Sprachen:
The SCCS opinion states:
6. Retrieval of evidence and classification of fragrance substances
For a systematic review, a structured approach of identifying, grading and aggregating available information should be used. Regarding the classification of substances as allergens, a number of approaches have been suggested (158-160). The categorisation of skin sensitisers according to sensitising potency has also been proposed (161, 162). For this opinion, these discussions were extended to reconcile different perspectives and to arrive at a strategy that is both consistent and applicable in practice. By default, positive human evidence (clinical data) overrides negative results obtained in animals. This implies that the observation of a sufficient number of positive clinical cases is more important than potency information derived from animal experiments (LLNA).
Cosmetovigilance information based on consumer complaints only is of limited value in the evaluation of sensitisation risk associated with cosmetic allergens, including fragrances, as it does not identify specific causative substances, and likely to severely under-estimate the frequency of contact dermatitis. An exception is the combination with qualified diagnostic work-up, as in the French REVIDAL/GERDA system (299); however, such data are generally published, thus publicly available, and considered in the present opinion.
6.1. Retrieval of evidence
A systematic search strategy was employed for the retrieval of clinical data, as outlined below. Experimental data are often not published hence the exact definition of the scope considered for the review is necessary and is given below. Additional LLNA data were reviewed, if identified by the search strategy, e.g. in chapter 8.1.2 and, as “additional information”, in Annex I of this opinion. This supplemental evidence was, however, not considered for the final categorisation in Table 13-2.
6.1.1. Search strategy for clinical data
Method of literature search:
- Manual search of the issues of the journal “Contact Dermatitis” (for the 26 “annex substances”, which were re-evaluated in the present opinion, starting 1999) up to October 2010, identifying all studies with fragrance substances.
- PubMed search of CAS numbers identified in the previous opinion, reviews and already identified clincial studies, respectively, and manual screening of identified publications (narrowed for the last 10 years for the 26 “annex substances”), if necessary narrowing the search results by adding “dermatitis” or “allergy”. For example, for citral: 5392-40- 5 AND (dermatitis or allergy), translated into "5392-40-5"[EC/RN Number] AND ( ("dermatitis"[MeSH Terms] OR "dermatitis"[All Fields]) OR ("hypersensitivity"[MeSH Terms] OR "hypersensitivity"[All Fields] OR "allergy"[All Fields] OR "allergy and immunology"[MeSH Terms] OR ("allergy"[All Fields] AND "immunology"[All Fields]) OR "allergy and immunology"[All Fields]) )
- Manual search of all RIFM reviews published in supplement issues of “Food and Chemical Toxicology2” in the past 20 years. In case of the least evidence on human sensitisation the substances were preliminarily selected and further research initiated.
- Consideration of the most important (“top 100”) fragrance compounds in terms of volumes used (disregarding functional additives such as solvents) as supplied by the International Fragrance Association IFRA (personal communication 2010).
- Consideration of fragrance compounds ranking 101 to 200 on the list of use volumes, if they were self-classified by manufacturers as skin sensitisers (R 43).
For the present systematic overview of available clinical data, only original studies were considered, as only these provide direct evidence, while other reviews, partly being based on the same original reports, only served to identify additional literature. In contrast, selected reviews, guidelines and similar publications were used as basis for methodological approaches (e.g., in section 11).
6.1.2. Collection of experimental (LLNA) data
The SCCS requested the International Fragrance Association (IFRA) to submit data on animal tests performed with fragrance substances, to be presented in a structured format. In response, industry submitted first a poster (163) and later a report consisting of LLNA protocol summaries on the 59 fragrance substances in the poster (164). No guinea pig studies were submitted. The SCCS has reviewed and analysed the report and the publications quoted in the report. A summary is given in chapter 8 and full data are given in Annex II. EC3 values on some additional fragrance substances in two published reviews (165, 166) have also been considered. Additional EC3 values may be available in the scientific literature and there may also be other unpublished data.
6.2. Grading of evidence
Assembled evidence has to be graded in two steps: (i) the quality of each single study, and (ii) the strength of evidence underlying the eventual classification as an allergen. Generally, studies (published or not) which are eligible for consideration will contribute to the final overall judgement to different degrees.
- Positive human data, if sufficiently demonstrated (point (i) below), will always over rule experimental (animal), in vitro or in silico data of similar internal validity, as they provide direct evidence on allergenicity in humans.
- Small study groups will contribute less precise information than larger studies of otherwise similar quality. As a minimum requirement, the size of the study groups and the numbers of events must be stated in the reports.
The following subsections will address special aspects of clinical and experimental studies, respectively.
