Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The majority of the available bacterial mutation studies shown that the pyrogallol has a genotoxicity activity both in Salmonella and E. Coli strain, especially in presence of exogenous metabolic activation. Thus, the results of studies in bacteria show that pyrogallol is a direct-acting mutagen.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
not specified
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Pyrogallol was purchased from Merck.
- 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: not specified
- 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: Pyrogallol was pro analysi grade,
- Preliminary purification step (if any): as reported
- Final dilution of a dissolved solid, stock liquid or gel:
- Final preparation of a solid:

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

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

OTHER SPECIFICS: not specified
Target gene:
L-histidine
Species / strain / cell type:
S. typhimurium TA 100
Additional strain / cell type characteristics:
not specified
Species / strain / cell type:
S. typhimurium TA 1537
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
Liver homogenate fraction mix (S9 mix)
Test concentrations with justification for top dose:
200, 50, 20 and 5 micro/g (Ames et al. 1975)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: water
- Justification for choice of solvent/vehicle: not specified
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
Remarks:
water (with and without metabolic activation)
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
N-ethyl-N-nitro-N-nitrosoguanidine
benzo(a)pyrene
other: 9-aminoacridine
Details on test system and experimental conditions:
The test was performed according to Ames et al. 1975 and no infomation related to eventual deviations were reported.

METHOD OF APPLICATION: in medium; in agar (plate incorporation); preincubation; in suspension; as impregnation on paper disk see above
- Cell density at seeding (if applicable): see above

DURATION
- Preincubation period: see above
- Exposure duration: see above
- Expression time (cells in growth medium): see above
- Selection time (if incubation with a selection agent): see above
- Fixation time (start of exposure up to fixation or harvest of cells): see above

SELECTION AGENT (mutation assays): see above

SPINDLE INHIBITOR (cytogenetic assays): see above

STAIN (for cytogenetic assays): see above

NUMBER OF REPLICATIONS: 2 for each strain

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: see above

NUMBER OF CELLS EVALUATED: see above

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): see above

CRITERIA FOR MICRONUCLEUS IDENTIFICATION: not relevant

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: see above
- Any supplementary information relevant to cytotoxicity: see above

OTHER EXAMINATIONS:
- Determination of polyploidy: see above
- Determination of endoreplication: see above
- Methods, such as kinetochore antibody binding, to characterize whether micronuclei contain whole or fragmented chromosomes (if applicable): see above

- OTHER: see above
Rationale for test conditions:
The Ames plate test was carried out as described by Ames et al.
Evaluation criteria:
Viable counts were carried out by spreading 0.2 ml of the diluted cultures on triplicate synthetic-agar plates containing in addition L-histidine (20 microg/ml) and biotin (3 microg/ml). The plates were incubated for 48 h at 37°C and the number of colonies was then determined.
Statistics:
not specified
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
not specified
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
not specified

Examination of the results obtained from the colicine-induction assay on pyrogallol shows that the substances induced

colicine E2 when tested directly on the test plates.

It has been suggested (Ben Gurion, 1978) that the colicine-induction test could be used as a screening test for mutagenic and carcinogenic substances.

To verify that pyrogallol is not only inducer of colicine but is mutagenic as well, it was also tested in the Ames test.

The observation that pyrogallol does not mutagenize strain TA98 when tested directly on the plates

(Rahimtula, 1977) was confirmed. But when pyrogallol was tested with additional tester strains, it was found to be mutagenic for strain TA1537 as well as for strain TA100. Pyrogallol mutagenicity was reduced in the presence of ratliver

microsomal preparation on the plates, probably as a result of an interaction with the mixture which reduced its activity towards the bacteria.

Conclusions:
Pyrogallol tested in Salmonella typhimurium (strains TA100 and TA1537) at doses 5-200 microg/plate (in presence and absence of metabolic activation) resulted as mutagenic.
Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
no guideline followed
Principles of method if other than guideline:
- Principle of test: to clarify the involvement of radical species in the genotoxicity of pyrogallol.
- Short description of test conditions: (i) induction of chromosomal aberrations in V79 cells, (ii) production of hydroxyl radicals at different pH values (6.0, 7.4 and 8.0), and (iii) analysis of the influence of antioxidant enzymes (catalase, superoxide dismutase) on the clastogenic effects
- Parameters analysed / observed: influence of antioxidant enzymes (catalase, superoxide dismutase) on the clastogenic effects
GLP compliance:
not specified
Type of assay:
in vitro mammalian chromosome aberration test
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: pyrogallol, hydrogen peroxide, superoxide dismutase from human erythrocytes (SOD), catalase, new-born calf serum and Ham’s F-10 medium
were obtained from Sigma Chemical Co. (St Louis, MO, USA). ethylenediaminetetracetic acid (EDTA), Tris, FeCl3, dimethyl sulfoxide (DMSO) and Giemsa dye were from Merck (Darmstadt, Germany).
Thiobarbituric acid (TBA) and colchicine were from Fluka (Buchs, Switzerland). Fungibact solution (penicillin: 10,000 IU/ml; streptomycin sulfate: 10,000g/ml; amphotericin B: 25 µg/ml) was from
Irvine Scientific.
- Expiration date of the lot/batch: not specified
- Purity test date: not specified

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

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: not specified
- 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: not specified
- Final preparation of a solid: not specified

FORM AS APPLIED IN THE TEST (if different from that of starting material) : not specified

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

OTHER SPECIFICS: not specified
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with
Metabolic activation system:
Catalase, SOD, SOD + catalase,S9 Mix
Test concentrations with justification for top dose:
0, 20, 40, 60, 80 µM
Vehicle / solvent:
not specified
Untreated negative controls:
yes
Negative solvent / vehicle controls:
not specified
True negative controls:
not specified
Positive controls:
not specified
Positive control substance:
not specified
Rationale for test conditions:
not specified
Evaluation criteria:
Ctg: chromatid gap;
Csg: chromosome gap;
Ctb: chromatid break;
Csb: chromosome break;
Dic: dicentric chromosome;
Rearr: rearrangements (trirradial, quadrirradial, and other complex rearrangements);
CAEG/cell: chromosomal aberrations excluding gaps per cell, corresponding to the sum of Ctb, Csb, Dic, Rings and Rearr per cell;
ACEG (%): percent of aberrant cells excluding gaps (average ± S.D.);
>10: multi-aberrant cells, corresponding to cells with more than 10 aberrations. These cells are included in the ACEG (%);
MI: mitotic index.
Statistics:
The statistical analysis of differences in chromosomal aberration frequency in the different experiments was carried out using the t-test. All analyses were performed with an SPSS statistical package (version 10.5) (SPSS Inc., Chicago, IL).
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
not specified
Untreated negative controls validity:
valid
Positive controls validity:
not specified
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: the clastogenic activity of the substance studied is clearly dependent on the pH: pyrogallol showed a clear clastogenic effect in a pH-dependent way.
- Effects of osmolality: not specified
- Evaporation from medium: not specified
- Water solubility: not specified
- Precipitation: not specified
- Definition of acceptable cells for analysis: not specified
- Other confounding effects: not specified

RANGE-FINDING/SCREENING STUDIES: not specified

CYTOKINESIS BLOCK (if used)
- Distribution of mono-, bi- and multi-nucleated cells: not specified

NUMBER OF CELLS WITH MICRONUCLEI
- Number of cells for each treated and control culture: 100 cells per dose were scored
- Indication whether binucleate or mononucleate where appropriate: not specified

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data:
- Negative (solvent/vehicle) historical control data:

ADDITIONAL INFORMATION ON CYTOTOXICITY: not specified
- Measurement of cytotoxicity used: [complete, e.g. CBPI or RI in the case of the cytokinesis-block method; RICC, RPD or PI when cytokinesis block is not used]
- Other observations when applicable: [complete, e.g. confluency, apoptosis, necrosis, metaphase counting, frequency of binucleated cells]

At pH 6.0, only pyrogallol at higher doses (60 and 80 µM) increases significantly the level of chromosomal aberrations in V79 cells.

At pH values of 7.4 and 8.0 pyrogallol induce significant levels of chromosomal aberrations at lower doses (< 8

µM).

Pyrogallol at higher doses and in a pH-dependent manner showed a significant induction of multi-aberrant cells (cells with more than 10 chromosomal aberrations), which represent in some cases more than 50% of the aberrant cells.

