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Description of key information

Skin sensitisation (OECDTG429): skin sensitising

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

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Endpoint:
skin sensitisation: in vivo (LLNA)
Remarks:
LLNA was performed before September 2016
Type of information:
experimental study
Adequacy of study:
key study
Study period:
3 September 2009 to 10 September 2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
LLNA was performed before September 2016.
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
24 April 2002
GLP compliance:
yes
Type of study:
mouse local lymph node assay (LLNA)
Species:
mouse
Strain:
CBA:J
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Jackson Laboratories, Bar Harbor, ME 04609
- Females (if applicable) nulliparous and non-pregnant: not specified
- Age at study initiation: 10 weeks at start of dosing
- Weight at study initiation: 19-25 grams at the outset (Day 1) of the study
- Housing: Animals were group housed 5 per cage upon receipt in compliance with National Research Council "Guide for the Care and Use of Laboratory Animals".
- Diet (e.g. ad libitum): Animals had access to Harlan Teklad Certified Rodent Chow 7012C ad libitum. No contaminants were known to be present in the certified diet at levels that would be expected to interfere with the results of this study.
- Water (e.g. ad libitum): Tap water was available ad libitum, via water bottles. No contaminants were known to be present in the water at levels that would be expected to interfere with the results of this study.
- Acclimation period: Study animals were acclimated to their housing for seven days prior to their first day of dosing.
- Prestudy Health Screen and Selection Criteria: All animals used in this study were assessed as to their general health by a member of the technical staff or other authorized personnel. During the acclimation period, each animal was observed at least once daily for any abnormalities or for the development of infectious disease. Only animals that were determined to be suitable for use were assigned to this study.
- Assignment to Study Groups: Animals were randomly assigned to study groups based on body weight and apparent good health.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22.2 - 30.6
- Humidity (%): 28 - 68
- Photoperiod (hrs dark / hrs light): 12/12
Vehicle:
other: Diethyl Phthalate
Concentration:
Test item at concentrations of 2.5, 5, 10, 25 and 50% v/v
No. of animals per dose:
Groups of five mice per dose
Details on study design:
Justification for dose selection:
In general, the doses were selected so that the highest concentration maximizes exposure while avoiding systemic toxicity and excessive local irritation. Doses were selected based on known reported uses of the material.

Dose preparation:
The test article was received at 50% and therefore the 50% dose group was dosed as received. On each day of dosing, the test article was prepared at 2.5%, 5%, 10%, or 25% (v/v) in volumetric flasks by mixing the appropriate amount of test article in the vehicle. All preparations were vortexed to mix. The test article dosing solutions were clear, colorless liquids.

MAIN STUDY:
Mice were treated topically on the dorsal surface of both ears, once daily for 3 consecutive days (Days 1, 2 and 3). Approximately 24 +/- 2 hours separated each application of test article. A volume of 25 µL/ear was applied using a micro pipette. On Day 6, the mice were injected i.v. with 20 µCi of 3H-thymidine in 250 µL of sterile saline. Five hours later the mice were euthanized by CO2 asphyxiation and the draining auricular lymph nodes were removed. At removal, the number of nodes collected per animal was recorded, and the nodes were examined for size/appearance and the data recorded. Any unexpected observations were noted in study records. A single cell suspension was prepared form the lymph nodes of each mouse. Cells were washed twice with phosphate buffered saline (PBS) and precipitated with 5% trichloroacetic acid (TCA) overnight at 2-8 oC. the pellets were recovered by centrifugation and resuspended in 1 mL of TCA and transferred to a vial containing scintillation fluid. An additional 1 mL of TCA was used to rinse the tube, and it was also transferred to the scintillation fluid. Incorporation of 3H-thymidine was measured in a β-scintillation counter.

OBSERVATIONS:
Mortality/morbidity:
Daily on days 1 to 6.

Clinical observations:
Observations were performed prior to dose administration and following dose administration. Clinical observations were performed once daily on Days 4-6. Particular attention was given to the application sites. Any significant alterations to the applications sites, and the general appearance of the pinnae, including build up of test article, was recorded.

Dermal irritation:
Animals were examined daily for signs of erythema and edema. Irritation was scored and recorded using the Draize scoring system. Scoring was performed prior to dosing on Days 1-3.

Body weight:
Animals were weighed on days 1 and 6.

DATA COLLECTION:
All data was collected manually except for the data generated by the scintillation counter (Beckman LS 6000 SC). SYSTAT version 9.01, developed by SPSS, Inc was used for statistical analysis. DPM for each group was determined. Increases in 3H-thymidine incorporation relative to the vehicle-treated control was derived for each group and recorded as stimulation indices (SI).

CRITERIA:
The criterion for a positive response was that one or more concentrations of a test article elicits a 3-fold or greater increase in isotope incorporation relative to the vehicle control.

Body weights on Days 1 and 6 and body weight changes were also evaluated using SYSTAT version 9.01, developed by SPSS, Inc.

