IPEMA

Endpoint summary

Description of key information

IPEMA is classified as sin sensitiser category 1B according to methods outlined in the Guideline on Defined Approaches (DA) for Skin Sensitization (Guideline 497) supplemented by Read-Across prediction models within OECD Toolbox (v4.5). 

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

June 2016

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

secondary literature

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 442B (Skin Sensitization: Local Lymph Node Assay: BrdU-ELISA)

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)

GLP compliance:

no

Type of study:

mouse local lymph node assay (LLNA)

Species:

mouse

Strain:

Balb/c

Sex:

female

Vehicle:

acetone/olive oil (4:1 v/v)

Concentration:

2 and 20 %w/v

No. of animals per dose:

3 mices

Positive control substance(s):

other: BC: Benzocaine (positive control)

Key result

Parameter:

other: ILW : the % increase in lymph node weight

Value:

1.68

Test group / Remarks:

20% test substance

The completed experiment results are in the attachement.

Interpretation of results:

other: The ILW obtained from the mice treated with IPEMA at the concentrations of 20% was 168%. This value was higher than the value obtained from 20%BC. So, IPEMA was considered to be a sensitizer under the conditions of this assay.

Conclusions:

Neither deaths nor signs of ill health were seen in the mice during this study. The ILW obtained
from the mice treated with 20%BC was high enough to detect such a very weak skin sensitizer as BC.
These results demonstrate the reliability of this assay to compare the strength of skin-sensitization
potential between chemical substances.

The ILW obtained from the mice treated with IPEMA at the concentrations of 20% was 168%.
This value was higher than the value obtained from 20%BC. So, IPEMA was considered to be a
sensitizer under the conditions of this assay.

Reference 2

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2021-06-23 to 2021-10-04

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

Details of test system:

Keratinoses transgenic cell line [442D]

Details on the study design:

see under "Any other information on materials and methods incl. tables"

Vehicle / solvent control:

DMSO

Negative control:

other: DMSO at a final concentration of 1% (v/v) in test item exposure medium was used as negative control.

Positive control:

cinnamic aldehyde [442D]

Positive control results:

Refer to the experiment 1-3 results in the pdf attachment.

Group:

test chemical

Run / experiment:

run/experiment 1

Parameter:

Imax [442D]

Value:

1.66

At concentration:

500 other: µM

Cell viability:

149.8%

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: The corresponding cell viability determined by MTT staining was 149.8%.

Remarks:

Microscopically, a clear cytotoxic effect was observed at the three highest test item concentrations (500, 1000 and 2000 µM) and a slight cytotoxic effect at a test item concentration of 250 µM. The increase in % viability may be caused by a slight MTT reducing potential of the test item, as determined in a MTT interference test. Therefore, the slight induction above the threshold of 1.5 may be caused by the cytotoxic effect.

Key result

Group:

test chemical

Run / experiment:

run/experiment 3

Parameter:

Imax [442D]

Value:

2.61

At concentration:

756.14 other: µM

Cell viability:

87.6 %.

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Group:

test chemical

Run / experiment:

run/experiment 2

Parameter:

Imax [442D]

Value:

1.41

At concentration:

500 other: µM

Cell viability:

87.4%

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: In the second experiment, no significant luciferase induction > 1.5 was found in the tested concentration range.

Remarks:

Therefore, no EC1.5 value could be calculated. To verify the results and as the test item induced the gene activity very close to the cytotoxic level in experiment 1, a third experiment with an adapted concentration range and a narrower dose-response analysis with dilution of 1.15-fold was performed to decide if induction is at cytotoxic levels or not.

Group:

test chemical

Run / experiment:

run/experiment 1

Parameter:

EC 1.5 [442D]

Value:

289.06 µM

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: The corresponding cell viability determined by MTT staining was 149.8%.

Remarks:

Microscopically, a clear cytotoxic effect was observed at the three highest test item concentrations (500, 1000 and 2000 µM) and a slight cytotoxic effect at a test item concentration of 250 µM. The increase in % viability may be caused by a slight MTT reducing potential of the test item, as determined in a MTT interference test. Therefore, the slight induction above the threshold of 1.5 may be caused by the cytotoxic effect.

