2-methoxy-4-propylphenol

Endpoint summary

Description of key information

Skin sensitisation: Sensitising, WoE (LLNA, GPMT, QSARs)

Respiratory sensitisation: The currently identified mechanisms of dermal and respiratory sensitization may be sufficiently different, and exposure levels are significantly lower than the probable minimum threshold level required for induction and/or elicitation to prevent any risk of respiratory sensitization. This conclusion is based on various elements of scientific evidence that together constitute a robust argument and obviate the need to conduct further specific studies to investigate the potential for respiratory sensitization of this substance. It is noted that, even if it was considered appropriate to conduct further testing, there is currently no available or suitable validated method that could be used.

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:

weight of evidence

Study period:

No data

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

documentation insufficient for assessment

Remarks:

Basic data given but study reported to be performed according to OECD 429 with deviations: purity of test item not reported, number of animals per group, positive controls and body weight not reported; no ear thickness measurement nor excessive irritation assessment. Considered sufficiently reliable in a weight of evidence for the purpose of hazard assessment.

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

details on environmental conditions of animal room, number of animals per group, positive controls and body weight not reported; no ear thickness measurement nor excessive irritation assessment

Principles of method if other than guideline:

Not applicable

GLP compliance:

not specified

Type of study:

mouse local lymph node assay (LLNA)

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Age at study initiation: 7-12 weeks

Vehicle:

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

Concentration:

5.1, 10.1 and 25.3 % in 4:1 acetone/olive oil

No. of animals per dose:

No data

Details on study design:

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Local Lymph Node Assay
- Criteria used to consider a positive response: A substance was classified as a skin sensitizer if, at one or more test concentrations, it induced a 3-fold or greater increase in local lymph node proliferative activity when compared with concurrent vehicle-treated controls (SI, ≥ 3).

TREATMENT PREPARATION AND ADMINISTRATION:
25 µL of control or test material was applied to the dorsum of both ears daily for three consecutive days. Five days after the initiation of exposure, all animals were injected via the tail vein with 250 µL of phosphate-buffered saline containing 20 µCi of tritiated thymidine. Animals were sacrificed 5 h later and the draining auricular lymph nodes were excised and pooled for each experimental group or each individual animal. The incorporation of tritiated thymidine measured by beta scintillation counting was reported in disintegrations per minute (DPM). Stimulation index (SI) was calculated for each treated group as the ratio of the DPM in the treated group (or mean DPM when individual animals were assessed) to the DPM or mean DPM of the concurrent vehicle control group.

Positive control substance(s):

not specified

Statistics:

No data

Positive control results:

No data

Key result

Parameter:

EC3

Value:

6.8

Parameter:

SI

Value:

2.7

Test group / Remarks:

5.1%

Parameter:

SI

Value:

3.6

Test group / Remarks:

10.1%

Parameter:

SI

Value:

7.8

Test group / Remarks:

25.3%

Cellular proliferation data / Observations:

CELLULAR PROLIFERATION DATA
Not reported

DETAILS ON STIMULATION INDEX CALCULATION
Stimulation index for test material at 5.1, 10.1 and 25.3 % were 2.7, 3.6 and 7.8, respectively.

EC3 CALCULATION
Calculated EC3 value for the test material was found to be 6.8 %

CLINICAL OBSERVATIONS:
Not reported

BODY WEIGHTS
Not reported

Interpretation of results:

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

Conclusions:

Under the test conditions, test material is classified as “Category 1B” moderate skin sensitiser according to the annex VI of the Regulation EC No. 1272/2008 (CLP) and to the GHS. Due to the lack of reported details (purity of test item, number of animals per group, positive controls and body weight not reported; no ear thickness measurement nor excessive irritation assessment reported), the study was not used in its own to classify the substance but it was considered sufficiently reliable in a weight of evidence for the purpose of hazard assessment.

Executive summary:

In a Local Lymph Node Assay (LLNA), groups of female CBA mice were topically applied with test material at the dose concentrations of 5.1, 10.1 and 25.3 % final concentration in 1:4 acetone:olive oil to the dorsum of both ears (25 µL/ear) daily for three consecutive days. A vehicle control group was treated with 1:4 acetone:olive oil alone in the same manner. Five days after the initiation of exposure, all animals were injected via the tail vein with 250 µL of phosphate-buffered saline containing 20 µCi of tritiated thymidine. Tritiated thymidine incorporation was quantified using a beta scintillation counter and stimulation index (Sl) was calculated for each dose group.

