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Administrative data

Description of key information

In a local lymph node assay, the concentration that would cause a 3-fold increase in proliferation (EC-3) was calculated to be 5.7% (NESIL = 1425 µg/cm²) which is consistent with moderate dermal sensitization potential.

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:
2006-06-26
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study was conducted according to GLP as well as Guidelines.
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Qualifier:
according to
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Qualifier:
according to
Guideline:
other: EC B.42 (Skin Sensitisation: Local Lymph Node Assay)
Principles of method if other than guideline:
not applicable
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:
Female CBA/J mice were obtained from Harlan (Indianapolis, Indiana), and were approximately 9-12 weeks at study start.
Each animal was evaluated to determine the general health status and acceptability for study purposes upon arrival at the laboratory. The animals were housed up to six per cage in clear plastic cages, in rooms designed to maintain adequate conditions (temperature, humidity, and photocycle), and acclimated to the laboratory for at least one week prior to the start of the study.

Before administration of test material began, animals were stratified by body weight and then randomly assigned to treatment groups using a computer program designed to increase the probability of uniform group mean weights and standard deviations at the start of the study. Animals placed on study were identified via subcutaneously implanted transponders that were correlated to unique alphanumeric identification numbers.

Animals were provided rodent diet in pelleted form. Feed and municipal water was provided ad libitum.
Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
0.3%, 1%, 3%, 10% or 30%) or vehicle (AOO, acetone/olive oil)
No. of animals per dose:
6
Details on study design:
Vehicle
AOO (4:1 acetone:olive oil), the preferred solvent in LLNA guidelines was selected based upon maximum miscibility of DER-331 while maintaining a solution suitable for application. Evaluation of DER-331 miscibility and selection were documented in the study file.

Dose Preparation and Analysis
Concentrations tested for the irritancy screen were selected based upon maximum miscibility in an appropriate LLNA vehicle while maintaining a solution suitable for application. Toxicity data regarding irritation potential were also taken into consideration.
Concentrations tested in the LLNA were based on this information and previous dermal sensitization data. DER-331 was combined with vehicle (acetone:olive oil at 4:1 (AOO)) to obtain concentrations of 0.3%, 1%, 3%, 10% or 30%. Solutions were prepared daily just prior to dosing. Preparation of the dosing materials was documented in the study file. The concentrations of the dosing solutions were not verified analytically.

Irritation Screen
Prior to the LLNA study, several concentrations of the test material were evaluated for irritation potential as measured by erythema of the ears. Mice (one female/concentration) received one application of DER-331 (0.5%, 1%, 5%, 10%, 20% or 30%), on the dorsal surface of each ear (25 l) on three consecutive days. Using an adjustable pipette with a disposable tip, the test solutions were spread over the dorsal surface of each ear in a manner to prevent material loss. Ears were inspected prior to application of the test material solutions, and erythema was evaluated on days 2, 3, and 6. All mice were weighed on days 1 and 6. Erythema scores and body weight data following test material applications were compared to the response of the animals treated with vehicle alone.

Dermal Sensitization via the Local Lymph Node Assay
The application of the test material (25 µl/ear) was made on the dorsal surface of both ears as described above. Six female mice/group received one of five concentrations of DER-331 (0.3%, 1%, 3%, 10% or 30%) or vehicle (AOO) once daily for three consecutive days. Ears were inspected prior to application of the test material solutions, and erythema was evaluated on days 2, 3, and 6. All mice were weighed on days 1 and 6. On day 6, all mice received a 250 µl intravenous injection (i.v.) via the lateral tail vein containing 20 µCi of 3H-thymidine (specific activity 2Ci/mmol) diluted in phosphate-buffered saline (PBS). Approximately five hours post administration, the mice were euthanized via CO2 asphyxiation and both auricular lymph nodes located at the bifurcation of the jugular veins were excised and placed in PBS. A single cell suspension of the auricular lymph nodes from one mouse was prepared by gentle mechanical disaggregation using a tissue homogenizer. The cells were washed two times and were suspended in 3 ml of 5% trichloroacetic acid (TCA) for approximately 18 hours. The suspended precipitates were centrifuged (200 x g for 10 minutes) and the supernatant removed. The pellet from each mouse was reconstituted and subsequently transferred to a scintillation vial containing Aquasol-2 scintillation cocktail. Two additional 2 ml aliquots of water were used to rinse the tubes and the rinses were added to the scintillation vials containing the pellet in TCA and cocktail. The radioactivity in each precipitate was measured using a scintillation counter and reported as disintegrations per minute (dpm) per mouse. A mean dpm value + SD (standard deviation) was calculated for each experimental group. The SI was calculated using the absolute dpm value for each mouse as the numerator and the mean dpm value from the vehicle control mice as the denominator; the mean SI + SD was calculated for each experimental group. Any test material that produces a SI of > 3 in the LLNA should be considered "positive" for contact sensitization (Kimber et al., 1994). While the criterion for a positive response (SI > 3) was originally developed empirically, a recent robust statistical evaluation indicated that it is an acceptable practical value for hazard identification (Basketter et al., 1999a). Furthermore, by determining EC3 values (estimated concentration resulting in a 3-fold SI), one can compare relative sensitization potency of chemicals and/or formulations (Basketter et al., 1999b). Based on the EC3 values derived from the LLNA, it has been proposed that contact allergens can be categorized as weak (10%-100%), moderate (1%-10%), strong (0.1%-1%), or extreme (0.1%); (ECETOC, 2003). The EC3 value is determined by interpolating between two values one above and one below the SI value of 3.
Statistics:
Statistics and Calculations
1. The Stimulation Index (SI) was calculated for each mouse using the following equation:
SI = (Disintegration per minute (dpm) of individual mouse)/
(Average dpm of the VH control mice)

