Reaction products of N-methyldiethanolamine and Boric Acid (1:1.5)

Endpoint summary

Key value for chemical safety assessment

Skin sensitisation

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

Three in vitro experimental studies were available which assessed the sensitising potential of the test item. Specifically, these three studies examine the ability of the test item to induce peptide/protein bonding (Adverse Outcome Pathway (AOP) Key Event 1, in a DPRA), a keratinocyte response (AOP Key Event 2, in an ARE Nrf2 LuSens assay) and a monocytic/dendritic cell response (AOP Key Event 3, in a h-CLAT). The following experimental findings are reported:

 

(1) Potential to induce peptide/protein bonding was evaluated in a Cysteine 1:10/Lysine 1:50 prediction model of the Direct Peptide Reactivity Assay (DPRA), according to the OECD Guideline 442C (2015).The mean cystein depletion was 98.29 % and the mean lysine depletion was 3.40 %. The mean peptide depletion was calculated as 50.84 %; and test item seems to induce peptide/protein bonding. Nevertheless, the medium malue was calculated between two very different results: one, the Cystein result, clearly negative, while Lysine result highly positive. Having a medium value between two so higly different result has just an apparent statistical value and it can not been taken as conclusive result.

 

(2) The test item's potential to activate the Nrf2 transcription factor was assessed in the genetically modified keratinocyte cell-line ARE Nrf2 Luciferase “LuSens” Assay (Bauch et al. 2012), according to the OECD Guideline 442D (2017). No substantial, reproducible, dose-dependent increase in luciferase induction above 1.5-fold was observed up to the maximal test item concentration of 2000 µg/mL. Therefore, the assay was negative and the test item is not considered to have the potential to activate Nrf2 transcription factor and does not induce a keratinocyte response.

 

(3) A Human Cell Line Activation Test (h-CLAT) was performed on the human monocytic leukaemia cell line (THP-1 cells), according to the OECD Guideline 442E (2018). Two out of three experiments were positive, therefore, the test item is considered to have the potential to activate a dendritic cell response.

 

The three in vitro tests, are controversial in their compared results: we have , in fact, the DPRA inconclusive, the LuSens negative and the h-CLAT clearly positive.

 

Three additional data on three analogue substances have been considered to complete the assessment:

 

Reaction products of monoethanolamine and boric acid (1:3) EC 701 -025 -6 (similar substance)

2,2'-methyliminodiethanol: EC 203 -312 -7 (precursor and potential methabolite)

Boric Acid 233 -139 -2 (precursor and potential methabolite)

 

For all three substances, a reliable test according to OECD 406 (Guinea pig maximidsation test) has been performed between 1994 and 1998 and in all three cases the result is negative. There is reason to think that the doubful results obtained in vitro are demonstrating a reactivity, which will not result in a positivity in vivo.

The OECD toolbox on the substance and the three considered analogues indicates that all the predicted properties connected with the skin sensitisation endpoints have the same behaviour.

 

In order to confirm the behaviour of the substance and the correct classification for skin sensitisation a LLNA has been performed.

 

The study was performed according to the Method B.42 and the OECD guideline 429 in compliance with GLP.

Concentrations of 25, 50 and 100 % (w/v) were chosen. Five animals per group were treated by topical application of the test substance concentrations, vehicle or positive control in the same manner as in the screen. DAE 433 was chosen as the vehicle, based on solubility. 
The values of SI for the test groups treated by the test item are below the threshold, stimulation index (SI) is < 3 [100% (1.27) 50% (0.97) and 25% (0.97)]. Therefore, the test substance is not a skin sensitizer in the LLNA.

Based on all performed tests considered in a weight of evidence, compaired also with the results of the similar substances it can be concluded that the substance is not a skin sensitizer

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

According to the CLP Regulation (EC) no. 1272/2008, a skin sensitiser is an agent that will lead to an allergic response in susceptible individuals following skin contact. As a consequence of a secondary - usually organ-specific - subsequent re-exposure, adverse health effects occur on the skin (allergic contact dermatitis). Skin sensitisers are classified in Category 1 with the signal word “warning” and the Hazard statement H317 “May cause an allergic skin reaction”. Where data are sufficient, skin sensitisers can be divided into sub-categories. If data are not sufficient for sub-categorisation, Category 1 must be chosen. The CLP (and UN GHS) criteria for classifying sensitisers are based on standard animal data and human data; data obtained from non-standard methods such as read-across or in vitro/in chemico test methods may be used in combination in a Weight of Evidence approach.

 

Indicators of potency of a substance can be obtained from in chemico/in vitro testing; specifically, the following tests may be accepted to fulfill the requirements of Annex VII:

 

(i) Direct Peptide Reactivity Assay (DPRA) addresses AOP Key Event 1: Peptide/protein binding

(ii) ARE-Nrf2 Luciferase Test Method (KeratinoSensTM) addresses AOP Key Event 2: Keratinocyte response

(iii) the Human Cell Line Activation Test (h-CLAT) addresses AOP Key Event 3: Monocytic /Dendritic cell response.

 

These test methods were developed to address specific events of the skin sensitisation AOP (OECD, 2012). The AOP for skin sensitisation describes the current understanding of key events linked to skin sensitisation. As each of the test methods only addresses a specific key event of skin sensitisation, currently they should not be used in isolation to identify a potential skin sensitiser but rather in combination in a Weight of Evidence approach.

Based on the inconclusive DPRA (molecular interaction with skin proteins), negative LuSens assay (inflammatory response in keratinocytes) and positive h-CLAT (dendritic cell activation), the data is not sufficient for a Weight of Evidence approach . All similar substances, precursors and potential metabolites are negative in reliable Guinea Pig maximisation test.

 

According to the CLP Regulation (EC) No.1272/2008, Annex I: 3.4.2.2.3.2.the following categories for skin sensitisation classification apply:

Category 1

Substances shall be classified as skin sensitizers in category 1 where data are not sufficient for sub-categorisation in accordance with the following criteria:

(a) if there is evidence in humans that the substance can lead to sensitisation by skin contact in a substantial number of persons; or

(b) if there are positive results from an appropriate animal test.

 

Sub-category 1A

Substances showing a high frequency of occurrence in humans and/or a high potency in animals can be presumed to have the potential to produce significant sensitisation in humans. Severity of reaction may also be considered.

 

Specific criteria:

Local lymph node assay - EC3 value ≤ 2 %

Guinea pig maximisation test - ≥ 30 % responding at ≤ 0.1 % intradermal induction dose or ≥ 60 % responding at > 0.1 % to ≤ 1 % intradermal induction dose

Buehler assay - ≥ 15 % responding at ≤ 0,2 % topical induction dose or ≥ 60 % responding at > 0.2 % to ≤ 20 % topical induction dose

 

Sub-category 1B

Substances showing a low to moderate frequency of occurrence in humans and/or a low to moderate potency in animals can be presumed to have the potential to produce sensitisation in humans. Severity of reaction may also be considered.

Local lymph node assay - EC3 value > 2 %

Guinea pig maximisation test- ≥ 30 % to < 60 % responding at > 0.1 % to ≤ 1 % intradermal induction dose or ≥ 30 % responding at > 1 % intradermal induction dose.

Buehler assay - ≥ 15 % to < 60 % responding at > 0.2 % to ≤ 20 % topical induction dose or ≥ 15 % responding at > 20 % topical induction dose.

The topical application of the test substances at 25% (w/v), 50 %(w/v) and at 100 % (w/v), resulted in SI values less than 3 (SI < 3.0); no EC3 was therefore determined. Based on the aforementioned criteria, the substance is not classified for skin sensitisation.