[1R-(1α,2β,5α)]-1-(isopropyl)-2-methoxy-4-methylcyclohexane

Endpoint summary

Description of key information

Skin Sensitisation (weight of evidence): not sensitising

OECD 429: not sensitising

OECD 442C: inconclusive

OECD 442D: not sensitising

OECD 442E: 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:

16 Mar - 12 Aug 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

The study was conducted according to GLP and OECD Guideline 442C. However, due to the limited solubility of the test substance precipitation was observed after 24 h incubation. Since a negative result was obtained for the test substance in this study, no conclusion on the lack of reactivity can be drawn according to OECD Guideline 442C.

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)

Remarks:

Landesamt für Umwelt, Wasserwirtschaft und Gewerbeaufsicht, Mainz, Germany

Type of study:

direct peptide reactivity assay (DPRA)

Details on the study design:

TEST SYSTEM
- Source: GenScript, Piscataway, US and RS Synthesis, Louisville, US
- Specification of the peptides:
Cysteine- (C-) containing peptide: Ac-RFAACAA-COOH (MW=751.9 g/mol)
Lysine- (K-) containing peptide: Ac-RFAAKAA-COOH (MW=776.2 g/mol)

TEST METHOD
The DPRA is an in chemico method which quantifies the remaining concentration of cysteine- or lysine-containing peptide following 24 h incubation with the test substance at 25 ± 2.5°C. The synthetic peptides contain phenylalanine to aid in the detection. Relative peptide concentration is measured by HPLC with gradient elution and UV detection at 220 nm. Cysteine- and lysine peptide percent depletion values are then calculated and used in a prediction model which allows assigning the test substance to one of four reactivity classes used to support the discrimination between sensitisers and non-sensitisers.

TEST SUBSTANCE PREPARATION
The test substance was prepared as a 100 mM preparation in acetonitrile. The solvent was chosen because the test substance was soluble in the vehicle.

INCUBATION CONDITIONS
- Temperature: 25 ± 2.5 °C
- Duration: 24 ± 2 h

HIGH PERFORMANCE LIQUID CHROMATOGRAPHY
- Specification of the device: Agilent HP 1100 with DAD (Software: Dionex Chromeleon)
- Column: Phenomenex Luna 3µ C18 (2), 100 mm x 2 mm with guard column „Security Guard“ C18, 4 mm x 2 mm
- Analytical balance: Accuracy 0.1 mg
- HPLC mobile phase:
A: 0.1% (v/v) trifluoracetic acid (99+%) in de-ionized water, HPLC grade
B: 0.085% (v/v) trifluoracetic acid (99+%) in acetonitrile, HPLC grade
- Flow: 0.35 mL/min
- Gradient:
Time (min): 0, 10, 11, 13, 13.5, 25
% B: 10, 25, 90, 90, 10, 10
- Wavelength: 220 and 258 nm
- Injection volume: 2 µL

NUMBER OF REPLICATIONS: samples were prepared in triplicates for each peptide

EXPERIMENTAL PROCEDURE
- Test substance solubility: Prior to the assay the solubility of the test substance was tested. A suitable non-reactive, water-miscible solvent which dissolves the test substance completely (no visible precipitation or cloudyness of the test-susbtance preparation) should be used. The preferred solvent was acetonitrile. When not soluble in acetonitrile solutions in water, isopropanol, acetone, propanol, methanol or mixtures of these solvents were tried.
- Preparation of peptide stock solutions: Peptide stock solutions in a concentration of 0.667 mM were prepared in pH 7.5 phosphate buffer (C-containing peptide) or pH 10.2 ammonium acetate buffer (K-containing peptide). The peptide stock solution was used for preparing the calibration samples and the test substance and control samples.
- Preparation of calibration samples: The calibration samples were prepared from the peptide stock solutions in 20% acetonitrile in the respective buffer (= dilution buffer) using serial dilution (please refer to Table 1). The analysis of the calibration samples was started before analysis of the test-substance samples.
- Preparation of test substance samples: The samples were prepared in suitable tubes, capped tightly and incubated at 25°C ± 2.5°C in the dark for 24 ± 2 h. The HLPC analysis of the batch of samples started about 24 h after sample preparation and the analysis time itself did not exceed 30 h.
- Preparation of vehicle controls: Several acetonitrile controls were prepared in triplicates in the same way as the test substance samples described above but with acetonitrile instead of the test substance. Set A was analyzed together with the calibration samples without incubation and serves as a performance control. Another three sets (two sets B and set C) were prepared and incubated with the samples. Sets B were placed at the very start and ending of the sample list and serve as stability control of the peptide over the analysis time. Set C was analyzed with the samples and serves for calculation of the peptide depletion of any chemical formulated in acetonitrile.
- Preparation of the co-elution control: The co-elution control was prepared in the same way as the test substance samples described above but without the peptide. Instead the respective peptide buffer was used. The samples were analyzed together with the calibration samples.

