4,4'-sulphonyldiphenol

Endocrine disrupter testing in aquatic vertebrates – in vivo

Administrative data

Endpoint:

fish: other

Remarks:

Zebrafish (Danio rerio) Extended One Generation Reproduction Test (ZEOGRT)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

21.01.2020 - 18.05.2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD TG 240 (Medaka Extended One Generation Reproduction Test (MEOGRT))

Version / remarks:

The test design was an adaptation of the OECD TG 240 (Medaka Extended One Generation
Reproduction Test) guideline and followed a draft protocol. The OECD TG 240
stipulates that “the specific methods and observational endpoints detailed in this guideline are
applicable to Japanese medaka alone. Other small fish species (e.g. zebrafish) may be
adapted to a similar test protocol”. However, there are recognized differences in biological life
history traits between the two species that must be considered when adapting the protocol.
The present test design considered these aspects of zebrafish biology and incorporated testing
recommendations at each life stage from corresponding validated OECD guidelines.
Nevertheless, the ZEOGRT procedure has not undergone validation and this study is not a
standard guideline test, but rather a research investigation. This study was based on the
following document:
Teigeler, M. Draft Standard Operating Procedure: Zebrafish Extended One Generation
Reproduction Test. Version 1.1, June 2017

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

4,4'-sulphonyldiphenol

EC Number:

201-250-5

EC Name:

4,4'-sulphonyldiphenol

Cas Number:

80-09-1

Molecular formula:

C12H10O4S

IUPAC Name:

4,4'-sulfonyldiphenol

Details on test material:

NA

Specific details on test material used for the study:

Batch identification: 03508136W0
Purity: 99.87 area-%

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Samples from all test groups were collected directly from the middle of the test vessel using a glass pipette and were placed into glass sample vials (20 mL nominal volume). The required volume of the sample was 10 mL. The samples were transferred to the analytical laboratory and analyzed generally directly after sampling. If this was not possible, the samples were stored in a freezer at the Laboratory ECO (at –20°C) until analysis. In this case care was taken
that any test item which precipitated during storage of the sample was analyzed as well. At each sampling time, two additional retained samples per test group were taken, frozen and sent later to the analytical laboratory if the values deviated from the nominal concentration by more than 20%. Additional samples were taken and analyzed, if the results were outside of ±20% of the expected nominal concentration. The dates for sampling, for transfer of the
samples to the Analytical Laboratory and the dates of the analytical determination can be derived from the archived raw data of this study and from the Analytical Report.
Additionally, samples of the stock solution were taken and analyzed (not under GLP, data is archived together with the raw data of this study) in order to adjust the delivery rate of the stock solution pumps.

F0-generation:
Concentration control samples were taken at the start of exposure (study day 0), before insertion of the fish from the pre-treatment spawning groups, and then once weekly from alternating replicates per test group of the F0-generation. Sampling started on day 0 and ended on day 49. On day 8, samples were collected from all test vessels at the same time to confirm the uniformity of the concentration within each test group. Since the fish of the F0-generation
were sacrificed on consecutive days, the samples for concentration control analyses were taken on the first day of sacrifice.

F1-generation:
Concentration control samples were taken at the start of the F1-generation exposure period (day 0), before insertion of fertilized eggs from the F0-generation, and then once weekly from alternating replicates per test group of the F1-generation. Sampling started on day 0 and ended on day 125. On day 7 and day 70, samples were collected from all test vessels at the same time to confirm the uniformity of the concentration within each test group. Additional samplesgeneration were sacrificed on consecutive days, the samples for concentration control
analyses were taken on the first day of sacrifice.
were taken and/or retained samples were analyzed, if the results were outside of ±20% of the expected nominal concentration.
Since the fish of the F1-generation were sacrificed on consecutive days, the samples for concentration control analyses were taken on the first day of sacrifice.

F2-generation
Concentration control samples were taken at the start of the F2-generation exposure period (day 0), before insertion of fertilized eggs from the F1-generation, from all test vessels at the same time to confirm the uniformity of the concentration within each test group.

