Registration Dossier

Toxicological information

Specific investigations: other studies

Currently viewing:

Administrative data

Link to relevant study record(s)

Referenceopen allclose all

Endpoint:
specific investigations: other studies
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
data from handbook or collection of data
Remarks:
Specific investigation not conducted according to regulatory test guidelines but providing relevant information related to biphenyl-4,4'-diol
Qualifier:
no guideline available
Principles of method if other than guideline:
Assessment of the anti-melanogenic activity of biphenyl-4,4'-diol in melanoma cells in vitro.
GLP compliance:
no
Type of method:
in vitro
Endpoint addressed:
other: inhibition of melanogenic acitivity
Species:
other: in vitro assay
Details on test animals and environmental conditions:
Cell culture system - B16 cells from Korean Cell Line bank were cultured in DMEM with 10% FBS and penicillin/streptomycin, in 24 well plates for each assay. Each assay was completed in triplicate and repeated three times for reproducibility.

GSH and GSSG assays were performed according to the methods of Pandey and Katiyar. 25% of the meta-phosphoric acid added cell pellets were centrifuged and the supernatant assayed. For GSH phosphate buffer and o-phthalaldehyde were added to the supernatant and fluorescence measured after 20 minutes. GSSG was assayed after pre-incubation with N-ethylmaleimide for 20 minutes and 0.1M NaOH .

Cyclic AMP quantitation involved use of an enzyme immunoassay kit to measure intracellular cAMP. B16 cells were lyzed in 0.1 M HCl. Supernatants were collected, neutralised and diluted and a fixed amount of cAMP conjugate was added to compete with the cell lysate cAMP. The cAMP substrate solutions was added to wells to determine binding enzyme activity. Colour development was stopped and absorbance read at 405 nm. Colour intensity is inversely proportional to the cAMP concentration in the cell lysate.

For protein preparation and Western blotting the B16 cells were plated on 24 well multidishes and incubated with or without 100 nm alpha-MSH. the cells were then incubated with or without 4,4'-dihydroxybiphenyl for 24 hours and then washed twice in PBS. Total cell lysates were lysed in lysis buffer and the supernatant collected. Protein concentrations were determined using protein assay reagents. For Western blotting, equal amounts of protein were boiled for 2 minutes, chilled on ice and then subject to SDS-Page gel electrophoresis and electrophoretically transferred to a PVDF membrane and the proteins were then visualised with an enhanced chemiluminescence detection system.
Route of administration:
other: in vitro
Vehicle:
not specified
Remarks:
cultured in DMEM with 10% FBS and penicillin/streptomycin
Details on exposure:
Cell culture system - B16 cells from Korean Cell Line bank were cultured in DMEM with 10% FBS and penicillin/streptomycin, in 24 well plates for each assay. Each assay was completed in triplicate and repeated three times for reproducibility.

GSH and GSSG assays were performed according to the methods of Pandey and Katiyar. 25% of the meta-phosphoric acid added cell pellets were centrifuged and the supernatant assayed. For GSH phosphate buffer and o-phthalaldehyde were added to the supernatant and fluorescence measured after 20 minutes. GSSG was assayed after pre-incubation with N-ethylmaleimide for 20 minutes and 0.1M NaOH .

CAMP quantitation involved use of an enzyme immunoassay kit to measure intracellular cAMP. B16 cells were lyzed in 0.1 M HCl. Supernatants were collected, neutralised and diluted and a fixed amount of cAMP conjugate was added to compete with the cell lysate cAMP. The cAMP substrate solutions was added to wells to determine binding enzyme activity. Colour development was stopped and absorbance read at 405 nm. Colour intensity is inversely proportional to the cAMP concentration in the cell lysate.

For protein preparation and western blotting the B16 cells were plated on 24 well multidishes and incubated with or without 100 nm alpha-MSH. the cells were then incubated with or without 4,4'-dihydroxybiphenyl for 24 hours and then washed twice in PBS. Total cell lysates were lysed in lysis buffer and the supernatant collected. Protein concentrations were determined using protein assay reagents. For Western blotting, equal amounts of protein were boiled for 2 minutes, chilled on ice and then subject to SDS-Page gel electrophoresis and electrophoretically transferred to a PVDF membrane and the proteins were then visualised with an enhanced chemiluminescence detection system.
Analytical verification of doses or concentrations:
no
Details on analytical verification of doses or concentrations:
no data
Duration of treatment / exposure:
24 hours
Frequency of treatment:
Single exposure
Post exposure period:
Not relevant
Remarks:
Doses / Concentrations:
5, 25 µM
Basis:
nominal conc.
No. of animals per sex per dose:
Each assay was completed in triplicate and repeated three times for reproducibility.
Control animals:
not specified
Details on study design:
The study included three primary investigations:
1) the effect of 4,4'-dihydroxybiphenyl on GSH and GSSG levels and the GSH/GSSG ratio
2) the effect of 4,4'-dihydroxybiphenyl on down regulation of tryosinase and MITF
3) the effect of 4,4'-dihydroxybiphenyl on cAMP and PKA activation
Examinations:
GSH and GSSG assays were performed according to the methods of Pandey and Katiyar. 25% of the meta-phosphoric acid added cell pellets were centrifuged and the supernatant assayed. For GSH phosphate buffer and o-phthalaldehyde were added to the supernatant and fluorescence measured after 20 minutes. GSSG was assayed after pre-incubation with N-ethylmaleimide for 20 minutes and 0.1M NaOH .

CAMP quantitation involved use of an enzyme immunoassay kit to measure intracellular cAMP. B16 cells were lyzed in 0.1 M HCl. Supernatants were collected, neutralised and diluted and a fixed amount of cAMP conjugate was added to compete with the cell lysate cAMP. The cAMP substrate solutions was added to wells to determine binding enzyme activity. Colour development was stopped and absorbance read at 405 nm. Colour intensity is inversely proportional to the cAMP concentration in the cell lysate.

For protein preparation and western blotting the B16 cells were plated on 24 well multidishes and incubated with or without 100 nm alpha-MSH. the cells were then incubated with or without 4,4'-dihydroxybiphenyl for 24 hours and then washed twice in PBS. Total cell lysates were lysed in lysis buffer and the supernatant collected. Protein concentrations were determined using protein assay reagents. For Western blotting, equal amounts of protein were boiled for 2 minutes, chilled on ice and then subject to SDS-Page gel electrophoresis and electrophoretically transferred to a PVDF membrane and the proteins were then visualised with an enhanced chemiluminescence detection system.
Positive control:
not applicable
Details on results:
Western blotting analysis revealed that tyrosinase protein levels were reduced by 4,4'-dihydroxybiphenyl.

