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Skin sensitisation

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Administrative data

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.

Data source

Reference
Reference Type:
publication
Title:
Evaluation of an Azo and Two Anthraquinone Dyes for Allergic Potential
Author:
DENISE M. SAILSTAD, JEFFREY S. TEPPER, DONALD L. DOERFLER, MOHAMMAD Qasim AND MARYJANE K. SELGRADE
Year:
1994
Bibliographic source:
FUNDAMENTAL AND APPLIED TOXICOLOGY 23, 569-577 (1994)

Materials and methods

Test guideline
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

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material: Solvent Red 1
- Molecular formula: C17H14N2O2
- Molecular weight: 278.31 g/mol
- Substance type: Organic
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

In vivo test system

Test animals

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.

Study design: in vivo (LLNA)

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.

Results and discussion

In vivo (LLNA)

Results
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.

Any other information on results incl. tables

Weak sensitizing effect were observed.

Applicant's summary and conclusion

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.