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Administrative data

Description of key information

Subacute and/or chronic toxicity studies are available for both the oral and dermal routes of administration. Based on the weight of evidence and considering the limited toxicological effects reported in subacute (Park, 2002; Jia et al., 2011) and chronic (Sharratt et al., 1964) toxicity studies and also for the BPO metabolite, benzoic acid (IPCS, 2000) , the NOAEL of ca. 200 mg/kg bw/day in rats derived from the 120 weeks dietary administration of benzoyl peroxide in rats can be considered as a conservative NOAEL. There were no findings indicative of systemic toxicity resulting from daily topical exposure of rats and mice to benzoyl peroxide gels at dose level of 100 and 833 mg/kg bw/d for 104 consecutive weeks, respectively. Skin irritation was observed at a dose level as low as 0.3 mg/cm²/day (1.67%) in rats but not at 0.17 mg/cm² (1%) in mice (CHPA, 2000, 2001).

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
repeated dose toxicity: oral
Remarks:
combined repeated dose and carcinogenicity
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Limitations in the design and reporting of the study. Howevet, it was considered and evaluated by WHO/JEFCA (Kroes et al., 2006) for the toxicological assessment of benzoyl peroxide as food additive.
Reason / purpose for cross-reference:
reference to same study
Qualifier:
no guideline followed
Principles of method if other than guideline:
Feeding toxicity and carcinogenicity study
GLP compliance:
no
Limit test:
no
Species:
rat
Strain:
other: Albino
Sex:
male/female
Details on test animals or test system and environmental conditions:
Diet composition: 100% whole meal flour, 56; dried skimmed milk, 20; fish meal, 11; alfalfa meal, 2; dried yeast and yeast extract, 1 each; cod liver oil, 3; salt mixture, 4. The remaining 2% was a triturate in whole meal flour of benzoyl peroxide added in the form of Novadelox, a commercial powder containing 18% benzoyl peroxide, 78% calcium sulphate and 4% magnesium carbonate.
Route of administration:
oral: feed
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
120 weeks
Frequency of treatment:
ad libitum
Remarks:
Doses / Concentrations:
0.0157, 0.157 and 1.57 % of Novadelox in diet
Basis:
other: nominal in diet (groups F3, F2 and F1, respectively)
Remarks:
Doses / Concentrations:
28, 280, 2,800 mg BPO/kg diet
Basis:
other: nominal in diet (groups F3, F2 and F1, respectively)
Remarks:
Doses / Concentrations:
Male: 1.9, 19 or 190 mg/kg bw/d
Basis:
other: actual ingested, calculation (Kroes et al., 2006)
Remarks:
Doses / Concentrations:
Female: 2.3, 23 and 230 mg/kg bw/d
Basis:
other: actual ingested, calculation (Kroes et al., 2006)
No. of animals per sex per dose:
25
Control animals:
other: yes, concurrent no treatment (FC group)
Observations and examinations performed and frequency:
Body wt gains of rats were noted weekly for 18 weeks, and then monthly. The animals were examined twice daily with regard to their general health
Sacrifice and pathology:
Animals that were moribund were killed. All animals that were killed or that died were autopsied. Benign and malignant tumours were analysed.
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 specified
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
not specified
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY
Some statistically significant differences were found between the mortality rates of control and experimental groups, but the importance of these findings is doubtful. The mortality of female rats fed on diet F2 was greater than the controls (group FC) at 78 weeks, whereas before and after this period no significant difference was found. There was no increased mortality in males receiving this diet or in males and females receiving the diet containing a higher concentration of Novadelox.

BODY WEIGHT AND WEIGHT GAIN
The rate of body weight gain was depressed (<10%), in several cases to a significant extent, in male and female rats consuming the flour diets containing the two highest levels of Novadelox. The effect, which was most noticeable during the first 8 months with diet F1, could have been due to (1) a diminished acceptability of the diet to rats, (2) a marginal nutritional deficiency caused by the action of benzoyl peroxide on flour, or to (3) the toxic action of Novadelox or its reaction products.

HISTOPATHOLOGY: NON-NEOPLASTIC
Many and varied gross and microscopic changes were found in all groups. Except for testicular atrophy, pathological abnormalities occurred to an equal extent in experimental and control animals or, because of a low incidence, occurred randomly in control or experimental groups. Infective changes were common, particularly in the lungs of both sexes of rat, and in the uterus of female rats. More than one lesion caused by infection was found per animal in rats. Local vascular damage frequently accompanied infective lesions. Degenerative changes were found most frequently in the liver, kidney, testes and adrenals of rats. Ulceration of the stomach was not found in control rats, while it occurred in 2 of the 96 examined which received the highest dosage level of benzoyl peroxide in flour diet.

HISTOPATHOLOGY: NEOPLASTIC (if applicable)
The overall tumour incidence was not significantly different between the experimental and control groups in rats. Most tumoun were found in rats which were examined after 100 weeks treatment, the occurrence being low before that time.
Dose descriptor:
NOEL
Effect level:
19 - 23 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No effect at this dose level
Dose descriptor:
NOAEL
Effect level:
190 - 230 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
histopathology: non-neoplastic
Critical effects observed:
not specified
Executive summary:

Sharratt et al. (1964) conducted a study on the treatment of whole meal flour with benzoyl peroxide. Albino rats (groups of 25 animals/sex/ species; strain and age were unspecified) were fed a diet with increasing amount of Novadelox, a commercial powder containing 18% benzoyl peroxide (purity unspecified). Control groups received a diet lacking Novadelox. According to the authors, treatment groups received 10, 100 or 1,000 times the daily intake, for 120 weeks. The estimated doses of benzoyl peroxide amounted to 28, 280 and 2,800 mg/kg diet (equal to approximately 1.9, 19 or 190 mg/kg bw for males, and 2.3, 23 and 230 mg/kg bw for females). Body weight gain was depressed in male and female rats consuming the diets containing the highest levels of benzoyl peroxide. The authors speculated that these weight depressions of about 10% were caused by marginal nutritional deficiencies, because an increased intake of food reversed the phenomenon. A part from the 280 mg/kg diet group, in which there was a large number of accidental deaths, there was no significant difference between experimental and appropriate control groups in the mortality rate of the animals. Testicular atrophy was increased at the highest concentratrion. The authors suggested that this atrophy was caused by benzoyl peroxide, which probably marginally decreased the amount of vitamin E in the diet. No evidence for benzoyl peroxide-related carcinogenicity was observed. Based on the limited and/or indirect effects observed, the highest concentration of 2,800 mg/kg diet, equivalent to ca. 200 mg/kg bw/day, can be considered as the NOAEL.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
200 mg/kg bw/day
Study duration:
chronic
Species:
rat

