3,4-dihydroxybenzaldehyde

Endpoint summary

Description of key information

First key event (QSAR prediction): Key study. Read across, executed by AW EC3 from LLNA or Skin sensitisation from GMP assays. Under the predicted conditions the test item may be classified as skin sensitizer.

Second key event (activation of keratinocytes): Key study. Test method according to the OECD 442D Guideline with GLP. Under the experimental conditions the test item may be classified as not skin sensitizer using the KeratinoSensTM test method. 

Conclusion: Based on a positive result obtained in one of
the key events of the skin sensitisation and also considering the
observational and practical experience, it was concluded to classify the substance for skin sentitisation as a precautionary measure (skin sens 1)

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 18 July to 29 July

Reliability:

1 (reliable without restriction)

Qualifier:

according to guideline

Guideline:

OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

ARE-Nrf2 luciferase KeratinoSens™ test method

Details of test system:

Keratinoses transgenic cell line [442D]

Details on the study design:

442D

PREPARATION OF TEST SOLUTIONS
- Preparation of the test chemical stock solution: the tets item was tested at 12 concentrations according to a geometric progression ratio of 2 from 0.98 μM to 2000 μM. Test item was diluted in DMSO.
- Preparation of the positive controls: for each culture plate, the cinnamaldehyde (SIGMA ALDRICH Ref W228613) was tested at 5 concentrations from 4 to 64 μM according to a geometric progression of ratio 2. Positive control was prepared in DMSO.
- Preparation of the solvent, vehicle and negative controls: 1 well by culture plate was left without cell and was filled with negative control (blanck).
Negative control: Treatment medium (DMEM 1 g/l glucose, Fisher Bioblock, batch: 2505790), 1% DMSO (Sigma Aldrich, batch n: L0840), 1% Non-heat inactivated foetal caf serum (Fisher Bioblock, batch n: 2337973).

APPLICATION OF THE TEST CHEMICAL AND CONTROL SUBSTANCES
- Number of replicates: 5
- Number of repetitions: 2.
- Test chemical concentrations: 0.98, 1.95, 3.91, 7.81, 15.63, 31.25, 62.5, 125, 250, 500, 1000 and 2000 μM.
- Application procedure: In 5 seeded plates, the medium was aspirated and replaced with 150 μl of treatment medium. Then, the 4-fold plate wax replicated 5 times: 50 μl from 4-fold plate were placed in each of the three white plates and in two trasnparent plates.
- Exposure time: The plates (1-fold) were incubated for 48 hours +/- 1 hour (37ºC, 5%CO2).
- Study evaluation and decision criteria used: The test item is identified as potential skin sensitizer if the 4 following conditions are met in 2 of 2 or in 2 of 3 repetitions. Otherwise the KEratinosens prediction is considered as negative:
-the Imax is equal to ot higher than 1.5 times and statistically significantly different as compared to the negative control
- thr EC1.5 value is strictly below 1000 μM.
- at the lowest concentration with a gene induction equal to or higher than 1.5, the cell viability must be strictly higher than 70%
- there is an apparent overall dose-response for luciferase induction, which is similar between the repetitions.
- Description on study acceptance criteria:
Positive control:
- The gene induction must be statistically significant above the threshold of 1.5 in at least one of the tested concentration.
- The EC1.5 value should be between IDEA Lab historial data.
Negative control:
- The average coefficient of variation of the luminescence reading for the solvent controls ( 3 x 6 wells) should be below 20% in each repetition. If for one repetition the validity criteria are not met, or in case of equivocal result additional repetitions should be considered. The validation of the results is carried out by the Study Director.


SEEDING AND INCUBATION
- Seeding conditions (passage number and seeding density): Cells were used at passage 21 in repetition 1 and passage 23 in repetiion 2. 125 μl of the cell suspension at 8.10^4 cells/ml (i.e. 10^4 cells per well).
- Incubation conditions: 24 hours +/- 1 hour at 37ºC, 5% CO2.


