Registration Dossier

Administrative data

Description of key information

Skin sensitisation:

Based on the available animal and human evidence for Isoeugenol, a classification as Skin Sens. 1A – H317: May cause an allergic skin reaction, is required for Isoeugenol. Isoeugenol has been already fully evaluated by HERA and a harmonised classification (CLH) is available for the registered substance for skin sensitisation Category 1A (RAC Opinion adopted on 10 March 2016, proposed future entry in Annex VI of CLP Regulation). Among all the data available, only one key study is included in this dossier, a GLP OECD 429 LLNA study on the supporting substance (mixture of trans- and cis-Isoeugenol) is reported (Rel.2). The results confirm the Skin Sensitization 1A classification.

Respiratory sensitisation:

The reliability of the two available studies cannot be evaluated. Indeed, the respiratory lymph node assay is a recently developed animal model and more research is needed to further validate this model (e.g. cut-off value). In addition, in one study it is not possible to determine if the level of irritation was excessive or not, but in some case animals died and showed signs of respiratory distress and therefore the level of irritation achieved was probably excessive. Moreover, human evidence does not indicate signs of 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
Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From June 06 to 12, 2001
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Remarks:
GLP study performed equivalent or similar to OECD test guideline No. 429 with deviations: Individual weights of animals at start of dosing and at scheduled kill not reported; animal room humidity level was outside the target range; no ear thickness measurement, no range-finding test performed.
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
yes
Remarks:
(individual weights of animals at start of dosing and at scheduled kill not reported; animal room humidity level was outside the target range; no ear thickness measurement, no range-finding test performed)
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes
Type of study:
mouse local lymph node assay (LLNA)
Species:
mouse
Strain:
other: CBA/J Hsd
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Sprague-Dawley, Inc., Indianapolis, IN.
- Age at study initiation: 6-8 weeks
- Weight at study initiation: 17.2-22.0 g (mean body weight: 20.0 g)
- Housing: Animals were housed individually in plastic shoebox-style cages.
- Diet: Purina Rodent Chow 5002, ad libitum
- Water: Tap water, ad libitum
- Acclimation period: 8 days

ENVIRONMENTAL CONDITIONS
- Temperature: 70-76 °F
- Humidity: 56-84 %
- Photoperiod: 12 h dark/12 h light

IN-LIFE DATES: From: June 06, 2001 To: June 12, 2001.
Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
0.25, 0.5, 1.0, 2.5, and 5.0 % in 4:1 acetone/olive oil
No. of animals per dose:
6
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 is considered a sensitizer if at least one concentration of the test material results in a statistically significant 3-fold or greater stimulation index (SI).

TREATMENT PREPARATION AND ADMINISTRATION:
- The vehicle and dilutions of the control and test materials were prepared on the bench top daily, prior to dosing. All suspensions were mixed by vortexing. 25 µL of control or test material was applied to the dorsum of each ear using a calibrated Finnpipet daily for three consecutive days. Animals were not treated on Days 4 and 5. On Day 6, animals were injected i.v. in the lateral tail vein with 0.25 mL containing 2 µCi of 125I-labelled Iododeoxyuridine and 10^-5 M FuDR in phosphate buffered saline (PBS). Approximately 5 h later, animals were euthanized by CO2 asphyxiation and the auricular lymph nodes were excised. The nodes were dissociated using the frosted ends of glass slides. The cell suspension was washed with Hanks' Balanced Salt Solution (HBSS) and then with PBS, prior to being resuspended in 5 % tricholoroacetic acid (TCA) and refrigerated at approximately 4°C. Approximately 18 h later the cells were centrifuged and resuspended in fresh 5 % TCA. The radioactivity was measured using a gamma counter (Packard Instruments).
Disintegrations per minute (DPM) and stimulation index (Sl) were calculated for each dose group.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
- To test if the compound was a sensitizer, a one-sample t test was performed for testing if the individual untransformed SI values of each dose level of each compound were different than 3.0.
- The natural log transformed DPM values for each test material concentration were compared against vehicle by first performing a Bartlett's Chi-Square test for variance homogeneity. If this was found to be non-significant, a one-way analysis of variance was used using dose (concentration). If this was found to be significant, then a Dunnett's t test was performed using an alpha of 0.05.
- If the Bartlett's Chi-Square was found to be significant, non-parametric analyses (specifically a Kruskal-Wallis test) were performed. If this was found to be significant, then a Jonckheere's-Terpera test was performed for dose-dependent trends.
- A confirmatory analysis was performed against the known standard hexylcinnamic aldehyde at two concentrations, 1 % and 25 % using the above methods.
- A fitted quadratic equation (a linear term and a square term of the concentration) was used to fit the data from the concentrations tested and to determine the concentration of test material required to elicit a stimulation index of 3 (EC3). The fitted quadratic equation was selected based on Loveless et al. (1996) and as stated the study protocol.
- A fitted linear equation was used to determine the concentration of hexylcinnamic aldehyde required to elicit a stimulation index of 3 (EC3) as only 3 doses [0 (vehicle), 1 and 25 %] of hexylcinnamic aldehyde were tested and the quadratic term was not significant.
- All calculations were performed using Microsoft Excel and SAS, version 6.12. PROCs GLM, FREQ, NPAR1WAY and MEANS were utilized.
Positive control results:
Stimulation index for positive control group treated with 25 % of hexylcinnamic aldehyde in acetone:olive oil (4:1) was found to be 6.3; classified as skin sensitizer.
Key result
Parameter:
EC3
Value:
< 2
Parameter:
SI
Value:
2.5
Variability:
± 0.5
Test group / Remarks:
0.25%
Parameter:
SI
Value:
1.2
Variability:
± 0.2
Test group / Remarks:
0.5%
Parameter:
SI
Value:
1.5
Variability:
± 0.3
Test group / Remarks:
1.0%
Parameter:
SI
Value:
4
Variability:
± 0.9
Test group / Remarks:
2.5%
Parameter:
SI
Value:
12.2
Variability:
± 1.9
Test group / Remarks:
5.0%
Cellular proliferation data / Observations:
CELLULAR PROLIFERATION DATA
DPM (as mean ± SE) for 0 (vehicle), 0.25, 0.5, 1.0, 2.5 and 5.0 % were 5.1 ± 1.7, 12.7 ± 2.5, 6.1 ± 1.1, 7.9 ± 1.7, 20.3 ± 4.6 and 62.1 ± 9.8, respectively.

