Trimethylamine, N-oxide

Endpoint summary

Description of key information

TMAO is not skin sensitising

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Endpoint:

skin sensitisation: in vitro

Type of information:

other: Weight-of-Evidence justification/conclusion

Remarks:

Weight-of-Evidence justification/conclusion

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: weight of evidence justification/conclusion

Justification for type of information:

Relevance of source information:
Charles River Laboratories (2018a) – DPRA – Reliability 1 (guideline study): The direct peptide reactivity assay (DPRA) addresses the molecular initiating event of the skin sensitization adverse outcome pathway (AOP) namely protein reactivity, by quantifying the reactivity of test chemicals towards model synthetic peptides containing either lysine or cysteine. The conclusions of a DPRA can contribute to skin sensitization hazard identification in a weight-of-evidence approach.

Charles River Laboratories (2018b) – Activation of keratinocytes - Reliability 1 (guideline study): The ARE-Nrf2 Luciferase assay addresses the second key event of the skin sensitization AOP; specifically, the activation of keratinocytes and includes inflammatory responses as well as changes in gene expression associated with specific cell signalling pathways such as the antioxidant/electrophile response element (ARE)-dependent pathways. The conclusions of a ARE-Nrf2 Luciferase assay can contribute to skin sensitization hazard identification in a weight-of-evidence approach.

Charles River Laboratories (2018c) – Activation of dendritic cells - Reliability 1 (guideline study): The U937 cell line activation test (U-SENS) assay addresses the third key event of the skin sensitization AOP; specifically, the activation of dendritic cells. The conclusions of a U937 cell line activation test (U-SENS) assay can contribute to skin sensitization hazard identification in a weight-of-evidence approach.

Weighting of the sources of information to reach an overall conclusion of the information requirement:
There is general agreement on the key biological events underlying skin sensitisation. The current knowledge of the chemical and biological mechanisms associated with skin sensitisation has been summarised as an AOP starting with a molecular initiating event through intermediate events to the adverse effect, namely allergic contact dermatitis. The first key event focuses on the molecular initiating event of the covalent binding of electrophilic substances to nucleophilic centres in skin proteins. The second key event takes place in the keratinocytes and includes inflammatory responses as well as changes in gene expression associated with specific cell signalling pathways such as the antioxidant/electrophile response element (ARE)-dependent pathways. The third key event is the activation of dendritic cells, typically assessed by expression of specific cell surface markers, chemokines and cytokines. The fourth key event is T-cell proliferation. Mechanistically-based in chemico and in vitro test methods addressing the first three key events of the skin sensitisation AOP have been adopted for contributing to the evaluation of the skin sensitisation hazard potential of chemicals. The fourth key event representing T-cell proliferation is indirectly assessed in the murine Local Lymph Node Assay (LLNA).

No single in vitro/in chemical/in silico method is sufficient for a determination of skin sensitisation potential. Therefore, non-animal assessment for skin sensitization is assessed in a weight-of-evidence considering results from the three key events of (Key Event 1) molecular interaction with skin proteins, (Key Event 2) inflammatory response in keratinocytes, and (Key Event 3) activation of dendritic cells. A case study assessing 54 test substances of known sensitising potential demonstrated that the in vitro/in chemical results of any 2 of the 3 key events provide very good predictivity (94%) for sensitising potential (Bauch et al, 2012).
The weight-of-evidence for the skin sensitisation of TMAO has been assessed with in vitro/in chemical methods addressing Key Events 1, 2 and 3. Valid results from GLP-compliant OECD approved methodologies from 2 of the 3 key events provide conclusive and consistent results allowing for a definitive conclusion.

Assessment of the uncertainty in the conclusion compared with the study normally required for the information requirement
This weight-of-evidence is based on the consideration of validated results from Key Events 1, 2, and 3. Non-borderline results for Key Events 1, 2 and 3 were derived in GLP-compliant OECD 442C, D, and E studies; respectively. Following the OECD 497 defined approach where 2-of-3 concordant non-borderline results are available a definitive conclusion can be derived. As the results are non-borderline, there is a low level of uncertainty (Kolle, et al., 2021).

