4,4a,5,9b-tetrahydroindeno[1,2-d]-1,3-dioxin

Endpoint summary

Description of key information

Skin sensitisation (OECD 442C and 442D): negative for key events peptide reactivity and activation of keratinocytes

Conclusion from in vitro skin sensitisation battery: not skin sensitising

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

key study

Study period:

01 Jun - 24 Aug 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))

Version / remarks:

adopted Feb 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 METHOD
The DPRA is an in chemico method which quantifies the remaining non-depleted concentration of cysteine- or lysine-containing peptides 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 SYSTEM
- Supplier: GenScript, Piscataway, US and RS Synthesis, Louisville, US
- Synthetic cysteine- (C-) containing peptide:
Alternative name: Ac-RFAACAA-COOH
Molecular weight: 751.9 g/mol
- Synthetic lysine- (K-) containing peptide:
Alternative name: Ac-RFAAKAA-COOH
Molecular weight: 776.2 g/mol

VEHICLE CONTROL
- Substance: acetonitrile

POSITIVE CONTROL
- Substance: ethylene glycol dimethacrylate
The positive control was prepared as a 50 mM preparation in acetonitrile.

CO-ELUTION CONTROL
- Sample prepared of the respective peptide buffer and test substance but without peptide.

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

PEPTIDE STOCK SOLUTION PREPARATION
C-containing peptide:
- Solvent: phosphate buffer (pH 7.5)
- Concentration: 0.667 mM
K-containing peptide:
- Solvent: ammonium acetate buffer (pH 10.2)
- Concentration: 0.667 mM
The peptide stock solution was used for preparing the calibration samples and the test-substance and control samples.

INCUBATION CONDITIONS
- Peptide to test substance ratios: C-containing peptide: 1:10 (0.5 mM peptide, 5 mM test substance); K-containing peptide: 1:50 (0.5 mM peptide, 25 mM test substance)
- Temperature: 25 ± 2.5 °C
- Duration: 24 ± 2 h
- Light conditions: in the dark

NUMBER OF REPLICATES
for each peptide in triplicates for treatment and controls

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

OTHER
Prior to HPLC analysis the samples were visually investigated for any precipitate that may have formed during the exposure period.

Parameter:

other: % mean depletion of cystein-peptide

Value:

-0.29

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Parameter:

other: % mean depletion of lysine-peptide

Value:

-0.69

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Parameter:

other: % overall mean depletion

Value:

0

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Table 5. 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	-0.29	0.13	-0.69	0.99	0.00
Positive control	55.00	4.60	6.11	0.44	30.55

The mean C-peptide depletion, caused by the test substance was determined to be -0.29%.

The mean K-peptide depletion, caused by the test substance was determined to be -0.69%.

Negative depletions were considered to be 0 for calculation of the mean peptide depletion, which was thus calculated to be 0.0%.

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

The acceptance criteria were met.

TEST-SPECIFIC CONFOUNDING FACTORS

- Precipitation: The test substance was soluble in acetonitrile. The samples of the test substance with the peptides were solutions. Visual observation after 24 -hour incubation time did not reveal precipitates in all samples of the test substance with both peptides.

- Co-elution: No co-elution of the test substance and peptides occurred as demonstrated by the consistent values of the area ratios 220 nm/ 258 nm.

 

COMPARISON WITH HISTORICAL CONTROL DATA

The positive control caused depletion of the cysteine-peptide comparable to historic data. The lysine-peptide caused a mean peptide depletion of 6.11 which is amongst the lowest values observed for this reaction in the lab. However, the lysine reaction is known to be minimal, only, and the validity of the test is not considered to be affected.

Interpretation of results:

other: DPRA prediction: negative (no or minimal reactivity) according to OECD 442C

Conclusions:

Based on the observed results it was concluded that the test substance revealed a minimal or no reactivity in the direct peptide reactivity assay under the test conditions chosen.

