Page 1 of 42 UNITED STATES ENVIRONMENTAL PROTECTION AGENCY WASHINGTON, D. C. 20460 OFFICE OF PREVENTION, PESTICIDES, AND TOXIC SUBSTANCES TXR Number:

MEMORANDUM DATE: July 6, 2004 SUBJECT: PHENOL/ SODIUM PHENATE: Toxicology Chapter for the AD Preliminary Risk Assessment Document. PC Code: 064001, 064002. DP Barcode: . Submission Number: .

FROM: Michelle M. Centra, Pharmacologist Regulatory Management Branch II Antimicrobials Division ( 7510C)

THROUGH: Timothy F. McMahon, Ph. D. Senior Toxicologist Antimicrobials Division ( 7510C)

TO: Najm Shamim, Ph. D. Science Coordinator Antimicrobials Division ( 7510C)

and Connie Welch, Branch Chief Ben Chambliss, Team Leader Antimicrobials Division ( 7510C)

Attached is the toxicology chapter for Phenol Reregistration Eligibility Decision ( RED).

The following supporting documents were used to generate the toxicology chapter of the Reregistration Eligibility Decision ( RED) for phenol:

1. Toxicity Profile of Phenol/ Sodium Phenate 2. USEPA. Toxicological Review of Phenol. Integrated Risk Information System ( IRIS). September, 2002. 3. Phenol Report of the Antimicrobials Division Toxicology End point Selection Committee ( ADTC). Page 2 of 42 PHENOL/ SODIUM PHENATE Reregistration Case Number: 4074 PC Code: 064001, 064002 Toxicology Disciplinary Chapter for the Reregistration Eligibility Decision Document Date completed: June 30, 2004 Contract Number: 68 W 01 036 Prepared for Antimicrobials Division Office of Pesticide Programs U. S. Environmental Protection Agency Arlington, VA 22202 Prepared by Samantha J. Jones, Ph. D., Toxicologist Versar, On Site Contractor for Versar 6850 Versar Center P, O. Box 1549 Springfield, VA 22151 Principal Reviewer _________________________________ Date ________________ Samantha J. Jones, Ph. D.

Disclaimer This data Summary may have been altered by the Antimicrobials Division subsequent to signing by Versar personnel.

form: FINAL June 21, 2000 Page 3 of 42 EPA Reviewer: Michelle M. Centra, Pharmacologist Signature: _______________________ Regulatory Management Branch II Date: ______________________ Antimicrobials Division 1.0 HAZARD CHARACTERIZATION Phenol ( hydroxybenzene, carbolic acid, phenyl hydroxide), is registered with the Office of Pesticides Programs ( OPP) as an active ingredient and used as an intermediate in the production of epoxy resins and various other products, as a general disinfectant, and in medicinal preparations. It is used to control Animal Pathogenic Bacteria ( G and G+ Vegetative), Pseudomonas SPP., Mycobacterium SPP. ( Tubercle Bacilli), Animal Pathogenic Fungi, Hydrophilic Viruses, Poliovirus Type 1, Parvovirus, Lipophilic Viruses, Vaccinia Virus, Influenza A2 ( Hong Kong, Japan, Japan 305/ 57 Asian Strain), HIV I ( Human Immunodeficiency Virus), and mold/ mildew. Phenol is manufactered by Sporicidin International and DR. Novis Smith & Company, Incorporated.

Formulated as a pressurized and ready to use liquid, phenol is registered for use as a sanitizer, bacteriostat, fungicide/ fungistat, tuberculocide, disinfectant, and virucide ( EPA Reg. No. 707 159). Phenol has a number of use sites including indoor food uses in eating establishments on equipment and utensils, non food indoor uses in commercial transportation facilities, institutional/ industrial floors, industrial premises/ equipment, laundry equipment, paints, latex, and specialty industrial products. Indoor residential uses of phenol encompass the bathroom premises, hard surfaces, diaper pails, dogs/ canines, household/ domestic dwellings, and solid waste containers ( garbage cans). Phenol also has indoor, medical uses on surgical instruments and pacemakers ( critical items), catheters and inhalation equipment ( semi critical items), bedpans and furniture ( noncritical items), non conductive floors, critical premises ( burn wards), noncritical premises, patient premises, and institution premises ( human/ veterinary). Additionally, phenol is used in aquatic non food residential areas for swimming pool water systems.

