7-methyl-2H-benzo-1,5-dioxepin-3(4H)-one

Endpoint summary

Description of key information

- LLNA, Not a skin sensitizer (OECD 429, GLP, K, Rel. 2);

- In chemico study (Peptide depletion, non OECD, non GLP, S, Rel.4): a very low adduct formation is expected between cysteine and the substance;

- In vitro study (ARE assay, non OECD, non GLP, S, Rel.4): not a skin sensitizer;

- OASIS TIMES prediction (S, rel. 2): no structural alert idenfied for the substance and its predicted metabolites.

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:

From August 12 to October 06, 2005

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

guideline study with acceptable restrictions GLP Study performed according to the old version of the OECD test guideline No. 429 (2002), therefore ear thickness measurements were not included in the pre-screen test. The substance being irritating to the skin, this deviation may have an impact on the reliability of the study results. However no visible signs of irritation were observed at any of the concentration tested, therefore the study results are considered as reliable. The rational for the choice of the maximal dose tested (30%) is not very clear and not well reported (technical reason linked to the physical state of the substance - solid - expected since no higher dose was tested in the pre-test).

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Adopted 24 April 2002

Deviations:

yes

Remarks:

tested only up to 30% (rational not clear), no ear thickness measurements, Age: 6-7 weeks old instead of 8-12 weeks old. Humidity: 30-95% instead of 30-70%, no duration of acclimatation period, single caging, use of hair dryer.

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

inspected on July 19 to 22, 2004 / signed on January 06, 2005

Type of study:

mouse local lymph node assay (LLNA)

Specific details on test material used for the study:

- Physical state: White solid

Species:

mouse

Strain:

other: CBA/CaOlaHsd

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Harlan Netherlands, Horst, The Netherlands.
- Age at study initiation: 6-7 weeks (beginning of acclimatization)
- Weight at study initiation: 19.0 ± 1.1 g (mean)
- Housing: Animals were individually housed in Makrolon Type I, with wire mesh top
- Diet: Pelleted standard diet, ad libitum
- Water: Tap water, ad libitum
- Acclimation period: no duration reported

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 3 °C
- Humidity: 30-95 %
- Photoperiod: 12 h dark / 12 h light

IN-LIFE DATES: From: August 24, 2005 To: September 27, 2005

Vehicle:

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

Concentration:

Main test: 1, 10 and 30 % w/v in acetone/olive oil 4:1 (v/v)

No. of animals per dose:

4

Details on study design:

RANGE FINDING TESTS:
- Compound solubility: Soluble at 30% in AOO 4:1.
- Irritation: No irritation effects were observed at the concentrations of 1, 10 and 30% after a single application.
- Systemic toxicity: 30% is the highest achievable concentration whilst avoiding systemic toxicity and excessive local irritation.
- Ear thickness measurements: not measured
- Erythema scores: not reported

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: pooled treatment group approach, using tritiated (3H)-methyl thymidine, according to the OECD 429 test guideline.
- Criteria used to consider a positive response: A test item is regarded as a sensitiser in the LLNA if the following criteria are fulfilled:
1) First, that exposure to at least one concentration of the test item resulted in an incorporation of 3HTdR at least 3-fold or greater than that recorded in control mice, as indicated by the stimulation index.
2) Second, that the data are compatible with a conventional dose response, although allowance must be made (especially at high topical concentrations) for either local toxicity or immunological suppression.
The decision to select a stimulation index (S.I.) of 3 as an arbitrary indication of sensitizing activity was made on the basis of investigations performed with a wide range of chemicals.

TREATMENT PREPARATION AND ADMINISTRATION:
- All formulations were prepared freshly before each dosing. Homogeneity of the test item in the vehicle was maintained during treatment with the magnetic stirrer.
- Groups of four mice were treated with the test material at concentrations of 1, 10 and 30 % w/v in acetone/olive oil 4:1. The mice were treated by daily application of 25 μL of the appropriate concentration of the test material to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3). A further group of four mice received the vehicle alone in the same manner. A hair dryer was used to dry the ear's surface as quickly as possible to avoid loss of test item applied.
Each animal was injected via the tail vein with 250 μL of phosphate buffered saline (PBS) containing 20.4 μCi of 3H-methyl thymidine (3HTdR) on Day 6.
After five hours, all animals were killed by intraperitoneal injection of Na-thiopental and the draining (auricular) lymph node of each ear was excised. The nodes from the four mice were excised and pooled for each experimental group. A single cell suspension of lymph node cells (LNC) was prepared in PBS by gentle mechanical disaggregation through gauze. LNC were washed two times with PBS. To precipitate out the radioactive material, the LNC were resuspended in 3 mL of 5 % Trichloroacetic acid (TCA) and incubated approximately 18 h incubation at approximately 4 °C. The precipitate was then resuspended in 1 mL of 5% TCA and transferred to 10 mL of scintillation fluid (Ultima gold) and thoroughly mixed. 3HTdR incorporation was measured bon a β-scintillation counter. Similarly background 3HTdR levels were also measured in two aliqots of 5% TCA.
The proliferation 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 3HTdR incorporation into lymph node cells of test nodes relative to that recorded for the control nodes (Stimulation Index).

