Registration Dossier
Registration Dossier
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EC number: 204-617-8 | CAS number: 123-31-9
Endpoint summary
Administrative data
Key value for chemical safety assessment
Genetic toxicity in vitro
Link to relevant study records
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- comparable to guideline study
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
- GLP compliance:
- no
- Type of assay:
- mammalian cell gene mutation assay
- Target gene:
- Thymidine kinase locus
- Species / strain / cell type:
- mouse lymphoma L5178Y cells
- Details on mammalian cell type (if applicable):
- - Type and identity of media: Fischer's medium with 5% heat-inactivated horse serum
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically "cleansed" against high spontaneous background: yes - Metabolic activation:
- with and without
- Metabolic activation system:
- S9 mix from Aroclor 1254 induced rat liver
- Test concentrations with justification for top dose:
- - S9: 1.56, 3.12, 6.25, 12.5, 25, 50 µg/mL
+ S9: 0.652, 1.25, 2.5, 5, 10 µg/mL - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: DMSO (+ S9), methanol (- S9)
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: - S9: EMS; + S9: EMS or 3-methylcholanthrene
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium
DURATION
- Exposure duration: 4 h
- Expression time (cells in growth medium): 2 d
- Selection time (if incubation with a selection agent): 11-14 d
- Fixation time (start of exposure up to fixation or harvest of cells): 13-16 d
SELECTION AGENT (mutation assays): trifluorothymidine
NUMBER OF REPLICATIONS: 2, two independent assays
DETERMINATION OF CYTOTOXICITY
- Method: relative total growth - Evaluation criteria:
- Positive response: dose-related trend and statistically significant response at one of the three highest doses
- Statistics:
- Dose trend test (Barlow et al., 1972); variance analysis (pairwise comparisons) with P < 0.05
- Species / strain:
- mouse lymphoma L5178Y cells
- Metabolic activation:
- with and without
- Genotoxicity:
- positive
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
- Conclusions:
- Positive with and without metabolic activation.
In the mouse lymphoma assay for induction of trifluorothymidine resistance in L5178Y/TK cells, HQ was positive at doses of 1.25 µg/mL and higher in the absence of S9, and at 2.5 µg/mL and higher in the presence of S9. - Executive summary:
Hydroquinone was tested in a mouse lymphoma mutation assay for induction of trifluorothymidine resistance in L5178Y/TK cells with a test protocol similar to OECD Guideline 476. Test concentrations of HQ ranged from 0.625-50 µg/mL without S9 mix and 0.625-10 µg/mL with S9 mix. Treatment time was 4 h, followed by a 2-day expression period and a 11 to 14-day selection period in the presence of TFT. Cytotoxicity was indicated by relative total growth. The lowest effective dose with a significantly increased mutant fraction was 1.25 µg/mL without S9 (relative total growth 32%), and 2.5 µg/mL with S9 (relative total growth 87%). The mutant fraction was statistically significantly and dose-dependantly increased from these concentrations up. HQ was positive in the mouse lymphoma assay both in the presence and absence of S9.
- Endpoint:
- in vitro cytogenicity / chromosome aberration study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- study well documented, meets generally accepted scientific principles, acceptable for assessment
- Remarks:
- Study meets basic scientific principles as a chromosome aberration study with the following deviations from guideline conditions: single assay with evaluation of only 100 cells per test concentration, no differentiated evaluation of types of chromosomal aberrations; sufficient documentation of test conditions and test results; study acceptable as key study
- Principles of method if other than guideline:
- Chromosome aberration test according to standard conditions single assay with evaluation of only 100 cells per test concentration, no differentiated evaluation of types of chromosomal aberrations
- GLP compliance:
- no
- Type of assay:
- in vitro mammalian chromosome aberration test
- Species / strain / cell type:
- Chinese hamster Ovary (CHO)
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9 mix from Aroclor 1254 pretreated rat liver
- Test concentrations with justification for top dose:
- - S9: 0, 5, 7.5, 10, 20 µg/mL
+S9: 0, 150, 450, 600 µg/ml - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: DMSO
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: - S9: mitomycin C; + S9: cyclophosphamide
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium
DURATION
- Exposure duration: -S9: 8.5 h; + S9: 2 h
- Fixation time (start of exposure up to fixation or harvest of cells): 10.5 h
SPINDLE INHIBITOR (cytogenetic assays): colcemid
STAIN (for cytogenetic assays): Giemsa
NUMBER OF REPLICATIONS: no
NUMBER OF CELLS EVALUATED: 100; 50 for the high dose -S9
DETERMINATION OF CYTOTOXICITY
- Method: mitotic index
OTHER EXAMINATIONS:
- Determination of endoreplication: yes - Evaluation criteria:
- Positive test result: total aberrations adjusted P-value <= 0.05 (Dunnett's t-test) for at least 2 test concentrations and additionally P-value for trend test P>=0.015, 0.015 > P >= 0.03, or P < 0.03
Weakly positive test result: total aberrations adjusted P-value <= 0.05 (Dunnett's t-test) for 1 test concentration and additionally P-value for trend test 0.015 > P >= 0.03, or P < 0.03
Questionable test result: total aberrations adjusted P-value <= 0.05 (Dunnett's t-test) for 1 test concentration and additionally P-value for trend test P>=0.015, or no significant effect for any test concentration but P-value for trend test P < 0.03
Negative test result: no significant effect for any test concentration and P-value for trend test P>=0.015, or 0.015 > P >= 0.03 - Statistics:
- Trend test by linear regression analysis of the percentage of cells with aberrations vs. log dose; statistical significance by Dunnett's t-test
- Species / strain:
- Chinese hamster Ovary (CHO)
- Metabolic activation:
- without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- higher test concentration reported to be cytotoxic (no further data)
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
- Species / strain:
- Chinese hamster Ovary (CHO)
- Metabolic activation:
- with
- Genotoxicity:
- positive
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- higher test concentration reported to be cytotoxic (no further data)
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
- Conclusions:
Negative without metabolic activation. Positive with metabolic activation
Hydroquinone was clastogenic in CHO cells after metabolic activation only as demonstrated by an increased frequency of cells with structural chromosomal aberrations.- Executive summary:
Hydroquinone was tested in a chromosome aberration assay with Chinese hamster ovary cells both with and without metabolic activation at test concentrations of 5-20 µg/mL and 150-600 µg/mL, respectively. Treatment times were 8.5 h without S9 and 2 h with S9 mix prepared from Aroclor 1254 induced rat liver. Cytotoxicity was reported to occur at the higher test concentrations (no further data).
In the absence of metabolic activation, the frequency of structural chromosomal aberrations was statistically significantly increased at 20 µg/mL, but the test result was negative due to lack of a significant dose-response relationship. With metabolic activation, increased frequencies of structural chromosomal aberrations occurred at 450 and 600 µg/mL (P ≤ 0.05) fulfilling the defined criteria for a positive test result.
- Endpoint:
- in vitro cytogenicity / chromosome aberration study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 11 October - 10 December 1999
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- in vitro mammalian chromosome aberration test
- Species / strain / cell type:
- lymphocytes: from human donor
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9 mix from Aroclor 1254 induced rat liver
- Test concentrations with justification for top dose:
- Test 1A: 0, 20, 30, 50 µg/mL (180, 270, 450 µM)
Test 1B: 0, 7.5, 10, 15 µg/mL (68, 90, 135 µM)
Test 2A: 0, 60, 80, 100 µg/mL (540, 720, 900 µM)
Test 2B: 0, 100 µg/mL (900 µM)
Test 2C: 0, 5, 7.5, 10 µg/mL ((45, 68, 90 µM)
Test 2D: 0, 10 µg/mL (900 µM) - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: none (test solution prepared in cell culture medium)
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- Positive controls:
- yes
- Positive control substance:
- other: - S9: mitomycin C; + S9: cyclophosphamide
- Details on test system and experimental conditions:
- Two separate tests were performed with diffferent combinations of presence/absence of metabolic activation, of exposure duration (short-term or continuous treatment), and harvest times (24 or 48 h).
CELLS: freshly isolated human lymphocytes from one healthy nonsmoking male donor
METHOD OF APPLICATION: in medium
METABOLIC ACTIVATION: - S9: 1A, , 1B, 2C, 2D; + S9: 1A, 2A, 2B
DURATION
- Exposure duration: short time treatments of 3 h: Test 1A, 2A, 2B; Continous treatments of 24 h: Test 1B, 2C; Continous treatment of 48h: Test 2D
- Harvest time (start of exposure up to fixation or harvest of cells): 24 h: Test 1A, 1B, 2A, 2C; 48h: 2B, 2D
SPINDLE INHIBITOR (cytogenetic assays): colcemid
STAIN (for cytogenetic assays): 2% Giemsa
NUMBER OF REPLICATIONS: 2
NUMBER OF CELLS EVALUATED: 100 per culture = 200 per test concentration
DETERMINATION OF CYTOTOXICITY
- Method: mitotic index - Evaluation criteria:
- Selection criteria:
If possible the highest test concentration should reduce the mitotic index by at least 50% but no more than 70% when compared to the negative contr.
ol.
The lowest test concentration should be on the borderline of mitotic inhibition.
Validity of chromosome aberration test:
If positive controls give statistically significant increase and if negative controls are in the range of historical control values, and if at least 160 cells are analyzable at the intended concentration level.
Positive response: Percentage of cells with structural aberrations significantly above background (p<0.05) and is at least 4%
Clastogenicity: Test substance considered to be clastogenic, if dose-related increase in the percentage of cells with structural aberrations over concurrent control frequencies, or if a single positive test point is observed in both assays (replicates).
Equivocal response: Positive response at a single test point in only the second assay, and if the positive response is higher than historical range for negative controls.
Negative response: neither a dose-related increase in the percentage of cells with structural aberrations over concurrent control frequencies, or a positive test result at a single test point in both assay or in the second assay only.
Cells excluded from assessment of clastogenicity:
Cells with only gaps, heavily damaged cells (cells with multiple aberrations), and cells with polyploid and endoreduplication recorded separately and not included in the assessment.
Both statistical significance and biological relevance are considered in the final evaluation. - Statistics:
- Test for statistical significant differences from negative controls by Fisher's exact probability test (two-sided)
- Species / strain:
- other: lymphocytes from a healthy male human donor
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- other: Test system: all strains/cell types tested
- Conclusions:
Negative with and without metabolic activation.
In neither of the chromosome aberration assays, both in the absence and presence of metabolic activation, did HQ produce a biologically relevant or statistically significant increase in the number of cells with structural aberrations at any test concentration and any timepoint analyzed, compared to negative control values.- Executive summary:
Hydroquinone was tested in a chromosome aberration assay with primary cultures of human lymphocytes from a healthy nonsmoking male donor according to OECD Guideline 473 (GLP study). Different combinations of test conditions were used with regard to metabolic activation (+/- S9), treatment time (short-term treatment for 3 h, continuous treatment for 24 and 48 h), and harvest time (24 or 48 h). Test concentrations of HQ ranged from 2-100 µg/mL for 3 h treatments, 5-15 µg/mL for 24 h treatments, and 10 µg/mL for 48 h treatments. Cytotoxicity was indicated at the higher test concentrations of the individual assays by reduction of relative mitotic indices to 57 to 43%. Frequencies of structural chromosomal aberrations ranged from 0% to 2.5% (single value of 4.0%) in the upper concentration range of HQ and up to 1.5% in negative controls, indicating absence of clastogenic effects.
In neither of the chromosome aberration assays, both in the absence and presence of metabolic activation, did HQ produce a biologically relevant or statistically significant increase in the number of cells with structural aberrations at any test concentration and any timepoint analyzed, compared to negative control values.
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- study well documented, meets generally accepted scientific principles, acceptable for assessment
- Remarks:
- Study report meets basic scientific principles of a standard mutation assay (Ames test) performed as micromethod: 10 concentrations up to cytotoxicity tested, valid negative and positive controls in the range of specified historical control data, standard evaluation criteria (comparable to OECD Guideline 471) were used. Tested bacteria strains included both indicators of reverse base pair substitutions and frameshift mutations (TA98, TA, TA1537) as well as an indicator of oxidative DNA damage (TA102). Test protocol and test results documented in detail; study acceptable as key study
- Qualifier:
- no guideline followed
- Principles of method if other than guideline:
- Screening assay performed in micromethod according to the principles of the standard Ames technique
- GLP compliance:
- no
- Type of assay:
- bacterial reverse mutation assay
- Species / strain / cell type:
- S. typhimurium, other: TA98, TA100, TA102, TA1537
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9 mix from Aroclor 1254 induced rat liver or kidney
- Test concentrations with justification for top dose:
- 0, 0.38, 1.14, 3.43, 10.29, 20.86, 92.59, 277.78, 833.33, 2500, 5000 µg/plate
- Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: solubility - Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: without S9: TA98, 2-nitrofluorene; TA100, MNNG; TA102, mitomycin C; TA1537, 9-aminoacridine; with liver S9: TA98, TA100, TA1537, 2-aminoanthracene; TA102, benzo[a]pyrene; with Kidney S9: TA98, TA100, TA102: streptozotocin; TA1537: artistolochic acids
- Remarks:
- Screening assay performed in micromethod
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in agar (plate incorporation on microplates); preincubation (on microplates)
DURATION
- Preincubation period: 90 min
- Exposure duration: 48 h
SELECTION AGENT (mutation assays): histidine
NUMBER OF REPLICATIONS: each dose in triplicate, no repetition of the assay
DETERMINATION OF CYTOTOXICITY
- Method: relative total growth - Evaluation criteria:
- Induction ratio: mean number of revertants per plate in presence of the concentration of the test compound / mean number of revertants per plate of the solvent control
Criteria for positive test results:
TA1537: dose-response for at least 3 test concentrations, and for the highest increase an induction ratio of at least 3
TA98, TA100, TA102: dose-response for at least 3 test concentrations, and for the highest increase an induction ratio of at least 2
Validity criteria for the test:
Solvent controls: the mean frequency of revertants of the actual solvent control is between the limits of the historical negative control values for each strain +/- S9 mix (historical control data included in the study report for January - December 2005)
Positive controls: the mean frequency of revertants induced by the reference substance for each strain +/- S9 mix must be higher than the lower limits of the historical positive control values (historical control data included in the study report for January - December 2005) - Statistics:
- Dunnett's t-test for statistical differences of revertant rates
- Species / strain:
- S. typhimurium TA 98
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- at 2500 and 5000 µg/plate (all strains)
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
- Species / strain:
- S. typhimurium TA 100
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- at 2500 and 5000 µg/plate (all strains)
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
- Species / strain:
- S. typhimurium TA 102
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- at 2500 and 5000 µg/plate (all strains); from 278 to 5000 µg/plate in TA102 without S9
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
- Species / strain:
- S. typhimurium TA 1537
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- at 2500 and 5000 µg/plate (all strains)
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
- Species / strain:
- E. coli WP2 uvr A
- Metabolic activation:
- not applicable
- Genotoxicity:
- not determined
- Cytotoxicity / choice of top concentrations:
- not determined
- Additional information on results:
- EVALUATION ACCORDING TO STATISTICAL SIGNIFICANCE AND THE DEFINED CRITERIA:
TA100 without metabolic activation: statistically significant increase of revertants (P<0.01) at 833 µg/plate. In the absence of a dose-relationship and an induction ratio of 1.2 criteria for a positive test result were not fulfilled, however. The increases were not attributable to a mutagenic effect.
TA102 without metabolic activation: statistically significant increases of revertants (all P<0.01) at concentrations ranging from 0.38 - 30.8 µg/plate with induction ratios of 1.5, 1.9, 1.7, 1.5, and 1.2. At higher test concentrations cytotoxicity was observed. In the absence of a dose-relationship (increase of revertant rate with increasing concentration) and a maximal induction ratio of 1.9 criteria for a positive test result were not fulfilled, however. The increases were not attributable to a mutagenic effect.
TA102 with metabolic activation: statistically significant increases of revertants (all P<0.01) at concentrations 3.43 and 10.29 µg/plate with induction ratios of 1.4 and 1.5. In the absence of a clear dose-relationship and a maximal induction ratio of 1.5 criteria for a positive test result were not fulfilled, however. The increases were not attributable to a mutagenic effect.
COMPARISON WITH HISTORICAL CONTROL DATA:
Actual values of negative and positive controls were within the limits of the historical controls. - Conclusions:
- Negative with and without metabolic activation.
Under the conditions of a screening micromethod assay of the Ames test there was no indication of a mutagenic activity in Salmonella typhimurium strains TA98, TA100, TA102, and TA1537 both in the absence or the presence of a metabolic activation system with liver or kidney S9 mix. - Executive summary:
Hydroquinone was tested in a screening micromethod assay of the Ames test with the Salmonella typhimurium strains TA98, TA100, TA102, and TA1537 at ten concentrations ranging from 0.38 to 5000 µg/plate. Metabolic activation systems included S9 mix from liver and kidneys of Aroclor 1254 pretreated rats. Preincubation was for 90 min and revertant rates were evaluated after 48 h. Solvent controls and strain-specific positive controls were within the historical control ranges of the testing laboratory. Cytotoxicity was generally observed at 2500 and 5000 µg/plate, and with strain TA102 in the absence of metabolic activation from 278 µg/plate up. There were no increases of revertant rates in any test strains that showed a clear dose response relationship and fulfilled the criteria of inductions rates of at least 2.0 for strains TA98, TA100, and TA102, and of 3.0 for strain TA1537.
Under the conditions of this assay there was no indication of a mutagenic activity of HQ in Salmonella typhimurium strains TA98, TA100, TA102, and TA1537 both in the absence or the presence of a metabolic activation system with liver or kidney S9 mix up to cytotoxic test concentrations.