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:
chronic toxicity: dermal
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study
Reason / purpose for cross-reference:
reference to same study
Guideline:
other: OECD Guideline 451 (Carcinogenicity Studies)
Principles of method if other than guideline:
See full RSS in section 7.7
GLP compliance:
yes
Limit test:
no
Species:
mouse
Strain:
B6C3F1
Sex:
male/female
Type of coverage:
open
Vehicle:
other: aqueous carbopol gel
Duration of treatment / exposure:
at least 104 weeks
Frequency of treatment:
7 days a week
Remarks:
Doses / Concentrations:
1, 5 and 25/15%
Basis:

Remarks:
Doses / Concentrations:
0.17, 0.83 and 4.17 mg/cm²
Basis:

Remarks:
Doses / Concentrations:
Males: 33, 166 and 833 mg/kg bw/d
Basis:
other: based on a bw of 0.03 kg
Remarks:
Doses / Concentrations:
Females: 40, 200 and 1000 mg/kg bw/d
Basis:
other: based on a bw of 0.025 kg
Control animals:
yes, concurrent vehicle
Dose descriptor:
NOAEL
Remarks:
systemic toxicity
Effect level:
> 833 mg/kg bw/day
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: No systemic toxicity was observed
Dose descriptor:
NOAEL
Remarks:
local effect
Effect level:
0.17 mg/cm² per day
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: The major microscopic findings were observed at the application site: hyperkeratosis, subepidermal subacute inflammation , and sebaceous gland hyperplasia. Minimal effects were observed at the concentration of 1%.
Critical effects observed:
not specified
Executive summary:

The toxicity of benzoyl peroxide gels was evaluated when administered daily by topical application to the dorsal skin of mice for at least 104 weeks. Male and female B6C3F1/Cr1BR mice were assigned to seven groups. Benzoyl peroxide in carbopol gel at concentrations of l, 5, and 25-15% (1, 5, and 25-15 mg/mouse/day) was applied topically once daily to a treatment area (approximately 2 x 3 cm) on the dorsal skin of mice in Groups 2, 3, and 4, respectively. Mice in Group 1 served as vehicle controls and received daily topical applications of the carbopol gel vehicle at a dose volume of 0.1 mL. Mice in Group 6 served as negative controls; the hair on the backs of these mice was dipped at the same intervals as the other mice on study; however, these mice were not treated. Sixty mice/sex were assigned to Groups 1, 2, 3, 4, and 6, with the first 10 mice/sex/group designated for interim sacrifice during Week 53 and the remaining 50 mice/sex/group designated for terminal sacrifice after 104 weeks of treatment. Fifty mice/sex in Group 5 served as recovery animals, in that they were treated with 25% benzoyl peroxide for 52 weeks, and then treated with the vehicle for the remainder of the study. Twenty mice/sex in Group 7 served as sentinel animals for pathogen screening at Weeks 26, 52, 78, and 104. Diet and water were provided ad libitum. Once weekly, each animal was removed from its cage and examined for abnormalities and signs of toxicity, specifically noting the location, size, and appearance of any grossly visible or palpable masses. The treated skin (or analogous site on the untreated control) was graded for irritation once weekly. Body weights were recorded weekly from Weeks l through 14 and every fourth week thereafter and at Weeks 53 and 105. Group 4 had additional body weights taken at Weeks 57 and 59. Food consumption was measured and recorded weekly for Weeks l through 13 and every fourth week thereafter and at Weeks 52 and 104. Blood smears were prepared from all moribund-, interim-, and terminal-sacrifice animals for possible evaluation of hematopoietic neoplasia. After 52 weeks (interim-sacrifice animals) or 104 weeks (terminal sacrifice animals) of treatment the animals were anesthetized, weighed, exsanguinated, and necropsied. At necropsy, macroscopic observations were recorded, and selected tissues were collected and preserved. The liver with gallbladder, kidneys, and brain were weighed from all animals at interim sacrifice. Selected tissues (treated skin, untreated skin, and livers) were examined microscopically from all interim-sacrifice mice (Groups 1, 2, 3, 4, and 6). All collected tissues were examined microscopically at all terminal-sacrifice mice in Groups 1, 2, 3, 4, and 6, whereas only treated and untreated skin was examined microscopically from the mice in Group 5 (high dose-discontinued). Tumors were statistically analyzed separately and combined for relationship.

Daily topical exposure of mice to benzoyl peroxide at concentrations of 1 and 5% continued for 104 weeks. However, due to findings (skin ulcerations at the application site) that 25% benzoyl peroxide exceeded the maximum tolerated dose, the concentration in the high-dose group was lowered to 15% at the beginning of Week 57. Due to further incidences of skin ulceration at the 15% benzoyl peroxide concentration, treatment of the high-dose animals was discontinued (with vehicle only) for the final 13 weeks of the study. With the exception of the findings of ulcerations at the application site, there were no treatment-related differences in clinical observations among any of the groups. Treatment did not affect survival, body weights, or food consumption. The major microscopic findings were observed at the application site. At the interim (Week 53) sacrifice, these findings consisted of hyperkeratosis (1, 5, and 25% benzoyl peroxide), subepidermal subacute inflammation (5 and 25% benzoyl peroxide), and sebaceous gland hyperplasia (males: 5 and 25% benzoylperoxide:females:1, 5, and 25% benzoyl peroxide). These findings were dose-dependent with regards to incidence and or group mean severity. Similar findings were noted at the terminal (Week 105-106) sacrifice with the exception that there were no findings for treated or untreated skin in the high-dose and high-dose-discontinued animals. The high-dose-discontinued animals, after being treated with 25% benzoyl peroxide for 1 year, were allowed 52 weeks of recovery. Although the high-dose animals were not originally intended to be a recovery group, they were net treated with benzoyl peroxide for the final 13 weeks of the study since the 25 -15% benzoyl peroxide concentrations had been found to exceed the maximum tolerated dose. In bath of these cases there was no residual effect of treatment.

