Pyrogallol

Endpoint summary

Description of key information

Pyrogallol was identified as a skin sensitizer in experimental animal models and by QSAR prediction. Moreover, a number of human studies in individuals exposed to hairdressing chemicals showed that pyrogallol is a contact sensitizer and, according to Banca Dati Sensibilizzanti (ISS, Italy), Pyrogallol is a substance for which a (skin) sensitizing potential cannot be excluded.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

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

Qualifier:

according to guideline

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)

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)

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.

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.

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

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.

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.

Reference 2

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Remarks:

Mouse Ear Swelling Test

Type of information:

experimental study

Adequacy of study:

supporting 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

Qualifier:

according to guideline

Guideline:

other: Gad et al., 1986; Auttachoat et al., 2011)

Deviations:

yes

Remarks:

with slight modifications.

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:

other: Mouse Ear Swelling Test

Justification for non-LLNA method:

The potential for induction of hypersensitivity responses following treatment with PYR was (also) evaluated using the MEST.

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)

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.

Route:

epicutaneous, open

Vehicle:

acetone/olive oil (4:l v/v)

Concentration / amount:

50 μl of PYR

Day(s)/duration:

days 1, 2 and 3.

Adequacy of induction:

highest technically applicable concentration used

Route:

epicutaneous, open

Vehicle:

acetone/olive oil (4:l v/v)

Concentration / amount:

total of 25 μl of Pyr or vehicle

Adequacy of challenge:

not specified

No. of animals per dose:

not specified

Details on study design:

To further distinguish between the irritant and sensitizing effects of PYR, the MEST was conducted

Challenge controls:

vehicle

Positive control substance(s):

yes

Positive control results:

The positive control group also exhibited significantly increased percent ear swelling when compared to the PCCO group. No significant changes were observed at either 24 or 48 hr time point

Reading:

2nd reading

Hours after challenge:

72

Group:

test chemical

Dose level:

5%

No. with + reactions:

17

Total no. in group:

8

Clinical observations:

A significant increase in percent ear swelling was observed at 72 hr after challenge in mice that had been sensitized with 5% PYR when compared to VHIC

Vehicle (acetone: olive oil)	11(%ear swelling)
vehicle, irritancy control	7(% ear swelling)
mice that had been sensitized with PYR at 1%	10(% ear swelling)
mice that had been sensitized with PYR at 5%	17(% ear swelling)*

* ≤ 0.05 when compared to the  vehicle, irritancy control

Interpretation of results:

Category 1 (skin sensitising) based on GHS criteria

Conclusions:

The MEST suggested that PYR was indeed a sensitizer at higher concentrations.
PYR, elicited dermal sensitization in female BALB/c mice, as demonstrated using the the MEST.

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.

Reference 3

Endpoint:

skin sensitisation, other

Remarks:

Immunofluorescent Staining of Lymph Node Cells

Type of information:

experimental study

Adequacy of study:

supporting 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

Qualifier:

no guideline available

Principles of method if other than guideline:

- Principle of test: immunofluorescent staining consists of labeling anti-bodies with a fluorescent dye, allowing the labeled antibodies to react with their specific antigen, and observing the reaction product under the fluorescence microscope.
- Short description of test conditions: Mice were exposed to 0.125–10% pyrogallol using the same treatment regimen as the LLNA and were sacrificedat 24, 72 and 96 h after the last pyrogallol treatment.
- Parameters analysed / observed: %B cells, %T cells and the ratioof %B cells in treated animals to %B cells in vehicle control ani-mals (“%B ratio”), with a cutoff value of 1.25 separating irritants(%B ratio < 1.25) and sensitizers (%B ratio ≥ 1.25). Cells (1.0 × 10^6) were labeled in a 96-well culture plate with 0.10 ml of the appropriate anti-mouse monoclonal antibodiesdiluted in PBS (pH 7.4, 1% BSA + 0.1% sodium azide). A Anti-mouse CD3Ɛ (145-2C11) conjugated tofluorescein isothiocyanate (FITC; 1:80) was used to label T cells. B cells were stainedwith FITC conjugated affinity purified anti-mouse immunoglobulin (1:80, BectonDickenson) and phycoerythrin (PE) conjugated antibody for CD86 (B7.2).

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:

other: Immunofluorescent Staining of Lymph Node Cells

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)

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.

Positive control results:

no data

Reading:

other: mean fluorescence intensity

Hours after challenge:

72

Group:

test chemical

Dose level:

5%

No. with + reactions:

2

Total no. in group:

8

Clinical observations:

%B cells

Reading:

other: mean fluorescent intensity

Hours after challenge:

72

Group:

test chemical

Dose level:

5%

No. with + reactions:

6

Total no. in group:

8

Clinical observations:

%T cells

Reading:

other: mean fluorescent intensity

Hours after challenge:

72

Group:

test chemical

Dose level:

5%

No. with + reactions:

4

Total no. in group:

8

Clinical observations:

%CD4+T cells

Cellular proliferation data / Observations:

not relevant

Mice were exposed to 0.125–10% PYR using the same treatment regimen as the LLNA and were sacrificed at 24, 72 and 96 hrs after the last PYR treatment. 24 hrs after the last exposure to PYR, the %B ratios exceeded 1.25 for the 5% and 10% PYR treatment groups, when compared to the VH control group. Consistent with the increase in %B cells, %T cells were decreased at these same PYR treatment levels. CD86 (B7.2) is a selective marker for modulation of B cell activation, and its expression in murine draining lymph node cells has been demonstrated following sensitizer, but not irritant, treatment (Gerberick et al., 1999). A significant increase in the expression (the mean fluorescence intensity) of CD86 was observed for B cells at 5% and 10% PYR. This was accompanied by a significant increase in the number of total lymph node cells. Similar results were observed for the number of total lymph node cells, %B cells, %T cells, and CD86 expression by B cells at 72 and 96 hr after the last treatment of PYR, in addition to a 10% decrease in %T cells at the 1% PYR treatment level at the 96 hr time point (data not shown). Based on these data, PYR would be classified as a sensitizer at 5% and above according to the criteria previously described (Gerberick et al. 1999, 2002). The positive control DNFB (0.15%) also decreased %T cells and increased the number of total lymph node cells, %B cells, and CD86 expression

Interpretation of results:

Category 1 (skin sensitising) based on GHS criteria

Conclusions:

Increases in auricular lymph node cellularity and in the percentage of B cells also support classifying PYR as a contact sensitizer.

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.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Pyrogallol was identified as a skin sensitizer in experimental animal models and by QSAR prediction. Moreover, a number of human studies in individuals exposed to hairdressing chemicals showed that pyrogallol is a contact sensitizer and, according to Banca Dati Sensibilizzanti (ISS, Italy), Pyrogallol is a substance for which a (skin) sensitizing potential cannot be excluded.

Based on the above information, the substance should be classified as H317: May cause an allergic skin reaction