Group:

test chemical

Run / experiment:

run/experiment 3

Parameter:

EC 1.5 [442D]

Value:

313.33 µM

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: The lowest tested concentration with a significant luciferase induction >1.5 (1.55) was found to be 326.90 µM. The corresponding cell viability was >70% (97.9%). The calculated EC1.5 was <1000 µM (313.33 µM).

Outcome of the prediction model:

positive [in vitro/in chemico]

Other effects / acceptance of results:

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
- Range of historical values if different from the ones specified in the test guideline: see table below

The in vitro KeratinoSens™ assay enables detection of the sensitising potential of a test item by addressing the second molecular key event of the adverse outcome pathway (AOP), namely activation of keratinocytes, by quantifying the luciferase activity in the transgenic cell line KeratinoSens™. The luciferase activity, assessed by luminescence measurement, compared to the respective solvent controls is used to support discrimination between skin sensitisers and non-sensitisers.

In the present study 1,3-di(3-Methyl-2-butenoxy)-2-hydroxypropane was dissolved in DMSO.

Based on a molecular weight of 228.33 g/mol a stock solution of 200 mM was prepared.

Based on the stock solution a set of twelve master solutions in 100% solvent was prepared by serial dilution using a constant dilution factor of 1:2. These master solutions were diluted 1:100 in cell culture medium. The following concentration range was tested in the assay:

2000, 1000, 500, 250, 125, 62.5, 31.25, 15.63, 7.81, 3.91, 1.95, 0.98 µM

Cells were incubated with the test item for 48 h at 37°C. After exposure cells were lysed and luciferase activity was assessed by luminescence measurement.

In the first experiment, a max luciferase activity (I_max) induction of 2.91 was determined at a test item concentration of 2000 µM. The corresponding cell viability was 93.9%. The lowest tested concentration with a significant luciferase induction >1.5 (1.80) was found to be 500 µM. The corresponding cell viability was >70% (121.0%). The calculated EC_1.5 was <1000 µM (256.25 µM).

In the second experiment, a max luciferase activity (I_max) induction of 5.55 was determined at a test item concentration of 2000 µM. The corresponding cell viability was 64.7%. The lowest tested concentration with a significant luciferase induction >1.5 (1.86) was found to be 500 µM. The corresponding cell viability was >70% (97.2%). The calculated EC_1.5 was <1000 µM (253.54 µM).

A dose response for luciferase activity induction was observed for each individual run as well as for an overall luciferase activity induction.

Under the condition of this study the test item is therefore considered as sensitiser.

The controls confirmed the validity of the study (see Table 6).

 

Luciferase Activity - Overall Induction

Table 5: Induction of Luciferase Activity – Overall Induction

Conc.	Experiment 1			Experiment 2			Conc.	Experiment 3
[µM]	mean	sd	Sign.	mean	sd	Sign.	[µM]	mean	sd	Sign.
0.98	1.22	0.12		1.13	0.32		214.94	1.44	0.07
1.95	1.30	0.10		1.01	0.09		247.18	1.38	0.18
3.91	1.22	0.13		0.98	0.08		284.26	1.39	0.09
7.81	1.34	0.19		1.05	0.11		326.90	1.55	0.27	*
15.63	1.24	0.09		1.10	0.13		375.94	1.54	0.22	*
31.25	1.27	0.11		1.15	0.18		432.33	1.75	0.21	*
62.50	1.31	0.01		1.24	0.10		497.18	1.58	0.21	*
125.00	1.38	0.08		1.19	0.15		571.75	1.88	0.25	*
250.00	1.47	0.34		1.37	0.38		657.52	1.96	0.56	*
500.00	1.66	0.05	*	1.41	0.11		756.14	2.61	0.51	*
1000.0	0.91	0.35		0.74	0.07		869.57	1.91	1.42
2000.0	0.02	0.01		0.52	0.80		1000.0	0.74	0.34

* = significant induction according to Student’s t-test, p<0.05; grey marked: viability >70%; sign.= significant

 

Additional Parameters

Table 6: Additional Parameters

Parameter	Experiment 1	Experiment 2	Experiment 3	Mean	SD
EC1.5	289.06	-	313.33	301.19	17.17
Imax	1.66	1.41	2.61	1.89	0.63
IC30	775.28	736.75	786.88	766.30	26.24
IC50	844.29	1086.52	821.81	917.54	146.77

n.a.=not applicable; [value] excluded due to cytotoxic effects (see discussion)