Positive control substance(s):

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

Statistics:

- The mean values and standard deviations were calculated in the body weight tables.
- A statistical analysis was conducted for assessment of the dose-response relationship, and the EC3 value was calculated according to the equation
EC3 = (a-c) [(3-d)/(b-d)] + c
where EC3 is the estimated concentration of the test item required to produce a 3-fold increase in draining lymph node cell proliferative activity; (a, b) and (c, d) are respectively the co-ordinates of the two pair of data lying immediately above and below the S.I. value of 3 on the local lymph node assay dose response plot.

Positive control results:

α-hexylcinnamaldehyde at 25 % induced skin sensitisation (SI = 4.0)

Key result

Parameter:

SI

Value:

< 3

Test group / Remarks:

Stimulation index for 1, 10 and 30 % were 0.83, 0.83 and 1.25, respectively.

Cellular proliferation data / Observations:

CELLULAR PROLIFERATION DATA
DPM / group for vehicle, 1, 10 and 30 % were 6293.56, 5225.82, 5253.89 and 7843.85, respectively.

DETAILS ON STIMULATION INDEX CALCULATION
Stimulation index for 1, 10 and 30 % were 0.83, 0.83 and 1.25, respectively.

EC3 CALCULATION
The EC3 value could not be calculated wince all SI's are below 3.

CLINICAL OBSERVATIONS:
Mortality / Viability: No deaths occurred during the study period.
Clinical signs (local / systemic): No symptoms of local toxicity at the ears of the animals and no systemic findings were observed during the study period.

BODY WEIGHTS
The body weight of the animals recorded was within the range commonly recorded for animals of this strain and age.

Table 7.4.1/1: Results of skin sensitisation

Test item Concentration %(w/v)	Group	Measurement DPM	Calculation			Result
			DPM-BG^a	number of lymph nodes	DPM per lymph node^b	S.I.
-	BG I	24.60	-	-	-	-
-	BG II	23.53	-	-	-	-
-	CG 1	6293.56	6269.5	8	783.7	NA
1	TG 2	5225.82	5201.8	8	650.2	0.83
10	TG 3	5253.89	5229.8	8	653.7	0.83
30	TG 4	7843.85	7819.8	8	977.5	1.25

BG = Background (1 ml 5% trichloroacetic acid) in duplicate

CG = Control Group

TG = Test Group

S.I. = Stimulation Index

a) = The mean value was taken from the figures BG I and BG II

b) = Since the lymph nodes of the animals of a dose group were pooled, DPM/node was determined by dividing the measured value by the number of lymph nodes pooled

The EC3 Value could not be calculated, since all SI’s were below 3.

Interpretation of results:

GHS criteria not met

Conclusions:

Under the test conditions, test material is not classified as a skin sensitiser according to the annex VI of the Regulation EC No. 1272/2008 (CLP) and to the GHS.

Executive summary:

A study was performed to assess the skin sensitisation potential of test material in the CBA/CaOlaHsd strain mouse following topical application to the dorsal surface of the ear. The method was conducted according to the OECD test guideline No 429 and in compliance with GLP.

Following a preliminary screening test in which no clinical signs of toxicity and no signs of local irritation were noted at the achievable concentrations of 0.5, 1, 10 and 30 % (w/v), the dose levels of 1, 10 and 30% (w/v) were selected for the main test.

 

The study comprised three groups, each comprising four female animals, were treated with 50 μl (25 μl per ear) of test material as a solution in acetone/olive oil 4:1 at concentrations of 1, 10 and 30 % (w/v) for 3 consecutive days. A further control group of four animals was treated with acetone/olive oil 4:1 alone. The proliferative response of the lymph node cells (LNC) from the draining auricular lymph nodes was assessed five days following the initial application, by measurement of the incorporation of 3H-methyl Thymidine (3HTdR) by β-scintillation counting of LNC suspensions. The proliferation response of lymph node cells was expressed as the number of radioactive disintegrations per minute per group and as the ratio of 3HTdR incorporation into lymph node cells of test nodes relative to that recorded for the control nodes (Stimulation Index).

 

The SI obtained for 1, 10 and 30% v/v test item were 0.83, 0.83 and 1.25, respectively, which indicates that test item did not show the potential to induce skin sensitization. The EC3 value was therefore not calculated. There were no deaths. No signs of systemic toxicity were noted in the test or control animals during the test.  Bodyweight changes of the test animals between prior to the first application and prior to treatment with 3HTdR were comparable to those observed in the corresponding control group animals over the same period.

