Registration Dossier

Administrative data

Description of key information

Key value for chemical safety assessment

Skin sensitisation

Endpoint conclusion
Additional information:

Hydroxycitronellal was found to be skin sensitizing in a broad variety of studies with animals and humans. Based on this broad evidence, exemplary studies have been compiled here in order to substantiate the classification decision for the given endpoint.

In the defined key study, i.e. a local lymph node assay comparable to OECD guideline 429, groups of four male CBA mice received doses of 0%, 1%, 3%, 10%, 30% and 50% hydroxycitronellal in diethyl phtalate (DEP); 1:3 Ethanol:DEP; 3:1 Ethanol:DEP and Ethanol for three consecutive days. On the sixth day after the first application, all mice were injected intravenously by the tail vein with [3H] methyl thymidine and incorporation of 3HTdR was measured. As result, the sensitization potential of hydroxycitronellal varied with the vehicle, resulting in SI values of 7.2 at 50% in ethanol and 5.3, 6.2 and 5.8 in DEP, 1:3 Ethanol:DEP and 3:1 Ethanol:DEP, respectively. The corresponding EC3 values were 19.7% (DEP), 19.3% (1:3 Ethanol:DEP), 22.2% (3:1 Ethanol:DEP) and 26.4% (Ethanol). Thus, hydroxycitronellal was found to be skin sensitizing in the LLNA under the chosen testing conditions.

 

A summary on the outcome of LLNAs performed with hydroxycitronellal is given in the HERA risk assessment (Human & Environmental Risk Assessment on ingredients of European household cleaning products - HYDROXYCITRONELLAL; 2005). There it is reported, that EC3 values in eight Local Lymph Node Assays varied between 19% and 33% with a mean around 22.8% (ca. 5.7 mg/cm2; see. Annex 1).

 

A guinea pig maximisation test reported in literature, acc. to Magnusson and Kligman has been chosen as an exemplary study for adjuvant tests in guinea pigs (Basketter 1992). Albino Dunkin-Hartley guinea-pigs were treated by a series of six intradermal injections (0.5% Hydroxycitronellal) followed by a 48-hr occluded epicutaneous administration (100% Hydroxycitronellal) 6 - 8 days later. After a resting period of 12 -14 days, animals were challenged on one flank by a 24-hr occluded patch at the maximum non-irritant concentration (50% hydroxycitronellal). It was reported, that 60% of the treated animals showed a dermal response and were judged to be positive. Thus, hydroxycitronellal was found to be a skin sensitizer in the GPMT under the chosen testing conditions.

 

Various other guinea pig maximization tests arereported in the HERA risk assessment (Human & Environmental Risk Assessment on ingredients of European household cleaning products - HYDROXYCITRONELLAL; 2005)and confirmed a skin sensitizing potential of hydroxycitronellal (see. Annex 1).

 

The allergenic potential of hydroxycitronellal is also evident from non-adjuvant tests.

In an exemplary Buehler skin sensitization study reported in literature, guinea pigs were topically treated with 30% hydroxycitronellal in ethanol for induction and subsequently topically treated with 3% or 10% hydroxycitronellal in acetone for challenge (Buehler 1985). Further animals were topically treated with 10% hydroxycitronellal in ethanol for induction and subsequently topically treated with 10% hydroxycitronellal in acetone for challenge. Induction with 30% followed by challenge with 10% hydroxycitronellal resulted positive allergic reactions in 2/8 animals (25%) of the test group, whereas induction/challenge with 10% hydroxycitronellal did not lead to any allergic skin reactions. Thus, hydroxycitronellal was found to be skin sensitizing in the Buehler test under the chosen testing conditions.

Further Buehler Tests confirmed, that hydroxycitronellal is a skin sensitizer (HERA risk assessment (Human & Environmental Risk Assessment on ingredients of European household cleaning products - HYDROXYCITRONELLAL; 2005) and produced positive results at concentrations of 25% and higher but gave negative results when tested at 5% and 2.5% (see. Annex 1).

 

The sensitization potential of hydroxycitronellal was also demonstrated in various other adjuvant tests such as the Cumulative Contact Enhancement Test and various other assays in guinea pigs and mice. Concerning other non-adjuvant methods, e.g. the modified Draize tests, hydroxycitronellal was found to be negative, although studies were carried out a low concentrations. Epicutaneous tests show that hydroxycitronellal produced no sensitisation even at open doses of 100% but when the test material was administered under occlusion (see. Annex 1).

 

Besides the studies with animals, several studies with human are be taken into account for assessment.

