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Toxicological information

Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
23-MAR-2005 To 06-JUL-2005
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
Data is from study report

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2005
Report Date:
2005

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Qualifier:
according to
Guideline:
other: Commission Directive 2004/73/EC, B.42: Skin Sensitization: Local Lymph Node Assay, 29 April 2004.
Principles of method if other than guideline:
The purpose of this Local Lymph Node Assay was to identify the contact allergenie potential of B 007 ([8-[(4-amino-3-nitrophenyl)azo]-7-hydroxy-2-naphthyl]trimethylammonium chloride) when administered to the dorsum of both ear lobes of mice.
GLP compliance:
yes
Type of study:
mouse local lymph node assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Test material form:
other: Dark brown powder
Details on test material:
- Name of test material (as cited in study report): Basic brown 17
- Molecular formula (if other than submission substance): C19H20N5O3.Cl
- Molecular weight (if other than submission substance): 401.852 g/mol
- Smiles notation (if other than submission substance):[N+](c1cc(\N=N\c2c3cc([N+](C)(C)C)ccc3ccc2O)ccc1N)([O])=O.[ClH]
- InChl (if other than submission substance): 1S/C19H19N5O3.ClH/c1-24(2,3)14-7-4-12-5-9-18(25)19(15(12)11-14)22-21-13-6-8-16(20)17(10-13)23(26)27;/h4-11H,1-3H3,(H2-,20,21,22,25);1H
- Structural formula attached as image file (if other than submission substance): No data available
- Substance type: Organic
- Physical state: Solid
- Analytical purity: No data available
- Impurities (identity and concentrations): No data available
- Composition of test material, percentage of components: No data available
- Isomers composition: No data available
- Purity test date: No data available
- Lot/batch No.: No data available
- Expiration date of the lot/batch: No data available
- Radiochemical purity (if radiolabelling): No data available
- Specific activity (if radiolabelling): No data available
- Locations of the label (if radiolabelling): No data available
- Expiration date of radiochemical substance (if radiolabelling): No data available
- Stability under test conditions: No data available
- Storage condition of test material: No data available
- Other: No data available
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Test Item Identity: B 007 ([8-[(4-amino-3-nitrophenyl)azo]-7-hydroxy-2-naphthyl]trimethylammonium chloride)
- Batch No.of test material: 64960101
- Expiration date of the lot/batch: 15-AUG-2024
- Purity : 95.6 area%

RADIOLABELLING INFORMATION
- Chemical: 3H-methyl Thymidine
- Source: Amersham Biosciences UK Limited. Buckinghamshire England HP7 9NA. UK
- Batch No.: 318
- Specific activity: 2 Ci/mmol; concentration, 1 mCi/ml)
- Storage conditions: In the original container at 5 °C ± 3°C.

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: In refrigerator (5°C ± 3°C), away from direct sunlight.
- Stability under test conditions: Stable under storage conditions.Stable during shipment without cooling.
- Solubility and stability of the test substance in the solvent/vehicle: No data
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: No data

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
The test item was placed into a volumetric flask on a tared Mettler balance and the vehicle (ethanol/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 du ring treatment with the magnetic stirrer.
The test item in the study was assayed at five consecutive concentrations.
Concentrations were in terms of material as supplied.

OTHER SPECIFICS:Safety precautions : Routine hygienic procedures (gloves, goggles, face mask).

In vivo test system

Test animals

Species:
mouse
Strain:
other: CBA/CaHsdRcc (SPF)
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: RCC Ltd CH-4414 Füllinsdorf / Switzerland.
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 8 - 12 weeks (beginning of acclimatization).
- Weight at study initiation: 16 g - 24 g (ordered).
- Identification: Each cage by unique cage card.
- Housing: Individual in Makroion type-2 cages with standard softwood bedding.
- Diet (e.g. ad libitum):Pelleted standard Kliba 3433, batch nos. 94/04 (for Groups 1-4) and 25/05 (tor Groups 5-7) mouse maintenance diet (Provimi Kliba AG, CH-4303 Kaiseraugst) available ad Iibitum.
- Water (e.g. ad libitum): Community tap water from Itingen, available ad libitum.
- Acclimation period: Under test conditions after health examination. Only animals without any visible signs of iIIness were used for the study.
- Indication of any skin lesions: No data

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3°C.
- Humidity (%): 30 - 70 %
- Air changes (per hr): 10 - 15 air changes per hour.
- Photoperiod (hrs dark / hrs light): 12 hour fluorescent light / 12 hour dark cycle with at least 8 hours music during the light period.
- IN-LIFE DATES: From: To:

Study design: in vivo (LLNA)

Vehicle:
other: ethanol/water (7/3, v/v)
Concentration:
Test 1
1 (Control Group') - 0
2 - 0.2%
3 - 0.5%
4 - 1%

Test 2
5 (Control Group') - 0
6 - 3%
7 - 6%
No. of animals per dose:
Test 1
1 (Control Group') - 4 animals
2 - 4 animals
3 - 4 animals
4 - 4 animals

Test 2
5 (Control Group') - 4 animals
6 - 4 animals
7 - 4 animals
Details on study design:
TREATMENT PREPARATION AND ADMINISTRATION:
PRE-TEST
In a non-GlP conform solubility pre-test, the test item was assayed at concentrations of 3 % and 6 % (w/v). 6 % (w/v) was the highest technically applicable concentration in the chosen vehicle.