DETAILS ON STIMULATION INDEX CALCULATION
Stimulation index (as mean ± SE) for test material at 0.25, 0.5, 1.0, 2.5 and 5.0 % were 2.5 ± 0.5, 1.2 ± 0.2, 1.5 ± 0.3, 4.0 ± 0.9 and 12.2 ± 1.9, respectively.

EC3 CALCULATION
Calculated EC3 value for the test material was found to be 1.54 % and EC-3 potency value was 385 µg/cm2.

CLINICAL OBSERVATIONS
No mortality, irritation or other adverse toxic effects were noted in any of the animals.

BODY WEIGHTS
Not reported
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 “Category 1A” skin sensitizer according to the annex VI of the Regulation EC No. 1272/2008 (CLP) and to the GHS since EC3 < 2%.
Executive summary:

In a Local Lymph Node Assay (LLNA), groups of female CBA/J Hsd mice (6 females/group) were topically applied with test material at the dose concentrations of 0.25, 0.5, 1.0, 2.5 and 5.0 % 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 and a positive control group was treated with hexylcinnamic aldehyde at the dose concentration of 1 and 25 % in acetone:olive oil (4:1) in same manner to confirm the sensitivity and reliability of the test method. Three days after the final auricular application (on Day 6), animals were injected intravenously with 125I- labelled luDR to label proliferating cells. 125I-incorporation was quantified using a gamma counter and disintegrations per minute (DPM) and stimulation index (Sl) were calculated for each dose group.

No mortality, irritation or other adverse toxic effects were noted in any of the animals. Mean DPM for 0 (vehicle), 0.25, 0.5, 1.0, 2.5 and 5.0 % were 5.1, 12.7, 6.1, 7.9, 20.3 and 62.1, respectively. Stimulation Index (SI Value) calculated for test material treated groups was found to be 2.5, 1.2, 1.5, 4.0 and 12.2 for the dose concentrations of 0.25, 0.5, 1.0, 2.5 and 5.0 %, respectively. Calculated EC3 value for the test material was found to be 1.54 % and EC-3 potency value was 385 µg/cm2. Stimulation index for positive control group treated with 25 % of hexylcinnamic aldehyde in acetone: olive oil (4:1) was found to be 6.3; classified as skin sensitizer and confirming the validity of the study.

Under the test conditions, test material is classified as “Category 1A” skin sensitizer according to the annex VI of the Regulation EC No. 1272/2008 (CLP) and to the GHS since EC3 < 2%.

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

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (sensitising)
Additional information:

A key study was available on an analogue substance (BRT, 2001, Rel.2). In this Local Lymph Node Assay (LLNA), groups of mice were treated with test material at the dose concentrations of 0.25, 0.5, 1.0, 2.5 and 5.0 % final concentration in 1:4 acetone: olive oil. The Stimulation Index values (SI Value) were 2.5, 1.2, 1.5, 4.0 and 12.2 for the concentrations of 0.25, 0.5, 1.0, 2.5 and 5.0 %, respectively. Calculated EC3 value for the test material was found to be 1.54 % and EC-3 potency value was 385 µg/cm2. The positive control treated with 25 % of hexylcinnamic aldehyde gave an SI of 6.3. The test system was therefore considered to be valid. No mortality, irritation or other adverse toxic effects were noted in any of the animals. Under the test conditions, the test material should be classified as skin sensitizer Category 1A since the EC3 < 2%.

In addition, Isoeugenol has been chosen for a full risk assessment by HERA (Human and Environmental Risk Assessment on ingredients of household cleaning products) program because of its known skin sensitising properties (HERA, 2005). A harmonised classification (CLH) is available for the registered substance for skin sensitisation Category 1A (RAC Opinion adopted on 10 March 2016 proposed future entry in Annex VI of CLP Regulation).

The summary and discussion of skin sensitisation of the CLH report (2015) is reported below:

Isoeugenol shows a definite skin sensitization potential in a wide variety of predictive test systems and is classified as a moderate skin sensitizer according to ECETOC standards. The evidences include the positive results obtained in GPMTs, in FCATs, in CCETs, in Buehler Tests, in OETs and CETs with guinea pigs, as well as in LLNAs.

Non-adjuvant tests in animals and maximized tests carried out on human subjects offer a sound basis for a “weight of evidence” judgment on exposure levels above the potency to induce allergy in naïve individuals during use of household products. The LLNA places this level at around 500 μg/cm2 (with some degree of variability) while the HRIP Test places this at around 260 μg/cm2 on the basis of two tests carried out on a total of 97 subjects. SCCS in its opinion on fragrance allergens in cosmetic products has pointed out that the EC3 value of Isoeugenol is 0.54% (M = 0.033), based on a report submitted by RIFM (2009). Studies on animals and humans demonstrate that Isoeugenol is a skin sensitizer of moderate allergenic potency. This is substantiated by clinical data that show possible allergy to Isoeugenol. However, very few cases of allergy are clearly attributable to the presence of Isoeugenol in any specific consumer products.

A number of experimental in vitro techniques provided indications of the positive allergenicity of Isoeugenol. The methods used in these studies have not been validated or related in any quantitative way to studies in animals or humans.

There are many published reports of studies in which Isoeugenol produces positive reactions in patients in routine diagnostic patch testing as summarized in the Table 7.4/1 (from CLH report of Isoeugenol, 2015)..

Table 7.4/1 Clinical patch testing of isoeugenol in “Fragrance Mix-sensitive” patients Patch test conditions Number tested Number reacting Scores Comments References

Patch test conditions

Number tested

Number reacting

Scores

Comments

References

No dose reported 24 hrs occlusion
Finn Chambers®

160

24

Not given

Not a primary study. Review of several studies or multicentre study.
Patients probably reacted to other test materials in the same study.

Temesvari et al. (2002)

No dose reported

32

9

Not given

Not a primary study. Review of several studies or multicentre study.
Patients probably reacted to other test materials in the same study.

