Registration Dossier

Administrative data

Description of key information

A guideline-compliant study of skin irritation in the rabbit is available for hexyl salicylate.

A guideline-compliant study of eye irritation in the rabbit is available for hexyl salicylate.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records
Reference
Endpoint:
skin irritation: in vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
February - June 1986
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 404 (Acute Dermal Irritation / Corrosion)
GLP compliance:
yes
Species:
rabbit
Strain:
New Zealand White
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: A Smith, Waringham, Surrey, England
- Housing: Individually housed in anodised aluminum cages with grid floors and no bedding material
- Diet (e.g. ad libitum): commercially available pelleted rabbit diet ad libitum
- Water (e.g. ad libitum): Tap water ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18-24
- Humidity (%): 40-67
- Photoperiod (hrs dark / hrs light): 12 hr light/ 12 hr dark

Type of coverage:
semiocclusive
Preparation of test site:
shaved
Vehicle:
unchanged (no vehicle)
Controls:
no
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.5 mL
Duration of treatment / exposure:
4 hours
Observation period:
Total 168 hours. Animals were observed at 1, 24, 48, 72, and 168 hours after patch removal
Number of animals:
4 animals per group
Details on study design:
TEST SITE
- Area of exposure: 2.5 cm square
- % coverage: 100%
- Type of wrap if used: Elastoplast elastic adhesive bandage

REMOVAL OF TEST SUBSTANCE
- Washing (if done): gentle swabbing with cotton wool soaked in warm water
- Time after start of exposure: after 4 hours

SCORING SYSTEM:

Erythema and escher formation:
No erythema: 0
Very slight erythema: 1
Well-defined erythema: 2
Moderate to severe erythema: 3
Severe erythema to slight eschar formation: 4

Oedema formation:
No odema: 0
Very slight oedema: 1
Slight oedema: 2
Moderate oedema: 3
Severe oedema: 4
Irritation parameter:
erythema score
Basis:
mean
Time point:
24/48/72 h
Score:
2
Max. score:
4
Reversibility:
not fully reversible within: 168 hours
Remarks on result:
other: For the 100% pure test substance
Irritation parameter:
edema score
Basis:
mean
Time point:
24/48/72 h
Score:
2.16
Max. score:
4
Reversibility:
not fully reversible within: 168 hours
Remarks on result:
other: For the 100% pure test substance
Irritation parameter:
erythema score
Basis:
mean
Time point:
24/48/72 h
Score:
2
Max. score:
4
Reversibility:
fully reversible within: 168 hours
Remarks on result:
other: For the 50% test substance in DEP
Irritation parameter:
edema score
Basis:
mean
Time point:
24/48/72 h
Score:
1.4
Max. score:
4
Reversibility:
fully reversible within: 168 hours
Remarks on result:
other: for the 50% test substance in DEP
Irritant / corrosive response data:
One hour after dosing well defined erythema and very slight oedema of the treated skin were observed in all four rabbits. Erythematous reaction was maintained twenty four hours after dosing when slight oedema was apparent in two rabbits and very slight oedema of the skin was maintained by one rabbit. Very slight oedema was apparent in two rabbits and slight oedema was observed in two rabbits at the forty eight and seventy two hour observations, well defined erythema being maintained by all four animals. Irritant reaction had declined within seven days of dosing, one rabbit exhibiting very slight erythema and oedema with slight desquamation and a second animal exhibiting only very slight oedematous reaction.
Well defined erythema of the treated skin was observed in all four rabbits one hour after dosing. Slight oedema was observed in one rabbit and very slight oedema was apparent in three rabbits at this time. Oedematous reaction had increased twenty four hours after dosing, slight oedema of the skin being observed in three rabbits and very slight oedema of the skin present in one rabbit. Well defined erythema was maintained by all four rabbits at the forty eight hour observation when moderate oedema of the skin was observed in two animals and slight oedematous reaction was noted in the remaining two animals of the group. Oedema of the skin showed slight decline in one rabbit within seventy two hours. Seven days after dosing well defined erythema and oedema were apparent in the remaining rabbit of the group. Desquamation from the surface of the treated skin was also observed in three of the four rabbits.
Other effects:
None

Mean scores

24-72 hours

Erythema

Oedema

24 hours

2

1.25

48 hours

2

1.5

72 hours

2

1.5

24-72 hours

Erythema

Oedema

24 hours

2

1.75

48 hours

2

2.5

72 hours

2

2.25

Interpretation of results:
Category 2 (irritant) based on GHS criteria
Conclusions:
Hexyl salicylate was found to be a moderate skin irritant under the conditions of this study. The mean scores for skin irritation do not trigger classification as a skin irritant; however as the reactions were not reversible within the study period, classification is appropriate.
Executive summary:

The irritation potential of hexyl salicylate was tested in a study by Haynes (1986), after exposure for 4 hours under semi-occlusive conditions in the rabbit, both as 100% and as 50% in DEP. The mean score for erythema over the 24-72 hour period was 2.0 for the 100% pure substance. The mean score for oedema over the 24-72 hour period was 2.16 for the 100% pure substance. Hexyl salicylate was found to be a moderate skin irritant under the conditions of this study. The mean scores for skin irritation do not trigger classification as a skin irritant; however as the reactions were not reversible within the study period, classification is appropriate.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records
Reference
Endpoint:
eye irritation: in vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
6th March 2000 - 13th March 2000.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 405 (Acute Eye Irritation / Corrosion)
Qualifier:
according to
Guideline:
EU Method B.5 (Acute Toxicity: Eye Irritation / Corrosion)
GLP compliance:
yes
Species:
rabbit
Strain:
other: SPF Albino
Details on test animals or tissues and environmental conditions:
TEST ANIMALS
- Source: BI Pharma KG, 88397, Biberach.
- Age at study initiation: Not documented
- Weight at study initiation: 2.5 - 3.0 kg b.w.
- Housing: The rabbits were housed individually in PPO cages (floor area: 2576 sq.cm) with perforated floor.
- Diet (e.g. ad libitum): A pelleted complete rabbit diet "Altromin 2123" was available ad libitum.
- Water (e.g. ad libitum): The animals had free access to bottles with domestic quality drinking water acidifed with hydrochloric acid to pH 2.5 to prevent microbial growth.
- Acclimation period: At least one week.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 ± 3°C
- Humidity (%): 55 ± 15%
- Air changes (per hr): 10 air changes / hour
- Photoperiod (hrs dark / hrs light): 12 hours light/12 hours dark

IN-LIFE DATES: From: To: Not documented
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent no treatment
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.1ml


Duration of treatment / exposure:
24 hours
Observation period (in vivo):
72 hours
Number of animals or in vitro replicates:
4 female rabbits
Details on study design:
REMOVAL OF TEST SUBSTANCE
- Washing (if done): Yes, with 20ml 0.9% sodium chloride solution.
- Time after start of exposure: After the first 24 hour reading

SCORING SYSTEM: Not documented

TOOL USED TO ASSESS SCORE: fluorescein and UV light to detect possible corneal damage.
Irritation parameter:
cornea opacity score
Remarks:
corneal opacity
Basis:
mean
Time point:
24/48/72 h
Score:
0
Reversibility:
fully reversible within: 72 hours
Irritation parameter:
iris score
Remarks:
iris lesion
Basis:
mean
Time point:
24/48/72 h
Score:
0
Reversibility:
fully reversible within: 72 hours
Irritation parameter:
conjunctivae score
Remarks:
redness of conjunctiva
Basis:
mean
Time point:
24/48/72 h
Score:
0.4
Reversibility:
fully reversible within: 72 hours
Irritation parameter:
chemosis score
Basis:
mean
Time point:
24/48/72 h
Score:
0.3
Reversibility:
fully reversible within: 72 hours
Irritant / corrosive response data:
One hour following application, one test animal showed markedly deepened folds, congestion and swelling of the iris, moderate circumcorneal injection, reaction to light of iris, diffuse red crimson conjunctiva with individual vessels not easily discernible, a swelling above normal and a discharge different from normal. A second animal showed a diffuse red crimson conjunctiva with individual vessels not easily discernible a swelling above normal and a discharge different from normal. A third animal showed markedly deepened folds, congestion and swelling of the iris, moderate circumcorneal injection and detectable reaction of light, a diffuse red crimson conjunctiva with individual vessels not easily discernible and a swelling above normal. the fourth test animal showed a diffuse crimson red conjunctiva with individual vessels not easily discernible and a swelling above normal.
24 hours following application of the test substance, all four animals displayed conjunctiva with some vessels definitely injected and a swelling above normal. 48 hours after application of the test substance, one test animal showed some conjunctival vessels definitely injected. The three other animals were free of any signs of eye irritation. 72 hours after application of the test article, all four animals were free of any signs of irritation.
Other effects:
No other effects documented

No additional information

Interpretation of results:
GHS criteria not met
Conclusions:
Under the conditions of this study, the test substance, Hexyl Salicylate, does not induce irritation of the eyes following its application. Based on these results, the test substance does not need to be classified according to Regulation EC No. 1272/2008.
Executive summary:

Hexyl salicylate was investigated for its ability to induce eye irritation. The test substance was applied undiluted to the left eye of each of 4 female SPF albino rabbits in a volume of 0.1ml. The right eye of each animal served as the untreated control. The eyes were examined and the changes graded according to a numerical scale at one, 24, 48 and 72 hours after dosing. The cornea, iris and conjunctivae were examined and scored based on their reactions to the test material. Slight to well-defined signs of irritation were observed in the treated eyes at the 1, 24 and 48 hour examination time-points. However, all effects were fully reversible and no signs of irritation were observed after 72 hours. Under the conditions of this study hexyl salicylate does not induce irritation of the eyes following its application. Based on these results, it does not need to be classified according to Regulation EC No. 1272/2008.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Additional information

Skin irritation

Hexyl salicylate was found to be a moderate skin irritant in a study in the rabbit. The mean scores for skin irritation do not trigger classification as a skin irritant; however the reactions were not reversible within the study period, which would normally result in classification.

