Phenol, 2,6-di-tert-butyl-, 4-(trans-4-propylcyclohexyl)-

Endpoint summary

Description of key information

Skin sensitisation was studied in vitro using the three key events, namely the DPRA, the Keratinosens and the hClat Assay.

In the DPRA assay, no indication for peptide depletion was shown. Due to precipitation during the experiment the prediction model did not apply and no conclusion could be obtained. In the Keratinosens assay, an increase of the luciferase activity was shown in the transgenic cells, indicating potential for skin sensitisation.

Therefore, the third key event, the hClat assay, was started to verify this result. In this OECD 442E study, there was no upregulation of the expression of the relevant cell surface markers CD86 and CD54 observed and the test item might be considered as non-sensitizer. On the other hand, the logP of the test item exceeds 3.5 and therefore, negative results cannot be considered for the overall interpretation of the endpoint of skin sensitization (cf. OECD 442E).

Given the fact that the results from two of the three key events cannot be used due to technical incompliance, the evaluation for skin sensitisation based on in vitro data is inconclusive.

Therefore a further in vivo study was conducted to conclude on hazard assessment for the endpoint of skin sensitisation. This study was performed according to GLP and the methods applied are fully compliant with OECD TG 429. The test material was negative in this assay and the test material was not a skin sensitiser under the test conditions of this study. According to Commission Regulation (EU) No 286/2011 as well as GHS (Globally Harmonized Classification System) the test material has no labelling requirement for skin sensitisation.

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:

key study

Study period:

Oct 30 2018 - Sep 19, 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

This study was performed according to GLP and the methods applied are fully compliant with OECD TG 429.

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Species:

mouse

Strain:

other: CBA/CaOlaHsd

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Envigo (formed partially from Harlan in September 2015), 5800 AN Venray, The Netherlands
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 8-9 weeks
- Weight at study initiation: (20 ± 2) g
- Housing: Full barrier in an air-conditioned room
- Diet (e.g. ad libitum): Altromin 1324 ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: at least five days
- Indication of any skin lesions: no

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 55 ± 10
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12 / 12

Vehicle:

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

Concentration:

6.25, 12.5, and 25 % (w/v)

No. of animals per dose:

5

Details on study design:

PRE-SCREEN TESTS:
- Compound solubility: Before the initiation of the prescreen test, a solubility test was performed to define the vehicle and the maximum concentration which is technically applicable to the animals.
The maximum technically applicable concentration of the test item in the vehicle was found to be 25% in AOO.

Preparation of the Test Item
Based on the results observed in the prescreen test the following test item concentrations were selected for the main study:
6.25, 12.5%, and 25% (w/v) The preparations were made immediately prior to each dosing.

Prescreen Test
Before the initiation of the prescreen test, a solubility test was performed to define the vehicle and the maximum concentration which is technically applicable to the animals.
The maximum technically applicable concentration of the test item in the vehicle was found to be 25% in AOO (Acetone, Sigma-Aldrich, lot no. STBJ1392, expiry date: 05/08/2020; olive oil highly
refined, Sigma-Aldrich, lot no.BCBW5235, expiry date: 17/06/2020).
In order to determine the highest tolerated and not excessively irritant test concentration a prescreen test was performed which was conducted under the same conditions as the main study, except there was no assessment of lymph node proliferation. The mice were observed daily for any clinical signs of systemic toxicity or local irritation at the application site. Body weights were recorded pre-test and prior to termination. Both ears were observed for erythema and scored. Ear thickness measurements were performed on day 1 (pre-dose), day 3 (approximately 48 hours after the first dose) day 6. Excessive local irritation was indicated by an erythema score ≥ 3 and/or ear swelling of ≥ 25%.

Controls
AOO was used as vehicle and served as negative control.
Positive controls are performed periodically. The recent results are included in the report.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: random
- Criteria used to consider a positive response: cf. OECD 429

Preparation of the Animals
The animals were randomly selected using the validated departmental computerised system E WorkBook (latest version, ID Business Solutions Ltd.).
Identification was ensured by cage number and individual marking (tail).

Clinical Observation
Prior to the application and once a day thereafter all animals were observed in order to detect signs of toxicity, including dermal irritation at site of application.

Weight Assessment
The animals were weighed prior to the application and at the end of the test period (prior to the treatment with 3HTdR).