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- comparable to guideline study with acceptable restrictions
- Remarks:
- Study is comparable to guideline study with the following acceptable restrictions: other positive control substance used for TA98; only 4 bacteria strains tested, no strain used sensitive to oxidative damage; sufficient documentation of testing procedure and test results; study acceptable as key study for mutagenicity testing in strains TA98, TA100, TA1535, and TA1537
- Reason / purpose for cross-reference:
- reference to same study
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Deviations:
- yes
- Remarks:
- (other positive control for TA98; only 4 bacteria strains tested, no strain used sensitive to oxidative damage)
- GLP compliance:
- not specified
- Type of assay:
- bacterial reverse mutation assay
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
- Metabolic activation:
- with and without
- Metabolic activation system:
- Aroclor 1254-induced S9 from male Syrian hamster liver or male Sprague-Dawley rat liver
- Test concentrations with justification for top dose:
- 0, 10, 33, 100, 333, 666 µg/plate
- Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: solubility - Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: without S9: 4-nitro-o-phenylenediamine with TA98, sodium azide with TA100 and TA1535, 9-aminoacridine with TA1537; with S9: 2-aminoanthracene with all strains
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in agar (plate incorporation); preincubation
DURATION
- Preincubation period: 20 min
- Exposure duration: 48 h
SELECTION AGENT (mutation assays): histidine
NUMBER OF REPLICATIONS: 3 plates/assay, and 2 assays
DETERMINATION OF CYTOTOXICITY
- Method: viability or reduced number of revertant colonies per plate - Evaluation criteria:
- Positive response indicated by reproducible, dose-related increase, whether it would be two-fold over background or not
- Species / strain:
- E. coli WP2 uvr A
- Metabolic activation:
- not applicable
- Genotoxicity:
- not determined
- Cytotoxicity / choice of top concentrations:
- not determined
- Species / strain:
- S. typhimurium TA 100
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Remarks:
- revertant rates similar to controls at all tested concentrations
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- in all strains without metabolic activation from 333 µg/plate up
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Species / strain:
- S. typhimurium TA 98
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Remarks:
- revertant rates similar to controls at all tested concentrations
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- in all strains without metabolic activation from 333 µg/plate up
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Species / strain:
- S. typhimurium TA 1537
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Remarks:
- revertant rates similar to controls at all tested concentrations
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- in all strains without metabolic activation from 333 µg/plate up
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Species / strain:
- S. typhimurium TA 1535
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Remarks:
- revertant rates similar to controls at all tested concentrations
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- in all strains without metabolic activation from 333 µg/plate up
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Conclusions:
Negative with and without metabolic activation.
There was no indication of a mutagenic activity in S. typhimurium strains TA98, TA100, TA1535, and TA1537 with and without metabolic activation.- Executive summary:
Hydroquinone (10 - 666 µg/plate) was tested in a Salmonella typhimurium reverse mutation assay with strains TA98, TA100, TA1535, and TA1537 applying a preincubation test protocol comparable to OECD Guideline 471. There was no indication of mutagenic activity up to cytotoxic concentrations (333 and 666 µg/plate) both in the absence and presence of metabolic activation (S9 mix from Aroclor 1254 -induced hamster or rat liver).
Referenceopen allclose all
Table: Results of the mouse lymphoma cell mutation assay without and with metabolic activation
Metabolic activation / trial |
Test concentration |
Cloning efficiency (%) |
Relative total growth (%) |
Mutant fraction |
Average mutant fraction |
- S9 / trial 1 |
0 |
47 |
68 |
73 |
74 |
35 |
57 |
96 |
|||
48 |
164 |
62 |
|||
60 |
110 |
65 |
|||
1.562 |
41 |
45 |
200 |
157* |
|
42 |
108 |
113 |
|||
3.125 |
25 |
23 |
454 |
333* |
|
42 |
50 |
213 |
|||
6.25 |
17 |
7 |
857 |
624* |
|
25 |
13 |
391 |
|||
12.5 |
TOX |
||||
TOX |
|||||
Positive control |
47 |
101 |
214 |
197* |
|
45 |
43 |
181 |
|||
- S9 / trial 2 |
0 |
76 |
96 |
19 |
22 |
61 |
100 |
28 |
|||
93 |
98 |
18 |
|||
85 |
106 |
24 |
|||
3.125 |
13 |
5 |
565 |
419* |
|
57 |
30 |
274 |
|||
6.25 |
48 |
13 |
542 |
542* |
|
37 |
9 |
541 |
|||
12.5 |
27 |
7 |
871 |
857* |
|
25 |
7 |
843 |
|||
25 |
28 |
6 |
863 |
774* |
|
30 |
7 |
685 |
|||
50 |
TOX |
||||
TOX |
|||||
Positive control |
120 |
114 |
108 |
108 |
|
- S9 / trial 3 |
0 |
93 |
114 |
19 |
38 |
62 |
109 |
47 |
|||
52 |
90 |
42 |
|||
90 |
87 |
45 |
|||
0.625 |
81 |
68 |
47 |
49 |
|
102 |
71 |
51 |
|||
1.25 |
36 |
22 |
164 |
179* |
|
27 |
16 |
195 |
|||
2.5 |
15 |
7 |
607 |
624* |
|
12 |
4 |
642 |
|||
5 |
TOX |
||||
12 |
1 |
1915 |
|||
10 |
TOX |
||||
TOX |
|||||
Positive control |
69 |
66 |
299 |
306* |
|
93 |
58 |
313 |
|||
+ S9 / trial 1 |
0 |
91 |
99 |
65 |
70 |
70 |
102 |
67 |
|||
68 |
98 |
70 |
|||
79 |
100 |
77 |
|||
0.625 |
93 |
116 |
67 |
72 |
|
90 |
133 |
78 |
|||
1.25 |
81 |
105 |
69 |
78 |
|
72 |
99 |
86 |
|||
2.5 |
75 |
81 |
138 |
134* |
|
98 |
93 |
130 |
|||
5 |
61 |
20 |
338 |
328* |
|
70 |
18 |
318 |
|||
10 |
43 |
7 |
485 |
464* |
|
44 |
7 |
582 |
|||
Positive control |
55 |
57 |
423 |
451* |
|
45 |
47 |
480 |
* Statistically significant increase compared to vehicle controls, P < 0.05
Table : Induction of chromosomal aberrations in CHO cells
Metabolic activation |
Test concentration (µg/mL) |
Total cells evaluated |
Number of aberrations |
Aberrations per cell |
Percent cells with aberrations |
||
Total aberrations |
Simple aberrations |
Complex aberrations |
|||||
- S9 |
0 |
100 |
3 |
0.03 |
3.0 |
0.0 |
3.0 |
100 |
3 |
0.03 |
3.0 |
0.0 |
3.0 |
||
5 |
100 |
2 |
0.02 |
2.0 |
1.0 |
1.0 |
|
7.5 |
100 |
2 |
0.02 |
2.0 |
1.0 |
1.0 |
|
10 |
100 |
4 |
0.04 |
4.0 |
2.0 |
1.0 |
|
20 |
50 |
5 |
0.10 * |
8.0 |
6.0 |
4.0 |
|
Positive control |
50 |
10 |
0.20 * |
20.0 |
12.0 |
8.0 |
|
+ S9 |
0 |
100 |
1 |
0.01 |
1.0 |
0 |
1.0 |
150 |
100 |
5 |
0.05 |
4.0 |
1.0 |
3.0 |
|
450 |
100 |
22 |
0.22 * |
17.0 |
12.0 |
9.0 |
|
600 |
100 |
29 |
0.29 * |
19.0 |
7.0 |
8.0 |
|
Positive control |
50 |
10 |
0.20 * |
18.0 |
12.0 |
8.0 |
|
* statistically significant increase (P <= 0.05, Dunnett's t-test)
Table: Overview of test conditions and test results of the different assays of the chromosome aberration tests in primary human lymphocytes
Test |
Treatment time (h) |
Harvest time (h) |
S9 |
Test concentration |
CAstr |
CAnum or gaps |
Relative MI (%) |
1A |
3 |
24 |
- |
0 |
0 |
0 |
100 |
20 |
1.0 |
0 |
86 |
||||
30 |
0.0 |
0 |
74 |
||||
50 |
0.5a |
0 |
57 |
||||
PC |
17.0* |
0 |
72 |
||||
+ |
0 |
0.5a |
0 |
100 |
|||
20 |
0.5a |
0 |
92 |
||||
30 |
0.5a |
0 |
68 |
||||
50 |
0.5a |
0 |
62 |
||||
PC |
22.0* |
0 |
71 |
||||
1B |
24 |
24 |
- |
0 |
0 |
0 |
100 |
7.5 |
0.5a |
0 |
95 |
||||
10 |
0.0 |
0 |
72 |
||||
15 |
1.0a |
0 |
45 |
||||
PC |
15.0* |
0 |
65 |
||||
2A |
3 |
24 |
+ |
0 |
1.5a |
0.5 (gap) |
100 |
60 |
0.0 |
1.0 (gap) |
67 |
||||
80 |
2.5b |
0.5 (gap) |
47 |
||||
100 |
4.0c |
0 |
52 |
||||
PC |
33.0* |
0 |
56 |
||||
2B |
3 |
48 |
+ |
0 |
0.0 |
0.5 (gap) |
100 |
100 |
2.5d |
2.5 (gap) |
45 |
||||
2C |
24 |
24 |
- |
0 |
0.0 |
0 |
100 |
5 |
0.5e |
1 (gap) |
68 |
||||
7.5 |
0.5e |
1 (gap) |
54 |
||||
10 |
2.0f |
1.5 (gap) |
43 |
||||
PC |
18.5* |
0 |
43 |
||||
2D |
48 |
48 |
- |
0 |
0.0 |
1 |
100 |
10 |
0.0 |
0.5 (gap) |
53 |
CAstr: structural chromosomal aberrations in 200 cells; CAnum or gaps: numerical chromosomal aberrations or gaps in 200 cells; MI: mitotic index, PC: positive control
* statistically significant increase (P<0.001)
Specification of structural chromosomal aberrations:
a chromosome break;
b 1% chromatid break and 1.5% chromosome break;
c 1.5% chromatid break, 0.5% chromatid exchange, and 2% chromosome break
d 0.5% chromatid break, 0.5% chromatid exchange, and 1.5% chromosome break
e 0.5% chromatid break;
f 0.5% chromatid break and 1.5% chromosome break
Genetic toxicity in vivo
Link to relevant study records
- Endpoint:
- in vivo mammalian cell study: DNA damage and/or repair
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- study well documented, meets generally accepted scientific principles, acceptable for assessment
- Remarks:
- Study meets basic scientific principles as a study on induction of DNA adducts in rat kidney at treatment conditions inducing nephrotoxic effects, study to investigate possible mechanisms of neoplastic effects at a target tissue; sufficient documentation; publication acceptable as key study for assessment of genotoxic effects
- Reason / purpose for cross-reference:
- reference to other study
- Qualifier:
- no guideline available
- Principles of method if other than guideline:
- DNA adducts in kidneys of F344 rats analyzed by 32P-postlabeling technique with separation by TLC and detection by audioradiography
- GLP compliance:
- no
- Type of assay:
- other: Identification of DNA adducts
- Species:
- rat
- Strain:
- Fischer 344
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Charles River, Stone Ridge, Kingston, N.Y.
- Age at study initiation: 10-12 w
- Weight at study initiation:
- Assigned to test groups randomly: yes
- Housing: group-housed
- Diet: Agway RMH Prolab 3000 or Agway RMH Prolab 3200 meal ad libitum
- Water: ad libitum
- Acclimation period: 5 d
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-23
- Humidity (%): 42-49
- Photoperiod (hrs dark / hrs light): 12 / 12 - Route of administration:
- oral: gavage
- Vehicle:
- - Vehicle(s)/solvent(s) used: water
- Concentration of test material in vehicle: 0.5, 5 or 10 mg/mL
- Amount of vehicle (if gavage or dermal): 5 mL/kg - Duration of treatment / exposure:
- 6 w
- Frequency of treatment:
- 5 d/w
- Post exposure period:
- sacrifice 2 h after last dose administration
- Dose / conc.:
- 0 mg/kg bw/day (actual dose received)
- Dose / conc.:
- 2.5 mg/kg bw/day (actual dose received)
- Dose / conc.:
- 25 mg/kg bw/day (actual dose received)
- Dose / conc.:
- 50 mg/kg bw/day (actual dose received)
- No. of animals per sex per dose:
- 4
- Control animals:
- yes, concurrent vehicle
- Positive control(s):
- not applicable
- Details of tissue and slide preparation:
- CRITERIA FOR DOSE SELECTION: high dose level of the carcinogenesis bioassay; 10-fold lower dose expected to a NOEL
SAMPLING TIMES: 2 h after last gavage dose
PREPARATION OF KIDNEY DNA:
Animals were sacrificed 2 h after last gavage dose and kidneys were excised and homogenized. The homogenate was homogenized and centrifuged at 600 g for 5 min to obtain a crude nuclear pellet. DNA was isolated by an abbreviated protocol (Reddy and Randerath, 1987), except that after ribonuclease and proteinase D treatments, the homogenate was transferred to the 341 DNA purification system (Applied Biosystems, USA) for automated extraction and precipitation. DNA precipitate contained on filter was extracted into water, and DNA concentration
deterined spectrophotometrically at 260 nm.
METHOD OF ANALYSIS:
32P-postlabeling performed by the monophosphate version of the nuclease P1-enhanced 32P-postlabeling assay (Randerath et al., 1989; Reddy et al., 1990). 32P-labeled adducts were resolved by multidirectional polyethyleneimine-cellulose TLC and detected by audioradiography. The audioradiograms of treated DNA samples were examined for the presence of extra spots or zones indicative of adducts by comparing with the audioradiograms of control DNA samples. In addition, background adducts were evaluated to ensure that there was no increase in their activity due to comigration of exposure-specific adducts.
To determine of adduct radioactivity, the PEI-cellulose was wet with ethanol, scraped from the plastic backing, and counted by Cerenkov asay (without addition of liquid scintillator fluid). Adduct levels expressed as Relative Adduct Level (RAL), calculated from count rates and specific activity (Reddy and Randerath, 1986).
Value of n for (RAL x 10exp9) corrrespondes to n adducts per 10exp9 DNA nucleotides or 0.003 x n femtomoles of adduct per microgram of DNA
Lower limit of detection: 1 adduct in 10exp9 to 10exp10 DNA nucleotides
Preparation of DNA reference samples: DNA modified with HQ in vitro was isolated from cultured rat Zymbal glands exposed to 0.7 mg of HQ per mL medium for 48 h. BQ-adducted DNA was prepared by in vitro reaction of BQ with untreated rat kidney DNA. Modification levels in terms of RAL x 10exp9 were 900-1300 adducts for HQ-treated DNA, and 1300-1700 adducts for BQ-treated DNA. - Statistics:
- Student's t-test for statistical significance of adduct levels between control and treated groups.
- Sex:
- male/female
- Genotoxicity:
- negative
- Toxicity:
- yes
- Remarks:
- mild nephrotoxic effect
- Vehicle controls validity:
- valid
- Positive controls validity:
- not applicable
- Additional information on results:
- 50 mg/kg dose-group (for details see Table):
- HQ-specific adducts: level in male F344 rats comparable to control, significant decrease in female F344 rats compared to vehicle controls
- BQ-specific adducts: level in male and female F344 rats comparable to controls
- Background adducts: significant decreases of adducts 2, 3 and 8 in male F344 rats compared to vehicle controls, significant decreases of adducts 4 and 8 in male F344 rats compared to vehicle controls
- Total adduct level: non-significant increase in treated rats compared to vehicle controls
- Clinical biochemistry measurements: mild nephrotoxic effect indicated by 36% increase in urinary excretion of NAG, statistically significant with P < 0.01; no other changes of urinary or serum parameters
5 and 25 mg/kg dose-groups:
- Adduct levels: As there was no effect at the high dose, analysis of DNA adducts was not performed at this dose level.
- Clinical biochemistry measurements: no significant changes of any parameter - Conclusions:
- Negative
There was no increased formation of HQ-specific or BQ-specific DNA adducts, as detected by the Nuclease P1-enhanced 32P-postlabeling assay, in male F344 rats at treatment conditions that showed evidence of concurrent kidney toxicity indicated by enzymuria, cell proliferation and histopathologic changes. Under these conditions, treated rats showed no new or increased levels of nuclear DNA adducts compared to control rats. On the contrary, there were significant reductions in levels of some endogenous adducts, possibly by virtue of the antioxidant properties of HQ. These data suggest that benign kidney tumours observed in the 2-year carcinogenesis bioassay with male F344 rats after gavage application of HQ are produced via a non-genotoxic mechanism. - Executive summary:
The formation of DNA adducts in kidneys of groups of 4 male and 4 female F344 rats was investigated by the Nuclease P1-enhanced 32P-postlabeling assay. Rats were treated by gavage with 0, 2.5, 25 and 50 mg HQ/kg bw/d for 6 wk, on 5 d/wk. Analysis of DNA adducts included HQ-specific and BQ-specific adducts as well as changes in the levels of endogenous background adducts. Additionally, in the same animals, indicators of nephrotoxicity (urinary enzymes, blood urea nitrogen, serum creatinine, microscopic analysis of urine) and hepatotoxicity (serum aspartate transaminase (AST), alanine transaminase (ALT)) were measured.
Adduct levels were only analysed at 50 mg/kg, as there was no increased formation of HQ-specific or BQ-specific DNA adducts in F344 rats. On the contrary, a significant decrease of HQ-specific DNA adducts was observed in female F344 rats, and there were significant reductions in some endogenous adducts in both sexes: Total adduct levels as detectible by the Nuclease P1-enhanced 32P-postlabeling assay were reduced compared to vehicle controls. At 50 mg/kg bw/d, a mild nephrotoxic effect was indicated by a 36% increase in urinary excretion of NAG (statistically significant, P < 0.01). Further, nephrotoxicity was indicated by additional findings from a simultaneous study with the same treatment schedule (English et al., 1994). There HQ was shown to induce cell proliferation in the kidneys of male F344 rats accompanied by mild toxicity as indicated by urinary parameters and histopathology. These effects were not observed in female F344 rats. The data suggest that benign kidney tumours observed in the 2-year carcinogenesis bioassay with male F344 rats after gavage application of HQ are produced via a non-genotoxic mechanism.