Prior to HPLC analysis the samples were visually investigated for any precipitate that may have formed during the exposure period. As the samples were visually turbid or displayed precipitates they were centrifuged prior to injection into the HPLC in order to remove any unsolved particles.

Positive control: ethylene glycol dimethacrylate

Key result

Parameter:

other: mean C-peptide depletion

Value:

-0.7 %

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Key result

Parameter:

other: mean K-peptide depletion

Value:

-0.97 %

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

OTHER EFFECTS:
No co-elution of test substance and peptides was noticed.

DEMONSTRATION OF TECHNICAL PROFICIENCY:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: The acceptance criteria were met with the exception of the vehicle control A of the K-peptide samples which is not availbale due to a technical error. However, the test is considered to be valid despite the lack of the performance control, as all other control samples (samples B and C) met the acceptance criteria.

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: The test substance was soluble in acetonitrile. The samples of the test substance with the peptides were homogeneous emulsions immediately after preparation. After 24 h precipitates were noticed in the samples of the C-peptide. The samples of the K-peptide were visually homogeneous emulsions after 24 h. Thus all samples were centrifuged prior to HPLC analysis.

COMPARISON WITH HISTORICAL CONTROL DATA
Historic control values of negative and positive controls, gathered over an appropriate time period, demonstrate the reproducibility of results and robustness of the procedures. They are used to derive suitable acceptance criteria for the test system.

Table 6. Mean peptide depletions of cysteine, lysine and both peptides.

	Cysteine-Peptide		Lysine-Peptide		Mean of both depletions (%)
	Mean depletion (%)	SD	Mean depletion (%)	SD
Test substance	-0.70	1.60	-0.97	0.22	0.00
Positive control	55.05	5.56	14.50	1.48	34.77

 

The mean C-peptide depletion, caused by the test substance was determined to be -0.70%.

The mean K-peptide depletion, caused by the test substance was determined to be -0.97%.

 

Negative depletions were considered to be 0 for calculation of the mean peptide depletion, which was thus calculated to be 0.00%.

Interpretation of results:

study cannot be used for classification

Conclusions:

Based on the observed results it was concluded that the test substance shows a minimal chemical reactivity in the Direct Peptide Reactivity Assay under the test conditions chosen. However, it should be noted that due to the limited solubility of the test substance the samples with both peptides were emulsions and that the result could therefore be under-predictive. According to OECD Guideline 442C a negative result should be considered inconclusive in this case.

Executive summary:

It was concluded that Menthylmethylether shows a minimal chemical reactivity in the DPRA under the test conditions chosen. However, it should be noted that due to the limited solubility of the test substance the samples with both peptides were emulsions and that the result could therefore be under-predictive.

Reference 2

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

16 Mar - 12 Aug 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 442E (In Vitro Skin Sensitisation: human Cell Line Activation Test (h-CLAT))

Principles of method if other than guideline:

The study was conducted based on the OECD draft test guideline: Draft Proposal for a New Guideline on In Vitro Skin Sensitization: human Cell Line Activation Test (h-CLAT), accessed on 01 Sep 2014.

GLP compliance:

yes (incl. QA statement)

Remarks:

Landesamt für Umwelt, Wasserwirtschaft und Gewerbeaufsicht, Mainz, Germany

Type of study:

activation of dendritic cells

Details on the study design:

TEST CELL LINE
- Source: American Type Culture Collection, Manassas, USA
- Passage number: from 5 until 30

TEST METHOD
The h-CLAT method is an in vitro method which quantifies changes of cell surface marker expression (CD86 and CD54) on a human cell line, THP-1 cells, following 24 h exposure to the test substance at 37°C. The changes of surface marker expression are measured by flow cytometry following cell staining with fluorescein isothiocyanate (FITC)-labelled antibodies. The relative fluorescence intensity of surface markers compared to solvent control are calculated and used in the prediction model to support the discrimination between sensitisers and non-sensitisers.

