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endocrine system modulation
Type of information:
experimental study
Adequacy of study:
supporting study
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment

Data source

Reference Type:
Report date:

Materials and methods

Test guideline
no guideline followed
Principles of method if other than guideline:
Rats were dosed orally on a daily basis during gestation day 14–18 (covering a critical programming period for male sexual differentiation). Ex vivo fetal testicular testosterone production and fetal testis gene expression analysis (targeting genes representing sexual determination and differentiation, steroidogenesis, gubernaculum development, and androgen signaling pathways) was conducted.
GLP compliance:
not specified
Type of method:
in vitro
Endpoint addressed:
other: Endocrine disrupting properities - mechanistic information

Test material

Constituent 1
Chemical structure
Reference substance name:
1,2-Benzenedicarboxylic acid, di-C9-11-branched alkyl esters, C10-rich
EC Number:
EC Name:
1,2-Benzenedicarboxylic acid, di-C9-11-branched alkyl esters, C10-rich
Cas Number:
Molecular formula:
C28 H46 O4
1,2-Benzenedicarboxylic acid, di-C9-11-branched alkyl esters, C10 rich
Test material form:
Details on test material:
CAS number: 68515-49-1
EC number 271-091-4
Specific details on test material used for the study:
CAS: 26761-40-0
Lot #: 1379769 23008238
Source: Fluka (Buchs, Switzerland)

Test animals

Details on test animals or test system and environmental conditions:
- Source: Harlan Laboratories (Indianapolis, IN)
- Age at study initiation: Time-pregnant Sprague Dawley (SD) rats were shipped on GD 1–2 (GD 0 ¼ sperm positive).
- Housing: housed individually in 20 x 25 x 47 cm clear polycarbonate cages with laboratory-grade pine shavings for bedding.
- Diet (e.g. ad libitum): fed NIH 07 breeding diet for rats ad libitum.
- Water (e.g. ad libitum): provided municipal drinking water (Durham, NC) ad libitum.

- Temperature (°C): 22°C–23°C
- Humidity (%): 50–60%
- Photoperiod (hrs dark / hrs light): 14L:10D light cycle (lights off at 9:00 P.M.)

Administration / exposure

Route of administration:
oral: feed
corn oil
2.5 ml vehicle/kg bw
Details on exposure:
Dosage levels were: corn oil (vehicle control), 500, 750, 1000, or 1500 mg DIDP/kg/day. DIDP was administered in corn oil (2.5 ml vehicle/kg bw). Pregnant rats were dosed orally on a daily basis during GD 14–18 (covering a critical programming period for male sexual differentiation)
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
Rats were dosed orally on a daily basis during GD 14–18
Frequency of treatment:
Rats were dosed orally on a daily basis during GD 14–18
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day
Vehicle control
Dose / conc.:
500 mg/kg bw/day
Dose / conc.:
750 mg/kg bw/day
Dose / conc.:
1 000 mg/kg bw/day
Dose / conc.:
1 500 mg/kg bw/day
No. of animals per sex per dose:
15 pregnant rats per block
Control animals:
yes, concurrent vehicle
Details on study design:
Pregnant rats were weight ranked and assigned to dosage groups (generally n = 3–4) to minimize differences in means and variance among treatment groups. Sample sizes were based on power calculations performed on previous similarly conducted studies which demonstrated 3–4 litters per dosage group with three individual males/litter for Testosterone (T) production, and the remaining males’ testes pooled for messenger RNA (mRNA) analyses was sufficient for detecting significant dose-related reductions in T production and gene expression.


Fetal necropsies.
Dams were rapidly euthanized by decapitation on GD18, and fetuses were removed, anesthetized via hypothermia on ice, decapitated, and dissected under a Leica MZ6 dissecting microscope (Wetzlar, Germany). For the experiment, a single testis from the first three males identified in a litter were removed and used for ex vivo T production. All remaining testes in each litter were pooled, immediately homogenized in TRI reagent (Sigma, St Louis, MO) on ice using a Kontes pestle homogenizer, and stored at 80°C until used to extract RNA. All necropsies began 1 h following administration of the final maternal dose and were conducted within a 2-h time frame between 08:00 and 10:00 A.M. Eastern Standard Time to avoid any potential confounding effects of fetal growth or time of day on the fetal endpoints.

