Registration Dossier

Administrative data

Description of key information

Non skin sensitising

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records
Reference
Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
June from 11th to 25th, 2003
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail
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 lymph node assay (LLNA)
Species:
mouse
Strain:
CBA/Ca
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Netherlands B.V. Postbus 6174.
- Females: nulliparous and non-pregnant.
- Age: 8-12weeks (beginning of acclimatization).
- Weight: 16.9 - 22.6 g (beginning of acclimatization).
- Housing: in groups of four in Makrolon type-3 cages with standard softwood bedding.
- Diet: pelleted standard Kliba 3433, batch no. 84102 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.

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 3 *C
- Humidity: 30-70 %
- Air changes: approximately 10-15 air changes per hour.
- Photoperiod: the animals were provided with an automatically controlled light cycle of 12 hours light and 12 hours dark.
- Other: music was played during the daytime light period.
Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
5, 10 and 25 %
No. of animals per dose:
4 female
Details on study design:
PRE-SCREEN TESTS:
- Compound solubility: 25 % (wlv) was the highest technically achievable concentration in acetone:olive oil, 4:1 (vlv).
- Irritation: a non-GLP conform pre-test in two mice, test item concentrations of 1, 5, 10 and 25 % (w/v) were tested on one ear each. No irritation effects were observed at these concentrations after a single application.

MAIN STUDY

TEST SOLUTION
The test item was placed into a volumetric flask on a tared Mettler balance and the vehicle (acetone:olive oil, 4:1 (v/v)) was quantitatively added. The weight/volume dilutions were prepared individually using a magnetic stirrer as homogenizer. Homogeneity of the test item in the vehicle was maintained during treatment with the magnetic stirrer.
The preparations were made freshly before each dosing occasion.

CONCENTRATION
The test item in the main study was assayed at three consecutive concentrations. The top dose is the highest technically achievable concentration whilst avoiding systemic toxicity and excessive local irritation. No severe irritant effects were tolerated choosing the test concentrations.
Concentrations were in terms of material as supplied.

TOPICALAPPLICATION
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 in acetone:olive oil, 4:1 (vlv). 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 ea/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.
5 days after the first topical apqlication, all mice were administered with 250 µl of 84.3 µCi/ml 'HTdR (equal to21.1uCi 3HTdR) by intravenous injection via a tailvein.

DETERMINATION OF INCORPORATED 3HTdR
Approximately five hours after treatment with 3HTdR all mice were euthanized by intraperitoneal injection of Vetabarcol (Veterinaria AG, Zürich).
The draining lymph nodes were rapidly excised and pooled for each experimentat 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) and incubated at approximately +4° C for at least 18 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 3HTdR incorporation was then measured on a β-scintillation counter. Similarly, background 3HTdR levels were also measured in two 1ml-aliquots of 5 % trichloroacetic acid.
The β-scintillation counter expresses 3HTdR incorporation as the number of radioactive disintegrations per minute (DPM).

INTERPRETATION OF RAW DATA
The proliferative response of lymph node cells is expressed as the number of radioactive disntegrations per minute per lymph node (DPM/node) and as the ratio of 3HTdR incorporated into lymoh node cells of test lymph nodes relative to that recorded for control lymph nodes (Stimulation Index - SI). Before DPM/node values were determined, mean scintillation-background DPM was subtracted from test and control raw data.

OBSERVATIONS
- Mortality / Viability: twice daily from acclimatization start to the termination of inlife phase.
- Body weights: at acclimatization start and prior to necropsy.
- Clinical signs: daily from acclimatization start to the termination of in-life phase. Especially the treatment sites were observed carefully.
Parameter:
SI
Value:
0.8
Variability:
-
Test group / Remarks:
test group 5 %
Parameter:
SI
Value:
1.1
Variability:
-
Test group / Remarks:
test group 10 %
Parameter:
SI
Value:
0.9
Variability:
-
Test group / Remarks:
test group 25 %
Cellular proliferation data / Observations:
Stimulation Indices of 0.8, 1.1 and 0.9 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 at the start of acclimatization period and prior to necropsy, was within the range commonly recorded for animals of this strajn and age.

Individul data

Test group Measurement dpm dpm - Background number of lymph nodes dpm per lymph node Stimulation Index
Background I 0 - - -
Background II 0 - - -
Control Group 5244 5244 8 656 -
Test item at 5 % 4372 4372 8 547 0.8
Test item at 10 % 5517 5517 8 690 1.1
Test item at 25 % 4951 4951 8 619 0.9
Interpretation of results:
other: not classified, according to the CLP Regulation (EC) No 1272/2008
Conclusions:
Not skin sensitising.
Executive summary:

In order to study a possible contact allergenic potential of test item, three groups each of four female mice were treated daily wilh 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 rignt) 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. All treated animals survived the scheduled study period.

A test item is regarded as a sensitizer in the LLNA if the exposure to one or more test concentrations resulted in 3 -fold or greater increase in incorporation of 3HTdR compared with concurrent controls, as indicated by the Stimulation Index (S.I.).

In the study Stimulation Indices of 0.8, 1.1 and 0.9 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).

Conclusion

Not skin sensitising

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

In order to study a possible contact allergenic potential of Fluorescent Brightener 367, three groups each of four female mice were treated according to the OECD guideline 429. The test item was administrated at the 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 rignt) for three consecutive days. 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. All treated animals survived the scheduled study period. In the study Stimulation Indices of 0.8, 1.1 and 0.9 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).

Justification for classification or non-classification

According to the CLP Regulation (EC) No 1272/2008, 3.4 Respiratory or skin sensitization 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 % because Stimulation Index resulted to be lower than 3 for all of the tested concentrations.

In conclusion, Fluorescent Brightener 367 does not meet the criteria to be classified as skin sensitizer, according to the CLP Regulation (EC) No 1272/2008.