TEST SUBSTANCE PREPARATION
The test substance preparations were prepared on a weight per volume basis within 4 h of application. Culture medium was used as vehicle because a good homogeneity of the preparation was achieved.
Concentrations:
Pre-Experiment 1:
0.5, 1, 5, 10, 50, 100, 500, 1000, 2000 and 5000 µg/mL
Pre-Experiment 2:
0.5, 1, 2, 4, 8, 16, 31, 63, 125, 250, 500, 1000, 2000 and 5000 µg/mL
Experiment 1:
82, 99, 119, 142, 171, 205, 246 and 295 µg/mL
Experiment 2:
512, 614, 737, 885, 1062, 1274, 1529 and 1834 µg/mL
Experiment 3 and 4:
33, 40, 48, 57, 69, 83, 99, 119, 143, 171, 206, 247, 296, 356, 427 and 512 µg/mL

POSITIVE SUBSTANCE
1-chloro-2,4-dinitrobenzene
- Concentration: 4.0 µg/mL in 0.2% DMSO in culture medium

NEGATIVE CONTROL
- Substance: lactic acid
- Concentration: 1000 µg/mL in culture medium

ISOTYPE CONTROL
In order to help distinguish non-specific staining from specific antibody staining each test-substance concentration and control is additionally incubated with mouse IgG1.

TEST CELL LINE
THP-1 cells
- Source: American Type Culture Collection, Manassas, USA
- Passage number: from 5 until 30

EXPOSURE CONDITIONS
- Method of application: in medium
- Exposure duration: 24 h

CELL CULTURE CONDITIONS
- Type and identity of media: RPMI 1640 supplemented with 10% FBS, 100 U/mL penicillin, 100 µg/mL streptomycin and 0.05 mM 2-mercaptoethanol
- Temperature (°C): 37
- Humidity (%): >90
-CO2 (%): ca. 5
- Cell counter: Casy 1 (Schärfe System)

NUMBER OF REPLICATIONS: duplicates each in two independent experiments

DETERMINATION OF CYTOTOXICITY
- Method: propidium iodide intercalation

DETERMINATION OF FLUORESCENCE
- Flow cytometer: FC 500 MPL with CXP Software (Beckman Coulter)
- Antibodies: FITC anti-human CD54 (DAKO/DAK-F714301); FITC mouse IgG1 (DAKO/DAK-X092701); FITC anti-human CD86 (BD Pharmingen 555657)
- Blocking solution: 0.01% Globulins Cohn fraction II,III (Sigma) with phosphate buffered saline (without Ca2+ / Mg2+)

Run / experiment:

other: other: Experiment 3

Parameter:

other: EC200 for CD54

Remarks:

in µg/mL

Value:

110

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Run / experiment:

other: other: Experiment 4

Parameter:

other: EC200 for CD54

Remarks:

in µg/mL

Value:

132

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Results of Experiment 1 and 2 are not given in detail due to high cytotoxicity (no evaluable concentration available).

Table 2. Summary of the main experiment (RFI above 150% (CD86) or 200% (CD54) with rel. viability ≥50% are indicated in bold).

	Concentration [µg/mL]	RFI CD86 mean (%)	RFI CD54 mean (%)	Rel. viability (%)
Experiment 3
Test substance	33	145.5	185.7	100.0
	40	112.0	108.0	100.0
	48	110.4	120.1	100.1
	57	101.5	157.5	100.4
	69	100.5	132.4	100.3
	83	106.5	108.1	100.0
	99	91.5	123.0	99.7
	119	93.6	261.0	99.6
	143	115.6	238.3	99.9
	171	120.3	217.8	99.3
	206	108.5	261.9	99.2
	247	110.5	225.2	99.1
	296	116.0	408.9	98.7
	356	103.4	529.2	97.2
	427	107.0	538.9	96.9
	512	221.1	2515.1	34.2
Vehicle control	-	100.0	100.0	100.0
Lactic acid	1000	70.8 71.1	91.1 94.2	100.1 100.0
DNCB	4.0	180.7 208.8	241.8 260.5	90.1 90.0
Experiment 4
Test substance	33	119.9	139.0	92.7
	40	118.5	177.7	92.6
	48	110.1	152.8	92.5
	57	108.4	141.7	92.8
	69	110.2	152.8	92.6
	83	100.1	155.3	92.4
	99	93.2	129.0	92.5
	119	93.4	101.3	92.6
	143	124.5	288.9	100.3
	171	111.7	125.7	100.5
	206	121.7	213.0	100.3
	247	93.8	136.2	100.4
	296	107.3	286.5	99.6
	356	100.6	640.0	94.5
	427	133.9	561.8	90.7
	512	333.7	1037.2	3.7
Vehicle control	-	100.0	100.0	100.0
Lactic acid	1000	77.1 78.7	100.3 96.1	100.1 100.2
DNCB	4.0	217.4 221.2	220.4 246.9	91.4 89.2

DNCB: 1-chloro-2,4-dinitrobenzene

 

The EC200 (the concentration resulting in a RFI of 200) for CD54 was calculated to be 110 µg/mL (Experiment 3) and 132 µg/mL (Experiment 4), respectively.

