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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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Data source

Reference
Reference Type:
publication
Title:
Unnamed
Year:
2013

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
other: ICCVAM-recommended protocol
Deviations:
no
Principles of method if other than guideline:
The objective of this study was to determine the contact sensitization potential of pyrogallol (PYR) when applied dermally to female BALB/c mice. Measurement of the contact hypersensitivity response was initially accomplished using the local lymph node assay.
GLP compliance:
not specified
Remarks:
refer to section "any other information on materials and methods" for remarks related to GLP compliance for a test performed after 2008, 1st June.
Type of study:
mouse local lymph node assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
impurity
Test material form:
not specified
Details on test material:
not specified
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material:
purchased from Sigma (St. Louis, MO).
- Expiration date of the lot/batch:
not specified
- Purity test date: not specified

RADIOLABELLING INFORMATION (if applicable)
not applicable
- Radiochemical purity:
-
- Specific activity: -
- Locations of the label: -
- Expiration date of radiochemical substance:
-

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material:
not specified
- Stability under test conditions:
not specified
- Solubility and stability of the test substance in the solvent/vehicle:
Initial studies on the solubility of PYR demonstrated that the compound was soluble at concentrations of 75% (w/v) and below in acetone:olive oil
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium:
not specified

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing:
not specified
- Preliminary purification step (if any):
not specified
- Final dilution of a dissolved solid, stock liquid or gel:
Test solutions were prepared daily in the vehicle 4:1 acetone:olive oil (AOO) as recommended by the Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM; Dean et al., 2001).
- Final preparation of a solid:
not relevant

FORM AS APPLIED IN THE TEST (if different from that of starting material)
liquid

TYPE OF BIOCIDE/PESTICIDE FORMULATION (if applicable)
not applicable

OTHER SPECIFICS: white odorless crystal (FW 126.11, CAS No.87-66-1)

In vivo test system

Test animals

Species:
mouse
Strain:
Balb/c
Sex:
female
Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Female BALB/c mice were obtained from Charles River Laboratories
- Females (if applicable) nulliparous and non-pregnant:not specified
- Microbiological status of animals, when known: The mice were determined to be hepatitis and Sendai virus free by serology testing.
- Age at study initiation: 6–8 wks old
- Weight at study initiation: 17–20 grams
- Housing: Mice were housed four animals per cage in plastic shoebox cages with hardwood bedding
- Diet (e.g. ad libitum): Food (Harlan Teklad laboratory diets NIH-07) was provided ad libitum in all experiments
- Water (e.g. ad libitum): tap water was provided ad libitum in all experiments
- Acclimation period: The animals were quarantined for at least 7 days prior to being placed on study
- Indication of any skin lesions: Animals were observed for signs of moribundity daily, weighed on the first and last days of each
study, and euthanized by CO2 inhalation

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21–24°C
- Humidity (%): 40–70% relative humidity
- Air changes (per hr):
- Photoperiod (hrs dark / hrs light): 12-h light/dark cycle
- IN-LIFE DATES: From: To:

Other information: The BALB/c mouse is the National Toxicology Program (NTP) strain of choice for conducting contact hypersensitivity testing in rodents.

Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
the highest exposure level of PYR selected for the LLNA was 50% in the first study (NIH, 1999; Dean et al., 2001).
Four additional exposure levels of PYR (2.5%, 5%, 10%, 25% and 50%).
The concentrations of PYR used in the second experiment were 0.5%, 1%, and 2.5%.
The concentrations of PYR used in the third experiment were 0.25%, 0.5%, 1%, 2.5%, 5%, and 10%
No. of animals per dose:
eight mice were assigned for each dose group.
Positive control substance(s):
other: 1-fluoro-2,4-dinitrobenzene
Statistics:
The data obtained in this study were first tested for homogeneity of variances using Bartlett’s Chi Square Test. Homogeneous data were evaluated by a parametric one-way analysis of variance. When significant differences occurred, treatment groups were compared to the appropriate control using Dunnett’s Test. If data were non-homogeneous, they were evaluated using a non-parametric analysis of variance. When significant differences occurred, treatment groups were compared to the appropriate control using the Wilcoxon Rank Test.
The positive control was compared to the PCCO using the Student’s t Test. The level of statistical significance was set at p ≤ 0.05. All values are presented as mean ± standard error of mean.