The addition of S9 Mix (P < 0.0003), SOD (P < 0.00002), SOD+catalase (P = 0.000002), but not catalase alone (P = 0.67), leads to a significant decrease in the frequency of chromosomal aberrations, suggesting that SOD alone is responsible for this effect. In these experimental

conditions, the decrease in the levels of chromosomal aberrations in the presence of S9 Mix, SOD and SOD + catalase is a consequence of the decrease in the levels of multi-aberrant cells (P = 0.001, 0.03 and 0.003, respectively), since the levels of significance are lower when the statistical analysis is carried out considering only the number of chromosomal aberration per cell (P = 0.08, 0.38 and 0.02, respectively).

The ability of the different phenolic compounds to generate hydroxyl radicals was measured by the deoxyribose assay, at different pH values in the presence of Fe3+/EDTA or in the presence of Fe3+.

The results obtained show that pyrogallol can generate OH• radicals in a pH-dependent way and this effect is more pronounced in the presence of Fe3+/EDTA.

The results obtained concerning the production of OH• by pyrogallol and the genotoxic activity of these compounds under our experimental conditions suggest that radical mechanisms, namely the production of OH•, could explain the genotoxicity of some of the compounds

studied.

Phenolic compounds can spontaneously oxidise at pH above neutrality, giving rise to the superoxide anion and semiquinone intermediates:

HP → P− + H+

(HP : protonated phenolic compound;

P−: unprotonated phenolic compound)

P− + O2 ⇒ SQ + O2•−

(SQ : semiquinone)

The superoxide anion produced this way can then initiate a free-radical chain reaction with simultaneous production of other reactive oxygen species, such as hydrogen peroxide and hydroxyl radical. The ultimate reactive oxygen species that interacts with DNA is considered to be the hydroxyl radical (OH•) generated from hydrogen peroxide by Fenton reactions. Since it has been demonstrated that superoxide anionand

hydrogen peroxide-generating systems are genotoxic to mammalian cells, namely V79 cells the genotoxicity of some of these compounds could be partially attributed to their auto-oxidation with concomitant production of radical species. Therefore, since phenolic compounds oxidise extracellularly with the production of superoxide anion, their genotoxicity can be attributed to either hydrogen peroxide formed in the

process that can enter the cells and give rise to OH• in the vicinity of DNA, or to the protonated form of superoxide anion (HO2 •) that is able to cross the cell membrane and give rise to hydrogen peroxide inside the cell with subsequent production of OH• in the vicinity of DNA

Conclusions:
Pyrogallol showed a clear clastogenic effect in a pH-dependent way: pH dependence on the genotoxic effects of the substance.
The genotoxic activity of pyrogallol seems to be mediated mainly by a radical mechanism (superoxide anion formation).
Executive summary:

Phenolic molecules are widely present in the environment and some of them are well known carcinogens. Some phenolic molecules are also genotoxic but the mechanisms involved in this process are not fully understood. We have studied the induction of chromosomal aberrations by phenol, catechol and pyrogallol in V79 cells at different pH values (6.0, 7.4 and 8.0). At the same pH values, the production of hydroxyl radicals was assessed by measuring the degradation of deoxyribose.

Apart from phenol, which only induces a non-significant increase in chromosomal aberration in this experimental system, catechol and pyrogallol showed clear clastogenic effect in a pH-dependent way. Experiments carried out at pH 7.4 in the presence of S9 Mix, SOD, catalase and catalase +SOD suggest that the formation of reactive oxygen species is not the main mechanism involved in the genotoxicity of catechol. However, concerning pyrogallol, our results suggest that its genotoxicity

is almost exclusively mediated by reactive oxygen species.

Taken together, these results suggest that, in spite of the structural similarity between the different molecules studied, the mechanisms of genotoxicity of these molecules could be considerably different.

The existence of several mechanisms of genotoxicity, partially shared by this class of compounds, could explain the synergistic effects observed between these compounds in several genotoxicity test systems. Accurate knowledge of their mechanisms of genotoxicity could improve considerably the assessment of their relevance to human health, since these compounds, once absorbed, are subject to a wide range of pH values in vivo.

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
according to
Guideline:
other: Tests were performed according to standard procedures as described in previous publications
Version / remarks:
Eckhardt. K., M.-T.. King. E. Gocke and D. Wild, Mutagenicity study of Remsen Fahlberg saccharin
and contaminants, Toxicol. Left., 7 (1980) 51-66.
Deviations:
not specified
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
not specified
Principles of method if other than guideline:
- Principle of test: The study shows mutagenic potential of a cosmetic compound by in vitro test (Ames Test) in order to predict the mutagenicity of the substance
- Short description of test conditions: An Ames test with different strain of Salmonella and two slightly different media were performed.
- Parameters analysed / observed: mugenic effects.
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: pyrogallol was obtained from Merck Co., Darmstadt (Germany);
- 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: not specified
- 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: not specified
- Final preparation of a solid: not specified

FORM AS APPLIED IN THE TEST (if different from that of starting material) not specified

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

OTHER SPECIFICS: not specified
Target gene:
Bacterial strains: 5 tester strains of Salmonella typhimurium designated TA1535, TA100, TA1538, TA98 and TA1537 were provided by Dr. B.N. Ames, University of California, Berkeley, CA (U.S.A.).
Species / strain / cell type:
S. typhimurium TA 1535
Remarks:
2 1535 strains were used
Additional strain / cell type characteristics:
not applicable
Species / strain / cell type:
S. typhimurium TA 1537
Additional strain / cell type characteristics:
not applicable
Species / strain / cell type:
S. typhimurium TA 1538
Additional strain / cell type characteristics:
not applicable
Species / strain / cell type:
S. typhimurium TA 98
Additional strain / cell type characteristics:
not applicable
Species / strain / cell type:
S. typhimurium TA 100
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
by the S9 liver fraction from Aroclor-pretreated rats
Test concentrations with justification for top dose:
In the Ames assay at least 5 doses, usually up to 3600 µg/plate as non-toxic and soluble substance
Vehicle / solvent:
the substance was tested on 2 slightly different minimal media: one (in the following named ZLM medium) is a modified minimal medium for E. coli, and the other
is the Vogel-Bonner (VB) medium. ZLM medium contained (in g/l): tri-Nacitrate.
2H20 (0.82), K2HPO4-3H20 (4.60), KH2PO 4 (1.50), (NH4)2SO 4 (1.00), MgSO4.7H20 (0.10) and glucose (17.0). The concentration of citrate was 3.5 times higher in VB medium than in ZLM medium. The concentrations of the other ions are up to 2-fold higher in VB medium.

Details on test system and experimental conditions:
refer to the method
Rationale for test conditions:
refer to the method
Evaluation criteria:
refer to the method
Statistics:
P ≤ 0.01
Species / strain:
S. typhimurium, other: 98, 100, 1535, 1537 and 1538
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
not specified
Conclusions:
pyrogallol showed mutagenic effects predominantly in the Ames tester strains sensitive for frameshift mutagens: in strains TA1537 (-S9), TA98 (± S9) and additionally in TA100 (±S9). The substance was mutagenic on both minimal media.
Executive summary:

As part of the investigation into mutagenic effects of environmental compounds, the Authors studied chemicals allowed as ingredients of cosmetics according to the guidelines of the Council of the European Communities. Three systems were used the Sahnonella/microsome test, the Basc test on Drosophila and the micronucleus test on mouse bone marrow. Of the 31 chemicals tested, 15 were mutagenic in the Ames test; and of these, 5 were also mutagenic in the Basc test and 2 in the micronucleus test.

