Methyl 2,4-dihydroxy-3,6-dimethylbenzoate

Endpoint summary

Description of key information

Skin sensitisation key studies:

In vivo skin sensitisation (LLNA, OECD 429, GLP): sensitising.

Human Repeated Insult Patch Test: negative at 10%

Respiratory sensitisation:

Not respiratory sensitising in absence of human data and absence of respiratory sensitisation alerts.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in vivo (LLNA)

Remarks:

LLNA was performed in 2004

Type of information:

experimental study

Adequacy of study:

key study

Study period:

08 April, 2003 - 15 April, 2003

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

information is sufficiently adequate

Justification for type of information:

LLNA was performed before 2016

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2002

Deviations:

yes

Remarks:

No details on test material, no details on clinical signs or local irritation at application site, positive control study performed 9 month before main study instead of 6 months, no dose relationship in response was observed in the positive control test.

GLP compliance:

yes

Type of study:

mouse local lymph node assay (LLNA)

Species:

mouse

Strain:

other: CBA/Ca/Ola/Hsd

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Harlan Interfauna UK Limited, Blackthorne, Bicester, Oxon, UK
- Females (if applicable) nulliparous and non-pregnant: not specified
- Age at study initiation: Young adults
- Weight at study initiation: 14.4 - 19.6 g
- Housing: A maximum of 4 mice was housed per cage, in cages suitable for animals of this strain and weight range.
- Diet: Free access to RM1 diet (supplied by Special Diets Services Limited, Witham, Essex, UK
- Water: Free access to mains water
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS set to maintain
- Temperature (°C): 22 ± 3
- Humidity (%): 30 - 70
- Air changes (per hr): A minimum of 15
- Photoperiod (hrs dark / hrs light): 12/12

Vehicle:

other: 25% ethanol/ 75% diethyl phthalate

Concentration:

Undiluted test item or the test item at concentrations of 1%, 2.5%, 5%, 10% or 25% w/v in vehicle.

No. of animals per dose:

Groups of four mice were treated.

Details on study design:

TREATMENT PROCEDURES:
TOPICAL APPLICATION:
Each test group of mice was treated by topical (epidermal) application to the dorsal surface of each ear lobe (left and right) with different test item concentrations of 1 %, 2.5%, 5%, 10 %, 25% and 100 % (undiluted) in 25% ethanol/ 75% diethyl phthalate (w/v). The application volume, 25 μL, was spread over the entire dorsal surface of each ear lobe once daily for three consecutive days. A further group of mice was treated with an equivalent volume of the relevant vehicle alone (control animals).
ADMINISTRATION OF 3H-METHYL THYMIDINE:
Three days after the third application, all mice were administered with approximately 250 μL of phosphate buffered saline (PBS) containing approximately 20 μCi of a 2.0 Ci/mmol specific activity 3H-methyl thymidine (3HTdR) by intravenous injection via the tail vein.
DETERMINATION OF INCORPORATED 3HTdR:
Approximately five hours after treatment with 3HTdR all mice were euthanized by inhalation of halothane vapour followed by cervical dislocation. The draining auricular lymph nodes were removed from each animal and, together with the nodes from the other animals in the group, were placed in a container of PBS.

A single cell suspension was prepared by mechanical disaggregation of lymph nodes through stainless steel gauze (200 μm mesh size). After washing three times with phosphate buffered saline (approx. 10 mL) the lymph node cells were resuspended in 5% trichloroacetic acid (approx. 3 mL) and incubated at approximately +4 °C overnight, the samples were pelleted by centrifugation and the supernatant was discarded. for precipitation of macromolecules. The precipitates were then resuspended in 5% trichloroacetic acid (1 mL) and transferred to glass scintillation vials with 10 mL of scintillation liquid (Optiphase) and thoroughly mixed.
The level of 3HTdR incorporation was then measured on a β-scintillation counter (Packard Tri-Carb 2500TR Liquid Scintillation Counter).

OBSERVATIONS:
Body weights: The bodyweight of each animal was recorded prior to dosing on day 1 and prior to injection of 3HTdR on day 6.
Clinical signs (local / systemic): Animals were checked at least once daily for signs of systemic toxicity.

