Reaction mass of 2-amino-2-methyl-1-propanol hydrochlorid (3207-12-3) and 2-amino-2-methyl-1-propanol (124-68-5)

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

09 May 2018 - 07 January 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))

Version / remarks:

2015

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

direct peptide reactivity assay (DPRA)

Test material

Test material information

1

Reference substance name:

(2-hydroxy-1,1-dimethylethyl)ammonium chloride

EC Number:

221-713-5

EC Name:

(2-hydroxy-1,1-dimethylethyl)ammonium chloride

Cas Number:

3207-12-3

Molecular formula:

C4H11NO.ClH

IUPAC Name:

1-hydroxy-2-methylpropan-2-aminium chloride

Test material form:

liquid

In chemico test system

Details on the study design:

The reactivity of a test chemical and synthetic cysteine or lysine containing peptides was evaluated by combining the test chemical with a solution of the peptide (reaction samples) and monitoring the remaining concentration of the peptide following 24 ± 2 hours of interaction time at room temperature (25 ± 2.5 °C). The peptide is a custom material containing phenylalanine to aid in detection and either cysteine or lysine as the reactive centre. Relative concentrations of the peptide following the 24 hour reaction time were determined by HPLC with gradient elution and UV detection at 220 nm. Samples were prepared and analysed in triplicates in batches to keep the total HPLC analysis time less than 30 hours.

Results and discussion

Positive control results:

The acceptance criteria were met in case of the positive control with the cysteine peptide depletion value of 70.09 % and a mean lysine peptide depletion value of 57.24 %. The SD of the percent peptide depletions of the positive control was 0.65 % and 0.84 % for the cysteine and lysine depletion respectively.

In vitro / in chemico

Results

Results 1

Key result

Run / experiment:

other: mean of three runs

Parameter:

other: % peptide depletion cysteine

Value:

3.9

Vehicle controls validity:

not examined

Negative controls validity:

not examined

Positive controls validity:

valid

Results 2

Key result

Run / experiment:

other: mean of three runs

Parameter:

other: % peptide depletion lysine

Value:

11.68

Vehicle controls validity:

not examined

Negative controls validity:

not examined

Positive controls validity:

valid

Other effects / acceptance of results:

The percent cysteine peptide depletion value of the test item (100 mM) was 3.93 % while of the neat test item (1g/mL) was 11.68 %. The percent lysine peptide depletion of the test item (100 mM) was 0.86 % while of the neat test item (1 g/mL) was 0.02 %.
The standard deviations for the test chemical replicates were 1.87 % and 1.23 % for the percent cysteine depletions and 0.42 % and 0.37 % for the percent lysine peptide depletions respectively.

Any other information on results incl. tables

Table 10 Mean peptide depletion values for the positive control and the test chemical

Name, replicate number	Obtained mean % cysteine peptide depletion	Obtained mean % lysine peptide depletion	Mean % obtained peptide depletion
(2-Hydroxy-1,1-dimethylethyl)ammonium chloride (mixture of free amine and HCl salt) (100 mM)	3.93	0.86	2.40
2-Hydroxy-1,1-dimethylethyl)ammonium chloride (mixture of free amine and HCl salt) (1 g/mL)	11.68	0.02*	5.85
CINNAMALDEHYDE	70.09	57.24	63.67

*Negative depletion values were substituted with zero when calculating the mean peptide depletion

The average percent peptide depletion was calculated for the test item. By using the cysteine 1:10 / lysine 1:50 prediction model (see table below), the threshold of 6.38% average peptide depletion was used to support the discrimination between a skin sensitiser and a non-sensitiser. On the basis of the prediction model, chemicals assigned to the minimal reactivity class should be classified as non-sensitisers whereas chemicals assigned to the low, moderate or high reactivity class should be classified as sensitisers. The test item had the average percent peptide depletion value of 2.40 %, thus the test item is classified as a non-sensitiser.

When the neat test item (1 g/mL) was tested, an average percent peptide depletion value of 5.85 % was obtained, which was under the minimal reactivity threshold value, as well.

Applicant's summary and conclusion

Interpretation of results:

other: negative

Conclusions:

Based on results of this in chemico Direct Peptide Reactivity Assay according to OECD 442C the test item has no or minimal reactivity towards the synthetic peptides and is thus not considered a potential skin sensitiser.

Executive summary:

This study was conducted to evaluate the skin sensitisation potential of the test item in chemico. The Direct Peptide Reactivity Assay (DPRA) proposed the molecular initiating event of the skin sensitisation Adverse Outcome Pathway (AOP), namely protein reactivity, by quantifying the reactivity of the test chemical towards cysteine and lysine model synthetic peptides. At the beginning of the assay the solubility of the test chemical was assessed and ultrapure water was chosen as the appropriate solvent.

Two individual tests were conducted with the cysteine peptide, since the first one did not meet the acceptance criteria and had to be rejected. The second run was valid and accepted. Only one individual run was needed with the lysine peptide, which has fulfilled all the acceptance criteria. All in all, the results of the two valid runs were used for the classification of the test item. The positive control replicates showed the expected percent peptide depletion values within acceptable limits with 70.09 % cysteine peptide depletion and 57.24 % lysine peptide depletion. The back-calculated values of the reference control replicates were within the expected molarity concentration range for the cysteine and lysine peptides, as well. Two stock solutions were prepared with the test item for both peptides: in addition to the 100 mM solutions, the test chemical was dissolved to its maximum soluble concentration (neat reaction sample) in the same solvent used to prepare the apparent 100 mM solutions. The percent cysteine peptide depletion value of the test item (100 mM) was 3.93 % while of the neat test item (1 g/mL) was 11.68 %. The percent lysine peptide depletion of the test item (100 mM) was 0.86 % while of the neat test item (1 g/mL) was 0.02 %.

The mean depletion value of the peptides was used to categorise the test chemical in one of the four classes of reactivity. No co-elution was observed with either cysteine or lysine peptides, therefore the Cysteine 1:10 / Lysine 1:50 prediction model was used for the discrimination between sensitisers and non-sensitisers. The mean peptide depletion of the test item was 2.40 % with the 100 mM test item stock solutions and 5.85 % with the 1 mg/mL test item stock solutions, thus none of them exceeded the 6.38 % threshold of the applicable prediction model. The results correspond to a negative outcome indicating that the test item has no or minimal reactivity towards the synthetic peptides, thus is not a potential skin sensitiser.