Sieben et al. (2001)

1% in petrolatum 48 hrs occlusion

226

45

Not given

Not a primary study. Review of several studies or multicentre study.
Patients probably reacted to other test materials in the same study.

Brites et al. (2000)

1% in petrolatum 48 hrs occlusion over 15 years

1112

231

Not given

Not a primary study. Review of several studies or multicentre study.
Patients probably reacted to other test materials in the same study.

Buckley et al. (2000b)

1% in petrolatum
Finn Chambers® or Scanport®, 48 hrs occlusion

40

8

Not given

Not a primary study. Review of several studies or multicentre study.
Patients probably reacted to other test materials in the same study.

Katsarma and Gawkrodger (1999)

1% in petrolatum
Finn Chambers® or Scanport®, 48 hrs occlusion

38

9

Not given

Not a primary study. Review of several studies or multicentre study.
Patients probably reacted to other test materials in the same study.

Katsarou et al. (1999)

Different concentrations (serial dilution study on isoeugenol - sensitive patients who had previously reacted to Fragrance-Mix)

19

18

Different scores recorded for different patients

Patients probably reacted to other test materials in the same study.

Johansen et al. (1996d)

1% or 2% in petrolatum 48 hrs occlusion in Finn Chambers® or Scanport®, tape

367

68

+ to +++ reactions

Not a primary study. Review of several studies or multicentre study.
Patients probably reacted to other test materials in the same study.

Johansen and Menne (1995)

No conditions given

50

3

Not given

Not a primary study. Review of several studies or multicentre study.
Patients probably reacted to other test materials in the same study.

Becker et al. (1994)

1%, 3% and 5% in petrolatum (serial dilutions)

6

1

Not given

Patients probably reacted to other test materials in the same study.

De Groot et al. (1993)

2% in petrolatum 48 hrs occlusion in Finn Chambers®

20

4

Not given

Patients probably reacted to other test materials in the same study.

Safford et al. (1990)

1% in petrolatum

162

27

Not given

Not a primary study. Review of several studies or multicentre study.
Patients probably reacted to other test materials in the same study.

Enders et al. (1989)

1% in petrolatum 48 hrs occlusion in Finn Chambers® or Scanpore®

54

12

Not given

Not a primary study. Review of several studies or multicentre study.
Patients probably reacted to other test materials in the same study.

Santucci et al. (1987)

Not given

42

19

Not given

Not a primary study. Review of several studies or multicentre study.
Patients probably reacted to other test materials in the same study.

Rudzki and Grzywa (1986)

1% in petrolatum

144

6

Not given

Not a primary study. Review of several studies or multicentre study.
Patients probably reacted to other test materials in the same study.

Angelini et al. (1985)

Not reported

80

7

Not given

Not a primary study. Review of several studies or multicentre study.
Patients probably reacted to other test materials in the same study.

Romaguera et al. (1983)

2% in Petrolatum

172

48

Not given

Not a primary study. Review of several studies or multicentre study.
Patients probably reacted to other test materials in the same study.

Calanan et al. (1980)

Not given

50

8

Not given

Not a primary study. Review of several studies or multicentre study.
Patients probably reacted to other test materials in the same study.

Bordalo et al. (2000)

1% in petrolatum 48 hrs patch tests

4900 consecutive patients

173

- 51 gave + reactions to 1% isoeugenol and to 8% Fragrance-Mix.
- 60 gave + reactions to 1% isoeugenol but ++ or +++ reactions to 8% Fragrance -Mix.
- 56 gave ++ or +++ reactions to both the Fragrance-Mix and Isoeugenol
- 6 gave ++ or +++ reactions to isoeugenol but only + reactions to Fragrance-Mix.

Not a primary study. Review of several studies or multicentre study.
Patients probably reacted to other test materials in the same study.

Schnuch et al. (2002)

5% isoeugenol in petrolatum

520

15

Not given

Not a primary study. Review of several studies or multicentre study.
Patients probably reacted to other test materials in the same study.

Ohela and Saramies (1983)

 

Although there have been numerous reports of patients giving frank allergic responses to Isoeugenol in clinical patch testing on dermatological patients, many of these studies do not establish a clear causal relationship according to currently accepted criteria. A publication has pointed out that reactions seen in dermatological clinics, while genuinely allergic in nature, may only occur under the severe conditions used in clinical diagnosis and may not relate to adverse effects from the use of consumer products. In a separate publication, the same authors have also defined criteria by which possible causality can be assessed. These criteria have been applied by the authors to a number of other proposed allergens. The same criteria have been used here to assess the strength of a causal link between the observed clinical reaction and everyday exposure to an Isoeugenol-containing product.

Isoeugenol is one of the eight components of the "Fragrance Mix" used by dermatologists to detect possible sensitivity to fragrances. This mix was first proposed, on the basis of the components of a fragrance used in a popular Tri-Adcortyl cream (Mycolog®, Squibb Corp.). It was concluded that the use of this ointment in treating eczematous and ulcerous skin may have contributed significantly to the cases of clinical dermatitis that had been ascribed to this substance. Clinical patch testing of patients who have already shown positive reactions to the "Fragrance Mix" frequently gives positive reactions to Isoeugenol although in such cases, it is rare that Isoeugenol is the only component of this "Fragrance Mix" to produce positive reactions. In a large multi-centre study covering nearly 60,000 patients tested in German clinics from 1996 to 2002, the frequency of reactions to Isoeugenol in patients reacting to the fragrance mix was reported to be about 13%. These patients have frequently reacted to other constituents of the fragrance mix (for instance 47.6% and 56.7% of patients reacting to chemically-dissimilar geraniol and amylcinnamic aldehyde respectively, also reacted to Isoeugenol).

It has been reported that while the proportion of patients reacting to the "Fragrance Mix" has been relatively constant over 17 years, there is a 5% yearly increase in the proportion of patients reacting to Isoeugenol (Buckley et al., 2000a) having reached an average 16.7% and 15.4% of "Fragrance Mix-sensitive" males and females respectively. However, the full significance of these findings has been questioned.