In conclusion, under the conditions of this study, there were no histologic findings indicative of toxicity resulting from daily topical exposure of mice to benzoyl peroxide gels at concentrations up to 25% ( (dose level ca. 833 mg/kg bw/d in males and ca. 1000 mg/kg bw/d in females)). The NOAEC for the dermal irritation was 0.17 mg/cm² (1%).

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
833 mg/kg bw/day
Study duration:
chronic
Species:
rat

Repeated dose toxicity: dermal - local effects

Link to relevant study records
Reference
Endpoint:
chronic toxicity: dermal
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study
Reason / purpose for cross-reference:
reference to same study
Guideline:
other: OECD Guideline 451 (Carcinogenicity Studies)
Principles of method if other than guideline:
See full RSS in section 7.7
GLP compliance:
yes
Limit test:
no
Species:
mouse
Strain:
B6C3F1
Sex:
male/female
Type of coverage:
open
Vehicle:
other: aqueous carbopol gel
Duration of treatment / exposure:
at least 104 weeks
Frequency of treatment:
7 days a week
Remarks:
Doses / Concentrations:
1, 5 and 25/15%
Basis:

Remarks:
Doses / Concentrations:
0.17, 0.83 and 4.17 mg/cm²
Basis:

Remarks:
Doses / Concentrations:
Males: 33, 166 and 833 mg/kg bw/d
Basis:
other: based on a bw of 0.03 kg
Remarks:
Doses / Concentrations:
Females: 40, 200 and 1000 mg/kg bw/d
Basis:
other: based on a bw of 0.025 kg
Control animals:
yes, concurrent vehicle
Dose descriptor:
NOAEL
Remarks:
systemic toxicity
Effect level:
> 833 mg/kg bw/day
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: No systemic toxicity was observed
Dose descriptor:
NOAEL
Remarks:
local effect
Effect level:
0.17 mg/cm² per day
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: The major microscopic findings were observed at the application site: hyperkeratosis, subepidermal subacute inflammation , and sebaceous gland hyperplasia. Minimal effects were observed at the concentration of 1%.
Critical effects observed:
not specified
Executive summary:

The toxicity of benzoyl peroxide gels was evaluated when administered daily by topical application to the dorsal skin of mice for at least 104 weeks. Male and female B6C3F1/Cr1BR mice were assigned to seven groups. Benzoyl peroxide in carbopol gel at concentrations of l, 5, and 25-15% (1, 5, and 25-15 mg/mouse/day) was applied topically once daily to a treatment area (approximately 2 x 3 cm) on the dorsal skin of mice in Groups 2, 3, and 4, respectively. Mice in Group 1 served as vehicle controls and received daily topical applications of the carbopol gel vehicle at a dose volume of 0.1 mL. Mice in Group 6 served as negative controls; the hair on the backs of these mice was dipped at the same intervals as the other mice on study; however, these mice were not treated. Sixty mice/sex were assigned to Groups 1, 2, 3, 4, and 6, with the first 10 mice/sex/group designated for interim sacrifice during Week 53 and the remaining 50 mice/sex/group designated for terminal sacrifice after 104 weeks of treatment. Fifty mice/sex in Group 5 served as recovery animals, in that they were treated with 25% benzoyl peroxide for 52 weeks, and then treated with the vehicle for the remainder of the study. Twenty mice/sex in Group 7 served as sentinel animals for pathogen screening at Weeks 26, 52, 78, and 104. Diet and water were provided ad libitum. Once weekly, each animal was removed from its cage and examined for abnormalities and signs of toxicity, specifically noting the location, size, and appearance of any grossly visible or palpable masses. The treated skin (or analogous site on the untreated control) was graded for irritation once weekly. Body weights were recorded weekly from Weeks l through 14 and every fourth week thereafter and at Weeks 53 and 105. Group 4 had additional body weights taken at Weeks 57 and 59. Food consumption was measured and recorded weekly for Weeks l through 13 and every fourth week thereafter and at Weeks 52 and 104. Blood smears were prepared from all moribund-, interim-, and terminal-sacrifice animals for possible evaluation of hematopoietic neoplasia. After 52 weeks (interim-sacrifice animals) or 104 weeks (terminal sacrifice animals) of treatment the animals were anesthetized, weighed, exsanguinated, and necropsied. At necropsy, macroscopic observations were recorded, and selected tissues were collected and preserved. The liver with gallbladder, kidneys, and brain were weighed from all animals at interim sacrifice. Selected tissues (treated skin, untreated skin, and livers) were examined microscopically from all interim-sacrifice mice (Groups 1, 2, 3, 4, and 6). All collected tissues were examined microscopically at all terminal-sacrifice mice in Groups 1, 2, 3, 4, and 6, whereas only treated and untreated skin was examined microscopically from the mice in Group 5 (high dose-discontinued). Tumors were statistically analyzed separately and combined for relationship.

