National Toxicology Program

V. TOXICOLOGICAL EFFECTS

A. Chemical Disposition

1. Human Data

dermal, human

• The penetration of isoeugenol through human epidermis has been studied. The lower abdominal epidermal skin from human cadavers was dosed with 10 mM carbon-14 labelled isoeugenol (specific activity of 10 mCi/ml) in various test vehicles for 24 hours. The vehicles tested were 5 liquid vehicles (ethanol, ethanol/water (85:15 v/v), ethanol/water (70:30 v/v), propylene glycol, and liquid paraffin), 6 cosmetic vehicles (lotion, milky lotion, o/w-type cream, w/o-type cream, o/w-type foundation, and oil type foundation (See Tables 3-8 [Table 3. Lotion Composition, Table 4. Milky Lotion Composition, Table 5. O/W type Cream Composition, Table 6 W/O Type Cream Composition, Table 7. O/W type Foundation Composition, Table 8. Oil type Foundation Composition] for the chemical composition for each of these vehicles); and 2 ointment vehicles (white petrolatum and macrogol ointment). For the liquid vehicles, 0.2 ml of each sample was applied. For the 5 cosmetic and 2 ointment vehicles, the test sample volume was 0.2 cm³. The percentage of penetration in liquid vehicles ranged from 0.29 ±0.04% (ethanol) to 4.31 ± 0.84% (liquid paraffin). The percent penetration was significantly greater (P<0.01) from liquid paraffin compared to the other 4 vehicles. The penetration of each compound showed a tendency to increase in direct proportion to the ratio of water to ethanol.

  The percentage of penetration in cosmetic and ointment vehicles ranged from 0.05 ± 0.01% (macrogol ointment) to 10.38 ± 0.63% (milky lotion). The percent penetration of isoeugenol from milky lotion was significantly (P<0.01) greater than from the other 7 cosmetic/ointment vehicles. The authors reported that the increased penetration afforded by this vehicle may be due to the water content of milky lotion [Jimbo et al., 1983].
  

2. Animal Data

No data were found on the chemical disposition of isoeugenol in animals.

B. Acute

1. Human Data

dermal, human

• Fifty adult male volunteers with no known allergies were subjected to dermal patch testing with isoeugenol in order to investigate the irritation potency of this compound. Lint patches with 0.05 grams of 32% isoeugenol in acetone were placed on the skin of the volunteers' backs for 48 hours. Based on readings at 48, 72, 96 and 120 hours, the irritation potential of isoeugenol was determined to be moderate [Motoyoshi et al., 1979].

2. Animal Data

oral, rats

• The acute LD₅₀ of isoeugenol was determined using groups of 10 (5 male, 5 female per dose level) adult Osborne-Mendel rats. Unspecified doses of neat isoeugenol were administered to the rats by stomach tube after 18 hours of fasting. The LD₅₀ for isoeugenol was determined to be 1560 (range 1290-1880) mg/kg. Prior to death, the rats appeared scrawny after one dose and became comatose. Death occurred within 1 hour to 7 days of administration [Jenner et al., 1964].

• A group of 6 (3 male, 3 female) adult Osborne-Mendel or Sherman rats was used to determine the acute hepatoxicity of isoeugenol. The rats were administered a dose of 520 mg/kg (1/3 the LD₅₀) via stomach tube daily for 4 days. On the fifth day, 5 of the 6 rats (one died prior to the fifth day) were sacrificed to determine whether any macroscopic liver lesions occurred. No macroscopic liver lesions were observed in the rats [Taylor et al., 1964].

• The acute oral LD₅₀ of isoeugenol for an unspecified strain and number of male and female guinea pigs was determined. Guinea pigs were given unspecified doses of isoeugenol by intubation after 18 hours of fasting. The LD₅₀ for isoeugenol was determined to be 1410 (range 1130-1780) mg/kg. Prior to death, the guinea pigs were observed to be depressed and became comatose. Death occurred within 3 to 6 days [Jenner et al., 1964].

• The acute oral LD₅₀ in quail, and the repellency-toxicity index (R₅₀), which is analogous to an LD₅₀ for redwing blackbirds, was determined for isoeugenol. An unspecified amount of isoeugenol suspended in propylene glycol was administered to an unspecified number of birds (redwing blackbirds and coturnix quail) via gavage. The LD₅₀ for isoeugenol in quail was determined to be greater than 316 mg/kg. The R₅₀ for redwing blackbirds was found to be greater than 1.00 percent [Schafer et al., 1983].