6.2.1. Quality of a clinical study
Two major types of clinical studies must be distinguished because they provide a different scope of information:
(i) Case reports or small case series, focusing on patients with positive (test) reactions to the target substance, sometimes including a set of non-exposed, possibly non- diseased “control patients”; these should present a concise summary of all relevant aspects of the patient's history, diagnostic procedures and possibly further outcomes. (ii) Clinical series in which results of a group of patients patch tested with the target substance, often combined with other substances, are presented. In the latter type of report, usually only a minority of patients tested show a positive reaction to the test substance. This implies that the majority of patients can be used to illustrate the proportion of irritant, doubtful and negative reactions. The degree of detail on the patients' histories is usually limited in such studies, compared to case reports.
Some of the basic quality criteria in clinical patch testing which should be considered are:
- Adherence to international patch test guidelines (32, 96).
- Material(s) tested should be characterised.
- Total number of patients tested must be given.
- Patient selection should be described.
- Relevance may be demonstrated either on a case-by-case basis, following pertinent guidelines, or in terms of a significant epidemiological association between sensitisation and exposure or valid markers of exposure.
Concerning relevance, it must be noted that while clinical relevance can provide important information (see 4.4.1), it is ideally based on comprehensive knowledge of prior exposures. Since the implementation of labelling 26 fragrances, previous exposure to these can often be ascertained in the assessment of relevance of a positive patch test reaction (44). However, exposure to substances not listed on a product ingredient label is obscure, except in very rare cases where elaborate diagnostics and chemical analyses are feasible (e.g. (167)). Thus, a lack of information on relevance (reported in studies) does not invalidate the impact of diagnosed contact sensitisation.
6.2.2. Quality of an experimental study
International guidelines such as the pertinent OECD guidelines for testing sensitisation have been developed and adopted. Experimental studies following these guidelines are considered as valid. However, a vast number of non-guideline studies are available and should be assessed on a case-by-case basis.
6.2.3. Quality of “other” evidence
Supporting evidence besides human and animal (experimental) data comprises in vitro test systems, in chemico experiments and structure activity relationships (SARs). SAR analysis has at present no formal regulatory validation for skin sensitisation, nevertheless it may provide useful indicative information on sensitising potential when no or limited clinical or animal data are available. SAR studies must consider a possible formation of haptens (allergens) from compounds able to act as prehaptens by, e.g. autoxidation outside the body as well as metabolic activation in the skin of compounds able to act as prohaptens (122, 168).
6.3. Aggregating evidence for a final conclusion
The criteria listed below are followed as a flow chart to arrive at a conclusion. This implies that if classification into one category is achieved, subsequent categories need not be considered. Based on the above criteria, fragrance substances were selected to be included in the present opinion if classified in one of the categories defined below.
6.3.1. Established contact allergen in humans
To qualify as an established contact allergen, the SCCS considers that at least one of the following two criteria must be met:
- At least two clinical series fulfilling the quality criteria from two different centres with cases of sensitisation, or at least three separate clinical series from different centres if a study, or studies, do not meet all quality criteria. (→ sufficient human evidence present) or
- Case reports from at least two independent centres describing more than two patients altogether in whom clinically relevant contact sensitisation had unequivocally been proven (→ sufficient human evidence present) or
- At least one clinical series fulfilling the quality criteria, together with at least one case report of clinically relevant contact sensitisation (→ sufficient human evidence present); or
- Experimentally induced sensitisation (e.g. unequivocally positive human maximisation tests/repeated insult patch test)3 (→ sufficient human evidence present).
6.3.2. Established contact allergen in animals
To qualify as an established contact allergen, the following criterion must be met:
- At least one positive animal study carried out according to accepted guidelines, providing evidence of a sensitisation potential (→ sufficient animal evidence present).
6.3.3. Likely contact allergen, if human, animal and other evidence is considered
To qualify as an likely contact allergen, if classification as “established ...” is not applicable, at least two of the following criteria must be met:
- Individual cases of allergic patch test reactions not fulfilling the requirements for sufficient evidence (→ limited human evidence present) or
- At least one positive non-guideline animal study, which should be evaluated on a case-by-case basis (→ limited animal evidence present) or
- Other evidence, e.g. results from in chemico experiments or in vitro tests or from structure-activity considerations based on sufficiently valid results for closely related compounds (→ other evidence present).
6.3.4. Possible contact allergen, if human, animal and other evidence is considered
To qualify as a posible contact allergen, if classification as “established ...” or as “likely ...” contact allergen is not applicable, at least one of the following criteria must be met:
- Individual cases of allergic patch test reactions not fulfilling the requirements for sufficient evidence (→ limited human evidence present) or
- At least one positive non-guideline animal study, which should be evaluated on a case-by-case basis (→ limited animal evidence present) or
- Other evidence, e.g. results from in chemico experiments or in vitro tests or from structure-activity considerations based on sufficiently valid results for closely related compounds (→ other evidence present).
6.4. Conclusions
The present opinion includes (i) a well-defined search strategy for retrieving pertinent evidence; (ii) a definition of criteria used to evaluate available evidence; and, finally (iii) a set of rules to categorise the substances with regard to the relevant toxicological endpoint, i.e. sensitisation in man, based on the evidence.