Stimulation Index (SI Value) calculated for test material treated groups were found to be 2.7, 3.6 and 7.8 for the dose concentrations of 5.1, 10.1 and 25.3 %, respectively. Calculated EC3 value for the test material was found to be 6.8 %.

Under the test conditions, test material is classified as “Category 1B” skin sensitiser according to the annex VI of the Regulation EC No. 1272/2008 (CLP) and to the GHS.

Due to the lack of reported details (purity of test item, number of animals per group, positive controls and body weight not reported; no ear thickness measurement nor excessive irritation assessment reported), the study was not used in its own to classify the substance but it was considered sufficiently reliable in a weight of evidence for the purpose of hazard assessment.

Reference 2

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

From August 17 to September 30, 1988

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

documentation insufficient for assessment

Remarks:

Pre-guideline and pre-GLP study. Only basic data given but the study is performed similarly to OECD TG 406 with some deviations: three injections instead of three pairs for intradermal induction and absence of positive control. Considered sufficiently reliable in a weight of evidence for the purpose of hazard assessment.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Deviations:

yes

Remarks:

no data available on positive control, patch site preparation and patch-technique not reported; three injections instead of three pairs for intradermal induction

Principles of method if other than guideline:

not applicable

GLP compliance:

not specified

Type of study:

guinea pig maximisation test

Justification for non-LLNA method:

At the time of study completion (1988), the LLNA OECD test method was not adopted.

Species:

guinea pig

Strain:

not specified

Sex:

male/female

Details on test animals and environmental conditions:

TEST ANIMALS
- Weight at study initiation: Males: 309-367 g; females: 317-351 g
- Diet: RGP pellets, hay and cabbage, ad libitum
- Water: Water, ad libitum

IN-LIFE DATES: From: August 17, 1988 To: September 30, 1988.

Route:

intradermal

Vehicle:

other: 0.01 % Dobbs in saline

Concentration / amount:

0.1%

Day(s)/duration:

Day1

Adequacy of induction:

highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically

Route:

epicutaneous, occlusive

Vehicle:

other: acetone/PEG400 for topical challenge

Concentration / amount:

100%

Day(s)/duration:

Days 6-7 / 48 hours

Adequacy of induction:

highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically

No.:

#1

Route:

epicutaneous, occlusive

Vehicle:

other: acetone/PEG400

Concentration / amount:

25%

Day(s)/duration:

12-14 days after the completion of topical induction application/24h

Adequacy of challenge:

highest non-irritant concentration

No.:

#2

Route:

epicutaneous, occlusive

Vehicle:

other: acetone/PEG400

Concentration / amount:

25%

Day(s)/duration:

12-14 days after the completion of topical induction application/24h

Adequacy of challenge:

highest non-irritant concentration

No.:

#3

Route:

epicutaneous, occlusive

Vehicle:

other: acetone/PEG400

Concentration / amount:

2.5%

Day(s)/duration:

24 hours

No. of animals per dose:

10 animals/test group (6 females and 4 males)
8 animals/control group (4 females and 4 males)

Details on study design:

RANGE FINDING TESTS:
- Intradermal injection ranging study: Test material at 0.1, 0.2, 0.5, 1.0 and 2.0 % in 0.01 % Dobbs/saline were injected intradermally into shoulder region of four females. Animal were observed for a period of 6-7 days and any response noted at each injection site. The concentration selected for the main study was 0.1 % in 0.01 % Dobbs/saline.
- Topical irritancy ranging study: Four animals were topically applied with test material at 10, 25 and 50 % in acetone/PEG-400 under occlusive patch for 48 h. Animals were evaluated for skin reactions at 24 and 48 h after dressing removal. Undiluted test material (100 %) was selected for topical induction as 50 % was not sufficiently irritant. Highest non-irritant concentration 25 % was selected for challenge application.

MAIN STUDY
A. INDUCTION EXPOSURE: INTRADERMAL
- No. of exposures: Three
Three intradermal injections were made; (1) Freund’s adjuvant in Dobbs saline, (2) test material (0.1%) in Dobbs saline, and (3) test material (0.1%) and Freund’s adjuvant in Dobbs

B. INDUCTION EXPOSURE: TOPICAL
- No. of exposures: One, Days 6-7
- Exposure period: 48 h
- Test groups: Undiluted test material (100 %) under occlusive patches.

