4,4'-isopropylidenediphenol

Endocrine disrupter testing in aquatic vertebrates – in vivo

Administrative data

Endpoint:

fish adult: (sub)lethal effects

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Remarks:

OECD 234 with minimal modifications

Data source

Reference

Reference Type:

publication

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD TG 234 (Fish Sexual Development Test)

GLP compliance:

no

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

Specific details on test material used for the study:

BPA obtained from Tokyo Chemical Industry Co., Ltd, Tokyo, Japan,
purity >99.0%

Sampling and analysis

Analytical monitoring:

yes

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION OF STOCK SOLUTIONS
14.4, 45, 144, 450 and 1440 mg of BPA were placed separately, each into 5-L glass media bottles (body diameter, 182 mm) and dissolved in 4.5 L of Milli-Q water with sonication for 120-180 minutes in an ultrasonic bath.

PREPARATION OF TEST SOLUTIONS
The stock solutions were diluted with dechlorinated tap water to the nominal concentrations for each group at a dilution ratio of 1 (aqueous stock solution) to 100 (dechlorinated tap water) by using a flow-through exposure system (SIS-1F; Shibata Scientific Technology (Tokyo, Japan). The water exchange rate was 5 volumes/day, and the stock solution was renewed every 3-4 days.

Test organisms

Aquatic vertebrate type:

fish

Test organisms (species):

Oryzias latipes

Study design

Test type:

flow-through

Water media type:

freshwater

Limit test:

no

Total exposure duration:

60 d

Test conditions

Test temperature:

25 +/- 2 °C

pH:

7.5 +/- 1

Nominal and measured concentrations:

nominal concentrations: control, 32, 100, 320, 1000 and 3200 μg/L
measured concentrations: 45, 92, 326, 1030 and 3406 μg/L

Details on test conditions:

FERTILIZATION AND HATCHING
Eggs were checked under a stereomicroscope, and the unfertilized, damaged or developmentally abnormal eggs were removed. After this selection process, fertilized eggs were exposed to BPA within 2 hours postfertilization (2hpf).
20 fertilized eggs from each concentration were distributed into 100-mL glass vessels. Observations were performed every 24 hours vira microscopic examination. Test solutions were exchanged once every 24 hours. Eight replicate 100-mL glass vessels were used for each concentration, for a total of 48 vessels.
0 - 30 DAYS POST HATCHING
After hatching, four replicate 5-L glass tanks were used for each concentration, for a total of 24 tanks. Thirty larvae were moved to each 5-L cubic glass tank (in total, 120 larvae per concentration) and cultured until 30 days posthatching (dph) using a flow-through system (water exchange rate, 5 volumes/day).
30 - 60 DAYS POST HATCHING
At 30 dph, 15 fish per tank (in total, 60 fish per treatment) were dissected. Thereafter, each 5-L cubic glass tank contained 15 fish (in total, 60 fish per treatment),which were cultured until 60 dph using the same flow-through system (water exchange rate, 5 volumes/day). A separate set of BPA exposure and control groups for the expression analysis of gonadal sex differentiation-related genes at stage 38 (n = 4) and 30 dph (n = 4) was set up additionally.

Dead individuals were removed as soon as they were observed.

Reference substance (positive control):

no

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

60 d

Dose descriptor:

LOEC

Effect conc.:

1.03 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

genetic/phenotypic sex ratio

Remarks:

sex reversal

Effect concentrations 2

Duration:

60 d

Dose descriptor:

NOEC

Effect conc.:

326 µg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

secondary sexual characteristics

Remarks:

or gonadal sex differentiation

Reported statistics and error estimates:

Test on homogeneity of variance of the data was performed with Bartlett's test (significance level, 5%) by using the open source statistical software R (http://www.R-project.org/) and the package Rcmdr (Fox & Bouchet-Valat, 2018). If homogeneity of variance was not rejected, differences in test results among treatments were performed using Dunnett's test. Otherwise Steel's test was performed.

Any other information on results incl. tables

Effect of BPA on gonadal sex differentiation

	Measured concentration		Genetic sex				Gonadal sex					Ratio of sex			Genetic sex	Gonadal sex		Ratio of sex
Age (dph)	μg/L		n		XX		Ovary		Testis-ova			Testis		reversal (%)	XY	Ovary	Testis-ova	Testis	reversal (%)
30	Control		40		21		21		0			0		0	19	0	0	19	0
	45		40		21		21		0			0		0	19	0	0	19	0
	92		40		22		22		0			0		0	18	0	0	18	0
	326		40		20		20		0			0		0	20	0	0	20	0
	1030		40		19		19		0			0		0	21	1	0	20	4.7
	3406		40		17		17		0			0		0	23	22	1	0	95.6
60	Control		60		32		32		0			0		0	28	0	0	28	0
	45		60		29		29		0			0		0	31	0	0	31	0
	92		60		22		22		0			0		0	38	0	0	38	0
	326		58		29		29		0			0		0	29	0	0	29	0
	1030		59		28		28		0			0		0	31	4	23	4	12.9
	3406		60		33		33		0			0		0	27	27	0	0	100

dph = days post hatching

Applicant's summary and conclusion

Validity criteria fulfilled:

not specified

Conclusions:

The effect of BPA on Japanese medaka using OECD TG234 with minor modifications, including analysis of the expression of gonadal sex differentiation and sex-determining genes at early developmental stages was studied by Horie et al. Testis-ova and sex reversal in XY males were observed in response to 1030 and 3406 μg/L BPA exposure at 30 and 60 dph from 2 hours postfertilization. Longer and higher concentrations of BPA exposure induced a greater rate of testis-ova and sex reversal.

Executive summary:

The effect of BPA on Japanese medaka using OECD TG234 with minor modifications, including analysis of the expression of gonadal sex differentiation and sex-determining genes at early developmental stages was studied by Horie et al. Testis-ova and sex reversal in XY males were observed in response to 1030 and 3406 μg/L BPA exposure at 30 and 60 dph from 2 hours postfertilization. Longer and higher concentrations of BPA exposure induced a greater rate of testis-ova and sex reversal.

The lowest observed effect concentration of BPA in the present study is LOEC < 3406 μg/L for mortality, embryo hatching and growth in O. latipes. In addition the no observed effect concentration of BPA on secondary sex characteristics or gonadal sex differentiation is NOEC = 326 μg/L