Positive control substance(s):

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

Statistics:

Not performed.

Positive control results:

The application of hexylcinnamaldehyde at concentrations of 2.5%, 5% or 10% w/v in acetone resulted in a greater than 3-fold increase in isotope incorporation at all three concentrations (no dose relationship in response was observed). Therefore, hexylcinnamaldehyde was shown to be a skin sensitiser, confirming the validity of this study. See section "Any other information on results incl. tables".

Key result

Parameter:

EC3

Remarks:

% w/v

Value:

19

Key result

Parameter:

other: NOEC %

Value:

10

Parameter:

SI

Value:

1.59

Remarks on result:

other: 1%

Parameter:

SI

Value:

2.25

Remarks on result:

other: 2.5%

Parameter:

SI

Value:

1.99

Remarks on result:

other: 5%

Parameter:

SI

Value:

1.41

Remarks on result:

other: 10%

Parameter:

SI

Value:

3.94

Remarks on result:

other: 25%

Cellular proliferation data / Observations:

CELLULAR PROLIFERATION DATA
See section "Any other information on results incl. tables"

DETAILS ON STIMULATION INDEX CALCULATION
See section "Any other information on results incl. tables"

EC3 CALCULATION
See section "Any other information on results incl. tables"

CLINICAL OBSERVATIONS: No data on results.

BODY WEIGHTS:
The body weight of the animals, recorded at the start of application and on day 6, was within the range commonly recorded for animals of this strain and age.

Skin sensitisation potential of Methyl atrarate:

Concentration of test substance (% w/v)	Number of lymph nodes assayed	Disintegration per minute (dpm)	dpm per lymph node	Test control ratio
0 (vehicle only)	8	3050	381	N/A
1	8	4846	606	1.59
2.5	8	6866	858	2.25
5	8	6052	757	1.99
10	8	4314	539	1.41
25	8	12008	1501	3.94
EC3	19% w/v

N/A: not applicable

Skin sensitisation potential of the positive control substance hexylcinnamaldehyde:

Concentration of hexylcinnamaldehyde (% w/v)	Number of lymph nodes assayed	Disintegration per minute (dpm)	dpm per lymph node	Test control ratio
0 (vehicle only)	8	2570	321	N/A
2.5	8	16088	2011	6.26
5	8	15659	1957	6.10
10	8	15611	1951	6.08

N/A: not applicable

Interpretation of results:

other: Sensitiser 1B

Remarks:

According to EU CLP (EC No. 1272/2008 and its amendments).

Conclusions:

The SI values calculated for the substance concentrations 1, 2.5, 5, 10 and 25 % were 1.59, 2.25, 1.99, 1.41 and 3.94, respectively. These results show that the test substance could elicit a SI ≥ 3. An EC3 value of 19% w/v was calculated. A NOEC of 10% is derived. The test isubstance was considered to be a sensitiser under the conditions of the test.

Executive summary:

The skin sensitisation potential of the substance has been tested according to OECD TG 429 test guideline and GLP principles. At 1, 2.5, 5, 10 and 25% the substance showed SI values of 1.59, 2.25, 1.99, 1.41 and 3.94, respectively. Reliable negative and positive controls were included. These results show that the test substance could elicit a SI ≥ 3. An EC3 value of 19% w/v was calculated. A NOEC of 10% is derived. Based on the results, the substance was considered to be a sensitiser and should be classified as skin sensitizer (Category 1B) and labeled as H317: May cause an allergic skin reaction according to Regulation (EC) No. 1272/2008 and GHS.

Reference 2

Endpoint:

skin sensitisation: in vitro

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Reference 3

Endpoint:

skin sensitisation: in vitro

Remarks:

SENS-IS test

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

2017

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

no guideline followed

Principles of method if other than guideline:

The SENS-IS test was used to evaluate sensitizing potential of the test item by assessing the induction of irritation and/or sensitisation biomarkers in an in vitro 3D skin model. The SENS-IS test is based on an analysis by RT-PCR (reverse transcription polymerase chain reaction) of two sets of genes, one specifically reflecting the irritant potential of a chemical after application to the skin ("IRRITATION" set of genes) and the ther set of genes correlating with the skin sensitisation potential (2 subsets, "SENS_IS" and "REDOX"). By measuring the level of expression of these two seperate sets of genes at a given time point after application and by comparison with internal negative, an irritant and a sensitiser positive control, the SENS-IS test can give an indication of the sensitiser potential of the test item.