 

In summary, after 24 h of exposure to test substance CD54 expression was induced in THP-1 cells affording at least 50% viability in at least two independent experiments.

 

TEST-SPECIFIC CONFOUNDING FACTORS

- Precipitation: At concentrations used in the main experiment the test substance was an emulsion in culture medium (2× stock preparations) at 119 µg/mL onwards. Lower concentrations were solved. In 0.2% DMSO in culture medium solutions were noticed at all concentrations (final concentrations). No precipitates were noticed in any concentration after 24 h.

 

RANGE-FINDING/SCREENING STUDIES:

Pre-Experiment 1: The CV75 value (= estimated concentration that affords 75% cell viability) was determined by linear regression from the concentration response curve to be 205 µg/mL.

Pre-Experiment 2: The CV75 value (= estimated concentration that affords 75% cell viability) was determined by linear regression from the concentration response curve to be 1274 µg/mL.

 

ACCEPTANCE CRITERIA

The acceptance criteria were met in all experiments.

 

COMPARISON WITH HISTORICAL CONTROL DATA

The positive and negative and vehicle control data is comparable to historic data.

Interpretation of results:

other: not skin sensitisting based on the key event "activation of dendritic cells"

Conclusions:

Based on the observed results it was concluded that the test substance induces dendritic cell activitation.

Executive summary:

In summary, after 24 hours of exposure to test substance Menthylmethylether CD54 expression was induced in THP-1 cells affording at least 50% viability in at least two independent experiments.

Reference 3

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

16 Mar - 12 Aug 2015

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)

Version / remarks:

2015

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Landesamt für Umwelt, Wasserwirtschaft und Gewerbeaufsicht, Mainz, Germany

Type of study:

activation of keratinocytes

Details on the study design:

TEST CELL LINE
- Cell type: HaCaT cells (human)
- Source: prepared in collaboration with Christoph J. Wruck, RWTH Aachen
- Passage number: from 5 until 15

TEST METHOD
The ARE-Nrf2 luciferase test method makes use of an immortalised adherent cell line derived from HaCaT human keratinocytes stably transfected with a selectable plasmid. The cell line contains the luciferase gene under the transcriptional control of a constitutive promoter fused with an ARE element from a gene that is known to be up-regulated by contact sensitisers. The luciferase signal reflects the activation by sensitisers of endogenous Nrf2 dependent genes. This allows quantitative measurement (by luminescence detection) of luciferase gene induction, using established light producing luciferase substrates, as an indicator of the activity of the Nrf2 transcription factor in cells following 48 h exposure to the test substance at 37 °C.

TEST SUBSTANCE PREPARATION
The test substance preparations were prepared on a weight per volume basis within 4 h of application. DMSO was used as vehicle because a good homogeneity of the preparation was achieved.
Concentrations:
Pre-Experiment:
0.5, 1.0, 5.0, 10, 50, 100, 500, 1000, 2000 µg/mL
Main Experiment:
558, 670, 804, 965, 1157, 1389, 1667 and 2000 µg/mL

POSITIVE SUBSTANCE
ethylene glycol dimethacrylate
- Concentration: 18 µg/mL in 1% DMSO in culture medium

NEGATIVE CONTROL
- Substance: DL-lactic acid
- Concentration: 450 µg/mL in 1% DMSO in culture medium

VEHICLE
DMSO with final concentration of 1%

EXPOSURE CONDITIONS
- Method of application: in medium
- Exposure duration: 48 h

CELL CULTURE CONDITIONS
- Type and identity of media: DMEM with 1% FBS
- Temperature (°C): 37
- Humidity (%): ≥ 90
-CO2 (%): ca. 5
- Cell counter: Casy 1 (Schärfe System)

NUMBER OF REPLICATIONS: triplicates each in three independent experiments

DETERMINATION OF CYTOTOXICITY
- Method: MTT assay
- Spectralphotometer: TriStar² Multimode reader LB 942 (Berthold)

