1,2-Benzenedicarboxylic acid, di-C9-11-branched alkyl esters, C10-rich

Endocrine disrupter mammalian screening – in vivo (level 3)

Administrative data

Endpoint:

endocrine disrupter mammalian screening – in vivo

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

2007

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Data source

Reference

Reference Type:

publication

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Qualifier:

no guideline followed

Principles of method if other than guideline:

The antiandrogenic effects of phthalate, di-isodecyl phthalate (DIDP) was investigated by Hershberger assay in castrated male SD rats. An androgen agonist, testosterone (0.4 mg/kg/d), was administered for 10 consecutive days by subcutaneous (sc) injection as a positive control. DIDP was administered at 20, 100, or 500 mg/kg body weight (bw)/d orally in combination with testosterone (0.4 mg/kg/d, sc) for 10 consecutive days, respectively.

GLP compliance:

not specified

Test material

Test material information

Constituent 1

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

Test material form:

liquid

Details on test material:

CAS number: 68515-49-1
EC number 271-091-4

Specific details on test material used for the study:

Expiration Date: Not specified
Purity: Not specified

Test animals

Species:

rat

Strain:

Sprague-Dawley

Remarks:

Crl:CD

Sex:

male

State:

castrated male

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Samtako Inc. (Osan, Korea)
- Age at study initiation: 4 wk old
- Weight at study initiation: 185 ± 10 g
- Housing: Housed under specific pathogen-free (SPF) conditions
- Diet (e.g. ad libitum): Pelleted rodent diet available ad libitum
- Water (e.g. ad libitum): Drinking water available ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23 ± 3°C
- Humidity (%): 55 ± 5%
- Air changes (per hr): 10–18 times per hour
- Photoperiod (hrs dark / hrs light): illuminated artificially for 12 h daily at 300–500 lux.

The procedure used for castration was based on the Organization for Economic Cooperation and Development (OECD) protocol for detecting endocrine disruptors (OECD, 2001). Castration (removal of testis and epididymis) was performed on 6-wk-old animals weighing 215 ± 16 g via a midline incision, and treatment with test compounds was commenced 1 wk later to allow the animals time for to recover completely.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

DIDP was dissolved in corn oil and administered at 20, 100, or 500 mg/kg bw/d to testosterone propionate (TP)-treated (0.4 mg/kg bw/d) castrated rats by oral gavage until d 10.

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

DIDP was administered at 20, 100, or 500 mg/kg bw/d to testosterone propionate (TP)-treated (0.4 g/kg bw/d) castrated rats by oral gavage until d 10.

Frequency of treatment:

DIDP was administered daily for 10 consecutive days

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

20 mg/kg bw/day

Doses / concentrations 2

Dose / conc.:

100 mg/kg bw/day

Doses / concentrations 3

Dose / conc.:

500 mg/kg bw/day

No. of animals per sex per dose:

Not specified

Control animals:

yes

Details on study design:

The test substance DIDP was administered at 20, 100, or 500 mg/kg bw/d to testosterone propionate (TP)-treated (0.4 mg/kg bw/d) castrated rats by oral gavage until gestation day 10. Through the experimental period, the animals were observed at least once a day, and body weights and abnormalities were recorded daily. All animals in each group underwent autopsy at the end of experiment. Vital organs including liver, kidneys, and adrenal glands and the following sex accessory tissues were dissected and weighed; testes, ventral prostates, combined seminal vesicles and coagulating glands, levator ani/bulbocavernosus (LABC), and Cowper’s glands.

Positive control:

An androgen agonist, testosterone (0.4 mg/kg/d), was administered for 10 consecutive days by subcutaneous (sc) injection as a positive control.

Examinations

Observations and examinations performed and frequency:

Through the experimental period, the animals were observed at least once a day, and body weights and abnormalities were recorded daily.

Sacrifice and pathology:

All animals in each group underwent autopsy at the end of experiment. Vital organs including liver, kidneys, and adrenal glands and the following sex accessory tissues were dissected and weighed; testes, ventral prostates, combined seminal vesicles and coagulating glands, levator ani/bulbocavernosus (LABC), and Cowper’s glands.

Other examinations:

Hormone Analysis: Blood was collected by exsanguination following ether anesthesia from the abdominal aorta and serum was prepared and stored at -80°C for hormone determinations.
Commercially available radioimmunoassay (RIA) kits were used to measure the serum concentrations of testosterone and luteinizing hormone (LH) (Amersham Corp., Arlington Heights, IL).