Phenol is produced through both natural and anthropogenic processes. It is naturally occurring in some foods, in human and animal wastes, and in decomposing organic material, and it is produced endogenously in the gut from the metabolism of aromatic amino acids. Phenol has been isolated from coal tar, but is now synthetically manufactured. Currently, the largest use of phenol is as an intermediate in the production of phenolic resins, which are used in the plywood, adhesive, construction, automotive, and appliance industries. Phenol is also used in the production of synthetic fibers such as nylon and for epoxy resin precursors such as bisphenol A. Phenol is toxic to bacteria and fungi, and it is used as a slimicide and disinfectant. Because of its anesthetic effects, phenol is used in medicines such as ointments, ear and nose drops, cold sore lotions, throat lozenges and sprays ( such as those sold under the Cepastat ® and Chloraseptic ® labels), and antiseptic lotions.

The acute toxicity database for phenol technical is considered complete. No additional studies are required at this time. Phenol has a moderate order of acute toxicity via the oral and dermal routes of exposure ( Toxicity Category II or III) and produces severe and marked irritation to the eyes and skin ( Toxicity Category I or II). Phenol concentrations used in acute inhalation studies failed to induce mortality in the study animals; therefore, toxicity endpoints and a toxicity category Page 4 of 42 could not be established. The acute toxicity data for phenol is summarized below in Table 2.

In the subchronic database, oral range finding studies in both the rat and the mouse were completed. Although the studies were non guideline ( due to the lack of subchronic parameters), systemic toxicities were noted at 10000 ppm in the rat and mouse, based on a decrease in mean body weight gain. In a two week inhalation study, rats had elevated plasma Mg2+ levels ( Hypermagnesaemia) and exhibited toxic effects in the central nervous system ( CNS).

Phenol was administered in two developmental guideline studies in the rat and mouse at concentrations of 30, 60, or 120 mg/ kg/ day and 70, 140, or 280 mg/ kg/ day, respectively. There was no evidence of toxicity in these animals at concentrations below the high dose. Fetal body weight was significantly reduced at 120 and 280 mg/ kg/ day in both rat and mouse studies. Additionally, female mice experienced increased mortality and clinical signs of CNS toxicity ( tremors, ataxia, lethargy) at the high dose ( 280 mg/ kg/ day). In a non guideline developmental study ( Kavlock, 1990), there were decreases in rat maternal body weight gain in maternal and offspring In a two generational reproductive study in rats exposed to 200, 1000, or 5000 ppm phenol in drinking water for 10 weeks/ generation, there were decreases in water and food consumption, body weight and body weight gain at the high dose ( potential reduced palatability). Offspring toxic effects including decreases in body weight and litter survival were observed at 5000. This occurred concurrently with maternal toxicity ( decreased maternal body weight); believed to be secondary to the animals' aversion to the flavor of the phenol treated water and resulted in decreased maternal as well as offspring body weight. In a non guideline reproductive study ( Bishop, et al. 1997) phenol was administered to mice at a concentration of 350 mg/ kg. There were no treatment related clinical signs or mortality observed in maternal, reproductive, and developmental parameters and the LOAEL was not established ( highest dose tested, 350 mg/ kg).

Two carcinogenicity studies performed by the National Cancer Institute did not exhibit an incidence of neoplasms in male and female mice or rats following administration of phenol, with the exception of a statistically significant increase in the occurrence of leukemia, lymphoma, or interstitial cell tumors in low dose male rats. Due to the lack of significant tumors in high dose males and the absence of significant neoplasms in mice and female rats, phenol was found to be non carcinogenic in the 2 year drinking water studies. Although phenol treated rats and mice experienced a decrease in mean body weight and body weight gain, the reduction was not significantly different from the respective controls and chronic toxicity was not observed at phenol concentrations up to 5000 ppm. A 20 week dermal study exhibited effects of chronic irritation and hair growth inhibition with administration of 3 mg phenol ( in 200 uL acetone). A single papilloma was found 7 weeks into the study, but there was no evidence that it was significantly increased or treatment related. In a special, mechanistic study there was no evidence of tumor initiation or hepatocyte GSH depletion following administration of 100 mg/ kg/ day phenol.