The historical positive control, α-Hexylcinnamaldehyde, gave a SI of 4.00, when tested at 25 % w/v. The test system was therefore considered to be valid.

 

Under the test conditions, test material is not classified as a skin sensitiser in the Local Lymph Node Assay according to the annex VI of the Regulation (EC) No. 1272/2008 (CLP) and to the GHS.

This study is considered as acceptable and satisfies the requirement for sensitisation endpoint.

Reference 2

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

No data

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

documentation insufficient for assessment

Remarks:

Non-GLP and non OECD TG442d compliant (not the recommended cell line (human MCF7 breast carcinoma cell line instead of HaCaT human keratinocytes) , no details on the test item, solvent control not reported and the nature of the solvent is not known, positive control is t-butyl-hydroquinone instead of cinnamic aldehyde)

Qualifier:

no guideline followed

Principles of method if other than guideline:

Test substance was tested for skin sensitization using in vitro Cell-based antioxidant response element (ARE) assay in AREc32 cell line derived from human MCF7 breast carcinoma cell line and assayed for luciferase activity.

GLP compliance:

not specified

Type of study:

other: in vitro Cell-based antioxidant response element (ARE)

Specific details on test material used for the study:

None

Details on the study design:

Skin sensitisation (In vitro test system)
Details on study design:
- Cell line: AREc32 cell line
- AREc32 is a stable cell line derived from the human MCF7 breast carcinoma cell line. The generation of the cell line was described by Wang et al. (2006) and the cell line has been licensed from CRX biosciences, Dundee, Scotland. AREc32 cells were maintained in Dulbecco’s modified Eagle’s medium containing glutamax (Gibco/Invitrogen, Basel, Switzerland) supplemented with 10% fetal calf serum and 500 lg/ml G418. AREc32 cell line was grown at 37°C in the presence of 5% CO2.
- Test chemical was dissolved in acetonitrile or dimethyl sulfoxide (DMSO) at a concentration of 100mM, further diluted in culture medium to a final concentration of either 10 or 2.5mM, and then serially diluted in culture medium containing an equal concentration of solvent in order to keep the solvent level constant at each test concentration.
- AREc32 cells were seeded in 96-well plates at a density of 50,000 cells per well in 180 µl of growth medium. Test chemical was added 40 h later dissolved in 20 µl of growth medium. Final solvent concentration was 0.25% in all experiments, unless a concentration range up to 1000 µM was tested: in this case solvent levels were at 1%. Cells were washed with phosphate buffered saline (PBS) without Ca2+ and Mg2+ 24 h after compound addition and then lysed by the addition of 20 µl of passive lysis buffer (Promega AG, Wallisellen, Switzerland).
- Luciferase activity was initiated by adding 50 µl of the luciferase assay substrate dissolved in luciferase assay buffer (both from Promega) to the cell lysate. Alternatively, 50 µl of assay reagent was made up according to the following recipe: 20mM tricine; 2.67mM MgSO4; 0.1mM ethylenediaminetetraacetic acid; 33.3mM dithiotreitol; 270µM coenzyme A; 470µM luciferin potassium salt (Synchem, Kassel, Germany); 530µM adenosine triphosphate; pH 7.8. Luciferase activity was measured with the GloMax luminometer (Promega).
- Tertbutyl-hydroquinone was included as a positive reference chemical in each assay plate.
- The screening on the AREc32 cell line was repeated three or four times, with duplicate analysis for each chemical at each test concentration in each repetition. In the first two repetitions four concentrations (2, 10, 50, and 250µM) were tested. In the third and fourth repetition, six binary dilutions covering the maximal noncytotoxic doses for each test chemical were selected. Wherever possible, tests up to a maximal dose of 1000µM were performed in these repetitions. For chemicals with contradictory results, further repetitions were made to clarify whether they are indeed ARE-inducers or not.
- Based on these experiments, for each test chemical the average maximal induction of gene activity (Imax) and (3) the average concentration inducing significantly enhanced gene activity above a certain threshold (EC1.5 for luciferase activity) were determined. The average relative variance between duplicates in this assay was 8.3%, and a luciferase expression of 50% above background values was statistically significant in all cases. Therefore, the threshold of 50% enhanced expression was selected as representative of significant induction. EC1.5 values were calculated accordingly with log-linear extrapolation from the values above and below the induction threshold.
- The results of the in vitro assays were scaled into five classes from 0 to 4 to give an in vitro score (non-sensitizer/weak/moderate/strong/extreme) (see Table 7.4.1/1).

Positive control results:

The positive control t-butyl-hydroquinone significantly induced luciferase activity in all assays with similar induction at the two dosing regimes: at 1% solvent and binary dilutions the average induction for t-butyl-hydroquinone compared with solvent control was 20.3-fold at 31µM and 41.4-fold at 62µM, whereas it was 31.5-fold at 50µM on the average in the experiments with 0.25% solvent.

