Registration Dossier

Administrative data

sub-chronic toxicity: dermal
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: well documented guideline study performed under GLP
Reason / purpose:
reference to same study

Data source

Reference Type:
Report Date:

Materials and methods

Test guideline
equivalent or similar to
OECD Guideline 411 (Subchronic Dermal Toxicity: 90-Day Study)
GLP compliance:
according to 21 CFR 58
Limit test:

Test material

Test material form:
solid: particulate/powder
migrated information: powder
Details on test material:
The HMB used in these studies was obtained from American Cyanamid Co. (Bridgewater, NJ), and samples were analyzed at Midwest Research Institute (Kansas City, MO). The infrared, ultraviolet/visible, and nuclear magnetic resonance spectra were consistent with the structure of HMB and with available literature references. Elemental analysis results for carbon were slightly high but agreed with theoretical values for hydrogen. Karl Fischer analysis for water indicated less than 0.04%. Analysis by two thin-layer chromatography systems indicated a single spot; no impurities with relative peak areas of greater than 0.1% of the major peak were detected by gas chromatography.

Test animals

other: rats and mice
other: Fischer 344 and B6C3F1
Details on test animals and environmental conditions:
Rats and mice used in the 13-week studies were obtained from Taconic Farms (Germantown, NY). Rats were housed 5/cage for the feed study and were housed individually for the dermal studies. Animals received NIH-07 diet (Zeigler Bros., Gardners, PA) and water ad libitum throughout the studies. Blood samples were collected, and the sera analysed for viral titers from 5 animals per sex and species at study start and at termination in the 13-week studies. Temp.: 72 ± 3°F; relative humidity--50 ±15%; fluorescent light 12 h/d; 10 air changes/h. Age of animlas at start of study was 6 weeks for males and 7 weeks for females.

Administration / exposure

Type of coverage:
not specified
other: one group acetone and another group in lotion
Details on exposure:
The dermal vehicles were formulated to concentrations of 0, 5, 10, 20, 40, or 80 mg/ml. A constant volume of 0.25 ml for rats and 0.1 ml for mice was applied over a fixed standard area (10%) of the interscapular region. The area was clipped 24 hours prior to the initial application, and weekly thereafter, with an electric clipper.
Analytical verification of doses or concentrations:
Details on analytical verification of doses or concentrations:
Dose formulation stability studies indicated that acetone solutions of HMB were stable for at least 3 weeks in the dark in sealed vials at room temperature, as were solutions stored for 3 hours open to light and air. HMB also was stable under similar conditions when formulated as part of a lotion comprised of lanolin oil (5.4%), white petrolatum (2.6%), stearic acid (4.3%), propyl paraben (0.05%), propylene glycol (5.4%), methyl paraben triethanolamine (0.1%), disodium EDTA (0.05%), and deionized water (80%). Stability studies conducted on a feed blend indicated a small but significant loss (~2%) after 3 weeks' storage in the dark in sealed containers at room temperature. No significant losses were observed with similar blends kept at 5¡C or -20¡C. Feed blends stored open to air and light for 3 days in a rat cage exhibited losses (~2%).
Duration of treatment / exposure:
for 13 weeks
Frequency of treatment:
5 days per week
Doses / concentrationsopen allclose all
Doses / Concentrations:
12.5 mg/kg bw/d
nominal per unit body weight
Doses / Concentrations:
25 mg/kg bw/d
nominal per unit body weight
Doses / Concentrations:
50 mg/kg bw/d
nominal per unit body weight
Doses / Concentrations:
100 mg/kg bw/d
nominal per unit body weight
Doses / Concentrations:
200 mg/kg bw/d
nominal per unit body weight
No. of animals per sex per dose:
10 males and 10 females per dose group
Control animals:
yes, concurrent vehicle
Positive control:
no positive control