In a European multicentre study a total of 1072 patients were patch tested in 9 different centres of which 20 out of 1072 patients (1.86%) had a positive reaction to Isoeugenol at a concentration of 1%. In another study, 20 perfume allergic patients were tested with several screening series of fragrances. Isoeugenol at a concentration at 2% gave a positive reaction in 5/20 (25%) of the patients. Other authors identified a causal link between cutaneous reactions in 713 patients and cosmetic products. In 578 out of 713 cases sensitisation were observed. In 10 out of 713 subjects Isoeugenol was found to be one of the causative ingredients as judged by patch testing. In another study in which 156 patients with contact allergy to cosmetic products were identified, Isoeugenol was one of the causative ingredients in 16 cases (10.3%), as determined by patch testing. In a European multicentre study involving 6 countries, 78 patients positive to one of two different fragrance mixes (both containing Isoeugenol), were tested with the individual constituents of the mixes. Results showed that 16/78 (20.5%) were positive to 2% Isoeugenol. Furthermore, the frequency of contact allergy to Isoeugenol in patients positive to the fragrance mix, is reported in a range of studies from different countries: 22% of the contact allergy reactions were due to Isoeugenol present in fragrance mix in Italy, 18.5 % in Denmark, 6% in Hungary, 16.6% in Germany and 17% in France. In addition, Isoeugenol has been found to cause sensitisation in 12-36% of healthy volunteers. Isoeugenol was restricted in the IFRA (International Fragrance Association) guideline to 0.2% until May 1998, where the concentration was lowered to 0.02%.

Most studies were performed with Isoeugenol without specification of the ratio between the cis and the trans isomer. Also very limited information is available on the skin sensitising potential of the specific isomers. However, the HMT with 8% Isoeugenol in petrolatum of which 90% was specified as cis-Isoeugenol shows (positive response in 21/31 patients) that the cis-isomer has skin sensitising potential. The clinical patch test with 1% Isoeugenol in petrolatum shows that the trans-isomer has the potential to induce an allergic reaction in sensitised people although a cross-reaction with cis-isomer cannot be excluded. In addition, there is a clear structural similarity between both isomers as can be expected for isomers. In addition, the double bond that differs between the two isomers is not expected to be relevant for the activation prior to protein binding. Therefore, the results obtained with Isoeugenol are considered relevant for the individual isomers and for the racemic mixture."

There is some information available that indicates that the skin sensitisation response might be dependent on the type of vehicle used. In a LLNA study Isoeugenol was tested using various vehicles, i.e. acetone/olive oil, dimethyl sulphoxide, methyl ethyl ketone, dimethyl formamide, propylene glycol, ethanol/water (50/50) and ethanol/water (90/10). These data show that the vehicle might affect the skin sensitisation response, though this is considered limited (up to a factor of 5). EC3 values ranged from 0.9% to 4.9% for Isoeugenol. Further, the CLP-regulation does not provide options to include vehicle-dependency in the classification itself or the setting of SCLs for skin sensitisation. Based on this, no full evaluation of the dependency of the skin sensitisation response on the type of vehicle is included in the discussion and conclusion of this endpoint.

Comparison with CLP criteria

The results of animal tests have showed that in LLNAs EC3 values of Isoeugenol is between 0.5 and 3.8 at applied concentrations, and a SI of three or more has been observed in LLNA of Isoeugenol from the test concentration of 1.3%. 100 % responding from 0.15 % intradermal induction dose of Isoeugenol have been detected in most of the GPMT studies. The above evidence supports that Isoeugenol is sub-category 1A skin sensitizer. The outcomes from most of the Buehler assays however indicate that Isoeugenol falls into sub-category 1B.

In human tests, a number of HRIPT give the evidence that Isoeugenol is sub-category 1A skin sensitizer (positive responses at ≤ 500 μg/cm2). Besides this, relatively high and substantial incidence of allergic contact dermatitis caused by Isoeugenol, and mixtures containing Isoeugenol, are observed in diagnostic patch tests and in epidemiological studies.

Overall there is clear evidence for classification in category 1A from animal tests (LLNA and GPMT) and human tests and human data. Only the results from the Buehler tests indicate category 1B. As human data is considered more relevant than animal data and the Buehler assay is considered less sensitive compared to the LLNA and the GPMT, classification in category 1A is warranted.

The GCL for Skin Sens. 1A substance is 0.1%. According to the ‘Guidance on the Application of the CLP Criteria’ (paragraph 3.4.2.2.5), specific concentration limits can be set based on potency. Tables 3.4.2-f/g/h of this CLP Guidance present the potency classes for the mouse LLNA-test, Guinea Pig Maximisation test and the Buehler assay, respectively. The results of the LLNA-studies and the GPMT-tests are sufficient for classification into category 1A. Based on the results of the LLNA-studies (EC3 0.5-3.8%), no EC3-value ≤0.2% (w/v) was observed. Thus according to the criteria in table 3.4.2-f of the CLP-guidance, this would correspond to a strong potency class. Further, the results of the GPMT tests (100% positive response following a 0.15% intradermal induction dose) also indicate a strong potency class following the criteria in table 3.4.2-g of the CLP-Guidance. For this potency class, the GCL of 0.1% applies (Table 3.4.2-I of the CLP-Guidance). However, when a 100% response in the GPMT is observed at 0.15% intradermal induction it can be expected that a response above 60% will occur at 0.1% induction. This would indicate an extreme sensitising potency.

Conclusions on classification and labelling

In March 2016, the Committee for Risk Assessment (RAC) decided to adopt a harmonised classification (CLH) for isoeugenol (racemic mixture as well as both isomers) as a strong skin sensitiser (Skin Sens. 1A; H317: May cause an allergic skin reaction). The RAC Opinion was adopted on 10 March 2016 and proposed future entry in Annex VI of CLP Regulation. RAC also decided to set a specific concentration limit (SCL) of 0.01%. Therefore, when present at concentrations equal to or greater than 0.001% in a mixture, the name of the substance must be indicated on the label.

References

CLH report. Proposal for Harmonised Classification and Labelling Based on Regulation (EC) No 1272/2008 (CLP Regulation), Annex VI, Part 2. Substance Name: 2-methoxy-4-(prop-1-enyl)phenol; 2-methoxy-4-((E)prop-1-enyl)phenol; 2-methoxy-4-((Z)prop-1-enyl)phenol, Dossier submitter: RIVM, May 2015 (version number: 1.0).