Test solutions

Vehicle:

no

Test organisms

Aquatic vertebrate type:

fish

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

The wild-type zebrafish strain was purchased from Fraunhofer IME laboratories (Schmallenberg, Germany) and was originally obtained from West Aquarium GmbH (Bad Lauterberg, Germany). The fish were bred in captivity specifically for scientific purposes. All fish originate from the same batch of fish and hatched October 2018.

Study design

Test type:

flow-through

Water media type:

freshwater

Limit test:

no

Total exposure duration:

170 d

Remarks on exposure duration:

F0-generation was exposed for 49-50 days. Fertilized eggs from F0 test groups were used to start F1generation which was exposed for 125 – 128 days. Viable eggs from the F1 test groups were collected to start the F2-generation which was exposed for 96 h.

Test conditions

Hardness:

100 mg/L CaCO3

Test temperature:

Approx. 26 ±1.5°C

pH:

7.5 – 8.5

Dissolved oxygen:

>60% saturation

Conductivity:

The dilution water used for the F0-generation had a specific conductivity of 264 μS/cm on day 0 and 255 μS/cm at the end of F0-generation exposure (day 50). The dilution water used for the F1-generation had a specific conductivity of 287 μS/cm at the end of exposure (study day128).

Nominal and measured concentrations:

Nominal concentrations:
0 mg/L (control), 2 µg/L, 10 µg/L, 50µg/L, 250 µg/L, 1250 µg/L

Measured concentrations (mean):
F0-generation: < LOQ, 1.75 µg/L, 9.86 µg/L, 45.33 µg/L, 220.39 µg/L, 1136.69 µg/L
F1-generation: < LOQ, 2.15µg/L, 10.03 µg/L, 50.74 µg/L, 228.38 µg/L, 1229.43 µg/L
F2-generation: < LOQ, 2.04 µg/L, 9.99 µg/L, 50.25 µg/L, 258.68 µg/L, 1332. 30 µg/L

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

128 d

Dose descriptor:

NOEC

Effect conc.:

0.25 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Male body lenght

Effect concentrations 2

Key result

Duration:

128 d

Dose descriptor:

LOEC

Effect conc.:

1.25 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Male body lenght

Any other information on results incl. tables

There was no adverse treatment-related effect on survival or any apparent signs of toxicity and abnormalities in fish of the F0-generation. However, from F0 day 32 until termination one fish with reduced growth was observed in test group 3 replicate B.

Among the F0-generation fish the body length and body weight on day 49/50 (termination of F0-generation) were not statistically significantly different in the treatment groups in comparison to the control group. There was no statistically significant difference on fertility, fecundity and sex ratio in any of the treatment groups in comparison to the control group.

There was no adverse treatment-related effect on hatching success, time to hatch, survival, or any apparent signs of toxicity and abnormalities in the F1-generation fish. However, during the F1-generation exposure, four fish with reduced growth were observed in test group 0 replicate E, test group 3 replicates E and G and in test group 5 replicate G from F1 day 29 until F1 day 35 (study day 51 – 57). One fish with a deformation was observed on F1 day 126 (study day 148, 2nd day of termination of F1-generation) in test group 4 replicate F. One fish showing a distended abdomen was observed in test group 5 replicate E from F1 day 99 until F1 day 105 (study day 121 – 127). This fish was sacrificed humanely by decision of the study director and according to the animal welfare act. Due to the low incidence, the longtime experience with the

test species and background incidence of deformations the findings in the F1-generation were considered to be incidental and not test substance related.

Among the F1-generation fish from test group 2 (10 μg/L nominal concentration) up to test group 5 (1250 μg/L nominal concentration) the body length at study day 35 (determined photographically) was statistically significantly reduced in comparison to the control group when using Williams test one-sided. This finding was transient and was not observed anymore

when the same fish were measured on study day 65 and the end of exposure. There was also no positive relationship between the severity of the effect and substance concentration and the length data in the test groups was all within the laboratory historical control range (1.79 to 2.37 cm ±0.10 to 0.40 cm). The body length among the F1-generation fish on day 65 (determined photographically) and length and weight at termination (F1 days 125-128; study days 147-150)

was not statistically significantly different in the test groups in comparison to the control group.

