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Experimental study was conducted by DENISE M. SAILSTAD et al.(Fundamental and applied toxicology ,1994) to evaluate the skin sensitizing potential of target substance 1-[(2-methoxyphenyl)diazenyl]-2-naphthol(1229-55-6) in mouse by LLNA mode.Skin senstization for 1-[(2-methoxyphenyl)diazenyl]-2-naphthol(1229-55-6) was observed in female Balb/c mice by using LLNA mode. Twenty-five microliters of the agent (acetone-dye solutions) or acetone (negative-vehicle control) was applied to the ears of Balb/c mice for 3 days. On the fourth day, mice were terminated by cervical dislocation and the auricular lymph nodes were removed.Aseptic techniques were used throughout the assay. Due to the sparse number of cells in each node, pools were made using the nodes of 3 mice. Three pools were used for each agent (dye or positive control) or acetone control group. Pooled nodes were homogenized to a single-cellsuspension using glass tissue grinders. Cell viabilities were evaluated using trypan blue exclusion and were above 90% for each pool. Cell mixtures of 1.2 X 10* cells were added to the wells of a 96-well microtiter plate in an RPMI 1640 media containing L-glutamine, 25 mM Hepes, 10% fetal bovine serum, and2%penicillinstreptomycin (Gibco Laboratories). Triated [3H]thymidine (2 ^Ci/well) (NEN Dupont, Boston, MA) was added to each well and incubated for 24 hr at 37°C and 5% CO2. The cells from each well were harvested onto filter disks using a Skatron cell harvester. Incorporation of 3H was determined using a beta scintillation counter . Experimental and control groups each contained nine mice. Groups were divided into three subgroups or "pools" of three mice each. Auricular lymph nodes from each pooled subgroup were processed as previously described. The counts per minute were obtained from triplicates of each pool. The average of these triplicate readings was used as input for the statistical evaluation using an analysis of variance (ANOVA). Analysis of the individual dye compounds, in which two separate experiments were performed, was adjusted for differences between the two replicates. Pairwise comparisons among different agents were performed as subtests of the overall ANOVA. Significance levels were adjusted for multiple comparisons using a modified Bonferroni correction . However 1-[(2-methoxyphenyl)diazenyl]-2-naphthol(1229 -55-6) was statistically different from the control. The response to 3% glutaraldehyde was used as a positive control in this experiment. Differences in acetone controls indicate normal variability. Therefore 1-[(2-methoxyphenyl)diazenyl]-2-naphthol(1229-55-6) was considered to be sensitizing in mice by LLNA mode.

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
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Justification for type of information:
Data is from publication.
Reference:
Composition 1
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Principles of method if other than guideline:
To evaluate the skin sensitizing potential of 1-[(2-methoxyphenyl)diazenyl]-2-naphthol in mice by LLNA mode.
GLP compliance:
not specified
Type of study:
mouse local lymphnode assay (LLNA)
Test material information:
Composition 1
Specific details on test material used for the study:
- Name of test material: Solvent Red 1 (1-[(2-methoxyphenyl)diazenyl]-2-naphthol )
- Molecular formula: C17H14N2O2
- Molecular weight: 278.31 g/mol
- Substance type: Organic
Species:
mouse
Strain:
Balb/c
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
Source: Charles River, Rayleigh, NC
Age at study initiation: 8-12 weeks old
Weight at study initiation: not specified
Housing: suspended polycarbonate cages with bedding made of pine shavings
Diet (e.g. ad libitum):standard diet, Purina rodent lab chowad libitum
Water (e.g. ad libitum): No data
Acclimation period: 4 weeks

ENVIRONMENTAL CONDITIONS
Controlled environmental condition.

