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Administrative data

developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Well described and documented guideline study, GLP study

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guidelineopen allclose all
according to guideline
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
according to guideline
other: EC Commission Directive 87/302/EEC of Nov. 18, 1987; Part B : Methods for the determination of toxicity: Teratogenicity study (rodent and non-rodent) ; Official Journal of the European Communities ; No. L 133, pp. 24 - 26 (1988)
according to guideline
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
GLP compliance:
yes (incl. QA statement)
Limit test:

Test material

Constituent 1
Chemical structure
Reference substance name:
Diisobutyl phthalate
EC Number:
EC Name:
Diisobutyl phthalate
Cas Number:
Molecular formula:
diisobutyl phthalate
Details on test material:
- Name of test material (as cited in study report): diisobutylphthalate
- Physical state: liquid/colorless - clear
- Analytical purity: 99,77% (GC)
- Lot/batch No.: 2527407 A
- Stability under test conditions: the stability under storage conditions was confirmed by reanalysis
- Storage condition of test material: room temperature

Test animals

Details on test animals or test system and environmental conditions:
- Source: time-mated Wistar rats (CrlGlxBrlHan:Wl) supplied by Charles River Laboratories, Germany which were free from clinical signs of disease, were used for the investigations
- Age at study initiation: female animals were supplied at an age of about 70 - 84 days
- Weight at study initiation: based on the pregnant animals the body weight on day 0 varied between 152.6 and 196.8 g
- Housing: housed singly from day 0 - 20 p.c. in type DK I II stainless steel wire mesh cages supplied by BECKER & CO., Castrop-Rauxel, Germany (height: 15 cm, length: 37,5 cm, width: 21 cm; floor area about 800 cm2).
- Diet (e.g. ad libitum): ground Kliba maintenance diet rat/mouse/hamster meal, supplied by PROVIMI KLIBA SA, Kaiseraugst, Switzerland. Food was available throughout the study (from the day of supply to the day of necropsy)
- Water (e.g. ad libitum): drinking water
- Acclimation period: between start of the study (beginning of the experimental phase) and first administration (day 6 p.c.) the animals were acclimated to the laboratory conditions

The animals were accommodated in fully air- conditioned rooms in which guaranteed central air conditioning
- Temperature (°C): 20-24
- Humidity (%): 30-70
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: feed
unchanged (no vehicle)
in diet
Details on exposure:

- Rate of preparation of diet (frequency): once
- Mixing appropriate amounts with (Type of food): for each concentration, the test substance was weighed out and thoroughly mixed with a small amount of food in a beaker. Then corresponding amounts of food, depending on the test group, were added to this premix in order to obtain the desired concentrations, and mixing was carried out for about 10 minutes in a laboratory mixer
- Storage temperature of food: room temperature
Analytical verification of doses or concentrations:
Details on analytical verification of doses or concentrations:
GC; analytical verifications of the stability of the test substance in the diet for a period of at least 15 days at room temperature were carried out before the study was initiated. The concentration control analyses of all concentrations revealed that the values were in the expected range of the target concentrations. The mean values were found to be in the range of 95.7 - 99.1% of the nominal concentration.
Details on mating procedure:
- Impregnation procedure: purchased timed pregnant
- Proof of pregnancy: vaginal plug referred to as day 0 post coitum (p.c.); the following day was designed "day 1" post coitum (p.c.).
Duration of treatment / exposure:
days 6 to day 20 of gestation
Frequency of treatment:
continuously via diet
Doses / concentrationsopen allclose all
Doses / Concentrations:
0, 1000, 4000 and 11000 ppm
nominal in diet
Doses / Concentrations:
0, 88, 363 and 942 mg/kg bw per day
other: calculated based on feed consumption
No. of animals per sex per dose:
Control animals:
yes, concurrent no treatment
Details on study design:
- Dose selection rationale: the following concentrations in the diet were chosen for the present full-scale toxicity study in Wistar rats: (1) 1000 ppm, as the expected no observed adverse effect level; (2) 4000 ppm, as intermediate concentration; and (3) 11000 ppm as the high concentration which should induce some developmental and/or maternal toxicity but not death or severe suffering.
Risk assessments undertaken for several phthalates indicate the oral uptake by food to be the major route of exposure for the general public. Thus, in the present prenatal developmental toxicity study in Wistar rats administration in the diet was selected. In general, oral administration of a test substance has proven to be appropriate for the detection of a toxicological risk.


Maternal examinations:
- Time schedule: a check was made twice a day on working days or once a day (Saturdays, Sundays or on public holidays) (days 0 - 20 p.c.).

- Time schedule: the animals were examined for clinical symptoms at least once a day, or more often when clinical signs of toxicity were elicited (days 0 - 20 p.c.).

