Hydroquinone

Genetic toxicity: in vivo

Administrative data

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

Cross-reference

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

publication

Title:

Measurement of nuclear DNA modification by 32P-postlabeling in the kidneys of male and female Fischer 344 rats after multiple gavage doses of hydroquinone.

Author:

English JC, Hill T, O'Donoghue JL, Reddy V

Year:

1994

Bibliographic source:

Fundam Appl Toxicol 23, 391 - 396

Materials and methods

Test guideline

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

Test material

Test material information

Constituent 1

Reference substance name:

Hydroquinone

EC Number:

204-617-8

EC Name:

Hydroquinone

Cas Number:

123-31-9

Molecular formula:

C6H6O2

IUPAC Name:

hydroquinone

Details on test material:

- Name of test material (as cited in study report): hydroquinone, HQ
- Analytical purity: > 99%

Test animals

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

Administration / exposure

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

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (actual dose received)

Doses / concentrations 2

Dose / conc.:

2.5 mg/kg bw/day (actual dose received)

Doses / concentrations 3

Dose / conc.:

25 mg/kg bw/day (actual dose received)

Doses / concentrations 4

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

Examinations

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.

Results and discussion

Test results

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

Any other information on results incl. tables

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 10^9 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

Applicant's summary and conclusion

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.