LUCIFERASE ACTIVITY MEASUREMENTS
- Choice of luminometer with demonstration of appropriate luminescence measurements based on control test: GloMax TM (Promega).
- Plate used: white cell culture 96-well plates for luminiscense reading, transparent cell culture 96-well pates for absorbance reading.
- Lysate preparation: Each well was gently washed one with 200 μl of PBS. Tehn, 100 μll of luciferase substrate (constitued by a mix of luciferase + ATP +lysing agent) were added in each well. The formation of foam was avoided by careful pippeting. The plates were incubated at least 15 minutes at room temperature to ensuere cells lysis.

DATA EVALUATION
- Cytotoxicity assessment: MULTISKAN EX plate reader (Thermo life sciences) - reading range 0 - 3.5 units of Ab
sorbance -linearity range 0 - 2.200 units of Absorbance. After 48 hours, the medium was aspirated and each well was gently washed once with 200 μl of PBS. Then, 225 μl of staining solution diluted at 0.6 mg/ml in treatment medium (from the 5mg/ml stock solution) were distributed in each well. The plates were covered with an adhesive plastic foil and incubated for 4 hours ± 30 minutes (37ºC, 5% CO2). After this contact time, the staining solution was eliminated and the cells were treated with 200 μl of 10% SDS (sodium dodecyl sulfate) one night in the dark (37°C, 5% CO2). After a 10 minutes homogenization, the absorbance was measured at 540 nm.

Vehicle / solvent control:

DMSO

Negative control:

other: 100 μl of DMSO were distributed in row G columns 1 to 6 and 12 in the well H12.

Positive control:

cinnamic aldehyde [442D]

Positive control results:

Repetition 1: The maximal average induction of luciferase activity (Imax) was 4.01 at a concentration of 64 μM. The mean value EC1.5 was 7.40 μM.
Repetition 2: The maximal average induction of luciferase activity (Imax) was 3.23 at a concentration of 64 μM. The mean value EC1.5 was 9.29 μM.

Key result

Group:

test chemical

Run / experiment:

run/experiment 1

Parameter:

Imax [442D]

Value:

1.09

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Key result

Group:

test chemical

Run / experiment:

run/experiment 2

Parameter:

Imax [442D]

Value:

0.95

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: No.

DEMONSTRATION OF TECHNICAL PROFICIENCY: yes, the 10 proficiency substances recomm ended in the OECD guideline 442D were performed, obtaining values that fall within the respective reference range for at least 8 out of the 10.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes, the CV of the luminescence readings for repetition
1 was 15.2% and for repetition 2 was 10.8% which are not higher than 20%.
- Acceptance criteria met for positive control: Yes, the luciferase activity induction was statistically
significant above the threshold of 1.5 in both repetitions. The average induction values at 64 μM were 4.01 and 3.23 in each repetition which are between 2 and 8. The EC1.5 values were 7.40 and 9.29 μM for each repetition which are within 2 SD of the historical mean of the testing facility and between 7 μM and 30 μM based on the OECD validation dataset.

Table 1. Reference items: 

Cinnamaldehyde	4 μM	8 μM	16 μM	32 μM	64 μM	EC1.5	Imax
Rep 1	1.41	1.52	1.71	2.49	4.01	7.40	4.01
Rep 2	1.28	1.47	1.68	2.12	3.23	9.29	3.23
Mean	1.34	1.49	1.70	2.30	3.62	8.29*	3.62

*geometric mean 

Control solvent	CV % control solvent
Rep 1	15.2
Rep 2	10.8

Table 2. Tet item summary results 

ID-22/09446	Viability		Induction
	IC50 μm	IC30 μm	Imax	Linear EC1.5 μm	EC1.5 Lin/Log μM
Rep 1	136.55	86.98	1.09	-	-
Rep 2	22.95	77.11	0.95	-	-
Mean	114.76	-	1.02	-	-
Geometric mean		81.90	-	-	-

