Hydroquinone

Repeated dose toxicity: oral

Administrative data

Endpoint:

chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

from August 1982 to August 1984

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

Study comparable to guideline study with acceptable restrictions: only 2 instead of 3 dose groups, no data on food consumption; hematology and clinical chemistry examinations only at 15 mo, no urinalysis data

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Cross-reference 2

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference 1

Reference Type:

publication

Title:

Toxicity and carcinogenicity of hydroquinone in F344/N rats and B6C3F1 mice.

Author:

Kari FW, Bucher J, Eustis SL, Haseman JK, Huff JE

Year:

1992

Bibliographic source:

Food Chem Toxicol 30, 737 - 747

Reference 2

Reference Type:

study report

Title:

Unnamed

Year:

1989

Report date:

1989

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies)

Deviations:

yes

Remarks:

(only 2 instead of 3 dose groups, no data on food consumption; hematology and clinical chemistry examinations only at 15 mo, no urinalysis data)

GLP compliance:

not specified

Limit test:

no

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
- Physical state: colourless, crystalline solid
- Analytical purity: > 99%
- Purity test date:
- Lot/batch No.: 56978
- Stability under test conditions: 2 weeks when stored in the dark at temperatures of up to 60 °C
- Storage condition of test material: bulk material was stored at 25 °C under nitrogen or argon and periodic characterization by infrared or ultravioloet spectroscopy and oxidative titration indicated no deterioration over the course of the studies

Test animals

Species:

mouse

Strain:

B6C3F1

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratories, Kingston, NY, USA
- Age at study initiation: males 8-9 w , females 9-10 w
- Fasting period before study: no
- Housing: 5 per cage
- Diet: NIH 07 rat ration (Zeigler Bros. Inc., Gardner, PA, USA) ad libitum
- Water: tap water ad libitum
- Acclimation period: males 18 d, females 26 d

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 - 27 °C (65 - 80 °F)
- Humidity (%): 40 - 79
- Air changes (per hr): 6 - 13 room air changes per hr
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS: appropriate amount of hydroquinone dissolved in deionised water by stirring with a magnetic stir bar; maximum storage time 21 d at room temperature in the dark in amber serum vials with Teflon-lined seals, sparged with argon or nitrogen before sealing

VEHICLE
- Concentration in vehicle: 5, 10 mg/mL
- Amount of vehicle (if gavage): 10 mL/kg

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

UV spectroscopy of acetonitrile extracts at 295 nm or methanol extracts at 293 nm; analyzed concentrations (data from 10 samples each): mean 4.88, range 4.72 - 5.09 mg/mL; mean 9.78, range 9.39 – 10.25 mg/mL; variation considered to be in acceptable range; stability investigations by HPLC after 21 days of storage of the dosing solution at room temperature in the dark indicated no notable decrease of hydroquinone concentration

Duration of treatment / exposure:

65 or 103 w , sacrifice after ca. 65 and 104 to 105 w

Frequency of treatment:

5 d/w

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 50, 100 mg/kg bw/d
Basis:
actual ingested

No. of animals per sex per dose:

64 or 65 males, 65 females (including 10 for 65 w treatment)

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: based on 13 week range-finding toxicity study (see separate endpoint study record)

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: 2 times per day

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: 2 times per day

BODY WEIGHT: Yes
- Time schedule for examinations: at initiation of dosing, weekly thereafter for 13 weeks, and monthly for the rest of the study period

FOOD CONSUMPTION: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes, at 15 mo
Investigated parameters: leukocytes, lymphocytes, segmented neutrophiles, monocytes, eosinophils, atypical lymphocytes, atypical mononuclear cells, bands, hematocrit, hemoglobin, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, mean cell volume, erythrocytes, reticulocytes

CLINICAL CHEMISTRY: Yes, at 15 mo
Investigated parameters: albumin, alkaline phosphatase, alanine aminotransferase, blood urea nitrogen, creatinine, sorbitol dehydrogenase, total protein

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes, all animals

ORGAN WEIGHTS: Yes, at 15 mo and 2 y

HISTOPATHOLOGY: Yes
Tissues examined in high-dose mice and controls: adrenal glands, brain, caecum, colon, duodenum, epididymis/prostrate/testes or ovaries/uterus, esophagus, gallbladder, gross lesions and tissue masses, heart, ileum, jejunum, kidneys, liver, lungs and mainstem bronchi, mammary gland, mandibular or mesenteric lymph nodes, nasal cavity and turbinates, pancreas, parathyroid glands, pituitary gland, rectum, salivary glands, skin, spleen, sternebrae and vertebrae including marrow, stomach, thymus, thyroid gland, trachea, and urinary bladder
Tissues examined in low-dose male mice: adrenal glands, gross lesions, liver, spleen, thyroid gland
Tissues examined in low-dose female mice: gross lesions, liver, lungs, ovaries, salivary glands, thyroid gland