- Endpoint:
- in vivo mammalian germ cell study: cytogenicity / chromosome aberration
- Remarks:
- Type of genotoxicity: chromosome aberration
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- comparable to guideline study with acceptable restrictions
- Remarks:
- Study with a test protocol similar to OECD Guideline 483 with an acceptable restriction: no positive control included in the assay; less well but sufficiently documented; study acceptable as key study for evaluation of genetic toxicity in vivo
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 483 (Mammalian Spermatogonial Chromosome Aberration Test)
- Deviations:
- yes
- Remarks:
- (no positive control included)
- GLP compliance:
- no
- Type of assay:
- mammalian germ cell cytogenetic assay
- Species:
- mouse
- Strain:
- other: (102/E1 X C3H/E1) F1
- Sex:
- male
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: no data
- Age at study initiation: 12-14 w
- Weight at study initiation: 26-28 g
- Assigned to test groups randomly: yes
ENVIRONMENTAL CONDITIONS: no data - Route of administration:
- intraperitoneal
- Vehicle:
- - Vehicle(s)/solvent(s) used: water
- Duration of treatment / exposure:
- single
- Post exposure period:
- 24 h until sacrifice
- Dose / conc.:
- 0 mg/kg bw/day (nominal)
- Dose / conc.:
- 40 mg/kg bw/day (nominal)
- Dose / conc.:
- 80 mg/kg bw/day (nominal)
- Dose / conc.:
- 120 mg/kg bw/day (nominal)
- No. of animals per sex per dose:
- 5
- Control animals:
- yes, concurrent vehicle
- Positive control(s):
- no
- Details of tissue and slide preparation:
- CRITERIA FOR DOSE SELECTION: not specified
SAMPLING: Intraperitoneal injection of colchicine (4 mg/kg) 3.5-5 h prior to sacrifice; at sacrifice 24 h after application testes were excised
DETAILS OF SLIDE PREPARATION: according to method of Adler (1984), stain with acetic orcein solution
EVALUATION:
Chromosome aberrations counted in 100 cells per test animal scoring for breaks, exchanges and gaps; calculation % of aberrant cells inclusive or exclusive gaps
Mitotic index = spermatogonial mitoses / spermatogonial mitoses + first and second meiotic divisions - Evaluation criteria:
- Positive test result: statistically significantly increase of % of aberrant cells exclusive gaps
- Statistics:
- Mann-Whitney-test
- Sex:
- male
- Genotoxicity:
- positive
- Toxicity:
- not examined
- Vehicle controls validity:
- valid
- Positive controls validity:
- not examined
- Additional information on results:
- Statistically significant increases of % aberrant cells (chromatide aberrations exclusive gaps) at all tested doses
- Conclusions:
- Positive
After intraperitoneal application of 40, 80 and 120 mg HQ/kg bw statistically significantly increased frequencies of aberrant cells (chromatide aberrations exclusive gaps) were observed in mouse spermatocytes. As the mode of application bypasses biotransformation and detoxification of HQ taking place in the liver, the biological significance of this finding for the human exposure situation is questionable. - Executive summary:
Groups of five male mice (strain (102/E1 X C3H/E1) F1) were treated with 0, 40, 80 and 120 mg HQ/kg by single intraperitoneal injection and induction of structural chromosomal aberrations in spermatocytes was scored 24 h after treatment (metaphase arrest with colchicine 4 mg/kg) in 100 cells per test animal. The test protocoll was principally similar to OECD Guideline 483. Statistically significantly increased frequencies of aberrant cells (chromatide aberrations exclusive gaps) were observed in the mouse spermatocytes. As the mode of application bypasses biotransformation and detoxification of HQ taking place in the liver, the biological significance of this finding for the human exposure situation is questionable.
- Endpoint:
- in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
- Remarks:
- Type of genotoxicity: chromosome aberration
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 1997
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- comparable to guideline study with acceptable restrictions
- Remarks:
- publication with limited details and some deviations from the test guidelines (2 or 3 animals/dose group).
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
- Deviations:
- yes
- Remarks:
- There was no positive controls. The controls consisted in the sampling in the same animal before administration. Only 3 males per dose group.
- GLP compliance:
- not specified
- Type of assay:
- micronucleus assay
- Species:
- mouse
- Strain:
- other: (102/E1xC3H/E1)F1
- Sex:
- male
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: colony of the GSF-Forschungszentrum für Umwelt und Gesundheit
- Age at study initiation: 10-12 weeks
- Weight at study initiation: approx. 28g
- Assigned to test groups randomly: no data
- Fasting period before study: no data
- Housing: no data
- Diet (e.g. ad libitum): commercial food pellets ad libitum
- Water (e.g. ad libitum): tap water ad libitum
- Acclimation period: no data
ENVIRONMENTAL CONDITIONS
- Temperature (°C): no data
- Humidity (%): no data
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): no data - Route of administration:
- intraperitoneal
- Vehicle:
- - Vehicle(s)/solvent(s) used: bidistilled water
- Details on exposure:
- PREPARATION OF DOSING SOLUTIONS:
injected volumes were 10 µl/g of body weight. - Duration of treatment / exposure:
- A single i.p. injection. Blood sampling took place at 0, 24, 40, 48, and 72 hours in the same animal.
- Frequency of treatment:
- A single i.p. injection.
- Post exposure period:
- Blood was sampled at 0, 24, 40, 48, and 72 hours after injection
- Remarks:
- Doses / Concentrations:
12.5, 25, 50, 75 mg/kg
Basis:
nominal conc. - No. of animals per sex per dose:
- 3
- Control animals:
- other: each animal served as its own control (T0)
- Positive control(s):
- no positive controls included in the study
- Tissues and cell types examined:
- erythrocytes from peripheral blood
- Details of tissue and slide preparation:
- CRITERIA FOR DOSE SELECTION: no data
TREATMENT AND SAMPLING TIMES : single ip injection, blood sampled at 0, 24, 40, 48, 72 hours
Sample of 100 µl per animal per sampling time was collected by retro-obital bleeding after light anaesthesia.
DETAILS OF SLIDE PREPARATION:
Triplicate samples of 5 µl bllod from each animal were layered each onto 1 ml Percoll solution (65% in phopshate-buffered saline (PBS)). The Percoll tubes were centrifuged for 20 min at 600g. The cells obtained were fixed and stained following the method described in Abramsson-Zetterberg et al., 1996 (Mutation Res. 350(2): 349-358).
METHOD OF ANALYSIS: PCE, MPCE and MNCE frequencies were analysed by flow cytometry methodology
FACStar Plus flow cytometer equipped with an argon ion laser operating at the 488 nm line, 1W as primary laser and a second argon ion laser operating at the 351-364 nm multiple UV lines at 200 mW.
For each sample dot plots representing DNA content versus RNA content were displayed using the DAS data analysis program. Regions of interest were defined for NCE and PCE, and MPCE.
The absolute number of events in the regions of interest were summed for the triplicate samples. The respective frequencies of MPCE and PCE were calculated and plotted as a function of time an concentration.
In order to minimize the effect of dose-related differential cell cycle delays, the area below the curve was plotted as a function of concentration.
For these calculations the fMPCE found at the time 0 for each animal was substracted from the fMPCE for the same animal at each subsequent time.
The values for the area under the curve were presented as (MPCExh)/1000 PCE. - Statistics:
- One-tailed Student's t test, corrected for multiple comparisons.
- Sex:
- male
- Genotoxicity:
- positive
- Remarks:
- at 25, 50, 75 mg/kg
- Toxicity:
- yes
- Vehicle controls validity:
- not applicable
- Negative controls validity:
- not applicable
- Positive controls validity:
- not applicable
- Additional information on results:
- The flow cytometry showed a bimodal pattern in the MPCE population.
The fluorescence index increased above control value with a peak at the 24h sampling time.
A small but significant increase was seen at 25 mg/kg at 24 and 40 hours, but not a 48 hours. The peak frequency was observed at 40 hours for all doses.
When results were integrated and the Area Below the Curve (ABC) was plotted as a function of doses, the shape of the curve was biphasic and for the entire concentration range the best fit was linear-quadratic. A good linear fit was observed for the doses 25-75 mg/kg.
The absence of significant elevation of fMPCE at 12.5 mg/kg and the shape of the curve suggest a no-effect threshold effect in the dose region between 12.5 and 25 mg/kg.. - Conclusions:
- Positive.
The frequency of micronucleated polychromatic erythrocytes was significantly increased at the doses 25, 50 and 75 mg/kg hydroquinone injected intraperitoneally. Statistical significance was observed at the 24-hours sampling time and the peak frequency was observed at 40 hours after treatment. No significant increase in micronucleated polychromatic erythrocytes was observed in mice administered 12.5 mg/kg. The data suggested a possible threshold in the dose region between 12.5 and 25 mg/kg. - Executive summary:
Frequency of Micronucleated Polychromatic erythrocytes was investigated by flow cytometry analysis in groups of 3 male mice administered intraperitoneally a single dose (12.5, 25, 50, or 75 mg/kg) of hydroquinone. Peripheral blood was sampled at times 0 (pre-treatment), 24, 40, 48, and 72 hours and processed to analyse micronucleated polychromatic and normochromatic erythrocytes.
Hydroquinone induced a significant increase in the frequency of MPCE at the dose 25 mg/kg and higher, at 24 hours and 40 hours sampling times.
The peak frequency was observed at 40 hours in all dose groups. No statistically significant increase was observed at the dose 12.5 mg/kg.
The Area below the curve plotted as a function of the dose showed a biphasic dose-response with a good linear fit between 25 and 75 mg/kg, and the data suggest that a No-effect threshold might exist in the region between 12.5 and 25 mg/kg.
- Endpoint:
- in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
- Remarks:
- Type of genotoxicity: chromosome aberration
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- comparable to guideline study with acceptable restrictions
- Remarks:
- Limited documentation, missing details, only one dose tested
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
- GLP compliance:
- not specified
- Type of assay:
- micronucleus assay
- Species:
- mouse
- Strain:
- Swiss
- Sex:
- male
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Charles River, Calco, Italy)
- Age at study initiation: no data
- Weight at study initiation: no data
- Assigned to test groups randomly: yes (no details)
- Housing: no data
- Diet : no data
- Water : no data
- Acclimation period: no data
ENVIRONMENTAL CONDITIONS: no data
IN-LIFE DATES: no data - Route of administration:
- other: intraperitoneal or oral gavage
- Vehicle:
- - Vehicle(s)/solvent(s) used: distilled water
- Concentration of test material in vehicle: no data
- Amount of vehicle (if gavage or dermal): no data - Details on exposure:
- PREPARATION OF DOSING SOLUTIONS:
no details. Hydroquinone was dissolved in sterile distilled water. - Duration of treatment / exposure:
- Single Dose: 80 mg/kg
Dose was chosen based on expected quantity of metabolite formed following benzene administration at 880 mg/kg
The other test substances included in the study were (nominal concentrations):
benzene, 880 mg/kg in olive oil
catechol, 40 mg/kg in distilled water
p-benzoquinone, 5 and 20 mg/kg in distilled water
phenol, 265 mg/kg in distilled water - Frequency of treatment:
- Single administration ip injection or by oral gavage
- Post exposure period:
- Various sampling times: 18, 24, 42 or 48 hours after administration
- Remarks:
- Doses / Concentrations:
80 mg/kg
Basis:
nominal conc.
Hydroquinone - No. of animals per sex per dose:
- 4 males per group
- Control animals:
- yes, concurrent vehicle
- Positive control(s):
- No. Hydroquinone effects were compared to other metabolites of benzene (p-benzoquinone, catechol) and benzene itself
- Tissues and cell types examined:
- polychromatic erythrocytes from bone marrow
- Details of tissue and slide preparation:
- DETAILS OF SLIDE PREPARATION:
smears were fixed in methanol (5 min) and stained with May-Grünwald and Giemsa in Sörensen's phosphate buffer, pH6.5.
METHOD OF ANALYSIS:
All slides were coded and scored blind.
The % PCE was calculated by counting both normochromatic erythrocytes (NCE) and PCE until 3000 PCE had been scored for the presence of micronuclei. The decrease in the PCE/NCE ratio compared to control was considered a consequence of the toxic activity of the test substance (bone marrow depression). - Statistics:
- Significance of Microncleus induction assessed by Kastenbaum-Bowman tables
Significance of induced cell toxicity (PCE/NCE ratio) assessed with t-test after arc-sin transformation - Sex:
- male
- Genotoxicity:
- positive
- Remarks:
- by oral administration
- Toxicity:
- yes
- Remarks:
- bone marrow depression at 42 hrs only
- Vehicle controls validity:
- not specified
- Negative controls validity:
- not applicable
- Positive controls validity:
- not applicable
- Sex:
- male
- Genotoxicity:
- positive
- Remarks:
- by intraperitoneal injection
- Toxicity:
- yes
- Remarks:
- bone marrow depression at 18-24hrs
- Vehicle controls validity:
- not specified
- Negative controls validity:
- not applicable
- Positive controls validity:
- not applicable
- Additional information on results:
- - Induction of micronuclei (for Micronucleus assay):
By oral gavage: weak increase (approx. 2x) (p< 0.05) at 18 (peak frequency), 42 and 48 h after treatment (not significant at 24h).
PCE/NCE ratio: Bone marrow depression is negligible until 42h after treatment when it is then induced (lower PCE/NCE ratio).
By intraperitoneal administration: large increase (8x) in micronuclei at 18 h (peak frequency) and 24 hours, not significant effects at 42 and 48 hours.
PCE/NCE ratio: Rapid induction of bone marrow depression with a peak at 18 hours, then declining to controls levels. - Conclusions:
- Positive.
Hydroquinone administered at a single dose by oral gavage at 80 mg/kg induced a weak micronucleus increase at 18, 42 and 48 hours, while a larger induction at 18, 24 hours was caused by intraperitoneal administration, with a concurrent bone marrow depression. - Endpoint:
- in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
- Remarks:
- Type of genotoxicity: chromosome aberration
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- comparable to guideline study with acceptable restrictions
- Remarks:
- Test protocol comparable to OECD Guideline 474 with sufficient documentation of experimental details and test results; study investigating different doses, single and multiple dosing, and different sampling times of bone marrow, no positive controls included in the assays; study acceptable as key study
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
- GLP compliance:
- no
- Type of assay:
- micronucleus assay
- Species:
- mouse
- Strain:
- other: (101/E1 X C3H/E1) F1
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: not specified
- Age at study initiation: 10-14 w
- Weight at study initiation: 25-28 g
ENVIRONMENTAL CONDITIONS: no data - Route of administration:
- intraperitoneal
- Vehicle:
- - Vehicle(s)/solvent(s) used: water
- Duration of treatment / exposure:
- Time intervals up to sampling of bone marrow:
Single dosing: 30 mg/kg: 24 h, 50 mg/kg: 6 and 24 h, 75 mg/kg: 18, 24, and 30 h, 100 mg/kg: 24 h
Multiple dosing: 24 h after the first, second and third application - Frequency of treatment:
- single dosing; or multiple dosing with daily application
- Post exposure period:
- no
- Remarks:
- Doses / Concentrations:
0, 30, 50, 75, 100 mg/kg bw
Basis:
nominal conc.
single dosing - Remarks:
- Doses / Concentrations:
0, 15, 75 mg/kg bw
Basis:
nominal conc.
multiple dosing - No. of animals per sex per dose:
- 5 males and females for single dosing; 5 males for multiple dosing
- Control animals:
- yes, concurrent vehicle
- Positive control(s):
- no data
- Tissues and cell types examined:
- polychromatic erythrocytes prepared from bone marrow
- Details of tissue and slide preparation:
- CRITERIA FOR DOSE SELECTION: dose ranges known to produce effects based on previous studies
DETAILS OF SLIDE PREPARATION: Bone marrow smears were air dried and stained with May-Grünwald / Giemsa.
METHOD OF ANALYSIS: 2000 PCE per test animal were evaluated for the frequency of micronuclei (number of micronuclei per cell not evaluated); ratio of PCE/NCE by counting the PE in the fields showing 2000 NE - Statistics:
- Individual group results by Mann-Whitney-Wilcoxon test; dose-response curve by least squares regression (Sachs, 1974); trend test for positive dose-related increase
- Sex:
- male/female
- Genotoxicity:
- positive
- Toxicity:
- not specified
- Vehicle controls validity:
- valid
- Negative controls validity:
- not specified
- Positive controls validity:
- not specified
- Additional information on results:
- RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (see Tables 1 and 2):
Single dosing: significant increases of micronucleus frequency for 50 mg/kg (24 h sampling time), 75 mg/kg (sampling times 18-30 h), 100 mg/kg (sampling time 24 h); maximum response for 24 h sampling time; dose-dependent increase for the 24 h sampling time assays
Multiple dosing: significant increases of micronuclei frequency for 3 doses of 15 mg/kg, and 1,2 or 3 doses of 75 mg/kg
- Ratio of PCE/NCE (see Tables 1 and 2): significant decreases for single application of 50 mg/kg and 24 h sampling time, and in one of two assays with single application of 50 mg/kg and 24 h sampling time; effects of questionable biological relevance
- Appropriateness of dose levels and route: Intraperitoneal application is an unphysiological route of uptake and is not representative of oral uptake as it evades detoxification of HQ by first-pass metabolism in the liver. - Conclusions:
- Positive
HQ induced micronuclei in polychromatic bone marrow erythrocytes after intraperitioneal application in mice. The lowest dose inducing a statistically significant increase of the frequency of micronuclei was a single dose of 50 mg/kg or three repeated doses of 15 mg/kg (45 mg/kg in total). - Executive summary:
A micronucleus assay in polychromatic erythrocytes of mouse bone marrow, with a test protocol comparable to OECD Guideline 474, was performed with the aim to investigate the frequencies of micronuclei at different doses after single (30, 50, 75, or 100 mg/kg) and multiple dosing (one to three daily doses of 15 and 75 mg/kg), and at different sampling times of the bone marrow. Statistically significant increases of micronucleus frequency were observed for sampling times of 24 h at 50-100 mg/kg with a dose-dependant increase, and at sampling times of 18-30 h at 75 mg/kg. The maximum responses were found for the 24 h sampling time. After multiple dosing, significant increases of micronuclei frequency were found for 3 doses of 15 mg/kg, and 1, 2 or 3 doses of 75 mg/kg. Two single observations of significant decreases of the ratio of PCE/NCE are of questionable biological relevance (one effect was not reproducible in a second assay at comparable conditions).
HQ induced micronuclei in polychromatic bone marrow erythrocytes after intraperitioneal application in mice. The lowest dose inducing a statistically significant increase of the frequency of micronuclei was a single dose of 50 mg/kg or three repeated doses of 15 mg/kg (45 mg/kg in total).