• Ten female Hartley albino guinea pigs were used to study the irritant potential of isoeugenol. The flank areas of the guinea pigs were shaved, and a dose of 20 mg of 0.2, 0.5, 1.0, 2.0, 5.0, and 10.0% isoeugenol in petrolatum was applied (in Finn chambers on Scanpor tape) for 48 hours. The degree of reaction was evaluated at 1, 24, and 48 hours after the isoeugenol was removed. The results indicated that isoeugenol produced an irritant response at concentrations of 5.0 and 10.0% only [Itoh, 1982].

• A group of 6 male Hartley guinea pigs were clipped in 2 areas (3x3 cm) on their backs. A dose of 0.1 grams of isoeugenol (100%) was applied to one of the areas, while the second area remained untreated. The irritation potential was read following 24 hours of contact with isoeugenol. Another application of isoeugenol was administered 30 minutes later. Every 24 hours the irritation potential was read, and a 0.1 gram dose of isoeugenol (100%) was applied (total of 3 applications, or 0.3 grams of isoeugenol). After 72 hours the animals were sacrificed and a thorough histopathological examination of the skin was performed to determine the relative irritancy. Isoeugenol was observed to be severely irritating to guinea pig skin [Motoyoshi et al., 1979].

dermal, rabbits

• The dorsal fur of 6 albino angora rabbits was clipped (3x3 cm areas). A dose of 0.1 grams of isoeugenol (100%) was applied to one of the exposed areas, and a second exposed area was treated with N-hexadecane (positive control). A third area was left untreated. The irritation potential was read following contact with isoeugenol for 24 hours, and another application was administered every 24 hours (for a total of 3 applications or 0.3 grams isoeugenol). After 72 hours, the animals were sacrificed, and the total irritation potential was scored. Isoeugenol was found to be severely irritating to rabbit skin upon histopathological examination [Motoyoshi et al., 1979].

dermal, miniature swine

• Six Pittman-Moore Improved miniature swine were used to study isoeugenol-induced dermal irritation. The hair on the swines' backs was clipped, and a dose of 0.05 grams of isoeugenol (100%) was applied under a 15 mm diameter patch. The patches were secured for a 48-hour exposure period. After 48 hours the patches were removed, and the total irritation potential was scored. The results indicated that isoeugenol does not induce dermal irritation in swine [Motoyoshi et al., 1979].

C. Prechronic

1. Human Data

dermal, human

• Standard patch testing and photopatch testing were performed on a 40-year-old rockblaster of 10 years, who suddenly developed a severe itchy, confluent vesiculo-bullous reaction on his hands while working with dinitrotoluene. He was patch tested in duplicate using Finn Chambers technique with the ICDRG standard test series, including isoeugenol (2% in petrolatum), for 48 hours. One of the test areas was subsequently irradiated with 5 joules of ultraviolet light, and the results were read 48 hours after illumination. The individual exhibited a severe contact sensitivity reaction to isoeugenol. However, the case subject did not exhibit a photocontact allergy to this compound [Emtestam and Forbeck, 1985].

• Standard patch testing was performed on a 46-year-old female cookie handler who presented with a 2-week history of eczema of her hands from handling "Thin Mint" cookies which developed after the employee's duties were changed to include handling the "Thin Mint" line. After 48 hours, the woman reportedly had a positive reaction to an unspecified amount of isoeugenol, one of the compounds included in the standard patch test series. She did not exhibit a positive reaction to peppermint oil which was originally suspected to be the primary irritant. The authors indicated that she was able to handle other cookies without problems, and postulated that the concentration of the sensitizing components may be higher in the mint cookies than in other cookies [Spencer and Fowler, 1988].
• Standard patch testing was performed on a 47-year-old female pharmacist who had developed constant rhinitis and mild dermatitis around her eyes which had persisted for 18 months. She had daily contact with coal tar and bitter almond oil. Before the appearance of the dermatitis around her eyes she had used crayons on her eyelids. She tested strongly positive to isoeugenol, although she had no occupational exposure to pure isoeugenol [Rudzki and Grzywa, 1977].

Many other patch test studies have been performed to determine the incidence of contact allergy to isoeugenol. Table 9 summarizes the results of these patch tests.