DETERMINATION OF LUMINESCENCE
- Luciferase reagent: SteadyGloLuciferase Assay (Promega)
- Luminometer: TriStar² Multimode reader LB 942 (Berthold)

Positive control substance(s):

yes

Run / experiment:

other: other: Experiment 1

Parameter:

other: EC1.50

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Run / experiment:

other: other: Experiment 2

Parameter:

other: EC1.50

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Run / experiment:

other: other: Experiment 3

Parameter:

other: EC1.50

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Table 2. Summary of the main experiment (concentrations with fold inductions above 1.50 with rel. viability ≥70% and with statistical significance are indicated in bold).

	Concentration [µg/mL]	Fold induction mean	Rel. viability (%) mean	t-test
				p-value	Markers
Experiment 1
Test substance	558	1.15 ± 0.05	101.9 ± 2.0	0.004	**
	670	1.08 ± 0.05	92.6 ± 3.1	0.043	*
	804	1.32 ± 0.12	93.8 ± 4.0	0.014	*
	965	1.37 ± 0.06	94.2 ± 1.0	0.000	**
	1157	1.59 ± 0.05	84.7 ± 2.9	0.000	**
	1389	1.38 ± 0.11	91.5 ± 6.0	0.006	**
	1667	1.67 ± 0.24	94.3 ± 3.2	0.017	*
	2000	0.01 ± 0.03	0.7 ± 0.2	0.000	**
Vehicle control	-	1.00 ± 0.13	100.0 ± 5.9	-	-
EGDMA	18	6.19 ± 0.44	99.1 ± 9.2	0.000	**
Lactic acid	450	0.90 ± 0.08	102.1 ± 4.0	0.032	*
Experiment 2
Test substance	558^1)	-	-	-	-
	670	1.21 ± 0.02	96.4 ± 3.8	0.000	**
	804	1.17 ± 0.15	96.0 ± 2.0	0.094	n.s.
	965	1.18 ± 0.14	99.9 ± 1.4	0.071	n.s.
	1157	1.23 ± 0.14	96.4 ± 4.0	0.042	*
	1389	1.45 ± 0.19	100.0 ± 5.3	0.022	*
	1667	1.38 ± 0.16	99.5 ± 4.5	0.020	*
	2000	-0.07 ± 0.05	0.5 ± 0.6	0.000	**
Vehicle control	-	1.00 ± 0.16	100.0 ± 4.1	-	-
EGDMA	18	8.42 ± 1.01	102.3 ± 4.5	0.000	**
Lactic acid	450	1.04 ± 0.09	108.6 ± 3.7	0.236	n.s.
Experiment 3
Test substance	558	1.36 ± 0.28	115.1 ± 4.5	0.077	n.s.
	670	0.89 ± 0.19	97.3 ± 14.7	0.223	n.s.
	804	1.06 ± 0.09	110.6 ± 3.1	0.186	n.s.
	965	1.03 ± 0.09	106.2 ± 6.2	0.308	n.s.
	1157	1.16 ± 0.09	106.5 ± 7.6	0.037	*
	1389	1.16 ± 0.26	108.7 ± 3.9	0.199	n.s.
	1667	1.36 ± 0.10	109.3 ± 6.1	0.005	**
	2000	0.04 ± 0.02	-11.5 ± 12.7	0.000	**
Vehicle control	-	1.00 ± 0.13	100.0 ± 11.2	-	-
EGDMA	18	7.91 ± 0.26	104.0 ± 8.1	0.000	**
Lactic acid	450	1.05 ± 0.09	107.4 ± 3.4	0.187	n.s.

1) Concentration is not reported due to a technical error

EGDMA: ethylene glycol dimethacrylate

n.s.: no statistical significance

Calculation of an EC 1.50 (the concentration resulting in a 1.50-fold luciferase induction) was not applicable.

 

In summary, after 48 h of exposure to test substance luciferase activity in LuSens cells was not induced in at least two consecutive concentrations with statistical significance affording at least 70% viability in at least two independent experiments.

TEST-SPECIFIC CONFOUNDING FACTORS

- Precipitation: At all concentrations used in the main experiment the test substance was an emulsion in 4% DMSO in the culture medium (4 x stock preparations) and a solution in 1% DMSO in culture medium (final concentrations). No precipitates were noticed in any concentration after 48 h.

