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Repeated dose toxicity: dermal

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Administrative data

Endpoint:
sub-chronic toxicity: dermal
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Comparable to guideline study with the following deviations: additional investigation of urinalysis and renal cell proliferation, addtional organ weights, histological examination of different tissues; no ophthalmoscopic examination; sufficient documentation of test conditions and test results
Cross-referenceopen allclose all
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study

Data source

Reference
Reference Type:
publication
Title:
Lack of nephrotoxicity and renal cell proliferation following subchronic dermal application of a hydroquinone cream.
Author:
David RM, English JC, Totman LC, Moyer C, O'Donoghue JL
Year:
1998
Bibliographic source:
Food Chem Toxicol 36, 609 - 616

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 411 (Subchronic Dermal Toxicity: 90-Day Study)
Deviations:
yes
Remarks:
(additional investigations: urinalysis, weights of brain, ovaries, lungs, thymus; histology of different tissues, renal cell proliferation; no ophthalmoscopic examination)
GLP compliance:
not specified
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): hydroquinone, HQ
- Composition of test material: HQ was supplied at the intended test concentrations in oil-in-water emulsion creams as vehicle
- Stability under test conditions: yes, for the duration of the study. Oxidation of HQ to benzoquinone was prevented by addition of sodium metabisulfite, a preservative routinely added to commerical preparations to prevent air oxidation of HQ. A petroleum jelly plug was used to seal the end of each tube. Individual tubes were used for no more than 3 days. If the cream became brown in colour it was not to be used.
- Storage condition of test material: no specific conditions specified

Test animals

Species:
rat
Strain:
Fischer 344
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Kingston
- Age at study initiation: 8 w
- Weight at study initiation: 175 g (males) or 134 g (females)
- Fasting period before study: no
- Housing: individually
- Diet: Agway Prolab RMH 3200 ad libitum
- Water: tap water ad libitum
- Acclimation period: not specified


ENVIRONMENTAL CONDITIONS
- Temperature (°C): no data
- Humidity (%): no data
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): no data

Administration / exposure

Type of coverage:
semiocclusive
Vehicle:
other: oil-in-water emulsion cream
Details on exposure:
TEST SITE
- Area of exposure: back skin, 4 x 6 cm x cm
- % coverage: not specified
- Type of wrap if used: semiocclusive, Vetrap (3 M Animal Care Products), secured with hypoallergenic tape
- Time intervals for shavings or clipplings: at least once weekly


REMOVAL OF TEST SUBSTANCE
- Washing: area wiped clean with saline-moistened gauze; no attempt was made to remove the HQ cream beyond wiping the skin surface
- Time after start of exposure: 6 h after daily application


TEST MATERIAL
- Amount(s) applied (volume or weight with unit): the amount applied was adjusted on the basis of the weekly mean body weight of each sex
- Concentration: 0, 2, 3.5 and 5%
- Constant volume or concentration used: no


VEHICLE
- Justification for use and choice of vehicle (if other than water): oil-in-water emulsion cream comparable to cream base of commercial cream preparations with HQ
- Amount(s) applied (volume or weight with unit): dosing volume 1.6 mL/kg bw
- Composition: HQ was applied in oil-in-water emulsion creams consisting of water (71.50%), propylene glycol (10.00%) stearic acid (7.00%), light mineral oil (3.00%), isopropyl myristate (2.50%), diethylaminocetyl phosphate (2.00%), cetyl alcohol (1.50%), Oleth-20 (1.50%), Oleth-2 (0.50%), hydroxyethyl cellulose (0.25%), sodium metabisulfite (0.20%) and disodium EDTA (0.05%); the water content was adjusted to compensate for the amount of hydroquinone; pH values were between 5.30 - 5.65