- Time schedule for examinations: All animals were weighed on days 0, 1, 3, 6, 8, 10, 13, 15, 17, 19 and 20 p.c.. The body weight change of the animals was calculated from these results. Furthermore, the corrected body weight gain was calculated after terminal sacrifice (terminal body weight on day 20 p.c. minus weight of the unopened uterus minus body weight on day 6 p.c.).

- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes


- Sacrifice on gestation day 20; on day 20 p.c., the dams were sacrificed in randomized order by cervical dislocation and the fetuses removed from the uterus.
- Organs examined: After the dams had been sacrificed, they were necropsied and assessed by gross pathology in randomized order to minimize bias. The uterus and the ovaries were removed and the following data were recorded: (1) weight of the unopened uterus; (2)
number of corpora lutea; (3) number and distribution of implantation sites (classified as live fetuses and dead implantations: a. early resorptions [only decidual or placental tissues visible or according to SALEWSKI from uteri from apparently non-pregnant animals and the empty uterus horn in the case of single-horn pregnancy]; b. late resorptions [embryonic or fetal tissue in addition to placental tissue visible]; and c. dead fetuses [hypoxemic fetuses which did not breathe spontaneously after the uterus had been opened]).
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
Fetal examinations:
- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: Yes: half per litter
- For food consumption, body weight, Simultaneous comparison, body weight change, corrected body weight gain (net maternal body weight change), carcass weight, weight of unopened uterus, number of corpora lutea, number of implantations, number of resorptions, number of live fetuses, proportions of preimplantation loss, proportions of postimplantation loss, proportions of resorptions, proportion of live fetuses in each litter, litter mean fetal body weight, litter mean placental weight : Simultaneous comparison of all dose groups with the control group using the DUNNETT-test (two-sided) for the hypothesis of equal means.
- For female mortality, females pregnant at terminal sacrifice, number of litters with fetal findings: pairwise comparison of each dose group with the control group using FISHER'S EXACT test (one-sided) for the hypothesis of equal proportions.
- For proportions of fetuses with malformations, variations and/or unclassified observations in each litter: pairwise comparison of each dose group with the control group using the WILCOXON-test (one-sided) for the hypothesis of equal medians
Calculations of conception rate and pre- and postimplantation losses were carried out: conception rate, preimplantation loss and postimplantation loss
Historical control data:
The test protocol is regularly used in the testing facility

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Details on maternal toxic effects:

There were no substance-related or spontaneous mortalities in any of the groups.

Only pregnant dams were used for the calculations of mean maternal food consumption, body weight, body weight change and substance intake. Only pregnant dams with scheduled sacrifice (day 20 p .c.) were taken for the calculation of mean gravid uterine weights, mean net maternal body weight change (corrected body weight gain) and summary of reproduction data. 2 females in each control and high dose groups as well as 3 in each low and mid dose groups were partially or totally excluded from the above mentioned calculations because they were not pregnant.

No clinical observations were noted in any of the dams of this study.

The mean food consumption of the high concentration dams (11000 ppm) was statistically significantly reduced on days 10-13 p.c. (about 8%) and 15-17 p.c. (about 6%) of the administration period. On all other intervals of the treatment phase (i.e. days 6-8, 8-10, 13-15, 17-19 and 19-20 p .c.) food uptake values of these dams was below the concurrent control values without attaining statistical significance. If calculated for the entire treatment phase (days 6-20 p.c.), the food consumption was about 5% below the concurrent control value.
The reduction in food consumption of the high concentration rats (11000 ppm) is considered to be substance-induced. This effect is in line with the impaired absolute and corrected body weight gains of these females.
Food consumption at the mid and low concentration (4000 and 1000 ppm) was not affected by the test substance administration. Food consumption values of these rats were similar to or even exceeded control values.

There were no statistically significant or biologically relevant differences between controls and the substance-treated dams in terms of mean body weights. The observable differences reflect the normal range of fluctuations for animals of this strain and age. Mean body weight gains of the 11000 ppm dams, however, showed statistically significant impairments between days 10-13 p.c. (value about 26% below the concurrent control value). If calculated for the entire treatment phase (days 6-20 p.c.) or the entire study period (days 0-20 p .c.), body weight gain of these females was also statistically significantly lower (about 11 %/10% below controls).
The substance-induced impairments on body weight gain at 11000 ppm have both, a maternal and an embryo-/fetal component and are in line with the reductions in food consumption of these rats. The corrected body weight gain (reflecting the maternal component) and the mean fetal body weight (reflecting the embryo-/fetal component) were both statistically significantly reduced at the highest concentration (11000 ppm).

Mean body weight gains of the dams of test groups 1 and 2 (1000 and 4000 ppm) were similar to those of the concurrent controls.

The corrected body weight gains (terminal body weight on day 20 p.c. minus weight of the unopened uterus minus body weight on day 6 p.c.) of the dams of test groups 1 - 2
(1000 and 4000 ppm) revealed no differences of any biological relevance to the corresponding control group. The net weight change of the 11000 ppm rats, however, was about 25% below the concurrent control value. As food consumption and absolute body weight of these rats were also diminished, the effect on net body weight at the high concentration is considered to be a substance-related sign of straight maternal toxicity.