Dose Groups
3 test groups (3 different concentrations) and 1 negative control group (vehicle) were tested

Topical Application
Each mouse was treated by topical application of 25 µL of the selected solution to the entire dorsal surface of each ear.
Topical applications were performed once daily over three consecutive days. The first treatment day is defined as study day 1

Administration of 3H-Methyl Thymidine
Five days after the first topical application all mice were dosed with 20 µCi 3H-methyl thymidine by intravenous injection (tail vein) of 250 µL of 3H-methyl thymidine, diluted with PBS to a working concentration of 80 µCi/mL.

Preparation of Cell Suspension
Approximately 5 hours after the injection of 3H-methyl thymidine all mice were sacrificed by cervical dislocation. The draining auricular lymph nodes were excised, individually pooled for each animal (2 lymph nodes per animal) and collected in phosphate buffered saline (PBS). A single cell suspension of pooled lymph node cells was prepared by gentle mechanical disaggregation through polyamide gauze (200 mesh size). After washing the gauze with PBS the cell suspension was pelleted in a centrifuge. The supernatant was discarded and the pellets were resuspended with PBS. This washing procedure was repeated.
After the final wash each pellet was resuspended in approx. 1 mL 5% TCA at approx. 4° C for approximately 18 hours for precipitation of macromolecules. Each precipitate was once washed again, resuspended in 1 mL 5% TCA and 7 mL scintillation fluid was added. Then this solution was transferred into scintillation vials and stored at room temperature overnight.

Determination of Incorporated 3H -Methyl Thymidine
The 3H-methyl thymidine – incorporation was measured in a ß-counter and expressed as the number of disintegrations per minute (DPM). Similarly, background 3H-methyl thymidine levels were also measured (5% TCA). Determination of radioactivity was performed individually for each animal.

Evaluation of Results
The proliferative response of lymph node cells was expressed as the number of radioactive disintegrations per minute per lymph node (DPM/NODE) and as the ratio of 3H-methyl thymidine - incorporation into lymph node cells of test group animals relative to that recorded for control group animals (STIMULATION INDEX). Before DPM/NODE values were determined, background values were subtracted.
EC3 values, calculated concentrations which induce stimulation indices of three, are determined by linear interpolation, EC3=c+[(3-d)/(b-d)]x(a c), between two points of the stimulation indices axis, one above (a,b) and one below (c,d) the stimulation index of three. If all measured points are above or below the stimulation index of three, no EC3 value can be stated.
A substance is regarded as a 'sensitiser' in the LLNA if at least one concentration of the test item results in a 3-fold or greater increase in 3H-methyl thymidine - incorporation into lymph node cells of the test group animals, relative to that recorded for the lymph nodes of control group animals (Stimulation Index equal to or greater than 3.0).
On the basis of the test results, the test substance may be classified into one of the following categories in conformity with the criteria given in Commission Regulation (EU) No 286/2011 as well as in GHS - Globally Harmonised System of Classification and Labelling of Chemicals, seventh revised edition, 2017:

Skin sensitiser
Category 1:
A substance is classified as a skin sensitiser
a) if there is evidence in humans that the substance can lead to sensitisation by skin contact in a substantial number of persons, or
b) if there are positive results from an appropriate animal test.
WARNING, exclamation mark. May cause an allergic skin reaction.
Sub-category 1A:
Substances showing a high frequency of occurrence in humans and/or a high potency in animals can be presumed to have the potential to produce significant sensitisation in humans. Severity of reaction may also be considered.
EC3 value ≤ 2%
WARNING, exclamation mark. May cause an allergic skin reaction.
Sub-category 1B:
Substances showing a low to moderate frequency of occurrence in humans and/or a low to moderate potency in animals can be presumed to have the potential to produce sensitisation in humans. Severity of reaction may also be considered.
EC3 value > 2%
WARNING, exclamation mark. May cause an allergic skin reaction.

Positive control substance(s):

other: shared control 1% phenylenediamine

Statistics:

Standard statistical methods have been applied for data processing.

Key result

Parameter:

SI

Value:

0.5

Variability:

0.1

Test group / Remarks:

6.25 %

Key result

Parameter:

SI

Value:

0.9

Variability:

0.1

Test group / Remarks:

12.5 %

Key result

Parameter:

SI

Value:

0.8

Variability:

0.5

Test group / Remarks:

25 %

Cellular proliferation data / Observations:

CELLULAR PROLIFERATION DATA

DETAILS ON STIMULATION INDEX CALCULATION

EC3 CALCULATION

CLINICAL OBSERVATIONS:
All animals survived throughout the test period without showing any significant clinical signs. From Day 4 until the end of the observation period (Day 6), all animals treated
with the test substance showed sticky fur at the application sites.