 

RANGE-FINDING/SCREENING STUDIES:

The CV75 value (= estimated concentration that affords 75% cell viability) of the test substance was determined by linear regression from the concentration response curve to be 1191 µg/mL. Generally, the highest tested concentration in the main experiment is 1.2³ fold of the CV75 value. However, as this calculated concentration was greater than the maximum concentration tested in the LuSens assay (= 2000 µg/mL) the top dose was chosen 2000 µg/mL.

 

ACCEPTANCE CRITERIA

The acceptance criteria were met in all experiments.

 

COMPARISON WITH HISTORICAL CONTROL DATA

The positive and negative and vehicle control data is comparable to historic data.

Interpretation of results:

other: not skin sensitisting based on the key event "inflammatory response in keratinocytes"

Conclusions:

Based on the observed results it was concluded that the test substance does not have a keratinocyte activating potential under the test conditions chosen.

Executive summary:

In summary, after 48 hours of exposure to test substanceMenthylmethyletherluciferase activity in LuSens cells was not induced in at least two consecutive concentrations with statistical significance affording at least 70% viability in at least two independent experiments.

Reference 4

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

06 July - 17 Aug 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2010

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

Version / remarks:

2003

Deviations:

no

GLP compliance:

yes

Type of study:

mouse local lymph node assay (LLNA)

Species:

mouse

Strain:

other: CBA/Jcr

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Weight at study initiation: 19.0-23.8 g
- Housing: 1 to 5 animals per cage in polycarbonate boxes, with bedding
- Diet: Formulab #5008 (PMI Feeds Inc.), ad libitum
- Water: municipal water supply, ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22-24
- Humidity (%): 44-91
- Air changes (per hr): minimum 10
- Photoperiod (hrs dark / hrs light): 12/12

Vehicle:

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

Concentration:

Test substance: 25, 50 and 100%
Positive control: 100%

No. of animals per dose:

5

Details on study design:

ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: 3H-methyl thymidine incorporation determined by ß-scintillation
- Criteria used to consider a positive response: A stimulation index (SI) was calculated for each group using the activity of each test group divided by the activity of the vehicle control group. The criterion for a positve response is that one or more concentrations of the test substance should elicit a 3-fold or greater increase in isotope incorporation relative to the vehicle control group.

TREATMENT PREPARATION AND ADMINISTRATION: The dorsal surface of both ears was topically treated (25 μL/ear) with the test substance on Day 1. The application was repeated on Day 2 and 3. Three days after the third application an injection of 250 µL phosphate buffered saline (PBS) containing 20 µCi of 3H-methyl thymidine (³HTdR) was made into the tail vein of each experimental mouse. Approximately five hours later, following injection of ³HTdR, the mice were sacrificed and draining auricular lymph nodes were excised and pooled for each individual animal. A single cell suspension was prepared by gentle separation through a 200 mesh stainless steel gauze. The cell suspensions were washed two times with an excess of PBS and precipitated with 5% trichloroacetic acid at 4°C for 18 h. The pellets were resuspended in 1 mL of trichloroacetic acid and transferred to 10 mL of scintillation fluid.

Positive control substance(s):

hexyl cinnamic aldehyde (CAS No 101-86-0)

Statistics:

A one-way parametric analysis of variance (ANOVA) with Dunnett's Multiple Comparisons Test, using GraphPad InStat version 3.06, was performed in DPM counts.
If test groups showed a SI >3, then an extrapolated EC3 value was calculated from SI values at low% and either mid or high% concentrations.
If all three groups show SI > 3, then the formula is: extrapolated EC3 = 2 exp {log2 (c) + [(3 - d)/(b - d)] x [log2 (a) - log2 (c)]}
If at least one concentration shows SI < 3, then the formula is: EC3 = [(3-d)/(b-d)] x (a-c) + c

Positive control results:

The SI value calculated for the positive control was 8.0.

Parameter:

SI

Value:

1.8

Test group / Remarks:

25%

Parameter:

SI

Value:

2.7

Test group / Remarks:

50%

Parameter:

SI

Value:

2.4

Test group / Remarks:

100%

Parameter:

EC3

Remarks on result:

other: As all SI values were < 3, an extrapolated EC3 value was not calculated.

Cellular proliferation data / Observations:

The lymph nodes of each individual animal were pooled and DPM values were measured from the pooled lymph node cell suspensions. Treatment with test substance concentrations of 25 and 50% in acetone/olive oil (4:1) and undiluted test substance resulted in DPM values per mouse of 2003, 3002 and 2631, respectively. The DPM value per mouse of the vehicle control was 1120.