Reference:
Bauch, C., Kolle, S.N., Ramirez, T., Eltze, T., Fabian, E., Mehling, A., Teubner, W., van Ravenzwaay, B. and Landsiedel, R., 2012. Putting the parts together: combining in vitro methods to test for skin sensitizing potentials. Regulatory toxicology and pharmacology, 63(3), pp.489-504

Charles River Laboratories (2018a). In Chemico Determination of the Skin Sensitization Potential of TMAO anhydrous using the Direct Peptide Reactivity Assay (DPRA). Unpublished report. Study number: 20143764. Study Sponsor: Plant Response Biotech

Charles River Laboratories (2018b). Evaluation of in vitro Skin Sensitization Potential of TMAO anhydrous with the KeratinoSensTM Assay. Unpublished report. Study number: 20143765. Study Sponsor: Plant Response Biotech

Charles River Laboratories (2018c). Evaluation of in vitro Skin Sensitization Potential of TMAO anhydrous with the U937 Cell Line Activation Test (U-SENS™) Assay. Unpublished report. Study number: 20143766. Study Sponsor: Plant Response Biotech

Kolle, S.N., Mathea, M., Natsch, A. and Landsiedel, R., 2021. Assessing experimental uncertainty in defined approaches: Borderline ranges for in chemico and in vitro skin sensitization methods determined from ring trial data. Applied In Vitro Toxicology, 7(3), pp.102-111.

Reason / purpose for cross-reference:

other: Weight-of-evidence source information

Qualifier:

no guideline required

Principles of method if other than guideline:

Weight-of-evidence justification

GLP compliance:

no

Type of study:

other: Weight-of-Evidence justification/conclusion

Specific details on test material used for the study:

Weight-of-evidence justification/conclusion

Group:

test chemical

Remarks on result:

no indication of skin sensitisation

Interpretation of results:

GHS criteria not met

Conclusions:

Not skin sensitising

Executive summary:

Consideration of animal welfare has resulted in the development of in vitro/in chemical/in silico methods to address skin sensitization. No single in vitro/in chemical/in silico method is sufficient for a determination of skin sensitisation potential. Therefore, non-animal assessment for skin sensitization is assessed in a weight-of-evidence considering results from the three key events of (Key Event 1) molecular interaction with skin proteins, (Key Event 2) inflammatory response in keratinocytes, and (Key Event 3) activation of dendritic cells. A case study assessing 54 test substances of known sensitising potential demonstrated that the in vitro/in chemical results of any 2 of the 3 key events provide very good predictivity (94%) for sensitising potential (Bauch et al, 2012).

 

The weight-of-evidence for the skin sensitisation of TMAO has been assessed with in vitro/in chemical methods addressing Key Events 1, 2 and 3.  Valid results from GLP-compliant OECD approved methodologies from 2 of the 3 key events provide conclusive and consistent results allowing for a definitive conclusion.

 

Key Event 1 – molecular interaction with skin proteins

The direct peptide reactivity assay (DPRA) was used to assess the skin sensitisation potential of
TMAO following the in chemico OECD TG 442C methodology and in compliance with OECD GLP criteria. Synthetic peptides containing cysteine or lysine were reacted with each purity grade of the registered substance (test articles) for 24 ± 2 hours. After the incubation period, the extent of peptide depletion was analysed using High Performance Liquid Chromatography (HPLC) coupled with ultra-violet (UV) spectrometric detection.

 

The skin sensitisation of the test articles was evaluated using the prediction model for cysteine and lysine as described in OECD TG 442C. The mean cysteine depletion was 20.4% and the mean lysine depletion was 0.2%. The overall mean peptide depletion of cysteine and lysine was 10.3%. Based on the OECD 442C prediction model, a conclusion of low reactivity for cysteine and lysine activity was reported.

 

The registered substance has low reactivity with skin proteins and is potentially sensitizing according to Key Event 1 as assessed according to OECD TG 442C.

 

Key Event 2 – inflammatory response in keratinocytes

The ARE-Nrf2 Luciferase assay was used to assess the skin sensitisation potential of TMAO following the in vitro OECD TG 442D methodology and in compliance with OECD GLP criteria. Making use of the immortalised adherent cell line derived from human keratinocytes stably harbouring a luciferase reporter gene under the control of the antioxidant response element of the AKR1C2 gene. The luciferase signal reflects the activation by sensitisers of endogenous Nrf2 dependent genes, and the dependence of the luciferase signal in the recombinant cell line on Nrf2 has been demonstrated. This allows quantitative measurement (by luminescence detection) of luciferase gene induction, using well established light producing luciferase substrates, as an indicator of the activity of the Nrf2 transcription factor in cells following exposure to electrophilic test substances. The cells are exposed to the test item for 48 hours, the cells are lysed and the lysate is then placed in the luminometer for reading.