BODY WEIGHTS
All animals showed the expected weight development, which allows for a weight loss of up to 2 g throughout the study.

Measurement of Ear Thickness

Directly prior to the first application, approximately 48 hours after the first application and shortly before excising the lymph nodes the thickness of both ears from all animals was measured.
The means of the ear thickness per group showed no relevant difference compared to the negative control.

Mean ear thickness on	Day 1	Day 3	Day 6
6.25% test group was	0.16 mm	0.17 mm	0.17 mm
12.5% test group was	0.17 mm	0.17 mm	0.17 mm
25% test group was	0.16 mm	0.17 mm	0.17 mm
negative control group	0.17 mm	0.17 mm	0.17 mm

Conclusion

The EC3 value (derived by linear interpolation) could not be calculated as the stimulation indices of all concentrations were below 3. The results of radioactivity determination were supported by the means of the ear thickness per group, which showed no relevant difference compared to the negative control.

Consequently, according to OECD 429, the test item, as described in this report is expected to have no sensitising properties and therefore, should not be regarded as a dermal sensitiser. According to Commission Regulation (EU) No 286/2011 as well as GHS (Globally Harmonized Classification System) the test item has no obligatory labelling requirement for skin sensitisation and is unclassified.

Interpretation of results:

GHS criteria not met

Conclusions:

The EC3 value (derived by linear interpolation) could not be calculated as the stimulation indices of all concentrations were below 3. The results of radioactivity determination were supported by the means of the ear thickness per group, which showed no relevant difference compared to the negative control.
Consequently, according to OECD 429, the test item, as described in this report is expected to have no sensitising properties and therefore, should not be regarded as a dermal sensitiser. According to Commission Regulation (EU) No 286/2011 as well as GHS (Globally Harmonized Classification System) the test item has no obligatory labelling requirement for skin sensitisation and is unclassified.

Executive summary:

This study was performed according to GLP and the methods applied are fully compliant with OECD TG 429. The test material was a skin sensitiser under the test conditions of this study. Based on the results of the prescreen test the test item was assessed for sensitising properties at concentrations of 6.25, 12.5%, and 25% (w/v), each dissolved in AOO 4:1 (v/v).

The EC3 value (derived by linear interpolation) could not be calculated as the stimulation indices of all concentrations were below 3. The results of radioactivity determination were supported by the means of the ear thickness per group, which showed no relevant difference compared to the negative control.

Consequently, according to OECD 429, the test item, as described in this report is expected to have no sensitising properties and therefore, should not be regarded as a dermal sensitiser. According to Commission Regulation (EU) No 286/2011 as well as GHS (Globally Harmonized Classification System) the test item has no obligatory labelling requirement for skin sensitisation and is unclassified.

Reference 2

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2018-10-26 to 2018-11-17

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))

Qualifier:

according to guideline

Guideline:

other: Direct Peptide Reactivity Assay (DPRA) for Skin Sensitization Testing, DB-ALM Protocol n°154, January 12, 2013

GLP compliance:

yes (incl. QA statement)

Remarks:

Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Germany

Type of study:

other: (in chemico) reactivity against synthetic peptides with a thiol or amino group

Details on the study design:

The in chemico direct peptide reactivity assay (DPRA) enables detection of the sensitising potential of a test item by quantifying the reactivity of test chemicals towards synthetic peptides containing either lysine or cysteine.

Positive control results:

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 65.68%.

Key result

Run / experiment:

other: cysteine run

Parameter:

other: mean peptide depletion [%]

Value:

2.16

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Key result

Run / experiment:

other: lysine run

Parameter:

other: mean peptide depletion [%]

Value:

0

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

Acceptance Criteria

The run meets the acceptance criteria if:
- the standard calibration curve has a r² > 0.99,
- the mean percent peptide depletion (PPD) value of the three replicates for the positive control is between 60.8% and 100% for the cysteine peptide and the maximum standard deviation (SD) for the positive control replicates is < 14.9%,
- the mean percent peptide depletion (PPD) value of the three replicates for the positive control is between 40.2% and 69.0% for the lysine peptide and the maximum SD for the positive control replicates is < 11.6%,
- the mean peptide concentration of the three reference controls A replicates is 0.50 ± 0.05 mM,
- the coefficient of variation (CV) of peptide peak areas for the six reference control B replicates and three reference control C replicates in acetonitrile is < 15.0%.