• Table 10 represents the compilation of results from patch testing collected by members of the Soap and Detergent Association (SDA). The results of the survey indicate that isoeugenol has a very low potential for either eliciting pre-existing sensitization reactions or inducing hypersensitivity in subjects exposed to isoeugenol-containing consumer products. Five case subjects exhibited an induced reaction following multiple exposures. Data on the sensitization reactions in these 5 subjects are presented in Table 11 [Thompson et al., 1983].

2. Animal Data

dermal, guinea pigs

• The sensitization potency of isoeugenol was studied using 10 female Hartley albino guinea pigs. Fifty mg of a 10% solution of isoeugenol in petrolatum was applied (via a cloth on Torri's patch plaster) to a shaved portion of the nape of the animals' necks. The isoeugenol was removed after a 48-hour exposure. This procedure was repeated 3 times a week for 2 weeks. After the 2-week induction period, the guinea pigs' flanks were shaved, and isoeugenol was applied at concentrations of 0.1% and 1.0% for 48 hours. The degree of reaction was read 1, 24, 48 hours, and 1 week after the isoeugenol was removed. The results indicated that 40% (8/20) and 80% (16/20) of the animals exhibited sensitization to 0.1% and 1.0% isoeugenol, respectively, 72 hours after application [Itoh, 1982].

D. Chronic/Carcinogenicity

1. Human Data

No data were found in the literature on carcinogenicity or other chronic effects in humans.

2. Animal Data

No data were found in the literature on carcinogenicity or other chronic effects in animals.

E. Reproductive Effects and Teratogenicity

1. Human Data

No data were found in the literature on reproductive effects and teratogenicity in humans.

2. Animal Data

No data were found in the literature on reproductive effects and teratogenicity in animals.

F. Genetic Toxicology

1. Human Data

The following study concerning the genetic toxicology of isoeugenol in humans has been described. No other data were found in the literature.
in vitro, humans

• Blood was collected from healthy, non-smoking volunteers for an in vitro study of the effects of cigarette smoke condensate upon sister-chromatid exchanges (SCE) in human lymphocytes. The weakly acidic semivolatile (WASV) fraction of cigarette smoke condensate, which was prepared using nonfiltered American blend cigarettes, was separated by preparative gel chromatography in to eleven subfractions. The chemical composition of these subfractions was determined, and the effects of the subfractions on SCE in cultured lymphocytes were investigated. Isoeugenol was found in subfraction F8, which contained as its main components phenol, 3-methylphenol, and 4-methylphenol.
  

  The F8 fraction was found to increase significantly SCE induction in human lymphocytes (P<0.01). However, this effect was more significant in the other fractions tested. The effects of the individual components of Fraction 8 on SCE induction were also determined. The SCE induction ability of isoeugenol was found to be moderate at condensate concentrations of 0.25 mM (P<0.01) and 0.5 mM (P<0.001) [Jansson et al., 1986].

2. Prokaryotic Data

Salmonella typhimurium

• Isoeugenol was tested in a preincubation modification of the standard Ames test in Salmonella typhimurium TA1535, TA1537, TA98, TA100, and TA97 with and without Aroclor 1254-induced rat and hamster metabolic activation. This compound was found to be non-mutagenic at all concentrations tested (3.3, 10.0, 33.0, 100.0, 250.0, 333.0, and 800.0 _g/plate) [Mortelmans et al., 1986].

• In the standard Ames test, isoeugenol was tested for mutagenicity in Salmonella typhimurium strains TA100, TA1535, TA98, TA1537, and TA1538 in the presence and absence of metabolic activation. Isoeugenol was tested at doses of 60.0, 120.0, 300.0 and 600.0 _g/plate. Isoeugenol was observed to be non-mutagenic in all of the Salmonella strains tested [Sekizawa and Shibamoto, 1982].

• Mutagenicity tests were performed on Salmonella typhimurium strains TA1535, TA100, TA1537, TA1538, and TA98 in the presence and absence of metabolic activation. Isoeugenol was non-mutagenic in these strains of Salmonella at a concentration of 0.8 mg per plate [Nestmann, et al, 1980].

