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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:
key study
Study period:
2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP/Guideline Study
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
GLP compliance:
yes
Type of study:
mouse local lymph node assay (LLNA)
Species:
mouse
Strain:
other: CBA/J
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Received from Harlan Laboratories, Indianapolis, IN
- Age at study initiation: 10-11 weeks
- Weight at study initiation: 19.2-25.0 grams
- Housing: The animals were individually housed in plastic solid bottom cages with
bedding during the dosing and resting phases of the study. After final weighing until
sacrifice, animals were housed in their respective dose groups in plastic cages with
bedding.
- Diet (e.g. ad libitum): Harlan Teklad Certified Global 16% Protein Rodent Diet® #2016C. The diet
was available ad libitum.
- Water (e.g. ad libitum): Filtered tap water was supplied ad libitum by an automatic water dispensing
system.
- Acclimation period: 14-28 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-22ºC
- Humidity (%): 45-65%,
- Air changes (per hr): 13
- Photoperiod (hrs dark / hrs light): 12-hour light/dark cycle

IN-LIFE DATES: September 19 - October 9, 2012
Vehicle:
propylene glycol
Concentration:
10%, 25% and 50%
No. of animals per dose:
5
Details on study design:
RANGE FINDING TESTS:
Nine test substance concentrations and the vehicle control were used for preliminary toxicity
testing. Test substance concentrations of 75%, 50%, 25%, 10%, 5%, 2.5%, 1%, 0.5% and 0.2%
in propylene glycol were tested to determine the highest achievable level that avoids overt
systemic toxicity and excessive local irritation. The top dose of 75% was selected based on
maximum solubility, compatibility, and viscosity of the test substance with the vehicle while
maintaining a pipettable solution that could be applied to the mouse ear (documented in the raw
data). Each group consisted of one mouse. The ears of each
mouse were scored for erythema and edema prior to dosing on Days 1, 2, 3, and prior to
termination on Day 6. Twenty-five microliters (25 µL) of the appropriate dilution of the test substance concentration or
the vehicle alone was applied to the dorsum of both ears of each mouse (50 L total) for three
consecutive days. Application was done using an appropriate size micropipette
to accurately deliver 25 µL. The dose was gently spread as evenly as possible over the dorsal
surface of the ear using the tip of the pipette. No treatment was made on Days 4 and 5. On Days
1, 2, 3 and 6, each site was evaluated for local irritation (erythema & edema).

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Criteria used to consider a positive response: Any test
material that produces a SI > 3 in the LLNA is normally considered “positive” for dermal
sensitization potential.

TREATMENT PREPARATION AND ADMINISTRATION:
Dilutions of the test substance were prepared as w/w mixtures in propylene glycol.
Concentrations of 10%, 25% and 50% were selected for the main test based on results of the
preliminary screening test. A single concentration of a 25% w/w mixture of HCA in propylene
glycol was prepared as a positive control. All dosage preparations were freshly prepared on the
day of administration.
Beginning on Day 1, a volume of 25 µL of the vehicle, the appropriate test substance
concentration, or the positive control substance was applied to the dorsum of both ears of each
mouse (50 µL total) once per day for three consecutive days (Days 1, 2, and 3) using a
micropipette. During application, the material was gently spread as evenly as possible 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:
Statistical analysis was performed on the body weight, body weight gain and dpm values.
Significance was judged at p <0.05. The treated group and vehicle control group were compared
using a One-Way Analysis of Variance (ANOVA), followed by comparison of the treated groups
to the vehicle control group by Dunnett’s t-test for multiple comparisons. Where variances are
considered significantly different by Bartlett’s test, groups were compared using a non-parametric
method (Kruskal-Wallis non parametric analysis of variance followed by Dunn’s test) (INSTAT
Biostatistics, Graph Pad Software, San Diego, CA). Outlier analysis was conducted using Grubbs
(1969).
Positive control results:
HCA elicited a proliferative response with a SI value of 6.27 relative to vehicle controls.
Parameter:
SI
Remarks on result:
other: Group 2 (positive control) - 6.27 Group 3 (10%) - 1.34 Group 4 (25%) - 2.33 Group 5 (50%) - 3.57
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: Group 1 (control) - 1852.28 ± 282.41 Group 2 (positive control) - 11605.91 ± 1945.93 Group 3 (10%) - 2491.27 ± 792.94 Group 4 (25%) - 4311.31 ± 1360.50 Group 5 (50%) - 6617.63 ± 1502.58
Interpretation of results:
sensitising
Remarks:
Migrated information Criteria used for interpretation of results: other: ECETOC 2003
Conclusions:
Based on the results of this study, Reaction product of 2-Hydroxyethyl methacrylate (HEMA)
with Polyphosphoric Acid (PPA) is considered positive for dermal sensitization potential in the
LLNA, with an EC3 value of 39%, which is consistent with weak sensitization potential as
described by an expert panel (ECETOC 2003).
Executive summary:

Based on the results of the screening assay, 10%, 25% and 50% w/w concentrations in propylene glycol and the vehicle alone were evaluated in the main study. All mice appeared active and healthy, and there were no consistent treatment-related effects on body weight over the course of the study. Very slight erythema was noted at two sites at the 10% concentration (Group 3) on Days 2 or 3 and most sites at the 25% concentration (Group 4) between Days 2 and 6. At the 50% concentration (Group 5), very slight to well-defined erythema was evident at most sites between Days 2 and 6, and slight edema was noted at three sites with desquamation and/or alopecia present at all sites on Day 6. No dermal irritation was observed at any site in the vehicle control group (Group 1). Very slight to well-defined erythema and slight to marked edema was noted in the positive control group (Group 2). Treatment of mice with 10%, 25% and 50% of Reaction product of 2-Hydroxyethyl methacrylate (HEMA) with Polyphosphoric Acid (PPA) resulted in stimulation index values of 1.34, 2.33 and 3.57, respectively, relative to vehicle control mice. As a stimulation index (SI) of greater than 3.0 was observed, Reaction product of 2-Hydroxyethyl methacrylate (HEMA) with Polyphosphoric Acid (PPA) was considered positive for dermal sensitization potential in the LLNA. The EC3 value calculated for the test substance was 39%, which is consistent with weak sensitization potential as described by an expert panel (ECETOC 2003). Proper conduct of the LLNA was

confirmed via a positive response with 25% HCA, a moderate contact sensitizer, which elicited a proliferative response with a SI value of 6.27 relative to vehicle controls.

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

Test material is considered to be a weak sensitizer based on results of the LLNA.


Migrated from Short description of key information:
Reaction product of 2-Hydroxyethyl methacrylate (HEMA) with Polyphosphoric Acid (PPA) is considered positive for dermal sensitization potential in the LLNA, with an EC3 value of 39%, which is consistent with weak sensitization potential as described by an expert panel (ECETOC 2003).

Justification for selection of skin sensitisation endpoint:
Only study conducted with evidence test material is a weak sensitizer.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification