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Ecotoxicological information

Biotransformation and kinetics

Currently viewing:

Administrative data

Endpoint:
biotransformation and kinetics
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Justification for type of information:
In order to assess any potential environmental impacts of resorcinol information on the uptake, metabolism and depuration processes in aquatic organisms is needed. Such data can be used to understand the possible relationships between exposure and acute and chronic toxic effects resulting from accumulated internal body burdens.

Therefore, information on the potential bioconcentration, biotransformation and kinetics of resorcinol (and other phenolics) has been collated from relevant peer reviewed studies.
Cross-reference
Reason / purpose for cross-reference:
reference to other study
Remarks:
This data will complement the existing information on the potential uptake and metabolism of resorcinol in mammals given in Section 5 of the dossier.

Data source

Reference
Reference Type:
publication
Title:
Parallel Biotransformation of Tetrabromobisphenol A in Xenopus laevis and Mammals: Xenopus as a Model for Endocrine Perturbation Studies
Author:
Fini, J. B., Riu, A., Debrauwer, L., Hillenweck, A., Le Mével, S., Chevolleau, S., ... & Zalko, D.
Year:
2012
Bibliographic source:
Toxicological Sciences

Materials and methods

Test guideline
Qualifier:
no guideline followed
Principles of method if other than guideline:
Xenopus laevis tadpoles were waterborne exposed for 72 h to 14C-TBBPA (10^-6M or 10^-7M, with daily renewal) with or without exogenously supplied T3 in aquarium water. Water samples were taken at different time points to establish TBBPA uptake kinetics.

All experiments were carried out using low-binding tips (VWR International) and glass material silanized with dimethylchlorosilane/toluene (5:95, vol/vol). Radioactivity in liquid samples was determined by direct counting on a Packard liquid scintillation counter (Model Tricarb 2200CA; Packard Instruments, Meriden, CT) using Packard Ultima Gold as the scintillation cocktail. Tadpoles from each group were pooled and immediately frozen in liquid nitrogen until extraction. Pools of tadpoles was homogenized with a Polytron homogenizer (Kinematica AG, Lucern, Switzerland) in pH 7.4 phosphate buffer and centrifugation at 250 x g (10 min, 4ºC). Then, a second extraction was carried out in the same conditions, but using water-saturated ethyl acetate. The organic and aqueous phases were separated and their radioactivity was measured by direct counting of aliquots using the scintillation counter. Residual radioactivity in the tadpoles’ pellets (non-extractable radioactivity) was determined by complete combustion using a Packard Oxidizer 306 (Packard Instruments). TBBPA and metabolites were quantified by integrating the area of the radio-chromatographic peaks.
GLP compliance:
no
Type of medium:
aquatic

Test material

1
Reference substance name:
2,2',6,6'-tetrabromo-4,4'-isopropylidenediphenol
EC Number:
201-236-9
EC Name:
2,2',6,6'-tetrabromo-4,4'-isopropylidenediphenol
Cas Number:
79-94-7
IUPAC Name:
4,4'-(2,2-Propanediyl)bis(2,6-dibromophenol)
Specific details on test material used for the study:
14C-TBBPA (10^-6M or 10^-7M, with daily renewal)
TBBPA -sulfate (10^-6M)
TBBPA- disulfate (10^-6M)

Results and discussion

Transformation products:
yes
Identity of transformation productsopen allclose all
No.:
#1
Reference
Reference substance name:
Unnamed
No.:
#2
Reference
Reference substance name:
Unnamed
No.:
#3
Reference
Reference substance name:
Unnamed
No.:
#4
Reference
Reference substance name:
Unnamed

Any other information on results incl. tables

Rapid Uptake and Extensive Metabolization of TBBPA by Tadpoles


Table 1 (taken from Supplementary material) summarises the results for the study in which waterborne Xenopus laevis tadpoles were exposed for 72 h to 14C-TBBPA (10-6M or 10-7M, with daily renewal) with or without exogenously supplied T3 in the aquarium water. Water samples were taken at different time points to establish TBBPA uptake kinetics.


