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

Endpoint summary

Description of key information

Based on the results of three in vitro and in chemico assays covering key event 1 to 3 the test item is considered to be not skin sensitising.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

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

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)

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.

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.

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

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.

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.

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.

Reference 2

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

9 May 2018 - 08 January 2019

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 Test Method)

GLP compliance:

yes (incl. QA statement)

Type of study:

activation of keratinocytes

Details on the study design:

Principle of the test:
The KeratinoSens™ method is an in vitro assay that quantifies the extent of luciferase gene induction following 48 hours incubation time of the KeratinoSens™ reporter cells with the test chemicals. Luciferase gene induction is measured in the cell lysates by luminescence detection using a light producing luciferase substrate (Luciferase Reagent). Cytotoxicity and the relative luminescence intensity of luciferase substrate in the lysates are measured and luciferase induction compared to solvent/vehicle control is calculated.
KeratinoSens™ cells were derived from HaCaT human keratinocytes and transfected with selectable plasmids containing luciferase gene under the transcriptional control of the AKR1C2 ARE gene sequence, upstream of the SV40 promoter. AKR1C2 is known to be one of the genes up-regulated upon contacting skin sensitisers in dendritic cells. Therefore, this method is able to mimic the activation of the Keap1-Nrf2-ARE regulatory pathway, and is relevant for the assessment of the skin sensitisation potential of test chemicals. A prediction model is used, to support the discrimination between sensitisers and non-sensitisers.

Procedure of the KeratinoSens™ method:
0. Preincubation of cells; Solubility assessment of test chemicals
1. Seeding of cells for testing - 24 h incubation
2. Preparation of the stock solution
3. Preparation of master plates
4. Exposure – 48 h incubation
5. Luciferase activation measurement; Cytotoxicity assessment

Positive control results:

The luciferase activity induction obtained with the positive control, Trans-Cinnamaldehyde was statistically significant above the threshold of 1.5 at two concentrations in the first test and at three concentrations in the other test. The EC1.5 value of the positive control was 16 µM and 13 µM in the two individual tests.

In addition, the average induction in the parallel plates for Trans Cinnamaldehyde at 64 μM was 9.40 and 11.42 fold. Although these values are out of the 2 – 8 fold induction domain, the dose response could be clearly seen with increasing luciferase activity induction at increasing concentrations for the positive control, therefore the tests were considered valid. Average fold induction values are presented in Table 3 and 4.

There was no cytotoxicity (cell viability lower than 70 %) induced by the positive control at any of the concentrations, thus no IC30 or IC50 values were determined.

Key result

Run / experiment:

other: 1

Parameter:

other: max. fold induction

Value:

1.07

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Key result

Run / experiment:

other: 2

Parameter:

other: max- fold induction

Value:

1.22

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Key result

Run / experiment:

other: both

Parameter:

other: EC1.5

Remarks on result:

not determinable

Other effects / acceptance of results:

DEMONSTRATION OF TECHNICAL PROFICIENCY:
Prior to routine use of the test method described in the OECD Test Guideline 442D, the laboratory demonstrated technical proficiency (Study Number: 392-442-4012), using the 10 Proficiency Substances listed in APPENDIX IA - ANNEX 1 of TG 442D. Moreover, a historical database of data generated with the positive control is maintained over time to confirm the reproducibility of the test method in the laboratory.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
- Acceptance criteria met for variability between replicate measurements: yes

Test item

There was no statistically significant induction above 1.5-fold observed at any of the concentrations in either of the runs therefore no EC1.5 values were determined for the test item. The average fold induction for each concentration is presented in Appendix III. The maximal fold induction was 1.07 for the first test and 1.22 for the second test. Based on the prediction model and the above described results, both tests were concluded negative. There was no cytotoxicity (cell viability lower than 70 %) induced by the test item at any of the concentrations, thus no IC30 or IC50 values were determined.

Positive and negative control

The coefficient of variation (CV%) of the luminescence reading for the negative control DMSO was not greater than 20 % in either of the tests (14.06 % and 19.69 % respectively).

The luciferase activity induction obtained with the positive control, Trans-Cinnamaldehyde was statistically significant above the threshold of 1.5 at two concentrations in the first test and at three concentrations in the other test. The EC1.5 value of the positive control was 16 µM and 13 µM in the two individual tests. In addition, the average induction in the parallel plates for Trans‑Cinnamaldehyde at 64 μM was 9.40 and 11.42 fold. Although these values are out of the 2 – 8 fold induction domain, the dose response could be clearly seen with increasing luciferase activity induction at increasing concentrations for the positive control, therefore the tests were considered valid. There was no cytotoxicity (cell viability lower than 70 %) induced by the positive control at any of the concentrations, thus no IC30 or IC50 values were determined.