2. EC3 Calculation:
EC3 = XL + [(3-YL)/(Yh-YL)](Xh-XL)
Where, YL = SI value below 3
XL = chemical concentration that elicits YL
Yh = SI value above 3
Xh = chemical concentration that elicits Yh

Means and standard deviation (SD) were calculated for body weight data (absolute and gain) and the LLNA response (dpm & SI values). These body weight and dpm data were analyzed by a one-way analysis of variance (Steele and Torrie, 1960). When differences were indicated by the ANOVA, a comparison of treated vs. control groups was done using a Dunnett's t-test (Steele and Torrie, 1960). The alpha level at which all tests were conducted was 0.05. The final interpretation of the biological significance of the responses was based on both statistical outcome and scientific judgment.
Positive control results:
not applicable
Parameter:
SI
Value:
0.9
Test group / Remarks:
0.3% in 4:1 acetone:olive oil
Parameter:
SI
Value:
1.1
Test group / Remarks:
1% in 4:1 acetone:olive oil
Parameter:
SI
Value:
1.5
Test group / Remarks:
3% in 4:1 acetone:olive oil
Parameter:
SI
Value:
5.4
Test group / Remarks:
10% in 4:1 acetone:olive oil
Parameter:
SI
Value:
11.8
Test group / Remarks:
30% in 4:1 acetone:olive oil
Parameter:
EC3
Value:
5.7

The Mean +/- SD DPM for the control, 0.3, 1, 3, 10 and 30% DER-331 groups was 1251 +/-525, 1117 +/- 247, 1381 +/- 465, 1886 +/- 646, 6807 +/-2893 and 14801 +/-4173, respectively.

During the screening study, mice were treated with three daily applications of 0.5%, 1%, 5%, 10%, 20% or 30% DER-331. Erythema was absent and body weights were unaffected in all dose groups.
 
Based on the results of the screening study, 0.3%, 1%, 3%, 10% or 30% DER-331 was tested in the LLNA to characterize the dose response.
 Erythema was absent, and body weights were unaffected in all dose groups.

A NOEL for the mouse LLNA was observed with a test concentration of 3% DER-331. The concentration that would cause a 3-fold increase in proliferation (EC3) was calculated to be 5.7% which is consistent with moderate dermal sensitization potential as described by an expert ECETOC panel (Technical Report No. 87, 2003).

Interpretation of results:
sensitising
Remarks:
Migrated information
Conclusions:
A NOEL for the mouse LLNA was observed with a test concentration of 3% DER-331. The concentration that would cause a 3-fold increase in proliferation (EC3) was calculated to be 5.7% which is consistent with moderate dermal sensitization potential.
Executive summary:

DER-331 was examined in the local lymph node assay (LLNA). Groups of mice were treated with dose levels of 0.3, 1, 3, 10 and 30% DER-331 in AOO (4:1 acetone:olive oil) for 3 consecutive days. On day 6, mice were injected with 3H-thymidine and 5 hours later incorporation of radiolabelled material in the auricular lymph nodes was determined. A NOEL for the mouse LLNA was observed with a test concentration of 3% DER-331. The concentration that would cause a 3-fold increase in proliferation (EC3) was calculated to be 5.7% which is consistent with moderate dermal sensitization potential.

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

DER-331 was examined in the local lymph node assay (LLNA). Groups of mice were treated with dose levels of 0.3, 1, 3, 10 and 30% DER-331 in AOO (4:1 acetone:olive oil) for 3 consecutive days. On day 6, mice were injected with 3H-thymidine and 5 hours later incorporation of radiolabelled material in the auricular lymph nodes was determined. A NOEL for the mouse LLNA was observed with a test concentration of 3% DER-331. The concentration that would cause a 3-fold increase in proliferation (EC3) was calculated to be 5.7% which is consistent with moderate dermal sensitization potential.

Other in vivo studies in guinea pigs, including studies done by the Buehler method and the maximization methods, produced results consistent with the above LLNA study. All produced evidence of dermal sensitization potential, although the response could not be quantified as to potency.

Has caused allergic skin reactions in humans.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

The LLNA study results indicate that BADGE can be classified as a sub-category 1B skin sensitizer (EC3 value >2%) according to the GHS system and according to Guidance to Regulation (EC) No. 1272/2008 on classification, labelling and packaging (CLP) of substances and mixtures.