• In the Ames test, the mutagenic effects of isoeugenol were tested in Salmonella typhimurium strains TA98, TA100, TA1535, and TA1537 in the presence and absence of S9 liver metabolic activation. Isoeugenol was observed to be non-mutagenic at a concentration of 3.0 mmol/plate in all of the Salmonella strains tested [Florin et al., 1980]

• In the Ames test, the mutagenic effects of isoeugenol were tested in Salmonella typhimurium strains TA100, TA98, TA1535, TA1537, and TA1538 with metabolic activation. The tests were conducted at dose levels of 2.0, 20.0, and 200.0 mg/plate isoeugenol. Isoeugenol was observed to be non-mutagenic at all of the doses tested [Hsia et al., 1979].

• The urinary metabolites of isoeugenol were tested for mutagenicity in the Ames test using Salmonella typhimurium TA98 and TA100 in the presence of metabolic activation. To obtain the isoeugenol urinary metabolites, 2 Sprague-Dawley rats were administered a 0.5 ml dose of isoeugenol via a gastric tube. The urine was then collected for the next 24 hours and sterilized. Two ml of urine was set aside for a direct urine assay. The remainder of the urine sample was diluted with a phosphate buffer at pH 7.0, and incubated with a beta-glucuronidase preparation for 4 hours. The incubation sample was then extracted with ether. The ether extract, aqueous fraction, direct urine, and control urine samples were used for the assays. No mutagenic activity was observed in Salmonella from the direct urine or from the aqueous fractions of the ether extractions [Rockwell and Raw, 1979].

• Mutagenicity tests were performed on Escherichia coli WP2- uvrA, trp¯ with and without S9 liver metabolic activation. Isoeugenol was tested at doses of 60.0, 120.0, 300.0, and 600.0 mg/plate. No mutagenic effects were observed [Sekizawa and Shibamoto, 1982].

• The SOS-inducing potency of 5.0 mM isoeugenol was tested in Escherichia coli strain PQ37 following nitrosation in a reaction mixture containing 10% ethanol. Isoeugenol was not found to be genotoxic in this test system in the absence of metabolic activation [Ohshima et al., 1989].

• In the DNA-repair test (rec assay) the mutagenic effects of isoeugenol were tested in Bacillus subtilis strains H17 Rec+ and M45 Rec-, without metabolic activation. Isoeugenol was tested at a dose of 0.8 mg/disk and was found to be genotoxic in this assay [Sekizawa and Shibamoto, 1982].

• In the DNA-repair test, isoeugenol was observed to be non-mutagenic in Bacillus subtilis strains H17 Rec+ and M45 Rec- at a dose of 22.0 mg/disk [Oda et al., 1978].

3. Eukaryotic Data

Chinese hamster ovary cells

• The effect of isoeugenol on SCE induction was tested in vitro at concentrations of 0, 10.0, 33.3, 100.0, and 333.0 mM in cultured Chinese hamster ovary cells (CHO K-1) with and without mitomycin C. Isoeugenol was found to be toxic at 333.0 mM. Isoeugenol did not have any influence on the cell cycle, nor did it have any effect upon SCE induction at any concentration tested [Sasaki et al., 1989].

• Isoeugenol was tested at an unspecified concentration for mutagenic effects in Saccharomyces cerevisiae strains D7 and XV185-14C without S9 metabolic activation. Isoeugenol did not induce tryptophan gene convertants in strain D7 or reversion of the histidine, homoserine, or tryptophan markers in strain XV185-14C, and was determined to be non-mutagenic in this test system [Nestmann and Lee, 1983].

G. Other Toxicological Effects

1. Immunotoxicity

a. Human Data

No data were found in the literature on immunotoxicity in humans.

b. Animal Data

dermal, mice

• Isoeugenol was applied to the backs of CBA/Ca mouse ears at concentrations of 0, 5, 10, and 25% for 3 consecutive days. The auricular (draining) lymph nodes were removed and weighed. The number of pyroninophilic cells were counted and lymphocyte proliferation was also determined, with and without interleukin-2. Isoeugenol (5, 10 and 25%) was found to elicit an immunological response in the draining lymph nodes which was characterized by lymphocyte proliferation [Kimber and Weisenberger, 1989].

2. Neurotoxicity

No data were found in the literature on neurotoxicity in humans or animals.

3. Biochemical Toxicology

a. Human Data

No data were found in the literature on the biochemical toxicology of isoeugenol in humans.

b. Animal Data

in vitro, rats, guinea pigs

• The UDP-Glucuronosyltransferase (UDPGT) activities of hydroxy-coumarins, monoterpenoid alcohols, and alkylphenols, including isoeugenol, were measured in liver microsomes from Wistar rats induced by either phenobarbital or 3-methylcholanthrene, or in non-induced controls which received sodium chloride or oil only. The UDPGT activity of 0.25 mM isoeugenol was 1.5 times the control value, in the presence of phenobarbital, and 2.39 times the control value in the presence of 3-methylcholanthrene.

