Registration Dossier

Administrative data

Description of key information

Not skin sensitizer

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records
Reference
Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
From October 29th to November 17th, 2003
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail
Justification for type of information:
The read across approach is detailed into the document attached to the IUCLID section 13.
Reason / purpose:
read-across source
Related information:
Composition 1
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
adopted 24 April 2002
GLP compliance:
yes (incl. certificate)
Type of study:
mouse local lymphnode assay (LLNA)
Test material information:
Composition 1
Species:
mouse
Strain:
CBA/Ca
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Netherlands.
- Females: nulliparous and non-pregnant.
- Age at study initiation: 8 - 12 weeks.
- Weight at study initiation: 16 - 24 g (ordered).
- Housing: individual in Makrolon type-2 cages with standard softwood bedding.
- Diet: pelleted standard Kliba 3433, batch no. 40/03 mouse maintenance diet (Provimi Kliba AG, CH-4303 Kaiseraugst) available ad libitum.
- Water: community tap water from ltingen, available ad libitum.
- Acclimation period: under test conditions after health examination. Only animals without any visible signs of illness were used for the study.
- Indication of any skin lesions:

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 3 °C
- Relative humidity: 30 - 70 %
- Air changes: 10 - 15 air changes per hour.
- Photoperiod: 12 hour fluorescent light / 12 hour dark cycle with at least 8 hours music during the light period.
Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
5, 10 and 25 %
No. of animals per dose:
2 females for the pre-test
4 females per group (the main test)
Details on study design:
PRE-SCREEN TESTS
- Number of animals: 2 mice.
- Concentration: 2.5, 5 and 25 % (w/v).
- Irritation: no severe irritant effects were tolerated choosing the test concentrations.
- Systemic toxicity: the top dose is the highest technically achievable concentration whilst avoiding systemic toxicity and excessive local irritation.

MAIN STUDY

TREATMENT PREPARATION
- Test solution: the test item was placed into a volumetric flask on a tared Mettler balance and the vehicle was quantitatively added. The weigh/volume dilutions were prepared individually using a magnetic stirrer as homogenizer.
- Preparation: test item formulations were made freshly before each dosing occasion and no more than 4 hours prior to application to the ears. Homogeneity of the test item in the vehicle was maintained during treatment with the magnetic stirrer.

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. The application volume, 25 µl, was spread over the entire dorsal surface (Ø ca 8 mm) of each ear lobe once daily for three consecutive days. A hair dryer was used to dry the ea's surface as quickly as possible to avoid loss of test item applied.
- Administration of 3H-METHYL THYMIDINE: 5 days after the first topical application, all mice were administered with 250 µl of 78.4 µCi/ml 3HTdR (equal to 19.6 µCi 3HTdR) by intravenous injection via a tail vein.

DETERMINATION OF INCORPORATED 3HTDR.
- Necropsy: approximately five hours after treatment with 3HTdR all mice were euthanized by intraperitoneal injection of VETANARCOL.
- Suspension: 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 two times with phosphate buffered saline (approx. 10 ml) the lymph node cells were resuspended in 5 % trichloroacetic acid (approx. 3 ml).
- Incubaction: at approximately +4 °C for at least 18 hours for precipitation of macromolecules.
- Precipitates: 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.
- Level of 3HTdR: measured on a β-scintillation counter. The β-scintillation counter expresses 3HTdR incorporation as the number of radioactive disintegrations per minute (DPM).
- Background 3HTdR level: measured in two 1ml-aliquots of 5 % trichloroacetic acid.

OBSERVATIONS
- Mortality / Viability: twice daily from acclimatization start to the termination of in-life phase.
- Body weights: prior to the 1st application and prior to necropsy.
- Clinical signs (local/ systemic): daily from acclimatization start to the termination of in-life phase. Especially the treatment sites were observed carefully.

INTERPRETATION OF RAW DATA
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 incorporated into lymph node cells of test group relative to that recorded for control group (Stimulation Index, S.I.). 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.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Parameter:
SI
Value:
1.2
Test group / Remarks:
5 %
Parameter:
SI
Value:
1.2
Test group / Remarks:
10 %
Parameter:
SI
Value:
0.7
Test group / Remarks:
25 %
Cellular proliferation data / Observations:
The Stimulation Iindicies of 1.2,1.2 and 0.7 were determined with the test item at concentrations of 5, 10 and 25 % (w/v) in acetone:olive oil, 4:1 (v/v).
The test item was found to be a non-sensitizer when tested at up to the highest achievable concentration of 25 % (w/v) in acetone:olive oil, 4:1 (v/v).

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 prior to the 1st application and prior to necropsy, was within the range commonly recorded for animals of this strain and age.