C. CHALLENGE EXPOSURE:
- No. of exposures: Four
- Day(s) of challenge: 12-14 days after the completion of topical induction application.
- Exposure period: 24 h
- Test groups:
Challenge 1 and 2: 25 % of test material in acetone/PEG-400 under occlusive patches.
- Control groups:
Challenge 1 and 2: Vehicle acetone/PEG-400 under occlusive patches.
- Evaluation (h after challenge): 24 and 48 h

OTHER: Skin reactions sites were examined 24 and 48 h after removal of patches and scored.

Challenge controls:

No data

Positive control substance(s):

no

Positive control results:

None

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

25 % in acetone/PEG-400

No. with + reactions:

8

Total no. in group:

10

Clinical observations:

Not reported

Remarks on result:

positive indication of skin sensitisation

Key result

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

25 % in acetone/PEG-400

No. with + reactions:

5

Total no. in group:

10

Clinical observations:

Not reported

Remarks on result:

positive indication of skin sensitisation

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

0 %

No. with + reactions:

0

Total no. in group:

4

Clinical observations:

Not reported

Remarks on result:

no indication of skin sensitisation

Key result

Reading:

2nd reading

Hours after challenge:

48

Group:

negative control

Dose level:

0 %

No. with + reactions:

0

Total no. in group:

4

Clinical observations:

Not reported

Remarks on result:

no indication of skin sensitisation

Key result

Reading:

rechallenge

Hours after challenge:

24

Group:

test chemical

Dose level:

25 % in acetone/PEG-400

No. with + reactions:

7

Total no. in group:

10

Clinical observations:

Not reported

Remarks on result:

positive indication of skin sensitisation

Key result

Reading:

rechallenge

Hours after challenge:

48

Group:

test chemical

Dose level:

25 % in acetone/PEG-400

No. with + reactions:

7

Total no. in group:

10

Clinical observations:

Not reported

Remarks on result:

positive indication of skin sensitisation

Key result

Reading:

rechallenge

Hours after challenge:

24

Group:

negative control

Dose level:

0 %

No. with + reactions:

0

Total no. in group:

4

Clinical observations:

Not reported

Remarks on result:

no indication of skin sensitisation

Key result

Reading:

rechallenge

Hours after challenge:

48

Group:

negative control

Dose level:

0 %

No. with + reactions:

0

Total no. in group:

4

Clinical observations:

Not reported

Remarks on result:

no indication of skin sensitisation

Key result

Reading:

rechallenge

Hours after challenge:

24

Group:

test chemical

Dose level:

2.5%

No. with + reactions:

3

Total no. in group:

10

Clinical observations:

Not reported

Remarks on result:

positive indication of skin sensitisation

Group:

positive control

Remarks on result:

not measured/tested

None

Interpretation of results:

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

Conclusions:

Under the test conditions, test material is classified as strong skin sensitiser (Category 1A) according to the annex VI of the Regulation EC No. 1272/2008 (CLP) and to the GHS since ≥ 30 % animals responded at ≤ 0,1 % intradermal induction dose. The study was not used in its own to classify the substance but it was considered sufficiently reliable in a weight of evidence for the purpose of hazard assessment.

Executive summary:

In a Magnusson & Kligman maximisation study (GPMT), sensitization was induced in guinea pigs by intradermal injections of 0.1 % test material and Freund's Complete Adjuvant. The induction process was supplemented 6-7 days later by an application of the undiluted (100 %) test substance to the shoulder injection sites under occlusive patches for 48 h. Twelve-14 days later, animals were challenged by 25 % of test material in acetone/PEG-400 under occlusive patches. Further challenges were made at weekly intervals. The test concentrations for the main study were determined from a range finding study.

Evidence of strong sensitisation was observed at challenge-1 that was further confirmed in challenge-2. Weak sensitisation response was observed when animals were challenged with 2.5 % of test material in subsequent challenge.

Under the test conditions, test material is classified as strong skin sensitiser (Category 1A) according to the annex VI of the Regulation EC No. 1272/2008 (CLP) and to the GHS since ≥ 30 % animals responded at ≤ 0,1 % intradermal induction dose. The study was not used in its own to classify the substance but it was considered sufficiently reliable in a weight of evidence for the purpose of hazard assessment.