The UDPGT activity of isoeugenol and the effect of induction by phenobarbital were also studied using Gunn rats, which have partially reduced UDPGT activity and completely lack UDPGT-bilirubin activity. The UDPGT activity of 0.25 mM isoeugenol was induced in the presence of phenobarbital to 3.84 times control value.

The UDPGT activity of isoeugenol was also studied in the presence and absence of phenobarbital using guinea pigs of unspecified strain. The UDPGT activity of 0.175 mM isoeugenol was 1.6 times control value in the presence of phenobarbital [Boutin et al., 1985].

4. Cytotoxicity

in vitro, humans

• Cultured human diploid embryonic lung fibroblasts (line MRC-5) were used to determine the damaging effects of tobacco smoke components, including isoeugenol. A dose of 25.0 mM isoeugenol was added to the cells for a 30-minute incubation period. The severity of damage to the plasma membrane was determined by the percentage of nucleotides released from the plasma membrane: high (>70%), moderate (70-48%), and no damage (<15%) nucleotide release. The study indicated that the plasma membrane was severely damaged by isoeugenol based on a 90% nucleotide release [Thelestam et al., 1980].
  

• The relative cytotoxicity of isoeugenol was determined by compiling the data from cytotoxicity studies involving the following 4 parameters: inhibition of cell growth, inhibition of oxidative metabolism, plasma membrane damage, and ciliotoxicity.

  Isoeugenol's ability to inhibit cell growth (CG) was determined using ascites sarcoma BP 8 cells. A dose of 1.0 mM of isoeugenol was added to the BP 8 cells and incubated for 48 hours. The rate of growth was then compared to a control to determine isoeugenol's ability to inhibit growth and expressed as a percentage. Isoeugenol was found to be a strong cell growth inhibitor (See A^CG, Table 12).
  

  Brown fat cells from hamsters were used to determine the ability of isoeugenol to inhibit oxidative metabolism (OM). The brown fat cells were treated with 0.6 _M norepinephrine to increase the rate of oxygen consumption. A dose of 1.0 mM of isoeugenol was added to the fat cells and incubated for 5 minutes. The toxicity was determined by comparing the norepinephrine-induced oxygen consumption, after addition of isoeugenol, to that of the control and expressing the ratio as a percentage. Isoeugenol was found to strongly inhibit oxidative metabolism (See A^OM, Table 12).
  

  The ability of isoeugenol to induce plasma membrane damage (MD) was determined by the percentage of cytoplasmic nucleotide leakage. Isoeugenol was added as a 25.0 mM solution in either ethanol or dimethyl sulfoxide (author did not specify which of these solvents was used) to cultured human diploid embryonic lung fibroblasts (line MRC-5) and incubated for a period of 30 minutes. Isoeugenol was found to have a strong damaging effect on plasma membranes (See A^MD, Table 12).
  

  The ability of isoeugenol to cause ciliostasis in embryo chicken trachea was used to measure ciliary activity (CA). A dose of 5.0 mM isoeugenol was added to the cultured trachea, and the time required for ciliostasis was recorded. The ciliotoxic effect was expressed as a percentage using time in minutes to ciliostasis when occurring within 60 minutes. Isoeugenol was determined to be a strong ciliary inhibitor (see A^CA, Table 12).
  The relative toxicity (AT) of isoeugenol was determined to be high (see Table 12). This value, as well as the individual cytotoxicity values are expressed on a 10-point scale (0: 0-9%, 1: 10-19%, 2: 20-29%, 3: 30-39%, 4: 40-49%, 5: 50-59%, 6: 60-69%, 7: 70-79%, 8: 80-89%, 9: 90-100%) [Curvall, et al., 1984].

in vitro, chickens

• The length of time required for ciliostasis in cultured embryo chicken trachea was measured to determine the ciliotoxicity of isoeugenol. A single dose of 5.0 mM isoeugenol was added to the culture and the time required for ciliostasis was reported. The study indicated that isoeugenol is a strong inhibitor of ciliary movement because ciliostasis occurred 6 minutes after dosing [Pettersson et al., 1982].