The EC3 was calculated using the following formula:
EC3 = c+ [(3-d)/(b-d)](a-c)
Where the data points lying immediately above and below the SI value of 3 have the co-ordinates (a,b) and (c,d) respectively.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
SYSTAT version 9.01, developed by SPSS, Inc was used for statistical analysis.
Body weights on Days 1 and 6 and body weight changes were also evaluated using SYSTAT version 9.01, developed by SPSS, Inc.
Individual DPM values were analyzed by log transformation (base 10) of the data. The evaluation of the equality of means for body weight data and DPM was made by a one-way analysis of variance using the F distribution to assess statistical significance. If statistically significant differences between the means are found, a Dunnett’s test was used to determine the degree of significance from the control means.
Positive control results:
The positive control item, HCA, gave a Stimulation Index of greater than 3 when tested at a concentration of 35%.
Key result
Parameter:
EC3
Remarks:
%
Value:
28.3
Key result
Parameter:
SI
Value:
1.8
Remarks on result:
other: dose 2.5%
Key result
Parameter:
SI
Value:
1.8
Remarks on result:
other: dose 5%
Key result
Parameter:
SI
Value:
1.7
Remarks on result:
other: dose 10%
Key result
Parameter:
SI
Value:
2.8
Remarks on result:
other: dose 25%
Key result
Parameter:
SI
Value:
4.3
Remarks on result:
other: dose 50%
Cellular proliferation data / Observations:
MORTALITY:
There was no mortality and all animals appeared normal throughout the study.

CLINICAL OBSERVATIONS:
No erythema or edema was noted in any of the mice in the vehicle group or in those dosed with the test article at 2.5%, 5%, 10%, 25% or 50% (v/v). Ears that appeared wet were observed in all mice treated with the vehicle and 2.5, 5, 10, 25 and 50% of the test article on Days 2-4. Mice treated with the test article at 25 and 50% also had ears that appeared wet on Days 5 and 6. There were no other findings.

BODY WEIGHTS:
The mean body weights and body weight changes of the mice treated with test substance on Days 1 and 6 showed no statistically significant differences. Therefore, administration of the test article did not appear to exert overt toxicity.

LOCAL LYMPH NODE ASSAY:
At termination, the lymph nodes from the mice in the vehicle group and the test article treated animals at 2.5%, 5%, 10%, 25% and 50% (v/v) were normal in size and appearance.
Exposure to test substance at 2.5%, 5%, 10%, 25% or 50% (v/v) resulted in stimulation indices of 1.8, 1.8, 1.7, 2.8 and 4.3, respectively. In addition, the response with 25% and 50% of the test article were both statistically significant (p<0.001) when the log DPM was compared to the vehicle group. Since the data indicated a positive response the EC3 was calculated and determined to be 28.3%.
Interpretation of results:
other: Skin sensitizer, Category 1B
Remarks:
in accordance with EU CLP Regulation (EC) No. 1272/2008 and its amendments
Conclusions:
Under the conditions of the test, exposure to the test substance resulted in an EC3 of 28.3%. The substance is therefore considered a skin sensitizer.
Executive summary:
The skin sensitisation potential of the test substance has been tested according to OECD TG 429: Local Lymph Node Assay" method and GLP principles. At 2.5, 5, 10, 25 and 50% (v/v) the substance showed SI values of 1.8, 1.8, 1.7, 2.8 and 4.3, respectively. No erythema or edema was noted in any of the mice. Ears that appeared wet were observed in all mice treated with the vehicle (Diethyl Phthalate) and 2.5, 5, 10, 25 and 50% of the test article on Days 2-4. Mice treated with the test article at 25 and 50% also had ears that appeared wet on Days 5 and 6. At termination, the lymph nodes from the mice in the vehicle group and the test article treated animals at 2.5%, 5%, 10%, 25% and 50% (v/v) were normal in size and appearance. An EC3 was calculated and resulted in an EC3 of 28.3%. The substance is therefore considered to be a skin sensitizer.
Endpoint:
skin sensitisation: in vitro
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
an in vitro skin sensitisation study does not need to be conducted because adequate data from an in vivo skin sensitisation study are available
Endpoint conclusion
Endpoint conclusion:
adverse effect observed (sensitising)
Additional information:
The skin sensitisation potential of the test substance has been tested according to OECD TG 429: Local Lymph Node Assay" method and GLP principles. At 2.5, 5, 10, 25 and 50% (v/v) the substance showed SI values of 1.8, 1.8, 1.7, 2.8 and 4.3, respectively. No erythema or edema was noted in any of the mice. Ears that appeared wet were observed in all mice treated with the vehicle (Diethyl Phthalate) and 2.5, 5, 10, 25 and 50% of the test article on Days 2-4. Mice treated with the test article at 25 and 50% also had ears that appeared wet on Days 5 and 6. At termination, the lymph nodes from the mice in the vehicle group and the test article treated animals at 2.5%, 5%, 10%, 25% and 50% (v/v) were normal in size and appearance. An EC3 was calculated and resulted in an EC3 of 28.3%. The substance is therefore considered to be a skin sensitizer.

Justification for classification or non-classification

Based on the results of an LLNA study, the substance is classified as skin sensitisation category 1B and shall be labelled with H317: May cause an allergic skin reaction according to Regulation (EC) No. 1272/2008 and its amendments.