The involvement of transcription factors in the regulation of melanogenesis by 4,4'-dihydroxybiphenyl was investigated. MITF protein levels were reduced in the B16 cells treated with 4,4'-dihydroxybiphenyl .

A suppressive effect of 4,4'-dihydroxybiphenyl on cAMP levels was also observed in alpha -MSH-treated cells, leading to the possibility that 4,4'-dihydroxybiphenyl modulates the cAMP-dependent melanogenesis likely by reducing cAMP.

4,4'-dihydroxybiphenyl effectively reduced pPKA, suggesting that alpha -MSH-induced pigmentation process was suppressed by 4,4'-dihydroxybiphenyl in B16 cells.
Conclusions:
It is proposed that the suppression of tyrosinase, MITF, cAMP, and PKA activation may be the major factors underlying 4,4'-dihydroxybiphenyl ’s antimelanogenic activity.
Executive summary:

Previous investigations have shown that 4,4'-dihydroxybiphenyl reduced melanin content via the inhibition of tyrosinase. In the current investigation, 4,4'-dihydroxybiphenyl treated B16 melanoma cells were used to measure several key cellular parameters known to be involved in melanogenic activity. Measurements included tyrosinase and microphthalmia transcription factor (MITF) protein levels, cyclic AMP levels, protein kinase A (PKA) activation, and reduced glutathione (GSH) and oxidized glutathione (GSSG) levels. The results showed that 4,4'-dihydroxybiphenyl effectively suppressed the amounts of tyrosinase and MITF proteins, cAMP levels, and PKA activation. 4,4'-dihydroxybiphenyl also enhanced the GSH/GSSG ratio. The study provided evidence that 4,4'-dihydroxybiphenyl suppressed several cellular key parameters in the melanogenic pathway by downregulating the cAMP-dependent PKA signaling pathway and decreasing MITF gene expression which in turn suppressed tyrosinase. It is concluded that the antimelanogenic action of 4,4'-dihydroxybiphenyl may be effected by a combination of its anti-oxidant property and its ability to enhance intracellular GSH levels.

Endpoint:
specific investigations: other studies
Type of information:
other: human case reports
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Remarks:
The report is an evaluation of 16 cases of human exposure to 4,4'-biphenol for possible signs of vitiligo.
Qualifier:
no guideline followed
Principles of method if other than guideline:
Examination of human case reports
GLP compliance:
not specified
Type of method:
in vivo
Endpoint addressed:
other: possible cases of vitilgo following 4,4'-biphenol exposure
Specific details on test material used for the study:
The cases of 16 employees were reviewed in detail, and the employees examined, to determine whether effects described following occupational exposure to 4,4'-biphenol were consistent with concerns raised about depigmentation of the skin.
Species:
other: human case reports
Strain:
not specified
Sex:
male/female
Details on test animals and environmental conditions:
Eighteen employees potentially exposed to 4,4'-biphenol were identified, each completed a medical questionnaire and was then examined by a physician. Two of the employees did not participate in the medical examination. The remaining 16 fell into four categories based on work type or location.
Manager and plant technology - 2; production plant workers - 8; laboratory staff - 4 and dispatch workers -2.
Route of administration:
other: possible occupational dermal exposure
Vehicle:
other: not applicable
Details on exposure:
Details of exposure are not available. The potential occupational exposure to individuals within the four groups identified varyed from sporadic product exposure (managers/despatch staff) to daily exposure during manufacturing process or laboratory uses. The sixteen employees had a duration of potential exposure varying between a few weeks to about two years.
Analytical verification of doses or concentrations:
no
Details on analytical verification of doses or concentrations:
Not applicable
Duration of treatment / exposure:
16 patients examined had a period of between a few weeks to circa two years during which symptoms had been observed.
Frequency of treatment:
Not applicable. Exposure varied from potentially daily to sporadic
Post exposure period:
not applicable
Remarks:
Doses / Concentrations:
Not quantified
Basis:
other: possible occupational exposure
No. of animals per sex per dose:
Not applicable. 16 human cases reviewed
Details on study design:
Eighteen potentially exposed employees were identified and each completed a questionnaire and were examined by the physician. Of the 18 potential exposed individuals, two did not participate in the medical examinations. The remaining sixteen participants were allocated to four general categories for assessment of potential exposure - manager/plant technologist (2); production plant (8); laboratory (4) and dispatch workers (2).
Examinations:
A physician completed a medical examination of 16 employees to assess the extent, severity and potential cause of various cases of dermatits or depigmentation.
Positive control:
not applicable
Details on results:
Neither the plant manager or plant techologist had any skin problems. Exposure was limited in both cases and one employee had only been working at the plant for three months. No evidence of vitiligo or adverse health finding associated with exposure to 4,4'-biphenol.

Among the production staff - four men had been working with 4,4'-biphenol for approximately two years. They had various degrees of dermal irritation but none showed any skin depigmentation. One employee presented with areas of pale skin around the eyes and areas of pallor and pigmented skin on the neck, with other symptoms. The diagnosis was potential melanotoxicity in this case. A second man, also exposed for upto two years, had indications of dermatitis affecting hands, shins, ears, knuckles and fingers, had doubtful loss of pigmentation and atypical shaped areas affected on the skin - the physician concluded melanotoxicity was not indicated in this case. One employee had areas of depigmentation on the chin and neck (barely discernible), indicative of vertiligo.


For the laboratory staff, three had various indications of dermatitis, urticaria, itchiness and one had no skin problems. Only two showed any indication of depigmentation , in one worker the associated loss of hair follicles, patches of dry discoloured skin, stining and burning sensations and sore, itchy eyes seemed to show some degree of resolution after a period without exposure to 4,4'-biphenol.

