Hydroquinone

Neurotoxicity

Administrative data

Endpoint:

neurotoxicity: sub-chronic oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

October 27, 1987 - October 31, 1988

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Remarks:

Comparable to guideline study with negligible restrictions: dosing on 5 d/w instead of 7 d/w as requested by OECD Guideline 424, no justification specified for this deviation, however, as the test substance has no potential to accumulate in the organism exposure-free weekends are not expected to influence the outcome of test results; ophthalmoscopic examination not performed, however, no effects on the eyes expected for oral exposure route by gavage

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference 1

Reference Type:

publication

Title:

Hydroquinone: Acute and subchronic toxicity studies with emphasis on neurobehavioral and nephrotoxic effects.

Author:

Topping DC, Bernard LG, O'Donoghue JL, English JC

Year:

2007

Bibliographic source:

Food Chem Toxicol 45, 70 - 78

Reference 2

Reference Type:

study report

Title:

Unnamed

Year:

1988

Report date:

1988

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

EPA OTS 798.6050 (Neurotoxicity Screening Battery)

Deviations:

no

Test guideline 2

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 424 (Neurotoxicity Study in Rodents)

Deviations:

yes

Remarks:

(dosing on 5 d/w instead of 7 d/w ; no opthalmoscopic examination)

GLP compliance:

yes

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, HQ
- Analytical purity: at least 99.6%
- Impurities (identity and concentrations): no data
- Purity test date: 9/2/87, 9/11/87
- Lot/batch No.: A13B
- Stability under test conditions: at least 7 d at room temperature; change of colour of the test solution by day 4; however, no decomposition product was found by HPLC analysis after spiking with 14C-hydroquinone; because of the colour change test solutions were prepared daily
- Storage condition of test material: room temperature

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Wilmington, MA, USA
- Age at study initiation: ca. 7 w
- Weight at study initiation: males 194 +- 7 g, females 153 +- 6 g
- Fasting period before study:
- Housing: single, males and females in separate rooms, no other studies housed in these rooms
- Diet: Agway Prolab Animal Diet (RMH 3200) ad libitum
- Water: drinking water ad libitum
- Acclimation period: 12 d


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 - 23 °C (70- 74 °F)
- Humidity (%): 32 - 53
- Air changes (per hr):
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: distilled degassed water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: daily

VEHICLE
- Concentration in vehicle: 5%
- Amount of vehicle (if gavage): 0.4, 1.28, 4.0 mL/kg, weekly adjustment for gain of body weight

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Mean concentration determined by HPLC and UV detection at 286 nm was 5.1 +- 0.3 % in males and 5.1 +- 0.2% in females being within acceptable variance.

Duration of treatment / exposure:

13 w

Frequency of treatment:

5 d/w

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 20, 64, 200 mg/kg bw/d
Basis:
actual ingested

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: high dose reported to produce clear behavioral effects and life-threatening toxicity during previous studies (NTP, 1986)

Examinations

Observations and clinical examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: 3 days prior to treatment (day -3) and at 1, 6 and 24 h, and 7, 14, 30, 60 and 91 d after administration of the initial dose; examinations conducted prior to dosing except on day 0 to avoid confounding with acute effects
- Parameters: home-cage activity / feces amount / feces color / feces consistency / urine amount / urine color (urine stains on papers under the home cages when left to stand overnight) / behavior while removing from cage

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: immediately after each dosing, daily check of mortality
- Physical examinations included hair coat, skin, eyes, mucus membranes, body temperature, respiratory rate, respiratory character, lacrimation, salivation, eye prominence, palpebral closure, convulsions, seizures, length of postictal period, biting, tremors, handling behavior.

BODY WEIGHT: Yes
- Time schedule for examinations: preexposure (day -3), day 0, 1, 4, 7, and at least once weekly thereafter

FOOD CONSUMPTION:
- Food consumption for each animal determined on day 0, 1, 4, 7, and at least once weekly thereafter and mean daily diet consumption calculated as g food/kg body weight/day: Yes

OPHTHALMOSCOPIC EXAMINATION: No

Neurobehavioural examinations performed and frequency:

FUNCTIONAL OBSERVATIONAL BATTERY: Yes
- Parameters examined: locomotor activity / rearing activity / tail elevation /static limb position / abnormal gait / hypotonic gait of hindlimb and forelimb / ataxic gait /head position / approach response / touch response / auditory orientation / olfactory orientation / righting reflex / proprioceptive positioning / tail pinch / abdominal tone / bladder tone / muscle tone / passive manipulation / pupillary size / eye position / nystagmus / visual orientation / palpebral reflex / pinna touch response / extensor postural thrust reflex / hopping reflex / visual placing / grip strength (semi-quantitative) / bizarre behavior / urination / defecation / spontaneous vocalizations
- Description of procedures: Modification of the method of McGrath (1956) adapting recommendations of Irwin (1968), Marshall et al. (1971), Deuel (1977), and Gad (1982)
- Minimization of bias:
- Same technicians used throughout testing: No data
- Technicians were blind to treatment status of animals: Yes
- Site of testing: cage-side, paper tray, animal in hand of observer
- Time schedule for examinations: 3 days prior to treatment (day -3) and at 1, 6 and 24 h, and 7, 14, 30, 60 and 91 d after administration of the initial dose; examinations conducted prior to dosing except on day 0 to avoid confounding with acute effects
- Environmental conditions:
- Noise level: minimal
- Other: minimization of distracting smells and lights
- Scoring criteria (if any): individually specified for each endpoint
- Duration of observation period for open field observations: 3 min