TBBPA was rapidly taken up by the exposed tadpoles (see Figure 1). After only 2 hours exposure, only 22.7 ± 1.3% or 25.0 ± 1.7% of radioactivity was found in water in the 10-6 M treatment  (with or without T3, respectively). Using 10-7 M TBBPA, maximal absorption was observed at 1 hour. A progressive release of radioactivity back into the water was observed in all groups, reaching 62.8 + 2.1 % and 75.4 + 3.5 % of the initial radioactivity applied after 23 hours for the 10-6 and 10-7 M groups respectively. Similar absorption kinetics were observed after the renewal of media at 24 hours. TBBPA uptake was lower for this second day of exposure but still high, with at least 57.0 ± 4.1% and 43.8 ± 1.5% for the 10-6 M and 10-7 M groups, respectively. T3 addition into the aquarium water did not significantly affect the uptake or release of radioactivity. Following the renewal of media at 48 h, a similar pattern was observed, with no significant difference in the uptake depending on TBBPA concentration. Taken together, these data demonstrated rapid absorption of 14C-TBBPA by tadpoles, followed by a gradual release of the parent TBBPA or metabolites into the water. For all groups, the 3-day radioactivity recovery ranged between 91.0 and 97.8%, indicating negligible loss. Around 15% of the total radioactivity administered was found in tadpoles (T) at the end of the experiment, regardless of the TBBPA concentration or T3 supply.


Four metabolites (TBBPA-glucuronide, -glucuronide-sulphate, -sulfate and -disulfate) were detected in water and tadpoles, and no significant amount of the parent TBBPA was detected in the water and tadpoles after 72 hours exposure (see Figure 2A and 2B). T3 additional did not significantly affect metabolic profile except for the disulfate measurement at 72 hours. In water, regardless of TBBPA concentration, almost all the radioactivity was recovered as metabolites, at 24, 48 and 72 hours respectively. Throughout the study TBBPA-monosulfate (M3) was identified as the major metabolite in water samples from both exposure concentrations (see Figure 2C). In tadpole extracts examined after 72 hours (see Figure 2D) the major metabolite detected was the monosulfate conjugate, as in the water.


 


TBBPA, and Not the Metabolites, Disrupt TH Signaling Disruption


Whether the antithyroidal effects observed in X. laevis were due to the parent TBBPA or its major metabolites was addressed. Effects on T3- induced morphological changes were addressed using a test based on T3-induced gill regression. T3 treatment significantly reduced tadpole head areas, due to gill regression and Meckel cartilage transformation, producing a triangular shape, with no effect in
controls. Head areas were measured after 6 days in tadpoles exposed to T3 (5nM), T3 + TBBPA, T3+ M3, or T3 + M4. TBBPA at 10-6 M, but none of its sulfate conjugates, repressed T3- induced GFP expression in vivo signaling. Again, as for the in vivo morphological gene assay, only TBBPA and not the metabolites had an antithyroidal effect. Using 14C-TBBPA, extremely high TBBPA uptake rates and extensive metabolism were observed. After 8 h, most of 14C-TBBPA disappeared from the water then reappeared, in very low amounts in water samples, strongly suggesting metabolization and deconjugation by tadpoles. The effects of TBBPA monosulfate and TBBPA-disulfate using two TH response assays were examined: a morphological test and a transgenic reporter gene assay. Neither metabolite had any effect in either test, corroborating the hypothesis that only the parent TBBPA antagonizes TH signaling.


 


 


TBBPA Displaces T3 From TRs


TBBPA, but none of its sulfated conjugates, displaced T3 from TRα in any species. TBBPA alone
also bound to the human TRα-LBD, activating transcription when applied alone at 3 and 10µM, whereas increased binding of TBBPA alone did not reach significance on X. laevis or zebrafish TR using nonparametric ANOVA. The data strongly suggest a direct action of TBBPA despite the active metabolism observed in X. laevis tadpoles, raising the question of the TH-disrupting mode of action. TBBPA displaces physiological concentrations of T3 from human, X. laevis, and zebrafish TRα. This displacement could well account for many of the in vivo antithyroid effects of TBBPA. Other actions could include crosstalk with other nuclear receptors.


