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Toxicological information

Genetic toxicity: in vivo

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Administrative data

Endpoint:
in vivo mammalian cell study: DNA damage and/or repair
Remarks:
Type of genotoxicity: 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:
other: Guideline study, GLP

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report Date:
2016

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
other: OECD guideline 489
Deviations:
no
GLP compliance:
yes
Type of assay:
mammalian comet assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
- Name of test material (as cited in study report): Hydroquinone
- Substance type: monoconstituent
- Physical state: powder
- Analytical purity: 99.9%
- Purity test date: 26 November 2014
- Lot/batch No.: FHQ1433021
- Expiration date of the lot/batch: 25 November 2016
- Storage condition of test material: sample stored at 15-25°C protected from light

Test animals

Species:
rat
Strain:
Fischer 344
Sex:
male/female
Details on test animals 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

Administration / exposure

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.
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 105, 210, 420 mg/kg bw
Basis:
nominal conc.
Main study
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.

Examinations

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.

Results and discussion

Test resultsopen allclose all
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.

Any other information on results incl. tables

Results of COMET assay - somatic tissues

Hydroquinone: Summary of Group Mean Data – Male Duodenum

Group / Treatment
(mg/kg/day)

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
(mg/kg/day) 

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
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 1 vs. Group 5)
A
                            ANOVA, Dose Response and Dunnett’s (Group 1 vs. Groups 2, 3, 4).

R                           Rank-Transformed Data

Hydroquinone: Summary of Group Mean Data – Male Kidney

Group / Treatment
(mg/kg/day) 

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
(mg/kg/day)

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
(mg/kg/day)

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
(mg/kg/day)

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
.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): 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.