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Toxicological information

Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2004
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was conducted according to test guidelines and in accordance with GLP.
Cross-referenceopen allclose all
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2004
Report Date:
2004

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
yes
Remarks:
Recommended strain of mice were not used - Balb/C used in place of CBA/Ca female mice and dosing solutions were not verfied analytically
Qualifier:
according to
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Deviations:
yes
Remarks:
Recommended strain of mice were not used - Balb/C used in place of CBA/Ca female mice and dosing solutions were not verfied analytically
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes
Type of study:
mouse local lymphnode assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): 1,2-Dichloropropane (Propylene Dichloride)
- Molecular formula - C3H6Cl2
- Physical state: colourless liquid
- Analytical purity: > 99 %
- Lot/batch No.: #06514HW

In vivo test system

Test animals

Species:
mouse
Strain:
Balb/c
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories
- Age at study initiation: 9 weeks
- Housing: housed in one per cage in suspended, stainless steel cages with wire-mesh floors and suspended above catch pans
- Diet (e.g. ad libitum): LabDiet Certified Rodent Diet #5002 (PMI Nutrition International, St. Louis, Missouri) in pellet form provided ad libitum
- Water (e.g. ad libitum): Municipal water provided ad libitum
- Acclimation period:


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 + 1°C
- Humidity (%): 40-70%
- Air changes (per hr): 12-15 times/hour
- Photoperiod (hrs dark / hrs light): 12- hour light/dark photocycle

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
other: not applicable
Vehicle:
other: not applicable
Concentration / amount:
not applicable
Challengeopen allclose all
Route:
other: not applicable
Vehicle:
other: not applicable
Concentration / amount:
not applicable
No. of animals per dose:
not applicable
Details on study design:
not applicable
Challenge controls:
not applicable
Positive control substance(s):
not specified

Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
All mice received one of three concentrations of PDC (5%, 20% or 80%, v/v) or vehicle (4:1 acetone: olive oil)
No. of animals per dose:
6 animals/dose
Details on study design:
MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Local Lymph Node Assay
- Criteria used to consider a positive response: The decision process with regard to a positive response includes a stimulation index ≥ 3, together
with consideration of dose-response and, where appropriate, statistical significance. Furthermore, by determining EC3 values (estimated concentration resulting in a 3- fold SI), one can compare relative sensitization potency of chemicals. The EC3 value is typically determined by extrapolating between two values immediately above and immediately below the SI value of 3.


TREATMENT PREPARATION AND ADMINISTRATION: 1,2-dicloropropane (PDC) was combined with AOO (4:1 acetone:olive oil) to obtain concentrations of 5%, 20%, or 80% PDC (v/v). In addition, HCA (a-hexylcinnamaldeyde) at 30% v/v was used as a positive control.

Ear Swelling Response - Prior to the administration of a dosing solution on the ear, the thickness of each ear was measured with a digital micrometer. Individual mice received one concentration of PDC (80%, 60%, 40%, 20%, or 10% v/v) or AOO on the dorsal surface of each ear (25 ml) daily for two consecutive days. The concentrations of PDC were based upon solubility of the test material and viscosity in AOO. The administration of the materials was made using an adjustable pipettor with a disposable tip. Ear thickness was determined 24 hours after the second application and the percent ear-swelling was calculated for each mouse. Ear thickness measurements following test material applications were compared to the response of the animal treated with vehicle alone.

Local Lymph Node Assay - The administration of the materials (25 μl/ear) was made on the dorsal surface of both ears using an adjustable pipettor with disposable tip (n = six mice/group). All mice received one of three concentrations of PDC (5%, 20% or 80%, v/v) or vehicle (4:1 acetone:olive oil),
once daily for three consecutive days. HCA (a-hexylcinnamaldehyde) at 30% v/v was run concurrently as a positive dermal sensitization control. On day 6, all mice received a 250 μl intravenous injection (i.v.) via the lateral tail vein containing 20 μCi of 3H-thymidine diluted in phosphate buffered saline (PBS). Approximately five hours later, the mice were sacrificed via CO2 asphyxiation and the auricular lymph nodes located at bifurcation of the jugular veins were excised and placed in PBS (phosphate buffer solution). Both lymph nodes from an individual animal were combined and analyzed as a pooled sample. A single cell suspension of lymph node cells from one mouse was prepared by gentle mechanical disaggregation using a tissue homogenizer. The cells were washed two times in PBS and were suspended in 3 ml of 5% trichloroacetic acid (TCA) for approximately 64 hours. The suspended precipitates were centrifuged (200 x g for 10 minutes) and the supernatant removed. The pellet from each mouse was reconstituted in 1 ml of 5% TCA and subsequently transferred to a scintillation vial containing 10 ml of 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 1 ml of pellet in TCA and cocktail. The radioactivity
in each precipitate was measured using a b-scintillation counter and reported as disintegration per minute (dpm) per mouse. A mean dpm value + SD (standard deviation) was calculated for each experimental group (six mice/group). In addition, a 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. A mean SI + SD was calculated for each experimental group six mice/group).
Positive control substance(s):
other: HCA (alpha-hexylcinnamaldehyde) at 30% v/v
Statistics:
Means and standard deviation (SD) were determined for body weights (absolute and gain), ear thickness, and the LLNA response (dpm & SI values). Data for absolute and body weight gains and dpm were analyzed by a oneway analysis of variance (ANOVA) (Steele and Torrie, 1960). Comparisons of treated vs.control groups were done by Dunnett’s t-test (Steele and Torrie, 1960) when ANOVA results suggested differences. The alpha level at which all tests were conducted was 0.05.

Results and discussion

Positive control results:
Sensitivity of the LLNA was demonstrated via the positive response from the positive control, a-hexylcinnamaldehyde (HCA), at 30% v/v, that elicited proliferation (SI) that was 14-fold greater than that of vehicle controls

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: Refer to tables 1 & 2
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: Refer to tables 1 & 2

Any other information on results incl. tables

None

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information
Conclusions:
PDC did not demonstrate any dermal sensitization potential, as it did not elicit lymphocyte proliferation greater than 3x threshold in the mouse LLNA.
Executive summary:

Three groups of Balb/C mice (6 female animals/dose) received one of three concentrations of 1,2-dichloropropane (PDC - 5%, 20%, or 80% w/v), or acetone/olive oil (AOO) on days 1-3 (six female mice/group). HCA (a-hexylcinnamaldehyde), a moderate contact sensitizer, was evaluated concurrently as a positive dermal sensitization control. The test materials were administered to the dorsal surface of both ears (25 ml/ear). On day 6, all mice received an intravenous injection of phosphate buffered saline containing 20 mCi of 3H-thymidine.Uptake of 3H-thymidine into the auricular lymph nodes draining the site of chemical application was measured five hours later.

Body weights were unremarkable. Topical applications of PDC at 5%, 20%, and 80% v/v dose levels elicited barely perceptible erythema (scores = 1) in 18/18 mice on the day of sacrifice.

PDC did not demonstrate LLNA results (dpm and stimulation index -SI) consistent with dermal sensitization as the lymph nodes draining the area of topical application did not demonstrate a proliferative response greater than the 3x threshold. SI values were consistently around 1.0 (equivalent to vehicle controls) at all doses tested. Sens itivity of the LLNA was demonstrated via a 30% HCA positive control group that elicited proliferation (SI) that was 14- fold greater than that of vehicle controls. Because the SI values for 5%, 20%, and 80% PDC, both individual animal values and dose group mean values, were all below 3, an EC3 value could not be determined. On the basis of these results, PDC did not demonstrate any contact sensitization potential.