 

The skin sensitisation of the test articles was evaluated following the defined approach as described in OECD TG 442D. TMAO showed no toxicity (no IC30 or IC50 values) and no biologically relevant induction of luciferase activity (no EC1.5 value).  The maximum luciferase activity induction (Imax) was 1.04-fold and 1.09 fold in experiments 1 and 2; respectively.. Based on the OECD 442D prediction model, a conclusion of negative for skin sensitization potential is reported for Key Event 2.

 

Key Event 3 – activation of dendritic cells

The U937 cell line activation test (U-SENS) assay was used to assess the skin sensitisation potential of TMAO following the in vitro OECD TG 442E methodology and in compliance with OECD GLP criteria. The U-SENS method quantifies the change in the expression of a cell surface marker associated with the process of activation of monocytes and dendritic cells (i.e., CD86), in the human histiocytic lymphoma cell line U937, following exposure to sensitisers. The measured expression levels of CD86 cell surface marker in the cell line U937 is then used for supporting the discrimination between skin sensitisers and non-sensitisers. The cells are exposed to the test item for approximately 45 hours, then stained with antibodies and the fluorescence intensity of the antibodies is recorded.

 

The skin sensitisation of the test articles was evaluated following the prediction model as described in OECD TG 442E. TMAO showed no toxicity (no CV70 value) and no biologically relevant induction of CD86 activity (no EC150 value).Based on the OECD 442E prediction model, a conclusion of negative for skin sensitization potential is reported for Key Event 3.

 

Weight-of-Evidence

Based on the consideration of a weight-of evidence approach considering the non-borderline results of GLP-compliant OECD methodologies for Key Events 1, 2, and 3 where 2 of the 3 Key Events are negative (Key Event 2 and 3) (Bauch et al, 2012; Kolle et al, 2021; OECD TG 497), TMAO is considered to be not skin sensitizing.

 

References:

Bauch, C., Kolle, S.N., Ramirez, T., Eltze, T., Fabian, E., Mehling, A., Teubner, W., van Ravenzwaay, B. and Landsiedel, R., 2012. Putting the parts together: combining in vitro methods to test for skin sensitizing potentials. Regulatory toxicology and pharmacology, 63(3), pp.489-504

Kolle, S.N., Mathea, M., Natsch, A. and Landsiedel, R., 2021. Assessing experimental uncertainty in defined approaches: Borderline ranges for in chemico and in vitro skin sensitization methods determined from ring trial data. Applied In Vitro Toxicology, 7(3), pp.102-111.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

Consideration of animal welfare has resulted in the development of in vitro/in chemical/in silico methods to address skin sensitization. No single in vitro/in chemical/in silico method is sufficient for a determination of skin sensitisation potential. Therefore, non-animal assessment for skin sensitization is assessed in a weight-of-evidence considering results from the three key events of (Key Event 1) molecular interaction with skin proteins, (Key Event 2) inflammatory response in keratinocytes, and (Key Event 3) activation of dendritic cells. A case study assessing 54 test substances of known sensitising potential demonstrated that the in vitro/in chemical results of any 2 of the 3 key events provide very good predictivity (94%) for sensitising potential (Bauch et al, 2012).

 

The weight-of-evidence for the skin sensitisation of TMAO has been assessed with in vitro/in chemical methods addressing Key Events 1, 2 and 3.  Valid results from GLP-compliant OECD approved methodologies from 2 of the 3 key events provide conclusive and consistent results allowing for a definitive conclusion.

 

Key Event 1 – molecular interaction with skin proteins

The direct peptide reactivity assay (DPRA) was used to assess the skin sensitisation potential of TMAO following the in chemico OECD TG 442C methodology and in compliance with OECD GLP criteria. Synthetic peptides containing cysteine or lysine were reacted with each purity grade of the registered substance (test articles) for 24 ± 2 hours. After the incubation period, the extent of peptide depletion was analysed using High Performance Liquid Chromatography (HPLC) coupled with ultra-violet (UV) spectrometric detection.

