Registration Dossier

Administrative data

Description of key information

Not skin sensitising

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Referenceopen allclose all

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:
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
The usage of information on Direct Blue 199_Na, which has the same main component and with a different counter ion, can be considered as suitable and appropriated because the difference in salification is expected to not influence the characteristics related to the specific end-point.
The impurity profile does not impact on the read across proposed. Details on the approach followed are included in the document attached to the IUCLID section 13.
Reason / purpose:
read-across source
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
adopted 24 June 2002
Qualifier:
according to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Version / remarks:
adopted by the Council on July 17, 1992
GLP compliance:
yes (incl. certificate)
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
Value:
2.6
Variability:
no deaths occurred
Test group / Remarks:
at 2.5 %
Parameter:
SI
Value:
2.2
Variability:
no deaths occurred
Test group / Remarks:
at 5 %
Parameter:
SI
Value:
2.1
Variability:
no deaths occurred
Test group / Remarks:
at 10 %

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:
skin sensitisation: in vivo (non-LLNA)
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
From March 6th to April 20th, 1989
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
The usage of information on Direct Blue 199_Na, which has the same main component and with a different counter ion, can be considered as suitable and appropriated because the difference in salification is expected to not influence the characteristics related to the specific end-point.
The impurity profile does not impact on the read across proposed. Details on the approach followed are included in the document attached to the IUCLID section 13.
Reason / purpose:
read-across source
Qualifier:
according to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Version / remarks:
aqdopted May 12, 1981
Qualifier:
according to
Guideline:
EU Method B.6 (Skin Sensitisation)
GLP compliance:
yes
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
Previously conducted, experiment already existing: LLNA study was also been conducted.
Species:
guinea pig
Strain:
Dunkin-Hartley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Kleintierfarm Madoerin AG.
- Age at study initiation: 7 weeks (males), 8 weeks (females).
- Weight at study initiation: 299 - 359 g (males), 288 - 321 females.
- Housing:individually in Makrolon type-3 cages with standard softwood bedding ("Lignocel", Schill AG, 4132 Muttenz, Switzerland).
- Diet: pelleted standard Kliba 342, Batch 50/89 and 51/89 guinea pig breeding/ maintenance diet ("Kliba", Klingentalmuehle AG, 4303 Kaiseraugst, Switzerland) ad libitum.
- Water: community tap water from Itingen, ad libitum.
- Acclimation period: one week under test conditions after veterinary examination.

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 3 °C
- Humidity: 40 - 70 %
- Air changes: 10 - 15 air changes per hour.
- Photoperiod: 12 hours artificial fluorescent light/12 hours dark.
- Other: music/light period.
Route:
intradermal
Vehicle:
water
Remarks:
oil
Concentration / amount:
1 %
Route:
epicutaneous, occlusive
Vehicle:
petrolatum
Remarks:
oil
Concentration / amount:
10 %
Day(s)/duration:
One week after the injections; duration: approximately 48 hours
No.:
#1
Route:
epicutaneous, occlusive
Vehicle:
petrolatum
Remarks:
oil
Concentration / amount:
5 %
Day(s)/duration:
Two weeks after the epidermal induction; duration: approximately 24 hours
Adequacy of challenge:
other: non-irritant concentration
No.:
#2
Route:
epicutaneous, occlusive
Vehicle:
petrolatum
Remarks:
oil
Concentration / amount:
5 %
Day(s)/duration:
Two weeks after first challenge; duration: approximately 24 hours
Adequacy of challenge:
other: non-irritant concentration
No. of animals per dose:
10 males and 10 females in the test groups; 5 males and 5 females in the negative control group
Details on study design:
RANGE FINDING TESTS
- Purpose: identify irritant test substance concentrations suitable concentrations for the induction phase of the main study. In addition, a suitable non-irritant concentration of the test substance, by the topical route of administration, was identified for the challenge application.
- Intradermal injections: intradermal injections (0.1 ml/site) were made into the clipped flank of two guinea-pigs at concentrations of 1 %, 3 % and 5 % of the test substance in distilled water. The resulting dermal reactions were assessed 24 hours later.
- Epidermal applications: patches of filter paper (2 × 2 cm) were saturated with concentrations of 3 %, 5 %, 10 % and 25 % of the test substance in petrolatum oil and applied to the clipped and shaved flanks of each of four guinea-pigs. The patches were covered by a strip of aluminium foil and firmly secured by elastic paper wound round the trunk and covered with impervious adhesive tape. This procedure ensured the intensive contact of the test substance. The dressings were removed after an exposure period of 24 hours and the reaction sites were assessed for erythema and edema. Further examination of the sites were performed 24 and 48 hours after removal of the dressings.