Key result

Parameter:

other: EC1.5

Remarks:

(µM)

Value:

1 000

Vehicle controls validity:

not specified

Negative controls validity:

not specified

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Key result

Parameter:

other: Imax

Value:

1.3

Vehicle controls validity:

not specified

Negative controls validity:

not specified

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 data

DEMONSTRATION OF TECHNICAL PROFICIENCY: no data

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: no data
- Acceptance criteria met for positive control: no data
- Acceptance criteria met for variability between replicate measurements: no data
- Range of historical values if different from the ones specified in the test guideline: no data

None

Interpretation of results:

GHS criteria not met

Conclusions:

Test substance did not show evidence of skin sensitisation in an in vitro experiment.

Executive summary:

Test substance was tested for skin sensitization using in vitro Cell-based antioxidant response element (ARE) assay in AREc32 cell line derived from human MCF7 breast carcinoma cell line. Test chemical was dissolved in acetonitrile or dimethyl sulfoxide (DMSO) at a concentration of 100mM, further diluted in culture medium to a final concentration of either 10 or 2.5mM, and then serially diluted in culture medium containing an equal concentration of solvent in order to keep the solvent level constant at each test concentration. AREc32 cells were seeded in 96-well plates at a density of 50,000 cells per well in growth medium. Test chemical was added 40 h later dissolved in 20 µl of growth medium. Final solvent concentration was 0.25% in all experiments, unless a concentration range up to 1000 µM was tested: in this case solvent levels were at 1%. Cells were washed with phosphate buffered saline 24 h after compound addition and then lysed. Luciferase assay substrate was added to the cell lysate. Luciferase activity was measured with the GloMax luminometer (Promega). The screening on the AREc32 cell line was repeated three or four times, with duplicate analysis for each chemical at each test concentration in each repetition. Based on these experiments, for each test chemical the average maximal induction of gene activity (Imax) and the average concentration inducing significantly enhanced gene activity above a certain threshold (EC1.5 for luciferase activity) were determined. Tertbutyl-hydroquinone was included as a positive reference chemical in each assay plate.

The positive control t-butyl-hydroquinone significantly induced luciferase activity in all assays with similar induction at the two dosing regimes: at 1% solvent and binary dilutions the average induction for t-butyl-hydroquinone compared with solvent control was 20.3-fold at 31µM and 41.4-fold at 62µM, whereas it was 31.5-fold at 50µM on the average in the experiments with 0.25% solvent.

The test item gave an Imax of 1.3 and an EC1.5 > 1000 µM. The two values are below the threshold for sensitiser potential (i.e. Imax ≥ 1.5 and EC1.5 ≤ 1000 µM).

Therefore no evidence of skin sensitisation potential of the test item was shown under the conditions of this in vitro assay

Reference 3

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

No data

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

documentation insufficient for assessment

Remarks:

Non-GLP and non OECD TG442c compliant (no details on the test item, incubation for 24h at 30°C instead of 25±2.5°C, no calibration study reported, controls not reported (suitability of the HPLC system before the test; stability of the reference controls over time; no influence of the selected vehicle on peptidic depletion), standard deviation between the replicates not reported); not clear which solvent was used and at which concentration. Lysine-depletion was not assessed because the Lys-peptide assay involves a large concentration of test compound (25mM) and there are solubility issues with many of the hydrophobic compounds included in the current study and not all chemicals were measured with the Lys-assay.)

Qualifier:

no guideline followed

Principles of method if other than guideline:

A key step in the skin sensitization process is the formation of a covalent adduct between the skin sensitizer and endogenous proteins and/or peptides in the skin. The modified peptides then trigger a specific T-cell mediated immune response. One possibility to predict skin sensitization based on in vitro data is therefore the evaluation of the reactivity of a test compound towards peptides and proteins. The test substance was therefore tested for skin sensitization using an in chemico peptide depletion assay, in which a peptide is incubated with an excess of a test chemical, and the unmodified peptide is detected with HPLC analysis, assuming that loss of the peptide signal is due to a covalent modification by the test chemical.