- Endpoint:
- in vivo mammalian germ cell study: cytogenicity / chromosome aberration
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 23 November 1983 - 9 March 1984
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- comparable to guideline study
- Remarks:
- Comparable to guideline study with acceptable deviation of extended exposure period; study report with detailed documentation of test conditions and test results; study acceptable as key study
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 478 (Genetic Toxicology: Rodent Dominant Lethal Test)
- Deviations:
- yes
- Remarks:
- (extended exposure period of 10 wks)
- GLP compliance:
- not specified
- Type of assay:
- rodent dominant lethal assay
- Species:
- rat
- Strain:
- other: COBS CD(SD)BR
- Sex:
- male
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Charles River Laboratories, Wilmington, MA, USA
- Age at study initiation: 63 d
- Weight at study initiation:
- Assigned to test groups randomly: [no/yes, under following basis: ]
- Fasting period before study:
- Housing: 5 per cage by sex
- Diet: Ourina Rodent Lboratory Chow 5001 ad libitum
- Water: tap water ad libitum
- Acclimation period: 12 d
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +- 2 (72 +- 2 °F)
- Humidity (%): 55 +- 15
- Air changes (per hr): no data (laminar flow rooms)
- Photoperiod (hrs dark / hrs light): 12 / 12 - Route of administration:
- oral: gavage
- Vehicle:
- - Vehicle(s)/solvent(s) used: water
- Justification for choice of solvent/vehicle: solubility
- Concentration of test material in vehicle: 5%
- Amount of vehicle (if gavage or dermal): - Duration of treatment / exposure:
- 10 w (study day 0 to 67)
- Frequency of treatment:
- 5 d/w
- Post exposure period:
- On the evening of study day 69, treated males were paired (1:1) with untreated females for 5 d/w for the following two weeks, a second female used in the second week's mating. Vaginal smears were examined daily, and the day sperm was seen was considered day 0 of gestation. Pregnant females were housed individually until Caeserean Section on day 14 of gestation.
- Dose / conc.:
- 0 mg/kg bw/day (actual dose received)
- Dose / conc.:
- 30 mg/kg bw/day (actual dose received)
- Dose / conc.:
- 100 mg/kg bw/day (actual dose received)
- Dose / conc.:
- 300 mg/kg bw/day (actual dose received)
- No. of animals per sex per dose:
- 25 males/group
- Control animals:
- yes, concurrent vehicle
- Positive control(s):
- Apholate (CAS No. 52-46-0)
- Justification for choice of positive control(s): substance known to induce dominant lethals
- Route of administration: intraperitoneal
- Doses / concentrations: 1.5 mg/kg on days 63 - 67 - Tissues and cell types examined:
- Female rats:
Examined parameters: insemination rate, pregnancy rate, counts of corpora lutea, of implantation sites with categorization as early deaths, late deaths or viable embryos, % pre-implantation loss, % post-implantation loss.
Examination of abdominal and thoracic organs of the dams for gross lesions.
Male rats:
During treatment body weights were recorded on day 0, 4, 7, and at least weekly thereafter; food consumption twice per week; clinical signs of toxicity on each treatment day during handling for dosing, cage side observations post-dose and at the end of work day. - Statistics:
- One-way analysis of variance (ANOVA), Duncan's multiple range test; reproductive parameters were subjected to Freeman-Tukey double arcsin transformation and analyzed by ANOVA, level of significance P <= 0.05
- Sex:
- male
- Genotoxicity:
- negative
- Toxicity:
- yes
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- FINDINGS IN MALE ANIMALS:
In all treated groups brown discoloration of urine was observed during the first 3 weeks on days following treatment (indicating excretion of the modified test substance).
300 mg/kg:
- Mortality: 2/25 in the 9th week of the treatment
- Mean body weights decreased from the 4th week to the end of the treatment period, significant effect on day 4, and days 29-69; total body weight gain 12.2% less than in controls
Mean food consumption: slight reduction (ca. 7%) compared to controls, statistically significant from day 4-14 and on days 45, 53, 60, and 66
- Clinical signs of toxicity: swollen eyelids, sialorrhea, spastic gait, tremors, convulsions
100 mg/kg: two deaths due to accidental instillation of test solution into the lungs during dosing
EVALUATION OF CESAERIAN SECTION FINDINGS:
HQ-treated groups: no effect on reproductive capacity of male rats (insemination rate, pregnancy rate), no indication of dominant lethal effects in male mice.
At 300 mg/kg in the first mating period, mean number of corpora lutea and implantations per dam were significantly greater compared to the negative controls. As this effect was not seen in the second mating period, it was considered to be of no toxicological significance.
Positive control substance: Evidence of dominant lethal effect in males
First mating: significantly reduced number of viable implantations, significantly increased number of early deaths, very high post implantation loss (61%)
Second mating: significant decreases of pregnancy rate, number of corpora lutea, implantation sites, and viable implants; significant increases of early deaths, and of pre- and post-implantation loss - Conclusions:
- Negative.
HQ administered to male rats in doses of 30, 100, or 300 mg/kg by gavage, 5 d/w over a ten week period (50 doses) did not produce dominant lethality as evidenced by the examination of the reproductive indices following mating to untreated virgin females during two weeks immediately following the initial treatment. - Executive summary:
In a dominant lethal assay with a protocol comparable to OECD Guideline 478, groups of 25 male CD rats were exposed to doses of 0, 30, 100, 300 mg HQ/kg bw/d by gavage (5% solution in water) applying an extended period of treatment of 10 weeks, 5 d/w, prior to mating. Control groups treated with the vehicle or the positive control substance apholate (1.5 mg/kg on study days 63-67) were also included in the study. During treatment of male rats, mortalities, body weights and food consumption, and clinical signs were recorded. On the evening of study day 69, treated males were paired (1:1) with untreated females for 5 d/w for the following two weeks (two matings with one female each yielding 50 matings per dose).
After Caeserean section on day 14 of gestation, the following parameters were examined: insemination rate, pregnancy rate, counts of corpora lutea, of implantation sites with categorization as early deaths, late deaths or viable embryos, % pre-implantation loss, % post-implantation loss.
HQ administered to male rats at doses of 30, 100, or 300 mg/kg by gavage, 5 d/w over a ten week period (50 doses) did not produce dominant lethality as evidenced by the examination ot the reproductive indices following mating to untreated virgin females during two weeks immediately following the initial treatment. Toxicity was indicated at 300 mg/kg by the observation of mortalities (2/25), significantly reduced body weights and food consumption, and significant clinical signs of toxicity (e.g. spastic gait, tremors, convulsions). The positive control substance clearly induced dominant lethality in male rats.
- Endpoint:
- in vivo mammalian germ cell study: gene mutation
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- no data
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Remarks:
- Good quality study conducted according to the OECD guideline 488, with sufficient documentation on test conditions and test results, with only a few details missing in the publication (details on body weight, food consumption, formulation preparation, total number of packaging reactions needed). Publication acceptable as key study for assessment of genotoxic effect
- Qualifier:
- according to guideline
- Guideline:
- other: OECD Guideline 488 (Transgenic Rodent Somatic and Germ Cell Mutation Assays) (28 July 2011)
- GLP compliance:
- yes
- Type of assay:
- transgenic rodent mutagenicity assay
- Species:
- mouse
- Strain:
- other: Muta(TM) mice
- Sex:
- male
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Muta(TM) mice model
- Target gene: lacZ bacteriophage
- Source: Japan Laboratory Animals, Inc. (Tokyo, Japan)
- Age at study initiation: nine-week old
- Weight at study initiation: no data (mean body weights on day 1: 25 to 25.5 g)
- Assigned to test groups randomly: no data
- Fasting period before study: not applicable
- Housing: no data
- Diet : basal diet CRF-1 (Oriental yeast)
- Water : ad libitum
- Acclimation period: 8 days
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-26
- Humidity (%): 35-70
- Air changes (per hr): 12
- Photoperiod (hrs dark / hrs light): 12 hrs/12 hrs
IN-LIFE DATES: no data - Route of administration:
- oral: gavage
- Vehicle:
- - Vehicle(s)/solvent(s) used: distilled water
- Amount of vehicle : 10 ml/kg - Details on exposure:
- PREPARATION OF DOSING SOLUTIONS: no details, except that distilled water served as vehicle.
The highest dose level was based on the results of the NTP 14-day gavage study in B6C3F1 mice in which 2 out of 5 mice died within 3 days at the dose of 250 mg/kg bw/day. - Duration of treatment / exposure:
- 4 weeks
- Frequency of treatment:
- once a day
- Post exposure period:
- Expression period: 3 days
- Dose / conc.:
- 0 mg/kg bw/day (nominal)
- Dose / conc.:
- 25 mg/kg bw/day (nominal)
- Dose / conc.:
- 50 mg/kg bw/day (nominal)
- Dose / conc.:
- 100 mg/kg bw/day (nominal)
- Dose / conc.:
- 200 mg/kg bw/day (nominal)
- No. of animals per sex per dose:
- 5
- Control animals:
- yes, concurrent vehicle
- Positive control(s):
- N-ethyl-N-nitrosurea
- 5 animals
- Route of administration: intraperitoneal, once a day, for 2 days
- Doses / concentrations: 100 mg/kg bw/day
- expression time: 10 days - Tissues and cell types examined:
- Liver, stomach, kidney, lung, (thyroid)
Rationale for tissue selection: target organs based on previous studies:
Liver: hepatocellular adenomals reported in female mice and male mice
Stomach: hyperplasia of the forestomach reported in mice (without tumor development); first site of contact
Kidneys: renal tubule adenomas in male rats, not in females
Thyroid gland: hyperplasia in males and female mice - Details of tissue and slide preparation:
- CRITERIA FOR DOSE SELECTION: the high dose was based on the result of the NTP 14-day gavage study in B6C3F1 mice in which HQ related death was observed in males treated t 250 mg/kg/day, and where treatment was associated with clinical signs such as tremors, convulsions and decreased body weight.
TREATMENT AND SAMPLING TIMES:
Liver, stomach, kidney, lung, thyroid were collected 3 days after the last treatment, and a gross pathological examination was conducted.
Positive control animals were sacrificed 10 days after the last treatment, and their organs were collected in the same manner.
Tissue samples were quickly frozen in liquid nitrogenand then stored at −80°C until analysis.
Genomic DNA was extracted from the liver and stomach at 0, 50, 100, and 200 mg/kg bw/day, and the lung and kidney at 0, and 200 mg/kg bw/day as follows. Frozen tissue was placed into a Dounce homogenizer and homogenized with a pestle. The homogenized tissue fragments were poured into an ice-cooled centrifuge tube containing sucrose solution. After centrifugation by a centrifuge (LC-122, TOMY) at 3000 r/min (1710 G) for 10 min, the organic layer was incubated with RNase and proteinase K at 50◦C for 3 h. A mixture of phenol andchloroform (1:1) was added, and the water layer was separated after centrifugation at 2500 r/min (1190 G) for 10 min; this operation was repeated two times. Chloroform and isoamyl alcohol (24:1) and the water layer were mixed, and similarly centrifuged. The water layer was added in another centrifuging tube, and ethanol was added to precipitate the DNA. DNA was washed by soaking in 70% ethanol for 10 min. The DNA collected following the evaporation of ethanol was dissolved in TE buffer (NIPPON GENE) at room temperature overnight. The DNA solution was stored in a refrigerator.
The DNA of the thyroid could not be extracted and, therefore, was excluded from the evaluation.
IN VITRO PACKAGING:
Packaging DNA packaging was performed according to the Instruction Manual of Transpack(Stratagene). The DNA solution (200–600 g/mL) was gently mixed with the Transpack packaging extract and incubated at 30◦C for 1.5 h twice, and SM buffer(NaCl, MgSO4·7H2O, Tris–HCl [pH: 7.5], and gelatin) was then added.
MUTANT FREQUENCY DETERMINATION
The phage solution absorbed Escherichia coli at room temperature for 20–30 min. An appropriately diluted E. coli solution was mixed with LB top agar for the titer plates. The remaining phage-E. coli solution was mixed with LB top agar containing P-gal (phenyl-beta-D-galactoside, Sigma–Aldrich) for the selection plates. These plates were then incubated overnight at 37°C. Packaging was repeated to reach a total number of 300,000 plaques. - Evaluation criteria:
- The mutant frequency (MF) was calculated by the following formula:
MF = total number of plaques on selection plates/total number of plaques on titer plates.
Mutation Frequencies in treated groups were compared to the control group by statistical analysis. - Statistics:
- Homogeneity of data: MFs in the treatment and negative control groups were analyzed with Bartlett’s test.
- homogeneous data were analyzed using the Dunnett test.
- The Steel test was used for non-homogenous data.
MFs between the negative and positive controls were compared by the Student’s t-test or Aspin–Welch’s t-test.
Five percent levels of probability were used as the criterion for significance. - Sex:
- male
- Genotoxicity:
- negative
- Remarks:
- in liver, stomach, kidney, and lung
- Toxicity:
- yes
- Remarks:
- decreased body weight in all treatment groups
- Vehicle controls validity:
- valid
- Negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Additional information on results:
- RESULTS OF DEFINITIVE STUDY
- Appropriateness of dose levels and route: mean body weights were decreased in all treated groups compared to the control group, indicating absorption and distribution of hydroquinone, and some degree of toxicity, although no clinical effects were reported as in previous NTP studies.
Positive controls were appropriate: N-ethyl-N-nitrosourea induced mutations at the above levels compared to negative controls for the same organs, with statistical significance:
Liver: 2-fold
Stomach: 11-fold
Kidney: 3-fold
Lung: 5-fold - Conclusions:
- Negative
When administered by oral gavage to groups of male transgenic Muta(TM) mice for 28 days at levels of 50, 100, or 200 mg/kg bw/day, Hydroquinone did not cause any significant increase in mutation frequency (lacZ) in liver, stomach, kidney, and lung, compared to vehicle-treated mice.
Under the conditions of the study Hydroquinone is considered non-mutagenic. - Executive summary:
In a lacZ transgenic mutation study conducted according to the OECD guideline 488, groups of 5 male Muta(TM) mice were treated by oral gavage with Hydroquinone administered in aqueous solution at levels of 0, 50, 100, or 200 mg/kg bw/day for 28 days, followed by an expression period of 3 days before sampling of the tissues which were quickly frozen and stored at -80°C until their processing for DNA extraction.
Body weight gain was decreased in all treatment groups compared to vehicle controls. There were no clinical signs.
Mutant frequencies (lacZ) in the liver, stomach, kidney and lung of treated mice were not significantly different from the negative control. N-Ethyl-N-nitrosourea was used as a positive control and was administered intraperitoneally 2 consecutive days at 100 mg/kg bw, followed by an expression period of 10 days. it was found to induce mutations in all four organs.
Under the conditions of the study, Hydroquinone is considered non-mutagenic.
- Endpoint:
- in vivo mammalian cell study: DNA damage and/or repair
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 04-Feb-2015 to 19-JAN-2016
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- other: OECD guideline 489
- Deviations:
- no
- GLP compliance:
- yes
- Type of assay:
- mammalian comet assay
- Species:
- rat
- Strain:
- Fischer 344
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source:
Fischer F344 were obtained from Harlan UK Ltd, Oxon, UK. This strain was used because carcinogenicity data were obtained in Fischer F344 with kidney tumours only seen in this strain of rats.
Because no historical control data were available in this laboratory for Fischer F344 rats additional groups of Sprague Dawley rats dosed with either vehicle control or positive control were included to allow for a comparison of strain specific background comet levels. Sprague-Dawley rats were obtained from Charles River (UK) Ltd, Margate, UK.
Male and female rats were included in the study due to different tumour profiles seen between male and female animals.
- Age at study initiation:
DRF study, Fischer F344: 7-9 weeks
Main study: see animal specification in Table 1
Females F344 were slightly under age, and under the lower limit of bw. They were considered to meet the guideline requirement of being young adult rats. None of the deviations affected the integrity or interpretation of the results (see details).
- Weight at study initiation:
DRF study, Fischer F344: Males: 171-216 g ; females: 119-154 g
Main study: see animal specification in Table 1.
- Assigned to test groups randomly: yes, under following basis: total randomisation was used, whereby animals were removed one at the time from the arrival crates and placed into separate cages. When all cages contained one animal, the procedure started again until all cages contained a maximum of 3 animals. Range-finder: randomiseed to groups of 3; Main experiment: randomised to groups of 6 (with the exception of positive control animals (Groups 5, 10, 15) allocated to groups of 3).
Checks were made to ensure the weight variation of Main Experiment animals prior to dosing was minimal and did not exceed ±20% of the mean weight of each sex (see deviations)
- Fasting period before study: no
- Housing: wire-topped, solid bottomed cages
- Diet (ad libitum): SQC rat and mouse maintenance diet No.1, Expanded (Special diets Services Ltd, William), or 5LF2 EU Rodent diet 14% (LabDiet, St Louis, USA
- Water: ad libitum
- Acclimation period: at least 5 days
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 45-65
- Air changes (per hr): 15-20
- Photoperiod (hrs dark / hrs light): 12 hours light (6:00 to 18:00), 12 hours dark
IN-LIFE DATES: From: 23 February 2015 To: 04 August 2015 - Route of administration:
- oral: gavage
- Vehicle:
- - Vehicle(s)/solvent(s) used: purified water
- Justification for choice of solvent/vehicle: the test substance is soluble in water. Stability of the formulations was shown in a stability study conducted as part of the study.
- Concentration of test material in vehicle: 1- 50 mg/ml
- Amount of vehicle (gavage): 10 ml/kg - Details on exposure:
- PREPARATION OF DOSING SOLUTIONS:
Formulations were freshly prepared prior to each dosing occasion by formulating Hydroquinone in purified water.
Prior to formulation preparation the vehicle control was degassed by purging with nitrogen for at least 15 minutes. The test article was weighed, the vehicle was added to the formulation container and the contents stirred to mix.
The exception to this was the 50 mg/mL formulation prepared for Group 3 of the Range finding study: this formulation was originally prepared at 100 mg/mL but was found to be insoluble. The formulation was therefore diluted with more vehicle to achieve the final concentration of 50 mg/mL.