USE OF RESTRAINERS FOR PREVENTING INGESTION: no; however, ingestion was prevented during the exposure by the semiocclusive wrap on the entire torso, and after removing the wrap by application of an Elizabethan collar
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analysis of the squeeze tubes by HPLC before use and at termination of the study indicated that HQ concentrations were within 10% of the nominal for the duration of the study, which is acceptable.
Duration of treatment / exposure:
13 w
Frequency of treatment:
5 d/w, 6 h/d
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
0, 2.0, 3.5, and 5.0% (w/w)
Basis:
other: nominal concentration in test material
Remarks:
Doses / Concentrations:
29.5 +- 2.4, 51.9 +- 4.3, and 73.9 +- 6.3 mg/kg bw/d in males; 43.8 +- 3.8, 77.0 +- 6.9, 109.6 +- 9.8 mg/kg bw/d in females
Basis:
other: based on concentration in test material, average weekly body weight, and the amount applied to the skin
No. of animals per sex per dose:
20 for toxicity study, additionally 5 for cell proliferation and urinalysis study
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: based on preliminary study to determine the highest concentration of HQ that could be tolerated when administered repeatedly under a semi-occlusive wrap using the criteria of the US EPA guidelines for determining a maximum tolerated dose. Concentrations higher than 5% applied under a semi-occlusive wrap produced severe irritation and parakeratosis after 4 weeks of treatment.

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least once daily


DETAILED CLINICAL OBSERVATIONS: Yes


DERMAL IRRITATION: Yes
- Time schedule for grading of the erythema and oedema scores according to Draize (1959): daily during weeks 1, 2, 6, 7, 12, and 13


BODY WEIGHT: Yes
- Time schedule for examinations: weekly


FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes


WATER CONSUMPTION: Yes
- Time schedule for examinations: 3 times per week during week 1, 2, 6, 7, 12, and 13


OPHTHALMOSCOPIC EXAMINATION: No


HAEMATOLOGY: Yes
- Time schedule for collection of blood: at termination of study
- Anaesthetic used for blood collection: Yes (carbon dioxide)
- Animals fasted: No data
- How many animals: 20 per group and sex
- Parameters examined: haemoglobin concentration, haematocrit, erythrocytes, total leukocytes, differential leukocyte count, platelet count, red blood cell indices, prothrombin time; examination of blood smears for cellular morphology


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at termination of study
- Animals fasted: No data
- How many animals: 20 per group and sex
- Parameters examined: alanine aminotransferase (ALT), sorbitol dehydrogenase (SDH), alkaline phosphatase, trigycerides, cholesterol, total bilirubin, total protein, albumin, albumin/globulin ratio, creatinine, urea nitrogen, sodium, potassium, chloride, calcium, phosphorus, glucose


URINALYSIS: Yes
- Time schedule for collection of urine: w 3, 6, 13
- Metabolism cages used for collection of urine: Yes
- Animals fasted: No data
- How many animals: 5 per group and sex and timepoint
- Parameters examined: acitivities of n-acetyl glucosaminidase (NAG), gamma-glutamyl transpeptidase (GGT), alanine aminopeptidase (AAP) activities, creatinine concentration, osmolality, specific gravity, colour and appearance; microscopic evaluation for blood cells, epithelial cells, bacteria, crystals, and amorphous sediment


NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes

ORGAN WEIGHTS: Yes
liver. kidneys, brain, testes or ovaries, lungs, thymus, adrenal glands

