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Description of key information

90d NOAEL oral of 393 mg/kg bw/d (females) and 429 mg/kg bw/d males) respectively was derived. The NOAEL found with mice was more than a magnitude higher (7579 mg/kg bw/d females and 5981 mg/kg bw/d males).

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1992
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: well documented guideline study performed under GLP
Reason / purpose for cross-reference:
reference to same study
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
GLP compliance:
yes
Remarks:
according to 21 CFR 58
Limit test:
no
Species:
rat
Strain:
Fischer 344
Sex:
male/female
Details on test animals or test system 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.
Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
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:
13 weeks
Frequency of treatment:
continuous through feed
Remarks:
Doses / Concentrations:
3125 ppm
Basis:
nominal in diet
Remarks:
Doses / Concentrations:
6250 ppm
Basis:
nominal in diet
Remarks:
Doses / Concentrations:
12500 ppm
Basis:
nominal in diet
Remarks:
Doses / Concentrations:
25000 ppm
Basis:
nominal in diet
Remarks:
Doses / Concentrations:
50000 ppm
Basis:
nominal in diet
No. of animals per sex per dose:
10 males and 10 females per dose
Control animals:
yes, plain diet
Positive control:
no positive control
Observations and examinations performed and frequency:
13-Week Studies, Dosed Feed: Observed 2 x d for mortality/moribundity; 1 x wk for clinical signs of toxicity; food consumption measured 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:
Dosed Feed: sperm morphology and vaginal cytology were evaluated in rats and mice exposed to 0, 3125, 12500, and 50000 ppm HMB.
Statistics:
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.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
one animal was killed accidentally but no other mortalities
Mortality:
mortality observed, treatment-related
Description (incidence):
one animal was killed accidentally but no other mortalities
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
significant for males as of 12500 ppm and for females as of 6250 ppm
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
see details
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
dark yellow to opaque urine
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
effects at highest dose group for females and two highest dose groups for males, mainly increased kidney
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
see details
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
effects observed, treatment-related
Description (incidence and severity):
see details
Details on results:
One low dose male rat was killed accidentally during the first week; there were no other deaths during the study. Dose-related decreases in growth (Figure 1) and in final body weights were noted in male and female rats. Chemically-related clinical signs were limited to urine-stained fur in the perineal area and a dark yellow to opaque-greenish urine.
At necropsy, chemically-related gross lesions were observed in the two highest dose groups of male rats and in the highest dose group of female rats. The kidneys were enlarged and had an abnormal shape and granular surface. In some animals both kidneys were affected, while in others, the changes were limited to one kidney. Absolute and relative right kidney weights were increased in males in the 50000 ppm group and in females in the 25000 and 50000 ppm groups.
Histopathologic lesions in the kidney of male and female rats were similar, consisting of dilatation of renal tubules, regeneration of tubule epithelial cells, papillary degeneration or necrosis, and inflammation. The most prominent lesion was dilatation of the renal tubules (Plate 3), the severity of which was greater in males than in females, occurring in at all exposure levels. The entire length of the nephron was generally affected, with dilated tubules present in the cortex, outer, and inner medulla. In kidneys with minimal to mild dilatation, there generally was a focal distribution of the lesion in which much of the kidney appeared relatively unaffected (Plates 3 and 5). The majority of the tubules from some of the more severely affected kidneys in the 50000 ppm groups of male and female rats were dilated and often contained protein casts and cell debris; more extensively dilated tubules were lined by a flattened epithelium. Renal tubule epithelial cell regeneration increased in severity and/or incidence in groups of rats with tubule dilatation. At the highest dose, particularly in male rats, there was mild to moderate inflammation with fibrosis in the renal interstitium.
The cellular infiltrate was a mixture of neutrophils, lymphocytes, and macrophages. Necrosis of the tip of the renal papilla was also present in highest dose males and females (Plate 4).
Adjacent to the area of papillary necrosis, there was minimal hyperplasia of the cuboidal epithelium on the surface of the renal papilla or the transitional epithelium of the renal pelvis.
There was no evidence of inflammation or hyperplasia of the transitional epithelium of the urinary bladder in male or female rats.
Both absolute and relative liver weights increased markedly in a dose-related fashion in males and females (Table 5, Appendix A3). There were no histopathologic changes associated with the weight increase, but changes in activities of hepatic enzymes in serum occurred (described below).
In hematologic evaluations, male rats given 25000 or 50000 ppm HMB exhibited significant increases in platelet counts beginning at day 3 of the study and persisting at day 15 and week 12. Similar responses occurred in the 12500 ppm dose groups at day 15 and week 12 as well as in the 2 lower dose groups at day 15 (Appendix B). Sporadic increases occurred during the study in counts of segmented neutrophils and reticulocytes. There were increases in serum concentrations of UN at 3 and 15 days (50000 ppm dose groups), and increases in activities of serum ALT at 3 days (6250 to 50000 ppm groups) and in serum GGT at all time points (50000 ppm and lower dose groups at 15 days). Urine volume in male rats was increased in animals at 15 days and 12 weeks (50000 ppm group), and specific gravity was increased at 3 days and decreased at 12 weeks (50000 ppm) (Appendix B).
In female rats, there were increases in HCT, HGB concentration, MCHC, and RBC count at 3 days in animals treated with 6250 to 50000 ppm HMB (25000 and 50000 ppm for MCHC). Other hematologic findings included decreases in HGB concentration at 15 days and decreases in MCV at 3 days and 12 weeks. Significant changes in serum enzyme activities in treated female rats included minimal to mild increases in ALT at 3 days (25000 and 50000 ppm) and in SDH at 12 weeks (all treatment groups). GGT increased in female rats at 15 days (25000 and 50000 ppm) and 12 weeks (all treatment groups). Urine volume was increased in female rats at 15 days and 12 weeks (50000 ppm), and specific gravity was increased at 3 days (25000 and 50000 ppm).
In reproductive system evaluations, a significant decrease in epididymal sperm density and a non-significant increase in the percentage of abnormal sperm were observed in male rats. An increase in the length of the estrous cycle was seen in female rats receiving the highest concentration of HMB in the diet (Appendix C).
Dose descriptor:
NOAEL
Effect level:
6 250 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: attributable to microscopic kidney lessions
Dose descriptor:
NOAEL
Effect level:
393 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: ppm values were converted to mg/kg bw/d by study authors based on actual chemical consumed and separately for females and males (converted from 6250 ppm)
Dose descriptor:
NOAEL
Effect level:
429 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: ppm values were converted to mg/kg bw/d by study authors based on actual chemical consumed and separately for females and males (converted from 6250 ppm)
Critical effects observed:
not specified

