Diisobutyl phthalate

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Non-GLP, near-guideline study, acceptable for assessment.

Data source

Reference

Reference Type:

publication

Title:

Developmental toxic effects of diisobutyl phthalate, the methyl-branched analogue of di-n-butyl phthalate, administered by gavage to rats

Author:

Saillenfait AM, Sabaté JP, Gallisot F

Year:

2006

Bibliographic source:

Toxicology Letters, 165, 39-46

Materials and methods

Principles of method if other than guideline:

Pre-natal developmental toxicity study. Dams were administered the test substance daily by gavage on GD 6-20, with foetuses subject to a standard developmental assessments on GD 21 (including degree of transabdominal testicular migration in males).

GLP compliance:

not specified

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Diisobutyl phthalate

EC Number:

201-553-2

EC Name:

Diisobutyl phthalate

Cas Number:

84-69-5

Molecular formula:

C16H22O4

IUPAC Name:

1,2-bis(2-methylpropyl) benzene-1,2-dicarboxylate

Details on test material:

Purity: ≥ 99%
Source: ACROS (Geel., Belgium)

Test animals

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: IFFA CREDO Breeding Laboratories (Saint-Germain-sur-l'Arbresle, France)
- Age at study initiation: Not reported, dams were primiparous
- Weight at study initiation: 180-200 g
- Fasting period before study: No
- Housing: Single. Clear polycarbonate cages with stainless steel wire lids and corn cob granules as bedding.
- Diet (e.g. ad libitum): Food pellets (UAR Alimentation Villemoissonm France) ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 1-2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 2°C
- Humidity (%): 50 ± 5%
- Air changes (per hr): Not reported
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

olive oil

Details on exposure:

No further details

Analytical verification of doses or concentrations:

not specified

Details on analytical verification of doses or concentrations:

No data

Details on mating procedure:

- Impregnation procedure: cohoused
- Verification of same strain and source of both sexes: yes
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy

Duration of treatment / exposure:

From GD 6 to 20 (termination on GD 21)

Frequency of treatment:

daily

Duration of test:

Termination on GD 21

Doses / concentrations

Remarks:

Doses / Concentrations:
0 (olive oil), 250, 500, 750, 1000 mg/kg b.w./day
Basis:
nominal conc.

No. of animals per sex per dose:

23-24 (20-22 pregnant)

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Based on range-finding study (0, 250, 500, 750 or 1000 mg/kg bw/d) in which maternal and foetal effects recorded at 750 mg/kg bw/d and above.

Examinations

Maternal examinations:

CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily

BODY WEIGHT: Yes
- Time schedule for examinations: Every 3 days

FOOD CONSUMPTION (if feeding study): Yes

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:

- Live foetal weight
- Sex ratio
- Trans-abdominal testicular migration
- External examinations: Yes: all per litter
- Visceral examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter

Statistics:

Post-implantation loss, dead fetuses, resorptions, TTM and alterations among litters was evaluated by using the Kruskal-Wallis test followed by the Mann-Whitney test where appropriate.
Rates of pregnancy and incidences of fetal alterations per dose analysed by using Fisher's test.
p < 0.05 deemed significant

Indices:

Maternal parameters: (corrected) body weight change, food consumption
Gestation parameters: Number of implantation sites per litter, post-implantation loss, dead/live foetuses, resporptions, sex distribution, foetal body weight
Various Malformations (external, visceral and skeletal)
Various Variations: (external, visceral and skeletal)

Historical control data:

Not used

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:

Maternal toxic effects:yes

Details on maternal toxic effects:
Significant deviations in body-weight gain seen at dose levels of 500 mg/kg b.w./day and above

Effect levels (maternal animals)

1

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

2

Dose descriptor:

NOAEL

Effect level:

250 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: developmental toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
Significant decrease in fetal body weight and degree of trans-abdominal testicular migration at dose levels of 500 mg/kg b.w./day and above.
Wide-ranging embryotoxic/tetragenic effects seen at dose levels of 750 mg/kg b.w./day and above.