Interpretation of results:

other: negative

Conclusions:

Based on these results and the KeratinoSens™ prediction model, the test item turned out to be negative and therefore having a non-sensitising potential under the experimental conditions of KeratinoSens™ method (ARE-Nrf2 Luciferase Test Method).

Executive summary:

In the course of this study the skin sensitisation potential of the test item was studied using the KeratinoSens™ method (ARE-Nrf2 Luciferase Test Method). For the test chemical and positive control substance, in order to derive a prediction two independent tests were sufficient to be conducted, since the results of those tests were concordant and both runs met the acceptance criteria. The luciferase activity induction obtained with the positive control, Trans-Cinnamaldehyde was statistically significant above the threshold of 1.5 at two concentrations in the first test and at three concentrations in the other test. The EC1.5 value of the positive control was 16 µM and 13 µM in the two individual tests. In addition, the average induction in the parallel plates for Trans‑Cinnamaldehyde at 64 μM was 9.40 and 11.42 fold and the dose response could be clearly seen with increasing luciferase activity induction at increasing concentrations for the positive control. There was no cytotoxicity (viability < 70 %) induced by the positive control at any of the concentrations. Moreover, the average coefficient of variation (CV) of the luminescence reading for the negative (solvent) control DMSO was not greater than 20 % in either tests. The CV% for the DMSO control was 14.01 % and 19.69 % in the first and second tests respectively. For the test item twelve doses were used in two independent runs between 2000 µM to 1 µM. There was no cytotoxicity (viability < 70 %) induced at any concentrations) by the test item in KeratinoSens™ cells compared to the solvent/vehicle control, thus no IC30 or IC50 values could be determined. The fold induction did not exceed the threshold of 1.5-fold at any concentrations compared to the respective negative controls in any of the independent runs. Therefore, both runs were negative for luciferase gene induction.

Table 1.               Summary of the KeratinoSens™ results for the test item

Name of the Test item	Significant induction above 1.5-fold (yes/no)	KeratinoSens™ result obtained (sensitiser/ non-sensitiser)
(2-Hydroxy-1,1-dimethylethyl)ammonium chloride (mixture of free amine and HCl salt)	no	non-sensitiser

 

Based on these results and the KeratinoSens™ prediction model, the test item was negative therefore having a non-sensitising potential under the experimental conditions of KeratinoSens™ method (ARE-Nrf2 Luciferase Test Method).

 

Reference 3

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2019-01-14 to 2019-03-21

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: IN VITRO SKIN SENSITISATION ASSAYS ADDRESSING THE KEY EVENT ON ACTIVATION OF DENDRITIC CELLS ON THE ADVERSE OUTCOME PATHWAY FOR SKIN SENSITISATION

Version / remarks:

2018

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

activation of dendritic cells

Details on the study design:

The h-CLAT method is an in vitro assay that quantifies changes of cell surface marker expression (i.e. CD86 and CD54) on a human monocytic leukemia cell line, THP-1 cells, following 24 hours exposure to the test item. These surface molecules are typical markers of monocytic THP-1 activation and may mimic DC activation, which plays a critical role in T-cell priming. The activation process through which DCs change from antigen processing to antigen presenting cells addresses the third key event of the skin sensitization Adverse Outcome Pathway.

The changes of surface phenotypic biomarker expression were measured and quantified by flow cytometry following cell staining with fluorochrome-tagged antibodies. The relative fluorescence intensity of surface markers compared to solvent/vehicle control were calculated and used in the prediction model, to support the discrimination between sensitisers and non-sensitisers. Cytotoxicity measurement was also conducted concurrently to assess whether upregulation of surface marker expression occurred at sub-cytotoxic concentrations.

Positive control results:

The positive control gave expected results for both markers, meaning that the RFI values of both CD86 and CD54 expression was over the positive criteria (CD86 ≥ 150 % and CD54 ≥ 200 %) and the respective cell viabilities were more than 50 % in each run.

Key result

Run / experiment:

other: 2 to 4

Parameter:

other: % increase in CD86 marker expression

Value:

150

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Key result

Run / experiment:

other: 2 to 4

Parameter:

other: % increase in CD54 marker expression

Value:

200

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: no

DEMONSTRATION OF TECHNICAL PROFICIENCY:
Prior to routine use of the test method described in Annex I of the Test Guideline 442E, the laboratory demonstrated technical proficiency by correctly obtaining the expected h-CLAT prediction for the 10 proficiency substances recommended in OECD 442E and by obtaining CV75, EC150 and EC200 values that fell within the respective reference ranges (Study Number: 392-442-3821). Moreover, a historical database of reactivity check results positive controls and solvent/vehicle controls was generated and has been maintained to confirm the reproducibility of the test method over time.


ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes, except run 1
- Acceptance criteria met for positive control: yes
- Acceptance criteria met for variability between replicate measurements: yes

Table 1 Outcome of the individual runs of the main tests

Test item	Obtained CV75 value (µg/mL)	Result of the individual runs for CD86 (positive/negative)				h-CLAT prediction for CD86 expression	Result of the individual runs for CD54 (positive/negative)				h-CLAT prediction for CD54 expression
(2-Hydroxy-1,1-dimethylethyl)ammonium chloride (mixture of free amine and HCl salt)	3817	i	p	p	p	positive	i	p	p	p	positive

n – negative outcome of a valid run / p – positive outcome of a valid run / i – invalid run

Since 3 out of 4 runs were positive for CD86 marker expression and 3 out of 4 runs were positive for CD54 marker expression the overall outcome of the study was positive.

Interpretation of results:

other: positive

Executive summary:

In the course of this OECD 442E compliant study the skin sensitization potential of the test item was studied.

 

The extent of cytotoxicity induced on THP-1 cells by the test item was studied in four dose finding tests. The default concentration used for test item dissolved in saline was set to the highest allowed concentration (5000 µg/mL) in the last two dose finding tests. An average CV75 value of 3817 µg/mL was calculated and it was used for setting the dose-range for measuring CD86 and CD54 expression in the main test. Eight doses were used in four independent runs between 4584 µg/mL – 1278 µg/mL. The increase in CD86 marker expression (RFI) was equal to or greater than 150 % at most of the tested doses (with >50 % of cell viability) compared to the respective negative controls in all of the independent runs. Therefore, all three valid runs were concluded positive for CD86 marker expression. However, effective concentration for CD86 expression (EC150) was not determined, since no clear dose response could be observed. The increase of CD54 marker expression (RFI) was greater than 200 % compared to the negative controls at several higher concentrations (with >50 % of cell viability) in all three independent valid runs. Based on the concordant results of the three individual runs for CD54 expression, prediction was concluded as positive. Thus, the effective concentration for CD54 expression (EC200) was determined by linear interpolation and log-linear extrapolation. The EC200 value for CD54 was 2640 µg/mL. Three out of four runs were considered valid in the main study, since the first run did not meet all acceptance criteria for the negative controls. Since both CD86 and CD54 marker expressions gave positive result, the overall h-CLAT prediction was concluded positive.

 

Table 1 Summary of the h-CLAT results for the test item

Name of the Test item	Obtained CV75 value (µg/mL)	h-CLAT result for CD86 (positive/negative)	h-CLAT result for CD54 (positive/negative) and obtained EC200 value (µg/mL)	h-CLAT result obtained (sensitizer/ non-sensitizer)
(2-Hydroxy-1,1-dimethylethyl)ammonium chloride (mixture of free amine and HCl salt)	3817	positive	positive (2640 µg/mL)	sensitizer

Based on these results and the h-CLAT prediction model, the test item “(2-Hydroxy-1,1-dimethylethyl)ammonium chloride (mixture of free amine and HCl salt)” demonstrated an in vitro sensitizing potential under the experimental conditions of human Cell Line Activation Test.

 

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

There are three in vitro/in chemico studies available with the test item assessing its skin sensitising potential. The studies were conducted according to GLP and the respective OECD TG (OECD 442C, OECD 442D, OECD 442E) and are considered valid. The available data is considered suitable and reliable to allow assessment of the test items properties in regards to skin sensitisation.

OECD 442C, DPRA

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 usedfor 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.

OECD 442D, KeratinoSens

In the course of this study the skin sensitisation potential of the test item was studied using the KeratinoSens™ method (ARE-Nrf2 Luciferase Test Method). For the test chemical and positive control substance, in order to derive a prediction two independent tests were sufficient to be conducted, since the results of those tests were concordant and both runs met the acceptance criteria. The luciferase activity induction obtained with the positive control, Trans-Cinnamaldehyde was statistically significant above the threshold of 1.5 at two concentrations in the first test and at three concentrations in the other test. The EC1.5 value of the positive control was 16 µM and 13 µM in the two individual tests. In addition, the average induction in the parallel plates for Trans‑Cinnamaldehyde at 64 μM was 9.40 and 11.42 fold and the dose response could be clearly seen with increasing luciferase activity induction at increasing concentrations for the positive control. There was no cytotoxicity (viability < 70 %) induced by the positive control at any of the concentrations. Moreover, the average coefficient of variation (CV) of the luminescence reading for the negative (solvent) control DMSO was not greater than 20 % in either tests. The CV% for the DMSO control was 14.01 % and 19.69 % in the first and second tests respectively. For the test item twelve doses were used in two independent runs between 2000 µM to 1 µM. There was no cytotoxicity (viability < 70 %) induced at any concentrations) by the test item in KeratinoSens™ cells compared to the solvent/vehicle control, thus no IC30 or IC50 values could be determined. The fold induction did not exceed the threshold of 1.5-fold at any concentrations compared to the respective negative controls in any of the independent runs. Therefore, both runs were negative for luciferase gene induction.