Daily topical exposure of mice to benzoyl peroxide at concentrations of 1 and 5% continued for 104 weeks. However, due to findings (skin ulcerations at the application site) that 25% benzoyl peroxide exceeded the maximum tolerated dose, the concentration in the high-dose group was lowered to 15% at the beginning of Week 57. Due to further incidences of skin ulceration at the 15% benzoyl peroxide concentration, treatment of the high-dose animals was discontinued (with vehicle only) for the final 13 weeks of the study. With the exception of the findings of ulcerations at the application site, there were no treatment-related differences in clinical observations among any of the groups. Treatment did not affect survival, body weights, or food consumption. The major microscopic findings were observed at the application site. At the interim (Week 53) sacrifice, these findings consisted of hyperkeratosis (1, 5, and 25% benzoyl peroxide), subepidermal subacute inflammation (5 and 25% benzoyl peroxide), and sebaceous gland hyperplasia (males: 5 and 25% benzoylperoxide:females:1, 5, and 25% benzoyl peroxide). These findings were dose-dependent with regards to incidence and or group mean severity. Similar findings were noted at the terminal (Week 105-106) sacrifice with the exception that there were no findings for treated or untreated skin in the high-dose and high-dose-discontinued animals. The high-dose-discontinued animals, after being treated with 25% benzoyl peroxide for 1 year, were allowed 52 weeks of recovery. Although the high-dose animals were not originally intended to be a recovery group, they were net treated with benzoyl peroxide for the final 13 weeks of the study since the 25 -15% benzoyl peroxide concentrations had been found to exceed the maximum tolerated dose. In bath of these cases there was no residual effect of treatment.

In conclusion, under the conditions of this study, there were no histologic findings indicative of toxicity resulting from daily topical exposure of mice to benzoyl peroxide gels at concentrations up to 25% ( (dose level ca. 833 mg/kg bw/d in males and ca. 1000 mg/kg bw/d in females)). The NOAEC for the dermal irritation was 0.17 mg/cm² (1%).

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
0.17 mg/cm²
Study duration:
chronic
Species:
mouse
Quality of whole database:
As the chronic study in rat did not allow to derive a NOAEL for the local irritation, the NOAEL issued from the mouse study is used for risk assessment.

Additional information

Oral route

In a study conducted under the GLP using OECD test guideline 422 (Park, 2002), rats were exposed to benzoyl peroxide at levels of 0, 250, 500 and 1,000 mg/kg/day for 29 days for males and for 41-51 days for females. No deaths were found in any animals including control groups during exposure period. Hematemesis was observed in one male receiving 500 mg/kg but this is not related to benzoyl peroxide exposure. No hematological effect attributable to benzoyl peroxide was observed in any treated groups. A significant dose-related decrease in the weight of testes and epididymis was observed in males treated with 1,000 mg/kg. In the males, receiving 1,000 mg/kg, testis degenerations were observed microscopically (5 animals of 10). In females at 1,000 mg/kg, slight effects in uterus such as epithelial vacuolation or hyperplasia were observed (control 0/10; 250 mg/kg 0/10, 500 mg/kg 2/10; 1000 mg/kg 3/10). There were no significant dose-related changes in any other pathological findings. Since reproductive organ weights were significantly changed in treated animals this study suggested the NOAEL as to 500 mg/kg for male rats.

Another study assessed the chronic toxicity of benzoyl peroxide by oral route (Sharratt et al., 1964). Even if this study was limited compared to actual guidelines, it was considered and evaluated by WHO/JEFCA (Kroes et al., 2006) and US FDA (Life Sciences Research Office, 1980) for the toxicological assessment of benzoyl peroxide as food additive.

Albino rats (groups of 25 animals/sex/ species; strain and age were unspecified) were fed a diet with increasing amount of Novadelox, a commercial powder containing 18% benzoyl peroxide (purity unspecified) used in the treatment of whole meal flour. Control groups received a diet lacking Novadelox. According to the authors, treatment groups received 10, 100 or 1,000 times the daily intake, for 120 weeks. The estimated doses of benzoyl peroxide amounted to 28, 280 and 2,800 mg/kg diet (equal to approximately 1.9, 19 or 190 mg/kg bw/day for males, and 2.3, 23 and 230 mg/kg bw/day for females). Body weight gain was depressed in male and female rats consuming the diets containing the highest levels of benzoyl peroxide. The authors speculated that these weight depressions of about 10% were caused by marginal nutritional deficiencies, because an increased intake of food reversed the phenomenon. A part from the 280 mg/kg diet group, in which there was a large number of accidental deaths, there was no significant difference between experimental and appropriate control groups in the mortality rate of the animals. Testicular atrophy was increased at the highest concentration. The authors suggested that this atrophy was caused by benzoyl peroxide, which probably marginally decreased the amount of vitamin E in the diet. No evidence for benzoyl peroxide-related carcinogenicity was observed. Based on the limited and/or indirect effects observed, the highest concentration of 2,800 mg/kg diet, equivalent to ca. 200 mg/kg bw/day, can be considered as the NOAEL (Sharratt et al., 1964).

Four groups of mice were gavaged daily with BPO at doses of 0, 50, 100, 200 mg/kg bw/d for 42 days, respectively (Jia et al., 2011). In liver tissue, superoxide dismutase (SOD) activity was significantly decreased, while the content of malondialdehyde (MDA) significantly increased following BPO exposure at 200 mg/kg b. w. BPO significantly decreased the Mg(2+) -ATPase and Ca(2+) -ATPase activities of the liver at 200mg/kg b. w. BPO, at all of the doses assayed, produced non-significant effects on glutathione peroxidase (GSH-Px) and Na(+) K(+) -ATPase activities. Experimental results suggested that BPO had certain adverse effects on antioxidant status and the activities of Mg(2+) -ATPase and Ca(2+) -ATPase of liver tissue.

Supporting data from benzoic acid/sodium benzoate

As discussed in section “Toxicokinetics, metabolism and distribution, benzoyl peroxide is rapidly converted to benzoic acid or sodium benzoate in the skin of both animals and humans, and these are absorbed into the circulation and rapidly cleared through the kidneys (Nacht et al., 1981; Morches and Holzmann, 1982; Yeung et al., 1983; Wepierre et al., 1986). Both benzoic acid and sodium benzoate are considered to be safe for direct ingestion as human food additives (http://ec.europa.eu/food/fs/sc/scf/out137_en.pdf) and are widely used antibacterials and antifungals and as such would not be expected to present a health risk to those exposed through topical use of benzoyl peroxide. Further, benzoic acids database includes long-term rodent carcinogenicity evaluation indicating a lack of tumorigenic activity after oral administration to Swiss mice in drinking water (Toth, 1984).