HISTOPATHOLOGY: Yes, high dose and control groups
After fixation in 10% neutral buffered formalin, staining with haematoxylin and eosin microscopic examination of:
samples of skin from treated and untreated areas on the back, bone marrow, kidneys, liver, lymph nodes, spleen, thymus, thyroid gland, Zymbal's gland
Other examinations:
RENAL CELL PROLIFERATION by BrdU incorporation method (Details taken from English et al. (1994) Fund Appl Toxicol 23: 397 - 406)
- Time schedule for examination: w 3, 6, 13
- How many animals: 5 per group and sex and timepoint
- Procedure: At the beginning of week 3, 6 and 13, osmotic pumps, that were loaded with bromodeoxyuridine (20 mg BrdU/mL) prepared in 0.1 M PBS, were implanted surgically, subcutaneously into the mid-dorsal lumbar area. Animals were dosed with HQ cream as usual (application site moved to the flank) and killed 3 days later. Kidneys were fixed in situ by whole-body perfusion with PBS followed by 2% paraformaldehyde and 1% glutaraldehyde in PBS. After 24 h, kidneys and a 2-inch length of duodenum (as experimental control for cell replication) were embedded and sectioned and were stained by immunohistochemically methods for detection of BrdU incorporation.
- Evaluation: The numbers of labelled cells per 500 renal cells were counted for the P1, P2, P3 and "other tubular cells" of the kidney.
Identification of cells on the basis of their morphology and topographic location:
P1 segment: origin at glomerulus and comprising about one half of the pars convoluta; location within the cortical labyrinth adjacent to the glomeruli; morphology tall with intermediate length microvilli
P2: remainder of the pars convoluta and initial part of the pars recta; location within the cortical labyrinth and at the tips of the medullary rays; morpholgy similar to P1 cells except that they were shorter in height with shorter microvilli
P3: remainder of the pars recta; location at the base of the medullary rays and in the outer stripe of the outer medulla; morphology cells with long microvilli and dark cytoplasm
Statistics:
Homogeneity of variances with a Bartlett's test at P<- 0.01; comparison of groups with analysis of variance followed by Duncan's test at P<-0.05

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Dermal irritation:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
not examined
Details on results:
CLINICAL SIGNS AND MORTALITY: No HQ-related changes

BODY WEIGHT AND WEIGHT GAIN: No HQ-related changes

FOOD CONSUMPTION: significant decrease in males at 3.5% and 5.0% test concentration compared to control only on day 4 (P<=0.05)
Evaluation: No toxicologically significant effect on food consumption


WATER CONSUMPTION:
Males: for all dose groups within 10% of the values of the control group, both during dosing period on workdays and on exposure-free weekends
Females: water consumption was comparable in all groups; however, in all groups including controls water consumption was 20-30% higher on weekends compared to workdays
Evaluation: No HQ-related changes


DERMAL EFFECTS:
Males:
- Brown discoloration: in all groups including controls appeared within first days of treatment; intensity of discoloration minimal, but exacerbated by HQ-treatment;
- Scaly skin (minimal ichthyosis): in 70-100% of treated males compared to 20% of controls;
- Brown spots: incidences were higher in HQ-treated groups without dose-related trend
Females:
- Brown discoloration: incidence and intensity was slightly higher at 3.5% and 5.0% test concentration compared to other groups, effect appeared within first days of treatment;
- Scaly skin (minimal ichthyosis): in 92-100% of treated females compared to 6% of controls indicating that females may be more sensitive than males- Brown spots: incidences were slightly higher in HQ-treated groups without dose-related trend
All groups: epidermal hyperplasia at application site of all groups, no difference in incidence or severity among groups; no evidence of exogenous ochronosis
Evaluation: no toxicologically significant skin effects


DERMAL IRRITATION:
Concentration-related increase of erythema in HQ-treated groups with highest score of 2 throughout the course of the study, effects abated during treatment-free weekends
Evaluation: no indication that maximal tolerable dose (MTD) was exceeded


HAEMATOLOGY:
Males: significant changes of leukocytes (decrease at 5% HQ), of platelets (increase at 3.5% HQ), and hemoglobin and hematocrit( both increased at 2 and 5% HQ)
Females: no differences among groups
Evaluation: Significant changes of haematogical parameters were within normal biological range for rats and were not considered to be biologically significant.

CLINICAL CHEMISTRY:
Males: significant increases of total protein, ALT and SDH at 5% HQ
Females: no differences among groups
Evaluation: ALT and SDH activities were increased in individual males from all groups including controls. The magnitude of the group mean enzyme acitivities was not clinically relevant. A possible effect in the liver, indicated by increased ALT and SDH, was not reflected in liver weights or treatment-related histological changes in the liver. Thus, significant changes of serum chemistry parameters were not considered to be an adverse or toxicologically meaningful effect of HQ.

URINALYSIS
3 wk data: significant changes of osmolality and specific gravity at 3.5% HQ, however, with increases in males and decreases in females (volume of urine higher in this group)
6 wk data: all male dose-groups sigificant decreases of GGT, AAP, creatinine; females showed sigificant decreases of GGT and AAP at 2% HQ only
13 wk data: males without significant changes; females showed significant increase of osmolality and specific gravity
Evaluation: There was no consistent trends among groups concerning urinary parameters which might indicate kidney damage.


