Linalyl acetate

Endpoint summary

Key value for chemical safety assessment

Skin sensitisation

Endpoint conclusion

Additional information:

The potential of Linalyl acetate for skin sensitization was investigated by several authors using different experimental approaches. Conflicting results have been obtained denpendent on the study protocol used. In order to integrate all available, no key study has been identified but the information has been evaluated in a weight of evidence approach in order to determine the skin sensitizing potential of Linalyl acetate. 

 

In vitro studies:

Linalylacetate has been found to be not sensitizing in an in chemico and an in vitro study.

In a Direct peptide reactivity assay (DPRA) according to OECD TG 442C, LINALYL ACETATE (purity 98.8%) was dissolved in acetonitrile and mixed with a Cysteine- and a Lysine-containing peptide according to the standard operating procedure of the DPRA (Givaudan 2017; RCR 153'697). One study with three replicates was conducted. After 24 h incubation time, peptide depletion induced by LINALYL ACETATE was determined by HPLC-UV.

The test substance gave 3.5 % depletion of the Cys-peptide and 0 % depletion of the Lys-peptide. The average peptide depletion is 1.8 % and below the threshold of 6.38%. Thus, LINALYL ACETATE was non-reactive and classified into the MINIMAL reactivity class according to the prediction model. It is therefore considered a non-sensitizer according to the prediction model of the DPRA.

In a KeratinoSens^TMassay according to OECD TG 442D, LINALYL ACETATE (purity 98.8%) was dissolved in DMSO and tested at 12 concentrations in three repetitions, each time in three replicates (Givaudan 2017; RCR 153'682). After 48 h incubation time, luciferase induction and cellular viability at each of the concentrations were determined.

LINALYL ACETATE was weakly toxic to the KeratinoSens™ cells. In two repetitions, it did not induce the luciferase gene above a threshold of 1.5 at a non-cytotoxic dose, while in one repetition a weak induction at a non-cytotoxic dose was noted. LINALYL ACETATE is considered a non- sensitizer according to the prediction model of the KeratinoSens™assay.

 

Local Lymph Node Assays:

Evidence for skin sensitizing effects of autooxidation products in different Linalyl acetate specifications were derived from local lymph node assays equivalent or similar to OECD TG 429 with female CBA/Ca mice (Sköld 2005). In three studies the potential allergenicity of Linalyl acetate as standard test material (97% purity), additionally purified (destilled) Linalyl acetate, and air-exposed Linalyl acetate (in Erlenmeyer flasks covered with aluminum foil, with stirring for one hour, four times per day for 10 weeks) was investigated. Standard Linalyl acetate, distilled Linalyl acetate, and oxidized Linaly acetate resulted in EC3 values of 20%, 25.4% and 3.6% respectively. The results obtained demonstrate a direct correlation of EC3 values and the presence of oxidation products in the test materials used. Furthermore, hydroperoxide derivatives of Linalyl acetate were identified, having a comparable potency in the LLNA like the air-exposed linalyl acetate sample (EC3 = 3.6%).

 

In an LLNA, performed according to OECD TG 429 and GLP, four groups of four female mice each were treated with LINALYLE ACETATE SYNTHETIC (purity 96.5%, no analytical verification of peroxide content performed) at concentrations of 1 %, 10%, 30% and 100% (undiluted) (w/v) in ethanol:water, 7:3 (v/v) by topical application to the dorsum of each ear lobe (left and right) on three consecutive days (RCC 2002). After intravenous injection 3H-methyl thymidine, draining auricular lymph nodes were excised and pooled per group.

No test item-related clinical signs were observed. All treated animals survived the scheduled study period. STIMULATION INDICES of 1.0, 6.4, 10.0 and 22.3 were determined with the test item at concentrations of 1%, 10%, 30% and 100% (undiluted) (w/v) in ethanol:water, 7:3 (v/v). Therefore, LINALYLE ACETATE SYNTHETIC showed an allergenic potency when tested at concentrations of 10%, 30% and 100% (undiluted) (w/v). In this study an EC3 of 4.3% (w/v) was calculated.

 

The skin sensitizing potential of Linalylacetate (99.8%) was assessed using the radioactive Murine Local Lymph Node Assays according to OECD 429 and GLP guidelines (BASF 2017).