GLP compliance:

not specified

Type of study:

direct peptide reactivity assay (DPRA)

Specific details on test material used for the study:

None

Details on the study design:

Skin sensitisation (In chemico test system)
Details on study design:
- The details on the tested peptide are as follows:
Name: CysReact
Sequence: Ac-RFAACAA
Sequence origin: Cys-peptide described by Gerberick et al. (2004).
- The peptide reactivity assay in HPLC vials was conducted using concentration of the test peptide of 0.25 mM and the concentration of the phosphate buffer was 20 mM.
- The samples were incubated for 24 h at 30°C with shaking at 140 rpm prior to analysis by HPLC with a DAD detector. Five microliter of the samples was injected onto a Waters polarity column, 2.1 mm ID x150 mm, 5-Micron. The mobile phase consisted of ammonium formiate (20 mM) in 5% formic acid (pH 5) (A) and methanol (B). A gradient with 85% (A) for 1 min and then from 85% (A) to 100% (B) over 17 min at a flow rate of 0.15 ml/min was used for separation. Chromatograms were extracted at 214 nm.
- Peptide depletion was expressed as the concentration of the peptide compared to control samples with the peptide in solvent only. To measure dose-response curves, reactivity assays were performed under equal conditions in microtiter plates in a final volume of 100 µl. The unreacted cysteine in the parent peptide was then determined by addition of 50 µl of a solution of monobromobimane (0.75 mM in 100 mM NaCO3, pH 8.8) and reading of the fluorescence at 385/480 nm.

Positive control results:

No data

Key result

Parameter:

other: Cysteine depletion

Remarks:

(%)

Value:

4

Vehicle controls validity:

not specified

Negative controls validity:

not specified

Positive controls validity:

not specified

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: no data

DEMONSTRATION OF TECHNICAL PROFICIENCY: no data

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: no data
- Acceptance criteria met for positive control: no data
- Acceptance criteria met for variability between replicate measurements: no data
- Range of historical values if different from the ones specified in the test guideline: no data

None

Interpretation of results:

GHS criteria not met

Conclusions:

Test substance did not show evidence of skin sensitisation in an in chemico experiment (cysteine depletion).

Executive summary:

The test substance was tested for skin sensitization potential using an in chemico peptide depletion assay, in which a peptide (CysReact) is incubated with an excess of a test chemical, and the unmodified peptide is detected with HPLC analysis, assuming that loss of the peptide signal is due to a covalent modification by the test chemical. The assay was conducted using concentration of the test peptide of 0.25 mM. The samples were incubated for 24 h at 30°C with shaking prior to analysis by HPLC. Chromatograms were extracted at 214 nm. Peptide depletion was expressed as the concentration of the peptide compared to control samples with the peptide in solvent only. To measure dose-response curves, reactivity assays were performed under equal conditions in microtiter plates in a final volume of 100 µl. The unreacted cysteine in the parent peptide was then determined by addition a solution of monobromobimane and reading of the fluorescence at 385/480 nm.

The test item gave a cysteine depletion of 4% thus lower than the threshold of 15% determined in this study. Therefore a very low adduct formation is expected between cysteine and the test item.

Therefore no evidence of skin sensitisation potential of the test item was shown under the conditions of this in chemico assay.

Reference 4

Endpoint:

skin sensitisation, other

Type of information:

(Q)SAR

Adequacy of study:

supporting study

Study period:

28 September 2016

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:

1. SOFTWARE
OASIS TIMES v.2.27.19

2. MODEL (incl. version number)
Skin sensitization with autoxidation v.21.26

3. SMILES OR OTHER IDENTIFIERS USED AS INPUT FOR THE MODEL
Cc1ccc2c(c1)OCC(=O)CO2

4. SCIENTIFIC VALIDITY OF THE (Q)SAR MODEL
See QMRF attached

- Defined endpoint:
Species : Mouse; guinea pigs
Endpoint : In vivo: skin sensitization
Endpoint units : LLNA – EC3, % ; GPMT - % of animals showing reaction of skin
Dependent variable : Obs. Skin Sensitization effect
Experimental protocol : LLNA (the murine local lymph node assay); GPMT (the guinea pig maximization test)
Endpoint data quality and variability : High quality. The model was derived from a data set compiled from chemicals tested in the LLNA, GPMT as well as from the BfR (formerly BgVV) list

