Registration Dossier

Diss Factsheets

Administrative data

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:
key study
Study period:
2007-03-02 to 2007-03-06
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
2002
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
2004
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of study:
mouse local lymph node assay (LLNA)
Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Age at study initiation: 8-12 weeks
- Weight at study initiation: 15-23 g
- Housing: individually
- Diet: certified rat and mouse diet, ad libitum
- Water: tap water ad libitum
- Acclimation period: at least five days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-25
- Humidity (%): 30-70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12
Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
1, 0.5, 0.25 %
No. of animals per dose:
4
Details on study design:
RANGE FINDING TESTS:
- Irritation: Seven mice were treated daily by application of 25 µL of the undiluted test material and concentrations of 50, 25, 10, 5, 2.5 and 1 %. Severe signs of systemic toxicity were observed in all dose groups except the 1 % dose group. The dose level selected for the main test was therefore 1 %.

MAIN STUDY

TREATMENT PREPARATION AND ADMINISTRATION:
Groups of four mice per concentration were treated by daily application of 25 µL to the dorsal surface of each ear for three consecutive days. Administration was carried out using an automatic micropipette and spread over the dorsal surface of the ear using the tip of the pipette.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
The proliferation response of lymph node cells was expressed as the number of radioactive disintegrations per minute per lymph node (disintegrations per minute/node) and as the ratio of 3HTdR incorporation into lymph node cells of test nodes relative to that recorded for the control nodes (Stimulation Index).

The EC3 value is the concentration of test material expected to cause a 3 fold increase in HTdR incorporation.
The equation used for the calculation of EC3 is:
EC3 = c + [(3-d/b-d) x (a-c)]
a = lowest concentration giving stimulation index >3
b = actual stimulation index caused by a
c = highest concentration failing to produce a stimulation index of 3
d = actual stimulation index caused by c
Positive control results:
The stimulation index (expressed as the mean radioactive incorporation for each treatment group divided by the mean radioactive incorporation of the vehicle control group) of a 5 % (v/v) concentration in acetone/ olive oil 4:1 was 2.5, the SI of a 10 % concentration was 4.03 and the SI of a 25 % concentration was 9.13. Therefore, the positive control substance was considerd to be a sensitiser.
Key result
Parameter:
EC3
Value:
0.77
Test group / Remarks:
in %
Parameter:
SI
Value:
3.75
Test group / Remarks:
Test substance concentration 1 %
Parameter:
SI
Value:
2.16
Test group / Remarks:
Test substance concentration 0.5 %
Parameter:
SI
Value:
2.11
Test group / Remarks:
Test substance concentration 0.25 %

Table 1: Disintegrations per Minute, Disintegrations per Minute/Node and Stimulation Index

Concentration (% v/v) in acetone/olive oil 4:1

DPM

DPM/Node

Stimulation Index

Result

Vehicle

7740.67

967.58

NA

NA

0.25

16328.71

2041.09

2.11

Negative

0.5

16749.20

2093.65

2.16

Negative

1

29046.08

3630.76

3.75

Positive

An EC3 value of 0.77% was calculated.

Interpretation of results:
Category 1A (indication of significant skin sensitising potential) based on GHS criteria
Conclusions:
The test substance was found to be skin-sensitising.
Executive summary:

A study was performed to assess the skin sensitization potential of the test material in the CBA/Ca strain mouse following topical application to the dorsal surface of the ear. The study was performed according to OECD Guideline 429 and GLP. Following a preliminary screening test, three groups, each of four animals, were treated with 50 µL (25 (µL per ear) of the test material as a solution in acetone/olive oil 4:1 at concentrations of 1 %, 0.5 % or 0.25 % v/v. A further group of four animals was treated with acetone/olive oil 4:1 alone. The stimulation indices of the concentrations 0.25, 0.5 and 1 % were 2.11, 2.16 and 3.75, respectively. An EC3 value of 0.77 % was calculated. Therefore, the test result of the substance at a concentration of 1 % was considered positive and the test substance was found to be a skin sensitiser (Cat. 1A).

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (sensitising)
Additional information:

Skin sensitization

Key study

A study was performed to assess the skin sensitization potential of the test material in the CBA/Ca strain mouse following topical application to the dorsal surface of the ear. The study was conducted according to OECD Guideline 429 and GLP. Three groups, each of four animals, were treated with 50 µL (25 µL per ear) of the test material as a solution in acetone/olive oil 4:1 at concentrations of 1 %, 0.5 % or 0.25 % v/v. The stimulation indices of the concentrations 0.25, 0.5 and 1 % were 2.11, 2.16 and 3.75, respectively. An EC3 value of 0.77 % was calculated. Therefore, the test result of the substance at a concentration of 1 % was considered positive and the test substance was found to be a skin sensitizer.

Supporting studies

110 male and female human volunteers participated in a study to assess the potential of the test material at a concentration of 0.5 % (in DEP/ethanol) to induce skin sensitization after repeated epidermal contact. The subjects were instructed to apply the test material to a treatment area on their upper backs. Patches were applied three times per week for a total of nine inductions. Two weeks after the final induction patch application, a challenge patch was applied to a new test site. This patch was removed and the test site scored after 24 and 72 hours. As a result, during induction phase, moderate skin reactions (erythema and edema) were observed after the 7th induction application. In one additional subject, barely perceptible skin reactions were observed after the induction applications 7, 8 and 9. No skin reactions were observed after the challenge exposure. Therefore it was concluded that the test material at a concentration of 0.5 % did not cause skin sensitization. For details please refer to IUCLID6, section 7.10.4.

107 male and female human volunteers participated in a study to assess the potential of the test material at a concentration of 0.05 % (in DEP/ethanol) to induce skin sensitization after repeated epidermal contact. Patches were applied three times per week for a total of nine inductions. Two weeks after the final induction patch application, a challenge patch was applied to a new test site. This patch was removed and the test site scored after 24 and 72 hours. As a result no skin reactions were observed either after the induction applications or after the challenge exposure. Therefore it was concluded that the test material at a concentration of 0.05 % did not cause skin sensitization. For details please refer to IUCLID6, section 7.10.4.

A human patch test was carried out with 10 volunteers to assess the skin-sensitising property of the test item at a concentration of 10 % in olive oil. The subjects were treated repeatedly (6 times) after intervals of 48 h. The seventh application was performed after 10 days. As a result, no skin reactions were observed and therefore the test substance at this concentration was considered to not be a skin-sensitizer at a concentration of 10 %. For details please refer to IUCLID6, section 7.10.4.

Conclusion

The animal study conducted according to OECD 429 was selected as the key study. It was done using reliable test methods and with GLP with a low concentration. The human tests were selected as supporting studies as the substance was also applied in most cases in an even lower concentration as in the animal test. Furthermore the human studies were mostly dependent on the proper application of the test material by the individual subjects and no control was established to evaluate this. Therefore no qualitative conclusion can be derived from the human tests. In addition the mouse study was selected as key for safety reasons, as it represents the worst case for this endpoint.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008

The available experimental test data is reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Based on available data on skin sensitisation, the test item is classified for skin sensitisation into category 1A and labeled with H317 (May cause an allergic skin reaction) according to Regulation (EC) No 1272/2008 (CLP), as amended for the eighth time in Regulation (EU) No 2016/918.  

Categories Display