TREATMENT PROCEDURES
TOPICAL APPLICATION
Each test group of mice was treated by topical (epidermal) application to the dorsal surface of each ear lobe (Ieft and right) with the test item at a concentration of 0.2 %, 0.5 %, 1 %, 3 % and 6 % (w/v) in ethanol/water (7/3, v/v). The application volume, 25 µl, was spread over the entire dorsal surface (0 - 8 mm) of each ear lobe once daily for three consecutive days.Two control groups each of four mice were treated with an equivalent volume of the relevant vehicle alone (control animals). A hair dryer was passed briefly over the ear's surface to prevent the loss of any of the test item applied.

ADMINISTRATION OF 3H-METHYL THYMIDINE"
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, all mice were administered 250 µl of 81.63 (for Groups 1-4) or 80.85 (for Groups 5-7) µCi/mI 3HTdR (equal to 20.4 (for Groups 1-4) or 20.2 (for Groups 5-7) 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 Iymph nodes were rapidly excised and pooled for each experimental group (8 nodes per group with the exception of Group 1, in which only 7 nodes were excised).
Single cell suspensions (phosphate buffered saline) of pooled Iymph 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 Iymph 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 'Irga-Safe Plus' scintillation liquid and thoroughly mixed.The level of 3HTdR incorporation was then measured on a ß-scintillation counter. Similarly,background 3HTdR levels were also measured in two 1 ml-aliquots of 5 % trichloroacetic acid .
The ß-scintillation counter expresses 3HTdR incorporation as the number of radioactive disintegrations per minute (dpm).
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.

Results and discussion

Positive control results:
RESULTS
CALCULATION AND RESULTS OF INDIVIDUAL DATA
The proliferative capacity of pooled Iymph node cells was determined by the incorporation of 3H-methyl thymidine measured on a ß-scintillation counter.

Test Item concentratlon % (w/v) S.I.
Group 2 5* 2.4*
Group 3 10* 3.6 *
Group 4 25 11.2
EC3 = 7.5 % (w/v)
A clear dose-response relationship was observed.
* This value was used in calculation of EC3.

VIABILITY / MORTALITY
No deaths occurred during the study period.

CLINICAL SIGNS
No clinical signs were observed in any animals of the control group. On the second application day, a slight ear swelling was observed at both dosing sites in all mice of Group 4 (25 %), persisting for the remainder of the in-life phase of the study. One day after the third local application, a slight ear swelling was observed at both dosing sites in all mice of Group 2 (5 %) and Group 3 (10 %), persisting for the remainder of the in-life phase of the study.

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.

CONCLUSION
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 this study STIMULATION INDICES of 2.4, 3.6 and 11.2 were determined with the test item at concentrations of 5 %, 10 % and 25 % (w/v), respectively, in acetone:olive oil, 4:1 (viv).ALPHA-HEXYLCINNAMALDEHYDE was therefore found to be a skin sensitizer and an EC3 value of 7.5 % (w/v) was derived.

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Value:
1
Test group / Remarks:
Test 1: Group2: 0.2 % (w/v) in ethanol/water (7/3, v/v).
Parameter:
SI
Value:
1
Test group / Remarks:
Test 1: Group3: 0.5 % (w/v) in ethanol/water (7/3, v/v).
Parameter:
SI
Value:
1.3
Test group / Remarks:
Test 1: Group4: 1 % (w/v) in ethanol/water (7/3, v/v).
Parameter:
SI
Value:
0.9
Test group / Remarks:
Test 2: Group6: 3 % (w/v) in ethanol/water (7/3, v/v).
Parameter:
SI
Value:
1.3
Test group / Remarks:
Test 2: Group7: 6 % (w/v) in ethanol/water (7/3, v/v).
Cellular proliferation data / Observations:
CELLULAR PROLIFERATION DATA : The proliferative capacity of pooled Iymph node cells was determined by the incorporation of 3H-methyl thymidine measured on a ß·scintillation counter.

CLINICAL OBSERVATIONS: No clinical signs were observed in any animals of the control groups, Group 2 (0.2 %), Group
3 (0.5 %) or Group 4 (1 %). On the third application day, a slight erythema was observed at
both dosing sites in all mice of Group 6 (3 %). On the second application day, a moderate or
slight erythema was observed at both dosing sites in all mice of Group 7 (6 %), persisting for
the remainder of the in·life phase of the study.

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.

VIABILITY/ MORTALITY: No deaths occurred during the study period.