MAIN STUDY
INDUCTION EXPOSURE - Intradermal injection
An area of dorsal skin from the scapular region (approximately 6 × 8 cm) was clipped free of hair. Three pairs of intradermal injections (0.1 ml/site) were made at the border of a 4 × 6 cm area in the clipped region as follows:
(1) Freunds' complete adjuvant 50:50 with distilled water for injection
(2) the test article, diluted to 1 % with distilled water.
(3) the test article at the concentration used in (2) above, emulsified in a 50:50 mixture of Freunds' complete adjuvant, and the vehicle used in (2).
The control group was treated in the same way, with omission of the test article.

INDUCTION EXPOSURE - Epidermal applications
One week after injections, the scapular area (approximately 6 × 8 cm) was again clipped and shaved free of hair. A 2 × 4 cm patch of filter paper was again saturated with the test article (10 % in petrolatum oil) and placed over the injection sites of the test animals. The patch was covered by an aluminium foil and firmly secured by an elastic plaster wound round the trunk of the animal and secured with impervious adhesive tape. The dressings were left in place for approximately 48 hours. The epidermal application ensured intensive contact of the test article.
The control group was treated in the same way, with omission of the test article.
Reaction sites were assessed for erythema and edema immediately, 24 and 48 h after removal of the dressing.

CHALLENGE EXPOSURE - Challenge
The test and control guinea pigs were challenged two weeks after the epidermal induction application.
Hair was clipped and shaved from a 5 × 5 cm area on the left and right flank of each guinea-pig. Two patches (2 × 2 cm) of filter paper were saturated with the non-irritant concentration (5 % in petrolatum oil) of the test article and with the vehicle alone, applied to the left flank and right flank respectively, using the same method employed in epidermal application.
The dressings were removed approximately 24 hours later. The sites were addressed for erythema and edema immediately, 24 and 48 hours after removal.
The control group was treated in the same way as described above.

CHALLENGE EXPOSURE - Re-challenge
A second challenge was performed two weeks after the first challenge. The method for the animals of the test article-treated group was equal to that described for the first challenge.
Control animals were treated with the vehicle alone on the left flank.

OBSERVATIONS
In addition to the sensitizing reactions the following observations and data were recorded during the test and observation period:
- Mortality/Viability: Once daily.
- Body Weights: at acclimatization start, start of application and end of test.
- Symptoms (local/ systemic): daily.
- Necropsy: no necropsy was performed in the animals killed at termination of observation.
- Sacrifice: all animals were killed at the end of the test period with an intraperitoneal injection of T61 (Hoechst AG) and discarded.

INTERPRETATION
The results evident in test animals at the challenge application(s) were compared with the results evident in control animals. An allergic reaction was defined by visible reddening of the challenge site.
The significance of difference in the number of animals with positive reactions between the treated group and the control group was assessed by the Fisher Test.
If the dermal reactions of test animals at challenge were more persistent or marked than those of the control animals, the animals were considered to show evidence of contact hypersensitivity.
If the dermal reactions of test animals at challenge were not clearly different from the reactions in control group animals, the results for the test animals were considered "inconclusive".
If the dermal reactions to the challenge application were identical to or less persistent/marked than the dermal reactions in control animals, the animal was considered to show no evidence of contact hypersensitivity.

INTERPRETATION - Reading of challenge reactions
The challenge site was evaluated immediately, 24 and 48 hours after removal of the patch. The readings were made under artificial fluorescent light (daylight spectrum).
Redness constitutes the minimum criterion of an allergic reaction. Strongly sensitized animals displayed a vivid redness, associated with indurated swelling. The reactions were scored on the basis of the Draize score.