Table 1: Body weights and DPM counts.

	Animal	Body weight (g)		DPM count	Mean DPM ± SD
		Day 1	Day 6
Vehicle Control Group	1	22.1	22.7	1193	1120 ± 453
	2	19.4	20.4	1268
	3	21.7	22.5	1418
	4	21.2	22.1	1394
	5	19.9	21.9	327
Test Group I - 25%	1	20.6	21.8	1731	2003 ± 1062
	2	20.8	23.4	255
	3	22.4	23.5	2697
	4	19.0	20.3	2759
	5	21.3	22.3	2573
Test Group II - 50%	1	22.5	23.0	2959	3002 ± 404
	2	22.7	25.3	2492
	3	20.5	21.8	2888
	4	19.9	20.7	3056
	5	20.9	21.9	3615
Test Group III - 100%	1	22.2	22.7	888	2631 ± 1754
	2	23.8	24.1	2127
	3	23.2	23.5	3188
	4	22.2	23.7	1568
	5	21.2	22.8	5384
Positive Control Group	1	22.4	23.9	17478	8907 ± 6480
	2	22.1	22.4	12977
	3	20.9	21.2	743
	4	21.2	22.4	5880
	5	22.3	22.8	7455

Observations

All animals appeared normal for the duration of the study.

 

Body weight

All test group animals exhibited weight gain during the study

Interpretation of results:

other: CLP/EU GHS criteria not met, no classification required according to Regulation (EC) No 1272/2008

Conclusions:

Under the conditions of the mouse Local Lymph Node Assay the test substance at concentrations of 25, 50 and 100% revealed no sensitising properties.

Executive summary:

Menthyl Methyl Ether produced a stimulation index < 3 in all groups of Test animals, and is not therefore considered a sensitizer (defined as producing a positive response).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

Three in vitro tests and two in vivo LLNA tests are available for the test substance. One LLNA test performed in 2004 was disregarded due to major methodological deficiencies. The study was conducted according to GLP and OECD Guideline 429. However, adequate proficiency with the LLNA was not demonstrated within the study report. The time interval between reliability check and study initiation was greater than 6 months. Furthermore, trypan blue exclusion instead of radioactive labelling was used for the assessment of cell proliferation. For this reason, the recorded lymph node cell counts varied noticeably within the groups and were therefore not conclusive. In conclusion, this study was disregarded and an in vitro/in chemical testing battery was performed in 2015. Since no conclusion on skin sensitisation was possible from the in vitro/ in chemico test method(s), a LLNA test was performed. The details of these tests are described in the following.

in chemico/vitro:

a) Molecular interaction with skin proteins

The reactivity of the test substance towards synthetic cysteine (C)- or lysine (K)-containing peptides was evaluated in the Direct Peptide Reactivity Assay (DPRA) according to OECD Guideline 442C and in compliance with GLP (2015). For this purpose the test substance was incubated with synthetic peptides for ca. 24 hours at ca. 25°C and the remaining non-depleted peptide concentrations were determined by high performance liquid chromatography (HPLC) with gradient elution and UV-detection at 220 nm. The test substance was dissolved at a 100 mM concentration in acetonitrile. Three samples of the test substance were incubated with each peptide in ratios of 1:10 (for C-peptide) or 1:50 (for K-peptide). Additionally triplicates of the concurrent vehicle control were incubated with the peptides. Further, a co-elution control was assessed in order to detect possible interference of the test substance with the peptides. The samples consisted of the test substance, vehicle and the respective peptide buffer but without peptide. Moreover the samples were analyzed by measuring UV absorbance at 258 nm and the area ratio 220 nm / 258 nm was calculated as a measure of peak purity.

The test substance was soluble in acetonitrile. The samples of the test substance with the peptides were homogeneous emulsions immediately after preparation. After 24 hours precipitates were noticed in the samples of the C-peptide. The samples of the K-peptide were visually homogeneous emulsions after 24 hours. Thus all samples were centrifuged prior to HPLC analysis.

The mean C-peptide depletion, caused by the test substance was determined to be -0.70%. The mean K-peptide depletion, caused by the test substance was determined to be -0.97%. Negative depletions were considered to be “zero” for calculation of the mean peptide depletion, which was thus calculated to be 0.00%. No co-elution of test substance and peptides was noticed.