Acceptance Criteria

Table 7: Acceptance Criteria

Criterion	Range	Exp. 1	pass/fail	Exp. 2	pass/fail	Exp. 3	pass/fail
CV Solvent Control [%]	< 20%	15.4	pass	9.6	pass	13.5	pass
No. of positive control concentration steps with significant luciferase activity induction >1.5	= 1	2.0	pass	2.0	pass	3.0	pass
EC1.5 PC [µM]	± 2 x SD of historical mean	20.57	pass	16.63	pass	15.16	pass
Induction PC at 64 µM	2 .00 < x < 8.00	2.05	pass	6.65	pass	5.27	pass

 

Historical Data

Table 8: Historical Data

Acceptance Criterion	Range	Mean	SD	N
CV Solvent Control	< 20%	11.6	3.4	186
No. of positive control concentration steps with significant luciferase activity induction >1.5	= 1	2.3	0.6	186
EC1.5 PC	7 < x < 34 µM	19.1	5.9	186
Induction PC at 64 µM	2.00 < x < 8.00	3.8	1.4	186

 

 

 

 

Interpretation of results:

Category 1 (skin sensitising) based on GHS criteria

Conclusions:

In this study under the given conditions the test item induced the luciferase activity in the transgenic KeratinoSens™ cell line in at least two independent experiment runs. Therefore, the test item might be considered as sensitiser.
The data generated with this test should be considered in the context of integrated approached such as IATA, combining the result with other complementary information, e.g. derived from in vitro assays addressing other key events of the skin sensitisation AOP.

Executive summary:

The in vitro KeratinoSens™ assay enables detection of the sensitising potential of a test item by addressing the second molecular key event of the adverse outcome pathway (AOP), namely activation of keratinocytes, by quantifying the luciferase activity in the transgenic cell line KeratinoSens™. The luciferase activity, assessed by luminescence measurement, compared to the respective solvent controls is used to support discrimination between skin sensitisers and
non-sensitisers.

In the first experiment, a max luciferase activity (I_max) induction of 1.66 was determined at a test item concentration of 500 µM. The corresponding cell viability determined by MTT staining was 149.8%. Microscopically, a clear cytotoxic effect was observed at the three highest test item concentrations (500, 1000 and 2000 µM) and a slight cytotoxic effect at a test item concentration of 250 µM. The increase in % viability may be caused by a slight MTT reducing potential of the test item, as determined in a MTT interference test. Therefore, the slight induction above the threshold of 1.5 may be caused by the cytotoxic effect.

In the second experiment, no significant luciferase induction > 1.5 was found in the tested concentration range. Therefore, no EC_1.5 value could be calculated.

To verify the results and as the test item induced the gene activity very close to the cytotoxic level in experiment 1, a third experiment with an adapted concentration range and a narrower dose-response analysis with dilution of 1.15-fold was performed to decide if induction is at cytotoxic levels or not.

In the third experiment, a max luciferase activity (I_max) induction of 2.61 was determined at a test item concentration of 756.14 µM. The corresponding cell viability was 87.6 %. The lowest tested concentration with a significant luciferase induction >1.5 (1.55) was found to be 326.90 µM. The corresponding cell viability was >70% (97.9%). The calculated EC_1.5 was <1000 µM (313.33 µM).

A dose response for luciferase activity induction was observed for experiment 1 and experiment 3.

Under the condition of this study the test item is therefore considered positive.

Reference 3

Endpoint:

skin sensitisation, other

Type of information:

(Q)SAR

Adequacy of study:

supporting study

Study period:

2021

Reliability:

1 (reliable without restriction)

Justification for type of information:

Software: QSAR Toolbox 4.5
Version: Data base version 4.5

SMILES OR OTHER IDENTIFIERS USED AS INPUT FOR THE MODEL: CC(C)=CCOCC(O)COCC=C(C)C

for further information see report

Interpretation of results:

Category 1B (indication of skin sensitising potential) based on GHS criteria

Executive summary:

Predicted endpoint: EC3, S M W N, Skin sensitisation; No effect specified; No species specified; No
duration specified; No guideline specified
Predicted value: Positive
Unit/scale: Skin sensitisation II (ECETOC)
Data gap filling method: Read-across analysis
Summary: 
The predicted Log Kow value is outside the domain of applicability. However, the target molecule does
contain structural elements with alerts for at least one key element for dermal sensitization - formation
of radial reactions and protein binding and is within the domain of applicability from at least one
metabolic profiler. Therefore, within the limitations of the available information, the target molecule is
predicted to be positive for dermal sensitization based on read-across.