- Unambiguous algorithm:
Type of model : (Q)SAR
Explicit algorithm : TIMES-SS model aims to encode structure toxicity and structure metabolism relationships through a number of transformations simulating skin metabolism and interaction of the generated reactive metabolites with skin proteins. The skin metabolism simulator mimics metabolism using 2D structural information. The autoxidation (abiotic oxidation) of chemicals is also accounted for. A training set of diverse chemicals was compiled and their skin sensitization potency assigned to one of three classes. These three classes were Strong, Weak or Non sensitizing.
Descriptors in the model : To assess the reactivity of specific alerting group requiring additional 3D QSAR models, the following descriptors were used:
- EHOMO - Energy of the Highest Occupied Molecular Orbital, [eV]
- ELUMO - Energy of the Lowest Unoccupied Molecular Orbital, [eV]
- Molecular weight (MW)
- Electronegativity – 0.5*(EHOMO –ELUMO), [eV]
- E_GAP – (EHOMO –ELUMO), [eV]
- Log Kow
- ACCEPT_DLC – Acceptor superdelocalizability
Descriptor selection : Descriptors were selected by using the probabilistic approach for identifying common stereoelectronic (reactivity) patterns of the chemicals – COREPA [3].
Algorithm and descriptor generation : The COREPA (COmmon REactivity PAttern) method [3] was used to derive the sub-models incorporated in the TIMES-SS models. It is a probabilistic technique for identifying common stereoelectronic (reactivity) patterns of structurally diverse chemicals which may exert similar or differential biological effects. All energetically reasonable conformers are used to establish conformer distributions across the global and local stereoelectronic descriptors associated with the activity of studied chemicals. The COREPA model is derived in the form of a decision tree. Its logic boxes consist of decision rules based on the reactivity patterns described by a combination of global descriptors of molecular steric and electronic structure and local reactivity parameters associated with specific alerting groups.
Two additional 3D QSAR models implemented into the TIMES-SS model were derived for predicting skin sensitization potential of Aldehydes and Michael acceptors:
1. 3D QSAR model distinguishing skin sensitizing from not skin sensitizing aldehydes:
- The chemicals used to derive the model were aldehydes;
- The decision tree is consisted of one node separating Strong from Weak and non-sensitizing aldehydes based on calculated EHOMO in the ranges [-11.7, -8.22] [-11.7, -11] and MW in the ranges [13.6, 184] [250, 254].
2. 3D QSAR model distinguishing skin sensitizing from not skin sensitizing Michael acceptors:
- The chemicals used to derive the model were Michael acceptor having a double bond adjacent to electron-withdrawing group.
- The decision tree is consisted of two nodes. The first one separates Strong from Weak and Non-sensitizing chemicals based on calculated ELECTRONEGATIVITY in the ranges [-5.13, -4.23] [-6.02, -5.42] and E_GAP in the ranges [8.07, 10.5] [10, 10.9]. The second one separates Weak from Non-sensitizing Michael acceptors based on calculated Log(Kow) in the ranges [0.68, 6.53] [-1.61, -0.583] and ACCEPT_DLC in the ranges [0.229, 0.275] [0.233, 0.242]
Software name and version for descriptor generation : COREPA-M software developed at Laboratory of Mathematical Chemistry

- Defined domain of applicability:
Description of the applicability domain of the model : The applicability domain of TIMES-SS model consists of the following layers:
1. General parametric requirements - includes ranges of variation of log KOW and MW. It specifies in the domain only those chemicals that fall in the range of variation of the MW and log Kow defined on the bases of the correctly predicted training set chemicals.
This layer of the domain is applied only on parent chemicals.
2. Structural domain - it is represented by list of atom - centered fragments extracted from the chemicals in the training set. The training chemicals were split into two subsets: chemicals correctly predicted by the model and incorrectly predicted chemicals. These two subsets of chemicals were used to extract characteristics determining the "good" and "bad" space of the domain. Extracted characteristics were split into three categories: unique characteristics of correct and incorrect chemicals (presented only in one of the subsets) and fuzzy characteristics presented in both subsets of chemicals.
Structural domain is applied on parent chemicals, only.
3. Mechanistic domain - in SS model it includes:
- Interpolation space: this stage of the applicability domain of the model holds only for chemicals for which an additional COREPA model is required. It estimates the position of the target chemicals in the population density plot built in the parametric space defined by the explanatory variables of the model by making use the training set chemicals. Currently, the accepted threshold of population density is 10%.
The mechanistic domain is applied on the parent structures and on their metabolites.
Methods used to assess the applicability domain : A stepwise approach for determining the applicability domain of the TIMES-SS model is proposed, distinguishing chemicals for which the model provides highly reliable predictions.
General parametric requirements are imposed in the first stage, specifying in the domain only those chemicals that fall in the range of variation of the physicochemical properties of the chemicals in the training set. The second stage defines the structural similarity between chemicals that are correctly predicted by the model. The structural neighborhood of atom-centered fragments is used to determine this similarity. The third stage in defining the domain is based on a mechanistic understanding of the modeled phenomenon. Here, the model domain combines the reliability of specific reactive groups hypothesized to cause the effect and the domain of explanatory variables determining the parametric requirements in order for functional groups to elicit their reactivity.
Software name and version for applicability domain assessment : Domain Manager v.1.11 developed at Laboratory of Mathematical Chemistry University, "Prof. Assen Zlatarov," 1 Yakimov Str., Bourgas 8010, BULGARIA
Limits of applicability : In order to belong to the model applicability domain a target structure must meet the requirements of all the domain layers.