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2005
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
not specified
GLP compliance:
yes
Remarks:
The tests were performed in laboratory in compliance with the Swiss Ordinance, which is based on the OECD Principles of Good Laboratory Practice.
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Bisdoxe (Rio de Janeiro, Brazil) and Phitoteraphia Biofitogenia Laboratorial Biota (Rio de Janeiro, Brazil) provided commercial samples of hair gels SunSet Alizador Negro (manufacturing date 03/29/2005), SunSet Alizador Negro Forte (12/21/2005), and Embelleze Heneˆ Gel (03/30/2005).
- Expiration date of the lot/batch: not specified
- Purity test date: not specified

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

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: not specified
- 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: The principal genetic characteristics relative to histidine and biotin dependence, uvrB deletion, rfa mutation, spontaneous genetic reversion in the histidine synthesis, and presence of pAQ1 (only TA102) or pKM101 plasmids were verified prior to the assays and the results agreed with published data.
- Preliminary purification step (if any): not specified
- Final dilution of a dissolved solid, stock liquid or gel: The chemicals were dissolved in either distillated water or dimethylsulfoxide analytical grade (>99.9%) purchased from Merck KgaA (Darmstadt, Germany).
- Final preparation of a solid: not specified

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

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

OTHER SPECIFICS: The content of pyrogallol in these gels (samples) varied from 0.5 to 1.5% w/w.
Target gene:
S. typhimurium strains TA98, TA100, TA102, TA1535, and TA1537 were obtained from MoltoxTM
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Test concentrations with justification for top dose:
Cosmetic samples were tested on the following dosages: 648, 1080, 1800, 3000, and 5000 µg/plate.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Cosmetic samples dissolved in dimethylsulfoxide
- Justification for choice of solvent/vehicle: not specified
Untreated negative controls:
yes
Remarks:
Negative controls were assayed with the same volume of the vehicle
Negative solvent / vehicle controls:
not specified
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
other: positive control in absence of S9: 4-NQO (0.5 µg/plate) for TA98; SA (5 µg/plate) for TA100 and TA1535; MC (0.5 µg/plate) for TA102 and 9-AAC (0.1 mg/ plate) for TA1537. Positive controls in presence of S92-AA (2.5 µg/plate)
Remarks:
Negative and cosmetic sample were assayed in triplicate and the positive ones in duplicate.
Evaluation criteria:
The mutagenicity index (M.I.) was calculated as the ratio between the numbers of histidine revertants induced per plate (His+) of the tests and negative control. The spontaneous His+ in negative and positive controls for each strain were compared to the range according to the literature (Maron and Ames, 1983;
Aiub et al., 2004). The results were certified as positive when the following requirements were fulfilled (Vargas et al., 1993; Vargas et al., 1995): (i) the
ratio between the tests and negative His+ corresponds, at least, to twice (M.I.P2) for TA98, TA100, and TA102 or to three time (M.I. P3) for TA1535 and TA1537; (ii) a significant response for analysis of variance (ANOVA, p < 0.05) is reached; and (iii) the His+ versus the concentrations reproduces a positive and representative (p < 0.01) dose–response curve
Statistics:
analysis of variance (ANOVA, p < 0.05)
Key result
Species / strain:
S. typhimurium, other: TA98, TA100, TA 102, TA1535, TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
not specified
Key result
Species / strain:
S. typhimurium, other: TA98, TA100, TA 102, TA1535, TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not determined
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
not specified
Remarks on result:
other: Result related to the cosmetic product "Alizador Negro"

mutation frequencies (M.I.) were lower than two in each tested Salmonella strain and respective contents, either in the absence or in the presence of S9. Based on these results, it was concluded that none of the tested hair gel samples exhibits mutagenic effect in prokaryotic cells.

The data obtained from TA100 and TA1535 (both +S9) tested with Heneˆ Gel and from TA1535 (-S9) and TA1537 (+S9) tested with Alizador Negro Forte apparently indicate a dose–response relationship. However, according to statistical analysis these responses were not significant (p > 0.01).

Statistical analysis also indicated no significant (p > 0.05) induction, except by the Alizador Negro Forte for the strains TA98 and TA100 in the presence of metabolic

activation and by the Alizador Negro for TA100 in the absence of S9. However, these exceptions do not seem to be relevant because no dose–response relationship was observed.

The initial decrease in the M.I. observed in the TA98 response by Alizador Negro Forte, in the presence of S9, suggests that more than one biological process occurred.

Within these results, one acute value of toxicity (M.I. < 0.7) was recorded, but the highest M.I. value was lower than two. Thus, possible biological effects were not covered by other independent assay because no evidence of mutagenicity was detected. The same was observed in TA1537 in the presence of S9.

Despite being statistically significant (ANOVA analysis), only a very low increase of M.I. (up to 1.06) by the Alizador Negro Forte for TA100 in the presence of S9 was noticed. The Alizador Negro seems to have significant (p < 0.05) increased the M.I. value (until 1.27) in TA100 in the absence of S9, but it did not show increase in the reversion of strain TA1535, which has the same hisG gene coding (Maron and Ames, 1983) and would response identically in the same conditions. A subsequent test using TA100 and lower quantity of the Alizador Negro per plate (at 8, 40, 200, 1000 and 2000 lg/plate) did not significantly (p = 0.062) induce mutagenesis and all M.I. values were lower than 1.1 (data not shown). Thus, the p-values lower than 0.05 do not indicate any significant induction on the bacterial reverse mutation.

Conclusions:
The p-values lower than 0.05 do not indicate any significant induction on the bacterial reverse mutation.
Pyrogallol does significantly induce the generation of DNA-damaging ROS only at pH > 5.
The cosmetic ingredient review expert panel cosmetic regulations, which restricts its use up to 5% w/w and no evidence of mutagenicity induced by each tested hair gels in both prokaryotes and eukaryotes was detected in this study, despite the presence of 0.5–1.5% w/w pyrogallol. The already known mechanism of ROS generation inhibition by acid medium can possibly be responsible for the observed lack of mutagenicity.
Executive summary:

In the present work, three commercial acid (pH 3.5–4) pyrogallol-containing hair gels, SunSet Alizador Negro (two formulations) and EmbellezeHeneˆ Gel, were tested for mutagenicity using two well-established assays. In the Salmonella mutagenicity assay using 648–5000 µg/plate of cosmetic samples, none of the samples reached a 2-fold increase in revertants relative to the controls. Both in the absence and in the presence of S9, the dose–response relation in strains TA98, TA100, TA102, TA1535, and TA1537 was not significant (p > 0.01). Thus, none of the products induced mutagenesis in eitherassay. Previous studies have shown pyrogallol is mutagenic in various test systems, including Salmonella. However studies have also shown that acidic conditions may repress the reactive-oxygen species (ROS) produced by pyrogallol, and ROS is considered the primary mechanism for the mutagenicity of pyrogallol. Consistent with this are our results, which show that acidic, commercially available pyrogallol-containing hair gels are not mutagenic in Salmonella.

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail
Qualifier:
according to
Guideline:
other: The first assay was performed following protocols reported by Zeiger et al. (1992)
Deviations:
no
Principles of method if other than guideline:
- Principle of test: This a bacterial reverse mutation test which uses amino-acid requiring different strains of Salmonella typhimurium to detect point mutations by base substitutions or frameshifts. The principle of this bacterial reverse mutation test is that it detects mutations which revert mutations present in the test strains and restore the functional capability of the bacteria to synthesize an essential amino acid.
- Short description of test conditions: Suspensions of bacterial cells are exposed to the test substance in the presence and in the absence of an exogenous metabolic activation system. 5 different analysable concentrations of the test substance were used.
- Parameters analysed / observed: the ability to restore the functional capability of the bacteria to synthesize an essential amino acid.
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 assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: not specified
- 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: the test article was sent to the laboratory as a coded aliquot from Radian Corporation (Austin, TX), and incubated with the bacterial tester strains for 20 minutes at 37° C.
- Stability under test conditions: not specified
- Solubility and stability of the test substance in the solvent/vehicle: not specified
- 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: not specified
- Final preparation of a solid: not specified

FORM AS APPLIED IN THE TEST (if different from that of starting material) not specified