OTHER-
female Balb/c mice (Charles River, Raleigh, NC)were group housed in suspended polycarbonate cages with bedding made of pine shavings in an environmentally controlled, American Association for Accreditation of Laboratory Animal Care (AAALAC)-accredited vivarium. Randomly selected animals were tested serologically on arrival, and sentinel mice that were monitored serologically throughout the study were free of Sendai virus, pneumonia virus of mice, mouse hepatitis virus, other murine viruses, and mycoplasma. Mice also were monitored for, and found to be free of, ectoparasites and endoparasites.
Vehicle:
other: Acetone
Remarks:
Twenty-five microliters
Concentration:
1.93mg/ml
No. of animals per dose:
9 female mice in test group
9 female mice in control group.
Details on study design:
PRE-SCREEN TESTS:
Prior to performing both the LLNA , baseline ear thickness was measured and saturated solutions of dye were applied on the ear. Subsequently, ear thickness was measured over a 24-hr period. None of the solutions used for contact-sensitivity testing were found to be irritating.

MAIN STUDY

ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: LLNA
- Criteria used to consider a positive response: Experimental and control groups each contained nine mice. Groups were divided into three subgroups or "pools" of three mice each. Auricular lymph nodes from each pooled subgroup were processed as previously described. The counts per minute were obtained from triplicates of each pool. The average of these triplicate readings was used as input for the statistical evaluation using an analysis of variance (ANOVA). Analysis of the individual dye compounds, in which two separate experiments were performed, was adjusted for differences between the two replicates. Pairwise comparisons among different agents were performed as subtests of the overall ANOVA. Significance levels were adjusted for multiple comparisons using a modified Bonferroni correction.


TREATMENT PREPARATION AND ADMINISTRATION:LLNA was used to evaluate the dye for proliferative and allergic potential. Twenty-five microliters of the agent (acetone-dye solutions) or acetone (negative-vehicle control) was applied to the ears of Balb/c mice for 3 days. On the fourth day, mice were terminated by cervical dislocation and the auricular lymph nodes were removed.
Aseptic techniques were used throughout the assay. Due to the sparse number of cells in each node, pools were made using the nodes of three mice. Three pools were used for each agent (dye or positive control) or acetone control group. Pooled nodes were homogenized to a single-cell suspension using glass tissue grinders. Cell viabilities were evaluated using trypan blue exclusion and were above 90% for each pool. Cell mixtures of 1.2 X 10* cells were added to the wells of a 96-well microtiter plate in an RPMI 1640 media containing L-glutamine, 25 mM Hepes, 10% fetal bovine serum, and 2% penicillinstreptomycin . Triated [3H]thymidine (2µ Ci/well) was added to each well and incubated for 24 hr at 37°C and 5% CO2. The cells from each well were harvested onto filter disks using a Skatron cell harvester . Incorporation of 3H was determined using a beta scintillation counter
Positive control substance(s):
other: Formalin [15%] and 3% glutaraldehyde were used as positive controls
Statistics:
Yes ,statistics was performed by using ANOVA.
Key result
Parameter:
other: counts per minute
Value:
ca. 0.05 - < 0.05
Variability:
p < 0.05.
Test group / Remarks:
test group values was statistically different from the control were observed.
Remarks on result:
other: positive indication of skin sensitization was observed.

Weak sensitizing effect were observed.

Interpretation of results:
other: Sensitizing
Conclusions:
1-[(2-methoxyphenyl)diazenyl]-2-naphthol(1229-55-6) was observed for its skin sensitizing potential in female mice by using LLNA mode.1-[(2-methoxyphenyl)diazenyl]-2-naphthol was considered to be sensitizing in female mice by using LLNA mode.
Executive summary:

Skin senstization for 1-[(2-methoxyphenyl)diazenyl]-2-naphthol(1229-55-6) was observed in female Balb/c mice by using LLNA mode. Twenty-five microliters of the agent (acetone-dye solutions) or acetone (negative-vehicle control) was applied to the ears of Balb/c mice for 3 days. On the fourth day, mice were terminated by cervical dislocation and the auricular lymph nodes were removed.Aseptic techniques were used throughout the assay. Due to the sparse number of cells in each node, pools were made using the nodes of 3 mice. Three pools were used for each agent (dye or positive control) or acetone control group. Pooled nodes were homogenized to a single-cellsuspension using glass tissue grinders. Cell viabilities were evaluated using trypan blue exclusion and were above 90% for each pool. Cell mixtures of 1.2 X 10* cells were added to the wells of a 96-well microtiter plate in an RPMI 1640 media containing L-glutamine, 25 mM Hepes, 10% fetal bovine serum, and2%penicillinstreptomycin (Gibco Laboratories). Triated [3H]thymidine (2 ^Ci/well) (NEN Dupont, Boston, MA) was added to each well and incubated for 24 hr at 37°C and 5% CO2. The cells from each well were harvested onto filter disks using a Skatron cell harvester. Incorporation of 3H was determined using a beta scintillation counter . Experimental and control groups each contained nine mice. Groups were divided into three subgroups or "pools" of three mice each. Auricular lymph nodes from each pooled subgroup were processed as previously described. The counts per minute were obtained from triplicates of each pool. The average of these triplicate readings was used as input for the statistical evaluation using an analysis of variance (ANOVA). Analysis of the individual dye compounds, in which two separate experiments were performed, was adjusted for differences between the two replicates. Pairwise comparisons among different agents were performed as subtests of the overall ANOVA. Significance levels were adjusted for multiple comparisons using a modified Bonferroni correction . However 1-[(2-methoxyphenyl)diazenyl]-2-naphthol(1229 -55-6) was statistically different from the control. The response to 3% glutaraldehyde was used as a positive control in this experiment. Differences in acetone controls indicate normal variability. Therefore

1-[(2-methoxyphenyl)diazenyl]-2-naphthol(1229-55-6) was considered to be sensitizing in mice by LLNA mode.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (sensitising)
Additional information:

Skin sensitization 

In different studies, 1-[(2-methoxyphenyl)diazenyl]-2-naphthol(1229-55-6) has been investigated for potential of skin sensitization to a greater or lesser extent. The key and supporting studies are based on in vivo experiments in mouse and human for target chemical1-[(2-methoxyphenyl)diazenyl]-2-naphthol(1229-55-6).

Experimental study was conducted by DENISE M. SAILSTAD et al.(Fundamental and applied toxicology ,1994) to evaluate the skin sensitizing potential of target substance 1-[(2-methoxyphenyl)diazenyl]-2-naphthol(1229-55-6) in mouse by LLNA mode.Skin senstization for 1-[(2-methoxyphenyl)diazenyl]-2-naphthol(1229-55-6) was observed in female Balb/c mice by using LLNA mode. Twenty-five microliters of the agent (acetone-dye solutions) or acetone (negative-vehicle control) was applied to the ears of Balb/c mice for 3 days. On the fourth day, mice were terminated by cervical dislocation and the auricular lymph nodes were removed.Aseptic techniques were used throughout the assay. Due to the sparse number of cells in each node, pools were made using the nodes of 3 mice. Three pools were used for each agent (dye or positive control) or acetone control group. Pooled nodes were homogenized to a single-cellsuspension using glass tissue grinders. Cell viabilities were evaluated using trypan blue exclusion and were above 90% for each pool. Cell mixtures of 1.2 X 10* cells were added to the wells of a 96-well microtiter plate in an RPMI 1640 media containing L-glutamine, 25 mM Hepes, 10% fetal bovine serum, and2%penicillinstreptomycin (Gibco Laboratories). Triated [3H]thymidine (2 ^Ci/well) (NEN Dupont, Boston, MA) was added to each well and incubated for 24 hr at 37°C and 5% CO2. The cells from each well were harvested onto filter disks using a Skatron cell harvester. Incorporation of 3H was determined using a beta scintillation counter . Experimental and control groups each contained nine mice. Groups were divided into three subgroups or "pools" of three mice each. Auricular lymph nodes from each pooled subgroup were processed as previously described. The counts per minute were obtained from triplicates of each pool. The average of these triplicate readings was used as input for the statistical evaluation using an analysis of variance (ANOVA). Analysis of the individual dye compounds, in which two separate experiments were performed, was adjusted for differences between the two replicates. Pairwise comparisons among different agents were performed as subtests of the overall ANOVA. Significance levels were adjusted for multiple comparisons using a modified Bonferroni correction . However 1-[(2-methoxyphenyl)diazenyl]-2-naphthol(1229 -55-6) was statistically different from the control. The response to 3% glutaraldehyde was used as a positive control in this experiment. Differences in acetone controls indicate normal variability. Therefore 1-[(2-methoxyphenyl)diazenyl]-2-naphthol(1229-55-6) was considered to be sensitizing in mice by LLNA mode.