Phenol is characterized as Group D; the not classified compounds for which there is inadequate or no human or animal evidence of carcinogenicity. The updated toxicological review in the EPA IRIS database ( USEPA, 2002) provides a summary of the weight of the evidence with respect to the carcinogenic potential of phenol. Page 5 of 42 The results of the mutagenicity studies indicated that phenol was not mutagenic in Salmonella typhimurium or Drosophila melanogaster and did not induce micronuclei or bone marrow chromosomal aberrations in mice. However, mutagenic effects were observed in Chinese Hamster Ovary cells and spermatocytes in mice and HL60 cells. The genotoxic potential of phenol appears to depend on the competing processes of activation to a genotoxic form and metabolic inactivation ( e. g., via conjugation). Phenol tended to be negative in bacterial gene mutation assays but was positive or equivocal in mammalian cell gene mutation assays. Phenol tended to induce micronuclei in mice when administered intraperitoneally but was negative ( or positive only at very high doses) when administered orally. This difference is likely due to the first pass conjugation and inactivation of orally administered phenol. Phenol was also positive in in vitro micronucleus tests with human lymphocytes and Chinese Hamster Ovary cells. Results from DNA damage assays are inconsistent, but they tend to show that phenol can cause sister chromatid exchanges or cell transformation if it is not metabolically inactivated. Overall, phenol did not exhibit strong mutagenic effects.

The ADTC committee noted neurotoxic signs from acute dermal toxicity studies ( Brown, et al.  convulsions; OTS 0515567 tremors; Conning, et al. stimulation of motor nerve endings or spinal motor centers) and a 15 day inhalation study ( tilting plane results showed an effect in treated rats). There were no neurotoxic signs of phenol noted from the oral studies using gavage or drinking water as the method of administration. As noted in the IRIS Toxicological profile, numerous CNS effects have been observed following phenol dosing. Tremors were observed in one animal that later died ( apparently of dehydration) following dosing in drinking water ( ClinTrials BioResearch, 1998). Tremors have also been observed in several gavage studies in rats and mice ( 43735402; Dow Chemical Co., 1994; Moser et al., 1995). However, in a specialized 13 week neurotoxicity study in male and female rats that included an FOB and a detailed neurohistopathology evaluation ( ClinTrials BioResearch, 1998), the only observed nervous system effects were tremors in one animal and decreased motor activity in females. A short term gavage screening study ( Moser, et al. 1995) found that the only effect in an FOB was a marginal decrease in motor activity and increased rearing post exposure. Part of this could be attributed to the dehydration observed in the study.

Phenol is essentially completely metabolized in 24 hours. Phenol was predominantly conjugated with sulfate and lower amounts of glucuronic acid and the metabolites were rapidly excreted in the urine. The major metabolites found are phenyl sulfate, quinol sulfate, phenyl glucuronide, and quinol glucuronide.