HERA (2005) Human and Environmental Risk Assessment on ingredients of Household Cleaning Products (Isoeugenol)

RAC report. Annex to news alert ECHA/NA/16/10. Helsinki, 15 March 2016. More information about the adopted opinions


Respiratory sensitisation

Link to relevant study records

Referenceopen allclose all

Endpoint:
respiratory sensitisation: in vivo
Type of information:
experimental study
Adequacy of study:
disregarded due to major methodological deficiencies
Study period:
No data
Reliability:
3 (not reliable)
Rationale for reliability incl. deficiencies:
unsuitable test system
Remarks:
The reliability of the study cannot be evaluated. Indeed, the respiratory lymph node assay is a recently developed animal model and more research is needed to further validate this model (e.g. cut-off value). No evaluation of irritation to the respiratory tract was made thus it cannot be determined if the level of irritation was excessive or not.
Qualifier:
no guideline followed
Principles of method if other than guideline:
In respiratory lymph node assay, mice were exposed with test material via the respiratory route on three consecutive days. The immune response was determined by measuring cell proliferation and cytokine responses in the lymph nodes draining the respiratory tract. In this model, the most pronounced effects were found in the mandibular lymph nodes, which drain the nasopharynx.
GLP compliance:
no
Species:
mouse
Strain:
Balb/c
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: RIVM institute’s own breeding colony
- Age at study initiation: 6-8 weeks
- Housing: Animals were housed in macrolon cages under conventional conditions.
- Diet: Hope Farms chow pellets (Woerden, The Netherlands), ad libitum
- Water: Water, ad libitum
Route of induction exposure:
inhalation
Remarks:
vaporized or nebulized
Vehicle:
other: vaporized (no vehicle) or nebulized (acetone)
Concentration:
Vaporization using maximum vapour pressure: 11 ppm
Nebulization in acetone: 300 ppm
No. of animals per dose:
6 males
Details on study design:
EXPOSURE
- No. of exposures: 3
- Exposure period: 3 days
- Test groups: Mice were exposed nose-only to test material for 45, 90, 180 or 360 minutes per day on day 0, 1 and 2.
- Control group: Controls were exposed to the vehicle for 360 minutes per day on day 0, 1, and 2.
- Frequency of applications: Three
- Duration: 5 days
- Concentrations: Vaporization using maximum vapour pressure: 11 ppm; Nebulization in acetone: 300 ppm
- Dermal control was included. Mice were topically exposed to 10% test material in acetone: olive oil 4:1 (AOO) on the dorsum of both ears (25 μL/ear) on day 0, 1 and 2. Control mice received the same treatment with the vehicle (AOO).
Challenge controls:
Not applicable
Positive control substance(s):
not specified
Negative control substance(s):
not specified
Results:
Effects of exposure to test material via maximum vapour pressure:
- Exposure to test material (11 ppm) increased cell number and cell proliferation in the mandibular LNs. The increase in cell proliferation did not show a time-dependent effect and the response was highly variable. None of the observed effects were statistically significant.
- Exposure to test material did not increase proliferation in the auricular LNs. However, the cell number in the control group was higher than normally observed. Therefore, the calculated SIs are below 1.
- Dermal application of test material (10%) resulted in a SI of the auricular LNs of 29.3.

Effects of exposure to aerosols of test material
- Exposure to aerosols of test material (300 ppm) resulted in toxic effects in the mice that were exposed for 360 min/day. After two days of exposure one mouse died and the other mice displayed several signs of distress. These mice were not exposed to test material on the third day. Effects of the two days exposure to test material were assessed on day 5. On the third day two mice died that were exposed for 180 minutes/day for 3 days. The other mice in this group appeared normal.
- Exposure to test material aerosols resulted in a significant increase of cell number and cell proliferation in the mandibular LNs. This increase was time-dependent, with the exception of the group that was exposed for 360 minutes/day. This group, however, was exposed for two days only. Effects of test material on cell number and cell proliferation were statistically significant for all exposure groups.
- Test material exposure for 90 minutes/day or longer increased proliferation in the auricular LNs. The mean SI in the auricular LNs was a factor 3-4 higher than in the mandibular LNs, but the variance was very high. Dermal exposure to 10% test material resulted in a SI of 18.8.
Positive control results:
None
Negative control results:
None

Effects of exposure to test material via maximum vapour pressure

 

Table 7.4.2/2: Effects of test material on mandibular LNs: cell number, cell proliferation and Sis

 

Group

Cell number

Proliferation

Stimulation index

Control

2.81 ± 1.44

1232 ± 596

1.0 ± 0.48

45 min/day

4.57 ± 1.49

3150 ± 1004

2.56 ± 0.81

90 min/day

5.69 ± 2.79

4383 ± 3530

3.56 ± 2.86

180 min/day

5.37 ± 1.20

2952 ± 711

2.40 ± 0.58

360 min/day

7.03 ± 2.19

4815 ± 1575

3.58 ± 1.40

 

Table 7.4.2/3: Effects of test material on auricular LNs: cell number, cell proliferation and Sis

 

Group

Cell number

Proliferation

Stimulation index

Inhalatory exposure

 

 

 

Control

6.46 ± 1.82

4406 ± 1766

1.0 ± 0.4

45 min/day

3.70 ± 1.34

1531 ± 414

0.35 ± 0.09

90 min/day

3.95 ± 0.98

1640 ± 477

0.37 ± 0.011

180 min/day

4.64 ± 1.35

2068 ± 919

0.47 ± 0.21

360 min/day

4.41 ± 1.63

1955 ± 584

0.43 ± 0.13

Dermal exposure

 

 

 

Control

3.92 ± 2.00

1278 ± 221

1.0 ± 0.17

10% test material

23.7 ± 6.82

37477 ± 13144

29.3 ± 16.7

 

Results are shown as mean ± SEM (n=6 per group). Cell number is expressed as 106 cells, proliferation is expressed as cpm per mouse. SIs are calculated by dividing the [3H]-thymidine incorporation of the experimental group with the mean [3H]-thymidine incorporation of the control group.