Based on the observed results it was concluded that the test substance shows a minimal chemical reactivity in the DPRA under the test conditions chosen. However, it should be noted that due to the limited solubility of the test substance the samples with both peptides were emulsions and that the result could therefore be under-predictive. Following OECD Guideline 442C a “negative” result should be considered “inconclusive” in this case.

b) Inflammatory response in keratinocytes

The keratinocyte activating potential of the test substance was evaluated in the LuSens assay in accordance with OECD Guideline 442D and in compliance with GLP (2015). For this purpose the test substance was incubated with a luciferase reporter cell line (LuSens cells) for 48 hours at 37°C and antioxidant response element (ARE) dependent luciferase activity was measured in a luminometer. In order to determine the concentrations suitable for the main experiment a pre-test was performed. Cells were exposed to 9 concentrations of the test substance and cytotoxicity was determined by MTT assay. The CV75 value (= estimated concentration that affords 75% cell viability) was determined by linear regression from the concentration response curve. In the main test luciferase activity was measured after 48 hour exposure. In parallel a MTT assay was performed to assess cytotoxicity of the test substance. A total of 3 valid experiments were performed. Relative luciferase fold induction and concurrent relative viability determined in the main experiments show only on two occasions in one experiment (2nd experiment at concentrations 1157 and 1667 µg/mL test substance) an increase above 1.50 with relative viability of ≥70%. Therefore, calculation of an EC1.50 (the concentration resulting in a 1.50-fold luciferase induction) was not applicable. In summary, after 48 hours of exposure to the test substance luciferase activity in LuSens cells was not induced in at least two consecutive concentrations with statistical significance affording at least 70% viability in at least two independent experiments. From this it has to be concluded that test substance does not have a keratinocyte activating potential under the test conditions chosen.

c) Activation of dendritic cells

The potential of the test substance to induce the cell membrane markers CD86 and CD54 expression was evaluated in the Human Cell Line Activation Test (h-CLAT) in accordance with OECD Guideline 442E and in compliance with GLP (2015). For this purpose the test substance was incubated with human pro-monocytic cell line THP-1 for ca. 24 hours at 37°C and membrane markers expression was measured by flow cytometry. In order to determine the concentrations suitable for the main experiment two pre-tests were performed. In the main test after 24 hour exposure THP-1 cells were stained with FITC labeled anti-human-CD86 / anti-human-CD54 antibody and propidium iodide and the fluorescence intensity was analyzed using flow cytometry. A total of 2 valid experiments (experiment 3 and 4) were performed. Relative fluorescence intensity (RFI) and concurrent relative viability showed no increases above 150% for CD86 but in experiment 3 from 119 to 427 µg/mL test substance an increase above 200% for CD54 were observed. In experiment 4 also increased RFI above 200% for CD54 in the concentrations range of 143 to 427 µg/mL were noticed. The EC200 (the concentration resulting in a RFI of 200) for CD54 was calculated to be 110 μg/mL (experiment 3) and 132 μg/mL (experiment 4), respectively. In summary, after 24 hours of exposure to the test substance CD54 expression was induced in THP-1 cells affording at least 50% viability in at least two independent experiments. From this it has to be concluded that the test substance induces dendritic cell activation.

Conclusion:

Based on the results described above and applying the evaluation criteria the test substance does not activate keratinocytes and activates dendritic cells. The peptide reactivity of the test substance could not be conclusively evaluated. Therefore, the skin sensitising potential of the test substance cannot conclusively be predicted based on the results of the in vitro/in chemico studies. A LLNA test was performed in order to finally evaluate the skin sensitising potential of the test substance.

in vivo

The skin sensitising potential of the test substance was evaluated in a LLNA test according to OECD 429 and in compliance with GLP (2015). The study was conducted on five female mice per dose group (25%, 50% and 100%). Each animal received 25 µL of the substance to the dorsum of each ear. Animals were treated once daily for three days. After a two-day rest period, all animals were injected with tritiated methyl-thymidine in the tail vein. Five hours later, animals were sacrificed, and the draining auricular lymph nodes removed and prepared for cell suspension and scintillation counting. A vehicle control (4:1 v/v acetone:olive oil) group and a positive control group (100% alpha-hexylcinnamaldehyde) of five females each were run concurrently, and verified that the test system is reliable. The test substance produced a stimulation index smaller than 3 in all groups and is therefore not considered to be a skin sensitiser.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

The available data on skin sensitisation of the test substance do not meet the criteria for classification according to Regulation (EC) No 1272/2008, and are therefore conclusive but not sufficient for classification.