Phenol is readily absorbed by the inhalation, oral, and dermal routes. The portal of entry metabolism for the inhalation and oral routes appears to be extensive and involves sulfate and glucuronide conjugation and, to a lesser extent, oxidation. Once absorbed, phenol is widely distributed in the body, although the levels in the lung, liver, and kidney are often reported as being higher than in other tissues ( on a per gram tissue basis). Elimination from the body is rapid, primarily as sulfate and glucuronide conjugates in the urine, regardless of the route of administration. Phenol does not appear to accumulate significantly in the body. Page 6 of 42 2.0 REQUIREMENTS Table 1. Requirements ( CFR 158.340) for Food/ Feed Use for Phenol/ Sodium Phenate Guideline Number and Toxicity Study Required Satisfied 870.1100 Acute Oral Toxicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 870.1200 Acute Dermal Toxicity . . . . . . . . . . . . . . . . . . . . . . . . . . . 870.1300 Acute Inhalation Toxicity . . . . . . . . . . . . . . . . . . . . . . . . . 870.2400 Primary Eye Irritation . . . . . . . . . . . . . . . . . . . . . . . . . . . . 870.2500 Primary Dermal Irritation . . . . . . . . . . . . . . . . . . . . . . . . . 870.2600 Dermal Sensitization . . . . . . . . . . . . . . . . . . . . . . . . . . . . yes yes yes yes yes yes yes yes yes yes yes yes 870.3100 Oral Subchronic ( Rodent) . . . . . . . . . . . . . . . . . . . . . . . . . 870.3150 Oral Subchronic ( Non Rodent) . . . . . . . . . . . . . . . . . . . . . 870.3200 21 Day Dermal . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 870.3250 90 Day Dermal . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . None 28 Day Inhalation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . yes yes yes yes yes yes yes yes yes yes 870.3700 Developmental Toxicity ( Rodent) . . . . . . . . . . . . . . . . . . 870.3700 Developmental Toxicity ( Non rodent) . . . . . . . . . . . . . . . 870.3800 Reproduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . yes yes yes yes yes yes 870.4100 Chronic Toxicity ( Rodent) . . . . . . . . . . . . . . . . . . . . . . . . 870.4100 Chronic Toxicity ( Non rodent) . . . . . . . . . . . . . . . . . . . . . 870.4200 Oncogenicity ( Rat) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 870.4200 Oncogenicity ( Mouse) . . . . . . . . . . . . . . . . . . . . . . . . . . . 870.4300 Chronic/ Oncogenicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . yes yes yes yes yes yes yes yes yes yes 870.5100 Mutagenicity   Gene Mutation  bacterial . . . . . . . . . . . . . 870.5300 Mutagenicity   Gene Mutation  mammalian . . . . . . . . . . 870.5375 Mutagenicity   Structural Chromosomal Aberrations . . . . 870.5385 Mutagenicity   Structural Chromosomal Aberrations . . . . 870.5500 Mutagenicity   Other Genotoxic Effects . . . . . . . . . . . . . . 870.5550 Mutagenicity   Other Genotoxic Effects . . . . . . . . . . . . . . yes yes yes yes yes yes yes yes yes yes yes yes 870.6100 Acute Delayed Neurotox. ( Hen) . . . . . . . . . . . . . . . . . . . . 870.6100 90 Day Neurotoxicity Hen) . . . . . . . . . . . . . . . . . . . . . . . 870.6200 Acute Neurotox. Screening Battery ( Rat) . . . . . . . . . . . . . 870.6200 90 Day Neuro. Screening Battery ( Rat) . . . . . . . . . . . . . . . 870.6300 Develop. Neuro . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . no no no no no

870.7485 General Metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 870.7600 Dermal Penetration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . yes yes yes yes 870.7200 Companion Animal Safety . . . . . . . . . . . . . . . . . . . . . . . . no no 3.0 DATA GAPS Based on the availability of the recent EPA IRIS report, in addition to open literature data, the ADTC considered the toxicity database for phenol adequate. Therefore, no data gaps were identified for the hazard characterization of phenol.

4.0 HAZARD ASSESSMENT 4.1 Acute Toxicity Page 7 of 42 Adequacy of database for Acute Toxicity: The acute toxicity database for phenol is considered complete. No additional studies are required at this time. Phenol has a moderate order of acute toxicity via the oral and dermal routes of exposure ( Toxicity Category II or III) and produces severe and marked irritation to the eyes and skin ( Toxicity Category I or II). Phenol concentrations used in acute inhalation studies failed to induce mortality in the study animals; therefore, toxicity endpoints and a toxicity category could not be established. The acute toxicity data for phenol is summarized below in Table 2.