Reference 3

Endpoint:

skin sensitisation: in chemico

Type of information:

(Q)SAR

Adequacy of study:

weight of evidence

Study period:

31 March 2015

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.2.27.16

2. MODEL (incl. version number)
Skin sensitization with autoxidation v. 20.23

3. SMILES OR OTHER IDENTIFIERS USED AS INPUT FOR THE MODEL
CCCc1ccc(O)c(OC)c1

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

Algorithm and descriptor generation :
The COREPA (COmmon REactivity PAttern) method 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]

- 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 logKow 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:
Ø 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 the applicability domain assessment: Domain Manager v.1.09 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: The training set consisting of 875 chemicals (not attached).
Available information for the training set: Chemical names, CAS numbers, SMILES and formula are available.
Data for each descriptor variable for the training set: Not provided
Data for the dependent variable for the training set:Not provided

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:
- 398 are Strong skin sensitizers
- 193 are Weak skin sensitizers
- 284 are Non skin sensitizers

Statistics for goodness-of-fit
Statistic of model: For 875 chemicals, the TIMES-SS model was able to predict correctly 91% of the strong sensitizers, 51% of the weak sensitizers and 69% of the non-sensitizers, i.e., an overall performance of 75 %.
Sensitivity: 77 %
Specificity: 69 %

Availability of the external validation set:External validation of the model was done by using a set of chemicals having skin sensitization data (Dimitrov et al., 2005)

- 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 (Mekenyan et al., 2004). 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: Analogue chemicals found in the training set are available in Appendix 5. No additional comments on structural analogues are provided by the author of prediction
- Other considerations (as appropriate): Comments on the mechanistic interpretation of the model prediction are not provided by the author of prediction.

6. ADEQUACY OF THE RESULT
OASIS TIMES evaluation showed an alert strong skin sensitisation. The substance is predicted to be a strong skin sensitiser in vivo. This prediction that the substance is a skin sensitiser supports the results of in vivo tests performed on the test substance (LLNA and GPMT).

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.20.23

GLP compliance:

no

Type of study:

other: QSAR

Key result

Parameter:

other: OASIS TIMES v. 2.27.16 prediction

Vehicle controls validity:

not applicable

Negative controls validity:

not applicable

Positive controls validity:

not applicable

Remarks on result:

positive indication of skin sensitisation

Predicted value (model result): Strong sensitiser

Concomitant predictions :

Transformation = Michael type addition on quinone methide (imine)

Active alert = Quinone methide(s)/imines, Quinoide oxime structure, Nitroquinones,

Transformation reliability = High, >= 60% (n>=5)

Alert performance = High, >= 60% (n>=5)

Vapour pressure, Pa = 0.2760

Active is = Metabolite

# activ transform = 90

AmountAduct/mol/ = 0.3166

 

Applicability domain (OECD Principle 3):

The chemical fulfils the general properties requirements

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%

Interpretation of results:

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

Conclusions:

OASIS TIMES evaluation showed skin sensitisation alert. The substance falls inside the applicability domain of the model.

Executive summary:

OASIS TIMES v. 2.27.16 software was used to predict the skin sensitisation of the substance.

Structural alert was identified for skin sensitisation (strong sensitiser). The substance falls inside the applicability domain of the model.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Additional information:

A weight-of evidence approach using the LLNA result and GPMT result on the registered substance and one QSAR was considered sufficiently robust to conclude on the skin sensitisation potential of the substance.

 

In a Local Lymph Node Assay (LLNA) (Gerberick, 2005, rel.4) performed according to OECD Test Guideline No. 429, groups of female CBA mice were topically applied with test material at the dose concentrations of 5.1, 10.1 and 25.3 % in 1:4 acetone:olive oil. A vehicle control group was treated with 1:4 acetone:olive oil alone in the same manner. Main deviations from the guideline include the absence of positive controls and ear thickness mesurements. However, the absence of positive control is not considered to reduce the reliability of this study since 211 chemicals were included in the trial, and Isoeugenol (used as a worst-case source substance for some endpoints - see Iuclid section 13 for read-across justification) was correctly identified as a Skin sensitizer 1A).

Stimulation Index (SI Value) calculated for test material treated groups were found to be 2.7, 3.6 and 7.8 for the dose concentrations of 5.1, 10.1 and 25.3 %, respectively. Calculated EC3 value for the test material was found to be 6.8 %.