In a prestudy (58V0138/16A054), groups of 5 female CBA/CaOlaHsd mice each were treated with 5%, 10% and 50% w/w preparations of the Linalyl acetate in methyl ethyl ketone (MEK) or with the vehicle alone. Linalylacetat has been found to show a skin sensitizing potential under the test conditions chosen. Based on 3H-thymidine incorporation, the stimulation indices were 12.04, 19.64 and 24.24 for 5%, 10% and 50% Linalyl acetate in MEK, respectively. The threshold concentration for sensitization induction was therefore <5%. In line, significant increases of stimulation indices based on cell counts (2.7, 3.8 and 3.61) and lymph node weights (1.75, 2.14, 2.15) were observed for 5%, 10% and 50% Linalyl acetate in MEK, respectively. When compared to the initial pretest performed (i.e. 1.35 and 2.1 after application of 10% and 50% Linalylacetate), the potency of the test substance appeared to increase concerning the SI based on lymph node weights. The test substance has been analyzed after completion of application. The undiluted test substance contained 11 mE peroxides /kg and for the formulations of 5%, 10% and 50 % in MEK, the maximum concentration of peroxides found were 5 mE/kg in the high dose group formulation. Therefore, a formation of peroxides in the test substance preparation during the study occurred and their contribution in the reactions observed cannot be excluded. No clinical signs and local findings have been observed during the course of the study.

In the final study (58V0138/16A162), the absence of relevant formation of peroxides in the test substance formulation was confirmed analytically. The test sample used (i.e. 0.5%-10% in MEK) contained 0.1 mE/kg peroxides or less. Groups of 5 female CBA/CaOlaHsd mice each were treated with 0.5%, 2.5% and 10% w/w preparations of Linalyl acetate in methyl ethyl ketone (MEK) or with the vehicle alone. Each test animal was treated with 25 μL per ear of the appropriate test-substance preparation, applied to the dorsal surfaces of both ears for three consecutive days. Lymph node response was evaluated by measuring 3H-thymidine incorporation, cell counts and weights of each animal’s pooled lymph nodes. In addition, a 0.8 cm diameter sample was punched out of the apical part of each ear and for each animal the weight of the pooled punches was determined in order to obtain an indication of possible skin irritation.

When applied as 10% and 2.5% preparations in MEK, Linalylacetate induced a statistically significant increase of 3H-thymidine incorporation into the cells from the auricular lymph nodes, which was above the cut off value for biological relevance (increase above the cut off stimulation index (SI) of 3). Concomitantly, the increase in the auricular lymph node cell counts at 10% and 2.5% was statistically significant and biologically relevant (above the cut-off value, i.e. increase to 1.5 fold or above of control value = stimulation index (SI) ≥ 1.5). In addition there was a statistically significant increase in lymph node weights at 10% and 2.5%. The Linalylacetate preparations did not cause any excessive increase in ear weights (> 25%), demonstrating the absence of excessive ear skin irritation. The increase at 10% was statistically significant.

Linalylacetat exhibits a skin sensitizing potential in the Murine Local Lymph Node Assay and the threshold concentration for sensitization induction was >0.5% <2.5%. The EC3 for 3H-thymidine incorporation was calculated to be 1.6% or 400 μg/cm2. Thus, it is concluded that Linalylacetate - containing no significant levels of peroxides – exhibits a skin sensitizing potential in the Murine Local Lymph Node Assay under the test conditions chosen.

 

In vivo guinea pig studies:

Conflicting results have been obtained in different studies with guinea pigs. Evidence for skin sensitization was found in two studies from secondary sources on guinea pigs with Linalylacetate without further information on the test substance purity.

In a delayed contact hypersensitivity study in Dunkin-Hartley guinea pigs, Linalylacetate was applied intradermally (10% in the presence of Freund´s complete Adjuvant) and topically (10% in acetone) for induction. Topical challenge using 5%, 10% and 20% Linalylacetate in acetone resulted in skin reactions in 0/10, 2/10 and 4/10 animals respectively (Takasago 1982).

In a guinea pig maximization test (GPMT), with limited information on test protocol and results, Linalylacetate was determined to be moderately sensitising at 10% (Ishihara 1986).

In contrast, no evidence for skin sensitization was found in an OET, skin painting test and a GPMT.

In an Open Epicutaneous Test using 6 guinea pigs per group, LINALYL ACETATE SYNTH. (3%, 10%, 30% in Ethanol or 100% - undiluted; no info on test substance purity provided) has been repeatedly applied daily for 3 weeks always using the same skin site for induction (Roche 1982). For challenge, all groups of guinea pigs previously treated for 21 days as described above, are tested on days 21 and 35 on the contralateral flank with the test material at the minimal irritating and some lower concentrations (no further specified) and the reactions have been read after 24, 48 and/or 72 hours. None of the 6 animals per dose group was determined to show induced allergic skin sensitization. The lowest irritant and the highest non-irritant concentration was 30% and 10% LINALYL ACETATE SYNTH. in Ethanol, respectively. Overall, single and repeated applications of a 10% alcoholic solution of LINALYL ACETATE SYNTH. were well tolerated. Under testing conditions used, no sensitizing potential of LINALYL ACETATE SYNTH. for the guinea pig has been detected.