The results of the test item meet the acceptance criteria if:
- the maximum standard deviation (SD) for the test chemical replicates is < 14.9% for the cysteine percent depletion (PPD),
- the maximum standard deviation (SD) for the test chemical replicates is < 11.6% for the lysine percent depletion (PPD),
- the mean peptide concentration of the three reference controls C replicates in the appropriate solvent is 0.50 ± 0.05 mM.

Both peptide runs and the test item results met the acceptance criteria of the test.

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	17.511	0.534	15.750	0.534
STD2	8.739	0.267	7.788	0.267
STD3	4.338	0.134	3.862	0.134
STD4	2.139	0.067	1.925	0.067
STD5	1.023	0.033	0.965	0.033
STD6	0.438	0.017	0.482	0.017
STD7	0.000	0.000	0.000	0.000

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	4.3460	0.1338	74.31	70.19	3.56	5.08
	5.3780	0.1652	68.21
	5.4020	0.1659	68.07
Test Item	16.5730	0.5054	2.03	2.16	0.21	9.59
	16.5700	0.5053	2.05
	16.5110	0.5035	2.39

 

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	5.7720	0.1969	61.18	61.16	1.52	2.48
	5.5520	0.1894	62.66
	6.0030	0.2047	59.63
Test Item	15.1060	0.5134	0.00	0.00	0.00	n/a
	15.0630	0.5119	0.00
	15.1160	0.5137	0.00

n/a:        not applicable

Prediction Model 1

Cysteine 1:10/ Lysine 1:50 Prediction Model ¹

Mean Cysteine andLysine PPD	Reactivity Class	DPRA Prediction²
0.00% ≤ PPD ≤ 6.38%	No or Minimal Reactivity	Negative
6.38% < PPD ≤ 22.62%	Low Reactivity	Positive
22.62% < PPD ≤ 42.47%	Moderate Reactivity
42.47% < PPD ≤ 100%	High Reactivity

1 The numbers refer to statistically generated threshold values and are not related to the precision of the measurement.

2 DPRA predictions should be considered in the framework of an IATA.

Prediction Model 2

Cysteine 1:10 Prediction Model

Cysteine PPD	ReactivityClass	DPRA Prediction²
0.00% ≤ PPD ≤ 13.89%	No or Minimal Reactivity	Negative
13.89% < PPD ≤ 23.09%	Low Reactivity	Positive
23.09% < PPD ≤ 98.24%	Moderate Reactivity
98.24% < PPD ≤ 100%	High Reactivity

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	1.08	Minimal Reactivity	-	2.16	Minimal Reactivity	-
Positive Control	65.68	High Reactivity	positive	70.19	Moderate Reactivity	positive

Interpretation of results:

study cannot be used for classification

Conclusions:

In this study under the given conditions the test item showed minimal reactivity towards both peptides. Due to the observed precipitation the prediction model does not apply and a prediction cannot be made.
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.

Reference 3

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2018-10-31 to 2018-12-04

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 Test Method)

Qualifier:

according to guideline

Guideline:

other: KeratinoSens™, EURL ECVAM DB-ALM Protocol No. 155, July 1st, 2015

GLP compliance:

yes (incl. QA statement)

Remarks:

Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Germany

Type of study:

activation of keratinocytes

Details on the study design:

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.

Positive control results:

The luciferase activity induced by the positive control at a concentration of 64 µM was between 2 and 8 (2.35 (experiment 1); 3.46 (experiment 2) .

Key result

Run / experiment:

other: 1

Parameter:

other: luciferase activity

Value:

1.98

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Concentration: 7.81 µM

Key result

Run / experiment:

other: 1

Parameter:

other: cell viability [%]

Value:

111.2

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Key result

Run / experiment:

other: 1

Parameter:

other: EC1.5 [µM]

Value:

2.08

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Key result

Run / experiment:

other: 2

Parameter:

other: luciferase activity

Value:

2.41

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Concentration: 7.81 µM

Key result

Run / experiment:

other: 2

Parameter:

other: cell viability [%]

Value:

203.4

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Key result

Run / experiment:

other: 2

Parameter:

other: EC1.5 [µM]

Value:

1.61

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other:

Other effects / acceptance of results:

Acceptance Criteria

The test meets acceptance criteria if:
- the luciferase activity induction of the positive control is statistically significant above the threshold of 1.5 (using a t-test) in at least one of the tested concentrations
- the average induction in the three technical replicates for the positive control at a concentration of 64 µM is between 2 and 8
- the EC1.5 value of the positive control is within two standard deviations of the historical mean
- the average coefficient of variation (CV; consisting of 6 wells) of the luminescence reading for the negative (solvent) control DMSO is <20% in each repetition.