INTERPRETATION - Rating of allergenicity
Based upon the percentage of animals sensitized (24-hour reading), the test article was assigned to one of the following five grades of allergenic potency, ranging from weak to extreme.
Grade 1, classificazion as week, sensitisation rate of range 0 - 8 %
Grade 2, classificazion as mild, sensitisation rate of range 9 - 28 %
Grade 3, classificazion as moderate, sensitisation rate of range 29 - 64 %
Grade 4, classificazion as strong, sensitisation rate of range 65 - 80 %
Grade 5, classificazion as extreme, sensitisation rate of range 81 - 100 %

READINGS AND SCORING - Irritation (Draize score)
The following parameters were recorded:
Erythema (E) - 0 to 4 numerical scores
Edema (0) - 0 to 4 numerical scores
Diameter (D) - mm

Erythema and edema were assessed using the following numerical grading system:
Erythema and eschar formation:
No erythema 0
Slight erythema (barely perceptible) 1
Well-defined erythema 2
Moderate erythema 3
Severe erythema (beet redness) to slight eschar formation (injuries in depth) 4

Edema formation:
No edema 0
Slight edema (barely perceptible) 1
Well-defined edema (edges of area well-defined by definite raising) 2
Moderate edema (raised approximately 1 millimeter) 3
Severe edema (raised more than 1 millimeter and extending beyond the area of exposure) 4

STATISTICAL ANALYSIS
Mean values with standard deviations.
Fisher-Test (The Exact Fisher Test for comparison of the basic probability of two binominal distributions. L. Sachs, Statistische Auswertungsmethoden, Georg Thieme Verlag, Stuttgart 1971).
For calculation of p-values the 24 hour readings of the animals from the control and test article-treated groups were used.
Positive control substance(s):
yes
Remarks:
dinitro-chloro-benzene
Positive control results:
67 % animal response. 0.5 % of the positive control substance in ethanol was used for the induction phase and 0.3 % of the positive control substance in ethanol was used for the challenge phase.
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
5 % in petrolatum oil
No. with + reactions:
2
Total no. in group:
20
Clinical observations:
No toxic symptoms were evident.
Remarks on result:
no indication of skin sensitisation
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
5 % in petrolatum oil
No. with + reactions:
2
Total no. in group:
20
Clinical observations:
No toxic symptoms were evident.
Remarks on result:
no indication of skin sensitisation

MAIN STUDY RESULTS

after 24 hrs after 48 hrs
positive/total % positive of total positive/total % positive of total
ERYTHEMA REACTION AFTER FIRST CHALLENGE PROCEDURE
Control group - left flank TS 0/10 0 0/10 0
Control group - right flank vehicle only 0/10 0 0/10 0
Test group - left flank TS 2/20 10 2/20 10
Test group - right flank vehicle only 0/20 0 0/20 0
ERYTHEMA REACTION AFTER SECOND CHALLENGE PROCEDURE
Control group - left flank vehicle only 0/10 0 0/10 0
Test group - right flank TS 1/20 5 0/20 0
Test group - left flank vehicle only 0/20 0 0/20 0

First Challenge: no positive reactions were observed after the 24 and 48 hours reading on the area treated only with vehicle; in the area trated with the test substance two positive reactions were observed after the 24 and 48 hours reading.

Second Challenge: one positive reaction was observed after the 24 hours reading on the flank treated with the test substance; no positive reactions were observed after the 24 and 48 hours reading on the flank treated only with vehicle.

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.

VIABILITY / MORTALITY

No death occurred.

SYMPTOMS, LOCAL - Vehicle control

Application area around the injection sites 1 and 3 were found to show erythema and edema from day 2 to 4 respectively 5. Necroses were observed from day 6 to 8 and from day 10 to 17. Exfoliation was observed from day 18 to 39 (termination of test). Scales were observed at day 5 of test.

Epidermal application: additionally discoloration was observed from day 23 to 26 of test.

SYMPTOMS, LOCAL - Test article

Application area around the injection site 1 was found to show erythema at day 2 to 4 and edema from day 2 to 5. Necroses were observed from day 6 to 8 and from day 10 to 17. Exfoliation was observed from day 18 to 39 (termination of test). Scales were observed at day 5 of test.

Application area around the injection sites 2 and 3 were found to show discoloration and edema from day 2 to 4 respectively 5. Necroses were observed from day 5 to 8 and from day 10 to 17. Exfoliation was observed from day 18 to 39 (temination of test).

Epidermal application: additionally discoloration was observed from day 10 to 18, 23 to 26 and day 37 to 39.

At day 9 of test no observation could be performed because the animals were treated semi-occlusively.

SYMPTOMS, SYSTEMIC

No systemic symptoms were observed.

BODY WEIGHTS

The body weight gain of all animals was not affected during the test.

