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Description of key information

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:
weight of evidence
Study period:
July 2004
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Additionally the study was reviewed by an external expert (R.J. Dearman, University of Manchester, Division of Infection, Immunity & Respiratory Medicine)
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
(2002)
GLP compliance:
yes (incl. certificate)
Type of study:
mouse local lymph node assay (LLNA)
Specific details on test material used for the study:
Purity: 99.8%, a certificate of analysis was provided to the test institute by the sponsor
- Stability under test conditions: No information on testing of stability of the test substance in the vehicle is provided in the report, however, all dose preparations were prepared freshly before dosing (all dose preparations were used within 24 hours of preparation).
Species:
mouse
Strain:
CBA/Ca
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Strain: CBA/Ca/Ola/Hsd
- Source:Harlan Interfauna UK Limited, Blackthorne, Bicester, Oxon, UK
- Age at study initiation: 8–12 weeks
- Weight at study initiation: 17.3-22.8 g
- Housing: a maximum of 4 mice was housed per cage
- Diet and water: ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C):22 +/- 3
- Humidity (%): 30-70
- Air changes (per hr): a minimum of 15 changes/hour
- Photoperiod (hrs dark / hrs light):12/12
Vehicle:
other: 1:3 Ethanol : Diethyl phthalate
Remarks:
Rational for the vehicle chosen: Betts CJ, Contact Dermatitis 56, 70-75, 2007
Concentration:
2.5, 5, 10, 25, and 50 % w/v
No. of animals per dose:
4
Details on study design:
Approximately 25µL of a 2.5, 5, 10, 25 or 50% w/v preparation of the test substance in 1:3 EtOH:DEP was applied, using a variable volume micro-pipette, to the dorsal surface of each ear. A vehicle control group was similarly treated using 1 : 3 EtOH:DEP alone. The procedure was repeated daily for 3 consecutive days.
Three days after the third application, all the animals were injected, via the tail vein, with approximately 250µL of phosphate buffered saline (PBS) containing 20 µCi of a 2.0 Ci/mmol
specific activity 3H-methyl thymidine. Approximately 5 hours later, the animals were humanely killed by inhalation of halothane vapour followed by cervical dislocation. The draining auricular lymph nodes were removed from each animal and, together with the nodes from the other animals in the group, were placed in a container of PBS.
A single cell suspension was prepared by mechanical disaggregation of lymph nodes through a 200-mesh stainless steel gauze. The cell suspensions were then washed three times by centrifugation with approximately 10 ml of PBS. Approximately 3 ml of 5 % w/v trichloroacetic acid (TCA) was added and, after overnight precipitation at 4 °C, the samples were pelleted by centrifugation and the supematant was discarded. The cells were then resuspended in approximately 1ml of TCA.
The lymph node suspensions were transferred to scintillation vials and 10 ml of scintillant (Optiphase) was added prior toP-scintillation counting using a Packard Tri-Carb Liquid Scintillation Counter.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Positive control results:
The application of hexylcinnamaldehyde at concentrations of 5 %, 10 %, and 25 % w/v on acetone:olive oil (4:1) resulted in a greater than 3-fold increase in isotope incorporation at both 10 % and 25 % concentrations. Therefore, hexylcinnamaldehyde was shown to be a skin sensitizer, confirming the validity of the protocol used for this study.
Parameter:
SI
Value:
1
Test group / Remarks:
2.5 % (w/v)
Parameter:
SI
Value:
0.9
Test group / Remarks:
5 % (w/v)
Parameter:
SI
Value:
0.5
Test group / Remarks:
10 % (w/v)
Parameter:
SI
Value:
0.6
Test group / Remarks:
25 % (w/v)
Parameter:
SI
Value:
1.2
Test group / Remarks:
50 % (w/v)
Parameter:
EC3
Remarks:
(%)
Value:
> 50
Test group / Remarks:
Estimated EC3, since test substance concentration up to 50 % caused no SI value increase greater than 3-fold.
Cellular proliferation data / Observations:
Benzyl alcohol in 1:3 EtOH:DEP did not have the capacity to cause skin sensitization at any of the doses applied. The EC3 value giving rise to a 3-fold increase in lymphocyte proliferation was estimated to be greater than 50 % w/v (greater than 12500 µg/cm²).

Effects on body weight/body weight gain or on general condition were not reported.

 Conc. of test substance (% w/v)  Number of lymph nodes assayed  dpm  dpm per lymph node  Test:control ratio
 0 (vehicle only)  8  2248  281  N/A
 2.5  8  2289  286  1.0
 5  8  1975  247  0.9
 10  8  1191  149  0.5
 25  8  1420  178  0.6
 50  8  2635  329  1.2
 EC3 Estimated to be greater than 50 % w/v (> 12500 µg/cm²)
Executive summary:

A LLNA on female CBA/Ca/Ola/Hsd-mice revealed no 3 -fold increase in lymphocytes after substance application at concentrations of 2.5, 5, 10, 25, and 50 % and thus no indication for a sensitising potential of the substance.

Hexyl cinnamaldehyde as positive control showed the expected positive result after application of a 10 or 25 % formulation in acetone:olive oil 4:1, confirming thus the validity of the protocol used for this study.

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

Klezak et al (1977) tested amongst several other fragance materials benzyl alcohol in different sensitisation tests on guinea pigs and yielded inconclusive results: Maximization Test (MT) and Draize Test (DT) revealed no positive skin reactions but the Open Epicutaneous Test (OET) and the Freund´s Completer Adjuvant Test (FCAT) showed positive results. The short descriptions of the test procedures used, led to the assumption that standard methods were used (in the case of MT similar to guideline), but the results were only reported in brief. Therefore the reliability of the data is restricted and no firm conclusion on the sensitizing potential of benzyl alcohol in animals can be made (OECD 2004). A more recent LLNA, fully compliant with the OECD 429 guideline, revealed a negative result (Betts 2005, Scognamiglio 2012). In this test on female CBA/Ca/Ola/Hsd-mice no 3 -fold increase in lymphocytes were revealed after substance application at concentrations of 2.5, 5, 10, 25, and 50 % and thus no indication for a sensitising potential of the substance (SI-values: 1.0 at 2.5 %, 0.9 at 5 %, 0.5 at 10 %, 0.6 at 25 %, 1.2 at 50%). Higher concentrations than 50 % were not tested, most probably due to the slight irritant properties of benzyl alcohol. Hexyl cinnamaldehyde, used as positive control in this test, showed the expected positive result after application of a 10 or 25 % formulation in acetone:olive oil 4:1, confirming thus the validity of the protocol used for this study.

According to Reg. (EU) 1272/2008 “evidence from animal studies is usually much more reliable than evidence from human exposure”. Nevertheless, human data should in general have precedence over experimental animal data. As a matter of fact, there is broad information regarding skin sensitization for benzyl alcohol from human experience, due to the widespread use of the substance since decades as fragrance ingredient and preservative in cosmetics, pharmaceuticals and food (cp. OECD Final Assessment Report 2004).

Published evidence of the last two decades reveal a very low positive reaction rate up to 0.3 % in dermatitis patients, e.g. Heisterberg et al., Contact Dermatitis 65, 266‐275, 2011; Schnuch et al., Contact Dermatitis 65, 167‐174, 2011; Cuesta et al., Contact Dermatitis 63, 77‐84, 2010; Uter et al., Contact Dermatitis 63, 254‐261, 2010; van Oosten et al., Contact Dermatitis 61, 217‐223, 2009; Schnuch et al., Allergo J 17, 631‐638, 2008; Schnuch et al., Contact Dermatitis 57, 1‐10, 2007; Hasan et al., Contact Dermatitis 53, 40‐45, 2005; Jappe et al., British Journal of Dermatology 149, 87‐93, 2003; older studies have to be interpreted with care, due to changes in testing standards, e.g. purity of the test substance, unsuitable test concentration or vehicle. Moreover, epidemiologically relevant decreases in sensitisation prevalences were found for benzyl alcohol (Schnuch, British Journal of Dermatology 164, 1316‐1325, 2011).

For a recent evaluation of the endpoint the evaluation of the German MAK Commission should be taken into account (i.e. German Commission for the Investigation of Health Hazards of Chemical Compounds in the Work Area; permanent Senate Expert Committee that recommends maximum workplace exposure values to the German Federal Institute for Occupational Safety and Health). A full documentation is provided for substances that are evaluated by MAK. The most recent toxicological evaluation of benzyl alcohol has just been published (Benzylalkohol, MAK Value Documentation in German language, 2017, The MAK Collection for Occupational Health and Safety, 2: 461–498; http://onlinelibrary.wiley.com/doi/10.1002/3527600418.mb10051kskd0063/pdf). As it could be seen in this documentation and consistent with usual practise at MAK also the endpoint sensitisation was

thoroughly evaluated and updated in the assessment of 2017.

The overall outcome with respect to skin sensitization is that “Sensitization is not expected as benzyl alcohol was not a contact sensitizer in a local lymph node assay and there were no conclusive positive clinical findings of sensitizing effects on the skin”. This is consistent with the 2006‐evaluation (Benzylalkohol, MAK Value Documentation in German language, 2006, The MAK Collection for Occupational Health and Safety, 1–24; http://onlinelibrary.wiley.com/doi/10.1002/3527600418.mb10051d0040/pdf). For information on the tasks of the Skin and Allergy working group at MAK see http://www.dfg.de/en/dfg_profile/statutory_bodies/senate/health_hazards/structure/working_groups/skin_allergy/index.html).

Overall, human evidence revealed a very low sensitization rate of at most 0.3 % in very large collectives of dermatitis patients over decades. Regarding animal data a recent guideline compliant LLNA showed no sensitizing potential. The other available animal studies from 1977 and 1978 with limited documentation and inconclusive results should be overruled by that newer test result. Therefore, benzyl alcohol should not be allocated as skin sensitizer. This conclusion is in line with current scientific evaluations (MAK 2017)

Migrated from Short description of key information:
In animal experiments with benzyl alcohol different methods yielded inconclusive results: Guinea pig maximization test (MT) = neg (Klezak 1977), Draize Test (DT) = neg (Klezak 1977), modified Draize test (DT) = neg (Sharp 1978), Open Epicutaneous Test (OET) = pos (Klezak 1977), Freund's Complete Aduvant's Test (FCAT) = pos (Klezak 1977), but human volunteers displayed no skin reactions when tested using MT (Kligmann 1970)

Justification for selection of skin sensitisation endpoint:
No study is selected here, since several studies including human data were evaluated based on a weight of evidence approach.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

Skin sensitisation

According to legal classification (Directive 67/548/EEC and Regulation (EC) No. 1272/2008 benzyl alcohol is not classified as skin sensitizer.

Based on the available data and according to the criteria of Regulation (EC) No 1272/2008, Annex I, classification as a skin sensitizer would not be justified. The available data indicate at most a very low sensitization potency of benzyl alcohol. Especially studies in large collectives of dermatitis patients have revealed only a low sensitisation rate of benzyl alcohol of about 0.3 %, despite the widespread use of the substance over decades. Thus, the criteria of Reg. (EU) 1272/2008 of a "substantial" number of persons that have to be sensitized in order to justify a classification as skin sensitizer is not achieved. This is in line with the result of the key experimental study, available for the substance (LLNA; Betts 2005, Scognamiglio 2012).

Respiratory Sensitization

There are no data available.