GLP compliance:

no

Type of study:

other: Induction of the expression of irritation and/or sensitization biomarkers in an in vitro 3D skin model

Specific details on test material used for the study:

After solubility assessment, the test item was dissolved in DMSO at 50% (w/v), 10% (w/v) and 1% (w/v) at room temperature and vortexed. The solubility of the test item was assessed visually for each preparation, if the test item was not fully dissolved it was further treated at 37°C and if needed at 65°C under agitation (no further details included in the report). Thirty µl (26.3 µl/cm2) of each dilution was applied on top of reconstituted epidermis (Episkin model), using a positive displacement pipette. The test item solution was gently spread on the epidermis surfaces to ensure it covered the full surface. After 15 minutes exposure, the Episkins were rinsed with PBS. Epidermis were then incubated at 37°C for 6 hours. After incubation, the complete epidermis was collected, placed in RNAzol solution and the total RNA was isolated by homogenization of the skin. After reverse transcription, quantitative gene expression was measured by RT-PCR using sybr green buffer using the LC480 Roche’s apparatus and specific biomarkers primers defined for the Sens-IS test. The test was also performed with dipropylene glycol (DPG) as solvent.
Vehicle treated skin was used as negative control (DMSO was used pure to the skin); Positive control for irritation: sodium lauryl sulfate (5% in PBS); Positive control for sensitization: TNBS (1M in water).
The test procedure and the controls were tested in at least 2 experiments (using different batches of Episkin models).

Acceptance criteria:
The IC50 value of SLS on Episkin must be ≥ 1.2 mg/ml. Over-irritation (or tissue destruction) induced by the test item is measured by the number of irritation biomarker genes over-expressed. I more than 20 genes are overexpressed for a given concentration of the test item, the sample is not accepted.

Evaluation criteria:
A test item is considered a sensitizer if it induces over-expression (>1.25 fold compared to the mean of the negative control) of at least 7 genes among two groups of respectively 21 and 17 genes named “SENS-IS” and “ARE”. To take into account non-specific genes overexpression due to cell stress no conclusion can be drawn if more than 20 genes are over-expressed in the “IRRITATION” group of genes. In the latter case the test item is analyzed at a lower concentration.

Positive control results:

Results of 23 control runs (including vehicle control and positive controls for irritation and sensitization) are included in the report. In all cases 5% SLS was correclty predicted to have skin irritant properties (17- 23 over-expressed genes in the "IRRITANT" group). TNBS was tested positive for sensitising properties in all tests, and in 3/23 also positive for irritant effects. Vehicle controls were negative for both endpoints.

Key result

Run / experiment:

other: 50%

Parameter:

other: Induction genes related to sensitization

Value:

10

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Key result

Run / experiment:

other: 10%

Parameter:

other: Induction genes related to sensitization

Value:

15

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Key result

Run / experiment:

other: 1%

Parameter:

other: Induction genes related to sensitization

Value:

3

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:

The test item induced over-expression of genes related to skin irritation for 15, 16 and 4 genes at respectively 50%, 10% and 1%. This indicates that the test item had an irritant effect on the epidermis, however it was not considered to be over-irritation or skin destruction.
Tests performed in DPG at 37°C at 50%, 10% and 1% gave similar results (positive at 50% and 10%), negative at 1%.

Schematic overview results:

Number of overexpressed genes	50% DMSO	10% DMSO	1% DMSO
IRRITATION	15	16	4
SENS_IS	2	7	3
ARE	8	8	0

Interpretation of results:

other: the result of this test is positive

Conclusions:

Based on the induction of irritation and/or sensitisation biomarkers in an in vitro 3D skin model (SENS-IS test), the test item is considered to be a moderate sensitiser.

Reference 4

Endpoint:

skin sensitisation: in chemico

Remarks:

DPRA

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

22 February 2016 - 12 July 2016

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Remarks:

Study is not conducted under GLP

Qualifier:

equivalent or similar to guideline

Guideline:

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

GLP compliance:

no

Type of study:

direct peptide reactivity assay (DPRA)

Specific details on test material used for the study:

The test item had a purity of 99.9%.