The controls fullfilled the validity criteria of the test.

 Cytotoxicity

Results of the Cytotoxicity Measurement

	Concentration [µM]	Relative Cell Viability [%]*
		Experiment 1	Experiment 2	Mean	SD
Positive Control	4.00	100.0	101.4	100.7	1.0
	8.00	93.9	98.7	96.3	3.4
	16.00	103.1	108.2	105.6	3.6
	32.00	105.8	104.9	105.3	0.6
	64.00	83.1	105.3	94.2	15.7
Test Item	0.98	90.5	133.5	112.0	30.4
	1.95	111.1	139.3	125.2	20.0
	3.91	112.5	171.1	141.8	41.4
	7.81	111.2	203.4	157.3	65.2
	15.63	77.2	81.3	79.3	2.9
	31.25	6.6	20.8	13.7	10.1
	62.50	8.9	9.6	9.3	0.5
	125.00	40.4	40.2	40.3	0.1
	250.00	77.6	169.3	123.4	64.8
	500.00	108.1	196.2	152.2	62.3
	1000.00	98.8	207.2	153.0	76.7
	2000.00	98.4	204.1	151.2	74.8

* Percentage of cell viability relative to the respective solvent control (i.e. set at 100%)

Induction of Luciferase Activity Experiment 1

Experiment 1	Concentration [µM]	Relative Fold Induction^1)					Significance
		Rep. 1	Rep. 2	Rep. 3	Mean	SD
Positive Control	4.00	1.13	0.99	1.07	1.07	0.07
	8.00	1.02	1.34	1.38	1.25	0.20
	16.00	1.37	1.26	1.69	1.44	0.23
	32.00	1.53	1.88	2.01	1.81	0.25	*
	64.00	2.28	2.25	2.52	2.35	0.15	*
Test Item	0.98	1.17	1.61	1.41	1.40	0.22
	1.95	1.31	1.52	1.58	1.47	0.14
	3.91	1.77	2.12	1.93	1.94	0.17	*
	7.81	1.74	2.23	1.96	1.98	0.24	*
	15.63	0.91	1.41	0.94	1.09	0.28
	31.25	0.14	0.21	0.15	0.16	0.04
	62.50	0.13	0.23	0.17	0.18	0.05
	125.00	0.58	0.74	0.57	0.63	0.10
	250.00	1.18	1.17	1.03	1.13	0.08
	500.00	1.05	1.57	1.35	1.32	0.26
	1000.00	1.54	1.56	1.49	1.53	0.04	*
	2000.00	1.56	2.06	2.41	2.01	0.43	*

¹⁾Percentage of fold induction is relative to the respective solvent control (i.e. set at 100%).

* = significant induction according to Student’s t-test, p<0.05

Induction of Luciferase Activity Experiment 2

Experiment 2	Concentration [µM]	Relative Fold Induction^1)					Significance
		Rep. 1	Rep. 2	Rep. 3	Mean	SD
Positive Control	4.00	1.23	1.06	1.00	1.09	0.12
	8.00	1.09	1.14	1.11	1.11	0.03
	16.00	1.28	1.36	1.41	1.35	0.07
	32.00	1.65	1.70	1.58	1.65	0.06	*
	64.00	3.37	3.99	3.02	3.46	0.49	*
Test Item	0.98	1.23	1.28	1.38	1.30	0.08
	1.95	1.34	1.83	1.66	1.61	0.25	*
	3.91	1.61	1.66	2.24	1.84	0.35
	7.81	2.29	2.14	2.80	2.41	0.35	*
	15.63	0.91	1.00	0.59	0.83	0.22
	31.25	0.17	0.23	0.19	0.20	0.03
	62.50	0.19	0.25	0.11	0.18	0.07
	125.00	0.41	0.88	0.34	0.54	0.29
	250.00	1.63	1.41	1.49	1.51	0.11	*
	500.00	2.17	1.88	1.72	1.92	0.23	*
	1000.00	2.34	1.88	2.12	2.11	0.23	*
	2000.00	2.16	2.23	2.43	2.27	0.14	*

¹⁾Percentage of fold induction is relative to the respective solvent control (i.e. set at 100%).