The two despatch staff had been working in the warehouse for approximately one year. One had symptoms of macular redness o the neck, chemical burns on the leg, general itching and sneezing but no evidence of depigmentation. The other reported transient redness and itching of skin and eyes, and sneezing, dermatitis was evident on the back of he hands (he had previous exposure to hydroquinone and substituted catechols). There was no evidence of depigmentation but irritant dermatitis was diagnosed.
Conclusions:
The examining physician concluded that in four of the sixteen cases evaluated, depigmentation was likely due to occupational vitiligo, caused by exposure to 4,4'biphenol.
Executive summary:

Two of the eight production staff and two of the four laboratory staff showed signs of slight depigmentation around the neck and head (and in one case the forearm). None of the cases were considered to be pronounced or extensive, but the examining physician did consider the depigmentation to represent occupational vitiligo due to exposure to 4,4'-biphenol (or possibly one of the minor manufacturing or product components). While an extensive literature search for reports of 4,4'-biphenol associated depigmentation, vitiligo or melanotoxicity failed to identify any information, vitiligo has been described as associated with occupational exposure to phenols and catechols e.g. p-tert-butylphenol (PTPB). The final product resulting from the Schenectady manufacturing process does contain very low concentrations of PTPB, and this may be confounding factor in the employee results, but the precautionary principle lead to the conclusion that the findings may be related to 4,4'-biphenol exposure and requisite precautions taken to avoid occupational risks.

Endpoint:
endocrine system modulation
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
data from handbook or collection of data
Remarks:
Published study; non-standard investigation
Qualifier:
no guideline available
Principles of method if other than guideline:
Investigation of oestrogen receptor binding in vitro
GLP compliance:
no
Type of method:
in vitro
Endpoint addressed:
other: oestrogen receptor binding
Specific details on test material used for the study:
>98% purity
Species:
mouse
Strain:
CD-1
Sex:
female
Details on test animals and environmental conditions:
Cytosol was prepared from the uteri of ovariectomised mice
Route of administration:
other: in vitro
Vehicle:
unchanged (no vehicle)
Details on exposure:
Uterine tissue was homogenized with a Polytron homogenizer for 10 sec at a ratio of 75 mg wet weight/ml of TEG buffer (10 mM Tris, 1.5 mM EDTA, 10% glycerol, pH 7.6). The homogenate was filtered through 125um Nitex and the filtrate was centrifuged at 1,000 x g for 10 min to separate nuclei. The supernatant was recentrifuged at 105,000 x g for 45 min at 4°C to form the cytosol fraction. Cytosolic estrogen receptor was used either directly or enriched by a 0-40% ammomum sulfate fractionation. Ammonium sulfate fractionation was achieved by stirring the uterine cytosol at 4°C and slowly adding ultrapure crystalline ammonium sulfate (Schwartz-Mann) to a final concentration of 40%. The solution was allowed to stir for an additional 60 min at 4°C and the resulting solution was centrifuged at 20,000 X g for 30 min at 4°C. The supernatant was decanted and the precipitate resuspended in TEG buffer, and aliquots were used directly for the binding assay. The precipitate containing ER was resuspended in TEG buffer. A 100 u l aliquot of ER preparation was incubated for 18 hr at 4°C with 5 nM [3H]estradiol and 2.5-25,000 nM concentrations of the unlabeled competitors. Receptor-bound fractions were assayed using the hydroxylapatite absorption procedure. Competitive binding data were analyzed and the C50 values (the concentration of unlabeled inhibitor yielding half-maximal specific binding relative to estradiol) were determined using a commercial CDATA computer program.
Analytical verification of doses or concentrations:
no
Details on analytical verification of doses or concentrations:
not applicable
Duration of treatment / exposure:
18 hours
Frequency of treatment:
Single exposure in vitro
Post exposure period:
Not applicable
No. of animals per sex per dose:
Not applicable; in vitro study
Examinations:
Receptor binding activity in vitro
Positive control:
Oestradiol and DES
Details on results:
EC50 binding values were determined from competitive binding curves - the lower the EC50 value the stronger the binding action to oestrogen receptors. The EC50 value for 4,4'-dihydroxybiphenyl was >5000 indicating weak binding activity in vitro.
Conclusions:
The receptor binding activity of 4,4'-dihydroxybiphenyl in vitro was very low
Executive summary:

4,4'-dihydroxybiphenyl was tested for binding activity to soluble uterine oestrogen receptor protein. The receptor binding activity of 4,4'-dihydroxybiphenyl in vitro was very low (EC50 >5000). The uterotrophic assay indicated a correlation between receptor binding in vitro and biological activity in vivo, from which it can be hypothesised that 4,4'-dihydroxybiphenyl is unlikely to elicit a biologically significant uterotrophic response.

Endpoint:
endocrine system modulation
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
data from handbook or collection of data
Remarks:
Assay of oestrogen receptor binding in vitro: non-standard pubished study
Qualifier:
no guideline available
Principles of method if other than guideline:
Investigation of oestrogenic activity in competition binding assays with ERa or ERb protein, and in stably transfected cells.
GLP compliance:
no
Type of method:
in vitro
Endpoint addressed:
other: endocrine dirsuption potential
Species:
other: in vitro
Strain:
other: not applicable
Sex:
not specified
Details on test animals and environmental conditions:
Various comparative assays were included in the non-guideline investigation including:
expression and generation of ERa and ERb protein extracts
non-separation solid phase ligand binding competition experiments
ligand binding experiments with solubilised receptor using gel filtration for separation of bound and free radioligand
transient gene expression assay in 293 human embryonal kidney cells
Route of administration:
other: in vitro
Vehicle:
not specified
Details on exposure:
Various in vitro exposure methods
Analytical verification of doses or concentrations:
not specified
Details on analytical verification of doses or concentrations:
not applicable
Duration of treatment / exposure:
Various in vitro exposure methods with differing treatment phases dependent on the nature of the assay
Frequency of treatment:
Various in vitro exposure methods with differing treatment phases dependent on the nature of the assay
Post exposure period:
No data
No. of animals per sex per dose:
In vitro assays, animal numbers not applicable
Details on study design:
Refer to attached document
Examinations:
The relative binding affinity of 4,4'-biphenol with oestrogen alpha and beta receptors was investigated in vitro. Transcriptional activation of the ostrogen receptors was also investigated in stably transfected human embryonic kidney cells.
Positive control:
17ß-estradiol used as the comparator for various test substances
Details on results:
Equilibrium binding of radioligands ERa and ERb proteins in the presence of different concentrations of unlabelled competitors provides information on the relative binding affinities of the competitors. The relative binding affinities of 4,4'-biphenol were <0.01 and 0.03 for ERa and ERb respectively (3000 to 10000 fold lower than 17beta-oestradiol). The relative transactivation activities for 4,4'-biphenol were 53 (ERa) and 72 (ERb), compared to a value of 100 for 17beta-oestradiol.