TYPE OF BIOCIDE/PESTICIDE FORMULATION (if applicable) NOT APPLICABLE

OTHER SPECIFICS: Top agar supplemented with L-histidine and d-biotin was added, and the contents of the tubes were mixed and poured onto the surfaces of minimal glucose agar plates. Histidine-independent mutant colonies arising on these plates were counted following incubation for 2 days at 37° C.
Target gene:
histidine-independent (revertant)
Species / strain / cell type:
S. typhimurium TA 98
Additional strain / cell type characteristics:
not specified
Species / strain / cell type:
S. typhimurium TA 100
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
buffer or S9 mix (metabolic activation enzymes and cofactors from Aroclor 1254-induced male Sprague-Dawley rat or Syrian hamster liver).
Test concentrations with justification for top dose:
Each trial consisted of triplicate plates of concurrent positive and negative controls and at least five doses of pyrogallol. The high dose was limited by toxicity.
Doses: 0, 3, 10, 33, 100 and 333 μg/plate for both TA100 and TA98
Vehicle / solvent:
not specified
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
Remarks:
0 μg/plate was the solvent control.
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
sodium azide
other: The positive controls in the absence of metabolic activation was 4 nitro-o-phenylenediamine (TA98). The positive control for metabolic activation with all strains was 2-aminoanthracene.
Details on test system and experimental conditions:
not specified
Rationale for test conditions:
not specified
Evaluation criteria:
In this assay, a positive response is defined as a reproducible, dose-related increase in histidine-independent (revertant) colonies in any one strain/activation combination. An equivocal response is defined as an increase in revertants that is not dose related, is not reproducible, or is not of sufficient magnitude to support a determination of mutagenicity. A negative response is obtained when no increase in revertant colonies is observed following chemical treatment. There is no minimum percentage or fold increase required for a chemical to be judged positive or weakly positive, although positive calls are typically reserved for increases in mutant colonies that are at least twofold over background.
Statistics:
Data are presented as revertants/plate (mean ± standard error) from three plates. The detailed protocol is presented by Zeiger et al. (1992).
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
positive
Remarks:
positive results were seen in S. typhimurium strain TA100 with and without 30% S9 derived from hamster or rat liver
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
negative results were obtained in strain TA98 tested with and without 30% S9 derived from hamster or rat liver
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
Positive controls validity:
valid
Conclusions:
In the study (concentration range 3 to 333 μg/plate), positive results were seen in S. typhimurium strain TA100 with and without 30% S9 derived from hamster or rat liver; negative results were obtained in strain TA98 tested under the same conditions.
The results of studies in bacteria show that pyrogallol is a direct-acting mutagen.
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail
Qualifier:
according to
Guideline:
other: The first assay was performed following protocols reported by Zeiger et al. (1992)
Deviations:
yes
Remarks:
this assay used a slightly modified protocol (activation only with rat liver S9)
Principles of method if other than guideline:
- Principle of test: This a bacterial reverse mutation test which uses amino-acid requiring different strains of Salmonella typhimurium ans Escherichia coli to detect point mutations by base substitutions or frameshifts. The principle of this bacterial reverse mutation test is that it detects mutations which revert mutations present in the test strains and restore the functional capability of the bacteria to synthesize an essential amino acid.
- Short description of test conditions: Suspensions of bacterial cells are exposed to the test substance in the presence and in the absence of an exogenous metabolic activation system. 5 different analysable concentrations of the test substance were used.
- Parameters analysed / observed: the ability to restore the functional capability of the bacteria to synthesize an essential amino acid.
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 assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Lot 010326
- 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: the test article was sent to the laboratory as a coded aliquot from Radian Corporation (Austin, TX), and incubated with the bacterial tester strains for 20 minutes at 37° C.
- Stability under test conditions: not specified
- Solubility and stability of the test substance in the solvent/vehicle: not specified
- 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: not specified
- Final preparation of a solid: not specified

FORM AS APPLIED IN THE TEST (if different from that of starting material) not specified

TYPE OF BIOCIDE/PESTICIDE FORMULATION (if applicable) NOT APPLICABLE

OTHER SPECIFICS: Top agar supplemented with L-histidine and d-biotin was added, and the contents of the tubes were mixed and poured onto the surfaces of minimal glucose agar plates. Histidine-independent mutant colonies arising on these plates were counted following incubation for 2 days at 37° C.

The chemical used was a white crystalline powder, was identified as pyrogallol using melting point analyses, infrared, ultraviolet, proton nuclear magnetic resonance, and mass spectroscopy. Karl Fischer titration results were inconclusive due to a reaction of pyrogallol with the reagent used, and weight loss on drying could not be determined due to the sublimation of the test article. Thin-layer chromatography indicated a single major spot and no impurities. High-performance liquid chroma-tography with ultraviolet detection (HPLC/UV) indicated one major peak and no impurities with areas equal to or greater than 0.05% of the total peak areas in four of six containers tested; one container had one impurity, and another had two impurities that had peak areas greater than or equal to 0.05% of the total peak area. The overall purity of lot 010326 was determined to be 99% or greater.
Target gene:
histidine-independent (revertant)
Species / strain / cell type:
S. typhimurium TA 98
Additional strain / cell type characteristics:
not specified
Species / strain / cell type:
S. typhimurium TA 100
Additional strain / cell type characteristics:
not specified
Species / strain / cell type:
E. coli WP2
Remarks:
WP2 uvrA/pKM101 was used as a bacterial tester strain
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
buffer or S9 mix (metabolic activation enzymes and cofactors from Aroclor 1254-induced male Sprague-Dawley rat or Syrian hamster liver).
Test concentrations with justification for top dose:
Each trial consisted of triplicate plates of concurrent positive and negative controls and at least five doses of pyrogallol. The high dose was limited by toxicity.
Doses: 0, 10, 50, 100, 250, 500, 750 and 1000 μg/plate for TA100, TA98 and Escherichia coli WP2 uvrA/pKM101.
Vehicle / solvent:
not specified
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
Remarks:
0 μg/plate was the solvent control.
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
sodium azide
methylmethanesulfonate
other: The positive controls in the absence of metabolic activation was 4 nitro-o-phenylenediamine (TA98). The positive control for metabolic activation with all strains was 2-aminoanthracene.
Details on test system and experimental conditions:
not specified
Rationale for test conditions:
not specified
Evaluation criteria:
In this assay, a positive response is defined as a reproducible, dose-related increase in histidine-independent (revertant) colonies in any one strain/activation combination. An equivocal response is defined as an increase in revertants that is not dose related, is not reproducible, or is not of sufficient magnitude to support a determination of mutagenicity. A negative response is obtained when no increase in revertant colonies is observed following chemical treatment. There is no minimum percentage or fold increase required for a chemical to be judged positive or weakly positive, although positive calls are typically reserved for increases in mutant colonies that are at least twofold over background.
Statistics:
Data are presented as revertants/plate (mean ± standard error) from three plates. The detailed protocol is presented by Zeiger et al. (1992).
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
positive
Remarks:
positive results were obtained over a concentration range of 10 to 1,000 μg/plate in S. typhimurium strain TA100
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
positive
Remarks:
positive results were obtained over a concentration range of 10 to 1,000 μg/plate in S. typhimurium strains TA98
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A pKM 101
Remarks:
positive results were obtained over a concentration range of 10 to 1,000 μg/plate in E. coli strain WP2 uvrA/pKM101 in the absence of S9
Metabolic activation:
without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A pKM 101
Metabolic activation:
with
Genotoxicity:
positive
Remarks:
With 10% rat liver S9, this sample of pyrogallol was mutagenic in the E. coli strain
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with
Genotoxicity:
ambiguous
Remarks:
small increases in revertants that were not well correlated with dose.
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with
Genotoxicity:
ambiguous
Remarks:
small increases in revertants that were not well correlated with dose.
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
Positive controls validity:
valid
Conclusions:
positive results were obtained over a concentration range of 10 to 1,000 μg/plate in S. typhimurium strains TA98 and TA100 and in E. coli strain WP2 uvrA/pKM101 in the absence of S9 (Table E2). With 10% rat liver S9, this sample of pyrogallol was mutagenic in the E. coli strain but gave equivocal responses in S. typhimurium strains TA98 and TA100 based on small increases in revertants that were not well correlated with dose. Thus, the results of studies in bacteria show that pyrogallol is a direct-acting mutagen.
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
not specified
Principles of method if other than guideline:
- Principle of test: The mutagenicity of Pyrogallol (in DMSO) was evaluated using strains TA1538 and TA98 of S. typhimurium by Ames Test (Bacterial Reverse Mutation Assay) The principle of this bacterial reverse mutation test is that it detects mutations which revert mutations present in the test strains and restore the functional capability of the bacteria to synthesize an essential amino acid and point mutations by base substitutions or frameshifts were detected.
- Short description of test conditions: Concentrations ranging from 20 to 1000 kg/plate were tested with and without metabolic activation.
- Parameters analysed / observed: Reverse mutation of the bacteria tested.
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay
Target gene:
TA 1538 and TA 98
Species / strain / cell type:
S. typhimurium TA 1538
Additional strain / cell type characteristics:
not specified
Species / strain / cell type:
S. typhimurium TA 98
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
Aroclor induced rat S-9 preparations supplied by Litton Bionetics, Kensington, Md. or Meloy Laboratories, Springfield, Va., were added to the agar overlay at levels 0.150-0.075 ml crude S-9/ml S-9 mix for Litton Bionetics/ Meloy Laboratories, respectively
Test concentrations with justification for top dose:
20, 50, 100, 250, 500, 1000 and 2000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used:Samples were dissolved or suspended in DMSO
- Justification for choice of solvent/vehicle: not specified
Positive controls:
yes
Remarks:
Dose responses for positive controls were determined during each experiment
Positive control substance:
other: For TA 1538 and TA 98, positive controls consisted of 2-nitropphenylenediamine (no metabolic activation) and m-phenylenediamine (with metabolic activation).
Details on test system and experimental conditions:
Each batch of S-9 enzymes was optimized for activation prior to use.
Rationale for test conditions:
Predictivity of mutagenicity in-vitro test for teratogenicity.
Evaluation criteria:
refer to Ames test.
Statistics:
For bacterial mutagenesis studies, Student’s T-test(121 and linear correlation with a rho null hypothesis to determine correlation were performed. or all statistical analyses, a level of probability of p = 0.05 was used for significance. When lower levels of probability were observed, these were noted.
Species / strain:
S. typhimurium TA 1538
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
not determined
Vehicle controls validity:
not specified
Untreated negative controls validity:
valid
Positive controls validity:
not examined
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
not determined
Vehicle controls validity:
not specified
Untreated negative controls validity:
valid
Positive controls validity:
not examined