ORGAN WEIGHTS: no significant differences


HISTOPATHOLOGY: NON-NEOPLASTIC
Kidneys:
Lesions observed after 3, 6 and 13 wk: not considered to be HQ-related, lymphocytic infiltration or tubular regeneration was present in occasional animals from all groups.
Terminal sacrifice: renal tubular regeneration in all control animals, in all high-dose males, and in 15/20 high-dose females; lymphocytic infiltration and degeneration of proximal tubule in few control and high-dose rats, incidence and severity not altered by exposure; mineralization of renal tubular epithelium both in controls and high-dose groups; minimal to minor hydropic degeneration of the proximal tubule epithelium more prominent in control rats than in high-dose rats.


OTHER FINDINGS: RENAL CELL PROLIFERATION ASSAY (Labelling index = LI)
3 wk data: males: significant increase of LI in P1 cells at 5% HQ, slight increases in other dose groups and in P2 and P3 cells
6 wk data: no changes in males or females
13 wk data: males without significant changes; females with significant increases of LI of other tubular cells at 3.5 and 5% HQ
Evaluation: There were no changes indicative of sustained cellular proliferation.

Effect levels

open allclose all
Dose descriptor:
NOAEL
Effect level:
73.9 mg/kg bw/day (actual dose received)
Sex:
male
Basis for effect level:
other: No signs of systemic toxicity or nephrotoxicity / highest tested dose
Dose descriptor:
NOAEL
Effect level:
109.6 mg/kg bw/day (actual dose received)
Sex:
female
Basis for effect level:
other: No signs of systemic toxicity or nephrotoxicity / highest tested dose

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
After dermal application of HQ for up to 13 weeks no signs of treatment-related nephrotoxicity or renal cell proliferation were found in F344 rats after exposure to doses of up to 73.9 mg/kg bw/d in male rats, and 109.6 mg/kg bw/d in female rats.
Executive summary:

Subchronic dermal toxicity with emphasis on aspects of nephrotoxicity and renal cell proliferation was investigated in groups of 20 male and 20 female F344 rats with a test protocol similar to OECD Guideline 411. Dermal application of 0, 2.0, 3.5, and 5.0% hydroquinone in an oil-in-water emulsion cream, comparable to cream base of commercial cream preparations with HQ, was performed on 5 d/w, for 6 h/d during 13 w. Ingestion of test substance from the application site was prevented using a semi-occlusive wrap, and during periods without treatment by application of an Elizabethan collar. Resulting dermal doses were 29.5, 51.9, and 73.9 mg/kg bw/d in male rats, and 43.8, 77.0, and 109.6 mg/kg bw/d in female rats applied on a skin surface area of 24 cm2 (dermal doses of ca. 1 to 4 mg/cm2). In additional groups of 5 rats per sex, that had been treated for 3, 6 and 13 w, urinalysis data were collected and renal cell proliferation in different proximal tubule segments (P1, P2, P3 and other cells) was investigated via a BrdU incorporation assay. Dermal irritation was scored by grading of the erythema and oedema scores according to Draize (1959) daily during weeks 1, 2, 6, 7, 12, and 13. There was a concentration-related increase of erythema in HQ-treated groups with highest score of 2 throughout the course of the study. These effects abated during treatment-free weekends. Consequently, there is no indication that the maximal tolerable dose (MTD) was exceeded.

Dermal application of 5% HQ procuded transient minor irritation of the skin but no evidence of exogenous ochronosis. Up to the highest tested dose, there were no clinical signs of intoxication, and no statistically significant or toxicologically relevant changes of body weights, organ weights, of food and water consumption, of haematological and clinical chemical parameters. Treatment-related nephrotoxicity was not indicated from urinalysis data and from kidney histology. There were no changes indicative of sustained cellular proliferation in renal tubuli. Consequently, NOAELs in this subchronic dermal toxicity study were 73.9 mg/kg bw/d in male rats, and 109.6 mg/kg bw/d in female rats.