In a skin painting test using 10 female white guinea pigs, induction treatement included 2 and 8 dermal (open) applications of 10% and 50% Linalylacetate (analytical purity: 95-99%) respectively over 2 weeks (BASF XIX/74). No dermal reactions were observed after topical challenge with or 5% and 10%.

In a guinea pig maximization test (GPMT) reported from secondary source, Linalylacetate was applied intradermally (0.5%) and topically (50% in acetone/PEG 400) on 10 Dunkin-Hartley guinea pigs for induction (Quest 1983). Three challenge applications (5% in acetone/PEG 400) resulted in faint erythema in single animals (1-3 of 10 animals) and control animals (1 of 8 animals). The authors concluded, that Linalylacetate is not a skin sensitizer under the chosen testing conditions.

 

Human data:

In a recent human Repeated Insult Patch Test (HRL 2017), Linalylacetate (purity 99.8%) has been repeatedly applied to human volunteers occlusively as a 2% formulation in 1:3 EtOH:DEP (2362 μg Linalylacetate/cm²). A total of 118 subjects were enrolled (age range: 19-70) and 99 (24 males, 75 females) subjects completed the test, however, no subject discontinued due to test material reaction.

For induction, the formulation on a webril/adhesive patch (25 mm Hill Top Chamber System®) was applied to the left side of the back for approximately 24 hours. An approximately 24-hour (or 48-hour) period, during which no test material was applied, followed the weekday (or weekend) patch removal. The identical test site was then repatched until nine induction patchings were completed over a period of approximately three weeks. No test material was applied approximately two weeks followed the last induction patching.

For challenge, the same formulation was applied as outlined for the induction patches. Challenge patch was applied to a virgin site for 24 hours. Readings were performed after challenge patch removal and 24h, 48h and 72h after challenge patch removal.

During the induction phase and challenge, no skin reactions were observed for the 2% Linalylacetate formulation in 1:3 EtOH:DEP. Accordingly, in this Repeated Insult Patch Test, the test material (Linalylacetate at 2 % w/v in 1:3 EtOH:DEP; 2362 μg/cm²) did not induce dermal sensitization in human subjects.

 

Various Repeated Insult Patch Tests are available as short references from secondary sources or as publications only.

In 2 human maximization studies involving 22 and 26 healthy voluteers, 10 % Linalylacetate (no data on purity) in petrolatum was applied occlusively for 5 alternate day 48 hour periods after SLS pretreatment (Epstein 1974). After challenge with the same test concentration, 2 volunteers (of 48 volunteers in total) was reported to show contact sensitization related reactions and 1 subject showed irritation reactions.

In 3 further human maximization studies of the same author involving 26,27 and 30 healthy voluteers, 10 % Linalylacetate (no data on purity) in petrolatum was applied in a similar fashion as described above (Epstein 1982). After challenge, 1 volunteer (of 83 volunteers in total) was reported to develop an allergic contact sensitization under the chosen testing conditions.

Other Repeated Insult Patch tests were reported, showing no indications of any sensitizing potential of Linalylacetate. After pretreatment with SLS, Linalylacetate with unknown purity was occlusively patched for 5 alternate-day 48 hour periods as 12% formulation (approx. 8300 µg/cm²) to 25 volunteers (Greif 1967). After challenge with the same test concentration, no skin reactions indicative of skin sensitization was observed.

In line, occlusive application of Linalylacetate with unknown purity as 20% formulation in petrolatum (approx. 13800 µg/cm²) for 5 alternate-day 48 hour periods to 25 volunteers revealed no skin reactions indicative of skin sensitization (Kligman 1975).

 

Several diagnostic patch tests have been reported in literature.

In an occlusive 48 hour patch test using 50 male volunteers without an history of allergy, Linalylacetate was tested at 32% formulation in acetone (approx. 8000 µg/cm²), and no dermal reactions were observed (Motoyoshi 1979).

A published multricentic study assessed a total of 1855 patients with a history of adverse reactions to frangrances in a patch test using 10% Linalylacetate (analytical purity 95 -99%) in petrolatum (Frosch 2002). Four patients with reactions indicative for skin sensitization (0.2%) were reported. Further 8 cases (0.4%) with doubtful positive reactions (only erythema) were observed after topical test substance application for 48 hours. Further, 100 male and female patients have been reported to show no positive skin reactions when patch tested with 1% and 5% Linalylacetate in petrolatum (Frosch 1995).

Patch testing of 22 with no personal history of fragrance sensitivity among consecutive dermatitis patients using 2%, 4% and 6% oxidized Linalylacetate for 48 hours resulted in no dermal reactions indicated as irritation (Hagvall 2008). When patch tested at 3 patients positive for patch test with oxidized Linalool, 1/3 patient reacted to 4% oxidized Linalylacetate, 1/3 reacted to 6% oxidized Linalylacetate, and 1/3 showed no reactions. Patch testing of oxidized Linalylacetate (6% in petrolatum, containing 1% Linalylacetate and 2% Linalylacetate hydroperoxides) resulted in positive skin reactions in 37/1717 (2.2%) consecutive patients, demonstrating a significant contribution of autooxidation products in the skin sensitizing potency of Linalylacetate (Hagvall 2015).

