Registration Dossier

Ecotoxicological information

Endocrine disrupter testing in aquatic vertebrates – in vivo

Some information in this page has been claimed confidential.

Administrative data

Endpoint:
amphibian: other
Remarks:
Larval Amphibian Growth and Development Assay with the African Clawed Frog (Xenopus laevis)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2019-2020
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2020
Report date:
2020

Materials and methods

Test guidelineopen allclose all
Qualifier:
equivalent or similar to guideline
Guideline:
OECD TG 241 (The Larval Amphibian Growth and Development Assay (LAGDA))
Version / remarks:
Adopte 28 July 2015
Deviations:
no
Qualifier:
equivalent or similar to guideline
Guideline:
other: U.S. EPA OCSPP 890.2300
Version / remarks:
2015
Deviations:
no
Guideline:
other: according to the Decision on Substance Evaluation letter for TBBPA
Deviations:
no
Principles of method if other than guideline:
In addition to the guidlines specified, he following endpoints ere measured: triiodothyronine (T3) and L-thyroxine (T4) concentrations in plasma and tissue homogenate
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Test material form:
not specified
Details on test material:
- Name of test material (as cited in study report): 3,3’,5,5’-Tetrabromo-bisphenol-A (TBBPA)
- Name of additional materials (as cited in study report): 3,3’,5,5’-Tetrabromo-bisphenol-A-monomethylether (TBBPA-MME) and 3,3’,5,5’-Tetrabromo-bisphenol-A-dimethylether (TBBPA-DME)
- Radiolabelled material: 3,3’,5,5’-Tetrabromo-bisphenol-A (ring-13C12)
Specific details on test material used for the study:
The test substance, a solid, was identified as Tetrabromobisphenol A; Batch number 651170360. The test substance contained >99% active ingredient and had an expiration date of 26 December 2020

Sampling and analysis

Analytical monitoring:
yes

Test solutions

Vehicle:
yes
Remarks:
UV sterilized well water

Test organisms

Aquatic vertebrate type:
frog
Test organisms (species):
Xenopus laevis
Details on test organisms:
Test Organism
African clawed frogs (Xenopus laevis) were used in this test. This species was selected as this was the recommended species for the test (1,2). Larvae were obtained from cultures maintained by Eurofins Easton. Adult frogs for the culture were obtained from Xenopus Express, Inc. of Brooksville, Florida, and were maintained under flow-through conditions at a target temperature of 22ºC for approximately 1.5 years for the purpose of providing tadpoles for the test. The photoperiod for adult frogs was 12 hours of light and 12 hours of dark per day, with a 30-minute transition period. The frogs were fed an adult Xenopus diet supplied by Zeigler Brothers, Inc. of Gardners, Pennsylvania at a rate sufficient to maintain the health of the culture. The culture frogs showed no sign of disease or stress prior to the test. Adult frogs used to supply embryos for use on test were confirmed to produce genetically identifiable offspring during non-GLP preliminary trials.

Study design

Test type:
flow-through
Water media type:
well water
Limit test:
no
Total exposure duration:
133 d

Test conditions

Nominal and measured concentrations:
TEST CONCENTRATIONS: Nominal Mean Measured
Negative Control < LOQ
5.7 µg/L 5.9 µg/L
18 µg/L 19 µg/L
59 µg/L 60 µg/L
188 µg/L 1 86 µg/L
600 µg/L 609 µg/L
50 mg/L PTU 51 mg/L PTU
1.0 µg/L T4 1.3 µg/L T4
Details on test conditions:
See table below
Reference substance (positive control):
yes

Results and discussion

Effect concentrationsopen allclose all
Key result
Duration:
133 d
Dose descriptor:
NOEC
Effect conc.:
609 µg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other:
Remarks:
overall
Remarks on result:
other:
Remarks:
Highest concentration measured
Key result
Duration:
133 d
Dose descriptor:
LOEC
Effect conc.:
> 609 µg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Overall
Remarks on result:
other:
Remarks:
Highest concentration measured
Details on results:
The LAGDA study was designed to evaluate a chemical’s ability to affect the hypothalamus, pituitary, and thyroid axis (HPT) and the hypothalamus, pituitary, and gonadal axis (HPG). In evaluations of the HPT axis there were no effects in thyroid histology, developmental morphology, time-to-reach Stage 62 at any of the TBBPA concentrations tested. There were slight increases and decreases in T3 and T4 measurements that occurred in the two highest treatment groups, but they were inconsistent with no observed effects on thyroid histology, developmental morphology, and time-to-reach Stage 62 that were observed at all TBBPA concentrations tested. In the evaluation of the HPG axis there were no effects on genetic sex, histology, and vitellogenin (VTG) at any of the TBBPA concentrations tested. The positive control responses were as expected with PTU showing the characteristic responses of delayed a thyroid hormone antagonist resulting in delayed development and a T4 responding as a thyroid hormone agonist characterized by accelerated development. The responses observed in the TBBPA groups were closer to the negative control than either of the positive controls which support no effects on thyroid endpoints.