As discolouration of the formulation was considered indicative of instability, the appearance of each formulation prior to dispatch to the animal room for dosing was verified and confirmed to be a clear colourless solution.
Ranger-finder:
1RF and 2RF, dose volume: 10 ml/kg
3RF and 4RF, dose volume: 20 ml/kg
5RF: not administered due to toxicity observed in 4RF
Main experiment: dose volume was 10 ml/kg for the three doses administered. - Duration of treatment / exposure:
- Two administrations by gavage: on day 1 and on Day 2
- Frequency of treatment:
- once a day
- Post exposure period:
- sampling times:
- somatic tissues: duodenum, liver, kidney were collected 30 minutes after the 2nd dose (day 2); this corresponded to the peak plasma time (Tmax) determined in previous toxicokinetic studies.
- male gonads: sampling time was 2 hours after the second treatment (day 2); there was an absence of specific kinetic information for that tissue following oral administration, however it was considered likely that there would be a delay in peak gonad exposure compared to somatic exposure. Therefore, the lower limit of the default sampling time recommended by OECD guideline 489, 2014 (2-6 hours after dosing) was selected. - Dose / conc.:
- 0 mg/kg bw/day (nominal)
- Dose / conc.:
- 105 mg/kg bw/day (nominal)
- Dose / conc.:
- 210 mg/kg bw/day (nominal)
- Dose / conc.:
- 420 mg/kg bw/day (nominal)
- No. of animals per sex per dose:
- 6
- Control animals:
- yes, concurrent vehicle
- Positive control(s):
- Ethylmethanesulphonate (EMS), 3 males/3 females
- Justification for choice of positive control(s): known mutagen. For gonadal cells, proficiency was demonstrated in an independent validation study (8322535) after validation of the test conditions.
- Route of administration: oral gavage
- Doses / concentrations: 150 mg/kg
Duodenum, liver, kidney (group 5 and 10): a single EMS administration on day 2, 3 hours prior to necropsy.
Gonads (group 15): 2 administrations, on day 1 and on day 2 at 21 hours after 1st dose. Sampling time was 3 hours post-dosing (i.e. at 24 hours after the 1st administration).
For the positive control, sampling time of 3 hours after treatment is the default sample time used for the laboratory's historical control data. - Tissues and cell types examined:
- TISSUES EXAMINED:
- Duodenum: first site of contact following oral administration, allowed for evaluation of direct mutagenic activity. There is also the potential for the duodenum to have been exposed further via enterohepatic circulation.
- Liver: adenomas were observed in this tissue in some studies and this is the major site of metabolism.
- Kidney: hyperplasia was reported in this tissue in particular in male Fischer rats
-Gonads: male gonad tissue was examined as this was considered a suitable substitute for germ cells in the comet assay.
Due to different tumour profiles seen between male and female animals the study was conducted in male and female animals, with the exception that males only were used for testing in gonad tissue. - Details of tissue and slide preparation:
- CRITERIA FOR DOSE SELECTION:
The known oral LD50 in rats was reported to be between 300-600 mg/kg bw. A dose range finding study was conducted starting at the dose 300 mg/kg bw/day. Subsequent higher doses were tested (420, 1000, 600 mg/kg/day) until an estimate of the MTD was determined.
From the results of the Range-Finder Experiment dose levels of 105, 210 and 420 mg/kg Hydroquinone (equivalent to 25% MTD, 50% MTD and MTD respectively) were tested in the Main Experiment.
TREATMENT AND SAMPLING TIMES :
For somatic tissues, duodenum, liver and kidney the sampling time was 30 min after the second treatment (day 2), i.e. 23.5 hours after the 1st administration. This corresponds to the peak in plasma (Tmax) identified in previous toxicokinetics studies.
For male gonads, sampling time was 2 hours after the second treatment (day 2), due to the absence of specific information for that tissue.
For positive controls, see details under "Positive controls"
DETAILS OF CELL SUSPENSIONS PREPARATION:
The duodenum samples were washed thoroughly in Merchant's solution and placed into fresh buffer. Each sample was vortexed in Merchant's solution for approximately 15 seconds. The tissue was removed from the Merchant's solution and the inner surface gently scraped (released material discarded) using the back of a scalpel blade. The tissue was vortexed in Merchant's solution for a further 15 seconds prior to gently scraping the inside of the duodenum with the back of a scalpel blade.
The kidney samples were cut into small pieces and washed thoroughly in Merchant's solution. The pieces were then pushed through bolting cloth (pore size of 150 µm) with approximately 4 mL of Merchant's solution to produce single cell suspensions.
The liver samples were washed thoroughly in Merchant's solution and placed in fresh buffer. The samples were cut into small pieces in Merchant's solution and the pieces of liver were then pushed through bolting cloth (pore size of 150 µm) with approximately 4 mL of Merchant's solution to produce single cell suspensions.
The testes were finely minced using a scalpel blade and tweezers and filtered through bolting cloth (pore size of 150 µm) with ice cold Merchant’s solution to produce single cell suspensions.
All cell suspensions were held on ice prior to slide preparation.
DETAILS OF SLIDE PREPARATION:
Three slides, labelled ‘A’, ‘B’ and ‘C’ were prepared per single cell suspension per tissue. Slides were labelled with the study number, appropriate animal tag number and tissue. Slides were dipped in molten normal melting point agarose (NMA) such that all of the clear area of the slide and at least part of the frosted area was coated. The underside of the slides was wiped clean and the slides allowed to dry. 30 µL of each single cell suspension was added to 300 µL of 0.7% low melting point agarose (LMA) at approximately 37°C. 100 µL of cell suspension/agarose mix was placed on to each slide. The slides were then coverslipped and allowed to gel on ice.
Procedure:
1-Cell Lysis
Once gelled the coverslips were removed and all slides placed in lysis buffer (2.5 M NaCl, 100 mM EDTA, 10 mM Tris, pH adjusted to pH 10 with NaOH, 1% Triton X 100, 10% DMSO) overnight at 2-8°C, protected from light.
2-Unwinding and Electrophoresis
Following lysis, slides were washed in purified water for 5 minutes, transferred to electrophoresis buffer (300 mM NaOH, 1 mM EDTA, pH>13) at 2-8°C and the DNA unwound for 20 minutes (duodenum) or 30 minutes (kidney, liver and gonad). At the end of the unwinding period the slides were electrophoresed in the same buffer at 0.7 V/cm for 20 minutes (duodenum) or 40 minutes (kidney, liver and gonad). As not all slides could be processed at the same time a block design was employed for the unwinding and electrophoretic steps in order to avoid excessive variation across the groups for each electrophoretic run; i.e. for all animals the same number of triplicate slides was processed at a time.
3-Neutralisation
At the end of the electrophoresis period, slides were neutralised in 0.4 M Tris, pH 7.0 (3 x 5 minute washes). After neutralisation the slides were dried and stored at room temperature prior to comet scoring.
4-Staining
Prior to scoring, the slides were stained with 100 µL of 2 µg/mL ethidium bromide and coverslipped.
METHOD OF ANALYSIS:
Slide scoring was carried out using fluorescence microscopy at an appropriate magnification and with suitable filters.
A slide from a vehicle and positive control animal were checked for quality and/or response prior to analysis. All slides were allocated a random code and analysed by an individual not connected with the dosing phase of the study.
All available animals (per sex) per group were analysed.
Measurements of tail moment and tail intensity (%DNA in tail) were obtained from 150 cells/animal/tissue. In general this was evenly split over two or three slides.
The number of ' hedgehogs' (a morphology indicative of highly damaged cells often associated with severe cytotoxicity, necrosis or apoptosis) observed during Comet scoring was recorded for each slide. To avoid the risk of false positive results 'hedgehogs' were not used for comet analysis. Each slide was scanned starting to the left of the centre of the slide.
The following criteria were used for analysis of slides:
1. Only clearly defined non overlapping cells were scored
2. Hedgehogs were not scored
3. Cells with unusual staining artefacts were not scored. - Evaluation criteria:
- VALIDITY CRITERIA:
The data were considered valid if the following criteria were met:
1. There was a marked increase in group mean positive control values compared to the concurrent vehicle control
2. The high dose was considered to be the MTD, the maximum recommended dose or the maximum practicable dose
Data from Group 1 F344 rats were compared to the data from the Group 1 Sprague Dawley rats. If comet values were considered comparable between the animals then the laboratory historical control data were used for assessment of data validity as follows:
3. Group mean vehicle control values were comparable to laboratory historical control data/in-house data for each tissue.
If there were clear differences between the strains no comparison to the historical control ranges was performed.
EVALUATION CRITERIA:
For valid data, the test article was considered to induce DNA damage if:
1. A least one of the test doses exhibited a statistically significant increase in tail intensity, in any tissue, compared with the concurrent vehicle control
2. The increase was dose related in any tissue.
The test article was considered positive in this assay if both of the above criteria were met.
The test article was considered negative in this assay if neither of the above criteria were met and target tissue exposure was confirmed.
Results which only partially satisfied the criteria were dealt with on a case by case basis. Biological relevance was taken into account, for example comparison of the response against the historical control data and consistency of response within and between dose levels.
A positive response was based on scientific judgment and included analysis of related, concurrent cytotoxicity information (such as ‘hedgehog’ assessment, histopathological changes and clinical pathology results) and the historical control data. - Statistics:
- Median tail intensity data were used for statistical analysis. Data from F344 (Sub group 1) animals only was analysed with statistical methods.
The positive control groups (5, 10 or 15) was compared to the vehicle control groups (1, 6 or 11 respectively) using a two-sample t test. The test was interpreted with one sided risk for increased response with increasing dose.
The vehicle control group (1, 6 or 11) and the treated groups (2-4, 7-9 or 12-14) were analysed separately using one-way analysis of variance (ANOVA). An overall dose response test was performed along with Dunnett’s test for pairwise comparisons of each treated group with the vehicle control. For all tissues the test was interpreted with a one-sided risk.
Levene's test for equality of variances between the groups was performed and where this showed evidence of heterogeneity (p=<0.01), the data was rank transformed prior to analysis. - Sex:
- male
- Genotoxicity:
- negative
- Remarks:
- duodenum, liver, kidney, gonads
- Toxicity:
- no effects
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
- Sex:
- female
- Genotoxicity:
- negative
- Remarks:
- duodenum, liver, kidney
- Toxicity:
- no effects
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- RESULTS OF RANGE-FINDING STUDY
- Dose range: 0, 300, 420, 1000, 600 mg/kg bw/day, in 2 administrations, at 0 and 23.5 hours.
- Clinical signs of toxicity in test animals:
No clinical signs at 300 mg/kg/day. At 420 mg/kg/day piloerection was observed solely in males on day 2 (1 and 2 h post-dose). Animals dosed at 600 mg/kg/day showed signs such as tremors, decreased activity, piloerection, hunched posture and tachypnoea and were either killed in-extremis or found dead. The high dose caused convulsions shortly after dosing.
Males treated with 300 mg/kg/day showed body weight gain, while a slight loss was observed at 420 mg/kg/day. No notable effect of treatment on body weights was observed in females.
necropsy of decedents identified no obvious cause of death.
The dose of 420 mg/kg/day was considered to be an appropriate estimate of the MTD for the main study.
RESULTS OF DEFINITIVE STUDY
- Appropriateness of dose levels and route:
Dose levels: 0, 105, 210 and 420 mg/kg Hydroquinone (equivalent to 25% MTD, 50% MTD and MTD respectively as determined in DRF study)
Route: same route as used in carcinogenicity studies
- Formulation analysis:
Formulations were homogeneuous and stable at room temperature for up to 7 days with no significant degradation. For dosing, the dosing solutions were prepared fresh each day. Animal dosing in the main experiment was completed within 2.5 hours of preparation of the test article formulations and on each dosing occasion the formulations were confirmed to be clear colourless solutions at the point of dispatch to the animal dosing room, and at the time of dosing the formulations, although it is acknowledged that this cannot be confirmed for two dosing occasions. It is considered the formulations were stable from preparation to completion of animal dosing.
Achieved concentrations : within +/-10% of the nominal test article concentrations, with the exception of the day 1 low dose (group 2 males) which was 88% from nominal. This was considered to have no impact on the validity of the study, as only the low dose was affected, and marginally outside acceptance.
- Rationale for exposure:
no clinical signs of toxicity following treatment with vehicle, hydroquinone or positive control.
Body weights: dose-related decrease in body weight gain, culminating with a slight weight loss at 420 mg/kg/day, compared to concurrent F344 vehicle control groups can be considered signs of systemic exposure in males and females.
KIDNEY: urine collected from all group 4 and 9 animals was reported to be darker in colour than the concurrent control group. This was attributed to metabolites of Hydroquinone and was considered evidence of kidney exposure following oral gavage dosing.
Microscopic examination showed acute tubular necrosis in males and females given 210 or 420 mg/kg/day, with a greater severity and/or incidence in males. Effects correlated with slightly higher levels of urea and/or creatinine compared to controls, in particular at the high dose providing also evidence of renal exposure to the test substance.
LIVER: increased hepatocyte mitosis was present in animals of all treated groups, with a generally dose-related effect. There was a decrease in glycogen vacuolation in males given 210 or 420 mg/kg/day and in females from all hydroquinone-treated groups.
Focal necrosis and degeneration/necrosis were present in one male treated at 420 mg/kg/day.
Those changes were accompanied by a slight increase in bilirubin, and slightly higher ALAT and ASAT activities in particular at the high dose, in males and females, which also indicate liver metabolic activity following hydroquinone administration and together with the indication of glycogen utilisation and mitosis provide evidence of organ exposure. Total bilirubin was slightly above controls in males of the high dose group.
TESTES:
A slight dose-related increase in absolute testes weight was observed in treated groups compared to vehicle controls. There were no microscopic findings in the gonads considered to be related to the tests substance.
- COMET ANALYSIS:
VALIDITY OF DATA
The data generated in this study confirm that:
1. There was a statistically significant increase in tail intensity in the positive control groups compared to the concurrent vehicle controls (only F344 groups analysed)
2. The high dose was considered to be the MTD
3. Data from Group 1, vehicle control F344 rats were compared to the data from the Group 1, vehicle control Sprague Dawley rats. Comet values were considered comparable between the animals (for all tissues and both sexes) therefore the comparison of the data to the laboratory historical control ranges was considered to be valid
4. Group mean vehicle control values were comparable to laboratory historical control data/in-house data for each tissue
5. The clinical chemistry and pathology data showed evidence of toxicity in the liver and kidney related to Hydroquinone and was therefore taken as evidence that the animals and the target tissue were systemically exposed to Hydroquinone. Exposure to the duodenum was assumed given the route of administration (oral gavage). There is no direct evidence of Hydroquinone exposure in the gonads which was expected as Hydrosuinone is not known to cause toxicity to gonads.
The assay data were therefore considered valid.
COMET RESULTS:
There was no dose-related increase in %hedgehogs in duodenum, kidney, liver or gonad cells following treatment with Hydroquinone, thus demonstrating that treatment with Hydroquinone did not cause excessive DNA damage that could have interfered with comet analysis.
Duodenum:
In male rats treated with Hydroquinone the group mean tail intensity was highly comparable with the concurrent vehicle control groups and all individual animals fell within the 95% reference ranges for this tissue. There were no statistically significant increases in tail intensity compared to the concurrent vehicle control group. This confirms that Hydroquinone did not induce any in DNA damage in the duodenum.
In female rats treated with Hydroquinone the group mean tail intensities were slightly elevated compared to the concurrent vehicle control, although at all dose levels this was less than two-fold and was not significantly different to the concurrent vehicle controls. For each dose group the increase in group mean tail intensity was attributed to isolated animals (notably Animals 215F, 219F and 226F) that had tail intensities that exceed the 95% reference ranges of the historical control data, however all animals were within the observed range of the historical control data. Furthermore all other Hydroquinone treated animals were highly comparable to the concurrent vehicle control and within the 95% reference ranges of the historical control data. It was therefore concluded that the female Hydroquinone data across all dose levels showed a normal degree of variation and there were no Hydroquinone related increases in DNA damage.
Kidney:
In male and female rats treated with Hydroquinone the group mean tail intensity was highly comparable with the concurrent vehicle control groups and all individual animals fell within the 95% reference ranges for this tissue. There were no statistically significant increases in tail intensity compared to the concurrent vehicle control group. This confirms that Hydroquinone did not induce any in DNA damage in the kidney.
Liver:
In male and female rats treated with Hydroquinone there were no statistically significant increases in tail intensity compared to the concurrent vehicle control groups. There were a small number of individual animals with slightly higher tail intensities (notably Animals 22M, 25M, 223F and 228F), which for the males resulted in a significant dose-response test. However, all animals (including those identified above) were considered to be generally comparable with concurrent vehicle controls and fell within the 95% reference ranges of the laboratory’s historical control data. The liver comet data for all animals were considered to be within normal biological variation and not indicative of any Hydroquinone induced DNA damage in the liver.
Gonads:
In male and female rats treated with Hydroquinone there were no statistically significant increases in tail intensity compared to the concurrent vehicle control groups. There were a small number of individual animals with slightly higher tail intensities (notably Animals 22M, 25M, 223F and 228F), which for the males resulted in a significant dose-response test. However, all animals (including those identified above) were considered to be generally comparable with concurrent vehicle controls and fell within the 95% reference ranges of the laboratory’s historical control data. The liver comet data for all animals were considered to be within normal biological variation and not indicative of any Hydroquinone induced DNA damage in the liver. - Conclusions:
- Negative.
When administered by oral gavage to groups of male and female Fischer F344 rats at levels of 0, 105, 210 or 420 mg/kg bw in 2 administrations approximately 24 hours apart, Hydroquinone did not cause any significant DNA damage in the Comet assay with cells prepared from duodenum, liver, kidney, and male testes, compared to vehicle-treated rats. Systemic exposure of the tissues was confirmed by biochemical changes and histopathology findings.
Under the conditions of the study Hydroquinone is not considered mutagenic. - Executive summary:
Groups of 6 males and 6 females Fischer F344 rats were administered by oral gavage Hydroquinone at 0, 105, 210 or 420 mg/kg bw in 2 administrations approximately 24 hours apart. The highest dose was the maximum tolerated dose determined in a dose-range finding study. Somatic tissues (duodenum, liver and kidneys) were sampled 30 minutes following the 2nd dosage, based on the peak plasma time determined in past toxicokinetic studies. Male gonads were sampled and examined as a substitute to germ cells. In the absence of specific kinetics data for male gonads and to account for potential peak delay, testes were sampled 2 hours following the 2nd dosage (default recommended sampling time in the test guideline).