Observations and examinations performed and frequency:
13-Week Studies, Dermal: Observed 2 x d for mortality/moribundity; 1 x wk for clinical signs of toxicity; site of application examined weekly; weighed initially,weekly, and at necropsy.
Sacrifice and pathology:
Necropsy performed on all animals; the following tissues were examined microscopically from all high dose and controls: Gross lesions, adrenal gland, brain, esophagus, eyes (if grossly abnormal), femur with bone marrow, gall bladder (mice), heart, intestine (duodenum, jejunum, ileum, cecum, colon, rectum), kidney, liver, lungs and bronchi, mandibular and mediastinal lymph nodes, nasal cavity with turbinates, ovary, pancreas, parathyroid, pituitary, preputial or clitoral gland (rats only), prostate, salivary gland, seminal vesicle, spinal cord (if neurologic signs present), spleen, stomach (forestomach and glandular), testis and epididymis, thymus, thyroid, trachea, urinary bladder, uterus. In addition to all gross lesions, the kidney was examined in all dose groups. Organ weights obtained from all core study animals include: liver, thymus, right kidney, right testis, heart, and lungs.
Other examinations:
Hematology, Clinical Chemistry, and Urinalysis: Hematology, clinical chemistry, and urinalysis were evaluated in rats, only, on day 3, day 15, and week 12. Sperm Morphology/Vaginal Cytology:
Dermal: sperm morphology and vaginal cytology were evaluated in rats exposed to 0, 12.5, 50.0, and 200.0 mg/kg HMB and in mice exposed to 0, 22.8, 91.0, and 364.0 mg/kg HMB.
Two approaches were employed to assess the significance of pairwise comparisons between dosed and control groups in the analysis of continuous variables. Organ and body weight data, which are approximately normally distributed, were analyzed using the parametric multiple comparisons procedures of Williams (1971, 1972) and Dunnett (1955). Clinical chemistry and hematology data, which typically have skewed distributions, were analyzed using the nonparametric multiple comparisons methods of Shirley (1977) and Dunn (1964). Jonckheere's test (Jonckheere, 1954) was used to assess the significance of dose-response trends and to determine whether a trend-sensitive test (Williams, Shirley) was more appropriate for pairwise comparisons than a test capable of detecting departures from monotonic dose-response (Dunnett, Dunn). If the P-value from Jonckheere's test was greater than or equal to 0.10, Dunn's or Dunnett's test was used rather than Shirley's or Williams' test.
The outlier test of Dixon and Massey (1951) was employed to detect extreme values. No value selected by the outlier test was eliminated unless it was at least twice the next largest value or at most half of the next smallest value.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Dermal irritation:
no effects observed
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):
not examined
Ophthalmological findings:
no effects observed
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:
effects observed, treatment-related
Description (incidence and severity):
increased kidney weights
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Details on results:
13-Week Dermal Studies in Rats, with the Acetone Vehicle
All animals survived to the end of the studies. No chemically-related changes were observed in body weight gains, food consumption, clinical observations, reproductive system evaluations, necropsy findings, or by histologic examination of skin samples from the site of application. Relative kidney weights were increased in a non-dose-related manner in female rats treated topically with 25 mg/kg or larger doses of HMB.
In male rats, there were no changes that were considered chemically-related in serum hematologic, biochemical, or urinary variables at any time point. In female rats, there were decreases in reticulocyte counts in animals in all dose groups at 12 weeks, increases in platelet counts in animals in the 50, 100, and 200 mg/kg dose groups at 15 days, and an increase in WBC count produced by a lymphocytosis in the 200 mg/kg group at 12 weeks. There were no relevant changes in biochemical or urinalysis variables in female rats at any time point.

Effect levels

Dose descriptor:
Effect level:
200 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: The dermal studies were limited by the reduced systemic exposure achievable by this route, but indicated that rats and mice are generally similarly affected by oral and dermal exposures. Thus, the high dose in this study is considered as NOAEL.

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

In the dermal 13-week part of the study (see also oral 13-weeks study) no NOAEL was derived by the study authors but in absence of adverse dose dependant effects seen, the high dose of 200 mg/kg bw/d can be considered as NOAEL.
Executive summary:

In the dermal 13-week part of the study (see also oral 13-weeks study) no NOAEL was derived by the study authors but in absence of adverse dose dependant effects seen, the high dose of 200 mg/kg bw/d can be considered as NOAEL. The study indicated that rats and mice are generally similarly affected by oral and dermal exposures.