Results and discussion

In vivo (LLNA)

Resultsopen allclose all
Key result
Parameter:
SI
Value:
> 3
Test group / Remarks:
dermal exposure to PYR produced a positive response in the LLNA at concentrations as low as 0.5%.
Remarks on result:
other: The SI SI was defined as the total DPM per mouse divided by vehicle DPM per mouse.
Remarks:
Disintegrations per minute (DPM) were calculated as (counts per minute (CPM) - background count)/(the efficiency of the Beta Counter, which was 71%).
Parameter:
SI
Value:
> 3
Test group / Remarks:
2,5% dose group - first experiment
Parameter:
SI
Value:
> 3
Test group / Remarks:
5% dose group - first experiment
Parameter:
SI
Value:
> 3
Test group / Remarks:
10% dose group - first experiment
Parameter:
SI
Value:
> 3
Test group / Remarks:
25% dose group - first experiment
Parameter:
SI
Value:
> 3
Test group / Remarks:
50% dose group - first experiment
Parameter:
SI
Value:
> 3
Test group / Remarks:
1% dose group - second experiment
Parameter:
SI
Value:
> 3
Test group / Remarks:
0,5% dose group - third experiment
Parameter:
SI
Value:
< 3
Test group / Remarks:
0,25% dose group - third experiment
Cellular proliferation data / Observations:
CELLULAR PROLIFERATION DATA : first experiment: PYR induced a significant increase in the proliferation of lymph node cells (SI > 3) at all concentrations; All groups except 0.25% PYR exhibited a significant increase (SI > 3)

DETAILS ON STIMULATION INDEX CALCULATION not specified

EC3 CALCULATION : EC3 value between 0.5–1% after combining all three LLNAs

CLINICAL OBSERVATIONS: not specified

BODY WEIGHTS not specified


Any other information on results incl. tables

Based on the ICCVAM-recommended criteria, a chemical would be classified as positive if at least one concentration reached a stimulation index (SI) of ≥ 3 with the results being statistically significant and dose-responsive. The SI was defined as the total DPM (Disintegrations per minute)  per mouse divided by vehicle DPM per mouse.

Applicant's summary and conclusion

Interpretation of results:
Category 1 (skin sensitising) based on GHS criteria
Conclusions:
The results demonstrate that PYR is both a sensitizer and an irritant in female BALB/c mice: dermal exposure to PYR produced a positive response in the LLNA at
concentrations as low as 0.5%.
Executive summary:

Hair dye components such as pyrogallol and cresol have been shown previously to promote allergic reactions such as rashes, dermal inflammation, irritation and dermatitis. The objective of this study was to determine the contact sensitization potential of pyrogallol (PYR) when applied dermally to female BALB/c mice. Measurement of the contact hypersensitivity response was initially accomplished using the local lymph node assay. For PYR, significant increases in the proliferation of lymph node cells were observed at concentrations of 0.5% (w/v) and higher. . Results from the irritancy assay suggested that PYR was an irritant. To further delineate whether PYR was primarily an irritant or a contact sensitizer, the mouse ear swelling test (MEST) was conducted. A significant increase in mouse ear thickness was observed at 72 hr following challenge with 0.5% PYR in mice that had been sensitized with 5% PYR. Additional studies examining lymph node subpopulations and CD86 (B7.2) expression by B cells further support the indication that PYR was a sensitizer in BALB/c mice. The results demonstrate that PYR is both a sensitizer and an irritant in female BALB/c mice.