Systemic exposure to benzoyl peroxide in humans does not appear to result from topical exposure, as evidenced by the lack of benzoyl peroxide in body fluids following application to the skin (Morches and Holzmann, 1982). Benzoyl peroxide itself has not been found to penetrate below the upper layers of human skin (Nacht et al., 1981; Morches and Holzmann, 1982). Importantly, in vivo percutaneous penetration and metabolism studies in Rhesus monkeys have found virtually complete renal excretion in the form of unconjugated benzoic acid and detected no hippuric acid formation after topical benzoyl peroxide exposure (Nacht et al., 1981; Yeung et al., 1983). This supports that benzoyl peroxide-derived benzoic acid did not reach the liver where it would have been conjugated with glycine to form hippurate. Since urinary hippurate would be expected if significant levels of parent benzoyl peroxide reached the systemic circulation, these Rhesus studies support the human studies indicating no evidence of parent benzoyl peroxide in the systemic circulation.

In the acid conditions of the stomach, more than 50% of benzoyl peroxide is hydrolysed to benzoic acid (Sydney, 2010). Should any residual benzoyl peroxide be absorbed by the gastro-intestinal tract, it would be destroyed by liver peroxidases. In the stomach the equilibrium moves to the undissociated benzoic acid molecule, which should be absorbed rapidly. Benzoate from sodium benzoate would change from the ionized form to the undissociated benzoic acid molecule. As a result, the metabolism and systemic effects of benzoic acid and sodium benzoate can be evaluated together.

 

In general, the database for benzoic acid and sodium benzoate is limited, and there are no studies available performed according to current guidelines. In addition, the documentation of these studies in most cases is insufficient. Detailed information is given in Text tables 1 and 2. From the available studies, it can be assumed that the toxicity of benzoic acid after short-term oral exposure is low. In high-dosed rats given approximately 2250 mg/kg body weight per day via diet over 5 days, excitation, ataxia, convulsions, and histopathological changes in the brain were seen. The mortality was about 50%; in some cases, bleeding into the gut was noted (Kreis et al., 1967). In two other studies with rats dosed with approximately 825 mg/kg body weight per day over 7–35 days (Kreis et al., 1967) or with 65–647 mg/kg body weight per day over 28 days (Bio-Fax, 1973), no clear treatment-related effects occurred. The reduced weight gain at 2250 and 825 mg/kg body weight per day may be attributed to reduced food intake in the study by Kreis et al. (1967). The relevance of the reduced relative kidney weight at 324 mg/kg body weight per day, which was not dose-related and not accompanied by changes in histopathological examinations, is unclear (Bio-Fax, 1973). As given in Table 1, both studies have several limitations (i.e., missing haematological and clinical chemical investigations, incomplete histopathological examinations); therefore, both of these studies were inadequate for derivation of a NO(A)EL. More information on dose–response can be gained from the study of Fujitani (1993), in which rats received sodium benzoate for 10 days in feed. At the lowest tested concentration of 1358 mg/kg body weight per day, changes in serum cholesterol levels occurred in females. At doses of 1568 mg/kg body weight per day and above, changes in further serum parameters and an increased relative liver weight were described. Histopathological changes of the liver, increased relative kidney weights, and disorders of the central nervous system (convulsions) were seen after dosing via diet with approximately 1800 mg/kg body weight per day. In several other studies listed in Table 1, adverse effects were seen only at higher doses after feeding sodium benzoate over periods from 10 to 42 days, so that a lowest-observed- (adverse-)effect level (LO(A)EL) of 1358 mg sodium benzoate/kg body weight per day for short-term exposure can be derived. With cats (Bedford & Clarke, 1972), also described in Table 1, the effect levels with benzoic acid were lower. However, due to the differences in the metabolism of benzoic acid in cats compared with other experimental animals and humans, this study was not taken into further consideration.

Text table 1. Toxicity of benzoic acid and sodium benzoate after short-term oral exposure (from IPCS, 2000)

Species; strain; number of animals per dosea

Treatment

Duration (days)

Organs examined in histopathology, clinical chemistry, haematology

Results a

Reference

 

Benzoic acid

 

 

 

 

 

 

cat; 4 m

0 or 0.5% in diet (~0 or 300–420 mg/kg body weight)

3–4

liver, kidney, heart, stomach, lung, brain, spinal cord (only animals that died were examined); blood samples were taken from surviving cats

mild hyperaesthesia, apprehension, and depression starting 48–92 h after uptake; duration of the syndrome: about 20–48 h; mortality rate: 50%; degenerative changes in liver, kidneys, and lung, but no pathological findings in brain or spinal cord; surviving cats: urea and serum alanine aminotransferase (S-ALAT) 8, indicating liver and kidney damage

Bedford & Clarke (1972)

 

cat; 4 m

a) 100 or 200 mg/kg body weight via diet

b) 0 or 0.25% in diet (~0 or 130–160 mg/kg body

weight)

a) 15

b) 23

only blood samples were taken

no adverse effects were reported

Bedford & Clarke (1972)

rat; Wistar; 5–15 m

0 or 3% in diet (~0 or 2250 mg/kg body weight)

1–5

heart, liver, spleen, kidney, brain

body weight gain 9; in rats dosed over 5 days, disorders of the central nervous system (excitation, ataxia, tonoclonic convulsions); mortality rate ~50%; in some cases, bleeding into the gut; brain damage (necrosis of parenchymal cells of the stratum granulosum of the fascia dentata and the cortex of the lobus piriformis) in most animals dosed over 3–5 days (still present after 35 days)

Kreis et al. (1967)

rat; Wistar; 5–10 m

0 or 1.1% in diet (~0 or 825 mg/kg body weight)

7–35

heart, liver, spleen, kidney, brain

body weight gain 9; no clinical signs of intoxication

Kreis et al. (1967)

rat; albino; 10 m

0, 760, 3800, or 7600 ppm via diet (~0, 65, 324, or 647 mg/kg body weight)