Statistics:

Survival probabilities estimated by product-limit procedure of Kaplan and Meier (1958), statistical analysis by methods of Cox (1972) and Tarone (1975);
Statistical significance of organ weights, hematological and clinical chemistry data analyzed by Dunn's test (Dunn, 1964) or Shirley's test (Shirley, 1977);
Data on tumor incidences analyzed by life-table analysis, logistic regression, Fisher's exact test, and Cochran-Armitage trend test

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

changes not biologically relevant

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

changes not biologically relevant

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

no effects observed

Description (incidence and severity):

see discussion in Section 7.7

Details on results:

CLINICAL SIGNS AND MORTALITY
No significant differences in survival were observed between any groups of either sex; no compound-related clinical signs


BODY WEIGHT AND WEIGHT GAIN (for details see Table 1)
100 mg/kg: mean body weights of male mice 5-8% lower between week 93 and 104; mean body weight of female mice 5-8% lower between week 20 to 44 and 10-14% lower between week 45 and 104
Evaluation: In females, the decrease of body weight of 10-14% between week 45 and 104 was considered to represent an adverse treatment-related effect.


HAEMATOLOGY (data from 15 mo interim sacrifice group, for details see Table 2)
100 mg/kg: male mice showed significant increases of hematocrit and erythrocytes of 13% compared to the vehicle control
Evaluation: Slight significant increases of hematocrit and erythrocytes were only found in high-dose males and are therefore considered to be of negligible biological significance.


CLINICAL CHEMISTRY (data from 15 mo interim sacrifice group, for details see Table 2)
100 mg/kg: male mice showed showed significant increases of total protein and serum albumin, of serum alkaline phosphatase and of sorbitol dehydrogenase; female mice showed significant increases of total protein and serum albumin, whereas a significantly lower activity was observed for alanine aminotransferase and sorbitol dehydrogenase
Evaluation: The statistically significant effects in general represented only slight changes. Changes in enzyme activites were contrary in both sexes. Therefore the observed changes of clinical chemistry are considered to be of negligible biological significance.


ORGAN WEIGHTS (data from 15 mo interim and terminal sacrifice group, for details see Table 1)
100 mg/kg: relative liver weights of both sexes showed significant increases after 65 and 103 w of administration, whereas relative brain and kidney weights of females showed significant increases only at interim sacrifice after 65 w
50 mg/kg: relative liver weight of males showed a significant increase after 103 w, relative kidney weight of females showed a significant increase after 65 w only
Evaluation: Increases of relative liver weights in both sexes at both doses correlated with histopathological non-neoplastic and neoplastic liver changes (see below). However, increases of relative weights of brains and kidneys in female mice were only observed at the interim but not at the terminal sacrifice and are therefore considered to be of negligible biological significance.


HISTOPATHOLOGY: NON-NEOPLASTIC (for details see Table 3)
LIVER 65-week interim sacrifice:
50 and 100 mg/kg, male mice: increased incidences of diffuse centrilobular fatty change, diffuse cytomegaly, occasional syncytial cells (multinucleated hepatocytes)
LIVER 105-week sacrifice:
100 mg/kg, male mice: increased incidence of anisokaryosis (variation in size of hepatocyte nuclei), syncitial alteration (hepatocytes with more than 5 nuclei per cell), and foci of cellular alteration (basophilic foci) compared to vehicle controls
Evaluation: Centrilobular fatty change and cytomegaly were observed at the 65-week interim sacrifice but not at the terminal sacrifice. This may be explained by the fact that after the 65-week administration necropsy occurred within 24 hrs after the last dose, whereas dosing was stopped 2 weeks before necropsy in the 2 year study. Presumably, fatty change and cytomegaly were reversible after cessation of hydroquinone administration and are considered not to represent a toxicologically relevant effect.


HISTOPATHOLOGY: NEOPLASTIC
For detailed evaluation see Section 7.7

Effect levels

1

Dose descriptor:

LOAEL

Remarks:

chronic non-neoplastic and neoplastic effects

Effect level:

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

Based on:

test mat.

Sex:

male

Basis for effect level:

other: liver: increase of weights, histopathologal findings (at 65-week interim sacrifice only)

2

Dose descriptor:

LOAEL

Remarks:

chronic non-neoplastic and neoplastic effects

Effect level:

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

Based on:

test mat.