In the study, after a 3-day exposure, 15% of theradioactivity administered in aqueous media for the 3 days persisted in tadpoles, TBBPA proportion being around 6% for the higher dose exposure and around 10% for the lower dose used. However, the registrant consider that these observed residues should be due to relatively high hydrophobicity of this chemical (i.e. log octanol/water partition coefficient is reported to be 5.9).  Such a highly hydrophobic chemical could be taken up to an aquatic organism rapidly as well as significantly and be partitioned to the adipose tissue. Such preservation in adipose tissue could relatively delay metabolism when compared with hydrophilic chemicals such as resorcinol.  Therefore, in the case of TBBPA, the small residues in tadpole should have been observed.  Significant uptake of TBBPA and its continuous release from adipose tissue should have also resulted in the toxic effects on thyroid system reported in the study.


TBBPA Metabolism in X. laevis Parallels That in Mammals


The authors discussed the similarities between the metabolic capabilities of X. laevis and that of mammals.


 


Any other information on results incl. tables


Table 1 (Supplementary material) and Figure 1 (see Attached background material section) summarise the key results from the study.


Table 1 - Uptake kinetic of 14C TBBPA. Results are given as a percentage of initial radioactivity put in to the water at time of treatment (t0 or renewal)
























































































































































































































































































































































































 Time (hours)TBBPA 10-6MTBBPA 10-6M +T3TBBPA 10-7MTBBPA 10-7M +T3
DAY 10100.0%  100.0%  100.0%  100.0%  
0.1775.4%+/-0.3%73.6%+/-2.7%76.8%+/-1.9%78.4%+/-1.0%
0.553.5%+/-1.1%52.7%+/-1.8%56.5%+/-0.9%54.6%+/-4.4%
133.0%+/-1.1%33.4%+/-1.7%43.3%+/-1.7%44.7%+/-1.5%
225.0%+/-1.7%22.7%+/-1.3%45.4%+/-0.9%43.9%+/-3.9%
432.4%+/-2.6%29.1%+/-0.7%52.9%+/-3.2%52.5%+/-5.4%
851.6%+/-1.8%47.4%+/-2.3%57.6%+/-5.3%57.8%+/-3.7%
2362.8%+/-2.1%65.2%+/-4.2%75.4%+/-3.5%77.2%+/-0.4%
DAY 224100.0%  100.0%  100.0%  100.0%  
24.1775.3%+/-4.2%73.9%+/-5.8%71.0%+/-1.4%70.2%+/-3.2%
24.561.7%+/-1.4%56.0%+/-0.4%57.9%+/-0.2%56.9%+/-3.7%
2550.5%+/-2.7%45.5%+/-1.6%52.2%+/-1.5%52.3%+/-2.4%
2643.0%+/-4.1%39.8%+/-0.5%56.7%+/-2.5%56.8%+/-1.2%
2851.2%+/-2.8%49.9%+/-2.4%63.5%+/-1.5%63.0%+/-0.8%
3263.1%+/-4.6%64.1%+/-1.9%68.5%+/-2.2%67.0%+/-4.9%
4776.5%+/-4.3%82.7%+/-1.3%78.3%+/-6.2%87.1%+/-4.7%
DAY 348100.0%  100.0%  100.0%  100.0%  
48.1765.7%+/-5.4%66.3%+/-3.5%71.4%+/-4.4%66.1%+/-2.4%
48.556.9%+/-4.4%54.9%+/-3.2%54.7%+/-3.8%50.0%+/-5.3%
4947.8%+/-6.0%45.8%+/-1.1%53.3%+/-2.0%47.2%+/-1.3%
5046.8%+/-2.3%40.2%+/-4.0%56.4%+/-1.7%51.5%+/-2.6%
5253.0%+/-1.7%45.6%+/-3.3%61.5%+/-2.1%61.3%+/-7.5%
5666.4%+/-5.3%61.7%+/-1.7%68.3%+/-2.7%74.2%+/-10.6%
7285.2%+/-4.4%101.9%+/-5.8%82.8%+/-10.1%94.2%+/-13.4%