28

liver, kidney, adrenals, testes

no deaths or signs of intoxication
324 mg/kg body weight: relative kidney weights 9; no further information available

Bio-Fax (1973)

Sodium benzoate

 

 

 

 

 

rat; F344/Ducrj; 6 m/f

0, 1.81, 2.09, or 2.4% in diet (~0, 1358, 1568, or 1800 mg/kg body weight)

10

liver, kidney; standard clinical chemistry

.1358 mg/kg body weight: changes in serum levels (cholesterol (f))
.1568 mg/kg body weight: relative liver weight (m); changes in serum levels (albumin (m), total protein (m))
1800 mg/kg body weight: 1/6 males died (hypersensitivity, convulsions); body weight (m/f); relative liver weight (f); relative kidney weights (m/f); absolute weights of spleen and thymus (m); absolute/relative weights of thymus (f); changes in serum levels (gamma-glutamyltranspeptidase (GGT) (m), albumin (f), cholinesterase (f)); eosinophilic foci around periportal vein and enlargement of hepatocytes with glassy cytoplasm in the periportal area of the liver (m); no changes in the kidney (m)

Fujitani (1993)

rat; Sherman; 6 m/f

0, 2, or 5% in diet (~0, 2200, or 6700 mg/kg body weight)

28

no data available

2200 mg/kg body weight: slight depression of body weight gain (m) 6700 mg/kg body weight: mortality 100% within 11 days; signs of intoxication included hyperexcitability, urinary incontinence, and convulsions no further information available

Fanelli & Halliday (1963)

rat; 28 (no further data)

0 or 5% in diet (~0 or 3750 mg/kg body weight)

28

no data available

mortality about 100% within 3 weeks; decreased feed intake, diarrhoea, intestinal haemorrhage and crusted blood in the nose; no further information available

Kieckebusch & Lang (1960)

rat; 5 (no further data)

0 or 5% in diet (~0 or 3750 mg/kg body weight)

$28

no data available

mortality 80% within 4–5 weeks; decreased body weight; no further information available

Kieckebusch & Lang (1960)

rat; F344; 10–11 m/f

0, 0.5, 1, 2, 4, or 8% in diet (~0, 375, 750, 1500, 3000, or 6000 mg/kg body weight)

42

histopathology performed, but not further specified

. 375 mg/kg body weight: hypersensitivity after dosing
. 3000 mg/kg body weight: mortality about 100% within 4 weeks; apart from atrophy of the spleen and lymph nodes, no other morphological changes were

Sodemoto & Enomoto (1980)

rat; Sherman; 5 m/f

0 or 16–1090 mg/kg body weight via diet

30

adrenals, upper intestine, kidney, liver, spleen

no adverse effects were reported; no further information available

Smyth & Carpenter (1948)

mouse; B6C3F1; 4–5 m/f

0, 2.08, 2.5, or 3% in diet (~0, 3000, 3750, or 4500 mg/kg body weight)

10

liver, kidney; standard clinical chemistry

. 3750 mg/kg body weight: changes in serum levels (cholinesterase (m)
. 4500 mg/kg body weight: hypersensitivity in all animals; convulsions 1/5 males and 2/5 females (both females died); absolute/relative liver weight (m/f); relative kidney weight (f); changes in serum levels (cholesterol (m), phospholipids (m)); enlarged hepatocytes, single cell necrosis and vacuolation of hepatocytes in all livers (m); no changes in the kidney (m/f)

Fujitani (1993)

mouse; albino Swiss; 4 m/f

0, 0.5, 1, 2, 4, or 8% via drinking-water (~0–12 000 mg/kg body weight)

35

survival, chemical consumption, histological changes (not further specified) (prestudy for carcinogenicity study)

. 3000 mg/kg body weight: “suitable for lifelong treatment” based on four parameters: survival, body weight, chemical consumption, and histology
. 6000 mg/kg body weight: mortality 75% in m/f; body weight of surviving mice (m/f)
. 12 000 mg/kg body weight: mortality 100% within 3 weeks

Toth (1984)

 

a  m = male; f = female.

 

In a 90-day study with rats dosed with 0, 1, 2, 4, or 8% sodium benzoate via diet, the mortality in the highest dose group (~6290 mg/kg body weight per day) was about 50%. Other effects in this group included a reduced weight gain, increased relative weights of liver and kidneys, and pathological changes (not further specified) in these organs (Deuel et al., 1954). In two studies with rats given 1.5% benzoic acid via diet (approximately 750 mg/kg body weight per day), the animals showed a reduced weight gain with decreased feed intake after dosing over 18 months. In one of these studies, mortality was increased (15/50 rats of both sexes versus 3/25 in controls) (Marquardt, 1960). No further information on these studies is available, as only provisional results were published. In a four- generation study with rats, no effects on life span, growth rate, or organ weights were reported after dosing with up to 1% in the diet (approximately 500 mg/kg body weight per day) (Kieckebusch & Lang, 1960). Only animals of the third generation were autopsied after 16 weeks, but it is not clear if a complete histopathological investigation was performed.

With sodium benzoate, two long-term studies with rats (administration of up to 1400 mg/kg body weight per day via diet over 18–24 months; Sodemoto & Enomoto, 1980) or mice (lifelong application of up to 6200 mg/kg body weight per day via drinking-water; Toth, 1984) are available. The results gave no indication of a carcinogenic effect in the tested animals. Although the study with mice was not performed according to current guide- lines, the results seem to be reliable, due to a sufficient number of animals and detailed histopathological examinations. However, the results from the study with rats are uncertain, due to a very high mortality in animals of all dose groups, including controls (from an “infection” after 16 months), no detailed information about dosing regimen (only mean values given), and the considerable differences in the body weight of male and female rats (the body weight of females was about twice that of males).