Details on the study design:

Synthetic peptides containing cysteine or lysine were reacted with test articles for 24 ± 2 hours. The test item was prepared at 100mM concentration in acetonitrile (soluble after 1 minute vortexing). Solvent controls (solvent with peptide in absence of the test item), a positive control (cinnamic aldehyde) and coelution controls (test item without peptide) were included. Triplicates were prepared for each test or control sample. Single samples were prepared for the coelution controls. After that incubation period the extent of peptide depletion was analyzed using High Performance Liquid Chromatography (HPLC) coupled with ultra-violet (UV) spectrometric detection. Peptide depletion was calculated as the relative difference of the test item (or positive control) peptide peak area and the mean peptide peak area of the control.
Criteria to be fulfilled for a valid test:
1. The standard curves for each peptide must have a rE2 value greater than 0.990. The mean peptide concentration of the solvent control must equal 0.50 ± 0.05 mM.
2. The positive control must have a mean percent depletion of 60.8% - 100.0% for cysteine and 40.2% - 69.0% for lysine among the three replicates. Also the positive control must have a standard deviation <14.9% for cysteine and <11.6% for lysine.
3. The peak area CV has to be <15% for cysteine and lysine for 3 solvent controls run at the beginning of the test and three solvent controls run at the end of the test.
4. The peak area CV has to be <15% for cysteine and lysine and a mean peptide concentration of 0.50 ± 0.05 mM for three solvent control samples run concurrently with the test item samples.
5. The standard deviations for the three replicates of the test item must meet the same criteria as the positive control for the peptide used.
6. There must be no overlap between the rest of the test item peak of the coelution control and the peptide peak. If the coelution is seen with with lysine, evaluation of the cysteine data only is acceptable. If the coelution is seen with both peptides, the assay is considered inconclusive.

Positive control results:

The positive control showed a mean peptide depletion of 75.5% (cysteine-containing peptide) and 61.7% (lysine).

Key result

Run / experiment:

other: mean of three

Parameter:

other: Cysteine depletion

Value:

3.3

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Run / experiment:

other: mean of three

Parameter:

other: Lysine depletion

Value:

47.2

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

not determinable because of methodological limitations

Remarks:

Coelution test item and lysine peptide

Other effects / acceptance of results:

The mean peptide depletion of lysine-containing peptides was 47.2%. However, coelution was seen with the test item and the lysine peptide. Because of the coelution only the cysteine data was used for determining the sensitization potential of the test item.

Interpretation of results:

other: the results are inconclusive

Conclusions:

In a DPRA study, the test item showed 3.3% mean peptide depletion with peptides containing cysteine residues. The mean peptide depletion with peptides containing lysine residues was 47.2%, however coelution was seen with the test item and the lysine peptide. Therefore the outcome of the DPRA is inconclusive.

Reference 5

Endpoint:

skin sensitisation, other

Remarks:

Human Repeated Insult Patch Test

Type of information:

experimental study

Adequacy of study:

key study

Study period:

May 12, 1999 - June 25, 1999

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

test procedure in accordance with national standard methods with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

other: HRIPT as described by Draize et al., 1944, Draize, 1959, following Voss-Griffith testing scheme (Voss, 1958, Griffith and Buehler, 1976; details: Principles of method if other than guideline)

Principles of method if other than guideline:

A Repeated Insult Patch Test was performed with 100 human subjects (23 males, 77 females, 19-70 years old). Test item: Methyl atratrate (tested as 10% (w/v) solution in 3:1 EtOH/DEP). A series of nine induction patching (occlusively applied for 24 hours) were completed over a period of three weeks. Rest period: 2 weeks. Challenge phase: Virgin test area treated with patch for 24 hours. Observation: 48, 72 and 96 hours post-patching. No re-challenge was done.

GLP compliance:

no

Type of study:

patch test

Justification for non-LLNA method:

The test was initiated to evaluate the potential of the test material to induce contact dermal sensitization as a result of repeated applications in human subjects.