Ex vivo fetal testicular T production.
Following removal, fetal testes were immediately transferred individually into a single well on a 24-well plate containing 500 ll M-199 media without phenol red (Hazelton Biologics, Inc., St Lenexa, KS), supplemented with 10% dextran-coated charcoal-stripped fetal bovine serum (Hyclone Laboratories, Logan, UT). Testes were incubated for 3 h at 37°C on a rotating platform. Following incubation, media was removed and stored at 80°C until used for T measurement. The level of T in the media samples was measured by radioimmunoassay according to the manufacturer’s instructions (Diagnostic Products Corporation Coat-A-Count kits; Siemens Corp., Los Angeles, CA). The intraassay coefficient of variation was 3.1% (based on variability of the standard curve), and the interassay coefficient of variation was 13.7%. Cross-reactivity of the T antibody with dihydrotestosterone (DHT) was 3.2%. The limit of detection was 0.2 ng/ml for T.

Fetal testis gene expression analysis.
RNA was extracted from the fetal testes homogenate as previously described (Wilson et al., 2004) and cleaned to eliminate any potential genomic DNA contamination using Qiagen RNeasy Mini Kit (Valencia, CA) with the on-column DNase treatment step according to the manufacturer’s instructions. RNA concentration and purity were determined with a NanoDrop 2000 Spectrophotometer (Thermo Scientific, Wilmington, DE). An additional genomic DNA elimination reaction and complementary DNA (cDNA) synthesis were performed on the RNA samples using the SABiosciences RT2 First Strand Kit according to the kit instructions. For each individual sample, 300 ng of RNA was added to a single reaction, to be used across a 96-well array plate. The template cDNA was then added to RT2 SYBR Green qPCR Master Mix (SABiosciences, Fredrick, MD), and 25 ll was added to each well of the plate. The 96-well gene array plate (purchased from SABiosciences) was custom designed to contain 89 individual target genes,
3 housekeeping genes (beta-actin, beta-glucuronidase, and lactate dehydrogenase), an interassay control, a genomic DNA control, a reverse transcription control, and a positive PCR control. To verify interassay consistency in CT cycling, aliquots of fetal testicular cDNA from males of a corn oil (control)–treated dam were added to a well containing primers for the housekeeping gene beta-2 microglobulin on all plates.

The PCR reaction was run on an iCycler iQ Real-Time Detection System (Bio-Rad, Hercules, CA) using the following cycling parameters: one cycle of 95°C for 10 min, followed by 40 cycles of 95°C for 15 s, and 60°C for 1 min. Product purity was verified by melting curve analysis. The DCT value for each gene was determined by dividing the gene CT value by the mean CT value of
three housekeeping genes. The 2DDC T method was used to analyze data and change in gene expression levels were reported as fold change.

Results and discussion

Details on results:
Maternal Body Weight and Litter Effects of DIDP
Maternal body weight gain and fetal mortality were not significantly affected by 5-day (GD 14–18) DIDP in utero exposure at any dose tested .

Ex vivo Fetal Testicular Testosterone (T) Production Dose-Response for DIDP
Dose-response studies were performed to assess the relationship between in utero exposure (GD 14–18) to DIDP and fetal testicular ex vivo T production. DIDP had no effect on ex vivo T production.

Gene Array Screening of DIDP Fetal Testes
The effect of DIDP on gene expression following 5-day in utero exposure was assessed using a PCR array containing 89 individual candidate target genes. DIDP did not induce any changes in gene expression other than the down-regulation of Wnt7a.

Any other information on results incl. tables


Maternal Weight Gain and Fetus Survival Following Gestational Exposure From Day 14–18 to Increasing Doses of DIDP


DIDP doses (mg/kg)












Weight gain

48.7 ± 2.5 41.9 ± 1.4 37.2 ± 3.9 38.6 ± 2.0 41.5 ± 1.2

Number of live fetuses

12.3 ± 2.2 13.7 ± 0.3 12.0 ± 2.1 13.3 ± 0.9 14.0 ± 0.6

Fetal survival

93.3 ± 6.7 97.8 ± 2.2 100.0 ± 0.0 95.1 ± 2.5 100.0 ± 0.0

Note. Values are means ± SEM.
aMaternal weight gain = Body weight GD 18 - Body weight GD 14.
bFetal survival (%) = Number of live fetuses/total fetuses.



Applicant's summary and conclusion

The authors concluded that it was demonstrated that DIDP was not an active antiandrogenic phthalate, as defined by the lack of an effect on fetal testosterone production.
Executive summary:

In this published journal article, pregnant rats were dosed orally on a daily basis during Gestation Day (GD) 14–18 (covering a critical programming period for male sexual differentiation) with increasing doses of the test substance DIDP (500, 750, 1000, or 1500 mg/kg/day). For all experiments, a single testis from the first three males identified in a litter were removed and used to assess ex vivo Testosterone (T) production. DIDP had no effect on ex vivo T production. Fetal testis gene expression analysis was also conducted using a microarray. Only a single gene of the 89 examined, Wnt7a, was down-regulated by DIDP. The authors concluded DIDP was not an active antiandrogenic phthalate, as defined by the lack of an effect on fetal testosterone production.