Any other information on results incl. tables

Positive Control Study

SUMMARY

In order to study a possible contact allergenic potential of ALPHAHEXYLCINNAMALDEHYDE,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 (vIv) by topical application to the dorsum of each ear lobe (Ieft and right) for three consecutive days.

A control group of four mice was treated with the vehicle (acetone:olive oil, 4:1 (vIv)) only.

Five days after the first topical application the mice were injected intravenously into a tail vein with radio-Iabelled thymidine (3H-methyl thymidine). Approximately five hours after intravenous injection, the mice were sacrificed, the draining auricular Iymph nodes excised and pooled per group. Single cell suspensions of Iymph node cells were prepared from pooled Iymph nodes which were subsequently washed and incubated with trichloroacetic acid overnight. The proliferative capacity of pooled Iymph node cells was determined by the incorporation of 3H-methyl thyrnidine measured in a ß-scintillation counter.

All treated animals survived the scheduled study period.

No clinical signs were observed in any animals of the control group. On the second application day, a slight ear swelling was observed at both dosing sites in all mice of Group 4(25 %), persisting for the remainder of the in-life phase of the study. One day after the third local application, a slight ear swelling was observed at both dosing sites in all mice of Group 2 (5 %) and Group 3 (10 %), persisting for the remainder of the in-life phase of the study.

The results obtained (STIMULATION INDEX (S.I.)) are reported in the following table. The estimated concentration of test item required to produce a S.I. of 3 is referred to as the EC3 value.

 

Test Item concentratlon % (w/v)

S.I.

Group 2

5*

2.4*

Group 3

10*

3.6 *

Group 4

25

11.2

EC3 = 7.5 % (w/v)

A clear dose-response relationship was observed.

* This value was used in calculation of EC3.

Applicant's summary and conclusion

Interpretation of results:
other: Not Sensitising
Remarks:
based on CLP criteria
Conclusions:
In this study STIMULATION INDICES of 1.0, 1.0, 1.3, 0.9 and 1.3 were determined with the test item at concentrations of 0.2 %, 0.5 %, 1 %, 3 % and 6 % (w/v) , respectively, in ethanoVwater (7/3, v/v).
The test item B 007 ([8-[(4-amino-3-nitrophenyl)azo]-7-hydroxy-2-naphthyl]trimethylammonium chloride) does not show an allergenic potential when tested up to the concentration of 6 % (w/v) in ethanol/water (7/3, v/v).Thus the test item B 007 ([8-[(4-amino-3-nitrophenyl)azo]-7-hydroxy-2-naphthyl]trimethylammonium chloride) is considered to be a non-sensitiser.
Executive summary:

In order to study a possible contact allergenic potential of B 007 ([8-[(4-amino-3-nitrophenyl)azo]-7-hydroxy-2-naphthyl]trimethylammonium chloride), five groups each of four female mice were treated daily with the test item at concentrations of 0.2 %, 0.5 %, 1 %, 3 % and 6 % (w/v) in ethanol/water(7/3,v/v) by topical application to the dorsum of each ear lobe (Ieft and right) for three consecutive days. 6 % was the highest technically applicable concentration in the vehicle. Two control groups each of four mice were 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 Iymph nodes excised and pooled per group. Single cell suspensions of Iymph node cells were prepared from pooled Iymph nodes which were subsequently washed and incubated with trichloroacetic acid overnight. The proliferative capacity of pooled Iymph node cells was determi ned by the incorporation of 3H-methyl thymidine measured in a ßscintillation counter.

All treated animals survived the scheduled study period .

No clinical signs were observed in any animals of the control groups, Group 2 (0.2 %), Group 3 (0.5 %) or Group 4 (1 %). On the third application day, a slight erythema was observed at both dosing sites in all mice of Group 6 (3 %). Since the second application day, a moderate or slight erythema was observed at both dosing sites in all mice of Group 7 (6 %), persisting for the remainder of the in-Iife phase of the study.

The results obtained (STIMULATION INDEX (S.I.)) are reported in the following table.

Test 1

Test Item concentratlon

% (w/v)

S.I.

Group2

0.2

1.0

Group3

0.5

1.0

Group4

1

1.3

 

Test2

Test Item concentratlon

% (w/v)

S.I.

Group 6

3

0.9

Group 7

6

1.3

 

No constant dose - dpm/LN level (indicated by S.I. values) relationship was observed in the study. The variations of S.I. values obtained and reported in the above table were within a narrow range. As no test item related findings, such as significant body weight loss or local/systemic findings, were observed up to the concentration of 1 %, this indicated that the test item does not show an allergenie potential at lower test concentrations. At the higher concentrations tested, i.e. 3 % and 6 %, some test item related signs, such as slight to moderate ear erythema, were observed at the local dosing sites but no clear change of dpm/LN was caused by this local irritant effect. This demonstrates that the test item caused skin irritation but does not show an allergenic potential at lower test concentrations.

Thus the test item B 007 ([8-[(4-amino-3-nitrophenyl)azo]-7-hydroxy-2-naphthyl]trimethylammonium chloride) is considered to be a non-sensitiser.