2-(4-Bromphenyl)-5-butyl-1,3-dioxane, trans-

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 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 obligatory labelling requirement for skin sensitisation and is classified into Category 1B.

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:

May 19 - Sep 26, 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: Harlan Laboratories GmbH, 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:

15.5, 25, and 50 % (v/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 50% 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:
12.5%, 25% and 50% (v/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 50% in AOO (Acetone, Sigma-Aldrich, lot no. STBJ1392, expiry date: 01/06/2020; olive oil highly refined).
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:

0.9

Variability:

0.4

Test group / Remarks:

12.5 %

Key result

Parameter:

SI

Value:

1.9

Variability:

0.3

Test group / Remarks:

25 %

Key result

Parameter:

SI

Value:

3.9

Variability:

0.8

Test group / Remarks:

50 %

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 signs of systemic toxicity or excessive local irritation. Minor local effects such as sticky or scrubby fur were observed in all animals treated with the test substance from day 2 (second dosing) until day 6 (last day of observation). Temporarily, all animals of the 25% test group showed desquamation on day 2.

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
12.5% test group was	0.16 mm	0.17 mm	0.17 mm
25% test group was	0.17 mm	0.17 mm	0.17 mm
50% 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) was calculated to be at a test item concentration of 38.81%.
The measurement of the ear thickness did not reveal any relevant increase of the ear thickness in any dosage group.
Consequently, according to OECD 429 solutions or preparations containing more than 38.81% test material are expected to have a stimulation index of >3 and are therefore considered to be dermal sensitisers.
According to Commission Regulation (EU) No 286/2011 as well as GHS (Globally Harmonized Classification System) the test material has obligatory labelling requirement for skin sensitisation and is classified into Category 1B.

Interpretation of results:

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

Conclusions:

The EC3 value (derived by linear interpolation) was calculated to be at a test item concentration of 38.81%. The measurement of the ear thickness did not reveal any relevant increase of the ear thickness in any dosage group. Consequently, according to OECD 429 solutions or preparations containing more than 38.81% test material are expected to have a stimulation index of >3 and are therefore considered to be dermal sensitisers. According to Commission Regulation (EU) No 286/2011 as well as GHS (Globally Harmonized Classification System) the test material has obligatory labelling requirement for skin sensitisation and is classified into Category 1B.

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 12.5%, 25% and 50% (w/v), each dissolved in AOO 4:1 (v/v).

There was no mortality and there were no significant clinical observations or effects on body weights. The EC3 value (derived by linear interpolation) was calculated to be at a test item concentration of 38.81%. The measurement of the ear thickness did not reveal any relevant increase of the ear thickness in any dosage group. Consequently, according to OECD 429 solutions or preparations containing more than 38.81% test material are expected to have a stimulation index of >3 and are therefore considered to be dermal sensitisers. According to Commission Regulation (EU) No 286/2011 as well as GHS (Globally Harmonized Classification System) the test material has obligatory labelling requirement for skin sensitisation and is classified into Category 1B.

Reference 2

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2018-04-18 to 2018-04-25

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:

direct peptide reactivity assay (DPRA)

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 68.60%.

Key result

Run / experiment:

other: cysteine run

Parameter:

other: mean peptide depletion [%]

Value:

1.54

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	16.6350	0.5340	14.9940	0.5340
STD2	8.4320	0.2670	7.5600	0.2670
STD3	4.1790	0.1335	3.7750	0.1335
STD4	2.0190	0.0667	1.8800	0.0667
STD5	0.9840	0.0334	0.9440	0.0334
STD6	0.4630	0.0167	0.4610	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	3.7430	0.1205	76.55	73.13	2.97	4.06
	4.5380	0.1459	71.57
	4.5860	0.1474	71.27
Test Item	15.2670	0.4887	4.36	1.54	2.44	158.75
	16.0240	0.5129	0.00
	15.9210	0.5096	0.26

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	8.0020*			64.06	0.41	0.64
	5.0950	0.1809	63.77
	5.0140	0.1781	64.35
Test Item	14.2780	0.5076	0.00	0.00	0.00	n.a.
	14.3850	0.5114	0.00
	14.1430	0.5028	0.00

n.a.:       not applicable

* This replicate was excluded from further evaluation as the chromatogram showed several small peaks which do not correlate with the expected lysine peak. An integration of the lysine peak was not possible.