Table 2. Acute Toxicity Profile for Phenol/ Sodium Phenate Guideline Number Study Type/ Test substance (% a. i.) MRID Number/ Citation Results Toxicity Category 870.1100 ( § 81 1) Acute Oral Rat Phenol purity > 99% Berman, et al. 1994 LD50 = 400 ( 297 539) mg/ kg/ day II 870.110 ( § 81 1) Acute Oral  Rat Phenol purity 100% OTS #  0515567 86 870001405 LD50 = 1030 ( 940 1120) mg/ kg/ day III 870.1100 ( § 81 1) Acute Oral Rat Phenol purity not reported Flickinger. 1976 LD50 = 650 ( 490 860) mg/ kg/ day III 870.1200 ( § 81 2) Acute Dermal Rat Phenol purity not reported Brown, et al. 1975 LD50 ( Non occluded) = 0.68 ( 0.57 0.78) mL/ kg LD50 ( Occluded) = 0.50 mL/ kg II 870.1200 ( § 81 2) Acute Dermal Rabbit Sodium Phenate purity 57% OTS #  0515564 86 870001402 LD50 = 2350 ( 1880 2940) mg/ kg/ day III 870.1200 ( § 81 2) Acute Dermal Rabbit Phenol purity 100% OTS #  0515567 86 870001405 LD50 = 0.63 ( 0.56 0.70) mL/ kg II 870.1200 ( § 81 2) Acute Dermal Rat Phenol purity laboratory reagent grade Conning, et al. 1970 LD50 = 669.4 mg/ kg/ day II 870.1200 ( § 81 2) Acute Dermal Rabbit Phenol purity not reported Flickinger. 1976 LD50 = 850 ( 600 1200) mg/ kg/ day II 870.1300 ( § 81 3) Acute Inhalation Rat Phenol purity 100% OTS #  0515567 86 870001405 No deaths occurred at 2.5 L/ min for 8 hours Not established 870.1300 ( § 81 3) Acute Inhalation Rat Phenol purity not reported Flickinger. 1976 No deaths occurred at 900 mg/ m3 for 8 hours Irritation and timerelated CNS effects Not established 870.2400 ( § 81 4) Acute Eye Irritation Rabbit Sodium Phenate purity 57% OTS #  0515564 86 870001402 15% solution caused corneal necrosis and conjunctiva lesions II 870.2400 ( § 81 4) Acute Eye Irritation Rabbit Phenol purity 100% OTS #  0515567 86 870001405 Severe damage to the cornea at 15% and lesser damage in 5% Not established Table 2. Acute Toxicity Profile for Phenol/ Sodium Phenate Guideline Number Study Type/ Test substance (% a. i.) MRID Number/ Citation Results Toxicity Category Page 8 of 42 870.2400 ( § 81 4) Acute Eye Irritation Rabbit Phenol purity not reported Flickinger. 1976 Dose not provided. Severe conjunctiva, iritis, corneal opacities and ulcerations with no improvement after 14 day observation period. I 870.2500 ( § 81 5) Acute Dermal Irritation Rabbit Sodium Phenate purity 57% OTS #  0515564 86 870001402 Mild to marked erythema and marked capillary injection were observed in 50% of animals tested II 870.2500 ( § 81 5) Acute Dermal Irritation Rabbit Phenol purity 100% OTS #  0515567 86 870001405 10% solution caused moderate to marked erythema Not established 870.2500 ( § 81 5) Acute Dermal Irritation Rabbit Phenol purity not reported Flickinger. 1976 Corrosive I 4.2 Subchronic Toxicity Adequacy of database for Subchronic Toxicity: The database for subchronic toxicity is considered complete. Oral range finding studies in both the rat and the mouse were completed. Although the studies were non guideline ( due to the lack of subchronic parameters), systemic toxicities were noted at 10000 ppm in the rat and mouse, based on a decrease in mean body weight gain. In a two week inhalation study, rats had elevated plasma Mg2+ levels ( Hypermagnesaemia) and exhibited toxic effects in the central nervous system ( CNS).