There was no significant substance related-effect on female body length at termination, whereas male body length was statistically significantly reduced in the highest tested concentration in comparison to the control group when using Williams test one-sided. However, this reduction was minimal and the variation of body length in all test groups was high and

overlapped. However, as male body weight was the lowest in test group 5 (statistically not

significant) and higher concentrations than 1250 μg/L have not been tested, this minor effect will be considered as worst-case scenario, resulting in a LOEC for male body length at

1250 μg/L.

Evaluating reproduction in the F1-generation, there was a statistically significant reduction in fecundity in test group 1 (2 μg/L nominal concentration; 45% in comparison to the control group) and test group 3 (50 μg/L nominal concentration; 38% in comparison to the control group) when using Wilcoxon test one-sided. Since test group 2 (10 μg/L nominal concentration) and test groups 4 and 5 (250 μg/L and 1250 μg/L, respectively, nominal concentration) were not

affected and a concentration dependent trend could not be identified (Jonkheere-Terpstra, onesided), this reduction in fecundity cannot be stated as a substance related effect. In addition, no substance related effect on fertility was observed.

A ratio of 59% females to 41% males was observed in controls, and thus met the sex ratio

validity criterion of 30 – 70%. There was no statistically significant effect on histological sex

ratio at termination of the F1-generation (study days 125-128) in any of the treatment groups in comparison to the control group. There were also no statistically significant differences between control and treatment groups in the gonad maturity index or any other adverse histopathological findings. For the biomarker vitellogenin concentration in head/tail homogenates no statistically significant differences between control and treatment groups were detected for female or male fish.

In the F2-generation the hatching rate in test groups 2, 4 and 5 were statistically significantly different when compared to the control, although there was a 5% reduction. This response is considered a statistical artifact rather than a substance-related effect, because there was 100% hatching in all control replicates. Various chronic fish test guidelines set the validity criterion for

control hatching success to ≥80%. So due to the interrupted statistical response and the high hatching rate in all treatment groups, these results are considered as biological variation and

are not linked to DHDPS exposure.

There was no adverse treatment related effect on time to hatch and larval survival.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

The results are consistent with all validity criteria and the test is valid according to the Draft Standard Operating Procedure used. No deviations or other incidents occurred during the course of the reported test which may have influenced the results.

Conclusions:

In conclusion the overall NOEC identified in this test was 250 μg/L (nominal concentration), the LOEC was 1250 μg/L (nominal concentration) based on reduced male body length. Since no other test substance-related effects on endocrine relevant endpoints were observed in this study up to the highest tested concentration of 1250 μg/L, this study does not indicate any evidence of endocrine activity for DHDPS.

Executive summary:

A zebrafish extended one generation reproduction test (ZEOGRT) was conducted to assess the potential effects of DHDPS on population-relevant apical endpoints and endocrine relevant mechanistic biomarkers during the exposure to the test item on different life stages andgenerations of the zebrafish (Danio rerio).

The experiment was conducted under flow-through conditions with continuous exposure to 5 concentrations of the test substance and a dilutionwater control: 0 (Control), 2, 10, 50, 250 and 1250 μg/L as nominal concentrations based on test substance.

No test substance-related effects on endocrine relevant endpoints (fertility, fecundity, sex ratio, vitellogenin content, gonad maturity)were observed up to the highest tested concentration of 1250 µg/L.

There was no adverse treatment related effect on time to hatch and larval survival.

Male body length was statistically significantly reduced in the highest tested concentration in comparison to the control group when using Williams test one-sided. However, this reduction was minimal and the variation of body length in all test groups was high and

overlapped. Nebvertheless, as male body weight was the lowest in test group 5 (statistically not significant) and higher concentrations than 1250 μg/L have not been tested, this minor effect will be considered as worst-case scenario, resulting in a LOEC for male body length at1250 μg/L.

In conclusion the overall NOEC identified in this test was 250 μg/L (nominal concentration), the LOEC was 1250 μg/L (nominal concentration) based on reduced male body length. Since no other test substance-related effects on endocrine relevant endpoints were observed in this study up to the highest tested concentration of 1250 μg/L, this study does not indicate any

evidence of endocrine activity for DHDPS.