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Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2002-01-30 to 2003-02-13
Reliability:
3 (not reliable)
Rationale for reliability incl. deficiencies:
other: Relevant methodological deficiencies (improper dose levels tested)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2002
Report date:
2002

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
Draft Version November 2000
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of study:
mouse local lymph node assay (LLNA)

Test material

Constituent 1
Chemical structure
Reference substance name:
Bis(2-ethylhexyl) carbonate
EC Number:
238-925-9
EC Name:
Bis(2-ethylhexyl) carbonate
Cas Number:
14858-73-2
Molecular formula:
C17H34O3
IUPAC Name:
bis(2-ethylhexyl) carbonate
Test material form:
other: liquid

In vivo test system

Test animals

Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Age at study initiation: 7 - 12 weeks (beginning of acclimatisation)
- Weight at study initiation: 17.1 g - 23.0 g (beginning of acclimatisation period)
- Housing: In groups of four in Makrolon type-3 cages with standard softwood bedding ("Lignocel", Schill AG, CH-4132 Muttenz).
- Diet: ad libitum, pelleted standard Kliba 3433, batch no. 119/01 mouse maintenance diet (Provimi Kliba AG, CH-4303 Kaiseraugst)
- Water: ad libitum, community tap water
- Acclimation period: 6 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 3
- Humidity (%): 30 - 70
- Air changes (per hr): 10 - 15
- Photoperiod (hrs dark / hrs light): 12 hour fluorescent light / 12 hour dark cycle with at least 8 hours music during the light period

Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
25, 50, and 75 % (w/w)
No. of animals per dose:
4
Details on study design:
RANGE FINDING TESTS:
In a non-GLP conform pre-test in two mice test item concentrations of 10 %, 25 %, 50 % and 100 % (undiluted) (w/w) were tested on one ear each.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Local Lymph Node Assay (LLNA)
- Criteria used to consider a positive response:
The proliferative response of lymph node cells is expressed as the number of radioactive disintegrations per minute per lymph node (DPM/NODE) and as the ratio of 3HTdR into lymph node cells of test lymph nodes relative to that recorded for control lymph nodes (STIMULATION INDEX). Before DPM/NODE values were determined, mean scintillation-background DPM was subtracted from test and control raw data.

A test item is regarded as a sensitizer in the LLNA if the following criteria are fulfilled:
- First, that exposure to at least one concentration of the test item resulted in an incorporation of 3HTdR at least 3-fold or greater than that recorded in control mice, as indicated by the STIMULATION INDEX.
- Second, that the data are compatible with a conventional dose response, although allowance must be made (especially at high topical concentrations) for either local toxicity or immunological suppression.

TREATMENT PREPARATION AND ADMINISTRATION:

- TOPICAL APPLICATION
Each test group of mice was treated by topical (epidermal) application to the dorsal surface of each ear lobe (left and right) with different test item concentrations of 25 %, 50 % and 75 % (w/w) in acetone:olive oil, 4:1 (v/v). The application volume, 25 µl, was spread over the entire dorsal surface (diameter approx. 8 mm) of each ear lobe once daily for three consecutive days. A further group of mice was treated with an equivalent volume of the relevant vehicle alone (control animals). A hair dryer was used to dry the ear's surface as quickly as possible to avoid loss of test item applied.

- ADMINISTRATION OF 3H-METHYL THYMIDINE
3H-methyl thymidine (3HTdR) was purchased from Amersham International (Amersham product code no. TRA 310; specific activity, 2 Ci/mmol; concentration, 1 mCi/ml). Five days after the first topical application, all mice were administered with 250 µ of 80.34 µCi/ml 3HTdR (equal to 20.1 µCi 3HTdR) by intravenous injection via a tail vein.

- DETERMINATION OF INCORPORATED 3HTDR
Approximately five hours after treatment with 3HTdR all mice were euthanized by intraperitoneal injection of NARCOREN (Rhone Merieux GmbH, D-88471 Laupheim) at a dose of at least 2 ml/kg body weight (equivalent to 320 mg sodium pentobarbitone/kg body weight). The draining lymph nodes were rapidly excised and pooled for each experimental group (8 nodes per group). Single cell suspensions (phosphate buffered saline) of pooled lymph node cells were prepared by gentle mechanical disaggregation through stainless steel gauze (200 µm mesh size). After washing three times with phosphate buffered saline (approx. 10 ml) the lymph node cells were resuspended in 5 % trichloroacetic acid (approx. 3 ml) and incubated at approximately +4 °C overnight for precipitation of macromolecules. The precipitates were then resuspended in 5 % trichloroacetic acid (1 ml) and transferred to glass scintillation vials with 10 ml of 'Ultima Gold' scintillation liquid and thoroughly mixed. The level of 3HTdR incorporation was then measured on a beta-scintillation counter. Similarly, background 3HTdR levels were also measured in two 1ml-aliquots of 5 % trichloroacetic acid. The beta-scintillation counter expresses 3HTdR incorporation as the number of radioactive disintegrations per minute (DPM).