Equilibrium binding of radioligands ERa and ERb proteins in the presence of different concentrations of unlabelled competitors provides information on the relative binding affinities of the competitors. The relative binding affinities of 4,4'-biphenol were <0.01 and 0.03 for ERa and ERb respectively (3000 to 10000 fold lower than 17beta-oestradiol). The relative transactivation activities for 4,4'-biphenol were 53 (ERa) and 72 (ERb), compared to a value of 100 for 17beta-oestradiol.

Conclusions:
Studies in vitro indicate oestrogenic activity for 4,4'-biphenol.
Executive summary:

The oestrogenicity of 4,4'-biphenol was investigated in vitro using isolated oestrogen receptors and stably transfected cells. A low binding affinity of 4,4'-biphenol for oestrogen receptor alpha and beta is reported; values of <0.01 and 0.03 are reported compared to 100 for the reference compound 17 -beta oestradiol. However further investigation in stably transfected cells indicated 4,4'-biphenol to be a relatively strong transcriptional activator of oestrogen receptor alpha and beta; values of 53 and 72 are reported compared to

100 for the reference compound 17 -beta oestradiol.

Endpoint:
endocrine system modulation
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
data from handbook or collection of data
Remarks:
Published paper; non-standard investigation
Qualifier:
no guideline followed
Principles of method if other than guideline:
Assessment of oestrogen-like potency by measuring various parameters in MCF-7 cells including induction of pS2 protein, induction of progesterone receptor, reduction of oestrogen receptor levels and stimulation of cell growth.
GLP compliance:
no
Type of method:
in vitro
Endpoint addressed:
other: oestrogen receptor binding
Specific details on test material used for the study:
>99% purity
Species:
other: MCF-7 cells
Strain:
other: human breast cancer cell line
Sex:
female
Details on test animals and environmental conditions:
Oestrogen-dependent mammary cells, MCF-7 (BUS-stock), were provided by Tufts University Schoool of Medicine, Boston, Massachusetts. The cells were grown in DME (Dulbecco's modified Eagle's medium (phenol red free DME)) supplemented with 5% FBS, to confluence and passaged every 4-5 days over trypsin-EDTA. Cells were used for 30 passages and then renewed from stock.
Route of administration:
other: in vitro
Details on exposure:
Oestrogen receptors were isolated from MCF-7 cells as described by Soto et al. Cells were grown to confluence in 150 cm² tissue culture flasks in DME with 5% FBS. One day before harvesting, the medium was changed to phenol red-free DME with 5% CD-FBS. Cells were trypsinized and washed once with phosphate buffered saline and resuspended at 50 x 10E6 cells/ml in buffer (500 mM KCl, 1.5 mM EDTA, 10 mM Tris-HCl, pH 7.4), at 4°C. The cells were frozen in liquid nitrogen and rapidly thawed before centrifugation at 100,000 g for 1 hr. The supernatant (cytosol) was stored at 70°C until use.

Oestrogen receptors (cytosols from 2.5 x 10E6 cells), were incubated for 2 hours at 4°C with 2 nM radiolabelled 17b-oestradiol alone or in combination with unlabeled competitors at different concentrations of 17beta-oestradiol (10E-12 to 10E-6 M) or test compounds (10E-7 to 10E-3 M).
To dissolve hydrophobic chemicals the reaction mixtures contained 15% dimethyl sulfoxide. This did not alter the shape of the competition replacement curve. Free hormone was removed by a 30 min. incubation with 0.5% charcoal/0.05% dextran at 4°C. The mixture was then centrifuged at 800 x g, and the remaining radioactivity of an aliquot of the supernatant was determined by liquid scintillation spectrometry.

In the cell proliferation phase MCF-7 cells (4 x 10E4) were seeded in 6-well plates in DME with 5% FBS. The following day the medium was changed to phenol red-free DME with 10% CD-FBS, and the cells were exposed to 17b-oestradiol (10E-13 M to 10E-9 M) or test chemicals (10E-11 M to 10E-5 M). The test chemicals were dissolved in ethanol (final concentration in the culture medium did not exceed 0.2%; a concentration that did not affect the cell growth).
Cell growth was measured as micrograms of DNA on day 6 of exposure by fluorometric quantitation in 0.02 M Na2EDTA/NaOH pH 12.5, of the Hoechst 33258 dye (1 microM) using an automated set-up and a Perkin Elmer LS50 luminescence spectrophotometer.

There was no effect of the solvent on the cell growth, measured as micrograms DNA after 6 days (0.58/0.23 and 0.63/0.17 for untreated and 0.2% ethanol, respectively). The maximal cell growth induced by 17b-oestradiol was 13– 24 times higher than the solvent control in the individual experiments.

The proliferative effect (PE) was calculated as the ratio between the cell yield obtained with the chemical, versus the cell yield in the hormone-free solvent control. The relative proliferative effect was calculated as 100 x ((PE -1 of the test compound)/(PE-1 of 17b-E)). The relative proliferative potential was calculated by taking the ratio between the concentration of 17b-oestradiol and of the test chemical needed for inducing 40% relative proliferative effect ( EC40 value). The 40% level was chosen to be able to compare the relative proliferative potential of the partial agonists as well as that of the agonists. To verify that induced cell growth was stimulated through the oestrogen receptor-signal pathway, cells were co-exposed with the lowest concentration of the chemical inducing maximal cell proliferation and 100 nM 4-OHTAM for 6 days.