 

The skin sensitisation of the test articles was evaluated using the prediction model for cysteine and lysine as described in OECD TG 442C. The mean cysteine depletion was 20.4% and the mean lysine depletion was 0.2%. The overall mean peptide depletion of cysteine and lysine was 10.3%. Based on the OECD 442C prediction model, a conclusion of low reactivity for cysteine and lysine activity was reported.

 

The registered substance has low reactivity with skin proteins and is potentially sensitizing according to Key Event 1 as assessed according to OECD TG 442C.

 

Key Event 2 – inflammatory response in keratinocytes

The ARE-Nrf2 Luciferase assay was used to assess the skin sensitisation potential of TMAO following the in vitro OECD TG 442D methodology and in compliance with OECD GLP criteria. Making use of the immortalised adherent cell line derived from human keratinocytes stably harbouring a luciferase reporter gene under the control of the antioxidant response element of the AKR1C2 gene. The luciferase signal reflects the activation by sensitisers of endogenous Nrf2 dependent genes, and the dependence of the luciferase signal in the recombinant cell line on Nrf2 has been demonstrated. This allows quantitative measurement (by luminescence detection) of luciferase gene induction, using well established light producing luciferase substrates, as an indicator of the activity of the Nrf2 transcription factor in cells following exposure to electrophilic test substances. The cells are exposed to the test item for 48 hours, the cells are lysed and the lysate is then placed in the luminometer for reading.

 

The skin sensitisation of the test articles was evaluated following the defined approach as described in OECD TG 442D. TMAO showed no toxicity (no IC30 or IC50 values) and no biologically relevant induction of luciferase activity (no EC1.5 value).  The maximum luciferase activity induction (Imax) was 1.04-fold and 1.09 fold in experiments 1 and 2; respectively.. Based on the OECD 442D prediction model, a conclusion of negative for skin sensitization potential is reported for Key Event 2.

 

Key Event 3 – activation of dendritic cells

The U937 cell line activation test (U-SENS) assay was used to assess the skin sensitisation potential of TMAO following the in vitro OECD TG 442E methodology and in compliance with OECD GLP criteria. The U-SENS method quantifies the change in the expression of a cell surface marker associated with the process of activation of monocytes and dendritic cells (i.e., CD86), in the human histiocytic lymphoma cell line U937, following exposure to sensitisers. The measured expression levels of CD86 cell surface marker in the cell line U937 is then used for supporting the discrimination between skin sensitisers and non-sensitisers. The cells are exposed to the test item for approximately 45 hours, then stained with antibodies and the fluorescence intensity of the antibodies is recorded.

 

The skin sensitisation of the test articles was evaluated following the prediction model as described in OECD TG 442E. TMAO showed no toxicity (no CV70 value) and no biologically relevant induction of CD86 activity (no EC150 value).Based on the OECD 442E prediction model, a conclusion of negative for skin sensitization potential is reported for Key Event 3.

 

Weight-of-Evidence

Based on the consideration of a weight-of evidence approach considering the non-borderline results of GLP-compliant OECD methodologies for Key Events 1, 2, and 3 where 2 of the 3 Key Events are negative (Key Event 2 and 3) (Bauch et al, 2012; Kolle et al, 2021; OECD TG 497), TMAO is considered to be not skin sensitizing.

 

References:

Bauch, C., Kolle, S.N., Ramirez, T., Eltze, T., Fabian, E., Mehling, A., Teubner, W., van Ravenzwaay, B. and Landsiedel, R., 2012. Putting the parts together: combining in vitro methods to test for skin sensitizing potentials. Regulatory toxicology and pharmacology, 63(3), pp.489-504

Kolle, S.N., Mathea, M., Natsch, A. and Landsiedel, R., 2021. Assessing experimental uncertainty in defined approaches: Borderline ranges for in chemico and in vitro skin sensitization methods determined from ring trial data. Applied In Vitro Toxicology, 7(3), pp.102-111.

Justification for classification or non-classification

Based on a weight-of-evidence considering 2 of the 3 key skin sensitisation events, the registered substance is not skin sensitising. Therefore, the test item does not meet the classification criteria of EC 1272/2008 (as amended) for skin sensitisation.