In male rats at 50000 ppm (3656 mg/kg bw/d) sperm count per 'g' caudal tissue was significantly decreased. There were no treatment related effects for sperm mobility and incidence of abnormal sperm. In females there was no apparent effect on estrual cyclicity. The length of the estruous cycle in the 50000 ppm (3261 mg/kg bw/d) dose group however was significantly longer than in the control group.

Conclusions:
The NOAEL in this study was set to 6250 ppm (i.e. 393 mg/kg bw/d for females and 429 mg/kg bw/d for males) and was mainly based on microscopic kidney lessions observed at mid dose range.
Executive summary:

In summary, administration of HMB was associated with effects on the liver, kidney, and reproductive organs of rats and mice. Although these effects were observed primarily in dietary studies at concentrations that also affected body weight gain, these effects are considered to be tissue specific and dose-responsive. A no-observed-adverse-effect level (NOAEL) for microscopic kidney lesions was 25000 ppm HMB in the feed for mice and 6250 ppm for rats. An apparently reversible enlargement and cytoplasmic vacuolization of the livers of rats and mice was noted at dietary concentrations of 6250 ppm and above.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
393 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
the 90d rat study was chosen for this endpoint as rats appeared more sensitive to oxybenzone than mice. However, in mice similar effects were seen, but at higher doses (NOAEL approx. 6000 mg/kg bw/d)

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: dermal
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1992
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: well documented guideline study performed under GLP
Reason / purpose for cross-reference:
reference to same study
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 411 (Subchronic Dermal Toxicity: 90-Day Study)
GLP compliance:
yes
Remarks:
according to 21 CFR 58
Limit test:
no
Species:
other: rats and mice
Strain:
other: Fischer 344 and B6C3F1
Sex:
male/female
Details on test animals or test system 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.
Type of coverage:
not specified
Vehicle:
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:
yes
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
Remarks:
Doses / Concentrations:
12.5 mg/kg bw/d
Basis:
nominal per unit body weight
Remarks:
Doses / Concentrations:
25 mg/kg bw/d
Basis:
nominal per unit body weight
Remarks:
Doses / Concentrations:
50 mg/kg bw/d
Basis:
nominal per unit body weight
Remarks:
Doses / Concentrations:
100 mg/kg bw/d
Basis:
nominal per unit body weight
Remarks:
Doses / Concentrations:
200 mg/kg bw/d
Basis:
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.
Statistics:
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.
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):
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.
Dose descriptor:
NOAEL
Effect level:
200 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
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.
Critical effects observed:
not specified
Conclusions:
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.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
200 mg/kg bw/day
Study duration:
subchronic
Species:
other: rats and mice
Quality of whole database:
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.

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed

Additional information

Oxybenzone was investigated by NTP (National Toxicology Program, US) for subchronic toxicity towards rats and mice by using oral and dermal exposure. Systemic effects were seen in rats and a NOAEL oral of 393 mg/kg bw/d (females) and 429 mg/kg bw/d males) respectively was derived. The NOAEL found with mice was more than a magnitude higher (7579 mg/kg bw/d females and 5981 mg/kg bw/d males) but also showed liver and kidneys being the target organ.

Also the dermal route was investigated for both species and the NOAEL was set to 200 mg/kg bw/d as systemic availability by dermal route was limited and hardly any effects were seen at the high dose of 200 mg/kg bw/d and also no local effects were observed in the subchronic dermal studies.

A 27 -day subacute study on rats by oral exposure resulted in a higher NOAEL but was limited in parameters observed.

A NOAEL of 200 mg/kg body weight was identified in the developmental study. Given the next highest dose in the study was 1000 mg/kg body weight the use of the NOAEL from the 90 day rat oral study (393 mg/kg body weight) for development of the DNELs is still considered appropriate as it lies between the two doses.


Justification for selection of repeated dose toxicity dermal - systemic effects endpoint:
only dermal subchronic study available

Repeated dose toxicity: via oral route - systemic effects (target organ) digestive: liver; urogenital: kidneys

Repeated dose toxicity: dermal - systemic effects (target organ) digestive: liver; urogenital: kidneys

Justification for classification or non-classification

Based on the result of a subchronic oral study with rats NOAELs of 393 mg/kg (females) and 429 mg/kg bw/d (males), respectively, for systemic effects were derived. Therefore, classification for Specific Target Organ System Toxicity (STOT) Repeat Exposure (RE) is not required according to CLP (Regulation (EC) No 1272/2008). A classification per DSD (Directive 67/548/EEC) is also not required.