Under the test conditions, the test material is a moderate skin sensitiser (Category 1B).

 

In a Magnusson & Kligman maximisation study (GPMT) (URL, 1988, rel. 4) performed similarly to OECD Test Guideline No. 406, guinea pigs were injected 0.1 % test material and Freund's Complete Adjuvant by intradermal injections. The induction process was supplemented 6-7 days later by an application of the undiluted (100 %) test substance to the shoulder injection sites under occlusive patches for 48 h. Twelve-14 days later, animals were challenged by 25 % of test material in acetone/PEG-400 under occlusive patches. Further challenges were made at weekly intervals. The test concentrations for the main study were determined from a range finding study. Main deviations from the current OECD Test Guideline No. 406 include three injections instead of three pairs for intradermal induction and the absence of positive control.

Evidence of strong sensitisation was observed at challenge-1 that was further confirmed in challenge-2. Weak sensitisation response was observed when animals were challenged with 2.5 % of test material in subsequent challenge.

Under the test conditions, the test material is a strong skin sensitiser (Category 1A) since ≥ 30 % animals responded at ≤ 0,1 % intradermal induction dose.

 

OASIS TIMES predictions is "strong sensitiser" (Rel. 2). It should be noted that aforementioned LLNA study is included in the training set of the model. The prediction complements the observed data with the substance falling inside the applicability domain of the model.

 

Other published studies were used to support the classification of the registered substance as skin sensitizer:

- A study evaluated a peptide reactivity assay to predict fragrance allergens in vitro (Natsch,2007). The peptide reactivity assay in HPLC vials was conducted as described by Gerberick et al. (2004a) with modifications as follows. The test peptide was used at a concentration of 0.25 mM. The concentration of the phosphate buffer was 20mM. These changes resulted in less observed peptide precipitation. Samples were incubated at 30°C for 24 h with shaking at 140 rpm prior to HPLC analysis with a DAD dector. A aliquot of 5 µl of test samples were injected onto a Waters polarity column (5-micron).

Chromatograms were extracted at 214 nm. Peptide depletion was expressed as the concentration of 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 with a final volume of 100 µl. Unreacted cysteine in the parent peptide was determined by adding 50 ul of a solution of monobromobimane (0.75 mM in 100 mM NaCO3, pH 8.8) and reading the fluorescence at 385/480 nm. GSH depletion assays were performed under equal incubation conditions as for other peptides, but unreacted GSH was detected with the enzymatic test described by Tietze (1969). To detect adducts of the peptides with the chemicals, LC-MS analysis was performed on a Finnigan LCQ classic Mass spectrometer (Thermo Finnigan, USA). Mass spectra were recorded from 200 to 2000 amu. Peptides Cor1-C420 and Cys-react were run on specialized columns. The test article was used undiluted. The test article caused 0 ± 0.8% GSH depletion and 0.8 ± 2.89% CysReact depletion. Under the test conditions, the prediction is negative. However, the peptide reactivity assay performed by Natsch was found to have high reproducibility and was a good positive predictor of moderate and strong allergens, but it is not currently optimized for weak sensitizers, which seems to be the case for Dihydroeugenol. Furthermore the OASIS TIMES (v2.27.17) prediction indicates that the skin metabolites, rather than the parental substance, contribute to the skin sensitisation alerts. Since the peptide reactivity assay do not support the metabolic activation system, it could not be precluded that the outcomes of the assay may have underestimated the intrinsic skin sensitisation potency of Dihydroeugenol.

- Another Magnusson-Kligman maximization test was conducted for a group of 10 guinea pigs (Barrat, 1992). Cross-challenges were carried out at weekly intervals on alternate flanks using test materials at their maximum nonirritant concentration This study was designed to evaluate possible reaction mechanisms between chemical haptens and skin protein. Cross reactions were seen with induction performed with dihydroeugenol and challenged with eugenol and isoeugenol. Evidence of sensitization in 80% of the test guinea pigs was observed at challenge with 25% dihydroeugenol. The mean erythema score in positive guinea pigs was 1.4 indicating dihydroeugenol as a moderate sensitizer in guinea pigs. Examination of the possible reaction mechanisms allows the speculation that dihydroeugenol reacts via a phenolic radical mechanism and via formation of an orthoquinone

- The frequency of cross reactions on patients sensitive to Isoeugenol was studied in a Human Patch test performed on 8 volunteers (Itoh,1982). Dihydroeugenol in petrolatum (0.2%, 1% or 5%)) was applied under a 48 hour closed patch on the upper backs of 8 patients who exhibited moderately strong or strong positive reactions to Isoeugenol. Reactions were graded 72 hours after application. Cross reaction was only observed at 5%, with 2/8 patients reacting to Dihydroeugenol.