Source & ©: SCCS, 
26-27 June 2012, 6. Retrieval of evidence and
classification of fragrance substances. p. 40-44
The SCCS opinion states:
7. Reported fragrance allergens from the clinical perspective
In this chapter, clinical evidence regarding sensitisation to individual fragrance chemicals and to natural extracts (essential oils) is tabulated. In this report “single chemicals” refers to chemicals of natural or synthetic origin whose chemical identity is fully known. The term “natural extracts” refers to plant or animal derived mixtures of natural chemicals, for example lavender oil, whose composition may be variable and may or may not have been fully or partly established. Full information, including possible synonyms, structural formulas (in the case of single chemicals only), a short summary of available evidence and further information, e.g. on regulatory status, is presented in Annex I.
7.1. Tabular summary of evaluated individual fragrance chemicals
Regarding nomenclature, INCI names are used wherever possible. If an INCI name is not available, the perfuming name as listed by CosIng is used. Detailed information on the publications identified and considered for this report can be found in Annex I. Several substances are currently banned from the use in cosmetic products by Annex II of the Cosmetics Directive, based on concerns regarding one or more toxicological endpoints. While available clinical evidence regarding this set of substances is listed in Annex I, these substances have not been further evaluated and are thus not included in this chapter.
In this section, a tabular overview on the classification of substances considered is presented in four tables listing:
- Established contact allergens in humans (→ sufficient human evidence present).
- Substances with positive human data, which are, however, not sufficient to categorise as “established contact allergen in humans” (→ limited human evidence present).
- Substances with negative human data, i.e. patch tests of patients with suspected contact allergy to fragrance ingredients which yielded negative results.
- Substances eligible for inclusion (see beginning of chapter 6) for which no human data are available.
A critical point in understanding this scheme is that there is publication bias in reporting allergens. This is due to the fact that once a substance has been reported and accepted as a contact allergen in humans, further reports are less likely to be published unless they are part of a epidemiological survey or when there is a novel source of exposure. Moreover, the number of patients displaying positive test reactions obviously not only depends on the underlying prevalence of sensitisation, but also on how often a substance is patch tested. This implies that inclusion of an allergen or allergen mixture in the baseline patch test series (as for Fragrance Mix I and II, Myroxylon pereirae and HICC, and partly also other substances/mixtures) will yield the maximum possible number of cases. In contrast, patch testing in “special” series, e.g. as a break-down of single constituents of the respective mix in case of a positive reaction to the latter, or with application only in the case of strongly suspected fragrance intolerance, will mostly result in higher relative numbers than testing the same compound consecutively, but also in lower absolute numbers.
In Table 7-1, the single substances are listed with a semi-quantification of their impact which were categorised as established contact allergens in humans according to the criteria given in chapter 6.3.
Established contact allergens in humans, according to the criteria outlined in chapter 6.3.1, were categorised according to the number of patients reacting positively and to the number of patients tested, based on the publications considered (see annex I for references). The following categories were used:
If a test allergen has been tested in less than 1,000 patients, “r.t.” (rarely tested) is added in the following tables. For this categorisation, absolute numbers of cases of sensitisation, and not the relative frequency of positive patch tests, were used, because relative frequencies depend heavily on the selection of patients for patch testing. Thereby, an important allergen tested routinely, in the baseline series, may yield 1 to 2% positive reactions (usually in several thousand patients), while an allergen tested in a selective fashion (in much fewer patients) may yield an even higher relative frequency. Moreover, case reports/series cannot be interpreted in terms of relative frequencies. The calculation of absolute numbers was based on all available literature, as detailed in the annex I to this opinion, i.e., regarding the 26 substances already listed in Annex III to the Cosmetics Directive includes data already evaluated in the previous opinion.
Those substances which were categorised as +++ or more, i.e. those with the most reported cases, were also the top ranking substances in large series of patients tested with the 26 labelled fragrance ingredients ((44, 74) and additionally (170)). Geraniol is an exception, as it was all negative in the Danish study (170), but was still among the top ten in the Dutch and German studies (44, 74), with prevalence of 0.5%-0.6% positives. Geraniol has, in addition, caused many cases of contact allergy in other areas of Europe (49).
The use of absolute numbers allows the pooling of studies with different selection criteria. Limonene and linalool were not tested in their oxidized forms in the three studies (44, 74, 170) and would not have been identified, if only these publications had been the basis of assessment.
It should be noted that oxidised fragrance terpenes with defined content of the major haptens formed after autoxidation have not been commercially available for testing in dermatology clinics. In the published clinical studies testing oxidised fragrance terpenes, the patch test preparations have been obtained specifically for the performed multicentre studies. From 2012, patch test preparations of oxidised limonene and oxidised linalool with defined content of the major allergens in the oxidation mixtures, i.e. the hydroperoxides, are commercially available (see also chapter 5). Table 7-2 lists those substances which gave rise to a few reported cases of contact sensitisation only, or where results have been reported from just one clinical department. Thus, the level of evidence concerning human data must be regarded as limited, according to the definitions given in chapter 6.3.
