2-propen-1-amine, N-2-propen-1-yl-, hydrochloride

Endpoint summary

Description of key information

The skin sensitisation potential of 2-propen-1-amine, N-2-propen-1-yl-, hydrochloride was assessed using an in vitro KeratinoSens assay according to OECD guidelines 442D during a GLP compliant study.

After 48 hours of exposure of cells with 12 concentrations of 2-propen-1-amine, N-2-propen-1-yl, hydrochloride, Luciferase measurements and MTT viability testing were performed.

Acceptance criteria for the positive control (cinnamic aldehyde) were met.

The test item did not induce a significant luciferase induction >1.5 and the mean Imax value was 0.949 uM.  The statistical significance, viability, dose response and dose acceptance criteria were all met.

2-propen-1-amine, N-2-propen-1-yl, hydrochloride was classified as neagtive according to the KeratinoSens prediction model.

 

The skin sensitisation potential of 2-propen-1-amine, N-2-propen-1-yl-, hydrochloride was assessed using an in vitro direct peptide reactivity assay according to OECD guidelines 442C during a GLP compliant study.

After 24 hours of the test item being incubated with either cysteine or lysine they were run on a HPLC system and assessed according to the Cysteine 1:10/Lysine 1:50 predicition mode.

Acceptance criteria for the positive control (cinnamic aldehyde) were met.

The test item showed no or minial reactivity and the DPRA prediction was negative.  All acceptance criteria were all met.

2-propen-1-amine, N-2-propen-1-yl, hydrochloride was classified as negative as per the prediction model.

 

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

January 2021 to June 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Type of study:

other: Direct Peptide Reactivity Assay

Specific details on test material used for the study:

66% purity
Aqueous solution

Details on the study design:

Test items were incubated for 24 h +/- 2 h at 25 oC in solution at 100 mM in combination with either cysteine or lysine containing peptides and then run on an HPLC system (20 minute run time) using gradient elution and UV detection at 220 nm to measure peptide concentration.

Test items were compared to reference controls containing the test item solvent in combination with either cysteine or lysine peptide in order to determine the relative percent peptide depletion. Relative percent peptide depletion values were used in a prediction model that assigns test items to one of four reactive classes.

Acceptance Criteria

-Standard curve must have an r2 value >0.99
-The mean percent depletion value of the three replicates for the positive control cinnamic aldehyde should be between 60.8% and 100% for the cysteine peptide and between 40.2% ad 69.4% for the lysine peptide.
-The standard deviation for the positive control replicates should be <14.9% for the percent cysteine depletion and <11.6% for the percent lysine depletion
-Mean peptide concentration of reference controls A should be 0.5 +/- 0.05 mM
-The coefficient of variation (CV) of peptide peak areas for the 9 reference controls B and C should be <15%.

The standard curve is used to assign concentrations to test items and controls.
The RefA controls are peptide plus acetonitrile, prepared to endure peptide concentration is within specified limits.
RefB controls are peptide plus acetonitrile, prepared to ensure peptide stability throughout the run is within the specified limits.
RefC controls are peptide plus acetonitrile and test item solvent, prepared as a control to assess percent peptide depletion.

Using the cysteine 1:10/lysine 1:50 prediction model to assess reactivity classes

0% < mean % depletion <6.38% - No or minimal reactivity - DPRA prediction is negative
6.38% < mean % depletion <22.62% - low reactivity - DPRA prediction is positive
22.62% < mean % depletion <42.47% - moderate reactivity - DPRA prediction is positive
42.47% < mean % depletion <100% - high reactivity - DPRA prediction is positive

Vehicle / solvent control:

water

Positive control:

cinnamic aldehyde [442D]

Positive control results:

Positive control had a cysteine depletion of 75.031% and is within the acceptance criteria

Positive control had a lysine depletion of 61.90% and is within the acceptance criteria

Key result

Group:

test chemical

Run / experiment:

mean

Parameter:

other: Mean% Peptide Depletion (Cys + Lys)

Value:

1.366 %

Vehicle controls validity:

valid

Positive controls validity:

valid

Outcome of the prediction model:

negative [in vitro/in chemico]

Other effects / acceptance of results:

All acceptance criteria met

Interpretation of results:

GHS criteria not met

Conclusions:

2-propen-1-amine, N-2-propen-1-yl-, hydrochloride did not meet the criteria for classification as a skin sensitiser.

Executive summary:

The skin sensitisation potential of 2-propen-1-amine, N-2-propen-1-yl-, hydrochloride was assessed using an in vitro direct peptide reactivity assay according to OECD guidelines 442C during a GLP compliant study.

After 24 hours of the test item being incubated with either cysteine or lysine they were run on a HPLC system and assessed according to the Cysteine 1:10/Lysine 1:50 predicition mode.

Acceptance criteria for the positive control (cinnamic aldehyde) were met.

The test item showed no or minial reactivity and the DPRA prediction was negative.  All acceptance criteria were all met.

2-propen-1-amine, N-2-propen-1-yl, hydrochloride was classified as negative as per the prediction model.