Text table 2: Results of studies concerning long-term oral exposure to benzoic acid and sodium benzoate (from IPCS, 2000)

Species; strain; number of animals per dosea

Treatment

Duration (days)

Organs examined in histopathology, clinical chemistry, haematology

Results a

Reference

 

Benzoic acid

 

 

 

 

 

 

rat; Wistar; dose group: 30 m/20 f; controls: 13 m/12 f

0 or 1.5% in diet (~0 or 750 mg/kg body weight)

18 months

no data available

reduced weight gain with decreased feed intake; increased mortality rate (15/50 vs. 3/25 in controls); no further information available (only provisional results are given)

Marquardt (1960)

 

rat; Wistar or Osborne-Mendel; dose group: 20 m; controls: 10 m

0 or 1.5% in diet (~0 or 750 mg/kg body weight)

18 months

no data available

reduced weight gain with decreased feed intake; no further information available (only provisional results are given)

Marquardt (1960)

rat; not given; 20 m/f

0, 0.5, or 1% in diet (~0, 250, or 500 mg/kg body weight)

generation 1 and 2: lifelong
generation 3: 16 weeks
generation 4: until breeding

histopathology in animals of generation 3 (not further specified)

no effects on growth and organ weights; feeding of 0.5% led to prolongation of survival compared with controls; no further information available

Kieckebusch & Lang (1960)

Sodium benzoate

 

 

 

 

 

rat; Sherman; 5 m/f

0, 1, 2, 4, or 8% in diet (~0, 640, 1320, 2620, or 6290 mg/kg body weight)

90 days

histopathology performed, but not further specified

6290 mg/kg body weight: mortality about 50%; weight gain 9; relative weights of liver and kidneys 8; pathological lesions (not further specified) in liver and kidneys

Deuel et al. (1954)

rat; F344; dose group: 50 m/52 f; controls: 25 m/43 f

0, 1, or 2% in diet (m: ~0, 700, or 1400 mg/kg body weight; f: ~0, 290, or 580 mg/kg body weight)

18–24 months

histopathology performed, but not further specified

average mortality rate of all animals during the first 16 months: 14.5% (all dead rats showed pneumonia with abscess); about 100 rats including controls died after 16 months due to haemorrhagic pneumonia (infection); no adverse clinical signs and no differences in average body weight and mortality in dosed animals compared with controls; non-carcinogenic effects not reported

Sodemoto & Enomoto (1980)

mouse; albino Swiss; dose group: 50 m/f; controls: 99 m/f

0 or 2% via drinking water (~0 or 5960–6200 mg/kg body weight)

lifelong

liver, spleen, kidney, bladder,

thyroid, heart, pancreas, testes, ovaries, brain, nasal turbinates, lung

no difference in survival rates in treated animals compared with controls; no pathological or statistical evidence of tumour induction

Toth (1984)

a m = male; f = female.

 

Conclusion

Based on the weight of evidence and considering the limited toxicological effects reported in subacute (Park, 2002; Jia et al., 2011) and chronic (Sharratt et al., 1964) toxicity studies with BPO and also with the BPO metabolite, benzoic acid/sodium benzoate, the NOAEL of ca. 200 mg/kg bw/day in rats derived from the 120 weeks dietary administration of benzoyl peroxide in rats can be considered as a conservative NOAEL.

Dermal route

The toxicity of benzoyl peroxide gels was evaluated when administered daily by topical application to the dorsal skin of rats for at least 104 weeks (CHPA, 2000; Minnema, 2000; Totman et al., 2000). Male and female CDF®(F-344) Cr1BR rats were assigned to seven groups. Benzoyl peroxide in carbopol gel at concentrations of 1.67, 5.0, and 15.0% (5, 15. and 45 mg/rat/day) was applied topically once daily to a treatment area (approximately 3.5 x 5 cm) on the dorsal skin of rats in Groups 2, 3, and 4, respectively. Rats in Group 1 served as vehicle controls and received daily topical applications of the carbopol gel vehicle at a dose volume of 0.3 ml. Rats in Group 6 served as negative controls; the hair on the backs of these rats was clipped at the same intervals as the other rats on study; however, these rats were not treated. Sixty rats/sex were assigned to Groups 1, 2, 3, 4, and 6, with the first 10 rats/sex/group designated for interim sacrifice during Week 53 and the remaining 50 rats/sex/group designated for terminal sacrifice after 104 weeks of treatment. Fifty rats/sex in Group 5 served as recovery animals, in that they were treated with 15% benzoyl peroxide for 52 weeks, then treated with the vehicle for the remainder of the study. Twenty rats/sex in Group 7 served as sentinel animals for pathogen screening at Weeks 26, 52, 78, and 104. Diet and water were provided ad libitum. Once weekly, each animal was removed from its cage and examined for abnormalities and signs of toxicity, specifically noting the location, size, and appearance of any grossly visible or palpable masses. The treated skin (or analogous site on the untreated control) was graded for irritation once weekly. Body weights were recorded weekly from Weeks 1 through 14 and every fourth week thereafter and at Weeks 53 and 105. Food consumption was measured and recorded weekly for Weeks 1 through 13 and every fourth week thereafter and at Weeks 52 and 104. Blood smears were prepared from all moribund-, interim-, and terminal-sacrifice animals for possible evaluation of hematopoietic neoplasia. After 52 weeks (interim-sacrifice animals) or 104 weeks (terminal sacrifice animals) of treatment, the animals were anesthetized, weighed, exsanguinated, and necropsied. At necropsy, macroscopic observations were recorded, and selected tissues were collected and preserved. The liver, kidneys, and brain were weighed from all animals at interim sacrifice. Selected tissues (treated skin, untreated skin, and kidneys) were examined microscopically from all interim-sacrifice rats (Groups 1, 2, 3. 4. and 6). All tissues were examined microscopically from all terminal-sacrifice rats in Groups 1, 2. 3. 4, and 6, whereas only treated and untreated skin was examined microscopically from the rats in Group 5.