For a number of substances negative patch tests results were obtained, usually in rather small patient samples (max. 313 patients). For some of these substances exposure is substantial, according to data submitted from IFRA. It should be noted that a negative result does not rule out a notable sensitisation prevalence, as the study size has to be larger than, e.g. n=298 to yield a 95% CI which excludes a prevalence of 1% and larger than n=597 to exclude a prevalence of 0.5%.
For yet another subset of substances, no human data were publicly available. However, exposure to these substances is important as they are used in high volumes (this being the sole criterion for inclusion in this list) and, therefore their hazard with regard to contact sensitisation should be examined.
7.2. Tabular summary of evaluated natural extracts/essential oils
Natural raw materials in terms of extracts are used in the fragrance and flavour industry for various reasons. Most importantly, several naturally occurring mixtures have a very complex composition and sensory nature which cannot (fully) be achieved by synthetic the demand for perfumes based on natural materials is considerable (189). The three main methods used to concentrate plant fragrance substances (190); distillation, mechanical separation (“pressing”), and solvent extraction, yield very different extracts. Essential oils are obtained by water steam, water, ethanol, or water/ethanol distillation. Essence oils are essential oils that separate from the aqueous phase in the distillation receiver during the distillative concentration of fruit, usually citrus, juices. Citrus peel oils, apart from distilled lime oil, are prepared in a special way by pressing the peel to release mostly volatile substances from the pericarp in small oil glands, mostly highly volatile terpene hydrocarbons. However, they also contain small amounts of non-volatile compounds such as dyes, waxes and furocoumarins. The method of solvent extraction is generally applied in the separation of heat-labile materials or if an essential oil can only be obtained in very low yield, e.g. from blossoms. It is also used if the non-volatile components are desired for their fixative properties, e.g. in the preparation of resinoids from exudates. The most important extracts are termed: (i) concretes, an extract of fresh plant material with nonpolar solvents, containing not only volatile, but also a large proportion of non- volatile substances such as waxes; and (ii) absolutes, which are prepared by taking up concretes in ethanol; compounds that precipitate on cooling are removed by filtration, yielding a wax-free residue called absolute. Resinoids, used for their fixative properties, are prepared by extracting plant exudates with alcohols or nonpolar solvents. The products are usually highly viscous and thus sometimes diluted, e.g. with phthalates or benzyl benzoate. Oleoresins are concentrates prepared from spices by solvent extraction (189). An ISO norm exists regarding the nomenclature of aromatic natural raw materials (ISO/DIS 9235 Aromatic raw materials - vocabulary; International Standardisation Organisation, Geneva, Switzerland). This nomenclature has been considered in Annex I, whereas in the present opinion, nomenclature is according to the CosIng database. Concerning extraction processes for many essential oils, ISO standards exist; for detailed information see Annex I to this opinion. Regarding clinical data in terms of contact allergy to essential oils and natural extracts, the main focus is on general dermatological patients with complaints related to use of cosmetics etc. However, series of cases with occupational exposure to essential oils with occupational allergic contact dermatitis have also been reported (e.g. masseurs, physiotherapists (191, 192), aromatherapists (193-197), beauticians performing massages (198). For further details, e.g. PT results with various essential oils, see Annex I.
In this section, a tabular overview on the classification of substances considered is presented in three tables listing:
- Extracts identified as established contact allergens in humans (→ sufficient human evidence present).
- Extracts with positive human data, which are, however, not sufficient to categorise as established contact allergen in humans (→ limited human evidence present).
- Extracts with negative human data, i.e. patch tests of patients with suspected contact allergy to fragrance ingredients which yielded negative results.
In Table 7-5, essential oils with sufficient human evidence to categorise these as established contact allergens in humans are presented.
Table 7-6 lists a number of essential oils, mostly tested in just one clinical department, and thus, or for other reasons, not satisfying the criteria for being categorised as established contact allergen in humans (i.e. limited human evidence present).
The final table is an indicative list of natural extracts which lack published human data, but which are of interest: (i) as high-volume exposure; (ii) due to published positive animal experiments; or (iii) because they contain well-known (established) contact allergens.
7.3. Conclusions
- According to the criteria described in chapter 6.3 a total of 54 individual chemicals and 28 natural extracts (essential oils) can be categorised as established contact allergens in humans, including all currently regulated substances.
- Of the 54 individual chemicals which are established contact allergens in humans, 12 are considered to be of special concern due to the high number of reported cases, (> 100, i.e. category +++ or ++++ in Table 7-1). These are further considered in chapter 5 (limonene and linalool) and the remainder in chapter 11. In particular one ingredient stands out, hydroxyisohexyl 3-cyclohexene carboxaldehyde, having been the cause of more than 1,500 reported cases since the 1999 opinion (see also chapter 4.3.1, chapter 11.3 and Annex I).