Further supported by experimentalstudy was conducted by DENISE M. SAILSTAD et al.(Fundamental and applied toxicology ,1994) to evaluate the skin sensitizing potential of target substance 1-[(2-methoxyphenyl)diazenyl]-2-naphthol(1229-55-6) in mouse by Mouse ear swelling test.Skin senstization for 1-[(2-methoxyphenyl)diazenyl]-2-naphthol(1229-55-6) was observed in female Balb/c mice by using MEST test.On the day prior to sensitization, mice were anesthetized with metafane and their backs were shaved using styling clippers. Mice were sensitized on the shaved area with 100µlof the test agent or vehicle for 2 consecutive days. After a rest period ,on day 5th mice were challenged with acetone (vehicle) on one ear and test material at the concentration of 4.9 mg/ mlon the other ear . Treated and vehicle ear thickness measurements were taken prior to challenge and 24 and 48 hr after the challenge using an Oditest D1000 hand-held caliper with reduced spring tension (~36 g of force) (The Dyer Co., Lancaster, PA). Three measurements were taken at the apex of each ear. Ear measurements were alternated between right and left to reduce compression artifacts. Measurements were performed at approximately the same time and under the same conditions each day. For each test animal, triplicate measurements of ear thickness were averaged and the ear thickness of the vehicle-challenged ear was subtracted from the thickness of the treated ear. To adjust for non treatment-related differences, the pre-challenge (0 hr) value was subtracted from the post challenge (24 and 48 hr) values. A repeated measures multivariate analysis of variance (MANOVA) was applied to the adjusted measurements comparing the responses of unsensitized and sensitized mice at the two post-challenge times.Using the MEST, 1-[(2-methoxyphenyl)diazenyl]-2-naphthol also caused an increase in ear thickness that was significantly different from the unsensitized control animals at both 24 and 48 hr post- challenge. Therefore 1-[(2-methoxyphenyl)diazenyl]-2-naphthol(1229-55-6) was considered to be sensitizing in mice by mouse ear swelling test(MEST).

 It is further supported by experimental study conducted by F. WANTKE (Contact Dermatitis ,1992) to evaluate the skin sensitizing potential of target substance 1-[(2-methoxyphenyl)diazenyl]-2-naphthol(1229-55-6)in human .1-[(2-methoxyphenyl)diazenyl]-2-naphthol(1229-55-6) was observed for its skin sensitizing potential in male human by using Patch test . A 69-year-old male had used a particular suntan cream for 10 years, on his body in summer and on his face from spring to autumn when hiking. 2 years ago, he had developed dermatitis on the cheeks and forehead, which appeared within hours of using the suntan cream, increasing in severity from application to application. 2 weeks before presentation, he applied the suntan cream again. Dermatitis occurred on the cheeks and forehead, both eyelids showing swelling several hours later.Patch testing was done with the European standard series. . Following the first series of patch tests, a second with the individual components of thetest material was applied .A group of 10 healthy controls were also patch tested with test material at the concentration of 0.0003% in petrolatum . The substances were applied, using Finn Chamberson Scanpor tape, to the upper back. Readings were made after 3 days. Reactions were scored as recommended by the ICDRG. In the second series of patch tests the patient gave+++ reactions. .All control results were negative. Thus 1-[(2-methoxyphenyl)diazenyl]-2-naphthol(1229-55-6) was considered to be sensitizing in human.

Thus based on the above key and supporting studies on 1-[(2-methoxyphenyl)diazenyl]-2-naphthol(1229-55-6)andit can be concluded that1-[(2-methoxyphenyl)diazenyl]-2-naphthol(1229-55-6)is a skin sensitizer. Thus comparing the above annotations with the criteria of CLP regulation, 1-[(2-methoxyphenyl)diazenyl]-2-naphthol(1229-55-6) can be considered as ‘classified’ for skin sensitization.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

Thus comparing the above annotations with the criteria of CLP regulation, 1-[(2-methoxyphenyl)diazenyl]-2-naphthol(1229-55-6) can be considered as ‘classified’ for skin sensitization.