Cells were isolated and lysed under alkaline conditions and DNA was submitted to electrophoresis.
There was no mortality and no clinical signs in the main study. There was a dose-related decrease in body weight gain, culminating with a slight weight loss at 420 mg/kg/day. Systemic exposure and metabolisation was also evidenced by dark-coloured urine collected at 24 hours after the 1st administration that was indicative of metabolite excretion, and acute tubular necrosis at microscopic examination in males and females given 210 or 420 mg/kg/day, with a greater severity and/or incidence in males. Effects correlated with slightly higher levels of urea and/or creatinine compared to controls. In the liver, increased hepatocyte mitosis was present in animals of all treated groups, with a generally dose-related effect, and accompanied by a slight increase in bilirubin, and slightly higher ALAT and ASAT activities in particular at the high dose, in males and females.
Hydroquinone did not cause any significant DNA damage in the Comet assay with cells prepared from duodenum, liver, kidney, and male testes, compared to vehicle-treated rats. Positive and negative controls in F344 rats were consistent with concurrent controls in Sprague-Dawley rats which served to strengthen the historical control data. Systemic exposure of the tissues was confirmed by biochemical changes and histopathology findings. Under the conditions of the study Hydroquinone is not considered mutagenic.
Referenceopen allclose all
Table: Mean adduct levels in nuclear DNA isolated from the kidneys of rats by the Nuclease P1-enhanced 32P-postlabeling assay
Adduct no. |
RAL x 109 a |
|||||
Male |
Female |
|||||
0 mg/kg |
50 mg/kg |
P-value |
0 mg/kg |
50 mg/kg |
P-value |
|
6: HQ-specific adduct |
0.95 ± 0.21 |
0.89 ± 0.12 |
0.661 |
0.83 ± 0.009 |
0.60 ± 0.11 |
0.018 * |
7: BQ-specific adduct |
3.55 ± 0.22 |
3.71 ± 0.42 |
0.530 |
2.50 ± 0.43 |
2.23 ± 0.36 |
0.415 |
Background adducts: |
||||||
1 |
3.04 ± 0.22 |
2.79 ± 0.23 |
0.160 |
3.10 ± 0.19 |
2.63 ± 0.42 |
0.088 |
2 |
1.33 ± 0.21 |
0.88 ± 0.11 |
0.009 * |
1.03 ± 0.10 |
0.94 ± 0.10 |
0.243 |
3 |
1.61 ± 0.13 |
1.07 ± 0.15 |
0.002 * |
1.35 ± 0.12 |
1.18 ± 0.20 |
0.200 |
4 |
1.92 ± 0.21 |
1.49 ± 0.31 |
0.062 * |
1.58 ± 0.17 |
1.21 ± 0.17 |
0.025 * |
5 |
23.55 ± 13.33 |
21.27 ± 2.18 |
0.747 |
61.21 ± 8.49 |
46.8 ± 20.60 |
0.244 |
8 |
34.96 ± 4.51 |
28.81 ± 1.83 |
0.045 * |
37.76 ± 1.27 |
27.59 ± 3.60 |
0.002 * |
9 |
- |
- |
2.82 ± 0.25 |
2.71 ± 0.36 |
0.629 |
|
Total adducts |
70.89 ± 18.14 |
60.89 ± 3.53 |
0.322 |
112.16 ± 9.10 |
85.88 ± 25.26 |
0.098 |
* statistically significant decrease compared to controls, P ≤ 0.05
a Mean ± standard deviation of data of 4 animals with duplicate postlabeling analyses of each DNA sample
Table: Chromatid aberrations in differentiating spermatogonia of mice 24 h after treatment with HQ
Dose (mg/kg) |
Number of cells scored |
Mitotic index (%) |
Aberrant cells (% ± SE) |
|
Inclusive gaps |
Exclusive gaps |
|||
0 |
600 |
51.3 |
5.8 ± 1.0 |
0.8 ± 0.3 |
40 |
500 |
62.8* |
11.0 ± 0.6 |
3.8 ± 0.7** |
80 |
500 |
50.0 |
11.4 ± 1.1 |
3.2 ± 0.6** |
120 |
500 |
68.2* |
15.4 ± 2.0 |
4.8 ± 0.5** |
Statistically different from vehicle controls: * P < 0.05; ** P < 0.01
Table 1: Results of the micronucleus assay after single i.p. treatment of male and female mice with HQ
Dose (mg/kg) |
Sampling time (h) |
Frequency of micronucleated PCE (MPCE/1000 PCE) |
PCE/NCE |
||
Group mean males ± SE |
Group mean females ± SE |
Mean of both sexes ± SE |
Mean of both sexes ± SE |
||
0 |
6-30 |
1.4 ± 0.2 |
1.3 ± 0.2 |
1.3 ± 0.1 |
0.94 ± 0.01 |
30 |
24 |
1.9 ± 0.2 |
1.7 ± 0.2 |
1.8 ± 0.1 |
0.94 ± 0.01 |
50 |
6 |
1.3 ± 0.2 |
0.8 ± 0.2 |
1.1 ± 0.2 |
0.93 ± 0.03 |
24 |
3.1 ± 0.3 |
3.8 ± 0.3 |
3.5 ± 0.2** |
0.87 ± 0.01* |
|
75 |
18 |
5.1 ± 0.6 |
4.2 ± 0.4 |
4.7 ± 0.4** |
0.90 ± 0.02 |
24 |
7.0 ± 0.8 |
7.1 ± 0.3 |
7.1 ± 0.4** |
0.91 ± 0.03 |
|
30 |
2.7 ± 0.3 |
3.4 ± 0.2 |
3.1 ± 0.2* |
0.96 ± 0.01 |
|
100 |
24 |
10.0 ± 0.8 |
12.1 ± 1.9 |
11.1 ± 1.1** |
0.91 ± 0.01 |
Table 2: Results of the micronucleus assay after single or multiple i.p. treatment of male mice with HQ
Dose (mg/kg) |
Number of doses |
Sampling time after last dose |
Frequency of micronucleated PCE (MPCE/1000 PCE) |
PCE/NCE |
Group mean ± SE |
Group mean ± SE |
|||
0 |
1-3 |
24 |
1.2 ± 0.2 |
0.98 ± 0.01 |
15 |
1 |
24 |
1.1 ± 0.4 |
0.97 ± 0.01 |
2 |
24 |
1.5 ± 0.4 |
0.97 ± 0.01 |
|
3 |
24 |
2.1 ± 0.4* |
0.99 ± 0.01 |
|
75 |
1 |
24 |
7.6 ± 0.9** |
0.93 ± 0.03* |
2 |
24 |
3.8 ± 0.5** |
0.97 ± 0.01 |
|
3 |
24 |
2.8 ± 0.2** |
0.98 ± 0.01 |
Statistically significantly different: * P < 0.05; ** P < 0.01
Table 1: Summary of the Mutation Frequencies in transgenic mice
Treatment |
Dose (mg/kg bw/day) |
Mutation Frequencies - mean + SD (x 10E-06 ) |
|||
|
|
Liver |
Stomach |
Kidney |
Lung |
Distilled water (negative control) |
0 |
75.0 + 11.5 |
39.6 + 7.5 |
53.4 + 14.9 |
56.3 + 10.9 |
|
|
|
|
|
|
Hydroquinone |
50 |
42.4 + 13.3 |
54.7 + 14.0 |
- |
- |
Hydroquinone |
100 |
44.1 + 8.5 |
46.7 + 9.0 |
- |
- |
Hydroquinone |
200 |
69.0 + 40.1 |
55.9 + 12.3 |
47.0 + 13.8 |
61.4 + 26.1 |
|
|
|
|
|
|
N-ethyl-N-nitrosourea (positive control) |
100 |
158.0 + 27.5 (a) |
473.0 + 31.3 (b) |
147.8 + 37.7 (a) |
260.2 + 67.8 (b) |
(a) p< 0.05 statistical significance in the Student's t-test
(b) p< 0.05 statistical significance in Aspin-Welch's t-test
Laboratory historical negative control data :
mean + SD: 47.6 + 17.2 (x 10E-06)
range: 0.00 - 99.2 (x 10E-06)
No statistically significant increase was observed in the treated groups compared to the control group.
One animal in the group treated at 200 mg/kg bw/day showed a higher mutation frequency in the liver compared to the vehicle control group. The value was above the historical control, but was considered to be an isolated finding because the other animals of the group did not show any significant increase. The authors of the study hypothesized that the increase could have occurred from a single mutation and clonal expansion.
Results of COMET assay - somatic tissues
Hydroquinone: Summary of Group Mean Data – Male Duodenum
Group / Treatment |
Sub Group |
Total Comets Scored |
Tail Intensity |
Tail Moment |
Mean %Hedgehogs |
|||
Mean |
SEM |
Mean |
SEM |
|||||
1M / Vehicle |
0 |
1 |
900 |
0.78 |
0.16 |
0.09 |
0.02 |
11.64 |
2M / HQ |
105 |
1 |
900 |
0.93 |
0.15 |
0.10 |
0.01 |
11.10 |
3M / HQ |
210 |
1 |
900 |
0.86 |
0.16 |
0.10 |
0.02 |
11.02 |
4M / HQ |
420 |
1 |
900 |
0.88 |
0.21 |
0.11 |
0.03 |
10.81 |
5M / EMS |
150 |
1 |
450 |
14.11** |
1.14 |
1.86 |
0.17 |
13.40 |
Statistics |
|
|
|
SR, A |
|
|
|
|
1M / Vehicle |
0 |
2 |
900 |
0.89 |
0.23 |
0.10 |
0.02 |
12.49 |
5M / EMS |
150 |
2 |
450 |
12.88 |
1.98 |
1.59 |
0.25 |
9.94 |
Hydroquinone: Summary of Group Mean Data – Female Duodenum
Group / Treatment |
Sub Group |
Total Comets Scored |
Tail Intensity |
Tail Moment |
Mean %Hedgehogs |
|||
Mean |
SEM |
Mean |
SEM |
|||||
1F / Vehicle |
0 |
1 |
900 |
1.77 |
0.59 |
0.19 |
0.07 |
21.90 |
2F / HQ |
105 |
1 |
900 |
3.02 |
0.94 |
0.36 |
0.11 |
22.82 |
3F / HQ |
210 |
1 |
900 |
3.26 |
1.10 |
0.39 |
0.14 |
19.05 |
4F / HQ |
420 |
1 |
900 |
2.97 |
1.02 |
0.34 |
0.12 |
21.40 |
5F / EMS |
150 |
1 |
450 |
11.41*** |
0.37 |
1.37 |
0.08 |
26.51 |
Statistics |
|
|
|
S, A |
|
|
|
|
1F / Vehicle |
0 |
2 |
900 |
1.71 |
0.57 |
0.21 |
0.07 |
16.16 |
5F / EMS |
150 |
2 |
450 |
11.47 |
1.03 |
1.35 |
0.16 |
16.69 |
M Male F Female *** p<0.001 |
R Rank-Transformed Data
Hydroquinone: Summary of Group Mean Data – Male Kidney
Group / Treatment |
Sub Group |
Total Comets Scored |
Tail Intensity |
Tail Moment |
||||
Mean |
SEM |
Mean |
SEM |
Mean %Hedgehogs |
||||
6M / Vehicle |
0 |
1 |
900 |
1.94 |
0.46 |
0.22 |
0.05 |
11.55 |
7M / HQ |
105 |
1 |
900 |
1.49 |
0.29 |
0.18 |
0.03 |
12.10 |
8M / HQ |
210 |
1 |
900 |
1.38 |
0.38 |
0.17 |
0.06 |
11.00 |
9M / HQ |
420 |
1 |
900 |
1.67 |
0.35 |
0.19 |
0.04 |
12.59 |
10M / EMS |
150 |
1 |
450 |
22.18*** |
0.35 |
3.50 |
0.07 |
13.87 |
Statistics |
|
|
|
S, A |
|
|
|
|
6M / Vehicle |
0 |
2 |
900 |
1.28 |
0.28 |
0.14 |
0.03 |
11.29 |
10M / EMS |
150 |
2 |
450 |
19.42 |
1.16 |
2.69 |
0.17 |
13.11 |
Hydroquinone: Summary of Group Mean Data – Female Kidney
Group / Treatment |
Sub Group |
Total Comets Scored |
Tail Intensity |
Tail Moment |
Mean %Hedgehogs |
|||
Mean |
SEM |
Mean |
SEM |
|||||
6F / Vehicle |
0 |
1 |
900 |
1.02 |
0.23 |
0.12 |
0.04 |
14.50 |
7F / HQ |
105 |
1 |
900 |
1.65 |
0.74 |
0.18 |
0.08 |
17.91 |
8F / HQ |
210 |
1 |
900 |
0.63 |
0.22 |
0.08 |
0.03 |
17.08 |
9F / HQ |
420 |
1 |
900 |
0.84 |
0.27 |
0.11 |
0.03 |
17.24 |
10F / EMS |
150 |
1 |
450 |
18.26** |
1.95 |
2.80 |
0.39 |
18.00 |
Statistics |
|
|
|
SR, AR |
|
|
|
|
6F / Vehicle |
0 |
2 |
900 |
1.93 |
0.46 |
0.23 |
0.05 |
15.74 |
10F / EMS |
150 |
2 |
300 |
18.38 |
2.26 |
2.63 |
0.41 |
14.12 |
M Male
F Female
SEM Standard
Error of Means
Sub-Group 1 F344
rats
Sub-Group 2 Sprague
Dawley rats
** p<0.01
*** p<0.001
S Two-sample
t-test (Group 6 vs. Group 10)
A ANOVA,
Dose Response and Dunnett’s (Group 6 vs. Groups 7, 8, 9)
R Rank-Transformed
Data.
Hydroquinone: Summary of Group Mean Data – Male Liver
Group / Treatment |
Sub Group |
Total Comets Scored |
Tail Intensity |
Tail Moment |
Mean %Hedgehogs |
|||
Mean |
SEM |
Mean |
SEM |
|||||
1M / Vehicle |
0 |
1 |
900 |
0.28 |
0.08 |
0.04 |
0.01 |
3.48 |
2M / HQ |
105 |
1 |
900 |
0.45 |
0.10 |
0.06 |
0.01 |
3.22 |
3M / HQ |
210 |
1 |
900 |
0.42 |
0.16 |
0.05 |
0.02 |
2.24 |
4M / HQ |
420 |
1 |
900 |
0.64 |
0.17 |
0.09 |
0.02 |
3.56 |
5M / EMS |
150 |
1 |
450 |
20.45*** |
2.04 |
3.26 |
0.45 |
5.31 |
Statistics |
|
|
|
DR, S, A |
|
|
|
|
1M / Vehicle |
0 |
2 |
900 |
0.35 |
0.13 |
0.05 |
0.02 |
4.43 |
5M / EMS |
150 |
2 |
450 |
15.02 |
0.35 |
2.07 |
0.06 |
2.98 |
Hydroquinone: Summary of Group Mean Data – Female Liver
Group / Treatment |
Sub Group |
Total Comets Scored |
Tail Intensity |
Tail Moment |
Mean %Hedgehogs |
|||
Mean |
SEM |
Mean |
SEM |
|||||
1F / Vehicle |
0 |
1 |
900 |
0.72 |
0.17 |
0.10 |
0.02 |
4.71 |
2F / HQ |
105 |
1 |
900 |
0.90 |
0.24 |
0.13 |
0.03 |
5.60 |
3F / HQ |
210 |
1 |
900 |
1.02 |
0.42 |
0.13 |
0.05 |
4.43 |
4F / HQ |
420 |
1 |
900 |
1.34 |
0.47 |
0.18 |
0.06 |
5.12 |
5F / EMS |
150 |
1 |
450 |
21.52** |
1.31 |
3.55 |
0.33 |
5.89 |
Statistics |
|
|
|
SR, A |
|
|
|
|
1F / Vehicle |
0 |
2 |
900 |
1.40 |
0.34 |
0.17 |
0.04 |
4.05 |
5F / EMS |
150 |
2 |
450 |
22.20 |
2.12 |
3.37 |
0.49 |
6.78 |
M Male
F Female
SEM Standard
Error of Means
Sub-Group 1 F344
rats
Sub-Group 2 Sprague
Dawley rats
*** p<0.001
** p<0.01
DR Significant
Dose Response test (Groups 1-4)
S Two-sample
t-test (Group 1 vs. Group 5)
A ANOVA,
Dose Response and Dunnett’s (Group 1 vs. Groups 2, 3, 4)
R Rank-Transformed
Data.
Additional information
IN VITRO STUDIES
The assessment of the mutagenic potential of HQ in vitro is based on five key studies (Details presented in Table 1) and test results from further supporting studies (Details see Table 2 and 3).
HQ showed no mutagenic activity in a bacterial test system in investigations comprising all Salmonella typhimurium strains required by OECD Guideline 471 and including also a strain sensitive to oxidising or crosslinking agents (Key studies: Haworth et al., 1983; NTP, 1989; Watzinger, 2007a).
HQ was positive in the mouse lymphoma mutation assay both in the absence and presence of metabolic activation at relative total growths of 32% and 87%,respectively) (test protocol similar to Guideline study; key study: McGregor et al., 1988; NTP, 1989).
In vitro chromosome aberration tests withprimary cultures of human lymphocytes from single healthy nonsmoking male donorswere negative up to cytotoxic test concentrations (Guideline and GLP studies; key study: de Vogel, 2000; Roza et al., 2003;supporting study: Van Delft and de Vogel, 1997). In chromosome aberration tests with mammalian cell lines positive effects were reported. With Chinese hamster ovary cells an increased frequency of structural chromosomal aberrations occurred only in the presence of a metabolic activation system and at the upper dose range with simultaneous cytotoxic effects (no guideline study; key study: Galloway et al., 1987; NTP, 1989). In contrast,in V79 cells HQ induced a significant increase of structural chromosomal aberrations only without metabolic activation. Presence of S9 or SOD reduced the clastogenic activity. An association of the clastogenicity of HQ with the formation of the semiquinone radical or quinone, or with the generation of superoxide anion or peroxide was discussed (Supporting study: Do Ceu Silva et al., 2003).