 

The Human Repeat Patch Test (HRIPT) has been extensively used to study the potency and possible induction thresholds of hydroxycitronellal . The results of 85 HRIPTs are reported in the HERA risk assessment (see. Annex 1).

 

In 33 of the HRIPTs carried out on hydroxycitronellal, a second phase was introduced in which subjects who had completed the first full test and had shown no reactions, were subjected to a new complete HRIPT. The HRIPT already maximizes normal consumer exposure significantly and it is not known to what extent this second maximized test produces an unrealistic departure from a realistic simulation of consumer exposure. In any case, this second test gave rise to clear reactions in 32 of the 354 previously negative subjects re-tested this way (i.e. in 9 of the 35 second-phase studies).

 

In the HRIP Test, the allergenic potency of hydroxycitronellal appears to be vehicle-dependent. The presence of ethanol as a major or sole component of the vehicle, lowers the apparent threshold of induction.

 

A variety of other human skin sensitization tests, i.e. the Human Maximization Tests are reported in the HERA risk assessment report. In 12 of these tests carried out using an induction concentration of 12% in petrolatum, positive reactions were seen in all but four of these tests. At this dose, a total of 26 subjects were sensitized out of 298 tested. At an induction concentration of 10%, reactions were seen to both enantiomers. Studies carried out at 5% and 4% were negative but the number of subjects tested was low.

Overall, a No Expected Sensitization Level, i.e a dose (expressed as quantities retained on unit areas of skin) that is not expected to give rise to sensitization of subjects under exaggerated test conditions, has been derived in the HERA risk assessment report, including the following non-inducing levels of the different test systems:

- 5.7 mg/cm2in animal tests (LLNA EC3)

- ca. 2.5 mg/cm2 in human maximization tests (HMT)

- up to 11.8 mg/cm2 in HRIPTs with non-ethanolic vehicles

- 2.95 mg/cm2 in HRIPTs with ethanol-containing vehicles

- 1.18 mg/cm2in HRIPTs (second-phase/ethanol-containing vehicles

 

On the basis of a weight of evidence approach, theNo Expected Sensitization Level for hydroxycitronellal has been set at 2.95 mg/cm2.

 

A no expected sensitization induction level (NESIL) for hydroxycitronellal has been derived in 2008 by the expert panel of the Research Institute for Fragrance Materials (RIFM), being the basis for the recommended concentration limits in final products, i.e. IFRA standard of the international fragrance association (see Annex 2). The RIFM Expert Panel reviewed the critical effect data for Hydroxycitronellal and, based on the weight of evidence, established the No Expected Sensitization Induction Level (NESIL) as 5000 μg/cm². The basis for this NESIL represents a newer HRIPT, not included in the former assessment by HERA.

 

In this HRIPT, 100 subjects ( 32 males and 68 females), were treated for induction with 4.2% hydroxycitronellal (approximately 5000 μg/cm²) in 1:3 ethanol:diethyl phthalate (EtOH:DEP) on a webril/adhesive patch (25 mm Hill Top Chamber) occlusively (Harrison Research Labs 2006). Patches were applied to the left side of the back of each subject for 24 hours, followed by a 24-48 hour period, during which no test materials were applied, The identical test site was then repatched until the nine induction patchings were completed over a period of approximately 3 weeks. A rest period of approximately two weeks followed the last induction patching. The challenge patches were applied to a virgin site only (usually the right side of the back) in the same manner as in the induction Phase. Scoring was made directly after patch removal (24 hours) and 48 hours, 72 hours and 96 hours post-patching. No dermal sensitization in human subjects has been observed under the chosen testing conditions.

 

 

Many published reports of studies are available in which hydroxycitronellal was found to produce positive reactions in patients in routine diagnostic patch testing (for an overview, see Annex 1). Hydroxycitronellal is one of the eight components of the "Fragrance Mix" used by dermatologists to detect possible sensitivity to fragrances. Although there have been numerous reports of patients giving frank allergic responses to hydroxycitronellal in clinical patch testing on dermatological patients, many of these studies do not establish a clear causal relationship. Cases establishing a causal link between clinical reactions and everyday exposure to a hydroxycitronellal-containing product are relatively rare.

 

Respiratory sensitisation

Endpoint conclusion
Additional information:

No data available

Justification for classification or non-classification

The present data on dermal sensitization fulfill the criteria laid down in 67/548/EEC and regulation (EU) 1272/2008, and therefore, a classification with R43 and "Skin sensitisation" (Category 1B) is warranted.