* = significant induction according to Student’s t-test, p<0.05

Additional Parameters

Parameter	Experiment 1	Experiment 2	Mean	SD
EC1.5[µM]	2.08	1.61	1.85	0.33
Imax	2.01	2.41	2.21	0.28
IC30[µM]	n.a.	n.a.	n.a.	n.a.
IC50[µM]	n.a.	n.a.	n.a.	n.a.

n.a.: not applicable

Acceptance Criteria

Criterion	Range	Experiment 1	pass/fail	Experiment 2	pass/fail
CV Solvent Control PC (1% DMSO)	< 20%	13.7	pass	10.8	pass
CV Solvent Control TI (1% THF)	< 20%	14.2	pass	12.8	pass
No. of positive control concentration steps with significant luciferase activity induction >1.5	≥ 1	2.0	pass	2.0	pass
EC1.5 PC	± 2 x SD of historical mean	18.54	pass	24.09	pass
InductionPC at 64 µM	2.00 < x < 8.00	2.35	pass	3.46	pass

Historical Data

Acceptance Criterion	Range	Mean	SD	N
CV Solvent Control	< 20%	11.6	3.5	96
No. of positive control concentration steps with significant luciferase activity induction >1.5	≥ 1	2.4	0.6	96
EC1.5 PC	± 2 x SD of historical mean	18.5	6.0	96
InductionPC at 64 µM	2.00 < x < 8.00	3.8	1.5	96

Interpretation of results:

other: The data generated with this test should be considered in the context of integrated approached such as IATA.

Conclusions:

In this study under the given conditions the test item did induce the luciferase activity in the transgenic KeratinoSens™ cell line in at least two independent experiment runs. Therefore, the test item can 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.

Reference 4

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2019-01-21 to 2019-02-26

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: OECD Guidelines for Testing of Chemicals, No. 442E: In Vitro Skin Sensitisation assays addressing the Key Event on activation of dendritic cells on the Adverse Outcome Pathway for Skin Sensitisation”, adopted 25 June 2018

Qualifier:

according to guideline

Guideline:

other: Human Cell Line Activation Test (h-CLAT) for Skin Sensitisation, DB-ALM Protocol n°158, July 1st, 2015

GLP compliance:

yes (incl. QA statement)

Remarks:

Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Germany

Type of study:

activation of dendritic cells

Details on the study design:

The in vitro human cell line activation test (h-CLAT) enables detection of the sensitising potential of a test
item by addressing the third molecular key event of the adverse outcome pathway (AOP), namely
dendritic cell activation, by quantifying the expression of the cell surface markers CD54 and CD86 in
the human monocytic cell line THP-1. The expression of the cell surface markers compared to the
respective solvent controls is used to support discrimination between skin sensitisers and
non-sensitisers.

Positive control results:

The positive control (DNCB) led to an upregulation of the expression of CD54 and CD86 in both
experiments. The threshold of 150% for CD86 (227% experiment 1; 280% experiment 2) and
200% for CD54 (341% experiment 1; 261% experiment 2) were clearly exceeded.

Key result

Run / experiment:

other: 1

Parameter:

other: relative fluorescence intensity CD86 [%]

Value:

123

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Concentration: 482.25 µg/mL

Key result

Run / experiment:

other: 1

Parameter:

other: relative fluorescence intensity CD54 [%]

Value:

130

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Concentration: 1000 µg/mL

Key result

Run / experiment:

other: 2

Parameter:

other: relative fluorescence intensity CD86 [%]

Value:

114

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Concentration: 482.25 µg/mL

Key result

Run / experiment:

other: 2

Parameter:

other: relative fluorescence intensity CD54 [%]

Value:

89

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Concentration: 694.44 µg/mL

Other effects / acceptance of results:

Acceptance criteria:

The test meets acceptance criteria if:
• the cell viability of the solvent controls is >90%,
• the cell viability of at least four tested doses of the test item in each run is >50%,
• the RFI values of the positive control (DNCB) is ≥150% for CD86 and ≥200% for CD54 at a cell viability of >50%,
• the RFI values of the solvent control is not ≥150% for CD86 and not ≥200% for CD54,
• the MFI ratio of CD86 and CD54 to isotype IgG1 control for the medium and DMSO control, is >105%.

The test mets the acceptance criteria.