Induction of pS2, oestrogen receptor and PgR. MCF-7 cells (2 x 10E6) were seeded in 75 cm² flasks in DME with 5% FBS. The next day the medium was changed to phenol red-free DME with 10% CD-FBS, and the cells were exposed to chemicals at the lowest concentration giving maximum growth in the proliferation experiment. The oestrogen regulated secretory protein pS2 was measured in the medium on day 3 after the start of exposure by ELISA. Radioactivity was measured by a auto-gamma counter. The control levels (100%) of pS2 ranged from 0.3 to 3.6 µg pS2 in the medium/mg cytosolic protein in the different experiments.
Cytosolic extracts were harvested from the same flasks on day 3 after exposure. Cells were washed once with PBS at room temperature, frozen in liquid N2, and harvested in 1 ml buffer (10 mM Tris, 1.5 mM EDTA, 0.05 M sodium molybdate and 1 mM monothioglycerol, pH 7.4) using a plastic scraper. The extract was centrifuged at 100,000 x g for 1 hr at 4°C, and the supernatant (cytosolic extract) was stored at -70°C until analyzed. The levels of oestrogen receptor and PgR were measured in aliquots of cytosolic extract by enzyme immunoassay. The colorimetric reaction was measured as absorbance in a Perkin Elmer UV/IL lambda 6 spectrophotometer. The control level (100%) of PgR ranged from 17 to 77 fmol/mg cytosolic protein in the different experiments. The control level (100%) of oestrogen receptor ranged from 111 to 458 fmol/mg cytosolic protein in the different experiment. Total protein in the cytosol was measured.
Analytical verification of doses or concentrations:
no
Details on analytical verification of doses or concentrations:
not applicable
Duration of treatment / exposure:
6 days
Frequency of treatment:
Single exposure
Post exposure period:
None
Remarks:
10E-11 to 10E-5 M (actual concenration)
No. of animals per sex per dose:
Not applicable - human breast cancer cell line used, oestrogen receptors were isolated from MCF-7 cells.
Examinations:
Binding to human ostrogen receptor and induction of cell growth.
Effects on the expresssion of oestrogen-regulated protein
Positive control:
17 beta-oestradiol
Details on results:
The affinity for binding to human oestrogen receptors was measured by substitution of radiolabelled 17 beta-oestradiol from human oestrogen receptor isolated from MCF-7 cells. RBA (relative binding affinity) for 4,4'-dihydroxybiphenyl was 0.085%.

The EC50 values - concentration of test compound required for 50% of radiolabelled 17beta-estradiol to be displaced from the oestrogen receptor was 3.35 µM for 4,4'-dihydroxybiphenyl (representing the lowest relative binding affinity).

Induction of cell growth - 4,4'-dihydroxybiphenyl induced a maximal cell growth stimulation of 43% in comparison with 17 beta-oestradiol. It was noted that higher test concentration often reduced the proliferative effects. The relative proliferative potential was calculated by comparing the test concentrations that induced 40% cell growth with the concentration of estradiol having the same effect. The RPP for 4,4'-dihydroxybiphenyl 240,000 fold lower than 17 beta-estradiol

Effects on expression of oestrogen-regulated protein were investigated using a small secretorial peptide, pS2, synthesised in MCF-7 cells in the presence of oestrogen. Following exposure of MCF-7 cells to the test compounds for three days, levels of pS2, PgR and oestrogen receptor were measured in medium or in cytosol. PgR and pS2 levels reach maxima after three days following oestrogen stimulation. The lowest concentration of test compouind that induced maximal cel growth was used in this assay. 17 beta-oestradiol iduced 3-11 fold pS2 levels commpared with controls. The induction by 4,4'-dihydroxybiphenyl was weak and did not differ significantly from the control.


Based on the data presented 4,4'-dihydroxybiphenyl is classified as a partial agonist at the oestrogen receptor.



Conclusions:
The results of this study indicate that 4,4'-dihydroxybiphenyl is a partial agonist at the oestrogen receptor.
Executive summary:

The effects of 4,4'-dihydroxybiphenyl on oestrogen receptor binding, cell proliferation and regulation of oestrogen sensitive proteins were investigated in vitro in the human breast cancer cell line MCF-7.  The investigation examined induction of pS2 protein and progesterone receptor, reduction of oestrogen receptor level, and stimulation of cell growth.

The substance 4,4'-dihydroxybiphenyl showed 'oestrogen-like' properties in MCF-7 cells; with weak receptor binding but no induction of oestrogen-specific protein expression.

The results of this investigation indicate that 4,4'-dihydroxybiphenyl does not mimic 17 beta-oestradiol in this test system but was shown to be a partial agonist of 17 beta-oestradiol for oestrogen receptor binding.

Endpoint:
endocrine system modulation
Type of information:
other: Assay review
Adequacy of study:
weight of evidence
Reliability:
other: not applicable
Rationale for reliability incl. deficiencies:
other: The publication evaluates the use of E-SCREEN and discusses the validity and reliability of the tool subjectively.
Qualifier:
no guideline available
Principles of method if other than guideline:
The E-SCREEN assay was developed to assess the estrogenicity of environmental chemicals using the proliferative effect of estrogens on their target cells as an end point. This quantitative assay compares the cell number achieved by similar inocula of MCF-7 cells in the absence of estrogens (negative control) and in the presence of 1 7,B-estradiol (positive control) and a range of concentrations of chemicals suspected to be estrogenic.

The novel E-SCREEN assay measures estrogen induced increase of the number of human breast MCF-7 cells and is recognized as biologically equivalent to the increase of mitotic activity in the rodent endometrium. The objectives of this study were to validate the E-SCREEN assay and to test the estrogenicity of chemicals released into the environment in large volumes.
GLP compliance:
not specified
Type of method:
in vitro
Endpoint addressed:
other: use of the E-SCREEN to identify environmental chemicals with potential oestrogenic activity
Specific details on test material used for the study:
One of the industrial chemicals assayed for xenobiotic oestrogenic activity was 4,4'-dihydroxybiphenyl.
Species:
other: in vitro screen using MCF-7 cells
Strain:
other: Human breast cancer oestrogen-sensitive MCF-7 cells
Details on test animals and environmental conditions:
Cell line and cell culture condition: Human breast cancer oestrogen-sensitive MCF-7 cells were obtained from the Michigan Cancer Foundation. For routine maintenance, cells were grown in Dulbecco's modification of Eagle's medium (DME) supplemented with 5% fetal bovine serum (FBS) at 37°C in an atmosphere of 5% CO2/95% air under saturating humidity.

Steroids, xenobiotics and growth factors tested: 17,-Estradiol (E2) was obtained from Calbiochem. Other steroids were purchased from Steraloids. R26008 (allenolic acid) was supplied by Roussel-UCLAF. Toxaphene (technical grade) and endosulfan (technical grade) were obtained from Chem Services. Endosulfan alpha and beta isomers, o,p'-DDT, p,p'-DDT, p,p'-DDD, p,p'-DDE, PCB congeners, methoxychlor, dieldrin, phthalate esters, and antioxidants were from Ultra Scientific. The hydroxylated biphenyls were obtained from National Institute of Environmental Health Sciences. DES metabolites were also obtained from NIEHS. Estradiol was stored as a 1-mM stock solution in ethanol at -20°C. Pesticides were dissolved in ethanol to a final concentration of 10 mM, except endosulfan mixed isomers, dieldrin, and toxaphene, which were dissolved in dimethyl sulfoxide (DMSO); they were all diluted to desired concentrations in phenol red-free DME immediately before using. The final solvent concentration in culture medium did not exceed 0.1%; this concentration did not affect cell yields. Human recombinant EGF, basic FGF, and IGF-1 were purchased from Collaborative Research.