Species:

other: Human volunteers

Sex:

male/female

Details on test animals and environmental conditions:

Test item: Methyl atratrate (tested as 10% (w/v) solution in 3:1 EtOH/DEP)
Patch preparation: 0.3 mL on 25 mm patch, applied 15 min prior to patching to allow for volatilization.
Procedure outline:
Induction phase: A series of nine induction patching (occlusively applied for 24 hours) were completed over a period of three weeks. Same test sites were repatched. Rest period: 2 weeks. Challenge phase: Virgin test area treated with patch for 24 hours. Observation: 48, 72 and 96 hours post-patching. No re-challenge was done.

Positive control results:

Not given.

Key result

Reading:

other: 24, 48, 72 and 96 hours after challenge

Group:

test chemical

Dose level:

10% (w/v) in 3:1 EtOH/DEP

No. with + reactions:

0

Total no. in group:

100

Clinical observations:

None

Remarks on result:

no indication of skin sensitisation

Interpretation of results:

other: no induction of dermal contact sensitisation by 10% test substance in human volunteers under the conditions of the test

Conclusions:

A Repeated Insult Patch Test was performed with 100 human subjects (23 males, 77 females, 19-70 years old). The test substance tested in a 10% (w/v) concentration in 3:1 EtOH/DEP did not evoke a skin sensitising response. It was considered that a panel of 100 persons is limited and no conclusion can be drawn for the general population. Furthermore, only one test concentration was tested, therefore no conclusions can be drawn on the skin sensitising properties of the test substance at a higher concentration.

Reference 6

Endpoint:

skin sensitisation: in vitro

Remarks:

keratinosens

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

26 January 2016 - 12 February 2016 (laboratory phase)

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Remarks:

Study was not conducted under GLP

Qualifier:

equivalent or similar to guideline

Guideline:

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

GLP compliance:

no

Type of study:

activation of keratinocytes

Details on the study design:

KeratinoSens cells (Givaudan, Switzerland) were used to report activation of the antioxidant/ electrophile response –element. Cytotoxicity was determined by measuring the relative conversion of MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) in test item-treated cultures compared to the solvent control. Three independent assays were performed to determine the maximal induction (Imax), the concentration for maximal gene induction (CImax), the EC1.5 value (concentration for a statistically significant induction of 50% above solvent controls), and a mean IC50 (concentration leading to 50% viability as compared to solvent controls) for the test item. For each assay, the KeratinoSens cells were cultured in quadruplicate plates for 24 hours, treated with the test item for 48 hours, and assessed for luciferase induction (3 plates) and cytotoxicity (1 plate).
As a positive control cinnamic aldehyde was used, at concentrations of 4, 8, 16, 32 and 64 µM. The solvent control, the positive control and the test item were tested at a final concentration of 1% DMSO. Test concentrations for the test item were 0.977, 1.95, 3.91, 7.81, 15.6, 31.3, 62.5, 125, 250, 500, 1000 and 2000 µM.
Criteria for determination of a valid definitive assay:
The KeratinoSens assay was accepted when the positive control caused an EC1.5 value that fell within two standard deviations of the historical mean. Additionally, the results of the three definitive trials for each plate are assessed using similar criteria outlined in the validation ring trial (Natsch et al., Toxicology in vitro (2011)). Those acceptance criteria included:
1) Variability in DMSO solvent control wells for each definitive assay was <20%;
2) The positive control produced a statistically significant induction above 1.5 fold below 64 µM in each definitive assay.
Evaluation of test results:
A test article was predicted to have sensitization potential if:
1) The EC1.5 value fell below 1000 µM in at least 2 of 3 repetitions;
2) At the lowest concentration with a gene induction above 1.5, cellular viability was greater than 70%;
3) There was apparent overall dose response which was similar between repetitions.

Positive control results:

The positive control had a mean EC1.5 value of 13.73 µM, with a mean IC50 of > 64 µM.

Key result

Run / experiment:

other: mean of 3

Parameter:

other: EC1.5

Value:

151.97

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Other effects / acceptance of results:

The test item had a mean EC1.5 value of 151.97 µM, a mean IC50 of 474.76 µM and an Imax of 1.65. The CI max (concentration for maximal gene induction) was 250 µM. For details see "any other information on results incl. tables".