Results of the Cell Batch Activation Test (Batch 3)

Sample	Concentration [µg/mL]	CD86			CD54			Activated	Pass /Fail
		Cell Viability [%]	RFI	Threshold OECD TG 442E	Cell Viability [%]	RFI	Threshold OECD TG 442E	yes/no
DNCB	4 µg/mL	85.4	306	>150	84.7	220	>200	yes	pass
NiSO4	100 µg/mL	81.8	251	>150	80.9	363	>200	yes	pass
LA	1000 µg/mL	94.0	93	≤150	93.9	93	≤200	no	pass

Results of the Cell Batch Activation Test (Batch 4)

Sample	Concentration [µg/mL]	CD86			CD54			Activated	Pass /Fail
		Cell Viability [%]	RFI	Threshold OECD TG 442E	Cell Viability [%]	RFI	Threshold OECD TG 442E	yes/no
DNCB	4 µg/mL	84.5	342	>150	85.8	334	>200	yes	pass
NiSO4	100 µg/mL	81.7	280	>150	81.7	578	>200	yes	pass
LA	1000 µg/mL	95.3	90	≤150	95.6	123	≤200	no	pass

Results of the Dose Finding Assay

Sample		Experiment 1
		Concentration applied [µg/mL]	Cell Viability [%]
Medium Control	--	--	94.50
Solvent Control	THF	--	94.50
Test Material	C8	7.81	95.30
	C7	15.63	95.20
	C6	31.25	95.20
	C5	62.50	95.10
	C4	125.00	95.00
	C3	250.00	95.80
	C2	500.00	94.70
	C1	1000.00	95.00
Calculated CV75 [µg/mL]		No CV75
Mean CV75 [µg/mL]		No CV75
SD CV 75 [µg/mL]		No SD

CD54 and CD86 Expression Experiment 1

Sample	Conc. [μg/mL]	Cell Viability [%]			Mean Fluorescence Intensity			corrected Mean Fluorescence Intensity		Relative Fluorescence Intensity (RFI)		Ratio Isotype IgG1 to [%]
		CD86	CD54	Isotype IgG1	CD86	CD54	Isotype IgG1	CD86	CD54	CD86	CD54	CD86	CD54
Medium Control	-	95.8	95.7	95.6	1304	956	572	732	384	84	132	228	167
Solvent Control 2 (THF)	0.20%	96.0	96.2	95.7	1564.0	860.0	528.0	1036	332	100	100	296	163
Solvent Control 1 (DMSO)	0.20%	96.8	96.1	96.5	1435	856	565	870	291	100	100	254	152
DNCB	4.00	90.0	89.9	89.9	2627	1644	653	1974	991	227	341	402	252
Test Material	1000	95.6	95.1	95.3	1471	1040	610	861	430	83	130	241	170
	833.33	95.6	95.5	95.7	1644	822	534	1110	288	107	87	308	154
	694.44	96.2	96.4	96.4	1641	934	667	974	267	94	80	246	140
	578.70	95.9	96.1	96.4	1716	913	590	1126	323	109	97	291	155
	482.25	95.1	95.0	94.8	1822	947	543	1279	404	123	122	336	174
	401.88	95.1	95.6	95.4	1688	851	562	1126	289	109	87	300	151
	334.90	95.1	95.3	95.4	1753	926	561	1192	365	115	110	312	165
	279.08	95.5	95.8	95.9	1621	978	574	1047	404	101	122	282	170

CD54 and CD86 Expression Experiment 2

Sample	Conc. [μg/mL]	Cell Viability [%]			Mean Fluorescence Intensity			corrected Mean Fluorescence Intensity		Relative Fluorescence Intensity (RFI)		Ratio Isotype IgG1 to [%]
		CD86	CD54	Isotype IgG 1	CD86	CD54	Isotype IgG1	CD86	CD54	CD86	CD54	CD86	CD54
Medium Control	-	93.2	94.0	92.9	2600	1206	707	1893	499	117	102	368	171
Solvent Control 2 (THF)	0.20%	94.2	94.3	94.1	2098	1313	728	1370	585	100	100	288	180
Solvent Control 1 (DMSO)	0.20%	94.4	94.9	89.4*	2305	1177	690	1615	487	100	100	334	171
DNCB	4.0	80.4	81.3	81.2	5228	1983	714	4514	1269	280	261	732	278
Test Material	1000.00	94.10	95.40	95.20	2075	1158	724	1351	434	99	74	287	160
	833.33	95.30	95.20	95.40	1895	1179	662	1233	517	90	88	286	178
	694.44	95.30	95.50	95.10	2038	1184	664	1374	520	100	89	307	178
	578.70	95.10	95.00	95.10	2000	1179	675	1325	504	97	86	296	175
	482.25	95.80	95.30	95.60	2236	1114	671	1565	443	114	76	333	166
	401.88	95.80	96.10	95.70	1690	1038	683	1007	355	74	61	247	152
	334.90	95.90	96.00	96.20	1720	1069	672	1048	397	77	68	256	159
	279.08	96.00	95.70	95.70	2029	1087	698	1331	389	97	67	291	156