 

Effects of exposure to aerosols of test material

 

Table 7.4.2/4: Effects of test material on mandibular LNs: cell number, cell proliferation and Sis

 

Group

Cell number

Proliferation

Stimulation index

Control

2.34 ± 0.57

1070 ± 325

1.0 ± 0.30

45 min/day

5.18 ± 0.77**

4329 ± 688*

4.04 ± 0.64*

90 min/day

6.06 ± 1.41***

5486 ± 2515**

5.13 ± 2.35**

180 min/daya

5.14 ± 1.32**

6555 ± 2423***

6.13 ± 2.26***

360 min/dayb

5.54 ± 1.19**

4864 ± 1532*

4.54 ± 1.43*

 

Table 7.4.2/5: Effects of test material on auricular LNs: cell number, cell proliferation and Sis

 

Group

Cell number

Proliferation

Stimulation index

Inhalatory exposure

 

 

 

Control

3.38 ± 0.50

1493 ± 229

1.0 ± 0.15

45 min/day

4.74 ± 1.06

2528 ± 863

1.69 ± 0.58

90 min/day

10.7 ± 7.18

29549 ± 39947

19.79 ± 26.76

180 min/daya

10.7 ± 4.06

33648 ± 38004

22.54 ± 25.46

360 min/dayb

8.1 ± 3.12

15581 ± 15115

10.44 ± 10.12

Dermal exposure

 

 

 

Control

3.31 ±0.87

1698 ± 472

1.0 ± 0.28

10% test material

20.0 ±5.01

31941 ± 21910

18.8 ± 8.3

 

Results are shown as mean ± SEM (n=6 per group).a n=4; exposure for 3 days;b n=5, exposure for 2 days. Cell number is expressed as 106 cells, proliferation is expressed as cpm per mouse. SIs are calculated by dividing the [3H]-thymidine incorporation of the experimental group with the mean [3H]-thymidine incorporation of the control group. Statistically significant differences were assessed with a one-way ANOVA with a Bonferonni’s post hoc test. Asterisks depict significant differences from the control group: * p<0.05, ** p<0.01, *** p<0.001.

Interpretation of results:
study cannot be used for classification
Conclusions:
The authors considered that the study provided evidence of respiratory sensitisation. The reliability of the study cannot be evaluated. Indeed, the respiratory lymph node assay is a recently developed animal model and more research is needed to further validate this model (e.g. cutoff value). In addition, no evaluation of irritation to the respiratory tract was made, although Isoeugenol is classified as skin and eye irritant and irritation has been observed in sub-acute inhalation toxicity studies. In the skin LLNA it is required to use dose levels that do not induce excessive levels of irritation. In this study it cannot be determined if the level of irritation was excessive or not, but in some case animals died and showed signs of respiratory distress. Furthermore, there is a potential weakness in the model in that the mandibular lymph node drain the mouth and nasophanrynx but not the tracheal and lung tissues, therefore the relevance to respiratory sensitisation is not proven.
Executive summary:

In a respiratory lymph node assay, groups of male Balb/c mice were exposed with isoeugenol (racemic mixture) through inhalation, either via vaporization using maximum vapour pressure (11 ppm) or via nebulization in acetone (300 ppm). Mice were exposed nose-only to test material for 45, 90, 180 or 360 minutes per day on day 0, 1 and 2. Controls were exposed to the vehicle for 360 minutes per day on day 0, 1 and 2. In this experiment a dermal control was included. Mice were topically exposed to 10% test material in acetone: olive oil 4:1 (AOO) on the dorsum of both ears (25 μL/ear) on day 0, 1 and 2. Control mice received the same treatment with the vehicle (AOO). At day 5, animals were killed and the auricular and mandibular lymph nodes were excised. The immune response was determined by measuring cell proliferation and cytokine responses in the lymph nodes draining the respiratory tract. 

 

Effects of exposure to test material via maximum vapour pressure: Exposure to test material (11 ppm) increased cell number and cell proliferation in the mandibular LNs. The increase in cell proliferation did not show a time-dependent effect and the response was highly variable. None of the observed effects were statistically significant. Exposure to test material did not increase proliferation in the auricular LNs. However, the cell number in the control group was higher than normally observed. Therefore, the calculated SIs are below 1. Dermal application of test material (10%) resulted in a SI of the auricular LNs of 29.3.

 

Effects of exposure to aerosols of test material: Exposure to aerosols of test material (300 ppm) resulted in toxic effects in the mice that were exposed for 360 min/day. After two days of exposure one mouse died and the other mice displayed several signs of distress. These mice were not exposed to test material on the third day. Effects of the two days exposure to test material were assessed on day 5. On the third day two mice died that were exposed for 180 minutes/day for 3 days. The other mice in this group appeared normal. Exposure to test material aerosols resulted in a significant increase of cell number and cell proliferation in the mandibular LNs. This increase was time-dependent, with the exception of the group that was exposed for 360 minutes/day. This group, however, was exposed for two days only. Effects of test material on cell number and cell proliferation were statistically significant for all exposure groups. Test material exposure for 90 minutes/day or longer increased proliferation in the auricular LNs. The mean SI in the auricular LNs was a factor 3-4 higher than in the mandibular LNs, but the variance was very high. Dermal exposure to 10% test material resulted in a SI of 18.8.

 

Test material elicited clearly a higher immune response as indicated by the cellular proliferation in the primary draining lymph node of the nasopharynx. The authors considered that the study provided evidence of respiratory sensitisation.

The reliability of the study cannot be evaluated. Indeed, the respiratory lymph node assay is a recently developed animal model and more research is needed to further validate this model (e.g. cutoff value). In addition, no evaluation of irritation to the respiratory tract was made, although Isoeugenol is classified as skin and eye irritant and irritation has been observed in sub-acute inhalation toxicity studies. In the skin LLNA it is required to use dose levels that do not induce excessive levels of irritation. In this study it cannot be determined if the level of irritation was excessive or not, but in some case animals died and showed signs of respiratory distress. Furthermore, there is a potential weakness in the model in that the mandibular lymph node drain the mouth and nasophanrynx but not the tracheal and lung tissues, therefore the relevance to respiratory sensitisation is not proven.

Therefore this study was not used for classification.

