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Description of key information

No skin sensitising

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records
Reference
Endpoint:
skin sensitisation
Remarks:
in vivo
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
October from 9th to 23th, 2002
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:
REPORTING FORMAT FOR THE ANALOGUE APPROACH
Justification for Read Across is explained in the endpoint summary and it is further detailed in the report attached to the IUCLID section 13.
Reason / purpose for cross-reference:
read-across source
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
adopted 24 June 2002
Qualifier:
according to guideline
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Version / remarks:
adopted by the Council on July 17, 1992
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: Harlan Netherlands.
- Age at study initiation: 8 - 12 weeks.
- Weight at study initiation: 18.0 -21.1 g.
- Housing: animals were housed in groups of four in Makrolon type-3 cages with standard softwood bedding (Lignocel, Schill AG).
- Water: community tap water from Itingen, ad libitum.
- Diet: pelleted standard Kliba 3433, batch no. 57/02 mouse maintenance diet (Provimi Kliba AG, Kaiseraugst), ad libitum.
- Acclimation period: 6 days, under test conditions after health examination. Only animals without any visible signs of illness were used for the study.
- Females: nulliparous and non-pregnant.

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 3 °C
- Humidity: 30 - 70 %
- Air changes: 10 - 15 air changes per hour.
- Photoperiod: 12 hours fluorescent light / 12 hours dark cycle.
- Others: at least 8 hours music during the light period.
Vehicle:
other: ethanol/water, 1:1 (v/v)
Concentration:
2.5 %, 5 %, 10 % (w/v)
No. of animals per dose:
4 females per group
Details on study design:
TEST ITEM PREPARATION
The test item was placed into a volumetric flask on a tared Mettler balance and the vehicle 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 shortly before each dosing occasion.

RANGE FINDING TESTS
To determine the highest non-irritant and technically applicable test item concentraqtion, a non-GLP pretest was performed in 2 mice with concentrations of 1, 2.5, 5 and 10 % (w/v).
The top dose is the highest non-irritant and technically achievable concentration whilst avoiding systemic toxicity and excessive local irritation. 10 % (w/v) was the highest technically applicable concentration that could sufficiently be dosed to achieve optimal skin contact.

MAIN STUDY
- Purpose: the purpose of this Local Lymph Node Assay was to identify the contact allergenic potential of the test item when administered to the dorsum of both ear lobes of mice.
- 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 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.
- Criteria used to consider a positive response: a test item is regarded as a sensitizer 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 Iindex.
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.
The decision to select a stimulation index of 3 as an arbitrary indication of sensitizing activity was made on the basis of investigations performed with a wide range of chemicals. This criteria is valid for the positive control.

TEST SYSTEM
- Number of test groups: 4
- Number of control (vehicle) group: 1

TREATMENT PROCEDURES
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 ~ 8 mm) of each ear lobe once daily for three consecutive days. The control group of four mice were treated with the vehicle alone. 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-methylthymidine
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, 250 µl of 82.43 µCi/ml 3HTdR (equal to 20.6 µCi 3HTdR) was intravenously administered to all mice via a tail vein.

Determination of incorporated 3H-methylthymidine
Approximately 5 hours after treatment with 3HTdR, 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 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 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 3HTdR incorporation was then measured on a β-scintillation counter. Similarly, background 3HTdR levels were also measured in two 1 ml-aliquots of 5 % trichloroacetic acid. The β-scintillation counter expresses 3HTdR incorporation as the number of radioactive disintegrations per minute (DPM).

OBSERVATIONS
In 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)
Positive control results:
No test item-related clinical signs were observed.
All treated animals survived the scheduled study period.
The Stimulation Indices of the positive control substance were 2.9, 2.6 and 7.1, at concentrations of 5 %, 10 % and 25 % (w/v) in acetone:olive oil, 4:1 (v/v) respectively. The positive control substance was found to be a non-sensitiser at 10 % (w/v) and showed allergenic potency at 25 % (w/v).
The EC3 of the test substance was 11.3 % (w/v), calculated from the stimulation indices 2.6 and 7.1, which resulted from testing at 20 % and 25 % (w/v) respectively.
Parameter:
SI
Remarks:
at 2.5 %
Value:
2.6
Parameter:
SI
Remarks:
at 5 %
Value:
2.2
Parameter:
SI
Remarks:
at 10 %
Value:
2.1

MAIN STUDY RESULTS

An exact EC3 value could not be calculated because the calculation requires data laying immediately above and below S.I. value of 3.

Calculations and results of individual data (main study)

Test item conc. % (w/v) Measurement dpm Calculation Stimulation Index
dpm Background* Number of lymph nodes dpm per lymph nodes
Background I 0
Background II 0
Control Group 1 1230 1230 8 154
2.5 Test group 2 3240 3240 8 405 2.6
5 Test group 3 2720 2720 8 340 2.2
10 Test group 4 2547 2547 8 318 2.1

Background was 1 ml of 5 % trichloroacetic acid in duplicate

*The mean value was taken from the figures background I and background II

** Since the lymph nodes of the animals of a dose group were pooled, DPM per node was determined by dividing the measured value by the number of lymph nodes pooled

Mortality / Viability: 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.