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	0.77	Minimal Reactivity	-	1.54	Minimal Reactivity	-
Positive Control	68.60	High Reactivity	sensitiser	73.13	Moderate Reactivity	sensitiser

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 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.

Executive summary:

In the present study the test item was dissolved in acetonitrile, based on the results of the pre-experiments.

Based on a molecular weight of 299 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.

For the 100 mM stock solution of the test item turbidity 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. Precipitation was observed for the samples of the test item and for the co-elution control of the test item. Samples were not centrifuged prior to the HPLC analysis.

For the 100 mM stock solution of the test item no turbidity or precipitation 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. Precipitation was observed for the samples of the test item and for the co-elution control of the test item. Samples were centrifuged prior to the HPLC analysis. Phase separation was observed for the samples of the positive control (including the co-elution control). Samples were not centrifuged prior to the HPLC analysis.

Since the acceptance criteria for the depletion range of the positive control were fulfilled, the phase separation was regarded as insignificant.

No co-elution of the test item with the peptide peaks was observed.

The stock solution of the test item showed minimal reactivity towards the synthetic peptides. The mean depletion of both peptides was ≤ 6.38% (0.77%). Since precipitation was observed for both peptide samples, a test item concentration of 100 mM as well as the full contact of peptide and test item is not guaranteed. According to the evaluation criteria in the guideline, no firm conclusion on the lack of reactivity should be drawn from a negative result if a test chemical is tested in concentration < 100 mM. Therefore, no prediction can be made.

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 68.60%.

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

 

Reference 3

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2018-04-18 to 2018-06-18

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 (3.98 (experiment 1); 3.21 (experiment 2)).

Key result

Run / experiment:

other: 1

Parameter:

other: luciferase activity

Value:

3.55

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Concentration: 31.25 µM

Key result

Run / experiment:

other: 1

Parameter:

other: cell viability [%]

Value:

83.4

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Key result

Run / experiment:

other: 1

Parameter:

other: EC1.5 [µM]

Value:

2.47

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Key result

Run / experiment:

other: 2

Parameter:

other: luciferase activity

Value:

2.76

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Concentration: 31.25 µM

Key result

Run / experiment:

other: 2

Parameter:

other: cell viability [%]

Value:

70.1

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Key result

Run / experiment:

other: 2

Parameter:

other: EC1.5 [µM]

Value:

2.04

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

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.

Table 1: Results of the Cytotoxicity Measurement

	Concentration [µM]	Cell Viability [%]
		Experiment 1	Experiment 2	Mean	SD
Solvent Control	-	100.0	100.0	100.0	0.0
Positive Control	4.00	101.8	97.8	99.8	2.8
	8.00	101.2	96.6	98.9	3.2
	16.00	104.9	97.1	101.0	5.5
	32.00	108.3	97.6	103.0	7.6
	64.00	104.1	88.9	96.5	10.7
Test Item	0.98	92.6	100.3	96.5	5.4
	1.95	102.3	102.6	102.4	0.2
	3.91	96.0	98.7	97.3	1.9
	7.81	95.3	89.0	92.1	4.4
	15.63	90.7	86.1	88.4	3.2
	31.25	83.4	70.1	76.7	9.4
	62.50	24.0	15.2	19.6	6.2
	125.00	2.0	2.5	2.3	0.3
	250.00	8.7	8.5	8.6	0.1
	500.00	54.7	32.2	43.4	15.9
	1000.00	58.0	51.0	54.5	4.9
	2000.00	56.1	58.5	57.3	1.7