Reference 4

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2021-03-22 to 2021-06-18

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: Direct Peptide Reactivity Assay (DPRA) for Skin Sensitization Testing, DB-ALM Protocol n°154

Version / remarks:

2013-01-12

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 442C (In Chemico Skin Sensitisation Assays addressing the Adverse Outcome Pathway key event on covalent binding to proteins)

Version / remarks:

adopted 2019-06-18

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

direct peptide reactivity assay (DPRA)

Details of test system:

cysteine peptide, (Ac-RFAACAA-COOH)

lysine peptide (Ac-RFAAKAACOOH)

Details on the study design:

PREPARATION OF TEST SOLUTIONS
- Preparation of the peptide/derivative stock solutions:
20.2 mg cysteine peptide with an amino acid sequence of Ac-RFAACAA were pre-weighed in a vial and dissolved in a defined volume (40.319 mL) of a phosphate buffer with pH 7.5 to reach a concentration of 0.667 mM.
20.55 mg lysine peptide with an amino acid sequence of Ac-RFAAKAA were pre-weighed in a vial and dissolved in a defined volume of ammonium acetate buffer with pH 10.2 (39.672 mL) to reach a concentration of 0.667 mM.
All peptides used for this study were stored at -80 °C and protected from light. Peptides were thawed only immediately prior to use.

- Preparation of the test chemical solutions:
The test item was pre-weighed into a glass vial and was dissolved in acetonitrile, which was found to be the appropriate solvent as determined in a pre-experiment. A stock solution with a concentration of 100 mM was prepared.
The test item solutions were incubated with the cysteine and lysine peptide solutions in glass vials using defined ratios of peptide to test item (1:10 cysteine peptide, 1:50 lysine peptide). The reaction solutions were left in the dark at 25 ± 2.5 °C for 24 ± 2 h before running the HPLC analysis. Reference controls, co-elution controls as well as the positive control were set up in parallel.

- Preparation of the positive controls, reference controls and co-elution controls:
Positive Control:
Cinnamic aldehyde ((2E)-3-phenylprop-2-enal) was solved in acetonitrile and was used as positive
control. A stock concentration of 100 mM was prepared and was included in every assay run for both peptides.
Co-elution Control:
Co-elution controls were set up in parallel to sample preparation but without the respective peptide solution. The controls were used to verify whether a test chemical absorbs at 220 nm and co-elutes with the cysteine or lysine peptide. The co-elution controls were prepared for every test item preparation and the positive control and were included in every assay run for both peptides. Reference Control:
Reference controls (RCs) were set up in parallel to sample preparation in order to verify the validity of the test run.
Reference control A was prepared using acetonitrile in order to verify the accuracy of the calibration curve for peptide quantification. Its replicates were injected in the beginning of each HPLC run.
Reference control B was prepared using acetonitrile in order to verify the stability of the respective peptide over the analysis time. Its replicates were injected in the beginning and in the end of each HPLC run.
Reference control C was set up for the test item and the positive control. RC C for the positive control was prepared using acetonitrile. RC C for the test item was prepared using the respective solvent used to solubilise the test item. The RC C was used to verify that the solvent does not impact the percent peptide depletion (PPD). Additionally, reference control C was used to calculate PPD. The RC C was included in every assay run for both peptides and was injected together with the samples.

INCUBATION
- Incubation conditions: 25 ± 2.5 °C for 24 ± 2 h
- Precipitation noted: No precipitation was observed

PREPARATION OF THE HPLC
- Standard calibration curve for both Cys and Lys:
A standard calibration curve was generated for both, the cysteine and the lysine peptide. Peptide standards were prepared in a solution of 20% acetonitrile: 80% buffer ( v / v ) using phosphate buffer (pH 7.5) for the cysteine peptide and ammonium acetate buffer (pH 10.2) for the lysine peptide (dilution buffer (DB)). A serial dilution of the peptide stock solution (0.667 mM) using the respective DB was performed, resulting in 7 calibration solutions covering the range 0.000 - 0.534 nM.