Treatment had no effect on: survival, body weights, food consumption, clinical observations, application site findings, gross pathology findings and organ weight changes at the interim sacrifice and gross pathology findings at terminal sacrifice. Microscopic evaluation revealed that the only treatment-related histopathologic findings noted at both the interim and terminal sacrifices were those seen at the site of topical dermal exposure. These consisted of hyperkeratosis, acanthosis, sebaceous gland hyperplasia, and chronic subepidermal inflammation. These changes occurred in all treatment groups (1.67, 5, and 15% benzoyl peroxide) and were mostly of a minimal to mild degree of severity. They progressed only slightly between 52 and 104 weeks of exposure. For the recovery animals (52 weeks of treatment with 15% benzoyl peroxide followed by 52 weeks of vehicle), hyperkeratosis was present approximately twice as frequently as it was in either the vehicle or negative control group, but less than half as frequently as in any other treatment group. Acanthosis was not appreciably increased in the recovery animals as compared to controls, and sebaceous gland hyperplasia was only equivocally present. These findings indicated good, although not complete, recovery from the effects of 52 weeks of treatment with 15% benzoyl peroxide, with remnants of hyperkeratosis remaining after 52 weeks of recovery.

In conclusion, under the conditions of the study, there were no findings indicative of systemic toxicity resulting from daily topical exposure of rats to benzoyl peroxide gels at concentrations up to 15% (dose level ca. 100 mg/kg bw/d in males and ca. 150 mg/kg bw/d in females) for 104 consecutive weeks. Skin irritation was observed at a dose level as low as 0.3 mg/cm²/day.

The toxicity of benzoyl peroxide gels was also evaluated when administered daily by topical application to the dorsal skin of mice for at least 104 weeks (CHPA, 2001; Minnema, 2001). Male and female B6C3F1/Cr1BR mice were assigned to seven groups. Benzoyl peroxide in carbopol gel at concentrations of l, 5, and 25-15% (1, 5, and 25-15 mg/mouse/day) was applied topically once daily to a treatment area (approximately 2 x 3 cm) on the dorsal skin of mice in Groups 2, 3, and 4, respectively. Mice in Group 1 served as vehicle controls and received daily topical applications of the carbopol gel vehicle at a dose volume of 0.1 mL. Mice in Group 6 served as negative controls; the hair on the backs of these mice was dipped at the same intervals as the other mice on study; however, these mice were not treated. Sixty mice/sex were assigned to Groups 1, 2, 3, 4, and 6, with the first 10 mice/sex/group designated for interim sacrifice during Week 53 and the remaining 50 mice/sex/group designated for terminal sacrifice after 104 weeks of treatment. Fifty mice/sex in Group 5 served as recovery animals, in that they were treated with 25% benzoyl peroxide for 52 weeks, and then treated with the vehicle for the remainder of the study. Twenty mice/sex in Group 7 served as sentinel animals for pathogen screening at Weeks 26, 52, 78, and 104. Diet and water were provided ad libitum. Once weekly, each animal was removed from its cage and examined for abnormalities and signs of toxicity, specifically noting the location, size, and appearance of any grossly visible or palpable masses. The treated skin (or analogous site on the untreated control) was graded for irritation once weekly. Body weights were recorded weekly from Weeks l through 14 and every fourth week thereafter and at Weeks 53 and 105. Group 4 had additional body weights taken at Weeks 57 and 59. Food consumption was measured and recorded weekly for Weeks l through 13 and every fourth week thereafter and at Weeks 52 and 104. Blood smears were prepared from all moribund-, interim-, and terminal-sacrifice animals for possible evaluation of hematopoietic neoplasia. After 52 weeks (interim-sacrifice animals) or 104 weeks (terminal sacrifice animals) of treatment the animals were anesthetized, weighed, exsanguinated, and necropsied. At necropsy, macroscopic observations were recorded, and selected tissues were collected and preserved. The liver with gallbladder, kidneys, and brain were weighed from all animals at interim sacrifice. Selected tissues (treated skin, untreated skin, and livers) were examined microscopically from all interim-sacrifice mice (Groups 1, 2, 3, 4, and 6). All collected tissues were examined microscopically at all terminal-sacrifice mice in Groups 1, 2, 3, 4, and 6, whereas only treated and untreated skin was examined microscopically from the mice in Group 5 (high dose-discontinued). Tumors were statistically analyzed separately and combined for relationship.

Daily topical exposure of mice to benzoyl peroxide at concentrations of 1 and 5% continued for 104 weeks. However, due to findings (skin ulcerations at the application site) that 25% benzoyl peroxide exceeded the maximum tolerated dose, the concentration in the high-dose group was lowered to 15% at the beginning of Week 57. Due to further incidences of skin ulceration at the 15% benzoyl peroxide concentration, treatment of the high-dose animals was discontinued (with vehicle only) for the final 13 weeks of the study. With the exception of the findings of ulcerations at the application site, there were no treatment-related differences in clinical observations among any of the groups. Treatment did not affect survival, body weights, or food consumption. The major microscopic findings were observed at the application site. At the interim (Week 53) sacrifice, these findings consisted of hyperkeratosis (1, 5, and 25% benzoyl peroxide), subepidermal subacute inflammation (5 and 25% benzoyl peroxide), and sebaceous gland hyperplasia (males: 5 and 25% benzoyl peroxide: females:1, 5, and 25% benzoyl peroxide). These findings were dose-dependent with regards to incidence and or group mean severity. Similar findings were noted at the terminal (Week 105-106) sacrifice with the exception that there were no findings for treated or untreated skin in the high-dose and high-dose-discontinued animals. The high-dose-discontinued animals, after being treated with 25% benzoyl peroxide for 1 year, were allowed 52 weeks of recovery. Although the high-dose animals were not originally intended to be a recovery group, they were net treated with benzoyl peroxide for the final 13 weeks of the study since the 25 -15% benzoyl peroxide concentrations had been found to exceed the maximum tolerated dose. In both of these cases there was no residual effect of treatment.

In conclusion, under the conditions of this study, there were no histologic findings indicative of toxicity resulting from daily topical exposure of mice to benzoyl peroxide gels at concentrations up to 25% (dose level ca. 833 mg/kg bw/d in males and ca. 1000 mg/kg bw/d in females)).The NOAEC for the dermal irritation was 0.17 mg/cm² (1%).

Justification for classification or non-classification

According to EU Regulation (EC) N0. 1272/2008 (CLP), the substance is not classified for repeated dose toxicity.