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Description of key information

The skin sensitization potential of target chemical 3-[(4,5-dihydro-3-methyl-5-oxo-1-phenyl-1H-pyrazol-4- yl)azo]-N,N,N-trimethylanilinium chloride/Basic Yellow 57 (CAS No. 68391-31-1) was assessedin various LLNA and Non-LLNA experimental studies which were conducted.Based on the available key data and supporting studies,it can be concluded thatchemical is unable to cause skin sensitization and considered as not sensitizing. Comparing the above annotations with the criteria of CLP regulation, it can be classified under the category “Not Classified”.

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:
12-May-2004 to 08 July 2004
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Principles of method if other than guideline:
The purpose of this Local Lymph Node Assay was to identify the contact allergenic potential of C 010 (3-[(4,5-dihydro-3-methyl-5-oxo-1-phenyl-1H-pyrazol-4-yl)azo]-N,N,N- trim ethylanilinium chloride) when administered to the dorsum of both ear lobes of mice.
GLP compliance:
yes
Type of study:
mouse local lymph node assay (LLNA)
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Test Item Identity: C 010 (3-[(4,5-dihydro-3-methyl-5-oxo-1-phenyl-1H-pyrazol-4-yl)azo]-N,N,N-trimethylanilinium chloride)
- Lot/batch No.of test material: 15
- Expiration date of the lot/batch: 30-DEC-2023
- Purity: 99.3% HPLC

RADIOLABELLING INFORMATION
- Chemical: 3H-methyl Thymidine
- Source: Amersham Biosciences UK Limited. Buckinghamshire England HP7 9NA. UK
- Batch No.: 314
- Specific activity:Amersham TRA 310, aqueous solution, sterilized 74 GBq/mmol (2 Ci/mmol), 37 MBq/ml (1 mCi/ml)
quantities: 9.25 MBq (250 )µCi), 37 MBq (1 mCi)
- Storage conditions: In the original container at room temperature (20°C ± 3°C), away from direct sunlight.

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: In the original container in refrigerator (5°C ± 3°C), avoid direct light.Stable during shipment without cooling.
- Stability under test conditions: Stable under storage conditions.
- 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 volumetrie flask on a tared Mettler balance and the vehicle (ethanol:water, 7:3 (v/v)) was quantitatively added. The weight/volume 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.

OTHER SPECIFICS:
Safety precautions: Routine hygienic procedures (gloves, goggles, face mask).
Species:
mouse
Strain:
other: CSAlCaOlaHsd
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Netherlands B.V. Postbus 6174 NL - 5960 AD Horst / The Netherlands
- Females (if applicable) nulliparous and non-pregnant: yes
- Microbiological status of animals, when known: No data
- Age at study initiation: 8 - 12 weeks (beginning of acclimatization)
- Weight at study initiation: 16 g - 24 g
- Identification: Each cage by unique cage card.
- Housing: Individual in Makroion type-2 cages with standard softwood bedding ("Lignocel", Schill AG, CH-4132 Muttenz).
- Diet (e.g. ad libitum): Pelleted standard Kliba 3433, batch no. 4/04 mouse maintenance diet (Provimi Kliba AG,CH-4303 Kaiseraugst) available ad libitum.
- Water (e.g. ad libitum): Community tap water from Itingen, available 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.
- 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 atleast 8 hours music during the light period.

- IN-LIFE DATES: From: 12-MAY-2004 To:26-MAY -2004
Vehicle:
other: ethanol:water, 7:3 (v/v)
Concentration:
1 (Control Group') - 0
2 - 2.5% (w/v)
3 - 5% (w/v)
4 - 10% (w/v)

No. of animals per dose:
1 (Control Group') - 4 animals
2 - 4 animals
3 - 4 animals
4 - 4 animals

Details on study design:
PRE-SCREEN TESTS:
In a non-GLP conform solubility pre-test, the test item was tested in different vehicles: ethanol:water, 7:3 (v/v) and acetone:olive oil, 4: 1 (v/v). A suitable vehicle (ethanol:water, 7:3 (v/v)) was selected and used in the main test.
In a non-GLP conform pre-test in two mice, the test item was tested at four different concentrations: 1 %,2.5 %,5 % and 10 % (w/v) , on one ear each.
24 hours after a single topical application, the pre-test results determined that 10 % (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 (left and right) with different test item concentrations of 2.5 %, 5 % and 10 % (w/v) in ethanol:water, 7:3 (v/v). The application volume, 25 µI, 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 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 with 250 µI of 83.0 µCi/ml 3HTdR (equal to 20.8 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). 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 two times 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 'Ultima Gold' 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 wejght tables.
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* 1.5
Group 3 10* 2.3 *
Group 4 25 * 8.4 *
EC3 = 11.7 % (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, Group 2 (5 %) or Group 3 (10 %). Approximately 90 minutes after the first topical applacation, a slight ear erythema was observed at both dosing sites in all mice of Group 4 (25 %).

