(2R)-4-hydroxybutan-2-aminium (2S)-hydroxy(phenyl)acetate

Endpoint summary

Key value for chemical safety assessment

Skin sensitisation

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

There are no studies available for (R)-3 -Ammonium-1-hydroxybutyl (S)-mandelat, but for (R)- madelic acid (Cas: 611-71-2) and for the racemat 3 -Amino-butan-1-ol (Cas: 2867-59-6).

3 -Amino-butan-1-ol (Cas: 2867-59 -6)

The skin sensitizing potential for 3 -Amino-butan-1-ol was investigated using the Local Lymph Node Assay (LLNA, OECD 429 ) in mice. Test item solutions at different concentrations were prepared in the vehicle DMF: 0.25, 0.5 and 1% (w/w). The highest concentration tested was the highest concentration that could be achieved whilst avoiding systemic toxicity and excessive local skin irritation. In this study Stimulation Indices (S.I.) of 1.21, 1.28 and 1.69 were determined with the test item at concentrations of 0.25, 0.5 and 1% (w/w) in DMF, respectively. The test item 3-Amino-butan-1-ol was thus not a skin sensitizer under the test conditions of this study. [BASF, 2014]

(R)-mandelic acid (Cas: 611-71-2)

The skin sensitizing potential for (R)-mandelic acid was investigated using the in vitro skin sensitization Turnkey Testing Strategy.

These in vitro studies are known to cover the three key steps of the adverse outcome pathway for skin sensitization as identified by the OECD (OECD Publication No.168; ENV/JM/MOMO(2012)10) and are validated in-house using 54 substances (Bauch et al. 2012 Regul Toxicol Pharmacol. 63: 489-504). Recently, a further validation performed with 213 substances has been published (Urbisch et al. 2015 Regul Toxicol Pharmacol. 71: 337-351).

Based on the results of the in house validation (Bauch et al., 2012 Regul Toxicol Pharmacol. 63: 489-504) the predictive capacity of the evaluation scheme using DPRA (peptide reactivity), LuSens/KeratinoSenx™ (keratinocyte activation) and (m)MUSST/h-CLAT (dendritic cell activation) is comparable to that of the local lymph node assay. This was confirmed in the validation study with more substances (Urbisch 2015): When compared to the sensitization potential of the substances in humans, the classification based on an evaluation scheme using results obtained from theDPRA, LuSens and mMUSST had a sensitivity of 90%, a specificity of 90% and an accuracy of 90%.

 

In the evaluation scheme used here any two of the three test results determine the overall classification, i.e. any two positive test results drive the prediction of a sensitizer, while any two negative test results drive the prediction of a test substance to be a non-sensitizer (Bauch et al. 2012; Table 1). If two assays (DPRA, LuSens or KeratinoSens, MUSST or h-CLAT) yield concordant results, the result of the third assay is not necessarily required to determine the overall outcome of the evaluation scheme.

 

Table 1: Decision matrix for combinations of DPRA, LuSens/KeratinoSens and MUSST/ h-CLAT assays.

DPRA	LuSens/ KeratinoSens^TM	MUSST/ h-CLAT	Test battery evaluation
positive	positive	positive	sensitizer
positive	positive	negative	sensitizer
positive	negative	positive	sensitizer
positive	negative	negative	non-sensitizer
negative	positive	positive	sensitizer
negative	positive	negative	non-sensitizer
negative	negative	positive	non-sensitizer
negative	negative	negative	non-sensitizer

Each individual assay was performed under GLP/ GLP like conditons and the cell based assays LuSens and MUSST consisted of at least two independent experiments. Positive and negative controls were included in each experiment and confirmed the functionality and validity of the individual assays. The DPRA and the keratinocyte activation assay follow the procedures of the respective OECD testing guidelines (OECD 442C and D). An OECD testing guideline for the dentritic cell activation assay is under preparation.