 

Based on the whole data, it can be concluded that the substance is a skin sensitizer. The LLNA data are sufficient to exclude Category 1A because a sufficiently low dose level was used in the study (5.1% gave SI of 2.7). So, on the basis that the LLNA is more quantitative than the GPMT and since it is the preferred study type for this endpoint, classification as moderate sensitiser (Category 1B) is considered relevant.

 

References:

Barratt M.D. and Basketter D.A. (1992) Possible origin of the skin sensitization potential of isoeugenol and related compounds. (I) Preliminary studies of potential reaction mechanisms. Contact Dermatitis, 27(2), 98-104.

Itoh M. (1982) Sensitization potency of some phenolic compounds - with special emphasis on the relationship between chemical structure and allergenicity. Journal of Dermatology, 9(3), 223-233.

Natsch A. , Gfeller H. , Rothaupt M. and Ellis G. (2007) Utility and limitations of a peptide reactivity assay to predict fragrance allergens in vitro. Toxicology In Vitro, 21(7), 1220-1226.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information:

Under the REACH regulation there is no legal standard information requirement in Annexes VII to X to perform any specific test for respiratory sensitisation. However, guidance document Chapter R.7.a endpoint specific guidance (paragraphs 7.3.5-7.3.9) describes how to use human and non-human data. As regards non-human data there is:

 

• No definitive guidance on use of QSARs
• No specific in vitro method
• The role on LLNA, cytokine fingerprints, total IgE/specific IgE methods are described. However, it is widely appreciated that, in both humans and animals, the accurate evaluation of antibody responses against chemical allergens in the form of hapten–protein conjugates can be technically demanding and highly variable between laboratories.
• Assessment should be case-by-case

Nonetheless, the registrant recognises that further information can be requested beyond the information mentioned in Annexes VII to X of REACH, if there is a concern that a given substance may constitute a risk to human health or the environment, and further information is needed to clarify such concern. The substance has been identified as a moderate skin sensitizer (Category B) and hence there is potential for a risk of respiratory sensitization effects following inhalation exposure; either as a cause of respiratory sensitization or as a route of exposure to elicit dermal and/or respiratory effects in an individual that is already sensitized via the dermal exposure route.

The case-by-case risk assessment for the potential for respiratory sensitization for this substance is addressed as follows.

 

Prevalence.

Compared with contact allergens, of which a few thousand have been identified, far fewer chemicals have been implicated as having the potential to cause sensitisation of the respiratory tract, the number being no more than 80, all are associated with occupational exposures (Kimber and Dearman, 1997; Bakerly et al., 2008; Health and Safety Executive, 2001; Baur, 2013; Baur and Bakehe, 2014).

 

Mechanistic Factors.

Most chemicals are too small in terms of molecular size to induce an adaptive immune response. To acquire immunogenic potential they must form stable associations with protein (hapten–protein conjugates). Mechanistic chemistry studies have revealed that chemical respiratory allergens can be assigned to one of six electrophilic mechanistic domains, with harder (stronger) electrophilic mechanisms such as acylation being more prevalent than softer (weaker) mechanisms, with the hypothesis being that the harder nucleophile lysine is the favoured biological nucleophile for sensitization of the respiratory tract (Enoch et al., 2012). The substance is not-predicted to react with skin proteins directly, but may act as a pro-hapten and no alerts for respiratory sensitisation has been predicted (OECD toolbox v3.4). The substance or its’ metabolites are expected to react via covalent binding with a cysteine peptide under the metabolic conditions of the peroxidase peptide reactivity assay (PPRA) (Gerberick, 2009), which is the first step of the adverse outcome pathway (AOP) of sensitization. The possible importance of lysine reactivity (in comparison to cysteine binding) in sensitization of the respiratory tract by chemical allergens is supported by some in chemico and in vitro studies (Hopkins et al., 2005; Lalko et al., 2011, 2012, 2013b). Hence, as the substance or its’ metabolites preferentially react with cysteine (skin sensitiser) it is unlikely to be a respiratory sensitizer, which seem to be those chemicals that are more likely to react with lysine.