- For an additional 33 individual chemicals (Table 7-2) and 14 natural extracts (Table 7-6), positive patch test results have been reported. However, they do not qualify for the above category, i.e.only limited human evidence is present.
- For a number of fragrance substances (n=18, Table 7-3) patch testing did not yield positive results. However, numbers of patients tested are generally too small to rule out the existence of clinical contact sensitisation with sufficient confidence. No clinical evidence has been identified for 39 individual chemicals that have been reported to be frequently used (Table 7-4).
- For the substances (and, if possible, also for the main constituents of the natural mixtures) with limited or no human evidence, additional animal data and/or SAR considerations are taken into account. Aggregated data for these substances are presented in chapter 13.
Source & ©: SCCS,
26-27 June 2012, 7. Reported fragrance allergens from the
clinical perspective. p. 45-57
The SCCS opinion states:
8. Animal data
8.1. Predictive tests and sensitising potency categories
The animal test methods used in harmonised classification of substances, according to their potential to cause skin sensitisation, are the guinea pig maximisation test (GPMT), the Buehler test6 and the local lymph node assay (LLNA)7. These methods are used in hazard identification and risk assessment for regulatory purposes under REACH8. For registration in REACH, the LLNA is the preferred method for measuring skin sensitisation potential in animals, and justification for the use of other methods needs to be provided. According to the directives on classification and labelling9, substances and preparations meeting positive criteria in these tests shall be classified as sensitising and assigned the symbol “Xi” and the risk phrase “R43: May cause sensitisation by skin contact”; or, according to the recent regulation on classification, labelling and packaging (CLP10) “H317: May cause an allergic skin reaction”.
As yet, there is no officially validated in vitro test method for skin sensitisation. Therefore, for cosmetic ingredients the LLNA, the GPMT and the Buehler test have also been used in risk assessment for regulatory purposes. Positive results from the OECD guideline animal tests mentioned above which are sufficient to classify a substance as a skin sensitiser (R43) are:
- GPMT; at least 30% of the animals have a positive response.
- Buehler test; at least 15% of the animals have a positive response.
- LLNA; at least a 3-fold increase in lymph node cell proliferative activity is induced, compared to vehicle-treated controls (stimulation index SI ≥3). For positive LLNAs, an EC3 value is calculated which gives the estimated concentration of a chemical necessary to give a 3-fold increase in proliferative activity compared to vehicle- treated controls.
Further categorisation of substances classified with R43 into three groups according to allergen potency (extreme, strong and moderate) has been proposed by a European Commission expert group on skin sensitisation (161, 205), and proposed also in the ECHA guidance document on application of the CLP criteria (162). Such categorisation is based on EC3 values in the LLNA, on intradermal induction concentration in the GPMT, and topical induction concentration in the Buehler test. The potency categories and their default concentration values based on EC3 values in the LLNA as defined in (161): extreme sensitiser (EC3 value ≤ 0.2); strong sensitiser (EC3 > 0.2 - ≤ 2); and moderate sensitiser (EC3 value > 2). When LLNA EC3 values are available from more than one study, the lowest value should normally be used. Where multiple animal data sets lead to different categorisation of the same substance, the higher potency category should apply (161, 205).
The potency categorisation of substances based on the LLNA is applied by the SCCP in risk assessment of cosmetic ingredients, particularly hair dye substances (206).
8.1.1. LLNA data
The SCCS requested the International Fragrance Association (IFRA) to submit data on animal tests performed with fragrance substances, to be presented in a structured format. In response, IFRA submitted first a poster (163) and later a report consisting of LLNA protocol summaries on the 59 fragrance substances in the poster (164). No guinea pig studies were submitted. The SCCS has reviewed and analysed the report and the publications quoted in the report.
Table 8-1 displays the EC3 values for fragrance substances in the report submitted by industry (164). EC3 values for some additional fragrance substances in two published reviews (165, 166) have also been included in Table 8-1. Table 8-2 presents LLNA results for oxidised substances. Full data are given in Annex II. Table 8-3 summarises the distribution of fragrance substances, by potency category, according to EC3 values. Additional EC3 values may be available in the scientific literature. Many more animal experiments may have been performed, but have not been published.
8.1.2. LLNA data on oxidised fragrance substances
For fragrance substances that can autoxidise upon air exposure, it is also important to investigate the sensitisation potency after air exposure. The oxidised compounds are clinically relevant as they represent what the consumers could come in contact with from perfumes and fragranced products. In Table 8-2 the LLNA data for some of the most commonly used fragrance substances, pure and after autoxidation, are presented. The EC3 values obtained for the pure substances are 5-10 times higher compared to those obtained for the same substances after air exposure. The experimental air exposure simulated air exposure that can take place during normal handling and storage. In the production process, some perfumes are “matured” aerobically, stirring included. During this process, some fragrance substances may be oxidised. It should be noted that, although only a few substances capable of oxidation have so far been investigated, structural alerts indicating possible autoxidation are common among the fragrance substances listed in this document (see chapter 9). It is important to further investigate this issue for increased understanding of the associated risk.