Reference 2

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

01 Jun - 24 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:

adopted Feb 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 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 CELL LINE
- Type: LuSens (human transgenic keratinocyte cell line derived from HaCaT cells)
- Source: prepared in collaboration with Christoph J. Wruck, RWTH Aachen
- Passage number: ≥ 5 - ≤ 15

CELL CULTURE CONDITIONS
- Type and identity of media:
Maintenance medium: D-MEM (Biochrom) with 10% FBS and 1% Penicillin/Streptomycin, Puromycin dihydrochloride (Sigma)
Assay medium: D-MEM (Biochrom) with 10% FBS
Test substance exposure medium: D-MEM (Biochrom) with 1% FBS
- Temperature (°C): 37
- Humidity (%): ≥ 90
- CO2 (%): ca. 5
- Cell counter: Casy 1 (Schärfe System)

TEST CONCENTRATIONS
In order to determine the concentrations suitable for the main experiment a pre-test with concentrations of 0.5, 1, 5, 10, 50, 100, 500, 1000 and 2000 µg/mL was performed and cytotoxicity was determined by MTT assay. 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 98 μg/mL. The highest tested concentration in main experiment 1was 1.23 fold of the CV75 value. The additional concentrations were obtained by a 1:1.2 serial dilution series of the maximum concentration.
Concentrations for the four main experiments was chosen as follows:
Experiment 1: 169, 141, 117, 98, 82, 68, 57, 47 μg/mL
As no decrease in relative cell viability below 70% was observed in the 1st experiment, higher concentrations were chosen for the 2nd experiment.
Experiment 2 and 3: 350, 292, 243, 203, 169, 141, 117, 98 μg/mL
As no decrease in relative cell viability below 70% was observed in the 3rd experiment, higher concentrations were chosen in the 4th experiment.
Experiment 4: plate1: 872, 727, 606, 505, 421, 350, 292, 243; plate 2: 203, 169, 141, 117, 98, 82, 68, 57

CONTROLS
Negative control:
- Substance: DL-Lactic acid
- Concentration: 450 μg/mL in 1% DMSO in test substance exposure medium
Positive control:
- Substance: Ethylene glycol dimethacrylate
- Concentration: 18 μg/mL in 1% DMSO in test substance exposure medium
Vehicle control:
- Substance: DMSO
- Concentration: 1%
Blank control: Test substance exposure medium without cells
Basal control: Test substance exposure medium with cells

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

NUMBER OF REPLICATIONS: triplicates each in four independent experiments

DETERMINATION OF CELL VIABILITY
- Method: MTT assay
- MTT concentration: 0.5 mg/mL
- Incubation time: 2 h
- Spectralphotometer: TriStar² Multimode reader LB 942 (Berthold)
- Wavelenght: 570 nm with reference wavelength 690 nm

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

Run / experiment:

other: Experiment 1

Parameter:

other: maximum luciferase activity induction

Value:

1.52

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: test item concentration: 169 µg/mL; cell viability: 85.4%

Run / experiment:

other: Experiment 2

Parameter:

other: maximum luciferase activity induction

Value:

1.29

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: test item concentration: 292 µg/mL; cell viability: 73.4%

Run / experiment:

other: Experiment 3

Parameter:

other: maximum luciferase activity induction

Value:

1.95

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: test item concentration: 350 µg/mL; cell viability: 73.4%

Run / experiment:

other: Experiment 4 (plate 1)

Parameter:

other: maximum luciferase activity induction

Value:

1.69

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: test item concentration: 243 µg/mL; cell viability: 64.1%

Run / experiment:

other: Experiment 4 (plate 2)

Parameter:

other: maximum luciferase activity induction

Value:

1.52

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: test item concentration: 203 µg/mL; cell viability: 69.3%