Applicant's summary and conclusion

Conclusions:
The study on the biotransformation and kinetics of tetrabromo bisphenol-A and the mechanism of TH signaling disruption of TBBPA in Xenopus resulted in the following conclusions:

1. TBBPA was rapidly taken up and metabolised extensively during 72 hours exposure to 10^-6 and 10^-7 M of TBBPA (with/without 5 x 10^-9 M of T3) under semi-static conditions.

2. Four metabolites (TBBPA-glucuronide, -glucuronide-sulphate, -sulfate and -disulfate) were detected in water and tadpoles, and no significant amount of the parent TBBPA was detected in the water and tadpoles after 72 hours exposure. The registrant considers that the low level of parent TBBPA at the end of exposure and the effects on thyroid system observed in this study are probably be due to high hydrophobicity of this chemical.

3. Only TBBPA and not the metabolites disrupted TH signaling, and

4. The effect of TBBPA on thyroid system was not due to indirect effects by the induction of UDGTs and/or SULTs, but should be direct effects on TRα.
Executive summary:

In order to assess any potential environmental impacts of resorcinol information on the uptake, metabolism and depuration processes in aquatic organisms is needed. Such data can be used to understand the possible relationships between exposure and acute and chronic toxic effects resulting from accumulated internal body burdens. Therefore, information on the potential bioconcentration, biotransformation and kinetics of resorcinol (and other phenolics) has been collated from relevant peer reviewed studies.


The study provides information on the biotransformation and kinetics of the phenolic flame retardant tetrabromobisphenol A (TBBPA) which is a high production chemical that would interfere with thyroid hormone (TH) signaling. Despite its rapid metabolism in mammals, TBBPA is found in significant amounts in different tissues. Such findings are considered by the authors to highlight first a need to better understand the effects of TBBPA and its metabolites and second the need to develop models to address these questions experimentally. The study used Xenopus laevis tadpoles to follow radiolabeled 14C-TBBPA uptake and metabolism. Extensive and rapid uptake of radioactivity was observed, tadpoles metabolizing > 94% of 14C-TBBPA within 8 h. Four metabolites were identified in water and tadpole extracts: TBBPA-glucuronide, TBBPA-glucuronide-sulfate, TBBPA-sulfate, and TBBPA-disulfate. These metabolites are identical to the TBBPA conjugates characterized in mammals, including humans. Most radioactivity (> 75%) was associated with sulfated conjugates. The antithyroid effects of TBBPA and the metabolites were compared using two in vivo measures: tadpole morphology and an in vivo tadpole TH reporter gene assay. Only TBBPA, and not the sulphated metabolites, disrupted thyroid signaling. Moreover, TBBPA treatment did not affect expression of phase II enzymes involved in TH metabolism, suggesting that the antithyroid effects of TBBPA are not due to indirect effects on TH metabolism. Finally, the study showed that only the parent TBBPA inhibits T3-induced transactivation in cells expressing human, zebrafish, or X. laevis TH receptor, TRα. It was concluded, that perturbation of thyroid signaling by TBBPA is likely due to rapid direct action of the parent compound.  In the case of TBBPA, these effects on thyroid system were observed despite its relatively rapid metabolism in amphibians.  However, the registrant considers that these observed residues should be due to relatively high hydrophobicity of this chemical (i.e. log octanol/water partition coeffcient is reported to be 5.9).  Such a highly hydrophobic chemical could be significantly, and rapidly, taken up by an aquatic organism and be partitioned to the adipose tissue. Such preservation in adipose tissue could delay metabolism when compared with hydrophilic chemicals such as resorcinol. Therefore, in the case of TBBPA, the small residues in tadpole should have been observed.  Significant uptake of TBBPA and its continuous release from adipose tissue should have also resulted in the toxic effects on the thyroid system reported in the study .