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Description of key information

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records
Reference
Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
From July 13th to 27th, 2005
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Study conducted according to internationally accepted testing guidelines and performed in compliance with Good Laboratory Practice. Justification for read across approach is given in the endpoint summary and in the read across justification report attached to the Section 13 of this dossier.
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
adopted 24 April 2002
Qualifier:
according to guideline
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
29 April 2004
GLP compliance:
yes (incl. QA statement)
Type of study:
mouse local lymph node assay (LLNA)
Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: RCC Ltd, CH·4414 Fiillinsdorf / Switzerland.
- Age at study initiation: 8 - 12 weeks (beginning of acclimatization). Nulliparous and non-pregnant.
- Weight at study initiation: 16 g - 24 g (ordered).
- Housing: individual accomodation in Makrolon type-2 cages with standard softwood bedding ("LignoceI", Schill AG, CH-4132 Muttenz).
- Diet : Pelleted standard Kliba 3433, batch no. 25/05 mouse maintenance diet (Provimi Kliba AG, CH-4303 Kaiseraugst) available ad Iibitum.
- Water: community tap water from ltingen, available ad Iibitum.
- Acclimation period: under test conditions after health examination. Only animals without any visible signs of illness were used for the study.

ENVIRONMENTAL CONDITIONS
- Temperature: ranges for room temperature 22 ± 3 °C.
- Humidity: 30 - 70 %.
- Air changes: 10 - 15 air changes per hour.
- Photoperiod: 12 hour fluorescent light / 12 hour dark cycle.
- Other: at least 8 hours music during the light period.
Room temperature and humidity were monitored continuously and values outside of these ranges occasionally occurred, usually following room cleaning. These transient variations are considered not to have any influence on the study and, therefore, these data are not reported but are retained at testing laboratory.
Vehicle:
other: ethanol/water (7/3, v/v)
Concentration:
5 %, 10 % and 25 % (w/v) in ethanol/water (7/3, v/v)
No. of animals per dose:
2 females for the pre-test (non GLP)
16 females for the main study, 4 per group
Details on study design:
RANGE FINDING TESTS
- GLP compliance: non-GLP conform pre-test.
- Test animals: two mice.
- Vehicle: the test item was tested in different vehicles, i.e. acetone/olive oil (4/1, v/v) and ethanol/water (7/3, v/v). A suitable vehicle ethanol/water (7/3, v/v) was selected and used in the main test.
- Concentration: 2.5 %, 5 %, 10 % and 25% were tested on one ear each.
- Compound solubility: 25 % was the highest technically applicable concentration in the chosen vehicle.
- Irritation: one day after a single topical application no irritation effects were observed at the tested concentrations.

MAIN STUDY
TEST ITEM FORMULATIONS PREPARATION
The test item was placed into a volumetric flask on a tared Mettler balance and the vehicle ethano/water (7/3, v/v) was quantitatively added. The weight/volume (w/v) dilutions were prepared individually using a magnetic stirrer as homogenizer.
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.
Concentrations were in terms of material as supplied.

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 the test item at concentrations of 5 %, 10 % and 25 % (w/v) in ethanol/water (7/3, 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).

ADMINISTRATION OF 3H-METHYL THYMIDlNE
Five days after the first topical application all mice were administered 250 µl of 81.87 µCi/ml 3HTdR (equal to 20.5 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 inhalation of CO2 (dry ice).
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 twice 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 ‘lrga-Safe Plus' 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 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.l.). 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, 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 (S.I.).
- Second, 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.