Although the weak response of TA 98 to pyrogallol was observed at three points, the lack of dose response questions the biological significance: the weak response of pyrogallol in the absence of metabolic activation agreed with the previous published results. The results with TA 98 did not exhibit a linear correlation and were abolished in the presence of metabolic S9 enzymes. Thus, the mutagenic activity of pyrogallol towards Salmonella typhimurium presents a dilemma to the toxicologist in extrapolation to animals. Since activity is present only in the absence of metabolic

enzymes, it is uncertain how to predict the behavior in whole animals.

Conclusions:
Pyrogallol exhibited a weak response that showed no linear correlation only in TA 98 in the absence of activation: Since activity is present only in the absence of metabolic enzymes, it is uncertain how to predict the behavior in whole animals.
Executive summary:

Three oxidative dyes were screened for mutagenic and teratogenic potential. A range of mutagenic response was observed using Salmonella typhimurium tester strains, TA 1538 and TA 98 in the presence or absence of metabolic activation. M-phenylenediamine exhibited activity with both strains while 4-chlororesorcinol showed no response in either

strain. Pyrogallol exhibited a weak response that showed no linear correlation only in TA 98 in the absence of activation.

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 472 (Genetic Toxicology: Escherichia coli, Reverse Mutation Assay)
Deviations:
not specified
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 assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Pyrogallol were purchased from Wako Pure Chemicals (Osaka, Japan).
- Expiration date of the lot/batch: not specified
- Purity test date: (reagent was of the best commercially available grade)

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: not specified
- 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: not specified
- Final preparation of a solid: not specified

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

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

OTHER SPECIFICS: not specified
Target gene:
Grow inhibition because of superoxide dismutase and catalase
Species / strain / cell type:
E. coli, other: katG katE double mutant E. coli UM255
Additional strain / cell type characteristics:
other: completely deficient in hydroperoxidase (both HP-I and HP-II) synthesis.
Metabolic activation:
not specified
Test concentrations with justification for top dose:
Pyrogallol in presence or not (into the plate) of ascorbic acid, naringin and quercetin.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used:
- Justification for choice of solvent/vehicle: not specified
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
not specified
True negative controls:
not specified
Positive controls:
not specified
Details on test system and experimental conditions:
see below
Rationale for test conditions:
not specified
Evaluation criteria:
pyrogallol cytotoxicity by colony-forming efficiency test
Statistics:
For assessing the differences in pyrogallol toxicity among Csa and Csb, student’s t-test and 2-way ANOVA were used.
Key result
Species / strain:
E. coli, other: katG katE double mutant E. coli UM255
Metabolic activation:
not specified
Genotoxicity:
not determined
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
not examined
Untreated negative controls validity:
not examined
Positive controls validity:
not examined

Growth of inhibition test showed that zone surrounding the paper disc of Csa was significantly smaller than that of Csb. The viability of Csa and Csb also decreased as the concentration of pyrogallol increased (p<0.001) and Csb was more susceptible to pyrogallol than Csa (p<0.05) (data not shown). The addition of SOD, catalase or ascorbic acid lessened the toxic effects of pyrogallol on both strains, and the differences in diameter of inhibition zone between Csa and Csb became smaller. In particular, the addition of 100 U/plate catalase abolished the toxicity caused by pyrogallol. It is known that catalase is supposed to play a critical role in detoxification of H2O in E. coli, since no glutathione peroxidase has been identified in E. coli (Smith and Schrift, 1979). The present results further provide evidence that H2O2 is one of mediators of pyrogallol-induced cytotoxicity. Pretreatment with SOD as well as ascorbic acid also inhibited pyrogallol toxicity dose-dependently in both strains, suggesting a possibility of O2•- formation in the process of pyrogallol toxicity. SOD is an enzyme that can catalyze the conversion of O2 •- to H2O2, while ascorbic acid is known to have the ability to act as a scavenger of O2•- and OH•

Conclusions:
The present results suggest that pyrogallol-induced cytotoxicity seems to be attributable to the formation of ROS. Both enzymatic (catalase, SOD) and
non-enzymatic (ascorbic acid, naringin) antioxidants played a protective role against the pyrogallol-induced cytotoxicity.
Executive summary:

Pyrogallol cytotoxicity was evaluated using Escherichia coli strains that express mammalian catalase gene derived from catalase mutant mice (Cs(b)) and wild-type (Cs(a)). Pyrogallol was more toxic to Cs(b) rather than to Cs(a) (p < 0.05), while catalase, superoxide dismutase and ascorbic acid decrease the toxic effect. Pyrogallol cytotoxicity seems to be attributable, at least in part, to reactive oxygen species formation.

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
not specified
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 assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Pyrogallol were purchased from Wako Pure Chemicals (Osaka, Japan).
- Expiration date of the lot/batch: not specified
- Purity test date: (reagent was of the best commercially available grade)

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: not specified
- 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: not specified
- Final preparation of a solid: not specified

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

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

OTHER SPECIFICS: not specified
Target gene:
histidine
Species / strain / cell type:
S. typhimurium TA 98
Additional strain / cell type characteristics:
not specified
Species / strain / cell type:
S. typhimurium TA 100
Additional strain / cell type characteristics:
not specified
Species / strain / cell type:
S. typhimurium TA 102
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9
Test concentrations with justification for top dose:
0, 1 nmol, 10 nmol, 100 nmol, 1 µmol, 2 µmol, 4 µmol, 6 µmol, 8 µmol and 10 µmol.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used:no data
- Justification for choice of solvent/vehicle: -
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
not specified
Evaluation criteria:
Mutagenic effect of pyrogallol was evaluated by Ames test: Results are expressed as Mutation index (MI) that indicates the revertants of pyrogallol divided by the revertants of negative control.
Statistics:
The differences among groups in relation to Ames test were analyzed by one-way analysis of variance (one-way ANOVA) following Bonferroni tests and trend analysis with a Windows version SPSS 11.0.
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not determined
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
Positive controls validity:
not specified
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
not determined
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
Positive controls validity:
not specified
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not determined
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
Positive controls validity:
not specified
Species / strain:
S. typhimurium TA 98
Metabolic activation:
without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
not determined
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
Positive controls validity:
not specified
Species / strain:
S. typhimurium TA 100
Metabolic activation:
without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
not determined
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
not specified
Species / strain:
S. typhimurium TA 102
Metabolic activation:
without
Genotoxicity:
ambiguous
Cytotoxicity / choice of top concentrations:
not determined
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
not specified

Data showed that pyrogallol was mutagenic to TA100 in the presence of metabolic activation, whereas in the absence of metabolic activation, pyrogallol was highly mutagenic to TA98 and TA100, and weakly mutagenic to TA102.