QUANTITATION OF GRIP STRENGTH
- Description of procedures: forelimb and hindlimb grip strength by procedure of Meyer et al. (1979) and modified by Mattson et al. (1986)
- Time schedule for examinations: 3 days prior to treatment (day -3) and at 1, 6 and 24 h, and 7, 14, 30, 60 and 91 d after administration of the initial dose; examinations conducted prior to dosing except on day 0 to avoid confounding with acute effects, determined three times at each time interval, the highest recorded value was used for statistical analysis
- Description of equipment: strength testing apparatus equipped with a dial push-pull gauge (0.5 kg or 1.0 kg Chatillon Modell DPP)

Sacrifice and (histo)pathology:

- Time point of sacrifice:
- Number of animals sacrificed: 10 per sex and dose group
- Parameters measured:
- Brain weight: yes
- Procedures for perfusion: Rats were anesthesized and perfused through the ascending aorta with 4% paraformaldehyde followed by 5% glutaraldehyde both in 0.1 M phosphate buffer, pH 7.4 at 4 °C. Following perfusion brain, spinal cord and sciatic-tibial nerves were removed. Tissues destined for plastic embedment were fixed for an additional 2 h in 5% glutaraldehyde. Tissues not destined for plastic embedment and all tissues from unperfused animals were immersed in 10% neutral buffered formalin.
- Number of animals perfused: 6 males and 6 females per dose
- Tissues evaluated: Cross sections through the forebrain, cerebrum, midbrain, cerebellum, pons, medulla oblongata, cervical spinal cord swelling, luminal spinal cord swelling, cervical and lumbar spinal cord ganglia, dorsal and ventral spinal roots (cervical and lumbar), sciatic nerve at mid-thigh, and tibial nerve including branches to the calf musculature
- Type of staining: Tissues from the high-dose and the control group: processed by standard neuropathological techniques for paraffin embedment, hematoxylin-eosin staining, and light microscopic examination; special stains (combined Luxol Fast Blue Stain-Bodian silver protargol impregnation) performed on perfused neural tissue; left sciatic-tibial nerve and medulla oblongata postfixed in Dalton's chrome-osmium solution, embedded in plastic, sectioned at 1 mm, stained with toluidine blue, and examined by light microscopy
- Methodology of preparation of sections:
- Thickness: 1 µm
- Embedding media: EPON
- Number of sections: not specified
- Number of animals evaluated from each sex and treatment group: 6

Other examinations:

Organ weights of brain and kidney; histopathological examination of kidney sections by light microscopy

Positive control:

Physostigmine (1 mg/kg bw/d s.c. in propylene glycol), 2,5-hexanedione (750 mg/kg bw/d p.o. by gavage), 3,3'-iminodipropionitrile (IDPN) (2000 mg/kg i.p.) administered 5 d/w for 28 d, 14 d and as single dose, respectively (dosing period until signs of subchronic neurotoxicity appeared for the different substances).

Statistics:

Statistical significance of subchronic toxicity study data evaluated by ANOVA (p<= 0.05), Bartlett's test (p<=0.01), Duncan's multiple range test (p<=0.05). FOB results analyzed using contingency table methods and Dunnett's t-test, modified for proportions. Loglinear models were fit to the three-way tables and significant effects were determined. Both godness of fit and statistical significance were detected (p<= 0.05). If significant or near significant dose-behavior interactions were detected, Dunnett's t-test (one-tail) was used to compare incidences at each observation time.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Mortality:

mortality observed, treatment-related

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Clinical biochemistry findings:

not examined

Behaviour (functional findings):

no effects observed

Gross pathological findings:

no effects observed

Neuropathological findings:

no effects observed

Details on results:

CLINICAL SIGNS AND MORTALITY
Depression of general motor activity (decreased movement primarily in the home cage) and appearance of tremors with a LOEL of 64 mg/kg; brown discoloration of urine stains with LOEL of 20 mg/kg; no mortality

200 mg/kg:
- minimal to mild tremors in all animals appearing between 30 and 60 min after dosing in males, and between 4 and 25 min in females; statistically significant increase of incidence of tremors during weeks 1 to 13 for both sexes and during week 14 for males only;
- statistically significant increase of incidence of depression of general activity for males (10/10) during week 1 to 13, and for females (5/10) during week 4 to 8
- minimal to moderate brown discoloration of urine stains
64 mg/kg:
- minimal to mild tremors in all females and in 7/10 males; statistically significant increase of incidence of tremors during weeks 6, 7 and 9 to 13 for males, and during weeks 2, 3, 5, 6, and 9 to 13 for the females;
- statistically significant increase of incidence of depression of general activity for males (1/10) during week 6 and (2/10) during week 8, and for females (2/10) during week 4
- minimal to moderate brown discoloration of urine stains
20 mg/kg:
- minimal to minor brown discoloration of urine stains

Evaluation: Tremors were considered to be related to hydroquinone exposure due to frequent appearance and statistically significance compared to controls. Brown discoloration of urine indicated systemic absorption and excretion of hydroquinone and its metabolites respectively.