Table 3. Test item mean viability percentage 

Concentration μM	0.98	1.95	3.91	7.81	15.63	31.25	62.5	125	250	500	1000	2000
Rep 1	72.65	85.35	93.65	85.86	88.91	91.79	82.47	50.64	43.86	32.01	14.73	8.81
Rep 2	91.23	82.31	80.57	74.48	84.60	80.90	77.85	44.26	43.06	25.77	9.57	4.68
Viability	81.9	83.8	87.1	80.2	86.8	86.3	80.2	47.4	43.5	28.9	12.2	6.7

Table 4. Test item mena induction

Concentration μM	0.98	1.95	3.91	7.81	15.63	31.25	62.5	125	25	500	1000	2000
Rep 1	0.82	1.01	1.09	0.91	0.79	0.68	0.55	0.62	0.87	0.55	0.27	0.12
Rep 2	0.95	0.91	0.89	0.91	0.76	0.61	0.43	0.44	0.58	0.40	0.21	0.03
Induction	0.89	0.96	0.99	0.91	0.78	0.64	0.49	0.53	0.73	0.48	0.24	0.07
SD	0.09	0.08	0.15	0.00	0.03	0.05	0.09	0.13	0.21	0.11	0.04	0.06

Interpretation of results:

other: KeratinoSensTM test result is considered as part of an integrated approach to testing and assessment (IATA) in accordance with OECD guideline 442D.

Conclusions:

Under the experimental conditions the test item may be classified as not skin sensitizer using the KeratinoSensTM test method.

Executive summary:

The KeratinoSensTM test method was performed for the test item as part of an integrated approach to testing and assessment (IATA) in accordance to OECD guideline 442D, under GLP conditions. A KeratinoSensTM cell culture was prepared, incubated for 24 hours ± 1 hour at 37ºC, 5% CO2 and distributed into 3 plates (96 wells) for the measurement of the induction of luciferase activity and 2 plates (96 wells) to assess the cytotoxicity. Test item at 12 concentrations from 0.98 μM to 2000 μM, positive control cinnamaldehyde at 5 concentrations from 4 to 64 μM and negative control 1% DMSO were placed in the seeded plates and incubated for 48 hours ± 1 hour at 37ºC, 5% CO2. 2 repetitions were performed on a different day with fresh stock solution. All validity criteria were fulfilled. In the first repetition the induction was lower than 1.5 and thus the EC1.5 was not determined. In the second repetition the induction was als lower than 1.5. The induction factor was less than 1.5 for all concentrations given more 70% viability and thus the EC1.5 was not determined either in agreement with the acceptance criteria. Bases on these findings, a negative result can be predicted for skin sensitization using the KeratinoSensTM test method.

Reference 2

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

(Q)SAR

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

results derived from a valid (Q)SAR model and falling into its applicability domain, with adequate and reliable documentation / justification

Justification for type of information:

See attached the Category Report and prediction report

Guideline:

other: REACH Guidance on QSARs R.6

Principles of method if other than guideline:

Read-across, executed by AW 'EC3 from LLNA or Skin sensitisation from GPMT assays'

Specific details on test material used for the study:

SMILES: Oc1ccc(C=O)cc1O

Key result

Parameter:

EC3

Remarks on result:

positive indication of skin sensitisation based on QSAR/QSPR prediction

Interpretation of results:

other: Skin sens. 1 (CLP Regulation EC no. 1272/2008)

Conclusions:

The test substance was predicted to be skin sensitizer (Read-across, executed by AW 'EC3 from LLNA or Skin sensitisation from GPMT assays').

Executive summary:

The test substance was predicted to be skin sensitizer (Read-across, executed by AW 'EC3 from LLNA or Skin sensitisation from GPMT assays').

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on the available data, the substance is classified as skin sens.1 according to CLP Regulation no. 1272/2008.