The mutagenicity of pyrogallol observed in the present study is decreased in the presence of S9 mix as metabolic

activation inducer

Conclusions:
The present results suggest that pyrogallol-induced mutagenicity seems to be attributable to the formation of ROS
Executive summary:

Pyrogallol mutagenicity was evaluated by Ames test. Pyrogallol showed mutagenic effect (mutagenic index = 3.8 for 10

micromol pyrogallol/plate). Pyrogallol mutagenicity seems to be attributable, at least in part, to reactive oxygen species formation

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

A micronucleus test (measuring frequency of micronucleated polychromatic erythrocytes in bone marrow) in male B6C3F1/N mice following three intraperitoneal injections of pyrogallic acid gave negative results. Those findings were confirmed by a second in vivo test in which no increase of micronucleated erythrocytes was observed in peripheral blood of male and female B6C3F1/N mice treated via dermal application of pyrogallic acid for 3 months. However, in this last study, results on male mice were equivocal because of a significant increase in micronucleated erythrocytes observed at single dose level (300 mg/kg) at the end of the 3-month study

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
according to
Guideline:
other: The micronucleus test was performed according to Schmid
Version / remarks:
Schmid, W., The mlcronucleus test for cytogenetic analysis, in: A. Hollaender (Ed.), Chemical
Mutagens, Vol. 4, Plenum, New York, 1976, pp. 31-53.
Deviations:
not specified
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
not specified
GLP compliance:
not specified
Specific details on test material used for the study:
- Source and lot/batch No.of test material:
pyrogallol was obtained from Merck Co., Darmstadt (Germany);
- 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:
not specified
- 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:
not specified
- Final preparation of a solid:
not specified

FORM AS APPLIED IN THE TEST (if different from that of starting material)
not specified

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

OTHER SPECIFICS: not specified
Species:
mouse
Strain:
NMRI
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: obtained from S. Ivanovas GmbH and Co., Kisslegg/Allgau (Germany),
- Age at study initiation: not specified
- Weight at study initiation: not specified
- Assigned to test groups randomly: [no/yes, under following basis: ] not specified
- Fasting period before study: not specified
- Housing: not specified
- Diet (e.g. ad libitum): standard chow (Altromin GmbH, Lage, Germany)
- Water (e.g. ad libitum): water ad libitum.
- Acclimation period: not specified

ENVIRONMENTAL CONDITIONS
- Temperature (°C): not specified
- Humidity (%): not specified
- Air changes (per hr): not specified
- Photoperiod (hrs dark / hrs light): not specified

IN-LIFE DATES: From: To: not specified
Route of administration:
intraperitoneal
Vehicle:
not specified
Details on exposure:
not specified
Duration of treatment / exposure:
The animals were treated at 0 and 24 h, and bone-marrow smears were prepared at 30 h.
Frequency of treatment:
not specified
Post exposure period:
not specified
Dose / conc.:
0 other: mg/kg
Dose / conc.:
68 other: mg/kg
Dose / conc.:
126 other: mg/kg
Dose / conc.:
252 other: mg/kg
No. of animals per sex per dose:
4 mice (2 male, 2 female) were used for each of 3 doses
Control animals:
yes
Positive control(s):
not specified
Tissues and cell types examined:
- N. Surviving/N.treated mice
- Micronucleated PE (%.)
Details of tissue and slide preparation:
Slides were coded, and 1000 polychromatic erythrocytes were scored per mouse.
Statistics:
Significance was calculated according to the Kastenbaum-Bowman tables (P<0.01)
Sex:
male/female
Genotoxicity:
positive
Remarks:
Significantly different from control @ 126 and 252 mg/kg
Toxicity:
not specified
Vehicle controls validity:
not specified
Negative controls validity:
not specified
Positive controls validity:
not specified
Conclusions:
In the mouse micronucleus test, up to 252 mg/kg pyrogallol was administered (i.p.) to four mice at 0 and 24 hours. An untreated group of four mice served as the control. Bone marrow smears were prepared at 30 hours. Pyrogallol significantly increased the percentage of micronucleated polychromatic erythrocytes.
Endpoint:
in vivo insect germ cell study: gene mutation
Remarks:
the Basc test on Drosophila detecting sex-linked recessive lethal mutations
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 477 (Genetic Toxicology: Sex-linked Recessive Lethal Test in Drosophila melanogaster)
Deviations:
not specified
Principles of method if other than guideline:
- Principle of test: The mutagenicity of Pyrogallol was evaluated using the recessive lethal mutations test in 2 strains of Drosophila melanogaster
- Short description of test conditions: One dose of Pyrogallol (in 5% saccharose) was fed to Drosophila melanogaster.
- Parameters analysed / observed: frequency of sex-linked recessive lethal mutations

GLP compliance:
not specified
Specific details on test material used for the study:
- Source and lot/batch No.of test material:
pyrogallol was obtained from Merck Co., Darmstadt (Germany);
- 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:
not specified
- 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:
not specified
- Final preparation of a solid:
not specified

FORM AS APPLIED IN THE TEST (if different from that of starting material)
not specified

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

OTHER SPECIFICS: not specified
Species:
Drosophila melanogaster
Strain:
other: The Berlin K (wild-type) and Basc strains were used.
Sex:
not specified
Details on test animals and environmental conditions:
TEST ANIMALS
- Source:
- Age at study initiation: not specified
- Weight at study initiation: not specified
- Assigned to test groups randomly: [no/yes, under following basis: ] not specified
- Fasting period before study: not specified
- Housing: not specified
- Diet (e.g. ad libitum):
- Water (e.g. ad libitum):
- Acclimation period: not specified

ENVIRONMENTAL CONDITIONS
- Temperature (°C): not specified
- Humidity (%): not specified
- Air changes (per hr): not specified
- Photoperiod (hrs dark / hrs light): not specified

IN-LIFE DATES: From: To: not specified

Route of administration:
oral: feed
Vehicle:
not specified
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: one dose close to the LD50 was applied by the adult feeding method in 5% saccharose, according to Wiirgler, F.E., F.H. Sobels and E. Vogel, Drosophila as assay system for detecting genetic changes, in: B.J. Kilbey, M. Legator, W. Nichols and C. Ramel (Eds.), Handbook of Mutagenicity Test Procedures, Elsevier, Amsterdam, 1977, pp. 335-373.

DIET PREPARATION
- Rate of preparation of diet (frequency): not specified
- Mixing appropriate amounts with (Type of food): see the reference
- Storage temperature of food: not specified
Duration of treatment / exposure:
not specified
Frequency of treatment:
not specified
Post exposure period:
not specified
Dose / conc.:
125 other: nM
Remarks:
one dose close to the LD50 was applied by the adult feeding method in 5% saccharose.
No. of animals per sex per dose:
3
Control animals:
not specified
Positive control(s):
not specified
Evaluation criteria:
About 1200 X-chromosomes were tested per experiment in each of 3 successive broods (3-3-4 days). In repeat experiments, sometimes only single broods were tested. F: progeny cultures with 2 or fewer wild-type males were routinely retested in the F 3 generation to confirm X-linked recessive lethal mutations (RLs). Mosaics were not counted. "Clusters" of 2 were included because their occurrence was compatible with statistical expectation of independent origin
Statistics:
P<0.05
Sex:
not specified
Genotoxicity:
positive
Remarks:
Sex-linked recessive lethals/chromosomes tested
Toxicity:
not specified
Vehicle controls validity:
not specified
Negative controls validity:
not specified
Positive controls validity:
not specified

Brood 1: 0,5%*

Brood 2: 0,13%

Brood 3: 0,26%

Conclusions:
The mutagenicity of Pyrogallol was evaluated using the recessive lethal mutations test. One dose of Pyrogallol (in 5% saccharose) was fed to Berlin K (wild-type) and Base
strains of Drosophila melanogaster. The dose administered was close to the LD50.
Approximately 1200 X-chromosomes were tested per experiment in each of three successive broods. F2 progeny cultures with two, or fewer, wild-type males were
routinely retested in the F3 generation to confirm X-linked recessive lethal mutations.
Pyrogallol significantly increased (P = 0.05) the frequency of sex-linked recessive lethal mutations.
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2005
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
not specified
GLP compliance:
yes (incl. certificate)
Remarks:
The tests were performed in laboratory in compliance with the Swiss Ordinance, which is based on the OECD Principles of Good Laboratory Practice
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material:
Bisdoxe (Rio de Janeiro, Brazil) and Phitoteraphia Biofitogenia Laboratorial Biota (Rio de Janeiro, Brazil) provided commercial samples of hair gels SunSet Alizador Negro (manufacturing date 03/29/2005), SunSet Alizador Negro Forte (12/21/2005), and Embelleze Heneˆ Gel (03/30/2005).
- Expiration date of the lot/batch:
not specified
- Purity test date:
not specified