- Uterus weight: the mean gravid uterus weights of the animals of test groups 1, 2 and 3 (1000, 4000 and 11000 ppm) were not influenced by the administration of the test substance. The differences between these groups and the control group are considered to be without any biological relevance.

- Necropsy findings: there were no substance-related observations at necropsy in any of the dams.

- Reproduction data of dams: the conception rate reached 88% in test groups 1 and 2 (1000 and 4000 ppm) and 92% in test groups 0 and 3 (0 and 11000 ppm). All rats which became pregnant had implantation sites at necropsy. Thus a sufficient number of females for the purpose of the study was available.
There were no substance-related and/or biologically relevant differences between the different test groups in conception rate, in the mean number of corpora lutea and implantation sites or in the values calculated for the pre- and the postimplantation losses, the number of resorptions and viable fetuses.
All differences observed appeared without a clear relation to dosing and reflect the normal range of fluctuations for animals of this strain and age and similar to the historical control data. This includes the statistically significantly increased mean percentage of early resorptions at 4000 ppm.

Effect levels (maternal animals)

Dose descriptor:
Effect level:
363 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: maternal toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Details on embryotoxic / teratogenic effects:
The scattered occurrence of the few observed external and skeletal malformations in single fetuses of all test groups including the controls (0, 1000, 4000 and 11000 ppm) without a consistent pattern, without a clear dose-response relationship and/or at incidences, which are similar to historical control rates does not suggest any substance-induced origin of these findings. The most salient malformations affected head (anophthalmia; malformed head; severely malformed skull bones), limbs (shortened scapula; misshapen or shortened humerus), ribs (branched) and sternum (malpositioned and bipartite sternebra). Soft tissue malformations, however, did not occur in this study.

In total one of the 208 examined control fetuses [= 0.5%] in one out of 23 litters [= 4.3%], 4 of the 197 examined low concentration fetuses [= 2.0%] in 4 out of 22 litters [= 18%], 2 out of 182 mid concentration fetuses [= 1.1 %] in one out of 22 litters [= 4.5%] and 2 out of 211 high concentration fetuses [= 0.9%] in 2 out of 23 litters [= 8.7%] showed malformations . The mean percentages of affected fetuses/litter with total malformations amounted to 0.5, 1 .9, 1.5 and 0.9% at 0, 1000, 4000 or 11000 ppm respectively. These incidences do not suggest any treatment-relationship.

External variations did not occur in any of the fetuses in this study. Soft tissue variations, exclusively in the form of dilated renal pelvis and ureter, occurred in all test groups including the controls without a clear relation to dosing and at incidences, which are fully within the historical control data range. From the broad spectrum of skeletal variations, which consisted primarily in transient delays in the ossification process, only one skeletal variation (i.e. of unilateral ossification of sternebra with unchanged cartilage) showed some relation to dosing and occurred at a statistically significantly increased rate in the high concentration fetuses
(11000 ppm) at an incidence, which was marginally above the upper historical control value.

Such slight retardations of the ossification process occur very frequently in gestation day 20 rat fetuses. This becomes obvious, if the overall rate of skeletal variations on a fetus/litter basis is taken into account: 92.2, 85.4, 89.2 and 97.3% at 0, 1000, 4000 or 11000 ppm. Furthermore, this type of minor ossification delay has to be regarded as a transient phenomenon that is fully reversible postnatally. Thus, although the affected fetus/litter value for this finding exceeded slightly the historical control range it is considered as an incidental finding and not relevant in terms of developmental toxicity.

If all variations are summarized, in total 111 of the 208 examined control fetuses [= 53%] in all 23 litters [= 100%], 94 of the 197 examined low concentration fetuses [= 48%] in all 22 litters [= 100%], 94 out of 182 mid concentration fetuses [= 52%] in all 22 litters [= 100%] and 114 out of 211 high concentration fetuses [= 54%] in all 23 litters [= 100%] showed variations. The mean percentages of affected fetuses/litter with total variations amounted to 53.6, 47.1, 52.7 and 54.1% at 0, 1000, 4000 or 11000 ppm respectively without attaining statistical significance.

A spontaneous origin is also assumed for the observed unclassified external and fetal skeletal observations. Distribution and type of these findings do not suggest any relation to treatment. Thus, the oral administration of the test substance with the diet up to a concentration of 11000 ppm (approx. 942 mg/kg body weight/day) to pregnant Wistar rats did not affect fetal morphology and gave in particular no indications for teratogenic properties.

Effect levels (fetuses)

open allclose all
Dose descriptor:
Effect level:
363 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: fetotoxicity
Dose descriptor:
Effect level:
> 942 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: teratogenicity

Fetal abnormalities

not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Applicant's summary and conclusion