Registration Dossier

Toxicological information

Skin sensitisation

Currently viewing:

Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
other: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
From October 09 to October 23, 2002
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Remarks:
The test was conducted by means of Read Across approach. The reliability of the source study report is 1. Further information was attached at section 13

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)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of study:
mouse local lymph node assay (LLNA)

Test material

Constituent 1
Reference substance name:
Acid Blue 025:1 - Similar Substance 03
IUPAC Name:
Acid Blue 025:1 - Similar Substance 03
Test material form:
not specified

In vivo test system

Test animals

Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source:Harlan Netherlands
- Age at study initiation: 8 - 12 weeks
- Weight at study initiation: 18.5 g -22.4 g
- Housing: ln groups of four in Makrolon type-3 cages with standard softwood bedding.
- Diet: Pelleted standard Kliba 3433, batch no. 57102 mouse maintenance diet ad libitum.
- Water: Community tap water from ltingen, available ad libitum.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3'C,
- Humidity (%): 30 -70 %
- Air changes (per hr): 10 - 15 air change per hour
- Photoperiod (hrs dark / hrs light): There was a 12 hour fluorescent light / 12 hour dark cycle with at least 8 hours music during the light period.

Study design: in vivo (LLNA)

Vehicle:
other: ethanol:water, 1:1 (v/v)
Concentration:
1 %, 2.5 %, 5 % and 10 % (w/v)
No. of animals per dose:
groups concentration %(w/v) N° per group
1 control / 4 females
2 2.5 4 females
3 5 4 females
4 10 4 females
Details on study design:
RANGE FINDING TESTS: ln a non-GLP conform pretest in two mice, test item concentrations ol 1%, 2.5 %, 5% and 10 % were tested on one ear each. No irritation effects were observed at the concentrations applied.
10 % (w/v) was the highest technically applicable concentration that could sufficiently be dosed to achieve optimal skin contact.

MAIN STUDY
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 2.5 %, 5 % and 10% (wlv) in ethanol:water, 1:1 (v/v). The application volume, 25 µl, was spread over the entire dorsal surface (Ø - 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
H-methyl thymidine (HTdR) was purchased from Amersham lnternational (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 µl of 82.49 µCi/mltHTdR (equalto 20.6 µCi HTdR) by intravenous injection via a tail vein.

DETERMINATION OF INCORPORATED HTDR
Approximately five hours after treatment with HTdR all mice were euthanized by intraperitoneal injection of VETANARCOL (Veterinaria AG, Zürich).
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 lymphnode cells were prepared by gentle mechanical disaggregation through stainless steel gauze (200 pm 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 for about 42 hours 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 HTdR incorporation was then measured on a p-scintillation counter. Similarly, background HTdR levels were also measured in two 1ml-aliquots of 5 % trichloroacetic acid.
The p-scintillation counter expresses HTdR incorporation as the number of radioactive disintegrations per minute (DPM).

OTHER:
ln addition to the sensitizing reactions the following observations and data were recorded during the test and observation period:
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)

Results and discussion

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Value:
ca. 1.8
Test group / Remarks:
Group 2
Remarks on result:
other: Test item concentration 2.5 % (w/v)
Parameter:
SI
Value:
ca. 1.5
Test group / Remarks:
Group 3
Remarks on result:
other: Test item concentration 5 % (w/v)
Parameter:
SI
Value:
ca. 1.7
Test group / Remarks:
Group 4
Remarks on result:
other: Test item concentration 10 % (w/v)

Any other information on results incl. tables

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: CLP criteria not met
Conclusions:
Non sensitising
Executive summary:

Method

The allergenic potential of the test substance was assessed according to the OECD guideline 429. Three groups of four female mice were used, each treated with the test item at concentrations of 2.5, 5 and 10 % (w/v) in ethanol:water, 1:1 (v/v) by topical application to the dorsum of each ear lobe (left and right) on three consecutive days. A control group of four mice was treated with the vehicle (ethanol:water, 1:1 (v/v)) only. Five days after the first topical application the mice were injected intravenously into a tail vein with radio-labelled thymidine (3H-methyl thymidine). Approximately five hours after intravenous injection, the mice were sacrificed, the draining auricular lymph nodes excised and pooled per group. Single cell suspensions of lymph node cells were prepared from pooled lymph nodes which were washed subsequently and incubated with trichloroacetic acid overnight. The proliferative capacity of pooled lymph node cells was determined by the incorporation of 3H-methyl thymidine measured in a pscintillation counter.

Results

No test item-related clinical signs were observed. All treated animals survived the scheduled study period. A test item is regarded as a sensitizer in the LLNA if the exposure to at least one concentration of the test item resulted in an incorporation of "HTdR at least 3-fold or greater than that recorded in control mice, as indicated by the STIMULATION INDEX (S.I.).

Test item concentration       %(w/v)        S.I

Group 2                                  2.5         1.8

Group 3                                  5           1.5

Group 4                                  10           1.7

An exact EC3 value could not be calculated because this calculation requires data lying immediately above and below S.l. value of 3.

 

Conclusion

The test item was found to be a non sensitizer when tested up to 10 % (wlv) in ethanol:water, 1:1 (v/v).