4,4'-isopropylidenediphenol

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:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: OECD protocols followed

Data source

Reference

Reference Type:

publication

Title:

Gene expression changes in the immature rat uterus: Effects of uterotrophic and sub-uterotrophic doses of bisphenol A

Author:

Ashby J, Odum J

Year:

2004

Bibliographic source:

Toxicological Sciences. 82(2): 458-467

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 440 (Uterotrophic Bioassay in Rodents: A Short-term Screening Test for Oestrogenic Properties)

GLP compliance:

not specified

Test material

Test material information

Constituent 1

Reference substance name:

4,4'-isopropylidenediphenol

EC Number:

201-245-8

EC Name:

4,4'-isopropylidenediphenol

Cas Number:

80-05-7

Molecular formula:

C15H16O2

IUPAC Name:

4-[2-(4-hydroxyphenyl)propan-2-yl]phenol

Details on test material:

- purity: >99 %

Test animals

Species:

rat

Strain:

other: Alpk:APfSD (Wistar derived)

Sex:

female

State:

immature female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: AstraZeneca breeding unit (Alderley Park)
- Age at study initiation: 19-20 days
2
- Weight at arrival (1 day before study initiation): 37-45 grams
- Fasting period before study: Not specified
- Housing: Up to 5 per cage in polypropylene cages
- Diet (e.g. ad libitum): Ad libitum, Rat and Mouse No. 1 diet (Special Diet Services Ltd., Witham,
Essex, UK)
- Water (e.g. ad libitum): Ad libitum
- Acclimation period: One day
ENVIRONMENTAL CONDITIONS
Not detailed in this study; noted that the conditions were according to in-house standards as
described by Odum et al., 2001. Toxicological Sciences. 61: 115-127.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

arachis oil

Details on oral exposure:

VEHICLE
Purchased from Sigma Chemicals (Poole, Dorset, UK).
All compounds given daily by gavage in 5 ml/kg arachis oil.

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

Time-course experiment: One day or three days
Dose-response experiments: One day or three days

Post exposure period
Time-course experiment: Animals sacrificed 4, 8, or 24 hours after one single dose or 24 hours after
the last of three daily doses (72 hours after the first dose).
Dose-response experiments: Animals sacrificed 4 hours after one single dose or 24 hours after the
last of three daily doses (72 hours after the first dose).

Frequency of treatment:

Once daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0.002 mg/kg bw/day

Doses / concentrations 2

Dose / conc.:

0.02 mg/kg bw/day

Doses / concentrations 3

Dose / conc.:

0.2 mg/kg bw/day

Doses / concentrations 4

Dose / conc.:

2 mg/kg bw/day

Doses / concentrations 5

Dose / conc.:

20 mg/kg bw/day

Doses / concentrations 6

Dose / conc.:

200 mg/kg bw/day

Doses / concentrations 7

Dose / conc.:

400 mg/kg bw/day

Doses / concentrations 8

Dose / conc.:

800 mg/kg bw/day

No. of animals per sex per dose:

Time-course experiment: 5
Dose-response experiment: 10

Control animals:

yes, concurrent vehicle

Positive control:

17beta-oestradiol (E2)

Examinations

Observations and examinations performed and frequency:

Uterine weight
Terminal body weight
Relative changes in uterine gene expression, as measured by RT-PCR analysis. Specific genes
investigated: PR (A and B isoforms), C3, and lipocalin (estrogen-regulated genes); RPB1 and 18S
rRNA (control genes)

Positive control
E2 dosing at 0.4 mg/kg-day. All tests performed as on BPA-treated rats.

Results and discussion

Any other information on results incl. tables

Time-course experiment: No effects on body weights. After 3 days of dosing, uterine weight was increased by 2.6-fold. Uterine weights were also significantly increased at 4, 8, and 24 hours postdosing. BPA significantly increased: PR expression (RNA) at 4 and 8 hours post-dosing (maximum expression at 4 hours post-dosing); lipocalin expression (RNA) at 4, 8 and 72 hours post-dosing (maximum expression at 4 hours post-dosing); C3 expression (RNA) at 4, 8, 24 and 72 hours postdosing (maximum expression at 72 hours post-dosing); RPB1 expression (RNA) at 8 hours post-dosing, and 18s rRNA expression (RNA) at 24 and 72 hours post-dosing. PR A and PR B isoforms (protein) were both increased from 4 hours onwards, with maximal protein expression at 72 hours.

Dose-response studies: No effects on body weights. No consistent increases in gene expression at doses of 2-20 mg/kg-day. At 4 hours after a single dose of 200-800 mg/kg BPA, there was a statistically significant increase in uterine weight. Three daily doses of BPA increased uterine weight at doses of 200-800 mg/kg-day, with statistical significance at 400-800 mg/kg-day. PR, C3, and lipocalin RNA expression was increased at 4 hours and/or 72 hours post-dosing at 800 mg/kg BPA. PR expression was increased 3-fold at 4 hours post-dosing with a dose of 200-800 mg/kg BPA. Lipocalin expression was increased 3 -4 fold at 4 hours after a single dose of 200 -800 mg/kg BPA. For C3 expression, there was an increase of 2.5-fold at 4-hours after a single dose of 20 mg/kg BPA, rising to ~6-fold for the 800 mg/kg dose at 4 hours. At 72 hours post-dosing, statistically significant increases in C3 expression were only observed at 400 and 800 mg/kg BPA.

Applicant's summary and conclusion

Conclusions:

The authors concluded that BPA does not produce reproducible changes in gene expression in the uterus of immature rats at dose levels that are not also uterotrophic.

Executive summary:

Expression levels of three estrogen-responsive uterine genes (complement component 3, lipocalin 2, and progesterone receptor) and two control genes (18S rRNA and RNA polymerase II large subunit) were determined using real-time RT-PCR. Observations of gene expression were made 4 h and 72 h after the first of three daily po administrations of BPA. Increases in gene expression were observed over the uterotrophic dose range (~200 - 800 mg/kg BPA). Over the dose range 2 -20 mg/kg BPA, there was no uterotrophic response and no increase in gene expression. The authors concluded that BPA does not produce reproducible changes in gene expression in the uterus of immature rats at dose levels that are not also uterotrophic. Therefore, in the present study, the no effect level for uterotrophic activity for BPA coincided with the no transcriptional effect level for uterine gene expression. (No content in EU, 2008)