- Verification of the suitability of the HPLC for test chemical and control substances: A single HPLC analysis for both the cysteine and the lysine peptide should be sufficient for a test chemical when the result is unequivocal. However, in cases of results close to the threshold used to discriminate between positive and negative results (i.e. borderline results), additional testing may be necessary. In situations where the mean percent depletion falls in the range of 3% to 10% for the cysteine, 1:10/lysine, 1:50 prediction model or the cysteine percent depletion falls in the range of 9% to 17% for the cysteine 1:10 prediction model, a second run should be considered, as well as a third one in case of discordant results between the first two runs.

DATA EVALUATION
- Cys and Lys peptide detection wavelength: 220 nm

Vehicle / solvent:

acetonitrile

Positive control:

cinnamic aldehyde

Positive control results:

Turbidity and phase separation was observed for the samples of the co-elution control of the positive control. Samples were not centrifuged prior to the HPLC analysis. Since the depletion range of the positive control were fulfilled, the observed turbidity and phase separation were regarded as not relevant.
The 100 mM stock solution of the positive control (cinnamic aldehyde) showed high reactivity towards the synthetic peptides. The mean depletion of both peptides was 67.16%.

Key result

Group:

test chemical

Parameter:

mean lysine depletion

Value:

3.33 %

At concentration:

100 mM

Vehicle controls validity:

not applicable

Negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Group:

test chemical

Parameter:

mean cystein depletion

Value:

39.28 %

At concentration:

100 mM

Vehicle controls validity:

not applicable

Negative controls validity:

not applicable

Positive controls validity:

valid

Outcome of the prediction model:

positive [in vitro/in chemico]

Other effects / acceptance of results:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for positive control: yes
- Acceptance criteria met for reference controls A to C: yes
- Acceptance criteria met for co-elution controls (Lysine and Cysteine): yes
- Acceptance criteria met for variability between replicate measurements: yes
- Range of historical values if different from the ones specified in the test guideline: see tables below

The in chemico direct peptide reactivity assay (DPRA) enables detection of the sensitising potential of a test item by addressing the molecular initiating event of the adverse outcome pathway (AOP), namely protein reactivity to form haptens, by quantifying the reactivity of test chemicals towards synthetic peptides containing either lysine or cysteine. The percentage depletion value of the cysteine and lysine peptide is used to categorize a substance in one of four reactivity classes to support discrimination between skin sensitisers and non-sensitisers.

In the present study 3-Methyl-3-butenyl 2-methyl-2-propenoate was dissolved in acetonitrile, based on the results of the pre-experiments. Based on a molecular weight of 154.21 g/mol a 100 mM stock solution was prepared. The test item solutions were tested by incubating the samples with the peptides containing either cysteine or lysine for 24 ± 2 h at 25 ± 2.5 °C. Subsequently samples were analysed by HPLC.

All test item solutions were freshly prepared immediately prior to use.

For the 100 mM stock solution of the test item no turbidity or precipitation was observed when diluted with the cysteine peptide solution. After the 24 h ± 2 h incubation period but prior to the HPLC analysis samples were inspected for precipitation, turbidity or phase separation. No precipitation, turbidity or phase separation was observed for any of the samples.

For the 100 mM stock solution of the test item phase separation was observed when diluted with the lysine peptide solution. After the 24 h ± 2 h incubation period but prior to the HPLC analysis samples were inspected for precipitation, turbidity or phase separation. Phase separation was observed for the samples of the test item (including the co-elution control).

Turbidity and phase separation was observed for the samples of the co-elution control of the positive control. Samples were not centrifuged prior to the HPLC analysis. Since the depletion range of the positive control were fulfilled, the observed turbidity and phase separation were regarded as not relevant.

Phase separation in the lysine experiment was observed. Therefore, the given peak areas and corresponding lysine peptide values can only be considered as an estimation of the peptide depletion and cannot be used for evaluation of this test substance.

Sensitising potential of the test item was predicted from the mean peptide depletion of the cysteine peptide by comparing the peptide concentration of the test item treated samples to the corresponding reference control C (RC Cacetonitrile).