BODY WEIGHT AND WEIGHT GAIN
200 mg/kg, only males: slightly decreased mean body weight compared to controls from day 1 until termination of the study, no significant effect; mean terminal body weight 7% lower than for control group


FOOD CONSUMPTION
200 mg/kg: significantly decreased compared to controls between days 0 to 4; thereafter comparable to controls


NEUROBEHAVIOUR
Statistically significant behavioral changes attributed to hydroquinone exposure observed at the following dose levels primarily during the 1 and 6 h FOB examinations:
200 mg/kg:
- decreased home-cage activity and locomotor activity for males at 6 h FOB
- decreased behavior while removing from the cage for females at 1 h FOB
- increased tremors for females at 1 h FOB
- brown discoloration of urine stains for males at 1 and 6 h, days 1, 30 and 60 FOB, and for females at days 30 and 60 FOB
64 mg/kg:
- brown discoloration of urine stains for males at days 1, 30 and 60 FOB, and for females at days 30 and 60 FOB
20 mg/kg:
- brown discoloration of urine stains for males at days 1 and 60 FOB, and for females at days 30 and 60 FOB

Evaluation: The behavioral changes listed above were attributed to be related to the exposure and represented acutely observable effects. In contrast, no changes attributable to long-term exposure were evident as other statistically significant changes appeared to be random in nature, were not related to dose level, or were not reproducible with time. Changes not considered related to HQ exposure included increased urination, increased or decreased defecation, increased spontaneous vocalizations, increased approach response, increased or decreased auditory orientation, increased olfactory orientation, increased visual orientation, decreased pinna touch response, increased tail pinch response, decreased quantitative grip strength. the positive control substances resulted in characteristic neurobehavioral effects.


ORGAN WEIGHTS
No effect on mean brain or kidney weights


GROSS PATHOLOGY
No treatment-related changes


HISTOPATHOLOGY: NON-NEOPLASTIC
Kidneys: no treatment-related changes
Neuropathologic findings: single findings in 3/20 controls and 2/20 animals in the 200 mg/kg group; effects not related to treatment

Effect levels

1

Dose descriptor:

NOAEL

Effect level:

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

Sex:

male/female

Basis for effect level:

other: no adverse effects

Remarks on result:

other:

2

Dose descriptor:

LOAEL

Effect level:

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

Sex:

male/female

Basis for effect level:

other: acute neurobehaviour other: acute CNS effects inidicated by clinical examination

Remarks on result:

other:

Any other information on results incl. tables

Number of tremors occurrences in males and females during the study

Doses		Males		Females			Males and females
mg/kg/day	N	Number of occurrences	%	Number of occurrences	%	N	Number of occurrences	%
0	680	0	0	0	0	1360	0	0
20	680	0	0	0	0	1352	0	0
64	680	74	11	88	13	1360	162	12
200	680	194	29	239	34	1360	461	35

N: number of observations during the study consists of the number of dosing days*(10 animals/sex)

Tremors occurred within an hour after each dosing.

Applicant's summary and conclusion

Conclusions:

Subchronic oral exposure of Sprague-Dawley rats to doses of 20, 64, and 200 mg/kg bw/d did not result in evidence of subchronic neurotoxicity as assessed by functional observation battery examinations, quantitative grip strength measurements, brain weights, or neuropathology examinations. Effects were restricted to acute responses of the central nervous system to dosing of hydroquinone.

Executive summary:

Groups of Sprague-Dawley rats (CD-SD, 10 males and 10 females/group) were exposed to doses of 0, 20, 64, and 200 mg/kg bw/d by gavage application of 5% aqueous hydroquinone solutions on 5 d/w for 13 w to investigate subchronic toxicity with emphasis on neurobehavioral and neurotoxic effects and on nephrotoxic effects. The study protocol was similar to OECD Guideline 424 (Neurotoxicity study in rodents) except for application on 5 d/w instead of 7 d/w and the absence of an ophthalmological examination.) Investigations included detailed clinical observations, a comprehensive functional observation battery, quantitative measurements of grip strength, and neuropathological examinations of the central and the peripheral nervous system as well as determination of brain weights.

Effects were restricted to acute responses of the central nervous system to dosing of hydroquinone, as depression of general motor activity (decreased movement primarily in the home cage) and appearance of tremors with a LOAEL of 64 mg/kg. Brown discoloration of urine stains in all dose groups indicated systemic absorption and urinary excretion of hydroquinone and its metabolites. Subchronic oral exposure did not result in evidence of subchronic neurobehavioral changes or morphologic changes in the CNS or PNS. Under the conditions of this study, the LOAEL was 64 mg/kg bw/d based on acute neurotoxic effects and the NOAEL was 20 mg/kg bw/d.