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 ± SD	Rel. viability (%) mean ± SD	t-test
				p-value	Markers
Experiment 1
Test substance	47	1.20 ± 0.21	93.3 ± 5.7	0.114	n.s.
	57	1.22 ± 0.07	93.1 ± 8.2	0.005	**
	68	1.22 ± 0.07	95.6 ± 14.4	0.005	**
	82	1.16 ± 0.05	92.4 ± 9.1	0.001	**
	98	1.19 ± 0.06	90.4 ± 7.0	0.003	**
	117	1.23 ± 0.13	79.5 ± 5.1	0.038	*
	141	1.08 ± 0.11	85.6 ± 5.6	0.182	n.s.
	169	1.52 ± 0.11	85.4 ± 1.4	0.002	**
Vehicle control	-	1.00 ± 0.14	100.0 ± 5.0	-	-
EGDMA	18	5.45 ± 0.94	105.5 ± 6.3	0.000	**
Lactic acid	450	0.87 ± 0.09	97.8 ± 7.0	0.021	*
Experiment 2
Test substance	98	1.23 ± 0.11	85.0 ± 9.7	0.019	*
	117	1.25 ± 0.11	86.7 ± 10.2	0.017	*
	141	1.23 ± 0.19	85.9 ± 8.4	0.078	n.s.
	169	1.11 ± 0.27	80.0 ± 8.1	0.278	n.s.
	203	1.13 ± 0.23	73.2 ± 1.0	0.224	n.s.
	243	0.98 ± 0.12	71.4 ± 5.5	0.426	n.s.
	292	1.29 ± 0.09	73.4 ± 7.0	0.005	**
	350	1.27 ± 0.06	62.2 ± 4.0	0.000	**
Vehicle control	-	1.00 ± 0.17	100.0 ± 6.1	-	-
EGDMA	18	7.51 ± 0.59	130.3 ± 7.1	0.000	**
Lactic acid	450	0.94 ± 0.24	116.7 ± 7.2	0.305	n.s.
Experiment 3
Test substance	98	1.22 ± 0.24	91.9 ± 8.8	0.126	n.s.
	117	1.11 ± 0.12	77.6 ± 6.6	0.122	n.s.
	141	1.15 ± 0.07	91.6 ± 8.2	0.015	*
	169	1.41 ± 0.16	86.5 ± 7.0	0.016	*
	203	1.36 ± 0.08	83.4 ± 6.2	0.001	**
	243	1.69 ± 0.16	76.1 ± 5.8	0.005	**
	292	1.46 ± 0.08	74.6 ± 1.7	0.000	**
	350	1.95 ± 0.24	73.4 ± 3.7	0.008	**
Vehicle control	-	1.00 ± 0.16	100.0 ± 7.6	-	-
EGDMA	18	6.64 ± 0.43	88.9 ± 8.8	0.000	**
Lactic acid	450	0.90 ± 0.13	108.2 ± 5.3	0.089	n.s.
Experiment 4 (plate 1)
Test substance	243	1.69 ± 0.21	64.1 ± 9.0	0.011	*
	292	1.55 ± 0.09	57.5 ± 6.5	0.000	**
	350	1.63 ± 0.05	53.1 ± 8.0	0.000	**
	421	1.45 ± 0.14	46.2 ± 5.0	0.007	**
	505	1.47 ± 0.34	38.1 ± 6.2	0.066	n.s.
	606	1.30 ± 0.20	33.8 ± 7.1	0.053	n.s.
	727	1.56 ± 0.22	24.5 ± 4.6	0.019	*
	872	1.51 ± 0.31	14.6 ± 2.4	0.050	n.s.
Vehicle control	-	1.00 ± 0.17	100.0 ± 5.5	-	-
EGDMA	18	7.54 ± 0.56	122.2 ± 8.4	0.000	**
Lactic acid	450	1.02 ± 0.10	117.8 ± 9.5	0.376	n.s.
Experiment 4 (plate 2)
Test substance	57	1.28 ± 0.24	82.0 ± 8.5	0.089	n.s.
	68	1.49 ± 0.09	75.6 ± 7.2	0.000	**
	82	1.33 ± 0.10	74.0 ± 7.3	0.004	**
	98	1.28 ± 0.08	74.0 ± 9.7	0.002	**
	117	1.23 ± 0.21	70.5 ± 6.3	0.100	n.s.
	141	1.46 ± 0.21	71.4 ± 7.8	0.027	*
	169	1.47 ± 0.07	63.2 ± 8.4	0.000	**
	203	1.52 ± 0.12	69.3 ± 6.9	0.003	**
Vehicle control	-	1.00 ± 0.17	1.00 ± 5.0	-	-
EGDMA	18	9.19 ± 0.44	123.3 ± 7.9	0.000	**
Lactic acid	450	1.06 ± 0.19	116.7 ± 4.1	0.273	n.s.