8.2. Methodological considerations
EC3 mean values
In the submitted poster (163) and the report by IFRA (164), the LLNA weighted mean EC3 values (μg/cm2) are presented. The SCCS considers it is misleading to present EC3 values as mean values from tests performed with different vehicles. It is generally agreed that the lowest EC3 value should be used if there is more than one study fulfilling the OECD guideline requirements (161, 205), and these have been introduced into Table 8-1. The EC3 values in the reviews by Gerberick et al. and Kern et al. (165, 166) were based on single representative experiments with a vehicle described in the OECD guideline 429 (see above), and preferably with acetone:olive oil. EC3 mean values, as in the submission by IFRA, were not presented in these two reviews.
Vehicle
The most frequently used vehicle in the submission by IFRA (164) was ethanol:diethyl phthalate (1:3), followed by acetone:olive oil (4:1). In some experiments, antioxidants were mixed with ethanol:diethyl phthalate. The vehicle was not reported in some of the references, and no rationale for using vehicles other than those recommended was given in the report (164). According to the OECD guideline 429 (see above), the recommended vehicles are acetone:olive oil (4:1), N,N-dimethylformamide, methyl ethyl ketone, propylene glycol, and dimethyl sulphoxide, but others may be used if sufficient scientific rationale is provided. It is well known that a difference in the EC3 value can be obtained for the same substance depending on which vehicle is used in the LLNA. Thus as an additional control, supplementary to the guideline based LLNA control, a clinically relevant solvent or the commercial formulation in which the test substance is marketed may be used.
Number of doses and animals
According to the OECD guideline 429 (see above), a minimum of three concentrations should be tested. The number of consecutive doses used in the reported data, was generally five, sometimes three and in few experiments two. The SCCS considers that too few concentrations were tested in four studies in which only two concentrations were used. Lower concentrations than those tested should have been used in experiments with five fragrance substances, in which the EC3 value could not be determined. Higher concentrations than those tested should also have been used in experiments with 12 substances, in which the EC3 value could not be determined. The number of animals per dose group was generally four plus a non-exposed control group, sometimes five, and in few experiments six; the minimum according to the OECD guideline being four.
Units for concentrations
In the submission by IFRA (164) the EC3 values are given in weight per area unit (μg/cm2). The SCCS considers that the EC3 values (%) are the values of primary interest in communicating risk assessment, as EU legislation, OECD guideline 429 and scientific literature refer to EC3 values (%). However, the SCCS recommends that molar (M) EC3 values should be considered, as they give the concentration based on the molecular weight of substances. They have thus been calculated and introduced into Table 8-1.
EC3 values (%) overestimate the intrinsic molecular sensitisation potency for low molecular weight compounds while compounds with a high molecular weight are underestimated. Regarding the differences in molecular weight between the studied fragrance substances, a variation is seen if the ranking list of the sensitisation potency is based on EC3 (%) or EC3 (M) since some substances have a molecular weight twice as high as others.
From comparisons in Table 8-1, we notice that, e.g. hydroxyisohexyl 3-cyclohexene carboxaldehyde (HICC) has an EC3 value of 17.1 %, or 0.81 M when the calculation includes its molecular weight, while for trans-2-hexenal the corresponding values are 2.6% and 0.26 M. The example shows that comparing the sensitisation potency between these two substances using the EC3 values in % exaggerates the sensitisation potency of trans-2- hexenal compared to that of HICC. When using the EC3 values in molar concentrations the difference is not so pronounced.
8.3. Summary of animal data by LLNA
The distribution of sensitising potency of fragrance substances compared to other substances, (e.g. biocides, dyes, plastic materials) taken from three references (164-166) as assessed by EC3 values in the LLNA, is shown in Figure 8-1 and Table 8-3. For 10 substances, no EC3-value could be established. These should have been tested at higher concentrations – some of these would most probably have generated an EC3 value. However, we reported here “No EC3 value established”. 5 substances should have been tested also at lower concentration and in these cases the EC3 value could have been lowered, meaning a more severe potency category could have been achieved. In all, approx 150 experiments were reported in (164), listed in Annex II. The median EC3 value of evaluable fragrance substances (5.9%) is similar to other substances tested (5.5%). However, very few fragrance substances have low EC3 values (≤ 2).
Substances with an EC3 value ≤2 may be categorised as strong or extreme sensitisers. Such potent sensitisers are comparatively rare among fragrance substances assessed in the LLNA. Nevertheless, fragrances are important allergens in humans, which points to repeated skin exposure to less potent sensitisers as a factor strongly determining sensitisation risk.