Sex:

female

Basis for effect level:

other: Significant increase of liver adenoma or carcinoma combined

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

Table 1: Body weights and relative organ weights after 65 and 103 w of gavage (mean ± standard error)

Application period	Parameter	Dose groups males			Dose groups females
		Vehicle control	50 mg/kg	100 mg/kg	Vehicle control	50 mg/kg	100 mg/kg
65 w	Body weight (g)	45.6±1.26	44.9±1.34	46.9±1.01	46.7±2.51	42.4±2.54	40.6±1.17
	Brain *	10.6±0.41	11.0±0.33	10.2±0.21	10.7±0.48	12.2±0.74	12.6±0.35^a
	Kidney *	18.0±0.63	19.6±0.41	19.2±0.57	11.2±0.30	13.1±0.44^b	13.3±0.26^b
	Liver *	44.6±2.32	52.5±5.35	54.8±3.88^b	40.5±0.95	40.9±1.15	45.2±1.25^b
103 w	Number of animals weighed	33	36	36	37	39	36
	Body weight	44.0±0.76	43.0±0.72	42.0 ±0.95	50.7±1.65	51.5±1.46	47.8±1.24
	Brain *	11.7±0.21	11.9±0.22	12.1±0.28	10.7±0.40	10.5±0.38	11.0±0.29
	Kidney *	11.8±0.38	11.7±0.23^§	12.4±0.32	7.5±0.44^#	7.4±0.31	7.3±0.22
	Liver *	67.2±4.80	76.4±4.82^a§	70.0±3.17^a	52.0±3.22^#	52.0±2.65	55.1±2.68^a

* relative organ weights as mg of organ per g body weight; ^§ N=35 mice weighed; ^# N=36 mice weighed

Statistically significant increased compared to vehicle control: ^a p<0.05

Table 2: Overview of statistically significant haematological and clinical chemical parameters in the 65-week gavage studies of hydroquinone

Affected sex	Endpoint	Parameter	Dose group
			Vehicle control	50 mg/kg	100 mg/kg
Male	Hematology	Hematocrit (%)	36.5±2.38	39.5±1.27	41.3±1.17^a
		Erythrocytes (10^6/µL)	7.3±0.52	8.0±0.29	8.3±0.28^a
	Clinical chemistry	Total protein (g/dL)	5.2±0.04	5.4±0.15	5.9±0.23^b
		Albumin (g/dL)	3.3±0.05	3.5±0.08	3.8±0.15^b
		Alkaline phosphatase (IU/L)	38.4±2.34	38.8±1.41	50.0±3.92^a
		Sorbitol dehydrogenase (SU/mL)	35.8±1.24	35.6±2.05	43.0±1.79^b
Female	Clinical chemistry	Total protein (g/dL)	5.3±0.07	5.5±0.12	5.7±0.05^b
		Albumin (g/dL)	3.5±0.06	3.6±0.10	3.9±0.04^b
		Alanine aminotransferase (IU/L)	38.9±6.62	31.6±6.19	23.7±1.33^b
		Sorbitol dehydrogenase (SU/mL)	35.6±1.09	33.7±1.37	32.4±0.73^a

statistically different from vehicle control: ^a p<0.05, ^b p<0.0.1

Table 3: Number of mice with selected lesions in the liver and thyroid gland in the 65 w and 103 w gavage study of hydroquinone

Application period	Site of lesion	Type of lesion	Dose groups males			Dose groups females
			Vehicle control	50 mg/kg	100 mg/kg	Vehicle control	50 mg/kg	100 mg/kg
65 w		Number of animals examined	10	10	10	10	10	10
	Liver	Diffuse centrilobular fatty change	1	0	7	0	0	0
		Diffuse fatty change	0	0	0	1	3	0
		Diffuse cytomegaly	0	8	10	0	0	0
		Syncytial cells	1	1	4	0	0	0
		Basophilic focus	0	0	1	0	0	0
		Clear cell focus	0	0	0	0	0	1
		Hepatocellular adenoma	1	1	4	0	1	0
		Hepatocellular carcinoma	2	1	1	0	0	0
		Hepatocellular adenoma or carcinoma	3	2	4	0	1	0
	Thyroid gland	Hyperplasia	0	0	0	0	0	2
103 w	Liver	Number of animals examined	55	54	55	55	55	55
		Anisokaryosis	0/55 (0%)	2/54 (4%)	12/55 (22%)	n.s.	n.s.	n.s.
		Syncytial alteration	5/55 (9%)	3/54 (6%)	25/55 (45%)	n.s.	n.s.	n.s.
		Basophilic focus	2/55 (4%)	5/54 (9%)	11/55 (20%)	2/55 (4%)	6/55 (11%)	3/55 (5%)
		Hepatocellular adenoma	9/55 (16%) P=0.018	21/54 (39%) P=0.008	20/55 (36%) P=0.015	2/55 (4%) P=0.007	15/55 (27%) P=0.001	12/55 (22%) P=0.005
		Hepatocellular carcinoma	13/55 (24%)	11/54 (20%)	7/55 (13%)	1/55 (2%)	2/55 (4%)	2/55 (4%)
		Hepatocellular adenoma or carcinoma ^a	20/55 (36%) P=0.223	29/54 (54%) P=0.053	25/55 (45%) P=0.250	3/55 (5%) P=0.009	16/55 (29%) P=0.002	13/55 (24%) P=0.007
	Thyroid gland	Number of animals examined	55	53	54	55	55	55
		Hyperplasia	5/55 (9%)	15/53 (28%)	19/54 (35%)	13/55 (24%)	47/55 (85%)	45/55 (82%)
		Adenoma	2/55 (4%)	1/53 (2%)	2/54 (4%)	3/55 (5%) P=0.186	5/55 (9%) P=0.397	6/55 (11%) P=0.233
		Carcinoma	0/55 (0%)	0/53 (0%)	0/54 (0%)	0/55 (0%)	0/55 (0%)	1/55 (2%)
		Adenoma or carcinoma ^b	2/55 (4%)	1/53 (2%)	2/54 (4%)	3/55 (5%) P=0.115	5/55 (9%) P=0.397	7/55 (13%) P=0.152