BODY WEIGHTS
The body weight of the animals, recorded prior to the 1st application and prior to necropsy, was within the range commonly recorded for animals of this strain and age.

CONCLUSION
In this study STIMULATION INDICES of 1.5, 2.3 and 8.4 were determined with the test item at concentrations of 5 %, 10 % and 25 % (w/v) , respectively, in acetone:olive oil, 4: 1 (v/v).
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.).
The test item ALPHA-HEXYLCINNAMALDEHYDE was found to be a skin sensitizer and an EC3 value of 11.7 % (w/v) was derived.
Parameter:
SI
Value:
1.2
Test group / Remarks:
Group 2: 2.5 % (w/v)
Parameter:
SI
Value:
1.5
Test group / Remarks:
Group 3: 5% (w/v)
Parameter:
SI
Value:
1.5
Test group / Remarks:
Group 4: 10 % (w/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.

Test Item concentratlon % (w/v) S.I.
Group 2 2.5 1.2
Group 3 5 1.5
Group 4 10 1.5

No clear dose-response relation was observed.
Calculation of the EC3 value was not done because no test concentrations produced a STIMULATION INDEX (S.I.) of 3 or higher.

EC3 CALCULATION
Calculation of the EC3 value was not done because no test concentrations produced a STIMULATION INDEX (S.I.) of 3 or higher.

CLINICAL OBSERVATIONS: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 on the test day 1 (prior to the 1st application) and on the test day 6, was within the range commonly recorded for animals of this strain and age.

VIABILITY / MORTALITY
No deaths occurred during the study period.

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 (v/v) 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 (v/v)) 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 thymidine measured in a ß-scintillation counter.

No clinical signs were observed in any animals of the control group, Group 2 (5 %) or Group 3 (10 %). Approximately 90 minutes after the first topical applacation, a slight ear erythema was observed at both dosing sites in all mice of Group 4 (25 %).

All treated animals survived the scheduled study period.

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 concentration

% (w/v)

S.I.

Group 2

5

1.5

Group 3

10*

2.3 *

Group 4

25 *

8.4 *

EC3 = 11.7 % (w/v)

A clear dose-response relation was observed.

• This value was used in calculation of EC3.

Interpretation of results:
other: Not Sensitising
Remarks:
based on CLP criteria
Conclusions:
In this study STIMULATION INDICES of 1.2, 1.5 and 1.5 were determined with the test item at concentrations of 2.5 %, 5 % and 10% (w/v) , respectively, in ethanol:water, 7:3 (v/v).
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.).
The test item C 010 (3-[(4,5-dihydro-3-methyl-5-oxo-1-phenyl-1H-pyrazol-4-yl)azo]-N,N,N-trimethylanilinium chloride) was found to be a non-sensitizer when tested at up to the highest applicable concentration of 10% (wlv) in ethanol:water, 7:3 (v/v).
Executive summary:

In order to study a possible contact allergenic potential of C 010 (3-[(4,5-dihydro-3-methyl-5-oxo-1-phenyl-1H-pyrazol-4-yl)azo]- N,N,N-trimethyl anilinium chloride), three groups each of four female mice were treated daily with the test item at concentrations of 2.5 %, 5 % and 10% (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. 10% 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-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 thymidine measured in a ß-scintillation counter.

To avoid loss of the test item applied to the outer ear surface the test item was carefully dosed. To avoid missing of the test item from the ears a hair dryer was used to immediately dry the wet ears.

All treated animals survived the scheduled study period.

No clinical signs were observed.

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

 

Test item concentration

S.I.

Group 2

2.5

1.2

Group 3

5

1.5

Group 4

10

1.5

No clear dose-response relation was observed.

 

Calculation of the EC 3 value was not done because no test concentrations produced a STIMULATION INDEX (S.I.) of 3 or higher.

 

In this study STIMULATION INDICES of 1.2, 1.5 and 1.5 were determined with the test item at concentrations of 2.5 %, 5 % and 10% (wlv) , respectively, in ethanol:water, 7:3 (v/v).

Thus, the test item C 010 (3-[(4,5-dihydro-3-methyl-5-oxo-1-phenyl-1H-pyrazol-4-yl)azo]-N,N,N-trimethylanilinium chloride) was found to be a non-sensitizer when tested at up to the highest applicable concentration of 10% (wlv) in ethanol:water, 7:3 (v/v).