Table 1 Summary of the KeratinoSens™ results for the test item

Name of the Test item	Significant induction above 1.5-fold (yes/no)	KeratinoSens™ result obtained (sensitiser/ non-sensitiser)
(2-Hydroxy-1,1-dimethylethyl)ammonium chloride (mixture of free amine and HCl salt)	no	non-sensitiser

 

Based on these results and the KeratinoSens™ prediction model, the test item was negative therefore having a non-sensitising potential under the experimental conditions of KeratinoSens™ method (ARE-Nrf2 Luciferase Test Method).

 

OECD 442E, h-CLAT

In the course of this OECD 442E compliant study the skin sensitization potential of the test item was studied. The extent of cytotoxicity induced on THP-1 cells by the test item was studied in four dose finding tests. The default concentration used for test item dissolved in saline was set to the highest allowed concentration (5000 µg/mL) in the last two dose finding tests. An average CV75 value of 3817 µg/mL was calculated and it was used for setting the dose-range for measuring CD86 and CD54 expression in the main test. Eight doses were used in four independent runs between 4584 µg/mL – 1278 µg/mL. The increase in CD86 marker expression (RFI) was equal to or greater than 150 % at most of the tested doses (with >50 % of cell viability) compared to the respective negative controls in all of the independent runs. Therefore, all three valid runs were concluded positive for CD86 marker expression. However, effective concentration for CD86 expression (EC150) was not determined, since no clear dose response could be observed. The increase of CD54 marker expression (RFI) was greater than 200 % compared to the negative controls at several higher concentrations (with >50 % of cell viability) in all three independent valid runs. Based on the concordant results of the three individual runs for CD54 expression, prediction was concluded as positive. Thus, the effective concentration for CD54 expression (EC200) was determined by linear interpolation and log-linear extrapolation. The EC200 value for CD54 was 2640 µg/mL. Three out of four runs were considered valid in the main study, since the first run did not meet all acceptance criteria for the negative controls. Since both CD86 and CD54 marker expressions gave positive result, the overall h-CLAT prediction was concluded positive.

 

Table 2 Summary of the h-CLAT results for the test item

Name of the Test item	Obtained CV75 value (µg/mL)	h-CLAT result for CD86 (positive/negative)	h-CLAT result for CD54 (positive/negative) and obtained EC200 value (µg/mL)	h-CLAT result obtained (sensitizer/ non-sensitizer)
(2-Hydroxy-1,1-dimethylethyl)ammonium chloride (mixture of free amine and HCl salt)	3817	positive	positive (2640 µg/mL)	sensitizer

Based on these results and the h-CLAT prediction model, the test item “(2-Hydroxy-1,1-dimethylethyl)ammonium chloride (mixture of free amine and HCl salt)” demonstrated an in vitro sensitizing potential under the experimental conditions of human Cell Line Activation Test.

Conclusion

In vitro studies covering three key steps of the adverse outcome pathway for skin sensitization as identified by the OECD (OECD Publication No.168; ENV/JM/MONO(2012)10) were performed. These study types have initially undergone validation using 54 substances (Bauch et al., 2012 Regul Toxicol Pharmacol. 63: 489-504). Recently, a validation performed with 213 substances has been published (Urbisch et al. 2015 Regul Toxicol Pharmacol. 71: 337-351).

 

Based on the results of this validation (Bauch et al., 2012 Regul Toxicol Pharmacol. 63: 489-504) the predictive capacity of the evaluation scheme using DPRA (peptide reactivity), LuSens/KeratinoSens™ (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 3). 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 3: 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

 

In accordance with the published evaluation scheme (Bauch et al., 2012 Regul Toxicol Pharmacol. 63: 489-504) and Sections 1.2 and 1.4 of Annex XI of EC regulation 1907/2006, the test substance is not a skin sensitizer.

 

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008
The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Based on available data on skin sensitising properties, the test item does not require classification skin sensitiser according to Regulation (EC) No 1272/2008 (CLP).