Detailed information on results

Concentration (µM)	Induction			Mean viability
	Replicate 1	Replicate 2	Replicate 3
2000	-0.02	0.00	-0.01	1.27
100	0.00	0.00	0.01	1.05
500	0.00	0.13	0.01	40.73
250	1.60	1.42	1.88	132.47
125	1.29	1.51	1.17	117.30
62.5	1.22	1.34	1.19	106.53
31.3	1.08	1.07	1.13	104.04
15.6	1.07	1.11	1.06	103.19
7.81	1.08	1.08	1.03	103.16
3.91	0.99	1.14	1.12	103.84
1.95	0.98	1.05	1.18	104.77
0.977	1.12	1.04	1.05	107.85

 

Interpretation of results:

other: the result of this test is positive

Conclusions:

In the KeratinoSens assay the test item had a mean EC1.5 value of 151.97 µM, a mean IC50 of 474.76 µM and an Imax of 1.65. The CI max (concentraton for maximal gene induction) was 250 µM. Based on these results it is concluded that the test item can activate the antioxidant-response-element, and it is predicted to be skin sensitizer.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Additional information:

The skin sensitisation is assessed with a number of methods.

Key study for deriving the hazard and the potency: LLNA (OECD TG 429)

The skin sensitisation potential of the substance has been tested according to OECD TG 429 test guideline and GLP principles. At 1, 2.5, 5, 10 and 25% the substance showed SI values of 1.59, 2.25, 1.99, 1.41 and 3.94, respectively. Reliable negative and positive controls were included. These results show that the test substance could elicit a SI ≥ 3. An EC3 value of 19% w/v was calculated. A NOEC of 10% is derived.

Key study for deriving assessment factor for interspecies extrapolation: HRIPT

A Repeated Insult Patch Test was performed with 100 human subjects (23 males, 77 females, 19-70 years old). The test substance tested in a 10% (w/v) concentration in 3:1 EtOH/DEP did not evoke a skin sensitising response. It was considered that a panel of 100 persons is sufficient to be used for deriving the interspecies assessment factors for workers and consumers.

In vitro skin sensitisation studies available

DPRA (OECD TG 442C)

In a DPRA study, the test item showed 3.3% mean peptide depletion with peptides containing cysteine residues. The mean peptide depletion with peptides containing lysine residues was 47.2%, however coelution was seen with the test item and the lysine peptide. Therefore the outcome of the DPRA is inconclusive.

KeratinoSens (OECD TG 442D)

In the KeratinoSens assay the test item had a mean EC1.5 value of 151.97 µM, a mean IC50 of 474.76 µM and an Imax of 1.65. The CI max (concentration for maximal gene induction) was 250 µM. Since the test item can activate the antioxidant-response-element the substance is positive in this test.

SENS-IS (Currently no guideline)

Based on the induction of irritation and/or sensitization biomarkers in an in vitro 3D skin model (SENS-IS test), the substance is considered positive in this test.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

The substance is not a respiratory sensitiser in absence of human data indicating such effects. In addition, the respiratory sensitisation is assessed using the integrated evaluation strategy for respiratory sensitisation data in the ECHA guidance (R7A, Fig. 7.3-2, 2014).

1)     The substance is a skin sensitiser;

2)     The substance does not belong to the di-isocyanates;

3)     The substance has no structural alerts or is structurally related to chemicals causing respiratory sensitisation as presented in Table R.7.3-1 in the ECHA guidance of 2008 or those provided in the following document: http://ec.europa.eu/health/scientific_committees/docs/annex6_respiratory.pdf

Therefore the substance is not considered to be a respiratory sensitiser.

Justification for classification or non-classification

Based on the results, the substance was considered to be a sensitiser and should be classified as skin sensitizer (Category 1B) and labeled as H317: May cause an allergic skin reaction according to EU CLP (EC no 1272/2008 and its amendment).

In absence human data indicating respiratory sensitisation and using the ITS in the ECHA guidance (R.7a, 2014) the substance is not considered to be a respiratory sensitiser in accordance with the criteria outlined in the EU CLP (EC 1272/2008 and its amendments).