Statistics:

All values are expressed as means ± SD. Quantitative differences between nontreated control groups and treated animal groups in terms of body and organ weights were analyzed by one-way analysis of variance (ANOVA). Dunnett’s test (significance criterion set at p < .05) was used for pairwise comparisons between control groups and treatment groups.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

not examined

Water consumption and compound intake (if drinking water study):

not examined

Clinical biochemistry findings:

not examined

Endocrine findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Significant increases in liver weights were observed at the highest doses (500 mg/kg/d) of DIDP, compared with testosterone treatment alone.
Ventral prostate and seminal vesicles weights were significantly reduced by DIDP at 500 mg/kg/d.

Gross pathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Effect levels

Key result

Dose descriptor:

dose level:

Effect level:

>= 500 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male

Basis for effect level:

organ weights

Any other information on results incl. tables

Table 1

Antiandrogenic Effects: Hershberger Assay Response to Phthalates in Castrated Male Rats After 10 d of Treatment

Experimental compounds^a and dose (mg/kg)	Initial b.w. (g)	Final b.w. (g)	Liver (g)	Kidneys (g)	Adrenals (g)
Con^b	174.7 ± 9.7	207.3 ± 10.5	8.28 ± 0.69	1.42 ± 0.11	42.4 ± 3.6
T^c	176.5 ± 11.5	211.8 ± 18.0	8.47 ± 1.13	1.67 ± 0.12	45.9 ± 5.7
DIDP
20	176.2 ± 8.9	216.4 ± 14.7	8.62 ± 0.52	1.60 ± 0.02	46.2 ± 2.5
100	172.7 ± 13.4	205.8 ± 11.0	8.13 ± 0.97	1.56 ± 0.08	45.5 ± 3.3
500	191.7 ± 18.6	236.9 ± 13.1	9.87 ± 0.49^d	1.74 ± 0.10	43.6 ± 3.0

Note. Data are expressed as means ± SD.

^aAbbreviation: di-isodecyl phthalate (DIDP)

^bCon, control.

^cT, testosterone.

^dSignificantly different from testosterone (T), p < .05.

 

Table 2

Antiandrogenic Effects on Sex Accessory Tissues: Hershberger Assay Response to Phthalates in SD Castrated Male Rats After 10 d of Treatment

Experimental compounds^a and dose (mg/kg)	Seminal vesicles^b (mg)	Ventral prostate (mg)	LABC (g)	Cowper’s glands (mg)	Glans penis (g)
Con^c	34.8 ± 2.2	12.9 ± 0.5	99.1 ± 4.0	4.2 ± 0.6	35.5 ± 0.6
T^d	513.8 ± 35.4	183.8 ± 4.0	441.6 ± 24.3	40.1 ± 4.7	78.3 ± 5.1
DIDP
20	485.2 ± 6.7	174.4 ± 24.1	407.5 ± 5.0	40.5 ± 3.5	76.1 ± 4.2
100	485.3 ± 10.2	175.5 ± 17.2	407.6 ± 39.8	40.6 ± 4.3	78.8 ± 6.3
500	469.2 ± 13.0^e	145.8 ± 20.4^e	449.8 ± 24.3	44.8 ± 3.3	82.5 ± 5.9

Note. Data are expressed as mean ± SD.

^aAbbreviation: di-isodecyl phthalate (DIDP)

^bSeminal vesicles were measured together with coagulating gland.

^cCon, control.

^dT testosterone.

^eSignificantly different from testosterone (T), p < .05.

Applicant's summary and conclusion

Conclusions:

The antiandrogenic effects of phthalate, di-isodecyl phthalate (DIDP) was investigated by Hershberger assay in castrated male rats. Seminal vesicle weight and ventral prostrate weight were both decreased in the high dose group (500 mg/kg bw/day). However no effects were observed on the weights of the levator ani/bulbocavernosus muscles (LABC), Cowper's glands, or glans penis.

Executive summary:

The antiandrogenic effects of phthalate, di-isodecyl phthalate (DIDP) was investigated by Hershberger assay in castrated male SD rats. An androgen agonist, testosterone (0.4 mg/kg/d), was administered for 10 consecutive days by subcutaneous (sc) injection as a positive control. DIDP was also administered at 20, 100, or 500 mg/kg body weight (bw)/day orally in combination with testosterone (0.4 mg/kg/d, sc) for 10 consecutive days. In the testosterone-treated groups, glans penis, seminal vesicles, ventral prostate, and levator ani/bulbocavernosus muscles (LABC) weights were found to be significantly increased, as expected. Ventral prostate weight and seminal vesicles weight were significantly decreased only in animals treated with 500 mg/kg bw/day DIDP. No effects were observed on the weights of the levator ani/bulbocavernosus muscles (LABC), Cowper's glands, or glans penis. 

 

these test results are inconsistent with a positive androgen antagonist result as specfied in the guideline, as a statsitically significant reduction (p<0.05) in any two or more of the five androgen dependent tissue weights was not accompanied by some degree of reduced growth in the reamining three target tissues.