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Toxicological information

Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Study period:
2008
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was conducted according to OECD TG 429, EPA OPPTS 870.2600, EU Method B.42 and in accordance with the Principles of Good Laboratory Practice.
Justification for type of information:
For the purposes of fulfilling the REACH requirements for propyl propionate a category of alcohol esters has been developed according to the principles laid down in the REACH guidance on grouping substances.
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:
2008
Report Date:
2008

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Deviations:
no
Principles of method if other than guideline:
not applicable
GLP compliance:
yes
Type of study:
mouse local lymph node assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): n-pentyl propionate
- Molecular formula: C8H16O2
- Molecular weight: 144.2
- Physical state: liquid, clear and colourless
- Analytical purity: Non-GLP certificate of analysis lists the purity of the test material as 99.96% wt. with 0.02% wt. water (Plant Quality Coordinator, 2007).
- Lot/batch No.: lot # VE10551G01
- Radiochemical purity (if radiolabelling): 98.3% (HPLC – GE Healthcare – Amersham, 2008)

In vivo test system

Test animals

Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan, Indianapolis, Indiana
- Age at study initiation: approximately 9-12 weeks
- Housing: Animals were housed up to six per cage in filter tubs containing corncob bedding.
- Diet (e.g. ad libitum): ad libitum, food pellets (LabDiet Certified Rodent Diet #5002 (PMI Nutrition International, St. Louis, Missouri) were provided
- Water (e.g. ad libitum): water bottle or a crock filled with water
- Acclimation period: at least 1 week prior to study

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22°C with a tolerance of ± 1°C (and a maximum permissible excursion of ± 3°C)
- Humidity (%): 40-70%
- Air changes (per hr): 12-15 times/hour
- Photoperiod (hrs dark / hrs light): 12 hours light/dark cycle

Study design: in vivo (non-LLNA)

Induction
Concentration / amount:
not applicable
Challenge
Concentration / amount:
not applicable
No. of animals per dose:
not applicable
Details on study design:
not applicable
Challenge controls:
not applicable

Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
5%, 25%, or 100% n-Pentyl Propionate
No. of animals per dose:
Six female mice/group
Details on study design:
RANGE FINDING TESTS: 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 n-Pentyl Propionate (1%, 5%, 25%, 50%, 75%, or 100%), on the dorsal surface of each ear (25 μl) on three consecutive days. Using an adjustable pipette with a disposable tip, the test solutions (25 μl/ear) 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. Erythema was absent and body weights were unaffected in all dose groups.

MAIN STUDY: The application of the test material (25 μl/ear) was made on the dorsal surface of both ears. Six female mice/group received one of three concentrations of n-Pentyl Propionate (5%, 25%, or 100%) or vehicle (4:1 AOO) once daily for three consecutive days. HCA at 30% (v/v) in vehicle was run concurrently as a positive dermal sensitization control. 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; Amersham code TRA310, Buckinghamshire, United Kingdom) diluted in phosphatebuffered 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 (Stomacher 80 Lab System, Seward Ltd., London, United Kingdom). 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 in 1 ml of 5% TCA and subsequently transferred to a scintillation vial containing 10 ml of Aquasol-2 scintillation cocktail (Packard Instrument Company, Meridan, Connecticut). 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 β-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 produced a SI of > 3 in the LLNA was considered “positive” for contact sensitization. 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). While a test material that produces a SI of > 3 in the LLNA should be considered “positive” for contact sensitization (Kimber et al., 1994), recent opinions have suggested circumstances in which the LLNA result and sensitization potential should be further considered in the context additional, scientific judgment (Ryan
et al., 2000; Basketter et al., 1998; Basketter et al., 2006). 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 and Calculations)

TREATMENT PREPARATION AND ADMINISTRATION: n-Pentyl Propionate was combined with vehicle (4:1 AOO) to obtain concentrations of 5%, 25%, and 100%. 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.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
The Stimulation Index (SI) and EC3 values were calculated using appropriate methods. Means and standard deviation (SD) were generated 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.

Results and discussion

Positive control results:
Proper conduct of the LLNA was demonstrated via the positive response from the positive control, 30% HCA, which elicited a stimulation index (SI) that was 7.4 in comparison to vehicle-treated mice.

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: Mice treated with 5%, 25% and 100% n-Pentyl Propionate exhibited proliferative responses with stimulation indices (SI) that were respectively 1.7, 1.1 and 1.8 in comparison to vehicle-treated mice.
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: Mice treated with 5%, 25% and 100% n-Pentyl Propionate exhibited values (dpm) that were respectively 1759.7, 1096.3 and 1845.5 in comparison to vehicle-treated mice.

Any other information on results incl. tables

None

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
n-Pentyl Propionate did not demonstrate dermal sensitization potential in the mouse LLNA as the lymph nodes draining the area of topical application did not demonstrate a 3-fold increase in proliferation when compared to vehicle-treated mice.
Executive summary:

The Local Lymph Node Assay (LLNA) study was conducted according to OECD TG 429, EU method B.49, EPA OPPTS 870.2600 and according to the Principles of Good Laboratory Practice (GLP), to assess the potential of n-Pentyl Propionate to cause contact sensitization by measuring lymphocyte proliferative responses from auricular lymph nodes following topical application of the test material to the mouse ear.

Screening Study: Three daily topical applications of 1%, 5%, 25%, 50%, 75%, or 100% n-Pentyl Propionate were given to one animal at each dose level. Erythema was absent and body weights were unaffected in all dose groups. Results from this study were used to determine the dosing concentrations for n-Pentyl Propionate in the LLNA.

LLNA: Six female mice/group received 5%, 25%, or 100% n-Pentyl Propionate, or vehicle (4:1 Acetone Olive Oil (AOO)) or 30% α-hexylcinnamaldehyde (HCA; positive control) on days 1-3. On day 6, uptake of 3H-thymidine into the auricular lymph nodes draining the site of chemical application was measured five hours post administration. Proper conduct of the LLNA was confirmed via a positive response using 30% α- hexylcinnamaldehyde (HCA), a moderate contact sensitizer, which elicited proliferation that was 7.4 in comparison to vehicle-treated mice.

Erythema was absent and body weights were unaffected in all dose groups.

Mice treated with 5%, 25% and 100% n-Pentyl Propionate exhibited proliferative responses with stimulation indices (SI) that were respectively 1.7, 1.1 and 1.8 in comparison to vehicle treated mice.

Therefore, n-Pentyl Propionate did not demonstrate dermal sensitization potential in the mouse LLNA as the lymph nodes draining the area of topical application did not demonstrate a 3-fold increase in proliferation when compared to vehicle-treated mice.