Hexyl salicylate is used as one of the non-irritant reference substances for validation of reconstructed human epidermis (RhE) models under OECD TG 439 (1) and also specified by ECVAM (2). OECD 439 includes four RhE models; EpiSkin™ (SM), EpiDerm™ (EPI-200), SkinEthic™ RHE and LabCyte EPI-MODEL24 SIT. Data from Cyprotex (3) performed using the EpiDerm™ (MatTek) model reports a mean viability of 106.2 (compared to 100.0 for controls), showing an absence of irritation. Validation data for LabCyte EPI-MODEL24 (4) (5) report mean viabilities for hexyl salicylate of 103.1, 110.8 and 101.1 in three independent assessments. These results are considered by the authors to be ‘false’ predictions (i.e. false negatives) based on the in vivo data for hexyl salicylate; however it is also noted that findings are consistent with those with EpiSkin™(6). A further validation study for the (not yet adopted) open source reconstructed epidermis model (OS-REp) reports mean viabilities of 95.6, 98.3 and 105.3 for 3-5 triplicate assays performed at three different laboratories (7) (8).

The negative responses seen in RhE models contrast with the irritation reported in rabbit studies in vivo; however it is also noted that the irritation seen in rabbit studies is not consistent with experience showing that hexyl salicylate is not an irritant in human skin in vivo (9). No classification is therefore proposed for hexyl salicylate, based on the weight of evidence from in vitro studies and the lack of consistency between the rabbit and human data.

(1) OECD Guidelines for the Testing of Chemicals, Section 4 Health Effects. Test No. 439: In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method. OECD, Paris; 28th July 2015.

(2) EUROPEAN COMMISSION JOINT RESEARCH CENTRE Institute for Health and Consumer Protection In-Vitro Methods Unit: European Centre for the Validation of Alternative Methods (ECVAM). Performance Standards for In-Vitro Skin Irritation Test Methods based on Reconstructed Human Epidermis (RhE). 24th August 2009.

(3) Cyprotex. Skin and Ocular Testing: Skin irritation, Background Information.

(4) Katoh M, Hamajima F, Ogasawara T & Hata K (2009). Assessment of human epidermal model LabCyte EPI-MODEL for in vitro skin irritation testing according to European Centre for the Validation of Alternative Methods (ECVAM)-validated protocol. Journal of Toxicological Sciences 34 (3): 327-334.

(5) JSAAE REPORT: VALIDATION STUDY OF IN VITRO SKIN IRRITATION TEST USING LABCYTE EPIMODEL 24 (FINAL REPORT). OCTOBER 5, 2009. LABCYTE VALIDATION MANAGEMENT TEAM.

(6) Liebsch M, Gamer, AO, Curren RD, Frank J, Genschow E, Tharmann J, Remmele M, Bauer B, Raabe H, Barnes N, Hilberer A, Wilt N, Lornejad-Schäfer MR, Schäfer C, Hayden P & Kandárová H. Follow-up Validation of the EpiDerm Skin Irritation Test (SIT): Results of a Multi-centre Study of Twenty Reference Test Substances.

(7) Mewes KR, Fischer A, Zöller NN, Laubach V, Bernd A, Jacobs A, van Rompay A, Liebsch M, Pirowd R & Petersohn D (2016). Catch-up validation study of an in vitro skin irritation test method based on an open source reconstructed epidermis (phase I). Toxicology in Vitro 36: 238-253.

(8) Groeber F, Schober L, Schmid FF, Traube A, Kolbus-Hernandez S, Daton K, Hoffmann S, Petersohn D, Schäfer-Korting M, Walles H & Mewes KR (2016). Catch-up validation study of an in vitro skin irritation test method based on an open source reconstructed epidermis (phase II). Toxicology in Vitro 36: 254-261.

(9) ESAC Peer review of submission of the follow-up validation study of the Modified EpiDermTM Skin Irritation Test (SIT) for hazard identification and labelling of chemicals according to EU classification system.

Eye irritation

In an eye irritation study, slight to well-defined signs of irritation were observed in the treated eyes at the 1, 24 and 48 hour examination time-points. However, all effects were fully reversible and no signs of irritation were observed after 72 hours.

Respiratory irritation

No evidence of local effects was seen in a 28 -day inhalation toxicity study with hexyl salicylate

Justification for classification or non-classification

Based a weight of evidence from the in vivo and in vitro data, hexyl salicylate does not require classification for skin irriation. No classification is required for eye irritation based on the results of a rabbit study. No evidence of local effects was seen in a 28 -day inhalation study; no classification is therefore proposed for respiratory irritation.