The 100 mM stock solution of the test item showed moderate reactivity towards the synthetic peptide. The mean depletion of the cysteine peptide was > 13.89% (39.28%). Based on the prediction model 2 the test item can be considered as positive in this assay.

The 100 mM stock solution of the positive control (cinnamic aldehyde) showed high reactivity towards the synthetic peptides. The mean depletion of both peptides was 67.16%.

The controls confirmed the validity of the study for both, the cysteine and lysine run.

Cysteine and Lysine Values of the Calibration Curve

Sample	Cysteine Peptide		Lysine Peptide
	Peak Area at 220 nm	Peptide Concentration [mM]	Peak Area at 220 nm	Peptide Concentration [mM]
STD1	17.0730	0.5340	14.2900	0.5340
STD2	8.6330	0.2670	7.2390	0.2670
STD3	4.3570	0.1335	3.5950	0.1335
STD4	2.1380	0.0667	1.7760	0.0667
STD5	1.0580	0.0334	0.8830	0.0334
STD6	0.4820	0.0167	0.4370	0.0167
STD7	0.0000	0.0000	0.0000	0.0000

Depletion of the Cysteine Peptide

Cysteine Peptide
Sample	Peak Area at 220 nm	Peptide Conc. [mM]	Peptide Depletion [%]	Mean Peptide Depletion [%]	SD of Peptide Depletion [%]	CV of Peptide Depletion [%]
Positive Control	4.8680	0.1517	69.47
	4.8600	0.1514	69.52	69.73	0.41	0.59
	4.7500	0.1480	70.21
Test Item	10.2800	0.3205	35.53
	9.6700	0.3015	39.36	39.28	3.71	9.45
	9.0960	0.2836	42.96

Depletion of the Lysine Peptide

Lysine Peptide
Sample	Peak Area at 220 nm	Peptide Conc. [mM]	Peptide Depletion [%]	Mean Peptide Depletion [%]	SD of Peptide Depletion [%]	CV of Peptide Depletion [%]
Positive Control	4.6890	0.1747	64.84
	4.6500	0.1732	65.13	64.58	0.72	1.12
	4.8330	0.1801	63.76
Test Item	12.9600	0.4831	2.82
	12.8930	0.4806	3.32	3.33	0.52	15.63
	12.8210	0.4779	3.86

Categorization of the Test Item

Prediction Model	Prediction Model 1 (Cysteine Peptide and Lysine Peptide / Ratio: 1:10 and 1:50)			Prediction Model 2 (Cysteine Peptide / Test Item Ratio: 1:10)
Test Substance	Mean Peptide Depletion [%]	Reactivity Category	Prediction	Mean Peptide Depletion [%]	Reactivity Category	Prediction
Test Item	--	--	--	39.28	Moderate Reactivity	positive
Positive Control	67.16	High Reactivity	positive	69.73	Moderate Reactivity	positive

Acceptance Criteria for Cysteine Peptide

Cysteine Peptide Run
Acceptance Criterion	Range	Value	pass/fail
coefficient of determination	R² > 0.99	0.9999	pass
mean peptide concentration of RC A	0.45 ≤ x ≤ 0.55 mM	0.5060	pass
mean peptide concentration of RC C (PC)	0.45 ≤ x ≤ 0.55 mM	0.4973	pass
mean peptide concentration of RC C (TI)	0.45 ≤ x ≤ 0.55 mM	0.4973	pass
CV of the peak area of RC B	< 15%	1.67	pass
CV of the peak area of RC C (PC)	< 15%	1.20	pass
CV of the peak area of RC C (TI)	< 15%	1.20	pass
mean peptide depletion of the PC	60.8% < x < 100%	69.73	pass
SD of peptide depletion of the PC replicates	< 14.9%	0.41	pass
SD of peptide depletion of the TI replicates	< 14.9%	3.71	pass