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

Skin sensitization

Various studieshas been investigated for the test chemical3-[(4,5-dihydro-3-methyl-5-oxo-1-phenyl-1H-pyrazol-4- yl)azo]-N,N,N-trimethylanilinium chloride/Basic Yellow 57 (CAS No. 68391-31-1)to observe the potential for skin sensitization to a greater or lesser extent. The studies are based on in vivo LLNA and Non-LLNA experiments for target chemical which have been summarized as below;

 

TheMouse Lymph node assay [LLNA] was performed for test chemical in order to study a possible contact allergenic potential of C 010 (3-[(4,5-dihydro-3-methyl-5-oxo-1-phenyl-1H-pyrazol-4-yl)azo]- N,N,N-trimethyl anilinium chloride), three groups each of four female mice were treated daily with the test item at concentrations of 2.5 %, 5 % and 10% (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. 10% 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-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 thymidine measured in a ß-scintillation counter. To avoid loss of the test item applied to the outer ear surface the test item was carefully dosed. To avoid missing of the test item from the ears a hair dryer was used to immediately dry the wet ears. All treated animals survived the scheduled study period. No clinical signs were observed. No clear dose-response relation was observed. Calculation of the EC 3 value was not done because no test concentrations produced a STIMULATION INDEX (S.I.) of 3 or higher. In this study STIMULATION INDICES of 1.2, 1.5 and 1.5 were determined with the test item at concentrations of 2.5 %, 5 % and 10% (wlv) , respectively, in ethanol:water, 7:3 (v/v). Thus, the test item C 010 (3-[(4,5-dihydro-3-methyl-5-oxo-1-phenyl-1H-pyrazol-4-yl)azo]-N,N,N-trimethylanilinium chloride) was considered to be a non-sensitizer when tested at up to the highest applicable concentration of 10% (wlv) in ethanol:water, 7:3 (v/v).

 

Similar Mouse Lymph node assay [LLNA] study was conducted by another safety assessment report for test chemical. The assay was performed according to OECD 429 Guidelines. No dose response relation was noted in this study. The positive control used affected an increase in stimulation index with an EC3 of 11.7%.Calculation of the EC3 value was not performed as the S.I. value did not reach or exceed 3 for any test concentration. Based on the criteria of the test system, Basic Yellow 57 was not a non-sensitizer when tested up to 10% in ethanol:water (7:3 v/v) in mice.

 

The overall result was further supported by Non-LLNA skin sensitization study carried out on Hartley Dunkin guinea pigs to determine the sensitizing potential of Basic Yellow 57. The test was performed according to OECD 406 guidelines. Basic Yellow 57 was prepared as a 0.1% w/v solution in water (injection 1). Freund’s complete adjuvant was diluted with an equal volume of water (injection 2). A 1:1 mixture of the material solution and Freund’s complete adjuvant solution was prepared (injection 3). The induction of sensitization was made through 3 pairs of 2 intradermal injections. One week after the injections a solution of 75% w/v of the material in distilled water was topically applied. The animals were challenged topically two weeks after the induction period using Basic Yellow 57 25% w/v followed by a further challenge one week later with 5% w/v in distilled water. Potential skin reactions were read 24, 48 and 72 hours after the final challenge. The intradermal injection caused an irritation response that was still present at the time of the topical induction. Following the first challenge, erythema was seen in 7 of the 10 animals. The subsequent challenge with 5% Basic Yellow 57 was therefore applied to determine whether the response was due to irritation or sensitization. Erythema was observed in 2 of the animals at 24 hours, but had resolved by 48 hours. Hence it was concluded that Basic Yellow 57 is not sensitizing to guinea pig skin.

 

Based on the available data for the target chemical and supporting studies,it can be concluded that thechemical 3-[(4,5-dihydro-3-methyl-5-oxo-1-phenyl-1H-pyrazol-4- yl)azo]-N,N,N-trimethylanilinium chloride/Basic Yellow 57 (CAS No. 68391-31-1)is unable to cause skin sensitization and considered as not sensitizing. Comparing the above annotations with the criteria of CLP regulation, it can be classified under the category “Not Classified”.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

The skin sensitization potential of test substance 3-[(4,5-dihydro-3-methyl-5-oxo-1-phenyl-1H-pyrazol-4- yl)azo]-N,N,N-trimethylanilinium chloride/Basic Yellow 57 (CAS No. 68391-31-1) wasobserved in various studies. From the results obtained from these studies it is concluded that the chemical is not likely to cause skin sensitization and hence can be classified as non-skin sensitizer.