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

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:
not specified
- 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:
The principal genetic characteristics relative to histidine and biotin dependence, uvrB deletion, rfa mutation, spontaneous genetic reversion in the histidine synthesis, and presence of pAQ1 (only TA102) or pKM101 plasmids were verified prior to the assays and the results agreed with published data.
- Preliminary purification step (if any):
not specified
- Final dilution of a dissolved solid, stock liquid or gel:
The chemicals were dissolved in either distillated water or dimethylsulfoxide analytical grade (>99.9%) purchased from Merck KgaA (Darmstadt, Germany).
- Final preparation of a solid:
not specified

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

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

OTHER SPECIFICS: The content of pyrogallol in these gels (samples) varied from 0.5 to 1.5% w/w.
Species:
mouse
Strain:
Swiss
Details on species / strain selection:
albino Swiss mice
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: not specified
- Age at study initiation: seven-weeks old
- Weight at study initiation: about 31 ± 3 g
- Assigned to test groups randomly: [no/yes, under following basis: ] not specified
- Fasting period before study: not specified
- Housing: not specified
- Diet (e.g. ad libitum): not specified
- Water (e.g. ad libitum): not specified
- Acclimation period: not specified

ENVIRONMENTAL CONDITIONS
- Temperature (°C): The environment temperature was adjusted to 20–22°C
- Humidity (%): relative humidity was kept at 72–84%.
- Air changes (per hr): not specified
- Photoperiod (hrs dark / hrs light): Artificial day light was continuously supplied by fluorescent bulbs for 12 h/day

IN-LIFE DATES: From: To: not specified
Route of administration:
intraperitoneal
Vehicle:
These samples were administered without dilution.
Dose / conc.:
2 000 other: mg/kg
Remarks:
A constant injection volume was adjusted for each mouse, which was weighed prior the administration
No. of animals per sex per dose:
12
Control animals:
yes
Positive control(s):
- Doses / concentrations:cyclophosphamide
- Justification for choice of positive control(s): not specified
- Route of administration: by oral via.
- Doses / concentrations: 2 x 30 mg/kg bw
Statistics:
The PE/NE ratios were calculated and statistically (p-ANOVA) compared to the value of the negative controls, which allowed the estimation of any possible toxicity.
Two-thousand PE per mouse were scored and the micronucleated cells of positive control and the experimental group was statistically compared to the negative control by p-ANOVA and the non-parametric v-square (v2) data analysis (Pereira, 1991). The micronucleated cell numbers by animal in the controls were according to our historical data in oral gavage tests
Sex:
male
Genotoxicity:
negative
Toxicity:
no effects
Remarks:
In a preliminary assay involving a group of four male albino Swiss mice receiving the maximum recommended dosages of the cosmetic samples
Vehicle controls validity:
not specified
Negative controls validity:
not specified
Positive controls validity:
not specified
Sex:
male
Genotoxicity:
negative
Toxicity:
not specified
Remarks:
The highest administered dose of pyrogallol can be estimated 30 mg/(kg body weight)/day based on its content in the tested hair gels
Vehicle controls validity:
not specified
Negative controls validity:
not specified
Positive controls validity:
not specified
Additional information on results:
no significant (p > 0.05) variation of the PE/NE ratios was observed.

No mortality was recorded and the animals were sacrificed after 48 h from the first administration

Conclusions:
The ANOVA and the non-parametric v-square analyses indicated that frequencies of micronucleated PE were not significant for the groups receiving the highest recommended dose of the hair gels products. No incidence of more than one micronucleus per cell was observed in the groups treated with hair gels. The micronucleus frequencies of the controls obtained in our experiment were lower than those observed by others.
Each tested hair gels did not significantly increase the micronucleated PE incidence in mouse bone marrow when the maximum recommended dose was administered by oral via. The highest administered dose of pyrogallol can be estimated 30 mg/(kg body weight)/day based on its content in the tested hair gels. Thus, no evidence of marrow bone cells mutagenicity could be expected in view of the lowest administered dosage (126 mg/(kg body weight)/day) previously detected as positive. Likewise, even cosmetics containing 5% w/w pyrogallol could not induce micronuclei in mouse marrow bone.
Executive summary:

In the present work, three commercial acid (pH 3.5–4) pyrogallol-containing hair gels, SunSetAlizador Negro (two formulations) and Embelleze Heneˆ Gel, were tested for mutagenicity using two well-established assays. In the mouse bone marrow micronucleus assay, 10 Swiss male mice were orally administered 2000 mg/kg of sample

per body weight/day. The ratio between polychromatic and normochromatic erythrocytes as well as the presence of micronuclei in bone marrow cells were determined. Equal numbers of micronucleated polychromatic erythrocytes were detected between the cells of each treated group and the negative control, using ANOVA and v-square analyses. Thus, none of the products induced mutagenesis in either assay. However studies have also shown that acidic conditions may repress the reactive-oxygen species (ROS) produced by pyrogallol, and ROS is considered the primary mechanism for the mutagenicity of pyrogallol. Consistent with this are our results, which show that acidic, commercially available pyrogallol-containing hair gels induce micronuclei in mouse bone marrow in vivo.

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration
Type of information:
experimental study
Adequacy of study:
weight of evidence
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:
other: The standard three-exposure protocol is described in detail by Shelby et al. (1993)
Deviations:
not specified
GLP compliance:
not specified
Type of assay:
mammalian erythrocyte micronucleus test
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material:
not specified
- 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:
not specified
- 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:
not specified
- Final preparation of a solid:
the substance was dissolved into the vehicle

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: not specifiedRMULATION (if applicable)
Species:
mouse
Strain:
B6C3F1
Remarks:
B6C3F1/N
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: not specified
- Age at study initiation: not specified
- Weight at study initiation: not specified
- Assigned to test groups randomly: [no/yes, under following basis: ] not specified
- Fasting period before study: not specified
- Housing: not specified
- Diet (e.g. ad libitum): not specified
- Water (e.g. ad libitum): not specified
- Acclimation period: not specified

ENVIRONMENTAL CONDITIONS
- Temperature (°C): not specified
- Humidity (%): not specified
- Air changes (per hr): not specified
- Photoperiod (hrs dark / hrs light): not specified

IN-LIFE DATES: From: To: not specified
Route of administration:
intraperitoneal
Vehicle:
- Vehicle(s)/solvent(s) used: pyrogallol was dissolved in phosphate-buffered saline.
- Justification for choice of solvent/vehicle: not specified
- Concentration of test material in vehicle: see below
- Amount of vehicle (if gavage or dermal): not relavant
- Type and concentration of dispersant aid (if powder): not specified
- Lot/batch no. (if required): not specified
- Purity: not specified
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:

DIET PREPARATION
- Rate of preparation of diet (frequency):
- Mixing appropriate amounts with (Type of food):
- Storage temperature of food:
Duration of treatment / exposure:
Male B6C3F1/N mice were injected intraperitoneally (three times at 24-hour intervals) for 3 days
Frequency of treatment:
once daily for 3 days (three times at 24-hour intervals)
Post exposure period:
The animals were killed 24 hours after the third injection.
Dose / conc.:
39 other: mg/kg
Dose / conc.:
78 other: mg/kg
Dose / conc.:
156 other: mg/kg
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Positive control(s):
The positive control animals received injections of cyclophosphamide.
- Justification for choice of positive control(s): not specified
- Route of administration: intraperitoneal
- Doses / concentrations: 20 mg/kg
Tissues and cell types examined:
polychromatic erythrocytes
Evaluation criteria:
2,000 polychromatic erythrocytes (PCEs, or reticulocytes) were scored for the frequency of micronucleated cells in each of five animals per dose group. In addition, the percentage of PCEs among a population of 200 erythrocytes in the bone marrow was scored for each dose group as a measure of toxicity
Statistics:
The results were tabulated as the mean of the pooled results from all animals within a treatment group plus or minus the standard error of the mean. The frequency of micronucleated cells among PCEs was analyzed by a statistical software package that tested for increasing trend over dose groups with a one-tailed Cochran-Armitage trend test, followed by pairwise comparisons between each dosed group and the solvent control group. In the presence of excess binomial variation, as detected by a binomial dispersion test, the binomial variance of the Cochran-Armitage test was adjusted upward in proportion to the excess variation. In the micronucleus test, an individual trial is considered positive if the trend test P value is less than or equal to 0.025 or if the P value for any single dosed group is less than or equal to 0.025 divided by the number of dosed groups. A final call of positive for micronucleus induction is preferably based on reproducibly positive trials (as noted above). Ultimately, the final call is determined by the scientific staff after considering the results of statistical analyses, the reproducibility of any effects observed, and the magnitudes of those effects.
Key result
Sex:
male
Genotoxicity:
negative
Toxicity:
not examined
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
In vivo, no significant increases in the frequencies of micronucleated polychromatic erythrocytes (reticulocytes) were observed in bone marrow of male B6C3F1/N mice injected intraperitoneally with pyrogallol (39 to 156 mg/kg) once daily for 3 days
Conclusions:
In vivo, no significant increases in the frequencies of micronucleated polychromatic erythrocytes (reticulocytes) were observed in bone marrow of male B6C3F1/N mice injected intraperitoneally with pyrogallol (39 to 156 mg/kg) once daily for 3 days
Executive summary:

The induction of micronuclei in bone marrow polychromatic erythrocytes of male B6C3F1/N mice were studied. Mice were administered pyrogallol by intraperitoneal injection once daily for 3 days from 39 to 156 mg/kg.