Endpoint:
respiratory sensitisation: in vivo
Type of information:
experimental study
Adequacy of study:
disregarded due to major methodological deficiencies
Study period:
No data
Reliability:
3 (not reliable)
Rationale for reliability incl. deficiencies:
unsuitable test system
Remarks:
The reliability of the study cannot be evaluated. Indeed, the respiratory lymph node assay is a recently developed animal model and more research is needed to further validate this model (e.g. cut-off value). No evaluation of irritation to the respiratory tract was made thus it cannot be determined if the level of irritation was excessive or not.
Qualifier:
no guideline available
Principles of method if other than guideline:
The respiratory LLNA was performed according to Arts et al., 2008. In respiratory lymph node assay, mice were exposed with test material via the respiratory route on three consecutive days. The immune response was determined by measuring cell proliferatio in the lymph nodes draining the respiratory tract (mandibular lymph nodes).
GLP compliance:
no
Species:
mouse
Strain:
Balb/c
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Housing: Animals were housed in polycarbonate cages under conventional conditions in light, humidity and temperature controlled rooms.
- Diet: Standard pellet diet (RM3 [E] SQC, Special Diet Service, Witham, UK), ad libitum
- Water: Unfluoridated tap water, ad libitum
Route of induction exposure:
inhalation
Remarks:
Nose-only
Vehicle:
other: acetone
Concentration:
Nebulization in acetone (vapour): 75 mg/m3
No. of animals per dose:
6 males
Details on study design:
MAIN STUDY
INDUCTION EXPOSURE
- No. of exposures: exposure nose-only 1/Day for 3 consecutive days
- Exposure period: 45, 90, 180, or 360 min/day
- Test groups: Groups of male BALB/c mice (six animals per group) were exposed nose-only to test item on three consecutive days for 45, 90, 180, or 360 min/day.
- Control group: Control mice were exposed nose-only to the vehicle for 360 min/day.
- Site: Nose-only
- Concentrations: 75 mg/m3. Test item was nebulized in acetone to produce vapour. The vapor in this mixture downstream of the filters was also sampled on activated charcoal. In addition, the test atmosphere was sampled at a flow rate of approximately 1 L/min for 5 min on activated charcoal and these were used for wet chemical determinations and used to calculate the average actual concentrations during the exposures. The actual air concentrations measured were close to the target concentration of 75 mg/m3. The fluctuations of all test atmospheres on the 3 days of exposure were less than 10% as indicated by continuous mass concentration measurements using a total carbon analyser (TEA).
- Mice were necropsied 3 days after the last exposure and mandibular lymph nodes (LN) were excised, pooled for each animal, and suspended in 5 mL RPMI 1640 with 5% heat inactivated fetal calf serum (FCS) 100 U/mL penicillin, and 100 mg/mL streptomycin (standard medium). Cell proliferation was measured ex vivo using [3H]-thymidine incorporation and is expressed per animal. Stimulation indices (SI) were calculated by dividing the [3H]-thymidine incorporation of the exposed mice with the mean [3H]-thymidine incorporation of the vehicle group.
Positive control substance(s):
none
Negative control substance(s):
other: vehicle (acetone)
Results:
- Inhalation exposure to the test item did neither induce any macroscopically visible toxic effects nor affected body weight gain.
- Test item significantly increases the cell number and proliferation in the mandibular lymph nodes. After 45 min/day exposure, the cell proliferation was already significantly increased more than four-fold compared with the control group. At the time points, 90 and 180 min/day SI values did not further increase and appear to reach a plateau of 3.5-fold. The variation within the experimental groups was relatively high, and these changes were not statistically significant. Prolonged exposure (for 360 min/day) did induce a further increase of cell proliferation to an SI value of 7.2. This increase was statistically significant compared with the vehicle control group.
- Dose–response information of test item was used to calculate the ED3 value, a measure for the potency of chemicals in the respiratory LLNA. The calculated ED3 value for test item was 415 µg which induces 3-fold increase in cell proliferation.
Positive control results:
None
Negative control results:
Not reported

See Figure 7.4.2/1. Stimulation index (SI) in the mandibular lymph nodes after inhalation exposure to Isoeugenol as attached background material

Interpretation of results:
study cannot be used for classification
Conclusions:
The reliability of the study cannot be evaluated. Indeed, the respiratory lymph node assay is a recently developed animal model and more research is needed to further validate this model (e.g. cutoff value). In addition, no evaluation of irritation to the respiratory tract was made, although Isoeugenol is classified as skin and eye irritant and irritation has been observed in sub-acute inhalation toxicity studies. In the skin LLNA it is required to use dose levels that do not induce excessive levels of irritation. In this study it cannot be determined if the level of irritation was excessive or not. Furthermore, there is a potential weakness in the model in that the mandibular lymph node drain the mouth and nasophanrynx but not the tracheal and lung tissues, therefore the relevance to respiratory sensitisation is not proven.
Executive summary:

A study was performed to assess the respiratory sensitisation potential of the test item in the Balb/c mouse following inhalation (vapour). Groups of male BALB/c mice (six animals per group) were exposed nose-only to test item on three consecutive days for 45, 90, 180, or 360 min/day. Control mice were exposed nose-only to the vehicle for 360 min/day. Test item was nebulized in acetone to produce vapour. The actual air concentrations measured were close to the target concentration of 75 mg/m3. The fluctuations of all test atmospheres on the 3 days of exposure were less than 10% as indicted by continuous mass concentration measurements using a total carbon analyser. Mice were necropsied 3 day after the last exposure and mandibular lymph nodes (LN) were excised. Cell proliferation was measured ex vivo using [3H]-thymidine incorporation and is expressed per animal. Stimulation indices (SI) were calculated by dividing the [3H]-thymidine incorporation of the exposed mice with the mean [3H]-thymidine incorporation of the vehicle group.

 

Inhalation exposure to the test item did neither induce any macroscopically visible toxic effects nor affected body weight gain. Test item significantly increases the cell number and proliferation in the mandibular lymph nodes. After 45 min/day exposure, the cell proliferation was already significantly increased more than four-fold compared with the control group. At the time points, 90 and 180 min/day SI values did not further increase and appear to reach a plateau of 3.5-fold. The variation within the experimental groups was relatively high, and these changes were not statistically significant. Prolonged exposure (for 360 min/day) did induce a further increase of cell proliferation to an SI value of 7.2. This increase was statistically significant compared with the vehicle control group. The ED3 value for test item was 415 µg, which induces 3-fold increase in cell proliferation.