POSITIVE CONTROL
Stimulation Indicies of 1.5, 3.2 and 6.9 were determined with the positive control, alpha-hexylcinnamaldehyde, at concentrations of 5,10 and 25 % (wlv) in acetone:olive oil, 4:1 (vlv).
The substance was found to be a skin sensitizer and an EC3 value of 9.4 % (w/v) was derived.

Calculation and results of test item

Test item concentration % (w/v) Measurement dpm dpm BG* number of lymph nodes dpm per lymph node** S.I.
Background I -- 0 -- -- -- --
Background II -- 0 -- -- -- --
Control Group -- 2505 2505 8 313 --
Test item group 2 5 3111 3111 8 389 1.2
Test item group 3 10 2908 2908 8 364 1.2
Test item group 4 25 1862 1862 8 233 0.7

Background (1 ml 5 % trichloroacetic acid) in duplicate.

*The mean value was taken from the figures backgroud I and 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.

No dose-response relation was observed.

Calculation of the EC3 value was not done because no test concentrations produced a Stimulation Index (S.I.) of 3 or higher.

POSITIVE CONTROL

Calculation and results of positive control, alpha-hexylcinnamaldehyde

Test item concentration % (w/v) S.I.
Group 2 5* 1.5*
Group 3 10* 3.2
Group 4 25 6.9

* The value was used in calculation of EC3.

A clear dose-response relation was observed.

Interpretation of results:
other: not classified, according to the CLP Regulation ((EC) 1272/2008)
Conclusions:
Not skin sensitizer.
Executive summary:

In order to study a possible contact allergenic potential of test substance, three groups each of four female mice were treated daily with the test item at concentrations of 5, 10 and 25 % (w/v) in acetone:olive oil, 4:1 (v/v) by topical application to the dorsum of each ear lobe (left and right) for three consecutive days. 25 % was the highest technically achievable concentration in the vehicle. A control group of four mice was treated with the vehicle (acetone:olive oil, 4:1 (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 subsequently washed 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 β-scintillation counter.

No test item-related clinical signs were observed. Alltreated animals survived the scheduled study period.

No dose-response relation was observed. Calculation of the EC3 value was not done because no test concentrations produced a Stimulation Index (S.I.) of 3 or higher.

Conclusion

No dose-response relation was observed and the calculation of the EC3 value was not done because no test concentrations produced a Stimulation Index (S.I.) of 3 or higher; thus, the test item does not meet the criteria to be classified as skin sensitizer, according to the CLP Regulation ((EC) No 1272/2008).

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:

There are no experimental studies on the skin sensitisation potential of the Fluorescent Brightener 002, therefore the available information on the structural analogous Similar Substance 01 have been taken into consideration. The read across approach can be considered as suitable and appropriate to investigate the skin sensitisation potential of Fluorescent Brightener 002 (the read across approach is detailed in the document attached to the IUCLID section 13).

Three experiments assessing the skin sensitisation potential of Smilar Substance 01 are available. They were all conducted following the mouse local lymphnode assay (LLNA), using lot with a different content of test item (in all the cases, the content was highern than 98 %).

In the key experiment, three groups each of four female mice were treated daily with the Similar Substance 01 at concentrations of 5, 10 and 25 % (w/v) in acetone:olive oil. 25 % was the highest technically achievable concentration in the vehicle. Five days after the first topical application the mice were injected intravenously into a tail vein with radio-labelled thymidine (3H-methyl thymidine). No test item-related clinical signs were observed. Alltreated animals survived the scheduled study period. No dose-response relation was observed; therefore, the calculation of the EC3 value was not done because no test concentrations produced a Stimulation Index (S.I.) of 3 or higher.

The supporting experiments were conducted under the same conditions. In one case, the the test item at concentrations was administrated in acetone:olive oil, while in the other it was administrated as in DMF (Dimethylformamide). All treated animals survived the scheduled study period; no clinical signs were observed. In both cases, the EC3 values could not be calculated because no test concentrations produced a Stimulation Index (S.I.) of 3 or higher.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

According to the CLP Regulation (EC) No 1272/2008, 3.4 Respiratory or skin sensitisation section, skin sensitizer means a substance that will lead to an allergic response following skin contact.

Based on the Local lymph node assay (LLNA) results, a substance in considered a skin sensitizer when the EC3 value resulted to be higher than 2 %.

The LLNA assay failed to calculate an EC value higher than 2 %; Stimulation Index resulted to be lower than 3 for all of the tested concentrations, in all the experiments available.

In conclusion, Fluorescent Brightener 002 does not meet the criteria to be classified as skin sensitizer, according to the CLP Regulation (EC) No 1272/2008.