No significant increases in the frequencies of micronucleated polychromatic erythrocytes (reticulocytes) were observed in bone marrow of male mice, suggesting that pyrogallol did not induce bone marrow toxicity over the dose ranges tested.

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration
Type of information:
experimental study
Adequacy of study:
weight of evidence
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:
other: The standard three-exposure protocol is described in detail by Shelby et al. (1993)
Deviations:
not specified
GLP compliance:
not specified
Type of assay:
mammalian bone marrow chromosome aberration test
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: not specified
- 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: not specified
- 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: not specified
- Final preparation of a solid: the substance was dissolved into the vehicle

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: not specified
Species:
mouse
Strain:
B6C3F1
Remarks:
B6C3F1/N
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: not specified
- Age at study initiation: not specified
- Weight at study initiation: not specified
- Assigned to test groups randomly: [no/yes, under following basis: ] not specified
- Fasting period before study: not specified
- Housing: not specified
- Diet (e.g. ad libitum): not specified
- Water (e.g. ad libitum): not specified
- Acclimation period: not specified

ENVIRONMENTAL CONDITIONS
- Temperature (°C): not specified
- Humidity (%): not specified
- Air changes (per hr): not specified
- Photoperiod (hrs dark / hrs light): not specified

IN-LIFE DATES: From: To: not specified
Route of administration:
dermal
Vehicle:
- Vehicle(s)/solvent(s) used: pyrogallol was dissolved in phosphate-buffered saline.
- Justification for choice of solvent/vehicle: not specified
- Concentration of test material in vehicle: see below
- Amount of vehicle (if gavage or dermal): not relavant
- Type and concentration of dispersant aid (if powder): not specified
- Lot/batch no. (if required): not specified
- Purity: not specified
Duration of treatment / exposure:
3 months
Frequency of treatment:
not specified
Post exposure period:
not specified
Dose / conc.:
38 other: mg/kg
Dose / conc.:
75 other: mg/kg
Dose / conc.:
150 other: mg/kg
Dose / conc.:
300 other: mg/kg
Dose / conc.:
600 other: mg/kg
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Positive control(s):
not performed
Tissues and cell types examined:
polychromatic erythrocytes
Evaluation criteria:
polychromatic erythrocytes (PCEs, or reticulocytes) were scored for the frequency of micronucleated cells in each of five animals per dose group.
Statistics:
Pairwise comparison of each dosed group with the vehicle control group is significant at P≤0.005.
Significance of micronucleated NCEs/1,000 NCEs tested by the one-tailed trend test; significant at P≤0.025
Key result
Sex:
female
Genotoxicity:
negative
Toxicity:
not examined
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
In a in vivo test, no significant increases in the frequencies of micronucleated erythrocytes, an indicator of chromosomal damage, were observed in peripheral blood of female B6C3F1/N mice treated with pyrogallol (38 to 600 mg/kg) via dermal application for 3 months
Conclusions:
No significant increases in the frequencies of micronucleated erythrocytes, an indicator of chromosomal damage, were observed in peripheral blood of female B6C3F1/N mice treated with pyrogallol (38 to 600 mg/kg) via dermal application for 3 months
Executive summary:

The induction of micronuclei in bone marrow polychromatic erythrocytes of female B6C3F1/N mice were studied. Mice were administered pyrogallol by dermal route, for 3 months (38 to 600 mg/kg).

No significant increases in the frequencies of micronucleated polychromatic erythrocytes (reticulocytes) were observed in bone marrow of female mice, suggesting that pyrogallol did not induce bone marrow toxicity over the dose ranges tested.

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration
Type of information:
experimental study
Adequacy of study:
weight of evidence
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:
other: The standard three-exposure protocol is described in detail by Shelby et al. (1993)
Deviations:
not specified
GLP compliance:
not specified
Type of assay:
mammalian bone marrow chromosome aberration test
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: not specified
- 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: not specified
- 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: not specified
- Final preparation of a solid: the substance was dissolved into the vehicle

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: not specified
Species:
mouse
Strain:
B6C3F1
Remarks:
B6C3F1/N
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: not specified
- Age at study initiation: not specified
- Weight at study initiation: not specified
- Assigned to test groups randomly: [no/yes, under following basis: ] not specified
- Fasting period before study: not specified
- Housing: not specified
- Diet (e.g. ad libitum): not specified
- Water (e.g. ad libitum): not specified
- Acclimation period: not specified

ENVIRONMENTAL CONDITIONS
- Temperature (°C): not specified
- Humidity (%): not specified
- Air changes (per hr): not specified
- Photoperiod (hrs dark / hrs light): not specified

IN-LIFE DATES: From: To: not specified
Route of administration:
dermal
Vehicle:
- Vehicle(s)/solvent(s) used: pyrogallol was dissolved in phosphate-buffered saline.
- Justification for choice of solvent/vehicle: not specified
- Concentration of test material in vehicle: see below
- Amount of vehicle (if gavage or dermal): not relavant
- Type and concentration of dispersant aid (if powder): not specified
- Lot/batch no. (if required): not specified
- Purity: not specified
Duration of treatment / exposure:
3 months
Frequency of treatment:
not specified
Post exposure period:
not specified
Dose / conc.:
38 other: mg/kg
Dose / conc.:
75 other: mg/kg
Dose / conc.:
150 other: mg/kg
Dose / conc.:
300 other: mg/kg
Dose / conc.:
600 other: mg/kg
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Positive control(s):
not performed
Tissues and cell types examined:
polychromatic erythrocytes
Evaluation criteria:
polychromatic erythrocytes (PCEs, or reticulocytes) were scored for the frequency of micronucleated cells in each of five animals per dose group.
Statistics:
Pairwise comparison of each dosed group with the vehicle control group is significant at P≤0.005.
Significance of micronucleated NCEs/1,000 NCEs tested by the one-tailed trend test; significant at P≤0.025
Key result
Sex:
female
Genotoxicity:
negative
Toxicity:
not examined
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
In a in vivo test, no significant increases in the frequencies of micronucleated erythrocytes, an indicator of chromosomal damage, were observed in peripheral blood of female B6C3F1/N mice treated with pyrogallol (38 to 600 mg/kg) via dermal application for 3 months
Conclusions:
In male mice, results of this assay were judged to be equivocal, based on a significant increase in micronucleated erythrocytes observed at a single dose level (300 mg/kg) at the end of the 3-month study period.
Executive summary:

The induction of micronuclei in bone marrow polychromatic erythrocytes of male B6C3F1/N mice were studied. Mice were administered pyrogallol by dermal route for 3 months from 38 to 600 mg/kg.

In male mice, results of this assay were judged to be equivocal, based on a significant increase in micronucleated erythrocytes observed at a single dose level (300 mg/kg) at the end of the 3-month study period. No significant alteration in the percentage of PCEs in bone marrow or blood was observed, suggesting that pyrogallol did not induce bone marrow toxicity over the dose ranges tested.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Justification for classification or non-classification

The harmonized classification for pyrogallic acid is Muta. 2, with the hazard statement H341 “Suspected to causing genetic defects”.

In the light of the results obtained through the in vitro and the in vivo genotoxicity tests, pyrogallol seems to act as an in vitro genotoxin. In fact, the findings are ambiguous because the positive results of the in vitro tests are not confirmed by the in vivo tests.