Table 2: Induction of Luciferase Activity Experiment 1

Experiment 1	Concentration [µM]	Fold Induction					Significance
		Rep. 1	Rep. 2	Rep. 3	Mean	SD
Solvent Control	-	1.00	1.00	1.00	1.00	0.00
Positive Control	4.00	1.08	1.05	1.07	1.07	0.01
	8.00	1.54	1.82	1.43	1.60	0.20	*
	16.00	1.96	1.69	1.57	1.74	0.20	*
	32.00	3.15	2.52	2.44	2.70	0.39	*
	64.00	4.34	4.16	3.45	3.98	0.47	*
Test Item	0.98	1.13	1.84	1.39	1.45	0.36
	1.95	1.38	1.40	1.46	1.41	0.04
	3.91	1.68	1.73	1.83	1.75	0.07	*
	7.81	2.22	2.51	2.40	2.38	0.15	*
	15.63	2.78	2.94	2.84	2.85	0.08	*
	31.25	3.65	3.39	3.60	3.55	0.14	*
	62.50	2.49	2.49	3.23	2.74	0.43	*
	125.00	1.00	0.72	0.78	0.84	0.15
	250.00	0.78	0.61	0.56	0.65	0.11
	500.00	1.00	1.17	1.25	1.14	0.13
	1000.00	1.12	1.16	1.28	1.19	0.09
	2000.00	1.27	1.44	1.44	1.38	0.10

Table 3: Induction of Luciferase Activity Experiment 2

Experiment 2	Concentration [µM]	Fold Induction					Significance
		Rep. 1	Rep. 2	Rep. 3	Mean	SD
Solvent Control	-	1.00	1.00	1.00	1.00	0.00
Positive Control	4.00	1.12	1.12	1.24	1.16	0.07
	8.00	1.21	1.26	1.10	1.19	0.08
	16.00	1.46	1.46	1.38	1.43	0.05
	32.00	1.93	1.91	1.71	1.85	0.12	*
	64.00	2.75	3.08	3.80	3.21	0.54	*
Test Item	0.98	1.30	1.37	1.54	1.41	0.13
	1.95	1.49	1.44	1.53	1.49	0.04
	3.91	1.72	1.66	1.85	1.74	0.09	*
	7.81	1.92	1.90	2.11	1.98	0.11	*
	15.63	1.92	2.13	2.28	2.11	0.18	*
	31.25	2.77	2.29	3.24	2.76	0.47	*
	62.50	1.67	1.45	1.79	1.63	0.17	*
	125.00	0.46	0.44	0.33	0.41	0.07
	250.00	0.29	0.23	0.30	0.27	0.04
	500.00	0.69	0.70	0.86	0.75	0.09
	1000.00	0.84	0.82	0.94	0.87	0.06
	2000.00	0.80	0.81	1.02	0.88	0.13

Table 4: Induction of Luciferase Activity – Overall Induction

	Concentration [µM]	Fold Induction				Significance
		Experiment 1	Experiment 2	Mean	SD
Solvent Control	-	1.00	1.00	1.00	0.00	-
Positive Control	4.00	1.07	1.16	1.11	0.07
	8.00	1.60	1.19	1.39	0.29
	16.00	1.74	1.43	1.59	0.22
	32.00	2.70	1.85	2.28	0.60
	64.00	3.98	3.21	3.60	0.55	*
Test Item	0.98	1.45	1.41	1.43	0.03
	1.95	1.41	1.49	1.45	0.05
	3.91	1.75	1.74	1.75	0.00	*
	7.81	2.38	1.98	2.18	0.28	*
	15.63	2.85	2.11	2.48	0.53
	31.25	3.55	2.76	3.16	0.55	*
	62.50	2.74	1.63	2.19	0.78
	125.00	0.84	0.41	0.62	0.30
	250.00	0.65	0.27	0.46	0.27
	500.00	1.14	0.75	0.95	0.28
	1000.00	1.19	0.87	1.03	0.23
	2000.00	1.38	0.88	1.13	0.36