The reliability of the study cannot be evaluated. Indeed, the respiratory lymph node assay is a recently developed animal model and more research is needed to further validate this model (e.g. cutoff value). In addition, no evaluation of irritation to the respiratory tract was made, although Isoeugenol is classified as skin and eye irritant and irritation has been observed in sub-acute inhalation toxicity studies. In the skin LLNA it is required to use dose levels that do not induce excessive levels of irritation. In this study it cannot be determined if the level of irritation was excessive or not. Furthermore, there is a potential weakness in the model in that the mandibular lymph node drain the mouth and nasophanrynx but not the tracheal and lung tissues, therefore the relevance to respiratory sensitisation is not proven.

Therefore this study was not used for classification.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:

No study was identified on the registered substance (trans-Isoeugenol). However, a study is available on the supporting substance (Isoeugenol, mixture of cis- and trans-Isoeugenol, see Iuclid section 13 for read-across justification). In a respiratory lymph node assay (RIVM, 2007, Rel.3), groups of male Balb/c mice were exposed with Isoeugenol through inhalation, either via vaporization using maximum vapour pressure (11 ppm) or via nebulization in acetone (300 ppm). Mice were exposed nose-only to test material for 45, 90, 180 or 360 minutes per day on day 0, 1 and 2. Controls were exposed to the vehicle for 360 minutes per day on day 0, 1 and 2. In this experiment a dermal control was included. Mice were topically exposed to 10% test material inacetone: olive oil 4:1 (AOO) on the dorsum of both ears (25 μL/ear) on day 0, 1 and 2. Control mice received the same treatment with the vehicle (AOO). At day 5, animals were killed and the auricular and mandibular lymph nodes were excised. The immune response was determined by measuring cell proliferation and cytokine responses in the lymph nodes draining the mouth and upper respiratory tract. 

 

Effects of exposure to test material via maximum vapour pressure: Exposure to test material (11 ppm) increased cell number and cell proliferation in the mandibular LNs. The increase in cell proliferation did not show a time-dependent effect and the response was highly variable. None of the observed effects were statistically significant. Exposure to test material did not increase proliferation in the auricular LNs. However, the cell number in the control group was higher than normally observed, but calculated SIs were below 1. Dermal application of test material (10%) resulted in a SI of the auricular LNs of 29.3.

 

Effects of exposure to aerosols of test material: Exposure to aerosols of test material (300 ppm) resulted in toxic effects in the mice that were exposed for 360 min/day. After two days of exposure one mouse died and the other mice displayed several signs of distress. These mice were not exposed to test material on the third day because of the excessive clnical signs induced after 2 exposures. Effects of the two day exposure to test material were assessed on day 5. On the third day two mice died that were exposed for 180 minutes/day for 3 days. The other mice in this group appeared normal. Exposure to test material aerosols resulted in a significant increase of cell number and cell proliferation in the mandibular LNs. This increase was time-dependent, with the exception of the group that was exposed for 360 minutes/day. This group, however, was exposed for two days only. Effects of test material on cell number and cell proliferation were statistically significant for all exposure groups. Test material exposure for 90 minutes/day or longer increased proliferation in the auricular LNs. The mean SI in the auricular LNs was a factor 3-4 higher than in the mandibular LNs, but the variance was very high. Dermal exposure to 10% test material resulted in a SI of 18.8.

Test material elicited clearly a higher immune response as indicated by the cellular proliferation in the primary draining lymph node of the nasopharynx. The authors considered that the study provided evidence of respiratory sensitisation, however, because of the excessive irritation that was induced by the exposures, and the doubts about the relevance of the mandibular lymph nodes for respiratory sensitization, this conclusion cannot be supported.

 

In another study that used a similar method (inhalation exposure to isoeugenol dissolved in acetone for 45, 90, 180 or 360 minutes) the authors concluded that isoeugenol caused respiratory sensitisation (Burg et al., 2014, Rel.3). However, there was no dose-related (period of exposure to a single concentration) increase in the stimulation index and there was no data presented on clinical signs or irritant effects seen in the animals. In addition, there is the methodological criticism that the mandibular lymph nodes do not drain the tracheal and lung tissues, which undermines the relevance of the observations for respiratory sensitisation.

 

The respiratory lymph node assay appears to be a model with some potential, but needs to be further validated with known skin and respiratory sensitizers, and also with non-sensitizers and irritants. Furthermore, there is a potential weakness in the model in that the mandibular lymph node drain the mouth and nasophanrynx but not the tracheal and lung tissues, therefore the relevance to respiratory sensitisation is not proven. The reliability of the available studies cannot be evaluated. Indeed, the respiratory lymph node assay is a recently developed animal model and more research is needed to further validate this model (e.g. cut-off value). In addition, no evaluation of irritation to the respiratory tract was made, although Isoeugenol is classified as skin and eye irritant and irritation has been observed in sub-acute inhalation toxicity studies. In the skin LLNA it is required to use dose levels that do not induce excessive levels of irritation. In this study it cannot be determined if the level of irritation was excessive or not, but in some case animals died and showed signs of respiratory distress.

 

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 strong skin sensitizer (Category 1A) 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 skin 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 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, see section 7.10.4). 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.

 

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Justification for classification or non-classification

Harmonized classification:

A harmonised classification (CLH) is available for the registered substance for skin sensitisation Category 1A,H317: May cause an allergic skin reaction. RAC Opinion adopted on 10 March 2016 proposed future entry in Annex VI of CLP Regulation.A specific concentration limit (SCL) of 0.01% has been set.

 

Self classification:

Based on the available animal and human evidence on the registered substance and cis-isomer, a classification as Skin Sens. 1A – H317: May cause an allergic skin reaction is required with EC3 value < 2%.

 

No classification for respiratory sensitisation is recommended at that time because the data available regarding respiratory sensitisation is not considered sufficiently reliable. Moreover, a scientific argument using mechanistic information and human evidence 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.