OBSERVATIONS
In addition to the sensitizing reactions the following observations and data were recorded during the test and observation period:
Mortality/Viability once daily from acclimatization start to the termination of in-life phase.
Body weights on the test day 1 (prior to the 1st application) and on the test day 6.
Clinical signs (local / systemic) once daily on the day of animals delivery and from test day 1 (after the 155 application) to the termination of in-life phase. Especially the treatment sites were observed carefully.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
The mean values and standard deviations were calculated in the body weight tables.
Parameter:
SI
Remarks on result:
other: Test item at 5 % (w/v): 0.9 Test item at 10 % (w/v): 1.1 Test item at 25 % (w/v): 1.6
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: Test item at 5 % (w/v): 1437 Test item at 10 % (w/v): 1895 Test item at 25 % (w/v): 2584

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

Test item conc. % (w/v) Measurement dpm Calculation Result
dpm - Background* N. of lymph nodes dpm per lymph nodes** S.I.
-- Background I 9 -- -- -- --
-- Background II 5 -- -- -- --
-- Control group 1664 1657 8 207 --
5 Test group 1437 1430 8 179 0.9
10 Test group 1895 1888 8 236 1.1
25 Test group 2584 2577 8 322 1.6

*The mean Background was calculated from the figures Background I and Background ll values.

** 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.

VIABILITY I MORTALITY

No deaths occurred during the study period.

CLINICAL SIGNS

No clinical signs were observed in any animals of the control group, Group 2 (5 %) or Group 3 (10 %). On the second application day, a slight ear erythema was observed at both dosing sites in all mice of Group 4 (25 %), persisting for a total of two days. After each topical application, some residues of test item were found at both dosing sites in all mice of Group 4 (25 %).

BODY WEIGHTS

The body weight of the animals, recorded prior to the first application and prior to necropsy, was within the range commonly recorded for animals of the strain and age.

RESULTS OF POSITIVE CONTROL

Vehicle: acetone/olive oil (4/1, v/v)

Test item conc. % (w/v) Measurement dpm Calculation Result
dpm - Background* N. of lymph nodes dpm per lymph nodes** S.I.
-- Background I 0 -- -- -- --
-- Background II 0 -- -- -- --
-- Control group 2257 2257 8 282 --
5 Test group 5310 5310 8 664 2.4
10 Test group 8055 8055 8 1007 3.6
25 Test group 25351 25351 8 3169 11.2

*The mean Background was calculated from the figures Background I and Background ll values.

** 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.

Test item conc. % (w/v) S.I.
Group 2 5 (a) 2.4 (b)
Group 3 10 (c) 3.6 (d)
EC3 = (a-c) [(3-d)/(b-d)] + c = 7.5 % (w/v)

EC3 = Estimated concentration for a Stimulation Index (S.I.) of 3.

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.

Interpretation of results:
not sensitising
Remarks:
Migrated information according to the CLP Regulation (EC 1272/2008) Criteria used for interpretation of results: EU
Conclusions:
The substance was found to be a non-sensitizer (S.I. = 1.6) when tested up to the highest applicable concentration of 25 % (w/v) in ethanol/water (7/3, v/v).
Executive summary:

In order to study a possible contact allergenic potential of the test item, three groups each of four female mice were treated daily at concentrations of 5 %, 10 % and 25 % (w/v) in ethanol/water (7/3, v/v) by topical application to the dorsum of each ear lobe (left and right) for three consecutive days. 25 % (w/v) was the highest technically applicable concentration in the vehicle. A control group of four mice was treated with the vehicle ethanol/water (7/3, 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 SH-methyl thymidine measured in a B- scintillation counter. All treated animals survived the scheduled study period. No clinical signs were observed in any animals of the control group, Group 2 (5 %) or Group 3 (10 %). On the second application day, a slight ear erythema was observed at both dosing sites in all mice of Group 4 (25 %), persisting for a total of two days. After each topical application, some residues of test item were found at both dosing sites in all mice of Group 4 (25 %).

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

In the study S.I. of 0.9, 1.1 and 1.6 were determined with the test item at concentrations of 5 %, 10 % and 25 % (w/v), respectively, in ethanol/water (7/3, v/v).

Conclusion

According to the CLP Regulation (EC 1272/208), a stimulation index of three or more is considered a positive response in the local lymph node assay and this positive response is considered suitable to classify a substance as skin sensitizer.

