Trometamol

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vitro

Type of information:

read-across based on grouping of substances (category approach)

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

GLP-guideline study, tested with the source substance 2-amino-2-methyl-1,3-propanediol (CAS 115-69-5). In accordance to the ECHA guidance document "Practical guide 6: How to report read-across and categories (March 2010)", the reliability was changed from RL1 to RL2 to reflect the fact that this study was conducted on a read-across substance.

Justification for type of information:

The jusitification for read-across has been attached in section 13 of this IUCLID.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Report date:

2011

Materials and methods

Principles of method if other than guideline:

To test direct peptide reactivity which is a key pathway leading to skin sensitisation, the test substance was investigated for peptide depletion by chemical reaction. The assay method established by Natsch and Gfeller (2008) was validated and improved in the testing facility and utilised in this study.

GLP compliance:

yes

Type of study:

other: peptide binding assay

Justification for non-LLNA method:

Use of an in vitro methos to assess senstization potential by assessment of peptide reactivity

Test material

Test material information

Constituent 1

Reference substance name:

2-amino-2-methyl-1,3-propanediol

IUPAC Name:

2-amino-2-methyl-1,3-propanediol

Details on test material:

- Name of test material (as cited in study report): XU-12398.00
- Molecular formula (if other than submission substance): C4H11NO2
- Molecular weight (if other than submission substance): 105.1
- Analytical purity: 99.9%

In vitro test system

Details on the study design:

Method developed by Natsch and Gfeller (2008) uses a single peptide and analyzes depletion of the peptide and formation of a the covalent adduct with LC-MS methods.Briefly, a standard hepta-peptide containing one cysteine, two lysines is incubated with a 10-fold excess of test article in neutral pH condition. After a 24 hour incubation, the reaction mixture is subjected to the HPLC-UV-MS analyses to measure remaining parent peptides. The amount of depleted peptide due to reaction with the test article will be calculated and used to determine protein reactivity of the test article which is then used as to evaluate the skin sensitization potential. Based on the results from previous studies, if there is more than 15% peptide depletion by the test article, the chemical is likely to have skin sensitization potential (Gerberick et al., 2004; Natsch and Gfeller 2008). Furthermore, formation of covalent adducts between the peptide and test articles would be considered as an indicator of potential skin sensitization.

Results and discussion

In vitro / in chemico

Results

Key result

Parameter:

other: Peptide depletion

Remarks:

Negative peptide reactivity

Value:

4.22

Vehicle controls validity:

valid

Remarks:

Negative control; vehicle control

Negative controls validity:

valid

Remarks:

Same as vehicle control

Positive controls validity:

valid

Remarks:

Diethyl malaete

Remarks on result:

other: Other: peptide reactivity

Remarks:

Negative for peptide reactivity

Any other information on results incl. tables

The test substance was completely soluble in acetonitrile and was not precipitated by mixing with peptide solutions. After 24 h incubation, there was no colour change or a precipitate observed from the test substance. The test article did not have any UV absorbance at 220 nm through entire HPLC chromatography and therefore there was no interference with HPLC-UV analyses for peptides. Furthermore, the test substance did not interfere with the MS detections used in the test system that were monitoring higher than 700.0 m/z. Using the established calibration curve, the concentrations of free peptide were calculated for each sample (Table 1). Average peptide depletion by the test substance was 4.22 ± 1.84%. Negative and positive controls resulted in 4.83 ± 1.66% and 96.13 ± 0.21% peptide depletion, respectively. These results confirmed the assay was valid. Because there was no peptide depletion by the test substance, no further analysis was performed to measure dimerized- or oxidized-peptide by the test substance.

 

Table 1: Individual data from free peptide quantitation and average peptide depletion

Group	Replicate#	Analyte Peak Area (counts)	Peptide conc. (mM)	Peptide depletion (%)	Average depletion (%)
Test substance	1	15300000	98.03	1.97	4.22 ± 1.84
	2	15200000	97.37	2.63
	3	14700000	94.07	5.93
	4	14700000	94.07	5.93
	5	14900000	95.39	4.61
Negative control	1	14600000	93.40	6.60	4.83 ± 1.66
	2	14900000	95.39	4.61
	3	15100000	96.71	3.29
Positive control	1	1030000	3.78	96.22	96.13 ± 0.21
	2	1080000	4.11	95.89
	3	1020000	3.71	96.29

Applicant's summary and conclusion

Interpretation of results:

other: Under the test conditions, there is no evidence that the test substance contains direct protein reactivity which would cause skin sensitisation.

Remarks:

Criteria used for interpretation of results: EU