 

The test battery applicability is limited when testing substances insoluble in the commonly used vehicles and highly volatile substances.Substances susceptible to base-catalyzed hydrolysis cannot be evaluated reliably for binding to lysine as the incubation is performed at pH 10.2. Also substances that interfere with the experimental measurements (e.g., co-elution with the peptide in the DPRA-assay) are not or only partially suitable for testing using in vitro methods. The substance under evaluation did not have any of the above mentioned limitations and the outcome of this assay is therefore considered adequate for hazard identification for the endpoint of skin sensitization.

A combination of several in vitro methods addressing key events of the adverse outcome pathway (AOP) for skin sensitization (OECD, 2012) as defined by the OECD, were part of this in vitro Skin Sensitization Turnkey Testing Strategy: protein reactivity (DPRA), activation of keratinocytes (LuSens) and activation of dendritic cells (h-CLAT). However, in the current case for (R)-mandelic acid the results derived with DPRA and LuSens were sufficient for a final assessment. Therefore further testing in h-CLAT was waived.

DPRA The test substance was dissolved in acetonitrile at a concentration of 100 mM. The samples of the test substance with the peptides were solutions. Visual observation after the 24-hour incubation time did not reveal precipitates in any samples of the test substance with the peptides. The mean C-peptide depletion, caused by the test substance was determined to be 1.57%. The mean K-peptide depletion, caused by the test substance was determined to be -0.69%. Negative depletions were considered to be “zero” for calculation of the mean peptide depletion, which was thus calculated to be 0.79%. No co-elution of test substance and peptides was noticed. Based on the observed results and applying the prediction model proposed in Gerberick et. al (2007) it was concluded that (R)-mandelic acid shows a minimal chemical reactivity in the DPRA under the test conditions chosen. LuSensIn order to determine the concentrations suitable for the main experiment a pre-test was performed. Cells were exposed to 9 concentrations of the test substance and cytotoxicity was determined by MTT assay. In the main test luciferase activity was measured after 48 hour exposure. In parallel a MTT assay was performed to assess cytotoxicity of the test substance. A total of 2 valid experiments were performed. At concentrations used in the main experiment the test substance was soluble in 4% DMSO in culture medium 3 (4 x stock preparations) and in 1% DMSO in culture medium 3 (final concentrations). No precipitates were noticed in any concentration after 48 hours. Calculation of an EC1.50 (the concentration resulting in a 1.50-fold luciferase induction) was not applicable. After 48 hours of exposure to test substance luciferase activity in LuSens cells was not induced in at least two consecutive concentrations with statistical significance affording at least 70% viability in at least two independent experiments. From this it has to be concluded that test substance (R)-mandelic acid does not have a keratinocyte activating potential.

Test Method	Test Result	Test Evaluation
Direct Peptide Reactivity Assay (DPRA)	0.79% mean peptide depletion (1.57% cysteine peptide depletion -0.69% lysine peptide depletion).^a	Negative
Keratinocyte Activation Assay – LuSens	In at least two independent experiments no biologically relevant ARE-dependent luciferase activity induction was observed.^b	Negative
Dendritic Cell Line Activation Assay Human Cell Line Activation Test (h-CLAT)	Not determined	Not determined

^aNegative depletions were considered to be “zero” for calculation of mean peptide depletion.

^bFor details of evaluation criteria see study report

Based on the results and applying the evaluation criteria, (R)-mandelic acid is not peptide reactive and does not activate keratinocytes. Applying the evaluation criteria (R)-mandelic acid is predicted not to be a skin sensitizer. [BASF, 2015]

Migrated from Short description of key information:
LLNA: not sensitizing (3-Amino-butan-1-ol)
in vitro skin sensitization test battery: not sensitizing (R-mandelic acid)
Sensitization involves a number of key steps in order to take place, and can be described in terms of an adverse outcome pathway (AOP). These include reactivity with skin proteins (peptide reactivity), activation of skin cells (keratinocyte activation) and immune cells (dendritic cell activation). The studies carried out for this substance address these three key steps. The results are then used in a predefined evaluation scheme to determine hazard classification as a sensitizer by a weight-of-evidence approach.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

No need for classification according to the EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.