Plasma-derived and blood derived human serum:
Plasma-derived human serum was prepared from outdated plasma supplied by the New England Medical Center Blood Bank, (Boston, MA). Calcium chloride was added to a final concentration of 30 mM to facilitate clot formation. Blood-derived serum was obtained using blood from healthy adult volunteers; blood was allowed to clot in glass centrifuge tubes for 2 to 4 hr to obtain serum. Plasma- and blood-derived serum were clarified by centrifugation (2000xg for 10 min), heat-inactivated (56°C for 30 min), centrifuged, charcoaldextran stripped, and stored in glass tubes at -20°C until use.

Charcoal (Norit A, acid washed; Sigma Chemical Co, St. Louis, MO) was washed twice with cold sterile water immediately before using. A 5% charcoal-0.5% dextran T70 (Pharmacia-LKB, Uppsala, Sweden) suspension was prepared. Charcoal-dextran (CD) suspension aliquots of a volume similar to the serum aliquots to be processed were centrifuged at 2500 rpm for 10 min. Supernatants were aspirated and serum aliquots were mixed with the charcoal pellets. This charcoal-serum mixture was maintained in suspension by rolling at 4 cycles/min at 37°C for 1 hr. This suspension was centrifuged at 2000xg for 20 min. The supernatant was then filtered through a 0.45-pm Nalgene filter. Over 99% of serum sex steroids were removed by this
treatment when determined by removal of 3H-E2; E2 levels after CD treatment were less than 0.01 pg/ml when measured by radioimmunoassay. CD sera were stored at -20°C until needed. Samples kept for 1 year in the freezer maintained their inhibitory properties on the proliferation of human estrogen-sensitive breast tumor MCF-7 cells; plasma- and blood-derived sera were equally effective.
Route of administration:
other: in vitro test
Details on exposure:
The E-SCREEN Test
The E-SCREEN assay was developed based on the following premises:
a) a human serum-borne molecule specifically inhibits the proliferation of human estrogen-sensitive cells; and
b) estrogens induce cell proliferation by canceling this inhibitory effect. Nonestrogenic steroids and growth factors did not abolish the proliferative inhibition by mammalian serum.

Cloned MCF-7 cells were trypsinized and plated into 12-well plates at initial concentrations of 20,000 cells per well. Cells were allowed to attach for 24 hr; then, the seeding medium (5% FBS in DME) was removed and replaced by the experimental medium [5% CD human serum supplemented to phenol red-free DME (CDHuS)]. A range of concentrations of the test compounds was added to this medium. The bioassay was terminated on day 6 (late exponential phase) by removing the media from the wells, adding a cell lysing solution (10% ethylhexadecyl-dimethylammonium bromide in 0.5% Triton X-100, 2 mM MgCl2, 15 mM NaCl, 5 mM phosphate buffer, pH 7.4) and counting the nuclei in a Coulter Counter Apparatus.

The best estimate of the proliferative behavior of a cell population is'td' or doubling time. Td is the time interval in which an exponentially growing culture doubles its cell number. Determining td requires measuring cell yields at several time intervals during the exponential proliferation phase. A less cumbersome alternative to measuring proliferation rates is comparing the cell yield achieved by similar cell inocula harvested simultaneously during the late exponential phase of proliferation. The proliferative effect (PE) is measured as the ratio between the highest cell yield obtained with the test chemical and with the hormone-free control. Under these experimental conditions, cell yield represents a reliable estimate of the relative proliferation
rate achieved by similar inocula exposed to different proliferation regulators.

In this experimental design, MCF-7 cell yields were measured 6 days after To; however, significant differences between control and estrogen-treated cultures are apparent after 4 days.
The estrogenic activity of xenobiotics was assessed
a) by determining their relative proliferative potency (RPP), which measures the ratio between the minimal concentration of estradiol needed for maximal cell yield and the minimal dose of the test compound needed to achieve a similar effect; and
b) by measuring their relative proliferative effect (RPE), which is 100 times the ratio between the highest cell yield obtained with the chemical and with E2. RPE is calculated as 100 x (PE-1) of the test compound/(PE-1) of E2. Thus, the RPE indicates whether the compound being tested induces a proliferative response quantitatively similar to the one obtained with E2, that is, a full agonist (RPE = 100), or a proliferative yield significantly lower than the one obtained with E2, that is, a partial agonist. For screening purposes, the range of xenobiotic concentrations was from 1 nM to 10 µM, and for E2 from 0.1 pM to 1 nM, measured at intervals of one order of magnitude.

For the progesterone receptor assay and oestrogen receptor processing MCF-7 cells were seeded in 25-cm2 flasks in 5% FBS-supplemented DME. Twentyfour hours later, the medium was changed to 5% CDHuS, and the chemicals to be tested were added. Control flasks were treated with vehicle. After 72 hr of exposure to the test xenoestrogens, medium was aspirated, the cell layer was rinsed with PBS, and the cells were frozen in liquid nnitrogen gas. To extract receptor molecules, cells were incubated with 1 ml of extraction buffer (0.5 M KCI, 10 mM potassium phosphate, 1.5 mM EDTA, and 1 mM monothioglycerol, pH 7.4) at 4°C for 30 min. After centrifugation to pellet the cell debris, receptor levels were measured in 100 µl extract aliquots by enzyme immunoassay using the Abbott estrogen and progesterone receptors kits.

pS2 assay- Culture media were harvested after 72 hr of exposure to the test chemicals and centrifuged to eliminate floating and detached cells; samples were kept frozen at -80°C until the immunoradiometric assay was performed following the manufacturer's protocol.