Figure 8-1: The distribution of fragrance chemicals and a variety of other chemicals (e.g. biocides, dyes, plastic materials), taken from the three references (164-166), are depicted as boxplots on a logarithmic scale. The bottom of the box denotes the 1st quartile (25% percentile), the thick line in the box the median, and the top of the box the 3rd quartile (75% percentile). Outliers, i.e. below the 25% and above the 75% percentiles, are shown as whiskers. Beyond the 1.5-fold interquartile range, single values are shown as circles instead of whiskers. The difference in distribution is not significant (Wilcoxon test: p=0.061).
Note: EC3 values for the five oxidised fragrances additionally examined (Table 8-2) range from 3.0 to 4.8 (median 4.4) and are lower by a factor of around 7 than EC3 values of the respective non-oxidised material.8.4. Conclusions
- In the event that human data are lacking, the LLNA provides important information on skin sensitising potential and potency.
- Animal data on fragrance substances submitted by IFRA (164) and assessed in this opinion were generated exclusively by LLNA. Other guideline methods are, however, also available.
- The vast majority of the submitted (164) and additional (165, 166) fragrance substances tested by the LLNA are skin sensitisers.
- Several studies in the IFRA report (164) were of insufficient quality, not following the OECD guideline.
- Fragrance substances that can be predicted to autoxidise upon air exposure should also be tested after air exposure, as oxidation may significantly increase their sensitising potency.
- It can be concluded that the skin sensitising potency, as assessed by the LLNA, is only one of several factors that are of importance for sensitisation to fragrance substances. This is illustrated by the fact that only a small fraction of sensitising fragrance substances can be categorised as an extreme allergen based on LLNA test results. Therefore, doses from repeated deposition onto skin must be considered a driving force of sensitisation risk.
Source & ©: SCCS,
26-27 June 2012, 8. Animal data. p. 58-65
The SCCS opinion states:
9. Structure activity relationships (SAR): grouping of substances based on expert judgement
Whether or not a particular chemical will be a sensitiser, and how potent it will be if it is a sensitiser, depends on its ability, either directly or after activation, to react with appropriate proteins in the skin. This fundamental concept was initially demonstrated by Landsteiner and Jacobs in 1936 (208) and subsequently validated by numerous studies with various types of chemicals (some key references: (209-213)). The ability to predict sensitisation potency, or lack of it, depends on being able to predict reactivity to skin proteins. This is the basis of SAR analysis for skin sensitisation. The prediction can often be made based on the chemical structure, recognising structural features (referred to as structural alerts) that are associated with reactivity.
The relationships between molecular structure and reactivity that form the basis for structural alerts are based on well established principles of mechanistic organic chemistry (214). Examples of structural alerts are aliphatic aldehydes (alerting to the possibility of sensitisation via a Schiff base reaction with protein amino groups), and α,β-unsaturated carbonyl groups, C=C-CO- (alerting to the possibility of sensitisation via Michael addition of protein thiol groups). Major mechanistic reactivity domains have been discussed in detail by Aptula and Roberts (215). Prediction of the sensitisation potential of compounds that can act via abiotic or metabolic activation (pre- or prohaptens) is more complex compared to that of compounds that act as direct haptens without any activation. The autoxidation patterns can differ due to differences in the stability of the intermediates formed, e.g. it has been shown that autoxidation of the structural isomers linalool and geraniol results in different major haptens/allergens. Moreover, the complexity of the prediction increases further for those compounds that can act both as pre- and prohaptens. In such cases, the impact on the sensitisation potency depends on the degree of abiotic activation (e.g. autoxidation) in relation to the metabolic activation.
These structural alerts can be applied by computerized expert systems, i.e. in silico or by estimations made by organic chemists (in cerebro) using their experience. When an organic chemist looks at a chemical structure, they recognise parts of the structure that they can associate with reactivity, the type of reactivity (i.e. assign the reaction mechanistic domain), and other features of the molecular structure that will affect the reactivity positively or negatively. Human experts should be aware of the complexities, and how structural modification can alter the reactivity associated with structural alerts, etc. Importantly, they can also recognise where there are unfamiliar structural features whose effects they cannot confidently predict. In such cases they can call for experimental chemistry work (in chemico) to be done to ascertain the presence or nature of, and degree of reactivity. In chemico methods include organic chemistry experimentation to identify chemical reaction products from oxidation and/or reaction with model nucleophiles, identification of mechanisms of reaction. In so called in chemico reactivity methods, the ability of a specific chemical to react with selected peptides is determined so as to predict the sensitisation potential of the chemical under investigation (216, 217). To make in chemico reactivity methods able to predict the activity of prohaptens, the addition of horseradish peroxidase and hydrogen peroxide oxidation system has been tested to model the enzymatic oxidation in the skin (218, 219).
Although computerized expert systems are derived from input by human experts, they are less well able to capture the subtleties of structure reactivity relationships, and they sometimes fail to detect aspects of chemistry that are obvious to organic chemists. Human experts should be aware of the complexities, as well as how structural modification can alter the reactivity associated with structural alerts, etc.