- FURTHER OBSERVATIONS:
-- Mortality/Viability: Twice daily from acclimatization start to the termination of in-life phase
-- Body weights: At acclimatization start and prior to necropsy
-- Clinical signs (local/systemic): Daily from acclimatization start to the termination of in-life phase. Especially the treatment sites were recorded carefully.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
A statistical analysis was conducted for assessment of the dose-response relationship, and the EC3 value was calculated according to the equation
EC3 =(a-c) [(3-d)/(b-d)] + c
where EC3 is the estimated concentration of the test item required to produce a 3-fold increase in draining lymph node cell proliferative activity; (a, b) and (c, d) are respectively the co-ordinates of the two pair of data lying immediately above and below the S.I. value of 3 on the local lymph node assay dose response plot.

Results and discussion

Positive control results:
In the study with positive control substance STIMULATION INDICES of 2.2,2.0 and 6.7 were determined with the test item ALPHA-HEXYLCINNAMALDEHYDE at concentrations of 5 %, 10 % and 25 % (w/w) in acetone:olive oil, 4:1 (v/v).
A test item is regarded as a sensitiser in the LLNA if the exposure to at least one concentration of the test item resulted in an incorporation of 3HTdR at least 3-fold or greater than that recorded in control mice, as indicated by the STIMULATION INDEX.
Based on these criteria, the test item ALPHA-HEXYLCINNAMALDEHYDE was found to be a non-sensitiser when tested up to 15 % (w/w) in acetone:olive oil, 4:1 (v/v).
ALPHA-HEXYLCINNAMALDEHYDE showed an allergenic potency when tested at concentration of 25% (w/w) in acetone:olive oil, 4:1 (v/v).

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: The test is considered to be unsuitable, because the results are likely to be due to the chemical´s irritating nature.
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: see table below

Any other information on results incl. tables

Test item concentration

% (w/w)

Measurement

Dpm*)

Calculation

Result

dpm - BG

Number of lymph nodes

dpm per lymph node**)

S.I.

--

BG I

3

--

--

--

--

--

BG II

6

--

--

--

--

--

CG 1

3056

3051

8

381

--

25 (a)

TG 2

12393

12388

8

1549

4.1 (b)

50 (c)

TG 3

12145

12140

8

1518

4.0 (d)

75

TG 4

10881

10876

8

1360

3.6

BG = Background (1 ml 5 % trichloroacetic acid) in duplicate

CG = Control Group

TG = Test Group

S.1. = Stimulation Index

*) = The mean value was taken from the figures BG I and BG II

**) = Since the lymph nodes of the animals of a dose group were pooled, DPM/Node was

determined by dividing the measured value by the number of lymph nodes pooled

EC3 = Estimated concentration for a STIMULATION INDEX of 3 =(a-c) [(3-d)/(b-d)] + c > 100 % (w/w)

a,b,c,d = Co-ordinates of the two pair of data lying immediately above and below the S.I.

value of 3 on the LLNA dose response plot.

Stimulation index at all tested concentrations was higher than 3, usually interpreted as demonstrating a sensitising potential. However, this result is most probably false positive because:

- According to the Australian NATIONAL INDUSTRIAL CHEMICALS NOTIFICATION AND ASSESSMENT SCHEME (NICNAS) January 2008, the test is considered to be unsuitable, because the results are likely to be due to the chemical´s irritating nature. The chemical is classified as a skin irritant with a cut off level of 25 % and above.

- The EC3 values were not consistent with the test results. EC3 is the estimated concentration for a STIMULATION INDEX of 3. In this study EC3 value of >100% (w/w) was theoretically calculated with STIMULATION INDICES of 4.1 and 4.0 at test item concentrations of 25 % and 50 % (w/w).

- The S.I. values in this study show a descending tendency with increasing concentrations applied and not the conventional dose response which means an increasing tendency with increasing concentrations. According to the study authors, one of the explanations for it is due to penetration limitations at higher dosing concentrations. However, this comment of the study authors is not conclusive. If the penetration at higher concentrations is limited, constant or slowed increase of the values for increasing concentrations can be expected but not an inverse dose response.

Further Results:

- VIABILITY / MORTALITY: No deaths occurred during the study period.

- CLINICAL SIGNS: No symptoms of local toxicity at the ears of the animals and no systemic findings were observed during the study period.

- BODY WEIGHTS: The body weight of the animals, recorded at the start of acclimatization period and prior to necropsy, was within the range commonly recorded for animals of this strain and age.

Applicant's summary and conclusion

Interpretation of results:
other: Test is invalid because of improper dose levels tested.
Conclusions:
The test is considered to be unsuitable, because the results are likely to be due to the chemical´s irritating nature.
Executive summary:

In a dermal sensitisation study with Bis(2-ethylhexyl) carbonate (at minimum 99.9% a.i.) in acetone:olive oil, 4:1 (v/v) groups of 4 female CBA mice were tested using the LLNA method according to the draft Version of OECD Guideline 429, November 2000. Positive control substance was alpha hexyl cinnamic aldeyde, which gave a positive result (STIMULATION INDEX of 6.7) at a concentration of 25 % in acetone:olive oil, 4:1 (v/v).

STIMULATION INDICES of 4.1, 4.0 and 3.6 were determined with the test item at concentrations of 25, 50, and 75 % (w/w) in acetone:olive oil, 4:1 (v/v), respectively.

Although the stimulation indices at all tested concentrations was higher than 3, usually interpreted as demonstrating a sensitising potential, this result is most probably false positive because the test is considered to be unsuitable, because the results are likely to be due to the test substance´s irritating nature. The test substance is classified as a skin irritant with a cut off level of 25% and above.

Thus, the test is considered to be unsuitable, because the results are likely to be due to the chemical´s irritating nature.

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