Determination of relative binding affinities
MCF-7 cells were grown in 150-cm² flasks in 5% FBS; they were harvested during the late exponential phase after 24 hr of exposure to 5% CDHuS. Cells were rinsed with PBS, and a suspension of 20 x 106E cells/ml of buffer (500 mM KCl, 1.5 mM EDTA, 10 mM Tris-HCl, pH 7.4, at 4°C) was sonified at 40°C (5-sec pulses with 30-sec intervals). The cell homogenate was centrifuged at 100,000 xg for 40 min, and supernatant aliquots were incubated with 2 nM 3H-E2 alone and in combination with unlabelled competitors at concentrations ranging from 1 pM to 1 µM E2 or 1 nM to 1 mM xenoestrogens for 16 hr at 40°C. The reaction mixture contained 15% DMSO to solubilize hydrophobic xenoestrogens.
This treatment did not alter the shape of the competition curve for E2 and nonylphenol, the only two compounds from which a competition curve could be obtained in the absence of DMSO. Separation of bound and free hormone was done by CD adsorption
Analytical verification of doses or concentrations:
not specified
Details on analytical verification of doses or concentrations:
No details
Details on study design:
Results were expressed as the mean ± SE. Proliferation yield experiments conducted in duplicate wells were repeated at least a minimum of 5 times. Mean cell numbers from each experiment were normalized to the steroid-free control (100%) to correct for differences in the initial plating density.
Differences between the diverse steroid treatment groups were assessed by analysis of variance and the a posteriori Shaffe's test (19). A p value of < 0.05 was
regarded as significant.
Details on results:
The E-SCREEN was used to identify estrogenic activity. Estrogenic effects were ranked according to relative proliferative effect based on the induction of a proliferative response in MCF-7 cells relative to estradiol (RPE = 100%). The RPE for 4,4'-dihydroxybiphenyl was 84% according to the E-SCREEN assay indicating partial agonist activity. The relative proliferative potency was calculated as a ratio of oestradiol and xenobiotic doses required to elicit maximal cell yields. RPP for 4,4'-dihydroxybiphenyl was 0.0003%, compared with 100% for oestradiol.
Conclusions:
Based on the results of this assay (the E-screen) it is concluded that 4,4'-dihidrixybiphenyldiol is classified as a partial agonist against the oestogen receptor and it is not classified for oestrogenic activity.
Executive summary:

This paper reports the development of the 'E-screen' assay for the assessment of the oestrogenicity of environmental chemicals, using the proliferative effect of oestrogens on target cells as an end point. This quantitative assay compares the effects of chemicals on the proliferative activity in oestrogen-sensitive MCF-7 cells in the absence of estrogens (negative control) and in the presence of 17 beta-oestradiol (positive control) at a range of concentrations. According to the E-screen assay, 4,4'-dihidroxybiphenyldiol is classed as a partial agonist at the oestrogen receptor and it is not classified for oestrogenic activity.

Endpoint:
endocrine system modulation
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
data from handbook or collection of data
Remarks:
Peer-reviewed publication
Qualifier:
equivalent or similar to
Guideline:
other: OECD in vivo uterotrophic screening assay
Principles of method if other than guideline:
OECD uterotrophic assay is an in vivo screen to detect oestrogenic effects with potentially endocrine disrupting chemicals, utilising the rapid and dramatic growth response in rat uterus during the natural oestrous cycle and the effect of oestrogen on the uterus. The receptor binding assay was developed as an in vitro pre-sceen to select potential chemicals with oestrogenic activity prior to conducting an in vivo study. While good correlation was established for results of the uterotrophic and receptor-binding assays, it was not possible to define the relationship between receptor-binding affinity and uterotrophic potency. The current report seeks to address that relationship.
GLP compliance:
not specified
Type of method:
in vivo
Endpoint addressed:
other: uterotrophic response
Specific details on test material used for the study:
Purity >99%; dissolved in olive oil.
Species:
rat
Strain:
Crj: CD(SD)
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source:Rat dams and their 10-day old female pups were obtained from Charles RIver Japan Inc
- Age at study initiation: The pups were weaned at 19 days old and study commenced the following day.
- Weight at study initiation: not stated
- Fasting period before study: not applicable
- Housing: Housed with dams in polycarbonate pens upto time of weaning and subsequently individually in wire mesh cages
- Diet (e.g. ad libitum): CRF-1 Oriental Yeast Co, Diet provided ad libitum to dams pre-weaning and MF diet oriental Yeast Co. also provided ad libitum to immature rats after weaning
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 9 days between arrival and weaning

ENVIRONMENTAL CONDITIONS
- Temperature (°C):23±2
- Humidity (%):55±5
- Air changes (per hr): not stated
- Photoperiod (hrs dark / hrs light): 12/12h

Route of administration:
subcutaneous
Vehicle:
olive oil
Details on exposure:
Biphenyl-4,4'-diol was subcutaneously injected into the back of 20-day-old female rats on three consecutive days.

As a positive control, ethynyl estradiol (EE, CAS no. 57-63-6, 98% purity, Sigma) in olive oil was also subcutaneously injected into the back of some rats in a dose of 0.6 µg/kg per day on three consecutive days after administration of biphenyl-4,4'-diol at the same dose.
A vehicle control group was injected with olive oil alone, and a positive control group was injected with EE after administration of olive oil.
A group injected with the estrogen-antagonist chemical tamoxifen in a dose of 1 mg/kg per day plus EE was also established to confirm the reliability of this study.
Analytical verification of doses or concentrations:
not specified
Details on analytical verification of doses or concentrations:
No information
Duration of treatment / exposure:
Three consecutive days of subcutaneous injection
Frequency of treatment:
Three times - daily injection
Post exposure period:
24 h after final dose administration
Remarks:
Doses / Concentrations:
For 4,4'-biphenol the doses were 0 (vehicle control), 60, 200 and 600 mg/kg bw/day. A second series used the same dose levels plus EE (ethynyl estradiol) in each case as a positive control and TMX+EE was included .
Basis:

No. of animals per sex per dose:
Six immature females
Control animals:
yes, concurrent vehicle
Details on study design:
Mean bodyweights for the 4,4'-biphenol groups ranged between 57.3 and 60.9g.
For the receptor binding affinity assay :
10mM stock solutions of each test substance and [2,4,6,7,16,17-3H] 17-estradiol were prepared with DMSO and diluted to ten fold higher concentration of the
final one with Tris–HCl (pH 7.4) containing 1mM EDTA, 1mM EGTA, 1mM NaVO3, 10% glycerol, 10 mg/ml gamma-globulin, 0.5mM phenylmethylsulfonyl fluoride, and 0.2mM leupeptin (binding buffer).

A solution 10µL dissolved in the binding buffer of approximately 10nM of recombinant human estrogen receptor ligand binding domain fused with glutathione
S-transferase and expressed in E. coli was dissolved in the binding buffer. After adding the sample solution (10 µl) of each chemical and 5 nM of [2,4,6,7,16,17-3H] 17-estradiol (10µl), the solution was incubated for 1 h at 25°C. Free radioligand was removed by incubation with 100µl of dextran-coated charcoal for 10 min at 4°C. After filtration, radioactivity in the filtrate was measured using liquid scintillation counter. Chemicals were tested over the 10E−11 to 10E−4M concentration range.

