females / dose
Control animals:
yes, concurrent vehicle
Details on study design:
Sex: male/female
Duration of test: gestation day 21

Results and discussion

Results: maternal animals

Effect levels (maternal animals)

Dose descriptor:
NOAEL
Effect level:
500 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
body weight and weight gain
food consumption and compound intake
other: maternal toxicity

Results (fetuses)

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

Effect levels (fetuses)

Dose descriptor:
NOAEL
Effect level:
500 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: fetal skeletal variations

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Any other information on results incl. tables

RS-Freetext:
Maternal toxicity was indicated by reductions in body weight gain and food consumption.  There was no
evidence of malformations or fetal toxicity.
Developmental effects:  There were no significant differences in mean foetal body weight and no statistically
significant increases in total or individual external, visceral or skeletal malformations in the treated group
when compared with controls.  The only visceral variation observed was a single incidence of dilated renal
pelves in the mid group.  Three controls, one low-dose, three mid-dose, and six high-dose foetuses were
stunted.  Those observations were considered incidental and unrelated to treatment.  There was a dose-
related increase in total foetuses with skeletal variations on both a per foetus basis (38/196, 35/177, 61/193,
123/196) and a per litter basis (18/25, 17/22, 20/24, 23/24) at a dose of 0-100-500-1,000 mg/kg, respectively.  
When compared with controls, rudimentary lumbar ribs and cervical ribs were dose-related significantly
increased (p 0.01) in the mid and high-dose groups on a per foetus basis (21%, 52% versus 8.2% in control
group and 6.2%, 9.2% versus 1% in control group, respectively; the historical control ranges are 3.7-21.6%
and 0.54-4.0%, respectively) and in the high-dose group on a per litter basis (23/24 vs. 10/24 for rudimentary
lumbar ribs and 10/24 vs. 2/25 for rudimentary cervical ribs).

Applicant's summary and conclusion

Conclusions:
The results indicate that DIDP was neither a selective developmental toxicant nor an embryotoxic agent.
Executive summary:

In regard with developmental effects, skeletal variations are observed in the developmental studies at 1,000 mg/kg/d concurrently with slight signs of maternal toxicity and lead to a NOAEL of 500 mg/kg/d.