The SAR evaluation made in the section below is based on in cerebro alerts applied by organic chemists.
Depending on the type of reactivity (the reaction mechanistic domain), it is sometimes possible to make a quantitative prediction of potency in the LLNA, which can be used to predict potency in humans relative to related known human sensitisers. These predictions use quantitative mechanistic models (QMMs) based on reactivity expressed quantitatively by model parameters, and sometimes in combination with hydrophobicity. For example, potency of aliphatic aldehydes and ketones (the Schiff base domain) in the LLNA is modelled by a combination of reactivity and hydrophobicity (220), whereas the LLNA potency of DNCB analogues (the SNAr domain) is well modelled by reactivity alone (221).
QMMs aiming not only to predict the potential to be a sensitiser but also to predict the potency, promise to be a useful tool in non-animal based risk assessment for skin sensitisation. However, in the field of fragrance substances there are major gaps in our present ability to apply QSAR/QMM. This is largely because many of the fragrance substances of interest have the potential to act via abiotic or metabolic activation (pre- and/or prohaptens, i.e. they themselves are only weak or non-sensitisers, but have the potential to be activated to form more potent sensitisers. Resulting sensitisation potency will depend on the extent of activation and the nature of the resulting products. It is possible to apply SAR analysis to identify these plausible possibilities, but QSAR modelling for these cases is not yet developed. However, much progress has been made in identifying structural alerts for the various activation mechanisms that have been recognised. This is reviewed by Karlberg et al. (122).
Chemicals with no structural alerts for direct reactivity, or for known activation mechanisms, and no unfamiliar structural features that might be associated with as yet unidentified activation mechanisms, can be predicted to be non-sensitising. Chemicals that do have alerts for reactivity (direct or via activation) are not necessarily sensitisers – they may be insufficiently reactive and/or insufficiently hydrophobic.
Substances meeting the inclusion criteria (see chapter 6), for which, however, no categorisation as established contact allergen in humans or established contact allergen in animals was possible, have been assessed for structural alerts. The results are presented in four tables based on the prediction made for the actual substance. The following SAR assessments have been used:
- Predicted sensitiser; structural alerts: Compounds containing structural alerts comprising direct reactive compounds and for compounds that after specific abiotic or metabolic activation (prohaptens and prehaptens) can be predicted to be sensitisers by structural comparison to known allergens.
- Possible sensitiser; structural alerts: Compounds containing structural alerts that by comparison to known allergens with similar structures were expected to be less reactive and hence less likely to be sensitising. Also compounds with structural alerts indicating a possible abiotic or metabolic activation (possible prehaptens or prohaptens) but with no structural data available for comparison, were included in this group. Consequently, a possible sensitiser may turn out to be a non sensitiser when tested in vivo.
- Predicted non-sensitiser (NS); no obvious structural alerts
- Not predictable due to insufficient/conflicting data
Table 9-1: Predicted sensitisers.
Table 9-2: Possible sensitisers.
Table 9-3: Predicted non-sensitisers with no obvious structural alerts.
9.1. General results
From this work with the included SAR predictions, the following observations can be made.
- For substances for which sufficient experimental/clinical evidence is missing, SAR analyses have been performed to predict a probable or possible risk of allergenic (sensitising) effect. These predictions are based on chemical reactivity and the recognition of structural features in a substance that are in common with the structural features that have been shown to cause sensitisation from other substances. In cases where the SAR analysis indicates a sensitisation potential, the substance should be investigated further to confirm or reject the conclusion drawn from the SAR analysis.
- Prediction of the sensitisation potential of compounds that can act via abiotic or metabolic activation (pre- or prohaptens) becomes more complex compared to that of compounds that act as direct haptens without any activation.
- The complexity of the prediction increases further for those compounds that can act both as prehaptens and prohaptens.
- Prediction of the sensitisation potential of compounds that can act as prehaptens is further complicated by the fact that the autoxidation patterns can differ due to differences in the stability of the intermediates formed, e.g. it has been shown that autoxidation of the structural isomers of linalool and geraniol results in different major haptens/allergens.
9.2. Conclusions
The SAR evaluation made in this section is based on in cerebro alerts applied by organic chemists.
- Applying only mechanism-based QSAR (QMM) as a tool in non-animal based risk assessment for skin sensitisation is of limited value for fragrance substances. This is due to major information gaps in the present model when addressing substances that act via abiotic or metabolic activation, and the high incidence of such substances in fragrances.
- Quantitative structure activity relationship (QSAR) models should be further developed, combining, as appropriate, information from in silico, in chemico and in vitro methods.
- SAR, as performed here, is only one consideration in the overall weight of evidence.
Source & ©: SCCS,
26-27 June 2012, 9. Structure activity relationships
(SAR): grouping of substances based on expert judgement. p. 66-71.
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