The percent ratio (B/B0 (%)) of standard ligand ([3H] 17beta-estradiol) bound to the receptor was calculated from the radioactivities of the solutions with and
without the test substance, subtracting the radioactivity due to non-specifically bound standard ligand to the receptor. The B/B0 values as a function of the concentration were fitted to the logistic equation and IC50 value of each chemical was calculated by least-squares method.

The binding abilities of test chemicals to the receptor were evaluated by relative binding affinity (RBA), ratio of IC50 values to l7beta-estradiol.
Examinations:
The relative binding affinity for 4,4'-biphenol was 0.0883% of E2.
Bodyweights were recorded daily throughout the assay. Clinical signs were also recorded daily.
At necropsy uterus weights were recorded for wet weights (absolute and relative) and for blotted weight (absolute and relative).
Positive control:
Ethynyl estradiol added to replicate treatment groups for use as a positive control.
Details on results:
Watery uterine fluid was grossly detected in rats given EE, 600 mg/kg bw/d 4,4'-biphenol and severa

The uterine weight of the rats given EE was higher than that of the rats given vehicle alone, and the uterine weight of the rats given tamoxifen plus EE was lower than that of the rats given EE, confirming the reliability of this study.

Uterine weight was significantly higher in the 60, 200, and 600 mg/kg bw/d 4,4'-biphenol groups. A significant decrease in uterine weight in the uterotrophic assay was detected at the low and middle doses of 4,4-biphenol plus EE, but no decrease in uterine weight was observed at the high dose of this chemical plus EE.

Uterotrophic Response

4,4’-biphenol dose level [mg/kg bw]

Bodyweight (g)

Uterine wet weight

Uterine blotted weight

mg

mg/100 g

mg

mg/100 g

0 [untreated control]

60.9

37.4

61.4

36.2

59.4

60

57.7

47.8*

82.8**

46.8*

81.0**

200

59.3

85.3**

143.0**

83.0**

139.1**

600

59.0

185.4**

314.7**

130.9**

222.3**

0 + EE [EE control]

59.8

148.4

249.4

121.2

203.4

60 + EE

60.1

118.0**

196.3**

103.0**

171.5*

200 + EE

58.8

107.4

180.1

92.4*

155.47*

600 + EE

57.3

230.8

400.8

126.7

220.8

TMX + EE

58.4

84.2**

144.7**

82.0**

140.9**

*significantly different to control (p<0.05); **p<0.01

EE: ethynyl estradiol

TMX: Tamoxifen

Conclusions:
Oestrogen receptor binding and uterotrophic activity were demonstrated for biphenyl-4-4'-diol under the conditions of this assay.
Executive summary:

A uterotrophic assay was performed in immature female rats to assess the relationship between uterotrophic potency in vivo and oestrogen receptor-binding affinity in vitro. 4,4'-biphenol showed oestrogen receptor binding and uterotrophic activity under the conditions of this assay; a uterotrophic response was apparent at all dose levels tested (60, 200 and 600 mg/kg bw); dose levels of 60 and 200 mg/kg bw reduced the uterotrophic response to the positive control ethinyl estradiol but a dose level of 600 mg/kg bw enhanced the response to ethinyl estradiol.

Description of key information

Additional information on biphenyl-4,4'-diol relates to the potential oestrogenicity and the influence of the substance on dermal pigmentation.

Additional information

Studies of oestrogen receptor binding

A number of published studies are available which investigate the potential of biphenyl-4,4’-diol to interact with the oestrogen receptor in vitro.

Olsen et al (2003) investigated the interaction of biphenyl-4,4’-diol with the oestrogen-dependent MCF-7 human breast cancer cell line based on oestrogen receptor binding, cell proliferation and the expression of oestrogen-sensitive proteins. Biphenyl-4,4’-diol had some affinity for the oestrogen receptor and was shown to induce cell growth, but did not induce the expression of specific proteins.  The results of this investigation indicate that 4,4'-dihydroxybiphenyl does not mimic 17 beta-oestradiol in this test system but was shown to be a partial agonist of 17 beta-oestradiol for oestrogen receptor binding. Korach et al (1989) similarly demonstrated a weak binding activity for biphenyl-4,4’-diol at the oestrogen receptor in mouse uterine tissue; the authors conclude that a significant uterotrophic response would be unlikely in vivo. Soto et al (1995) report the development of the ‘E-screen’ assay for the assessment of oestrogenic activity in environmental chemicals and conclude, based on the results of this screening assay, that biphenyl-4,4’-diol is a partial agonist at the oestrogen receptor and it is not classified for oestrogenic activity. In a study in isolated receptors and stably transfected cells, Kuiper et al (1998) report a low binding affinity of biphenyl-4,4’-diol for the oestrogen alpha- and beta-receptors; however a relatively string transcriptional activation is reported. In a study in vivo, Yamasaki et al (2004) note a uterotrophic response in immature rats at dose levels of 60, 200 and 600 mg/kg bw/d, correlating with the extent of binding in vitro to recombinant human oestrogen receptor.

The results of the studies therefore indicate a possible oestrogenic effect of biphenyl-4,4’-diol. However it is notable that no effects on reproductive endpoints were noted in an OECD 422 screening study at dose levels of up to 200 mg/kg bw/d. No effects on developmental parameters were observed in the same study at any dose level.

No effects were either observed in an pre-developmental toxicity study in Wistar rats (OECD 414, 2018).

Finally, in a 90-day repeated toxicity study in Wistar rats (OECD 408, 2018) none test item-related effect was observed on oestrus cycles and sperm analysis, and not treatment-related effect have reported after microscopic observation of mammalian gland, ovaris, oviducts, testis and epididymis.

Effect on melanogenesis

In a study in vitro, No et al (2006) conclude that the demonstrated suppression of melanogenesis by biphenyl-4,4'-diol in B16 cells was likely to be related to its antioxidant capacity, coupled with its ability to enhance intracellular glutathione levels.

Report of vitiligo

Reported cases of possible vitiligo following occupational exposure to biphenyl-4,4’-diol (Jackson Hocking Ltd., 2003) are consistent with the in vitro mechanistic study on the inhibition of melanin production; however it is unclear whether effects are due to the substance itself or to the presence of impurities.