Endpoints NOEC LOEC
Survival 609 µg/L >609 µg/L
NF Stage 58 Growth 609 µg/L >609 µg/L
NF Stage 62 Growth 609 µg/L >609 µg/L
Time to NF Stage 62 609 µg/L >609 µg/L
Juvenile Growth 609 µg/L >609 µg/L
Liversomatic Index 609 µg/L >609 µg/L
Histopathology 609 µg/L >609 µg/L
Sex Ratio 609 µg/L >609 µg/L

The overall NOEC was 609 µg/L and the overall LOEC was > 609 µg/L, the highest concentration tested.
Results with reference substance (positive control):
The positive control responses were as expected with PTU showing the characteristic responses of delayed a thyroid hormone antagonist resulting in delayed development and a T4 responding as a thyroid hormone agonist characterized by accelerated development

Any other information on results incl. tables

Endpoints

NOEC

LOEC

Survival

609 µg/L

>609 µg/L

NF Stage 58 Growth

609 µg/L

>609 µg/L

NF Stage 62 Growth

609 µg/L

>609 µg/L

Time to NF Stage 62

609 µg/L

>609 µg/L

Juvenile Growth

609 µg/L

>609 µg/L

Liversomatic Index

609 µg/L

>609 µg/L

Histopathology

609 µg/L

>609 µg/L

Sex Ratio

609 µg/L

>609 µg/L

 

The overall NOEC was 609 µg/L and the overall LOEC was > 609 µg/L, the highest concentration tested.

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
CONCLUSIONS
African clawed frogs (Xenopus laevis) were exposed to five test concentrations of TBBPA, a negative (dilution water) control, one concentration of L-thyroxine (T4) and one concentration of 6 n propyl-2-thiouracil (PTU) under flow-through conditions from embryo-larval development through metamorphosis, and then through early juvenile development. The five concentrations selected were 5.7, 18, 59, 188 and 600 µg TBBPA/L.    The LAGDA study was designed to evaluate a chemical’s ability to affect the hypothalamus, pituitary, and thyroid axis (HPT) and the hypothalamus, pituitary, and gonadal axis (HPG). In evaluations of the HPT axis there were no effects in thyroid histology, developmental morphology, time-to-reach Stage 62 at any of the TBBPA concentrations tested. There were slight increases and decreases in T3 and T4 measurements that occurred in the two highest treatment groups, but they were inconsistent with no observed effects on thyroid histology, developmental morphology, and time-to-reach Stage 62 that were observed at all TBBPA concentrations tested. In the evaluation of the HPG axis there were no effects on genetic sex, histology, and vitellogenin (VTG) at any of the TBBPA concentrations tested. The positive control responses were as expected with PTU showing the characteristic responses of delayed a thyroid hormone antagonist resulting in delayed development and a T4 responding as a thyroid hormone agonist characterized by accelerated development. The responses observed in the TBBPA groups were closer to the negative control than either of the positive controls which support no effects on thyroid endpoints. The overall NOEC was 609 µg/L and the overall LOEC was > 609 µg/L, the highest concentration tested.
Executive summary:

SUMMARY

 African clawed frogs (Xenopus laevis)were exposed to five test concentrations of, a negative (dilution water) control, one concentration of L-thyroxine (T4) and one concentration of 6‑n‑propyl-2-thiouracil (PTU) under flow-through conditions from embryo-larval development through metamorphosis, and then through early juvenile development. The five concentrations selected were5.7, 18, 59, 188 and 600 µg TBBPA/L. 

 LENGTH OF EXPOSURE:       132/133 Days

 AGE OF TEST ORGANISMS:       Tadpoles (NF Stage 8)

 

TEST ENDPOINTS: Stage of development, growth (wet weight,snout-to-vent and hind-limb length), liver somatic index (LSI), genetic/phenotypic sex ratios, as well as histology of the thyroid, gonad, reproductive duct, kidney and liver, plasma vitellogenin (VTG),triiodothyronine (T3) and L-thyroxine (T4) concentrations in plasma and tissue homogenateas well as general observations of health.

                                                                           

  RESULTS:   

 The LAGDA study was designed to evaluate a chemical’s ability to affect the hypothalamus, pituitary, and thyroid axis (HPT) and the hypothalamus, pituitary, and gonadal axis (HPG). In evaluations of the HPT axis there were no effects in thyroid histology, developmental morphology, time-to-reach Stage 62 at any of the TBBPA concentrations tested. There were slight increases and decreases in T3 and T4 measurements that occurred in the two highest treatment groups, but they were inconsistent with no observed effectson thyroid histology, developmental morphology, and time-to-reach Stage 62 that were observed at all TBBPA concentrations tested. In the evaluation of the HPG axis there were no effectsongenetic sex, histology, and vitellogenin (VTG) at any of the TBBPA concentrations tested. The positive control responses were as expected with PTU showing the characteristic responses of delayed a thyroid hormone antagonist resulting in delayed development and a T4 responding as a thyroid hormone agonist characterized by accelerated development. The responses observed in the TBBPA groups were closer to the negative control than either of the positive controls which support no effects on thyroid endpoints. 

 

Endpoints

NOEC

LOEC

Survival

609 µg/L

>609 µg/L

NF Stage 58 Growth

609 µg/L

>609 µg/L

NF Stage 62 Growth

609 µg/L

>609 µg/L

Time to NF Stage 62

609 µg/L

>609 µg/L

Juvenile Growth

609 µg/L

>609 µg/L

Liversomatic Index

609 µg/L

>609 µg/L

Histopathology

609 µg/L

>609 µg/L

Sex Ratio

609 µg/L

>609 µg/L

The overall NOEC was 609 µg/L and the overall LOEC was > 609 µg/L, the highest concentration tested.