Lysine Peptide

Lysine Peptide Run
Acceptance Criterion	Range	Value	pass/fail
coefficient of determination	R² > 0.99	1.0000	pass
mean peptide concentration of RC A	0.45 ≤ x ≤ 0.55 mM	0.4998	pass
mean peptide concentration of RC C (PC)	0.45 ≤ x ≤ 0.55 mM	0.4971	pass
mean peptide concentration of RC C (TI)	0.45 ≤ x ≤ 0.55 mM	0.4971	pass
CV of the peak area of RC B	< 15%	0.31	pass
CV of the peak area of RC C (PC)	< 15%	0.12	pass
CV of the peak area of RC C (TI)	< 15%	0.12	pass
mean peptide depletion of the PC	40.2% < x < 69.0%	64.58	pass
SD of peptide depletion of the PC replicates	< 11.6%	0.72	pass
SD of peptide depletion of the TI replicates	< 11.6%	0.52	pass

Historical Data Cysteine Peptide

Cysteine Peptide
	mean	SD	N
linearity of the calibration curve	0.9996	0.0009	75
mean peptide concentration of reference A [mM]	0.5096	0.0156	75
mean peptide concentration of reference C [mM]	0.5042	0.0151	75
CV of the peak area of control B [%]	1.71	1.45	75
CV of the peak area of control C [%]	1.17	1.05	75
mean peptide depletion of the PC [%]	71.07	4.21	75
SD of peptide depletion of the PC replicates [%]	1.10	4.82	75
SD of peptide depletion of the test items [%]	1.17	2.26	251

Historical Data Lysine Peptide

Lysine Peptide
	mean	SD	N
linearity of the calibration curve	0.9999	0.0001	64
mean peptide concentration of reference A [mM]	0.4989	0.0110	64
mean peptide concentration of reference C [mM]	0.4978	0.0115	64
CV of the peak area of control B [%]	0.77	1.08	64
CV of the peak area of control C [%]	0.59	0.74	64
mean peptide depletion of the PC [%]	60.17	4.23	65
SD of peptide depletion of the PC replicates [%]	1.29	1.44	65
SD of peptide depletion of the test items [%]	1.17	6.97	215

Results of the Reference Controls for the Cysteine Peptide

Cysteine Peptide Run
Sample	Peptide Peak Area				Peptide Concentration [mM]
	PA	Mean	SD	CV [%]	Peptide Concentration	Mean	SD	CV [%]
Reference A 1	16.4220	16.2250	0.1708	1.05	0.5122	0.5060	0.0053	1.05
Reference A 2	16.1350				0.5032
Reference A 3	16.1180				0.5027
Reference B 1	15.5440	15.6530	0.2608	1.67	0.4848	0.4882	0.0081	1.67
Reference B 2	16.1020				0.5022
Reference B 3	[9.758]				-
Reference B 4	15.6450				0.4879
Reference B 5	15.4460				0.4817
Reference B 6	15.5280				0.4843
Reference C 1 (PC solvent)	16.1340	15.9457	0.1905	1.19	0.5032	0.4973	0.0059	1.20
Reference C 2 (PC solvent)	15.9500				0.4974
Reference C 3 (PC solvent)	15.7530				0.4913
Reference C 1 (TI solvent)	16.1340	15.9457	0.1905	1.19	0.5032	0.4973	0.0059	1.20
Reference C 2 (TI solvent)	15.9500				0.4974
Reference C 3 (TI solvent)	15.7530				0.4913

Results of the Reference Controls for the Lysine Peptide

Lysine Peptide Run
Sample	Peptide Peak Area				Peptide Concentration [mM]
	PA	Mean	SD	CV [%]	Peptide Concentration	Mean	SD	CV [%]
Reference A 1	13.3930	13.4103	0.0551	0.41	0.4992	0.4998	0.0021	0.41
Reference A 2	13.4720				0.5021
Reference A 3	13.3660				0.4982
Reference B 1	13.4160	13.3627	0.0413	0.31	0.5001	0.4981	0.0015	0.31
Reference B 2	13.3670				0.4982
Reference B 3	13.4080				0.4998
Reference B 4	13.3210				0.4965
Reference B 5	13.3310				0.4969
Reference B 6	13.3330				0.4970
Reference C 1 (PC solvent)	13.3400	13.3360	0.0154	0.12	0.4972	0.4971	0.0006	0.12
Reference C 2 (PC solvent)	13.3190				0.4964
Reference C 3 (PC solvent)	13.3490				0.4976
Reference C 1 (TI solvent)	13.3400	13.3360	0.0154	0.12	0.4972	0.4971	0.0006	0.12
Reference C 2 (TI solvent)	13.3190				0.4964
Reference C 3 (TI solvent)	13.3490				0.4976