In short database entries from a secondary source, no dermal reactions have been reported after an an occlusive application of 20% Linalylacetate in petrolatum for 24 hours (Smith 2001). A closed patch test of 20 patients (perfume-sensitive) and 8patients (with Mycolog cream sensitivity)using 5% Linalylacetate in petrolatum for 48 hours showed no skin reactions (Larsen 1977; Larsen 1979). Patch tests were reported on patients with and without cosmetic dermatitis and in patients with facial melanosis (not further specified) and application of 5% Linalylacetate showed no skin effects (Ishihara 1981). Application of 3% Linalylacetate in petrolatum to cosmetic sensitive patients (not further specified) resulted in an allergic skin reaction in 1 patient (deGroot 1988). No skin reactions were reported in 19 patients with eyelid dermatosis and in 70 patients with dermatitis at other sites when patched with 1% Linalyl acetate in petrolatum (Nethercott 1989). In a 24 to 48 hour occluded patch test, 1 of 82 patients reacted to 0.05% - 0.5% Linalylacetate in EtOH or base cream (not further specified; Takenaka 1968; Fuji 1972).

Occupational patch testing of 26 workers of a perfume factory according ICDRG guidelines with 1% Linalylacetate in petrolatum resulted in no positive reactions (Schubert 2006).

In order to identify the skin irritating potential, Linalylacetate (undiluted) has been applied occluded for 4 hours in a patch test (Basketter 2004). According to the authors Linalyl acetate was not considered to be an irritant based on 1/31 positive reactions.

Several case studies have been reported as short database entries from a secondary source. Patching of 5% Linalylacetate to a 35-year-old female teacher (with a 2-month history of dermatitis on the face, hands and feet) and patching of 10% Linalylacetate to a 32 year old female patient (with allergic contact dermatitis on the fingers after exposure to lemon oil and lemon peel) did not result in dermal effects (DeLeeuw 1987; Hausen 1990). Patching of two professional aromatherapists with suspected allergic contact dermatitis resulted in positive skin reactions with 5% and 10% Linalylacetate in 1/2 patients (Dharmagunawardena 2002).

 

Conclusion

Conflicting results concerning the skin sensitization potential of Linalylacetate have been obtained. In chemico and in vitro studies did not reveal protein reactivity or the potential to activate keratinocytes. Some guinea pig studies (with limited documentation) involving intradermal induction treatment) revealed skin reactions indicative for a skin sensitizing potential, whereas repeated epicutaneous application of Linalylacetate did not result in such skin reactions. Different Local lymph node assays identified Linalylacetate as a skin sensitizer and the formation of skin sensitizing autoxidation products appears to play an important role. Testing of Linalylacetate in a LLNA with a confirmed low peroxide concentration identified Linalylacetate as skin sensitizer. The respective EC3 values observed were 20% (standart Linalylacetate in AOO), 25.4% (purified Linalylacetate in AOO), 4.3% (standart Linalylacetate in Ethanol/water) and 1.6% (peroxide free Linalylacetate in MEK), resulting in a weighed EC3 of 9.5%* (2375 µg/cm2).

*((20%+25.4%/2) + 4.3% + 1.6%)/3

Human data including repeated insult patch tests and patch tests on volunteers, unselected and selected patients are reported in literature with limited documentation. The majority of these reports did not identify skin sensitization reactions of Linalyl acetate, however, sporadic reactions have been observed in repeated as well as single patch tests. In a published multricentic study, reactions indicative for skin sensitization was observed in 0.2% of the patients patch tested with 10% Linalylacetate (Frosch 2002). In a recent human repeated insult patch test, performed under current standart protocol and documented with sufficient detail confirmed the absence of a skin sensitizing potential when applied at 2% (2362 μg/cm²) in 1:3 EtOH:DEP (HRL 2017).

Overall, in a weight of evidence approach, Linalylacetate is considered to be a skin sensitizer. According to the weighed mean of the EC3 value >2% and 3/4 LLNAs with an EC3 value >2%, the absence of skin sensitization in a well conducted HRIPT at a concentration >500 µg/cm², a low/moderate frequency (<1%) of positively patch tested unselected/consecutive patients in diagnostic patch tests and a low number (<100) of published cases, a classification as skin sensitizer Cat. 1B is warranted.

Respiratory sensitisation

Endpoint conclusion

Additional information:

No information is available.

Justification for classification or non-classification

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