- Appropriate measures of goodness-of-fit and robustness and predictivity:
Availability of the training set : Yes, but not attached
Available information for the training set : Training set consists of 875 chemicals (not attached)
CAS RN: yes
Chemicals Name: yes
SMILES: yes
Formula: yes
INChi: no
MOL file: no
Other information about the training set : The current skin sensitization model was developed using a dataset of 875 chemicals tested by Local Lymph Node Assay (LLNA), Guinea Pig Maximization Test (GPMT) and chemicals from the BfR list.
A unifying scale was derived evaluating the correlation and concordance of those chemicals that existed in all three datasets:
Unified skin sensitization scale LLNA GPMT BfR
Strong Extreme, Strong & Moderate Strong & Moderate Category A
Weak Weak Weak Category B
Non Non Non Category C


The distribution of training set chemicals having skin sensitization experimental data among the sensitization classes is as follows:
- 401 are Strong skin sensitizers
- 193 are Weak skin sensitizers
- 281 are Non skin sensitizers
Statistics for goodness-of-fit
Statistic of model: For 875 chemicals, the TIMES-SS model was able to predict correctly 90% of the strong sensitizers, 55% of the weak sensitizers and 77% of the non-sensitizers, i.e., an overall performance of 78 %.
Sensitivity: 78 %
Specificity: 77 %

- Mechanistic interpretation:
Mechanistic basis of the model
The TIMES-SS (Tissue Metabolism Simulator for skin sensitization) model integrates a simulator of skin metabolism together with a number of “local” QSAR models for assessing the reactivity of specific alerts. A skin metabolism simulator was developed based on empirical and theoretical knowledge (not enough reported observed skin metabolism data). The transformation probabilities (defining the priority of their execution) were parameterized to reproduce skin sensitization data. The simulator comprises of about 420 transformations, which can be divided into four main types: abiotic transformations, covalent interaction with proteins, Phase I and Phase II reactions. Autoxidation (AU) of chemical is also accounted for. Interactions with skin proteins are grouped into three types: leading to strong or weak skin sensitization effect and interactions requiring QSAR models to quantify the potency of sensitization of the alerting groups. The QSAR models were developed by the COmmon
PAttern Recognition (COREPA) approach [3]. The skin sensitization model predicts skin sensitization effect in three classes: strong, weak and non-sensitizers.
Reliability of alerts in the TIMES-SS model has been also evaluated to provide transparent mechanistic reasoning for predicting sensitization potential. Alert performance was defined as the ratio between the number of correct (positive and negative) predictions and the total number of chemicals within the local training set that triggered the alert. The alert performance was assessed based on the predictions on parents, autoxidation products simulated by the external AU simulator and metabolites as simulated by the skin metabolism simulator embedded in TIMES-SS model. Four different categories of reliability were defined:
- High reliability – alert performance higher than 60% and more than 5 chemical in local (transformation/alert) training set
- Low reliability – performance less than 60% and more than 5 chemicals in training set
- Undetermined reliability – less than 5 chemicals in training set
- Undetermined (theoretical) – there are no chemicals supporting the alert in the local training set

5. APPLICABILITY DOMAIN
See QPRF attached

- Descriptor domain: The chemical fulfils the general properties requirements

- Structural and mechanistic domains: The following ACF are identified:
Fragments in correctly predicted training chemicals – 100.00%
Fragments in non-correctly predicted training chemicals – 0.00%
Fragments not present in the training chemicals – 0.00%
The chemical is in the interpolation structural space

- Similarity with analogues in the training set: not reported

- Other considerations (as appropriate): Model predictions are based on simulation of skin metabolism and covalent interactions with proteins.

6. ADEQUACY OF THE RESULT
OASIS TIMES evaluation showed no alerts for skin sensitisation. The substance and its metabolites (based on simlation of skin metabolism) are predicted to be not skin sensitiser in vivo. This result fits well with the results of an in vivo test performed on the substance (LLNA, RCC, 2005, Rel.2) as well as in silico (In vitro peptide depletion, Natsch, 2009, Rel.4) and in vitro tests (In vitro ARE, Natsch, 2009, Rel.4).

Reason / purpose for cross-reference:

reference to other study

Reason / purpose for cross-reference:

reference to other study

Reason / purpose for cross-reference:

reference to other study

Principles of method if other than guideline:

TIMES (TIssue MEtabolism Simulator) model for Skin sensitization (TIMES-SS model) - Skin sensitization with autoxidation v.21.26

GLP compliance:

no

Type of study:

other: QSAR

Justification for non-LLNA method:

In vivo data available

Key result

Parameter:

other: Fragments in correctly predicted training chemicals

Value:

100

Remarks on result:

no indication of skin sensitisation

Outcome: Not a skin sensitiser within applicability domain

Conclusion: Not a skin sensitiser

Applicability domain: The chemical fulfils the general properties requirements.

Fragments in correctly predicted training chemicals – 100.00%

Fragments in non-correctly predicted training chemicals – 0.00%

Fragments not present in the training chemicals – 0.00%

The chemical is in the interpolation structural space.