The substance showed a S.I. of 1.6 when tested up to the highest applicable concentration of 25 % (w/v) in ethanol/water (7/3, v/v).

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

There are no experimental data available on the Leucophor 1111X, thus a read across approach with the structural analogous substance 01 and the analogous substance 02 has been proposed.

Both analogous substance 01 and analogous substance 02 are Stilbene derivatives Fluorescent Whitening Agents salts. They display similar structural and physicochemical properties; all of them exhibit high degree of dissociation in water and very low octanol/water partition coefficients because to a higher affinity with water phase than the octanol one. The water solubility is due to the sulphonated groups in the molecules. The differences occurring in the structure formulas (i.e. substituents) are expected to not significantly impact the toxicological characterisation.

Further details about the justification for read across approach are given in the report attached to the Section 13 of this dossier.

SKIN SENSITISATION

The skin sensitisation potential of analogous substance 01 has been investigated by the Local Lymph Node Assay (LLNA) in Mice, while the analogous substance 02 has been assessed by Guinea Pig Maximization test.

In order to study a possible contact allergenic potential of the analogous substance 01, three groups each of four female mice were treated daily at concentrations of 5 %, 10 % and 25 % (w/v) in ethanol/water (7/3, v/v) by topical application to the dorsum of each ear lobe (left and right) for three consecutive days. 25 % (w/v) was the highest technically applicable concentration in the vehicle. All treated animals survived the scheduled study period. No clinical signs were observed in any animals of the group treated at 5 % or in the group treated at 10 %. On the second application day, a slight ear erythema was observed at both dosing sites in all mice of the group treated at 25 %, persisting for a total of two days. After each topical application, some residues of test item were found at both dosing sites in all mice of treated at 25 %.

In the study S.I. of 0.9, 1.1 and 1.6 were determined with the test item at concentrations of 5 %, 10 % and 25 % (w/v), respectively, in ethanol/water (7/3, v/v) (Wang-Fan, 2005).

The potential of the analogous substance 02 to provoke delayed skin sensitization reactions was assessed after intradermal injection and topical skin insult applications of the test item. All guinea pigs appeared active and healthy throughout the test period. There were no signs of gross toxicity, adverse pharmacologic effects or abnormal behaviour. All animals gained weight. Barely perceptible to mild erythema was observed at all sites 24 hours after the topical booster application. By 48 hours all sites were clear. There was no evidence of erythema at any sites on any of the test animals 24 or 48 hours after challenge. Similarly, no irritation occurred on any of the dosed sites on the naive Guinea Pigs (Ventura and Shapiro, 1989).


Migrated from Short description of key information:
non skin sensitizer

Justification for selection of skin sensitisation endpoint:
Study conducted according to internationally accepted testing guidelines and performed in compliance with Good Laboratory Practice.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

According to the CLP Regulation (EC 1272/2008), 3.4 Respiratory or skin sensitisation section, skin sensitizer means a substance that will lead to an allergic response following skin contact.

The criteria to classify a substance as skin/respiratory sensitizer are reported into the second adaptation to technical progress*. A substance is considered a skin sensitizer when:

- an adjuvant type test method for skin sensitisation is used and a response of at least 30 % of the animals is considered as positive;

- for a non-adjuvant Guinea pig test method a response of at least 15 % of the animals is considered positive;

- a stimulation index of three or more is considered a positive response in the Local Lymph Node Assay.

A S.I. of 1.6 was recorded when similar substance was tested up to the highest applicable concentration of 25 % (w/v) in ethanol/water (7/3, v/v) in the Local Lymph Node Assay and less than the 30 % of animals showed a reaction in the Guinea pig maximisation test.

In conclusion, Leucophor 1111X does not meet the criteria to be classified as skin sensitizer, according to the CLP Regulation (EC 1272/2008).

*Commission Regulation (EU) No 286/2011 of 10 March 2011, amending, for the purposes of its adaptation to technical and scientific progress, Regulation (EC) No 1272/2008 of the European Parliament and of the Council on classification, labelling and packaging of substances and mixtures