Table 5: Additional Parameters

Parameter	Experiment 1	Experiment 2	Mean	SD
EC1.5[µM]	2.47	2.04	2.25	0.30
Imax	3.55	2.76	3.16	0.55
IC30[µM]	38.30	31.29	34.80	4.96
IC50[µM]	n/a	n/a	n/a	n/a

Table 6: Acceptance Criteria

Criterion	Range	Experiment 1	pass/fail	Experiment 2	pass/fail
CV Solvent Control	< 20%	11.6	pass	8.9	pass
No. of positive control concentration steps with significant luciferase activity induction >1.5	≥ 1	4.0	pass	2.0	pass
EC1.5 PC	7 < x < 34 µM	7.28	pass	18.59	pass
Induction PC at 64 µM	2.00 < x < 8.00	3.98	pass	3.21	pass

Table 7: Historical Data

Acceptance Criterion	Range	Mean	SD	N
CV Solvent Control	< 20%	11.3	3.3	41
No. of positive control concentration steps with significant luciferase activity induction >1.5	≥ 1	2.3	0.6	41
EC1.5 PC	7 < x < 34 µM	20.4	6.7	41
Induction PC at 64 µM	2.00 < x < 8.00	3.3	1.1	41

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 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:

In the present study the test material was dissolved in DMSO. Based on a molecular weight of 299 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 and a stable suspension was formed. 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 3.55 was determined at a test item concentration of 31.25 µM. The corresponding cell viability was 83.4%. The lowest tested concentration with a significant luciferase induction >1.5 (1.75) was found to be 3.91 µM. The corresponding cell viability was >70% (96.0%).The calculated EC_1.5was <1000 µM (2.47).

In the second experiment, a max luciferase activity (I_max) induction of 2.76 was determined at a test item concentration of 31.25 µM. The corresponding cell viability was 70.1%. The lowest tested concentration with a significant luciferase induction >1.5 (1.74) was found to be 3.91 µM. The corresponding cell viability was >70% (98.7%).The calculated EC_1.5was <1000 µM (2.04).

A dose response for luciferase activity induction was observed for each individual run as well as for an overall luciferase activity induction up to a concentration of 31.25 µM. Under the condition of this study the test item is therefore considered as sensitiser.

It should be noted, that concentration levels above 31.25 µM led to a decrease of cell viability up to a concentration of 250 µM. Further increase of the tested concentration levels led to a re-increase of the measured absorption during the MTT determination. This was found to be contradictory to the microscopically observed cell viability which clearly indicates strong cytotoxicity with increasing test item concentrations. For the concentration levels of 1000 µM and 2000 µM a slightly blue coloured supernatant was noted, which was ascribed to effects of the test item. These effects might influence the absorption measurement, leading to a false increase of the MTT signal and consequently cell viability. However, this effect was only observed at higher concentration levels. The final conclusion remains unaffected by this observation

Reference 4

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2018-10-02 to 2018-11-16

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 (324% experiment 1; 389% experiment 2) and 200% for CD54 (527% experiment 1; 430% experiment 2) were clearly exceeded.

Key result

Run / experiment:

other: 1

Parameter:

other: relative fluorescence intensity CD86 [%]

Value:

148

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Concentration: 85.25 µg/mL

Key result

Run / experiment:

other: 1

Parameter:

other: relative fluorescence intensity CD54 [%]

Value:

135

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Concentration: 85.25 µg/mL

Key result

Run / experiment:

other: 2

Parameter:

other: relative fluorescence intensity CD86 [%]

Value:

132

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Concentration: 147.31 µg/mL

Key result

Run / experiment:

other: 2

Parameter:

other: relative fluorescence intensity CD54 [%]

Value:

151

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Concentration: 212.13 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 1)

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	87.5	373	>150	88.1	358	>200	yes	pass
NiSO4	100 µg/mL	83.5	295	>150	82.1	603	>200	yes	pass
LA	1000 µg/mL	95.7	81	<=150	95.8	100	<=200	no	pass

Results of the Cell Batch Activation Test (Batch 2)

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	83.4	364	>150	82.3	419	>200	yes	pass
NiSO4	100 µg/mL	79.1	351	>150	77.9	688	>200	yes	pass
LA	1000 µg/mL	96.2	77	<=150	95.9	105	<=200	no	pass

 

Results of the Dose Finding Assay

Sample		Experiment 1		Experiment 2
		Concentration applied [µg/mL]	Cell Viability [%]	Concentration applied [µg/mL]	Cell Viability [%]
Medium Control	--	--	94.30	--	96.40
Solvent Control	THF	--	94.40	--	96.50
Test item	C8	7.81	94.00	7.81	97.30
	C7	15.63	93.00	15.63	96.70
	C6	31.25	82.90	31.25	96.60
	C5	62.50	79.80	62.50	96.70
	C4	125.00	75.20	125.00	78.90
	C3	250.00	74.80	250.00	76.80
	C2	500.00	72.50	500.00	79.10
	C1	1000.00	72.80	1000.00	78.80
Calculated CV75 [µg/mL]		176.78		No CV75
Mean CV75 [µg/mL]		176.78
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 Flourescence Intensity (RFI)		Ratio Isotype IgG1 to [%]
		CD86	CD54	Isotype IgG1	CD86	CD54	Isotype IgG1	CD86	CD54	CD86	CD54	CD86	CD54
Medium Control	-	96.8	96.6	96.0	1438	908	557	881	351	78	89	258	163
Solvent Control 2 (THF)	0.20%	96.3	96.0	95.9	1444.0	953.0	573.0	871	380	100	100	252	166
Solvent Control 1 (DMSO)	0.20%	95.8	95.3	95.3	1686	955	559	1127	396	100	100	302	171
DNCB	4.00	83.2	83.6	82.9	4226	2657	571	3655	2086	324	527	740	465
Test item	212.13	41.8	41.3	40.1	2169	1235	475	1694	760	194	200	457	260
	176.78	40.5	42.7	41.2	1971	1077	465	1506	612	173	161	424	232
	147.31	51.4	51.2	49.7	1756	980	499	1257	481	144	127	352	196
	122.76	59.8	60.7	60.1	1698	969	509	1189	460	137	121	334	190
	102.30	62.8	60.6	61.3	1790	951	515	1275	436	146	115	348	185
	85.25	50.9	50.7	50.0	1790	1013	501	1289	512	148	135	357	202
	71.04	64.3	65.2	64.8	1454	1005	516	938	489	108	129	282	195
	59.20	65.8	66.9	65.9	1516	1036	543	973	493	112	130	279	191

CD54 and CD86 Expression Experiment 2

Sample	Conc. [μg/mL]	Cell Viability [%]			Mean Fluorescence Intensity			corrected Mean Fluorescence Intensity		Relative Flourescence Intensity (RFI)		Ratio Isotype IgG1 to [%]
		CD86	CD54	IgG Isotype	CD86	CD54	Isotype IgG1	CD86	CD54	CD86	CD54	C86	CD54
Medium Control	-	95.4	95.1	95.2	2241	1250	618	1623	632	95	94	363	202
Solvent Control 2 (THF)	0.20%	95.5	94.9	94.5	2066	1245	595	1471	650	100	100	347	209
Solvent Control 1 (DMSO)	0.20%	94.6	94.6	94.2	2302	1268	596	1706	672	100	100	386	213
DNCB	4.0	83.7	83.3	82.8	7245	3494	607	6638	2887	389	430	1194	576
Test item	212.13	57.90	58.50	57.60	2328	1543	561	1767	982	120	151	415	275
	176.78	64.50	65.30	65.80	2377	1376	580	1797	796	122	122	410	237
	147.31	63.40	63.00	63.40	2515	1374	569	1946	805	132	124	442	241
	122.76	75.90	77.90	76.60	2041	1280	585	1456	695	99	107	349	219
	102.30	66.20	65.50	65.30	2300	1371	595	1705	776	116	119	387	230
	85.25	80.50	79.30	81.40	1941	1265	586	1355	679	92	104	331	216
	71.04	87.00	86.60	87.00	1816	1250	592	1224	658	83	101	307	211
	59.20	79.90	79.80	79.50	2019	1338	581	1438	757	98	116	348	230