Interpretation of results:

GHS criteria not met

Conclusions:

OASIS TIMES evaluation showed no skin sensitisation alert. The substance falls inside the applicability domain of the model.

Executive summary:

OASIS TIMES v. 2.27.19 software was used to predict the skin sensitisation potential of the substance and its predited metabolites (based on simulation of skin metabolism).

No structural alert was identified for skin sensitisation (not skin sensitiser). The substance falls inside the applicability domain of the model.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

A key study was identified (RCC, 2005, rel.2). This Local Lymph Node Assay was conducted according to the OECD test guideline No 429 and in compliance with GLP. Following a preliminary screening test in which no clinical signs of toxicity and no signs of local irritation were noted at the technically achievable concentrations of 0.5, 1, 10 and 30 % (w/v) [limited by the physical state of the substance - solid], the dose levels of 1, 10 and 30% (w/v) were selected for the main test. Three groups, each of four females, were treated with the test material as a solution in acetone/olive oil 4:1 at concentrations of 1, 10 and 30 % v/v for 3 consecutive days. A further group of four animals was treated with acetone/olive oil 4:1 alone.

The historical positive control, α-Hexylcinnamaldehyde, gave a SI of 4.00, when tested at 25 % w/v. The test system was therefore considered to be valid. Stimulation index for 10, 25 and 50 % v/v in acetone/olive oil 4:1 were 0.83, 0.83 and 1.25, respectively. There were no deaths. No signs of systemic toxicity or skin irritation were noted in the test or control animals during the test. Bodyweight changes of the test animalsbetweenprior to the first application and prior to treatment with 3HTdR were comparable to those observed in the corresponding control group animals over the same period. Under the test conditions, the test material is not a skin sensitizer.

Two supporting in chemico/in vitro studies were also identified as reported in a scientific publication (Natch, 2009, rel.4).

- In the in chemico assay, the test substance was tested for skin sensitization potential using a cysteine depletion assay, in which a peptide (CysReact) is incubated with an excess of a test chemical, and the unmodified peptide is detected with HPLC analysis, assuming that loss of the peptide signal is due to a covalent modification by the test chemical. Peptide depletion was expressed as the concentration of the peptide compared to control samples with the peptide in solvent only. The test item gave a cysteine depletion of 4% thus lower than the threshold of 15% determined in this study. Therefore a very low adduct formation is expected between cysteine and the test item.

- In the in vitro assay, the test substance was tested for skin sensitization using a Cell-based antioxidant response element (ARE) assay in AREc32 cell line derived from human MCF7 breast carcinoma cell line. Test chemical was dissolved at a concentration of 100mM, further diluted in culture medium to a final concentration of either 10 or 2.5mM, and then serially diluted in culture medium containing an equal concentration of solvent in order to keep the solvent level constant at each test concentration. AREc32 cells were seeded and test chemical was added 40 h later and incubated for 24 h. After washing and lysis of the cells, Luciferase assay substrate was added to the cell lysate and Luciferase activity was measured with the GloMax luminometer (Promega). The screening on the AREc32 cell line was repeated three or four times, with duplicate analysis. Based on these experiments, for each test chemical the average maximal induction of gene activity (Imax) and the average concentration inducing significantly enhanced gene activity above a certain threshold (EC1.5 for luciferase activity) were determined. Tertbutyl-hydroquinone was included as a positive reference chemical in each assay plate. The positive control t-butyl-hydroquinone significantly induced luciferase activity in all assays . The test item gave an Imax of 1.3 and an EC1.5 > 1000 μM. The two values are below the threshold for sensitiser potential (i.e. Imax ≥ 1.5 and EC1.5 ≤ 1000 μM).

Therefore no evidence of skin sensitisation potential of the test item was shown under the conditions of those two in chemico/in vitro assays.

The OASIS TIMES v. 2.27.19 software was used to predict the skin sensitisation of the substance and its predicted metabolites (based on simulation of skin metabolism). No structural alert was identified for skin sensitisation (not skin sensitiser). The substance falls inside the applicability domain of the model. Moreover, although the metabolic transformation of the substance in liver produces metabolites which could damage proteins and cause skin sensitization, these metabolites cannot be generated in skin due to much lower activities of enzymes in skin as compared with liver. The metabolite of the substance in skin does not possess reactive functionality which could damage proteins and cause skin sensitization (See the attached document " Expert review of capability of Calone to metabolise in skin and liver").

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Harmonized classification:

The substance has no harmonized classification according to the Regulation (EC) No. 1272/2008.

Self classification:

Based on the available data, the substance is not classified as skin sensitiser according to the Regulation (EC) No. 1272/2008 (CLP) and to the GHS.

No data was available for respiratory sensitisation. However, this substance is not a skin sensitizer, therefore according to Figure R.7.3 -2 of the Chapter R.7 (V 4.1 - October 2015) the chemical is not considered as a respiratory sensitizer.