PRETEST RESULTS

Intradermal induction - vehicle: distilled water

Animal no. Sex Concentration Reaction readings after 24 hours
% Erythema Oedema Diameter
321 M 5 -- 2 --
322 F 5 -- 2 --
321 M 3 -- 2 --
322 F 3 -- 1 --
321 M 1 -- 1 --
322 F 1 -- 1 --

According to Magnusson – Kligman and to the findings observed, the concentration selected for the main study was 1 %.

Application area blue discoloured, therefore, a possible erythema could not be observed.

Epidermal induction - vehicle: petrolatum oil

Animal no. Sex Concentration % Reaction readings after removal of bandage
Immediately After 24 hours After 48 hours
Erythema Oedema Erythema Oedema Erythema Oedema
323 M 25 1 0 1 0 0 0
10 1 0 0 0 0 0
5 0 0 0 0 0 0
3 0 0 0 0 0 0
324 F 25 0 0 0 0 0 0
10 0 0 0 0 0 0
5 0 0 0 0 0 0
3 0 0 0 0 0 0
325 M 25 1 0 1 0 1 0
10 0 0 0 0 0 0
5 0 0 0 0 0 0
3 0 0 0 0 0 0
326 F 25 0 0 1 0 1 0
10 0 0 1 0 1 0
5 0 0 0 0 0 0
3 0 0 0 0 0 0

According to Magnusson - Kligman, and to the findings observed, the concentration selected for the induction period was 10 % and for the challenge procedure 5 %.

Prior to observation the skin of all animals was depilated to enable the observation of a possible erythema.

Interpretation of results:
other: not classified, according to the CLP Regulation (EC 1272/2008)
Conclusions:
Under the test conditions reported, after the first challenge, the erythema reaction of animals to the test substance was 10 % at 24 h and 10 % 48 h. After the second challenge, the erythema reaction of animals to the test substance was 5 % at 24 h and 0 % at 48 h.
Therefore, the test substance is not a skin sensitizer.
Executive summary:

The purpose of the study was to assess the allergenic potential the test item when administered to the skin of male and female albino guinea pigs. For this purpose the Maximization-Test of B. Magnusson and A.M. Kligman (1969) was used. 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. Prior to the first reading of the reactions, the skin was depilated to clean the application site from staining produced by the test article, so that the reactions (erythema) were clearly visible at that time.

In the first challenge, no positive reactions were observed after the 24 and 48 hours reading on the area treated only with vehicle; in the area trated with the test substance two positive reactions were observed after the 24 and 48 hours reading. The second challenge showed one positive reaction after the 24 hours reading on the flank treated with the test substance; no positive reactions were observed after the 24 and 48 hours reading on the flank treated only with vehicle.

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. No death occurred.

Conclusion

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 described by Magnusson B. and Kligman A.M. (1969).

However, according to the CLP Regulation (EC 1272/2008), the substance does not meet the criteria to be classified as a sensitizer.

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

A possible allergenic potential of Direct Blue 199 was investigated using the already existing data on the Na form (i.e. Direct Blue 199_Na), both in Local Lymph Node Assay (LLNA) in Mice and in Albino Guinea Pigs Maximization Test (GPMT). The usage of information on Direct Blue 199_Na, which has the same main component and with a different counter ion, can be considered as suitable and appropriated because the difference in salification is expected to not influence the characteristics related to the specific end-point. The impurity profile does not impact on the read across proposed. Details on the approach followed are included in the document attached to the IUCLID section 13.

 

In the LLNA assay no test item-related clinical signs were observed. All treated animals survived the scheduled study period. 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.

 

In the GMPT during the first challenge, no positive reactions were observed after the 24 and 48 hours reading on the area treated only with vehicle; in the area treated with the test substance two positive reactions were observed after the 24 and 48 hours reading. The second challenge showed one positive reaction after the 24 hours reading on the flank treated with the test substance; no positive reactions were observed after the 24 and 48 hours reading on the flank treated only with vehicle. 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. No death occurred.

 

According to the outcomes from the two tests performed, the allergenic potency of the test article is considered to be low and, according to the CLP Regulation (EC 1272/2008), the substance does not meet the criteria to be classified as a sensitizer.

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.

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 %. Based on the Guinea pig maximisation test (GPMT) results, a substance in considered a skin sensitizer when: equal or more than 30 % to 60 % responding at intradermal induction dose > 0.1 % to ≤ 1 %; or equal or more than 30 % responding at intradermal induction dose > 1 %.

The LLNA assay failed to calculate an EC value higher than 2 %; furthermore, in the GPMT a response lower than 30 % was recorded.

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