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EC number: 204-617-8 | CAS number: 123-31-9
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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.
Reproduction or further distribution of this information may be subject to copyright protection. Use of the information without obtaining the permission from the owner(s) of the respective information might violate the rights of the owner.

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