EGDMA: ethylene glycol dimethacrylate

n.s.: no statistical significance

 

Calculation of an EC1.50 (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 concentrations used in the main experiment the test substance was soluble in 4% DMSO in the culture medium (4 x stock preparations) and in 1% DMSO in culture medium (final concentrations). No precipitates were noticed in any concentration after 48 h.

 

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. The minimal deviation in the positive control of experiment 4, plate 2 (fold induction = 9.19) is not considered to have an influence on the validity of the data.

Interpretation of results:

other: activation of keratinocytes negativ according to OECD 442D

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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information:

Sensitisation

 

The skin sensitizing potential of the test substance was evaluated in a combination of non-animal methods (in chemico and in vitro). Two key events of skin sensitisation a) molecular interaction with skin proteins and b) inflammatory response in keratinocytes were addressed.

 

a) Molecular interaction with skin proteins

The protein reactivity of the test substance towards synthetic cysteine (C)- or lysine (K)-containing peptides was investigated in a Direct Peptide Reactivity Assay (DPRA) (2015) performed according to OECD Guideline 442C and in compliance with GLP. 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 in acetonitrile to prepare a 100 mM solution. Three samples of the test substance and the positive control ethylene glycol dimethacrylate were incubated with each peptide in ratios of 1:10 (for cysteine) or 1:50 (for lysine). 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 analysed by measuring UV absorbance at 258 nm and the area ratio 220 nm / 258 nm was calculated as a measure of peak purity.

Visual observation after the 24-hour incubation time did not reveal precipitates in all samples of the test substance with both peptides. In addition, no co-elution of test substance and peptides was noticed. All acceptance criteria were met and the positive control proved the validity of the test. In presence of the test substance the mean depletion of cysteine and lysine peptide was -0.29% and -0.69%, respectively. The overall mean percent cysteine and lysine depletion is 0% and therefore the test substance is allocated to the “no or minimal reactivity” class (mean % depletion <6.38%) and considered to be negative for molecular interaction with skin proteins.

 

b) Activation of keratinocytes

The activation of keratinocytes of the test substance was investigated in an ARE-Nrf2 Luciferase Test in human transgenic keratinocyte cell line derived from HaCaT cells according to OECD Guideline 442D and in compliance with GLP (2015). In a preliminary cytotoxicity assessment a CV75 value of 98 µg/mL was determined. In the main experiment cells were incubated with test substance concentrations of 47 - 872 µg/mL in DMSO for 48 h at 37°C. After exposure cells were lysed and antioxidant response element (ARE) dependent luciferase activity was assessed by luminescence measurement. Additionally a MTT assay was performed to assess cytotoxicity of the test substance. The study was conducted in four independent experiments, whereas experiment 4 included 2 plates. No precipitates were noticed in any concentration after 48 h. The acceptance criteria were met in all experiments. The test substance did not induce luciferase activity in at least two consecutive concentrations with statistical significance affording at least 70% viability in at least two independent experiments. Thus calculation of an EC1.50 was not applicable. Under the conditions of the ARE-Nrf2 Luciferase test the test substance did not induce luciferase activity in the transgenic keratinocyte cell line.

 

Conclusion

Based on two negative results in the in vitro test battery according to the Adverse Outcome Pathway defined for skin sensitisation, the test substance is not considered to be a skin sensitiser.

Justification for classification or non-classification

The available data on skin sensitisation do not meet the criteria for classification according to Regulation (EC) 1272/2008, and are therefore conclusive but not sufficient for classification.