In a micronucleus assay in mouse lymphoma L5178Y cells, HQ induced the formation of micronuclei both in the absence and presence of metabolic activation by liver or kidney S9 mix (Supporting study: Watzinger, 2007). HQ was found to be negative in a micronucleus assay with V79 cells in the absence of an exogenous metabolising system. However, the formation of micronuclei in the presence of arachidonic acid indicated that HQ can be activated by prostaglandin-H-synthase to reactive compounds (Supporting study: Dobo and Eastmond, 1994). Micronucleus assays with primary cultures of peripheral blood human leukocytes yielded both positive test results (single donor and 15 donors, respectively; supporting studies: Yager et al., 1990; Do Ceu Silva et al., 2004) and negative test results (8 donors; supporting study: Doepker et al., 2000). Formation of HQ-induced MN depended both on chromosome breakage and chromosome loss indicating both a clastogenic activity and an aneugenic activity by disturbing microtubule assembly and spindle formation (Dobo and Eastmond, 1994; Yager et al., 1990).
Significant increases of the frequencies of SCE (test conditions similar to OECD Guideline 479) were observed in CHO cells (Supporting study: Galloway et al., 1987) and in primary cultures of peripheral blood human leukocytes (donor numbers 15, 1, and 1, respectively, supporting studies: Do Ceu Silva et al., 2004; Erexson et al., 1985; Morimoto et al., 1980). Effects in CHO cells were found to be lower after metabolic activation by liver S9 compared to values without activation. In the study with lymphocytes from 15 donors a possible influence of GST polymorphism (glutathione-S-transferase) on the induction of MN and SCE was investigated. While the presence of GSTT1 and GSTP1 had no effect on induction of micronuclei, metabolism of HQ by GSTM1 significantly reduced the formation of MN. In contrast, there was no influence of GST polymorphism on SCE induction (Do Ceu Silva et al., 2004).
Several studies indicate that HQ may induce oxidative DNA damage or covalent DNA binding in peroxidase-proficient cells in vitro but not in vivo (see also “IN VIVO STUDIES” below).
The formation of oxidative DNA damage could be demonstrated in cultures of human HL60 cells, a promyelocytic cell line with significant activity of myeloperoxidase. After short 30 min incubations with 10 µM HQ a doubling of the frequency of 8OHdG compared to controls was observed which returned to background levels on extended incubation presumably due to the rapid repair of this DNA damage. In contrast, in vivo no increase of 8OHdG was found in bone marrow of B6C3F1 mice after i.p. application of HQ (Supporting study: Kolachana et al., 1993). In a further study with human HL60 cells, HQ specific DNA adducts were identified by P1 enhanced32P-postlabeling. There was a single DNA adduct which was identical with the adduct formed by exposure to BQ, but higher concentrations of HQ and longer treatment times were required to achieve same adduct levels as by BQ exposure (e.g. 7.8 HQ specific adducts per 107nucleotides at 500 µM HQ for 8 hrs). As different adducts are formed in the cells compared to direct reaction of HQ with purified DNA, either chromatin structure or enzymatic processes, probably by activation via myeloperoxidase, are influencing DNA adduct formation within cells (Supporting study: Levay et al., 1991). The same HQ-specific adduct as in HL60 cells was also formed in cultures of human bone marrow cells and in mouse bone marrow macrophages in vitro. There was a significant correlation between cellular activity of peroxidase and adduct levels observed in the cells. No adducts were detected in peroxidase deficient leukaemia cell lines U-937 and Raji. The formation of DNA adducts by HQ may reflect a balance between activation by cellular peroxidase with formation of the semiquinone radical, and inactivation by cellular antioxidants (Supporting study: Levay et al., 1993). In cultured rat Zymbal glands from female Sprague-Dawley rats, the formation of several HQ-derived adducts was detected with total adduct levels of 10.8 and 12.5 per 107nucleotides (750 and 1500 µM HQ for 24 hrs). Rat Zymbal gland cells contain peroxidases which can oxidize HQ to reactive metabolites. However, since only a minor fraction (<20%) of HQ-derived adducts corresponded to BQ-derived DNA modifications, it appears that in Zymbal gland cultures BQ is a minor reactive intermediate of HQ, while the semiquinone radical may predominate (Supporting study: Reddy et al., 1989). In contrast, HQ-specific adducts were not detected in the Zymbal gland, bone marrow, liver and spleen in vivo after oral exposure of rats to a mixture of HQ and phenol (Supporting study: Reddy et al., 1990).
Table 1: Overview on key studies for assessment of in vitro genotoxicity
Test system |
Cells |
Metabolic activation |
Test concentration |
Test result |
Remarks |
Reference |
Salmonella typhimurium reverse mutation assay |
S typh. TA98, TA100, TA1535, TA1537 |
-/+ a |
10 - 666 µg/plate |
neg/neg |
Cytotoxicity at³ 333 µg/plate |
Haworth et al., 1983; NTP, 1989 |
Salmonella typhimurium reverse mutation assay (screening micromethod assay) |
S. typh.TA98, TA100, TA102, TA1537 |
-/+ b |
0.38 - 5000 µg/assay |
neg/neg |
Cytotoxicity at³2500 µg/assay and with strain TA102, -S9 at³278 µg/assay |
Watzinger, 2007a |
Chromosome aberration assay |
Primary cultures of human lymphocytes from a healthy nonsmoking male donor |
-/+ c |
2-100 µg/mL for 3 h treatment; |
neg/neg |
Up to 48 hrs of continuous treatment; |
de Vogel, 2000; Roza et al., 2003 |
Chromosome aberration assay |
CHO cells |
-/+ c |
-S9: 150-600 µg/mL |
neg/pos§ |
Treatment for 8.5 h -S9 and 2 h +S9; |
Galloway et al., 1987; NTP, 1989 |
Mouse lymphoma mutation assay |
L5178Y/TK cells |
-/+ c |
-S9: 0.625-50 µg/mL |
pos/pos* |
Treatment time 4 h; |
McGregor et al., 1988; NTP, 1989 |
a S9 mix from livers of Aroclor 1254 treated hamsters or rats
b S9 mix from liver and kidneys of Aroclor 1254 treated rats
c S9 mix from liver of Aroclor 1254 treated rats
# single trial with only 100 metaphases evaluated per test concentration, only 50 metaphases at 20 µg/mL, –S9; cytotoxicity was reported to occur at the higher test concentrations (no further data)
§ -S9: statistically significant increase of the frequency of structural chromosomal aberrations at 20 µg/mL (only 50 metaphases evaluated), negative due to lack of a significant dose-response relationship; +S9: statistically significant increase of the frequency of structural chromosomal aberrations at 450 and 600 µg/mL (P ≤ 0.05, cytotoxic concentrations)
* significant and dose-dependant increase of mutant fraction at³1.25 µg/mL -S9 (relative total growth 32%), and³2.5 µg/mL +S9 (relative total growth 87%)
Table 2: Overview of genotoxicity studies to be used as supporting information (Reliability 2)
Test system |
Cells |
Test conditions |
Test results |
Remarks |
Reference |
Chromosome aberration assay (Similar to OECD GL 473) |
V79 |
0, 20, 40, 60, 80 µM 0, 80 µM at pH 7.4 with S9, SOD, CAT or SOD + CAT |
pH 6.0: -S9 neg pH 7.4: -/+ S9 pos pH 8.0: -S9 pos at 80 µM |
Presence of S9 or SOD, or SOD + CAT significantly decreased CA at 80 µM HQ compared to assays without metabolic activation MI at 80 µM (% of control): 72% at pH 6.0, 53% at pH 7.4, 38% at pH 8.0 |
Do Ceu Silva et al., 2003 |
Chromosome aberration assay (OECD GL 473, GLP) |
human lymphocytes (1 male donor) |
0, 8, 16 µg/mL without additional metabolic activation |
Neg (no additional metabolic activation) |
Cytotoxicity: MI 64% and 54% at 8 and 16 µg/mL |
Van Delft and de Vogel, 1997 |
Micronucleus assay (Micromethod screening assay similar to standard conditions) |
mouse lymphoma L5178Y |
-S9: 0, 1.25, 2.5, 5.0 µg/mL without and with a 20-hr recovery period +S9: 0, 2.44, 4.88, 9.77 µg/mL |
Pos: both –S9 and +S9 |
-S9: sign. increase of MN at ≥ 1.25 µg/mL without recovery and at ≥ 2.5 µg/mL with recovery; + liver S9: sign. increase of MN at 9.77 µg/mL + kidney S9: sign. increase of MN at ≥ 4.88 µg/mL Cytotoxicity: -S9/without recovery: only slight at 5 µg/mL with 78% survival + kidney S9: 50% survival at 9.77 µg/mL |
Watzinger, 2007 |
Micronucleus assay with differentiation of CREST positive and negative MN (Standard conditions) |
V79 |
0, 17.5, 35, 70, 105, 140 µM without additional metabolic activation Specific investigations on role of oxidative metabolism: + catalase and -/+ AA + GSH and -/+ AA |
Without additions: neg + AA: pos + AA + catalase: neg + AA + GSH: neg |
Without additions: no sign. dose-related increase of MN In the presence of AA sign. dose-related increase of MN Sign. inhibition of MN induction by AA/PHS in the presence of catalase or GSH |
Dobo and Eastmond, 1994 |
Micronucleus assay with identification of kinetochore-positive MN (Standard conditions) |
human lymphocytes (1 male donor) |
0, 2 – 150 µM without additional metabolic activation |
Pos (no additional metabolic activation) |
Sign increase of total MN and kinetochore positive MN at ≥ 75 µM Cytotoxicity: cell viability 68% at 75 µM and 42% at 150 µM |
Yager et al., 1990 |
Micronucleus assay (Standard conditions) |
human lymphocytes from 15 donors with different GST genotypes |
0, 40, 80 µM |
Pos (no additional metabolic activation) |
Sign. increase of overall frequency of MN; |
Do Ceu Silva et al., 2004 |
Micronucleus assay (Standard assay conditions) |
human lymphocytes from 8 male and female donors |
0, 12.5 – 200 µM without additional metabolic activation |
Neg (no additional metabolic activation) |
Cytotoxicity: reduction of replication index was maximally about 30% at 200 µM HQ |
Doepker et al., 2000 |
SCE (Similar to OECD GL 479) |
CHO |
-S9: 0, 0.5, 1.67, 5.0 µg/mL |
-/+S9: pos |
Dose-related increase of SCE at all doses of > 20% compared to controls |
Galloway et al., 1987 |
SCE (Standard conditions) |
human lymphocytes from 15 donors with different GST genotypes |
0, 80 µM |
Pos (no additional metabolic activation) |
Sign. increase of overall frequency of SCE |
Do Ceu Silva et al., 2004 |
SCE ((Similar to OECD GL 479) |
human lymphocytes (1 male donor) |
0, 5, 50, 70, 100, 300 µM |
Pos (no additional metabolic activation) |
Sign. dose-related increase of SCE at 50 – 100 µM |
Erexson et al., 1985 |
SCE (Similar to OECD GL 479) |
human lymphocytes (1 male donor) |
0, 1.6, 8, 40, 200, 1000 µM |
Pos (no additional metabolic activation) |
Positive response: increase of SCE at 40 and 200 µM |
Morimoto et al., 1980 |
AA: arachidonic acid; CAT: catalase; CHO: Chinese hamster ovary cells; CREST assay: labelling of micronuclei with CREST antibody indicates micronuclei formed by loss of whole chromosomes; GST: glutathione-S-transferase; MI: mitotic index; MN: micronuclei; PHS: prostaglandin-H-synthase; SCE: sister chromatid exchange; Sign.: significant; SOD: superoxide dismutase
Table 3: Overview of studies on formation of DNA adducts to be used as supporting information (Reliability 2)
Type of DNA lesion |
Assay |
Test conditions |
Resulta |
Remarks |
Reference |
Oxidative DNA damage |
8OHdG adducts (HPLC detection) Human HL60 cells |
10 µM HQ |
Pos |
Frequency of 8OHdG was doubled compared to background after 30 min of exposure: 0.160 vs. 0.080 pmol 8OHdG/µg DNA. For longer exposures, adduct levels returned to background. Cytotoxicity: none up to 6 hrs exposure |
Kolachana et al., 1993 |
DNA damage by formation of HQ-specific DNA adducts |
DNA adducts by P1 enhanced32P-postlabeling Human HL60 cells |
Standard technique 0, 50, 100, 250, 500 µM |
Pos |
Identification of a single DNA adduct being identical with adduct formed by exposure to BQ; adduct levels: 0.5 – 7.8 per 107nucleotides (500 µM HQ for 8 hrs) Cytotoxicity: cell viability reduced to 25% at 500 µM HQ for 8 hrs |
Levay et al., 1991 |
DNA adducts by P1 enhanced32P-postlabeling Human HL60 cells |
Standard technique (optimised) 0 - 500 µM HQ |
Pos in HL60, HBM and MBMM Neg in leukaemia cell lines) |
Identification of a single identical DNA adduct in HL60, HBM and MBMM, no adduct in leukaemia cell lines; adduct level: HL60 > HBM > MBMM: In contrast, BQ formed two adducts in all cell types. |
Levay et al., 1993 |
|
DNA adducts by P1 enhanced32P-postlabeling cultured rat Zymbal glands from female Sprague-Dawley rats |
Standard technique 750, 1500 µM HQ |
Pos |
Formation of several HQ-derived adducts, total adduct level 10.8 and 12.5 per 107nucleotides (750 and 1500 µM HQ for 24 hrs) |
Reddy et al., 1989 |
|
DNA adducts by P1 enhanced32P-postlabeling cultured rat Zymbal glands from female Sprague-Dawley rats |
Standard technique 750 µM HQ |
Pos |
Formation of several HQ-derived adducts, total adduct level 13.4 per 107nucleotides (750 µM HQ for 24 hrs) |
Reddy et al., 1990 |
aAll assays without additional metabolic activation system as e.g. S9
Cells: Human HL60 cells: human promyelocytic cell line; HBM: freshly isolated human bone marrow cells; MBMM: primary cultures of mouse bone marrow macrophages
IN VIVO STUDIES
The assessment of the mutagenic potential of HQ in vivo is based on key studies (see Table 4) and supporting studies (see Table 5) with either intraperitoneal or oral application by gavage and both with somatic and germ cells as target cells.
HQ, at lowest effective doses of 50 - 80 mg/kg bw, induced both micronuclei and structural and numerical chromosomal aberrations in the bone marrow of male or female mice after intraperitoneal application while tests for induction of polyploidy or SCE were negative. Effects were found to be highly dependant on the sampling time of the bone marrow (Key study: Adler et al., 1990; supporting studies: Xu and Adler, 1990; Paccheriotti et al., 1991). There was also an increase of structural chromosomal aberrations in mouse spermatocytes with a lowest effective intraperitoneal dose of 40 mg/kg bw (Key study: Ciranni and Adler, 1991). HQ was negative in a dominant lethal assay with oral exposure by gavage for 10 weeks (comparable to OECD GL 478) (Key study: Krasavage et al., 1984).
In studies, investigating the formation of oxidative or specific DNA damage in somatic cells in vivo, HQ treatment was without effect.
Measurement of levels of 8-hydroxydeoxyguanosine adducts and of HQ-specific or BQ-specific DNA adducts in kidneys of HQ treated F344 rats indicated that HQ does not induce oxidative DNA damage or covalent binding to DNA after multiple gavage doses (0, 50 mg HQ/kg bw/d,6 wks, on 5 d/wk). On the contrary, there were significant reductions in levels of some endogenous adducts, possibly by virtue of the antioxidant properties of HQ. Consequently, there was a lack of formation of any HQ-treatment related DNA adducts, at exposures conditions up to dose levels producing mild kidney toxicity and significant cell proliferation in F344 male rats, thus favouring the expression of damage due to the compromised condition of the kidneys (Key studies: English et al., 1994, 1997). There was no increase of8-OHdG adducts in bone marrow of male B6C3F1 mice after intraperitoneal injection of 75 mg/kg bw, although increased adduct levels were found in vitro in cultures of human HL60 cells, a promyelocytic cell line (Supporting study: Kolachana et al., 1993). Also, there was no in vivo formation of typical HQ-derived adducts inliver, spleen, bone marrow and Zymbal gland after oral treatment (gavage of 75 mg/kg bw) offemale Sprague-Dawley rats which is in contrast to findings after in vitro exposure of Zymbal gland cultures to HQ (Supporting study: Reddy et al., 1990).
Finally, in the in vivo alkaline comet assay conducted in Fischer F344 rats, there was no DNA strand breaks in cells isolated from the liver (major site of HQ metabolisation), duodenum (secondary site of absorption and metabolisation), kidney (as target tissue in male Fischer F344 rats carcinogenicity studies), and male gonads as a surrogate for germ cells (Beevers, 2016). There was no increase in % tail DNA in these tissues sampled at peak plasma time and up to the maximum tolerated dose, although there was evidence of systemic toxicity and tissue exposure as shown by the clinical and biochemical effects, and histopathological alterations in liver and kidney. The early sampling time after the 2nd administration, consistent with peak plasma concentration before elimination (and 24 hours after the initial dose) ensures appropriate conditions of detection, and it is unlikely that potential damages would have been repaired within this short time-frame. Organ exposure was consistent with previous toxicokinetic data that had showed 14C labelling in all the tissues examined
The study was conducted in the same rat strain tested in long-term repeated dose toxicity studies, and the results indicate that the renal tubular tumors observed in Fischer F344 rats are not likely originating from direct DNA damage occurring in this tissue.
In another in vivo mutation assay, no mutagenic effects were observed in a transgenic rodent assay (TGR) in male Muta mice treated by oral gavage for 28 days, followed by an expression period of 3 days, with analysis of liver, stomach, kidney, and lung (Matsumoto et al., 2014). The model responds to mutation at the lacZgene and allows the detection primarily of base pair substitution mutations, frameshift mutations and small insertions/deletions.