Acceptance Criteria

Acceptance Criterion	Range	Experiment 1			pass/fail	Experiment 2			pass/fail
cell viability solvent controls [%]	>90	95.3	-	96.8	pass	94.2	-	95.5	pass
number of test dosed with viability >50% CD86	≥4	6			pass	8			pass
number of test dosed with viability >50% CD54	≥4	6			pass	8			pass
number of test dosed with viability >50% IgG1	≥4	5			pass	8			pass
RFI of positive control of CD86	≥150	324			pass	389			pass
RFI of positive control of CD54	≥200	527			pass	430			pass
RFI of DMSO solvent control of CD86	<150	128			pass	105			pass
RFI of DMSO solvent control of CD54	<200	113			pass	106			pass
RFI of THF solvent control of CD86	<150	99			pass	91			pass
RFI of THF solvent control of CD54	<200	108			pass	103			pass
MFI ratio CD86/IgG1 for medium control [%]	>105	258			pass	363			pass
MFI ratio CD86/IgG1 for DMSO control [%]	>105	302			pass	386			pass
MFI ratio CD86/IgG1 for THFcontrol [%]	>105	252			pass	347			pass
MFI ratio CD54/IgG1for medium control [%]	>105	163			pass	202			pass
MFI ratio CD54/IgG1for DMSO control [%]	>105	171			pass	213			pass
MFI ratio CD54/IgG1for THF control [%]	>105	166			pass	209			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 THF solvent control of CD86	102.9	17.4	36
RFI of THF solvent control 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:

other: The result should be considered in the context of integrated approach such as IATA.

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. Therefore, the test item might be considered as non-sensitiser. However, the log KOW is above 3.5 and according to OECD 442E a prediction cannot be made.

The data generated with this method may be not sufficient to conclude on the absence of 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.

A CV75 of 176.78 µg/mL was derived in the dose finding assay.

Based on the CV75, the main experiment was performed covering the following concentration steps:

212.13, 176.78, 147.31, 122.76, 102.30, 85.25, 71.04, 59.20 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.

Cytotoxic effects were observed for the cells treated with the test item. Relative cell viability at the highest test item concentration was reduced to 41.8% (CD86), 41.3% (CD54) and 40.1% (isotype IgG1 control) in the first experiment and to 57.90% (CD86), 58.50% (CD54) and 57.60% (isotype IgG1 control) in the second experiment.

The expression of the cell surfacemarker CD86 wasupregulated to 194% (212.13 µg/mL) and 173% (176.78 µg/mL) and the cell surface marker CD54 was upregulated to 200% (212.13 µg/mL) only in the first experiment. Due to cytotoxic effects (cell viability < 50%) of the test item in the respective concentrations, the upregulation of the cell surface markers CD86 and CD54 by the test item is considered to be not relevant for skin sensitisation.

The expression of the cell surface markers CD86 and CD54 was not upregulated above the threshold of 150% (CD86) and 200% (CD54) in the second experiment.

Therefore, the test item can be considered as non-sensitiser.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

According to Commission Regulation (EU) No 286/2011 as well as GHS (Globally Harmonized Classification System) the test material has obligatory labelling requirement for skin sensitisation and is classified into Category 1B.