Reference 2

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

January 2021-June 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 luciferase KeratinoSens™ test method)

GLP compliance:

yes (incl. QA statement)

Type of study:

ARE-Nrf2 luciferase KeratinoSens™ test method

Specific details on test material used for the study:

66% purity
Aqueous solution

Details of test system:

Keratinoses transgenic cell line [442D]

Details on the study design:

442D

PREPARATION AND ADIMISTRATION OF TEST SOLUTIONS
- test item: Per plate, a single application of 12 concentrations (2000, 1000, 500, 125, 62.5, 31.25, 15.625, 7.813, 3.906, 1.953 and 0.977 uM) of the test item was applied in cell culture medium (dilution factor of 2) with a final concentration of ethanol of 1%. The top concentration was previously determined by solubility testing.

- Positive controls: Per plate, a single application of 5 concentrations (8, 16, 32, 64 and 128 uM) of the positive control (cinnaminc aldehyde) was applied in cell culture medium (dilution factor of 2) with a final concentration of ethanol of 1%.

- Preparation of the solvent and negative controls: Per plate, a single application of culture medium with 1% ethanol was applied as the negative control (6 wells per plate). One well per plate was left empty (no cells).


DOSE RANGE FINDING ASSAY:
The test concentrations of 2-propen-1-amine, N-2-propen-1-yl, hydrochloride used in the study were selected on the basis of solubility testing carried out during the study. Solubility of the test item was confirmed up to 200 mM in 1% ethanol in cell culture medium. Subsequent dilution in cell culture medium gave a top concentration of 2000 uM.

APPLICATION OF THE TEST CHEMICAL AND CONTROL SUBSTANCES
- Number of repetitions: Four repetitions were performed. Each repetition consisted of 3 x 96-well plates for luminescence (n=12 overall) and 2 x 96-well plates for MTT (n=8 overall).

- Exposure time: Cells were incubated with the test or reference item for 48 +/- 2 hours prior to endpoint measurements.

- Study evaluation and decision criteria used: A test item is considered positive if the following conditions are met in 2 of 3 repetitions:
1. The Imax is greater than or equal to 1.5-fold and statistically significantly different as compared to the solvent negative control (as determined by a two-tailed, unpaired student's T-test)
2. The cellular viability is higher than 70% at the lowest concentration with induction of luciferase activity greater than or equal to 1.5-fold. Test items that only induce gene activity at cytotoxic levels are not rated positive, as in the case for some non-sensitising skin irritants.
3. The EC1.5 value is < 1000 uM or < 200 ug/mL for test items with no defined MW.
4. There is an apparent overall dose-response for luciferase induction (or biphasic response).

- Description on study acceptance criteria: Test results are acceptable if:
1. The positive control (cinnamic aldehyde) produced positive results, i.e the luciferase gene induction produced by this control is above the threshold of 1.5 in at least on of the tested concentrations and this induction is statistically significant compared to the solvent control.
2. The Imax and the EC1.5 for cinnamic aldehyde is calculated and meet either or both of the following targets:
-average induction in three replicates for cinnamic aldehyde at 32 uM is within the historical range (currently 1.6 and 3).
-EC1.5 value for cinnamic aldehyde is within the historical range (currently 6 uM and 39 uM).

SEEDING AND INCUBATION
Day 1 - Cell seeding (3 x 96-well plates for luminescence; 2 x 96-well plates fir MTT); 10,000 cells per well, passage number 17 for repetition 1 to 3, passage number 21 for repetition 4.

Day 2 - 24 hours after seeding, the test and control items were applied to plates and were incubated at 37 oC, 5% CO2, >95% relative humidity for 48 +/- 2 hours.

Day 4 - Evaluation of luciferase activity by luminescence (3 plates) and cell viability by MTT testing (2 plates).

Vehicle / solvent control:

cell culture medium

Negative control:

other: Ethanol

Positive control:

cinnamic aldehyde [442D]

Positive control results:

The positive control produced an induction at >1.5-fold in 4 out of 5 concentrations meeting the acceptance criteria.

The EC1.5 value was 10.868. The meets the acceptance criteria of between 6 and 39 uM.

The CV% of blank values was 11.2626%. This meets the acceptance criteria of <20%.

The average induction of the positive control was 3.269. This failed the acceptance criteria as it should be between 1.6 and 3.0. However, as other acceptance criteria were met, and there was a dose-dependant increase of induction with the positive control the results were considered valid.

Key result

Group:

test chemical

Run / experiment:

mean

Parameter:

Imax [442D]

Value:

0.949 µM

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

The test item did not induce a statistically significant luciferase induction >1.5 in either of the two valid repetitions. The statistical significance, viability, dose response and does acceptance criteria were all met and therefore the test item was classed as negative using the KeratinoSens prediction model.

Interpretation of results:

GHS criteria not met

Conclusions:

2-propen-1-amine, N-2-propen-1-yl-, hydrochloride did not meet the criteria for classification as a skin sensitiser.

Executive summary:

The skin sensitisation potential of 2-propen-1-amine, N-2-propen-1-yl-, hydrochloride was assessed using an in vitro KeratinoSens assay according to OECD guidelines 442D during a GLP compliant study.

After 48 hours of exposure of cells with 12 concentrations of 2-propen-1-amine, N-2-propen-1-yl, hydrochloride, Luciferase measurements and MTT viability testing were performed.

Acceptance criteria for the positive control (cinnamic aldehyde) were met.

The test item did not induce a significant luciferase induction >1.5 and the mean Imax value was 0.949 uM.  The statistical significance, viability, dose response and dose acceptance criteria were all met.

2-propen-1-amine, N-2-propen-1-yl, hydrochloride was classified as neagtive according to the KeratinoSens prediction model.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)