Table 4: Overview on key studies for assessment of in vivo genotoxicity
Target cells |
Test system |
Test animals and treatment |
Route |
Test result |
Remarks |
Reference |
Somatic cells |
Micronucleus assay in polychromatic erythrocytes from bone marrow |
Mouse (101/E1 X C3H/E1) F1 |
Intraperitoneal injection |
Posa |
Lowest effective dose 50 mg/kg bw or 3times 15 mg/kg bw/d (45 mg in total) |
Adler et al., 1990 |
Somatic cells |
Micronucleus assay in polychromatic erythrocytes from peripheral blood |
Mouse (101/E1 X C3H/E1) F1 |
Intraperitoneal injection |
Posb |
Lowest effective dose 25 mg/kg bw Peak frequency at 40 h Possible threshold between 12.5 and 25 mg/kg bw |
Grawé et al., 1997 |
Somatic cells |
Micronucleus assay in polychromatic erythrocytes from bone marrow |
Mouse (Swiss CD-1) |
Intraperitoneal injection or oral gavage |
Pos |
Weakly positive by oral gavage (2x); significant effects by ip route Peak frequency at 18 h |
Ciranni et al., 1988 |
Germ cells |
Assay for structural chromosomal aberrations in spermatocytes |
Mouse (102/E1 X C3H/E1) F1 |
Intraperitoneal injection |
Posc |
Lowest effective dose 40 mg/kg bw |
Ciranni and Adler, 1991 |
Germ cells |
Dominant lethal assay |
Rat CD |
Gavage |
Negd |
Toxicity at 300 mg/kg (mortalities 2/25, clinical signs of intoxication) |
Krasavage et al., 1984 |
Somatic cells |
Formation of DNA adducts in kidney cells by Nuclease P1-enhanced 32P-postlabeling assay |
F344 rat |
Gavage |
Nege |
Evidence of concurrent kidney toxicity indicated by enzymuria, cell proliferation and histopathologic changes |
English et al., 1994 |
Somatic cells |
Oxidative DNA damage in kidney cells |
F344 rat |
Gavage |
Nege |
Evidence of concurrent kidney toxicity indicated by enzymuria, cell proliferation and histopathologic changes |
English et al., 1997 |
Somatic cells |
lacZ transgenic mutation study in somatic cells from liver, stomach, kidney, and lung (OECD 488) |
LacZ Muta(TM)mouse |
Gavage 0, 25, 50, 100, 200 mg/kg bw 4 wks, once a day |
Neg |
Expression period: 3 days before tissue sampling No clinical signs but decrease in body weight gain in all treatment groups. |
Matsumoto et al., 2014 |
Somatic cells |
Single cell gel/comet assay in rodents for detection of DNA damage in liver, kidney, and duodenum (OECD 489) |
F344 rat |
Gavage 0, 105, 210, 420 mg/kg bw 2 administrations 24 hours apart Sampling time: 30 min after 2nddosing |
Neg |
Systemic exposure evidenced by urine coloration and signs of acute tubular necrosis at 210 and 420 mg/kg (MTD), increased hepatocyte mitosis and ALAT, ASAT activities, bilirubin. |
Beevers, 2016 |
Gonads/ germ cells |
Single cell gel/comet assay in rodents for detection of DNA damage in male gonads (OECD 489) |
F344 rat |
Gavage 0, 105, 210, 420 mg/kg bw 2 administrations 24 hours apart Sampling time: 2 hours after 2nddosing |
Neg |
No microscopic findings in testes at MTD |
Beevers, 2016 |
a Single application: statistically significant increases in micronucleus frequency for sampling times of 24 h at 50-100 mg/kg with a dose-dependant increase, and at sampling times of 18-30 h at 75 mg/kg, maximum responses for 24 h sampling time; multiple dosing: significant increases of micronuclei frequency for 3 doses of 15 mg/kg, and 1, 2 or 3 doses of 75 mg/kg.
b Single application: statistically significant increase in frequency of micronucleated polychromatic erythrocytes for sampling times 24 and 40 hours at 25 -75 mg/kg, and for sampling time 48 hours at 50-75 mg/kg. The peak response was 40 hours in all dose groups. No significant effect at 12.5 mg/kg. The plot of the Area below the curve versus doses showed a biphasic response suggesting a no-effect threshold.
c Statistically significant increase of frequencies of aberrant cells (chromatide aberrations exclusive gaps)
d no effects on insemination rate, pregnancy rate, counts of corpora lutea, of implantation sites with categorization as early deaths, late deaths or viable embryos, % pre-implantation loss, % post-implantation loss; the positive control substance showed a positive test result
e no increased or modified formation of HQ-specific or BQ-specific DNA adducts compared to control rats; on the contrary, there were significant reductions in levels of some endogenous adducts
Table 5: Overview of in vivo genotoxicity studies to be used as supporting information (reliability 2)
Target cells |
Test system |
Test animals and treatment |
Route |
Test result |
Remarks |
Reference |
Somatic cells |
Oxidative DNA damage in mouse bone marrow |
Mouse B6C3F1 |
Intraperitoneal injection |
Neg |
No significant increase compared to background frequency; while also 75 mg/kg phenol was also negative, a combination of 75 mg/kg HQ and 75 mg/kg phenol induced a significant increase of8-OHdG |
Kolachana et al., 1993 |
Somatic cells |
Formation of DNA adducts in liver, spleen, bone marrow and Zymbal gland by Nuclease P1 enhanced32P-postlabeling |
Sprague-Dawley rat |
Gavage Other test substance: 75 mg HQ + 75 mg phenol4 d |
Neg |
No in vivo formation of the typical HQ-derived adducts identified after in vitro incubation with Zymbal gland cultures |
Reddy et al., 1990 |
Somatic cells |
Assay for structural chromosomal aberrations in bone marrow |
Mouse(101/E1 X C3H/E1) F1 |
Intraperitoneal injection |
Pos |
Lowest effective dose 75 mg/kg bw, 24 h sampling time |
Xu and Adler, 1990 |
Somatic cells |
Assays for numerical chromosomal aberrations, micronuclei and SCE in bone marrow |
Mouse (C57B1/Cne X C3H/Cne) F1 |
Intraperitoneal injection |
CAnum: pos
MN: pos
SCE: neg |
At 80 mg/kg and 18 h sampling time sign. increase of hyperploidy and MN, no dose related effect no effect on polyploidy or SCE. A significant increase of average generation time was observed at a sampling time of 18 h and doses of 80 and 120 mg/kg. HQ was discussed to affect a chromosomal component rather than a spindle component of chromosomal segregation. |
Paccheriotti et al., 1991 |
CAnum: numerical chromosomal aberrations; MN: micronuclei; SCE: sister chromatid exchange
SUMMARY AND DISCUSSION
In the key in vitro studies, HQ showed no mutagenic activity in a bacterial test system in investigations comprising all Salmonella typhimurium strains required by OECD Guideline 471 and including also a strain sensitive to oxidising or crosslinking agents. An in vitro chromosome aberration test with primary cultures of human lymphocytes from a healthy nonsmoking male donor was negative up to cytotoxic test concentrations (Guideline and GLP study), while in a further test with Chinese hamster ovary cells (no guideline study) an increased frequency of structural chromosomal aberrations occurred only in the presence of a metabolic activation system and at the upper dose range with simultaneous cytotoxic effects. HQ was positive in the mouse lymphoma assay both in the absence and presence of metabolic activation at relative total growths of 32% and 87%, respectively) (test protocol similar to Guideline study).
Based on the total evidence including supporting studies, HQ was found to be a direct acting genotoxic agent in cultures of mammalian cell lines and in human peripheral lymphocytes inducing mutations, DNA adducts, chromosome aberrations, micronuclei and sister chromatid exchange. Effects mostly occurred at test concentrations associated with moderate to significant cytotoxic effects. There are clues that the outcome of genotoxicity assays with HQ is dependent on the routes of metabolisation available in the applied test system. HQ can be activated by prostaglandin-H-synthase and peroxidases to reactive compounds as e.g. semiquinone radical and benzoquinone. A metabolizing system based on hepatic enzymes (liver S9) can favour deactivation of the genotoxic activity of HQ while induction of genotoxic activity in the presence of S9 was reported in other assays. Metabolism by pathways yielding the semiquinone radical and/or reactive oxygen species was found to be a condition supporting clastogenic effects. In test systems applying human peripheral lymphocytes, test results may be dependent on the individual genotypes of metabolizing enzymes, as e.g. GST. The important effect of metabolic routes on genotoxicity testing results in vitro is further supported by the outcome of genotoxicity testing in vivo.
In vivo, at lowest effective doses of 50 - 80 mg/kg bw, HQ induced both micronuclei and structural and numerical chromosomal aberrations in the bone marrow of male or female mice after intraperitoneal application while tests for induction of polyploidy or SCE were negative. Effects were found to be highly dependent on the sampling time of the bone marrow. Micronucleus frequency was only weakly positive in mice administered HQ by oral gavage, compared to intraperitoneal injection. In another study investigating micronucleus formation in polychromatic erythrocytes from the peripheral blood of male mice treated intraperitoneally, the lowest effective dose was found at 25 mg/kg, with a peak frequency at sampling time 40 hrs. Integration of the dose-dependent frequencies over 72 hours and plotting area under the curve as a function of the dose showed a biphasic shape which suggested a threshold of effect between 12.5 mg/kg and 25 mg/kg. There was also an increase of structural chromosomal aberrations in mouse spermatocytes with a lowest effective intraperitoneal dose of 40 mg/kg bw. In contrast, no in vivo genotoxicity was found after oral HQ exposure (bolus application via gavage) for extended periods of 6 to 10 weeks in germ cells, or in liver, spleen, kidney, bone marrow and the Zymbal gland of rats or mice. A dominant lethal assay with oral treatment of male CD rats was negative. Measurement of levels of 8-hydroxydeoxyguanosine adducts and of HQ-specific or BQ-specific DNA adducts in kidneys of HQ treated F344 rats indicated that HQ does not induce oxidative DNA damage or covalent binding to DNA after multiple gavage doses. On the contrary, there were significant reductions in levels of some endogenous adducts, possibly by virtue of the antioxidant properties of HQ. Also, there was no in vivo formation of typical HQ-derived adducts in liver, spleen, bone marrow and Zymbal gland after oral treatment off male Sprague-Dawley rats which is in contrast to findings after in vitro exposure of Zymbal gland cultures to HQ. There was no increase of 8-OHdG adducts in bone marrow of male B6C3F1 mice after intraperitoneal injection, although increased adduct levels were found in vitro in cultures of human HL60 cells, a promyelocytic cell line.
Consequently, there was a lack of formation of any HQ-treatment related DNA adducts, at exposures conditions up to dose levels producing mild kidney toxicity and significant cell proliferation in F344 male rats, thus favouring the expression of damage due to the compromised condition of the kidneys. Additionally, the upper dose of 50 mg/kg bw/d was the high dose level of the NTP rat bioassay. These observation suggest that the kidney toxicity observed with HQ in male F344 rats is not likely due to direct (i.e. adducts) or indirect (i.e. oxidative) DNA damage, and that benign kidney tumours observed in the 2-year carcinogenesis bioassay with male F344 rats are produced via a non-genotoxic mechanism.
This is further supported by two complementary in vivo guideline studies (reliability 1) investigating genotoxic effects in tissues: a Transgenic rodent assay in lacZ transgenic male Muta mice exposed for 28 days, and an in vivo alkaline Comet assay in Fischer F344 rats treated by oral gavage (2 administrations 24-hr apart with sampling at peak plasma time).
The in vivo alkaline comet assay was used to investigate the potential formation of DNA strand breaks in cells isolated from liver, duodenum, kidney in male Fischer, and male gonads as a surrogate for germ cells. There was no increase in % tail DNA in these tissues sampled at peak plasma time and up to the maximum tolerated dose, although there was evidence of systemic toxicity and tissue exposure as shown by the clinical and biochemical effects, and histopathological alterations in liver and kidney. The early sampling time after the 2nd administration, consistent with peak plasma concentration before elimination (and 24 hours after the initial dose) ensures appropriate conditions of detection.
The study was conducted in the same rat strain tested in long-term repeated dose toxicity studies, and the results indicate that the renal tubular tumors observed in Fischer F344 rats are not likely originating from direct DNA damage occurring in this tissue.
Matsumoto et al. also reported no mutagenic effects in the lacZ gene a transgenic rodent assay in male Muta mice treated by oral gavage for 28 days following analysis of liver, stomach, kidney, and lung.
Intraperitoneal injection is a mode of application through which unmodified HQ may reach targets of in vivo genotoxicity studies, as e.g., the bone marrow and germ cells, as biotransformation and detoxification of HQ taking place in the liver after oral uptake is bypassed. That hepatic detoxification is substantially reflected in the lower acute toxicity of orally vs. intraperitoneally applied HQ. The comprehensive investigations on the metabolism and toxicokinetics of HQ (see IUCLID Section 7.1.1) have shown, that HQ is quickly metabolised after uptake via the gastro-intestinal tract. A different spectrum of metabolites is available after intraperitoneal application than after oral application presumably with a higher amount of the most reactive metabolites, in particular glutathione-conjugates. Additionally, pharmacokinetic modelling showed a fundamental difference in metabolism between humans and rats as a representative for other experimental animals. Deactivation steps predominate in human liver cells, and bioactivation steps predominate in rat liver cells. Body burdens of higher substituted glutathione HQ conjugates, which are considered to be the reactive metabolites, will be much higher in rats than in humans. Additionally, as oral or dermal exposure are the relevant exposure routes for humans, a finding of in vivo genotoxicity after intraperitoneal application only, is expected to be of no biological significance for the human exposure situation.
Furthermore, in vitro investigations show that the outcome of in vitro genotoxicity studies investigating the genotoxic potential of HQ is dependent on the activity of metabolic pathways leading either to activation or deactivation of genotoxic effects. Consequently, the biological relevance of positive genotoxicity findings in vitro is questionable with regard to the human exposure situation.
Results of toxicokinetics studies by intratracheal instillation and PBPK models refined to predict metabolite formation from inhalation exposure (using intratracheal data as input parameters) also showed a rapid absorption and metabolisation of hydroquinone, despite the absence of local pulmonary metabolic activity.
Refinement of PBPK models for dermal exposure indicate a slower absorption due to passage through the multiple skin layers. However, the major metabolites detected were also primarily glucuronide conjugates with only very low levels of glutathione conjugates.
Both for inhalation and dermal exposure, the major HQ metabolites identified were glucuronide conjugates, and sulfate conjugates similar to the metabolites detected by the oral route, and contrasting with the high levels of glutathione conjugates observed in intraperitoneal studies.
Justification for selection of genetic toxicity endpoint
Genotoxic effects were observed in several in vitro assays, and in vivo, in mouse micronucleus assays but essentially following intraperitoneal administration. In contrast, in vivo TGR assay in mouse, and an in vivo Comet in rats treated by oral gavage showed no DNA damage (mutations or DNA strand breaks) in all organs examined, including male rat gonads (as a surrogate to germ cells). The dataset information suggest the positive effects are route-specific and toxicokinetic-dependent.
Short description of key information:
IN VITRO
- S. typhimurium reverse mutation assay (OECD471 or screening micromethod assay): negative in the absence and presence of metabolic activation (Haworth et al., 1983; NTP, 1989; Watzinger, 2007)
- chromosome aberration assay in human lymphocytes (OECD473, GLP): negative with or without metabolic activation (de Vogel, 2000; Roza et al., 2003)
- chromosome aberration assay in CHO cells (no guideline study): negative in the absence of metabolic activation, positive in the presence of metabolic activation (Galloway et al., 1987; NTP, 1989)
- mouse lymphoma mutation assay (similar to OECD476): positive with or without metabolic activation (McGregor et al., 1988; NTP, 1989)
IN VIVO
- micronucleus assay in polychromatic erythrocytes of mouse bone marrow (comparable to OECD474): positive after single intraperitoneal application (Adler et al., 1990)
- micronucleus assay in polychromatic erythrocytes from mouse peripheral blood (comparable to OECD474 with modifications) by flow cytometry analysis: positive after single intraperitoneal application, with indication of a threshold (Grawé et al., 1997)
- structural chromosomal aberrations in mouse spermatocytes (similar to OECD483): positive after single intraperitoneal application (Ciranni and Adler, 1991)
- dominant lethal assay with treatment of male rats (comparable to OECD478): negative after oral application for 10 w (Krasavage et al., 1984)
- formation of DNA adducts in kidney cells, F344 rats: negative after oral application for 6 w (English et al., 1994)
- formation of 8-OHdG adducts in kidney cells, F344 rats: negative after oral application for 6 w (English et al., 1997)
- Transgenic mutation assay in male Muta(TM) mice (OECD488): negative in liver, kidney, stomach, lung after oral gavage for 4 w (Matsumoto et al., 2014)
- Alkaline Comet assay in male and female F344 rats (OECD489): negative in liver, kidney, intestine, male gonads, after 2 oral treatments (Beevers, 2016)
Endpoint Conclusion: Adverse effect observed (positive)
Justification for classification or non-classification
Based on the criteria of the CLP Regulation (EC) 1272/2008, HQ has been classified to Germ cell mutagenicity category 2, H341 suspected of causing genetic effects (genotoxic effects observed in animal experiments with intraperitoneal application or in in vitro studies).
Additional studies in rats (in vivo alkaline comet assay) and mice (transgenic rodent assay) treated by oral gavage showed no DNA damages in the main tissues investigated (liver, kidney, stomach or duodenum, lung and male gonads) indicating that the tissue findings in long-term repeated dose toxicity studies were not directly related to genotoxic activity in those tissues. Information provided by toxicokinetics data and PBPK models indicate that the main metabolites resulting from dermal and inhalation exposure would be essentially similar (identity and proportions) to those observed following oral administration, and which substantially differ from the metabolic profile detected after intraperitoneal injection.
Overall, it can be concluded that the adverse effects reported in repeated oral dose toxicity studies, in particular in rat kidneys, are occuring through a non-genotoxic mechanism.
The available data provide evidence that a more severe GHS hazard category is not justified.Information on Registered Substances comes from registration dossiers which have been assigned a registration number. The assignment of a registration number does however not guarantee that the information in the dossier is correct or that the dossier is compliant with Regulation (EC) No 1907/2006 (the REACH Regulation). This information has not been reviewed or verified by the Agency or any other authority. The content is subject to change without prior notice.
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