Interpretation of results:

Category 1B (indication of skin sensitising potential) based on GHS criteria

Conclusions:

In this study under the given conditions the test item showed moderate reactivity towards the cysteine peptide. The test item is considered as positive in this assay.
The data generated with this test should be considered in the context of integrated approached such as IATA, combining the result with other complementary information, e.g. derived from in vitro assays addressing other key events of the skin sensitisation AOP.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

IPEMA is classified as sensitizers according to methods outlined in the Guideline on Defined Approaches (DA) for Skin Sensitization (Guideline 497). 

This conclusion was based on a positive response in the Direct Peptide Reactivity Assay (DPRA) (OECD 422C)  and the Kerotinosens® (OECD 422E) assay and applying the 2o3 DA logic. 

The DPRA assay assesses the potential for a test substance to react with proteins and form haptens.  This is Key Element (KE) 1 of the Adverse Outcome Pathway for dermal sensitization.  In the DPRA assay, depletion of target peptides signals chemical reactivity which may lead to immunoreactivity.  Higher depletion levels signifies greater potency.  IPEMA depletion of the cysteine peptide was 39,3%.  This values exceeds the minimum cutoff of 6.38% and has moderate reactivity according to the test guidelines for the DPRA. 

The Kerotinose® assay assesses the potential to activate keratinocytes, KE2 in the AOP for dermal sensitization.  In the Kerotinsens® assay activation of keratinocytes, is  measured by luminescence from luciferase in a transgenic cell line.  The luminescence activity is defined as the concentration of the test substance causing a 50% increase above background.  This concentration is referred to and the EC1.5.  Test substances with lower EC1.5 values are more potent in this assay than those with higher EC1.5 values.  The EC1.5 concentrations for IPEMA was 313 micromolar.  THis is less than the 1000 micromolar cutoff and therefore positive in this assay. 

As a result of these two assays, IPEMA is considered sensitizer. The studies used are all Klimisch Category 1 (reliable without restrictions), internally consistent with the positive and negative controls used and all had clearly positive results (no borderline findings). IPEMA was negative in the h-CLAT assay, however, two positive assays lead to the conclusion that DPNG is a sensitiser.

We supplemented support for these findings by using Read-Across prediction models within OECD Toolbox (v4.5).  By Read-Across, IPEMA is predicted to be sensitizer.  It should be noted that this prediction is limited because the Kow value for IPEMA is outside the range of Kow values for the read-across categories generated for each molecule. However, by using the metabolic profilers in the OECD Toolbox, reactive subgroups were identified that identified relevant structural alerts that were in common with the analogs in the respective categories and a prediction could be made – see attached prediction reports.

To determine potency according to the DA, the combined normalized scores from the DPRA and hCLAT scores are used to discriminate between Cat 1A and 1B.  For h-CLAT, the minimum induction threshold (MIT) is converted to a score from 0 to 3 based on the cutoffs of 10 and 150 μg/ml. For DPRA, the mean percent depletion for the cysteine and lysine peptides, or the cysteine peptide alone, is converted to a score from 0 to 3, based on the threshold values associated with reactivity.  The scores to be assigned are identified in Table 3.1 (page 25 of 54) in the DA.  A positive prediction from the OECD Toolbox application generates a score of 1 and a negative prediction would generate a score of 0.

Based on the updated DIP, a total battery score is assigned into three ranks:

• score of 6-7 is defined as a strong (UN GHS Cat. 1A) sensitiser;


• score of 2-5 as moderate/weak (UN GHS Cat. 1B) sensitiser;


• score of 1 or 0, as not classified (i.e. a non-sensitiser).

The combined normalized scores for IPEMA;

 

Endpoint	IPEMA
h-CLAT (mg/mL)	NC
Normalized Score	0
DPRA (Mean Cysteine & Lyseine % Depletion)	NC
Normalized Score	NC
DPRA (Cysteine % Depletion)	39.28
Normalized Score	2
In Silico Prediction (OECD Toolbox v 4.5)	1
Total Normalized Score	3

 

Based on the normalized scores from the DA, IPEMA is classified as 1B sensitizers.