Acceptance Criteria

Acceptance Criterion	Range	Experiment 1			pass/fail	Experiment 2			pass/fail
cell viability solvent controls [%]	>90	95.6	-	96.8	pass	89.4	-	94.9	fail*
number of test dosed with viability >50% CD86	≥4	8			pass	8			pass
number of test dosed with viability >50% CD54	≥4	8			pass	8			pass
number of test dosed with viability >50% IgG1	≥4	8			pass	8			pass
RFI of positive control of CD86	≥150	227			pass	280			pass
RFI of positive control of CD54	≥200	341			pass	261			pass
RFI of DMSO solvent control of CD86	<150	119			pass	85			pass
RFI of DMSO solvent control of CD54	<200	76			pass	98			pass
RFI of THF solvent control of CD86	<150	142			pass	72			pass
RFI of THF solvent control of CD54	<200	86			pass	117			pass
MFI ratio CD86/IgG1 for medium control [%]	>105	228			pass	368			pass
MFI ratio CD86/IgG1 for THF control [%]	>105	254			pass	334			pass
MFI ratio CD86/IgG1 for DMSO control [%]	>105	296			pass	288			pass
MFI ratio CD54/IgG1for medium control [%]	>105	167			pass	171			pass
MFI ratio CD54/IgG1for DMSO control [%]	>105	152			pass	171			pass
MFI ratio CD54/IgG1for THF control [%]	>105	163			pass	180			pass

Historical Data

Criterion	mean	SD	N
cell viability solvent controls [%]	96.2	1.6	216
number of test doses with viability >50%	-	-	555
RFI of positive control of CD86	358.9	90.1	36
RFI of positive control of CD54	435.7	285.8	36
RFI of solvent control (THF) of CD86	102.9	17.4	36
RFI of solvent control (THF) of CD54	102.0	15.4	36
MFI ratio IgG1/CD86 for medium control [%]	285.6	124.3	36
MFI ratio IgG1/CD86 for THF control [%]	270.8	105.0	36
MFI ratio IgG1/CD54 for medium control [%]	308.6	137.1	36
MFI ratio IgG1/CD54 for THF control [%]	158.2	28.4	36

Interpretation of results:

study cannot be used for classification

Conclusions:

In this study under the given conditions the test item did not upregulate the expression of the cell surface markers in at least two independent experiment runs. Since the log KOW is higher than 6.5, the result has to be considered as inconclusive.
The data generated with this method may be not sufficient to conclude on skin sensitisation potential of chemicals and should be considered in the context of integrated approach such as IATA.

Executive summary:

In the present study the test material was dissolved in THF. For the dose finding assay stock solutions with concentrations ranging from 500 mg/mL to 3.91 mg/mL were prepared by a serial dilution of 1:2. Cells were incubated with the test item for 24 h at 37°C. After exposure cells were stained with propidium iodide and cell viability was measured by FACS analysis.

Due to a lack of cytotoxicity, no CV75 could be derived. Therefore, the main experiment was performed covering the following concentration steps:

1000, 833.33, 694.44, 578.70, 482.25, 401.88, 334.90, 279.08 µg/mL

In all experiments precipitation of the test item was observed for all concentration steps when mixing the test item stock solutions with cell culture medium.

Cells were incubated with the test item for 24 h at 37°C. After exposure cells were stained and cell surface markers CD54 and CD86 were measured by FACS analysis. Cell viability was assessed in parallel using propidium iodide staining.

No cytotoxic effects were observed for the cells treated with the test item. Relative cell viability at the highest test item concentration was 95.6% (CD86), 95.1% (CD54) and 95.3% (isotype IgG1 control) in the first experiment and 94.10% (CD86), 95.40% (CD54) and 95.20% (isotype IgG1 control) in the second experiment.

The expression of the cell surface marker CD86 was not upregulated above the threshold of 150% in any of the experiments. The expression of the cell surface marker CD54 was not upregulated above the threshold of 200% in any of the experiments.

In spite of the results, the test item has to be considered as inconclusive because of the log K_OW> 6.5.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

According to Regulation (EC) No 1272/2008 as well as GHS (Globally Harmonized Classification System) the test material has no labelling requirement for skin sensitisation.