Body weights at the start of acclimatization (day 1) and prior to necropsy (day 6)

Group Animal Weight day 1 (g) Weight day 6 (g) Mean day 1 (g) Standard deviation day 1 Mean day 6 (g) Standard deviation day 6
Control 1 20.1 20.4 19.4 1.4 20.5 1.4
2 21.1 20.1
3 18.3 22.5
4 18.1 19.1
2.50% 5 18.7 19.6 18.9 0.9 22 1.6
6 18.0 22.9
7 18.9 22.9
8 20.1 22.6
5% 9 19.4 21.6 20.1 0.9 22.2 0.6
10 19.3 22.8
11 20.5 21.9
12 21.1 22.6
10% 13 18.0 20.2 19.5 1.3 22 2
14 19.2 24.3
15 21.0 20.5
16 19.9 22.9

POSITIVE CONTROL RESULTS - ALPHA-HEXYLCINNAMALDEHYDE

Calculations and results of individual data (positive control study)

Test item conc. % (w/v) Measurement DPM* Calculation Stimulation Index
dpm Background Number of lymph nodes dpm per lymph node (y)
Background I 3
Background II 3
Control Group 1 4232 4229 8 529
5 Test group 2 12169 12166 8 1521 2.9
10 (a) Test group 3 10977 10974 8 1372 2.6
25 (c) Test group 4 29853 29853 8 3732 7.1

Background was 1 ml of 5 % trichloroacetic acid in duplicate

*The mean value was taken from the figures background I and background II

**Since the lymph nodes of the animals of a dose group were pooled, DPM per node was determined by dividing the measured value by the number of lymph nodes pooled

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

In order to study a possible allergenic potential of the test substance three groups of four female mice were 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 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 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.).

The S.I. resulted to be 2.6, 2.2 and 2.1 in the groups treated at the test item concentration of 2.5, 5 and 10 %, respectively.

An exact EC3 value could not be calculated because the calculation requires data laying immediately above and below S.I. value of 3.

Conclusion

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

The EC3 value could not be calculated because the calculation requires data laying immediately above and below S.I. value of 3, thus the substance does not meet the criteria to be classified as skin sensitizer, according to the CLP Regulation (EC 1272/2008).

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

There are no information about the skin sensitisation potential of Basic Blue 140, thus the available data on structural analogues Similar Substance 03 and Similar Substance 01 have been taken into account. The read across approach can be considered as reliable and suitable for the purpose; details and explanations are detailed in the report attached to the IUCLID section 13.

Similar Substance 03 has been investigate in both LLNA and GPMT assays.

The LLNA experiment was conducted according to the OECD guideline 429; three groups of four female mice were each treated with the test item in ethanol:water. No test item-related clinical signs were observed. All treated animals survived the scheduled study period. The Stimulation Index (S.I.) resulted to be 2.6, 2.2 and 2.1 in the groups treated at the test item concentration of 2.5, 5 and 10 %, respectively. The EC3 value could not be calculated because the calculation requires data laying immediately above and below S.I. value of 3, thus the substance does not meet the criteria to be classified as skin sensitizer, according to the CLP Regulation (EC 1272/2008).

The Similar Substance 03 was also investigated using the Maximization-Test of Magnusson and Kligman (1969). Ten animals (5 males, 5 females) were treated with the vehicle alone (petrolatum oil and distilled water) and twenty animals (10 males, 10 females) were treated with the test article. No positive reactions were evident after the first and second challenge application either on the area treated with vehicle only nor on the area treated with the substance, in the control group. No toxic symptoms were evident in the guinea pigs of either the control nor test group and no death occurred. According to the results described the allergenic potency of the test article is considered to be of a weak to mild grade when followed the rating of allergenicity; however, according to the CLP Regulation (EC 1272/2008), the substance does not meet the criteria to be classified as a sensitizer.

Similar Substance 01 was investigated by the modified Local Lymph Node Assay (IMDS), involving female NMRI mice to determine if there is any specific (sensitizing) or non-specific (irritant) stimulating potential of the test item. The study does neither point to a non-specific (irritant) nor to a specific immunostimulating (sensitizing) potential of the test item. This applies to NMRI mice, for weight and cell count indices of the draining lymph nodes as well as ear swelling and ear weight indices evaluated after application of test item. Compared to vehicle-treated animals, none of the parameters measured in the substance treated groups, i.e. cell counts and weights of the draining lymph nodes, ear weights and ear swelling, reached or exceeded the "positive levels" defined for this assay. These results show that there is no indication for a skin sensitizing effect after administration of a concentration up to and including 30 % test item in this test system. In conclusion, these results show that the test item has no sensitizing potential in mice after dermal application of up to and including a 30 % concentration. No indication for a non-specific (irritant) activation was detected, too. These results are verified by the comparison with the results of the positive control group (Alpha Hexyl Cinnamic Aldehyde).

BBl140, Similar Substanc e 03 and Similar Substance 01 share the same phthalocyaninic scaffold structure.

BBl140 and Similar Substance 01 present a zwitterionic moiety formed by sulphonic acid, which conducts an acid function, and a tertiary amine on the sulphonaminic chain, characterized by a basic function. BBl140 presents a further sulphonaminic functionalization, of which chain ends with a tertiary amine that, based on the stechiometrical ratio reported in the analytical characterization, in most of the cases can be salified with acetic acid.

Similar Substance 03 is functionalized by sulphonamine and two sulphonic acids, salificated by sodium.

Structural differences are expected to not significantly impact the skin sensitisation potential. The read across approach has been further detailed in the report attached to the IUCLID section 13.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

According to the CLP Regulation (EC 1272/2008), 3.4.2.2 skin sensitisation section, skin sensitizer means a substance that will lead to an allergic response following skin contact. Effects seen in either humans or animals will normally justify classification in a weight of evidence approach for skin sensitizers.

The test results on the analogous substance show that the test item has no sensitizing potential in mice after dermal application of up to and including a 30 % concentration. No indication for a non-specific (irritant) activation was detected, too.

In conclusion, the substance under registration is expected to be non skin sensitising.