Antigenic Response.

In any in vivo sensitisation test such as LLNA or GPMT, a positive response does not imply that a chemical will cause respiratory sensitisation because the immune responses induced by contact allergens and chemical respiratory allergens begin to diverge in a qualitative sense after the initial activation of T lymphocytes (Cochrane et al., 2015). Chemical respiratory allergens result in the development of a selective Th2-type immune response characterised by the increased expression of type 2 cytokines such as IL-4, IL-5 and IL-13. In contrast, under the same conditions, skin sensitising chemicals elicit Th1-selective immune responses.

 

Threshold Effects.

There is evidence that thresholds of elicitation can be defined for IgE mediated allergies and in the case of human respiratory sensitisation to proteins there is evidence for thresholds even if it is not currently possible to be specific in numerical terms (Basketter et al., 2010, 2012; Peters et al., 2001; Sarlo, 2003).

As an example for chemical respiratory sensitizers, over the past 30 years evidence has accumulated of occupational asthma associated with the use of glutaraldehyde, including the involvement of IgE antibody, and in particular in endoscopy, radiography and pathology suites. The evidence suggests that brief exposures to high levels of glutaraldehyde are required to induce allergic sensitization, this is consistent with the probability that peak exposures to chemicals may drive sensitisation (Arts et al., 2006; Vyas et al., 2000).

 

Experimental Evidence.

A study was conducted with patients with confirmed contact allergy to isoeugenol (an analogue of Dihydroeugenol) or hydroxyisohexyl-3-carboxaldehyde (HICC). These patients were exposed to the chemical to which they were sensitized by inhalation using an exposure chamber, with skin contact being shielded by protective clothing. No significant changes in lung function were observed suggesting the absence of respiratory sensitization (Schnuch et al., 2010). This study provides evidence that individuals already sensitised to a substance via the dermal route do not experience symptoms of sensitization when exposed via the inhalation route. Also of relevance is a study of lung function among employees in the fragrance industry (Dix, 2013). A group of workers exposed to fragrance materials during production or similar operations was compared with a non-exposed control group of office workers. There were no significant differences in lung function as determined by measurement of forced expiratory volume, forced vital capacity of peak expiratory flow (Dix, 2013). The relevance of these studies and other work has been recently reviewed (Basketter and Kimber, 2015).

 

Conclusion.

The currently identified mechanisms of dermal and respiratory sensitization may be sufficiently different, and exposure levels are significantly lower than the probable minimum threshold level required for induction and/or elicitation to prevent any risk of respiratory sensitization. This conclusion is based on various elements of scientific evidence that together constitute a robust argument and obviate the need to conduct further specific studies to investigate the potential for respiratory sensitization of this substance.

 

References

Arts, J.H.E., Mommers, C., de Heer, C., 2006. Dose-response relationships and threshold levels in skin and respiratory allergy. Crit. Rev. Toxicol. 36, 219–251.

Bakerly, N.D., Morre, V.C., Vellore, A.D., Jaakkola, M.S., Robertson, A.S., Burge, P.S., 2008. Fifteen-year trends in occupational asthma: data from the shield surveillance scheme. Occup. Med. 58, 69-174.

Baur, X., 2013. A compendium of causative agents of occupational asthma. J. Occup. Med. Toxicol. 8, 1-8.

Baur, X., Bakehe, P., 2014. Allergens causing occupational asthma: an evidence based evaluation of the literature. Int. Arch. Occup. Environ. Health 87, 339-363.

Basketter, D.A., Broekhuizen, C., Fieldsend, M., Kirkwood, S., Mascarenhas, R., Maurer, K., Pedersen, C., Rodriguez, C., Schiff, H.-E., 2010. Defining occupational consumer exposure limits for enzyme protein respiratory allergens under REACH. Toxicology 268, 165–170.

Basketter, D.A., Berg, N., Kruszewski, F.H., Sarlo, K., Concoby, B., 2012. Relevance of sensitization to occupational allergy and asthma in the detergent industry. J. Immunotoxicol. 9, 314–319.

Basketter, D.A. and Kimber, I., 2015. Fragrance sensitisers: Is inhalation an allergy risk? Regul. Toxicol. Pharmacol. 73(3): 897-902.