Fetal abnormalities

Abnormalities:

not specified

Overall developmental toxicity

Developmental effects observed:

not specified

Any other information on results incl. tables

Maternal parameters:

- no maternal deaths;

- body weight gain significantly reduced in high dose dams (GD 6-21), however weight gain after correction for gravid uterine weight unremarkable (i.e. weight gain reduction due to lower number of liver foetuses and foetal body weight);

- gravid uterine weight statistically significantly decreased in the 500, 750 and 1000 mg/kg bw/d groups (-19%, -27% and -61%, respectively);

Litter parameters:

- pregnancy rate and the number of implantations unaffected by treatment;

- incidence of resorptions statistically significantly increased at 750 mg/kg bw/d (28%) and 1000 mg/kg bw/d (59%) relative to controls (7%);

- number of liver foetuses per litter statistically significantly decreased at 750 mg/kg bw/d (-21%) and 1000 mg/kg bw/d (-51%);

- sex ratio unremarkable

Foetal parameters:

- mean foetal body weight statistically significantly reduced 7-24% at 500 mg/kg bw/d and above;

- external malformations (neural tube closure defects, anophthalmia) statistically significantly increased at 750 and 1000 mg/kg bw/d;

- skeletal malformations (primarily fused sternebrae) statistically significantly increased at 750 and 1000 mg/kg bw/d;

- skeletal variations (delayed ossification, supernumerary ribs) statistically significantly increased at 750 and 1000 mg/kg bw/d;

- total visceral malformations (urinary tract and vascular defects) were statistically significantly increased at 750 and 1000 mg/kg bw/d;

- visceral variations involved primarily the urinary tract, ureter and male reproductive system

- unilateral or bilateral undescended testes occurred in 3/55 male foetuses (2/20 litters) at 500 mg/kg bw/d, in 30/55 male foetuses (16/20 litters) at 750 mg/kg bw/d, and in 30/34 male foetuses (16/17 litters) at 1000 mg/kg;

- transabdominal testicular migration in relation to the bladder was significantly increased at 500 mg/kg bw/d and above, and in high dose males around two third of the testes (right and left) were located in the upper half of the abdominal cavity;

- alterations of the male reproductive system occurred at lower doses than those producing structural malformations/variations and embryotoxicity.

Applicant's summary and conclusion

Conclusions:

Diisobutyl phthalate was found to exhibit developmental toxicity in rats at dose rates of 500 mg/kg b.w./day and above. The overall NOEAL was 250 mg/kg b.w./day

Executive summary:

The prenatal developmental toxicity of DIBP was investigated in pregnant Sprague-Dawley rats administered doses of 0 (olive oil), 250, 500, 750 and 1000 mg/kg bw/d by gavage on GD 6-20. Dams and foetuses were subject to a standard range of assessments on GD 21, with the degree of transabdominal testicular migration determined additionally in male foetuses. Maternal parameters were generally unremarkable, with an apparent reduction in body weight secondary to statistically significant reductions in gravid uterine weight in the 500, 750 and 1000 mg/kg bw/d groups. Pregnancy rate and the number of implantations were unaffected by treatment, however the incidence of resorptions was statistically significantly increased, and the number of liver foetuses per litter decreased, at 750 mg/kg bw/d and above. Foetal sex ratio was unremarkable, but mean foetal body weight was significantly reduced at 500 mg/kg bw/d and above. The incidence of external malformations, total visceral malformations, skeletal malformations and skeletal variations was significantly increased at 750 and 1000 mg/kg bw/d. Visceral variations involved primarily the urinary tract, ureter and male reproductive system, and included significantly increased incidences of unilateral or bilateral undescended testes and significantly increased transabdominal testicular migration relative to the bladder at 500 mg/kg bw/d and above. No evidence foetal effects was found at the 250 mg/kg dose level. The NOAEL for maternal toxicity was therefore 1000 mg/kg bw/d, and the NOAEL for foetal effects 250 mg/kg bw/d.