Historical incidences in control groups (means±SD):

^a male mice: water vehicle controls: 106/347 (31±6 %), range 20 to 38%; untreated controls 609/2,032 (30±8%), range 16 to 58%

  female mice: water vehicle controls: 29/348 (8±5 %), range 0 to 14%; untreated controls 184/2,032 (9±5%), range 2 to 20 %

^b female mice: water vehicle controls 10/337 (3±2%), range 0 to 6 %; untreated controls 49/1,937 (3±3%), range 0 to 15%

Applicant's summary and conclusion

Conclusions:

The LOAEL for chronic toxicity was 50 mg/kg bw/d both in male B6C3F1 mice based on liver weight effects and liver histopathology at the 65-week interim sacrifice, and in female mice based on increased incidences of hepatocellular adenomas and of hepatocellular adenomas or carcinomas combined (detailed evaluation of neoplastic histopathology in Section 7.7). Female mice showed non-neoplastic findings (decrease of body weights) at 100 mg/kg bw/d only.

Executive summary:

HQ was administered to groups of 65 male and 65 female B6C3F1 mice for 103 weeks on 5 days per week via gavage at doses of 0, 50, and 100 mg/kg bw/d (vehicle water). The study protocol was similar to OECD Guideline 453 with several minor deviations. There were only 2 instead of 3 dose groups, and food consumption and urinalysis were not examined. Hematology and clinical chemistry examinations were performed at a single timepoint after 65 w of dosing in subgroups of 10 mice of each sex and dose. Mice were sacrificed for gross and histopathological examination at about 66 and 104 to 105 w after start of dosing.

There were no significant differences in survival between any groups of either sex and no compound-related clinical signs. There were no biologically significant changes of haematological or clinical chemistry parameters. Toxicity was indicated at 100 mg/kg by decreased body weights in males (5-8% lower than those of vehicle controls between weeks 93-104), and in females (5-8% lower between week 20-44, 10-14% lower between week 45-104). Relative brain weights (at 100 mg/kg) and kidney weights (at 50 and 100 mg/kg) showed significant increases in female mice at the interim sacrifice only and were considered to be of negligible biological significance. Significant increases of relative liver weights were found at 100 mg/kg in both sexes after 65 and 103 w of administration, and at 50 mg/kg in males after 103 w. Centrilobular fatty change and cytomegaly were observed in male mice at the 65-week interim sacrifice but not at the terminal sacrifice. This may be explained by the fact that after the 65-week administration necropsy occurred within 24 hrs after the last dose, whereas dosing was stopped 2 weeks before necropsy in the 2 year study. Presumably, fatty change and cytomegaly were reversible after cessation of hydroquinone administration. Findings in livers of male mice at 100 mg/kg after 105 w were characterized by increased incidences of anisokaryosis (variation in size of hepatocyte nuclei), syncitial alteration (hepatocytes with more than 5 nuclei per cell), and foci of cellular alteration (basophilic foci) compared to incidences in vehicle controls. Female mice showed increased incidences of hepatocellular adenomas and of hepatocellular adenomas or carcinomas (combined) at 50 and 100 mg/kg bw/d (for detailed presentation and discussion of neoplastic findings see Section 7.7).

The LOAEL for chronic toxicity was 50 mg/kg bw/d both in male B6C3F1 mice based on liver weight effects and liver histopathology at the 65-week interim sacrifice, and in female mice based on increased incidences of hepatocellular adenomas and of hepatocellular adenomas or carcinomas combined. Female mice showed non-neoplastic findings (decrease of body weights) at 100 mg/kg bw/d only.