Cochrane, SA., Arts, J.H.E., Ehnes, C., Hindle, S., Hollnagel, H.M., Poole, A., Suto, H., and Kimber, I., 2015. Thresholds in chemical respiratory sensitisation. Toxicology, 333, 179-194.

Dix, G.R., 2013. Lung function in fragrance industry employees. Occup. Med. (Lond.) 63, 377-379.

Enoch, S.J., Seed, M.J., Roberts, D.W., Cronin, M.T., Stocks, S.J., Agius, R.M., 2012. Development of mechanism-based structural alerts for respiratory sensitization hazard identification. Chem. Res. Toxicol. 25, 2490–2498.

Gerberick GF1, Troutman JA, Foertsch LM, Vassallo JD, Quijano M, Dobson RL, Goebel C, Lepoittevin JP. Investigation of peptide reactivity of pro-hapten skin sensitizers using a peroxidase-peroxide oxidation system. Toxicol Sci. 2009, 112(1):164-74.

Hopkins, J.E., Naisbitt, D.J., Kitteringham, N.R., Dearman, R.J., Kimber, I., Park, B.K., 2005. Selective haptenation of cellular and extracellular protein by chemical allergens: association with cytokine polarization. Chem. Res. Toxicol. 18, 375–381.

HSE (Health and Safety Executive), 2001. Asthmagen? Critical Assessments of the Evidence for Agents Implicated in Occupational Asthma. UK Health and Safety Executive.

Kimber, I., Dearman, R.J., 1997. Chemical respiratory allergy: an introduction. In: Kimber, I., Dearman, R.J. (Eds.), Toxicology of Chemical Respiratory Hypersensitivity. Taylor & Francis, London, UK, pp. 1-6.

Lalko, J.F., Kimber, I., Dearman, R.J., Gerberick, G.F., Sarlo, K., Api, A.M., 2011. Chemical reactivity measurements: potential for characterization of respiratory chemical allergens. Toxicol. In Vitro 25, 433–445.

Lalko, J.F., Kimber, I., Gerberick, G.F., Foertsch, L.M., Api, A.M., Dearman, R.J., 2012. The direct peptide reactivity assay: selectivity of chemical respiratory allergens. Toxicol. Sci. 129, 421–431.

Lalko, J.F., Dearman, R.J., Gerberick, G.F., Troutman, J.A., Api, A.M., Kimber, I., 2013a. Reactivity of chemical respiratory allergens in the peroxidase peptide reactivity assay. Toxicol. In Vitro 27, 651–661.

Lalko, J.F., Kimber, I., Dearman, R.J., Api, A.M., Gerberick, G.F., 2013b. The selective peptide reactivity of chemical respiratory allergens under competitive and noncompetitive conditions. J. Immunotoxicol. 10, 292–301.

Peters, G., Johnson, G.Q., Golembiewski, A., 2001. Safe use of detergent enzymes in the workplace. Appl. Occup. Environ. Hyg. 16, 389–396.

Sarlo, K., 2003. Control of occupational asthma and allergy in the detergent industry. Ann. Allergy Asthma Immunol. 90, 32–34.

Schnuch, A., Oppel, E., Oppel, T., Rommelt, H., Kramer, M., Riu, E., Darsow, U., Przybilla, B., Nowak, D., Jorres, R.A., 2010. Experimental inhalation of fragrance allergen in predisposed subjects: effects on skin and airways. Br. J. Dermatol. 162, 598-606.

Vyas, A., Pickering, S.A.C., Oldham, L.A., Francis, H.C., Fletcher, A.M., Merrett, T., Niven, R.M., 2000. Survey of symptoms respiratory function, and immunology and their relation to glutaraldehyde and other occupational exposures among endoscopy nursing staff. Occup. Environ. Med. 57, 752–759.

Justification for classification or non-classification

Harmonized classification:

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

Self-classification:

The substance is classified as Skin Sens. 1B, H317 (May cause an allergic skin reaction) according to the Regulation (EC) No. 1272/2008 (CLP) and to the GHS, since EC3 is > 2% (6.8%) in the LLNA.

No direct scientific data are available on the substance to address respiratory sensitisation. However, a scientific argument using mechanistic information has been constructed and is detailed in the discussion of the endpoint summary. As a result, the substance does not meet the criteria for classification according to Regulation (EC) No 1272/2008, Annex I section 3.4.