Non Guideline Subchronic ( Oral) Range Finding Toxicity Rat In a range finding subchronic 90 day oral toxicity study ( NIH PB# 80 1759), groups of 10 F344 rats/ sex/ dose received 0, 100, 300, 1000, 3000, or 10000 ppm phenol ( purity not reported) in drinking water for 13 weeks. There were no treatment related clinical signs or increases in mortality ( 100% survival) in rats. At necropsy no tissue, organ, or histomorphologic alterations were associated with administration of phenol. Rats experienced a significant depression in mean body weight and body weight gain when compared to control. Animals administered phenol at concentrations of 3000 ppm or less exhibited weight gains which were similar to control; indicating that animals were unaffected by treatment. However, the 10000 ppm dose group exhibited decreases in mean body weight that were 16 and 26% less than the controls in male and female treated rats, respectively ( animals weighed in week 12). There were no changes observed in feed consumption following phenol administration. Water was rejected by the rats at the highest concentration of phenol ( 10000 ppm); males and females consumed 50 and 67%, respectively, of control water levels. Considering the test article was administered in the drinking water, palatability at the high dose might have affected the water consumption. This resulted in depressed water intake in the subchronic studies for both male and female rats. The low and high doses for the chronic study were set at 2500 and 5000 ppm, respectively, based on the rejection of water and unacceptable decreases in mean weight gain at the 10000 ppm dose level. The Subchronic toxicity NOAEL is 3000 ppm. The Subchronic toxicity LOAEL is 10000 ppm, based on significant decreases in mean body weight gain. Page 9 of 42 Non Guideline Subchronic ( Oral) Range Finding Toxicity Mouse In a range finding subchronic 90 day oral toxicity study ( NIH PB# 80 1759), groups of 10 B6C3F 1 mice/ sex/ dose received 0, 100, 300, 1000, 3000, or 10000 ppm phenol ( purity not reported) in drinking water for 13 weeks. There were no treatment related clinical signs or increases in mortality ( 100% survival) in mice. Animal mean body weight and body weight gain were unaffected by treatment with all phenol concentrations except at 10000 ppm. The high dose treated mice exhibited decreased weight gain that was 80% less than control for males and 33% less for females. The weight loss that resulted in depressed mean body weights for both male and female mice was observed during the first 7 weeks of treatment, although the mice exhibited small weight gains for the remainder of the study. Feed consumption for treated males and females was unaffected by phenol but water consumption at 10000 ppm was 60 and 20% of controls for males and females, respectively. Considering phenol was administered in the drinking water, palatability at the high dose might have affected the water consumption; resulting in the depressed water intake observed in the subchronic studies for both male and female mice. The range finding study results indicated the dose levels for the carcinogenicity study. The high and low doses were set at 2500 and 5000 ppm, respectively, based on animal rejection of the water and unacceptable decreases in mean weight gain at the 10000 ppm dose level. The Subchronic toxicity NOAEL is 3000 ppm. The Subchronic toxicity LOAEL is 10000 ppm, based on significant decreases in mean body weight gain.

Non Guideline Two Week Inhalation Toxicity ( Special Study) Rat In an inhalation study ( Dalin and Kristoffersson, 1984), rats were exposed to phenol ( purity not reported) in an inhalation chamber ( air flow, 600 ± 10 L/ hr) at a concentration of 100 mg/ m3 for 15 days. General activity, behavior, and locomotion were noted frequently throughout the exposure period. Immediately prior to and following the experiment CNS effects were studied with the " tilting plane" method ( based on the ability of the animal to cling to a tilted plane, measured in degrees of an angle). There were treatment related effects observed in the animal activity one day after the start of exposure. On days 3 and 4, rats exhibited clear motor disorders, such as impaired balance and disordered walking rhythm. Additionally, sitting positions were labile, grooming behavior was disturbed, and involuntary neck twitches were observed. All of these symptoms were relatively mild and from the external appearance of the rats the animals appeared to be in good condition. By day 5 of exposure the above symptoms faded and animals appeared sluggish in behavior when compared to controls. There were no treatment related effects in food and water consumption or body weight in animals exposed to phenol. However, the CNS effects measured by the " tilting plane" method showed significant decreases in the value of the sliding angle after exposure to phenol. The mean sliding angle was 71.2 ± 2.4 degrees prior to phenol administration and following treatment there was a significant decrease ( 6%) to 66.8 ± 1.7 degrees. Hemoglobin, hematocrit, plasma Na+, Ca2+, Cl, and free phenol levels were not affected by treatment with phenol. However, K+ and Mg2+ levels were significantly increased with 1.3 and 1.2 fold increases, respectively, after phenol exposure. The activities of plasma enzymes LDH, GOT, GPT, and GLDH were markedly greater in the phenol treated animals with 5.0, 6.5 , 2.8, and 2.2 fold increases, respectively, over controls. Rats exposed to 100 mg/ m3 phenol showed excitement with typical muscular twitching and later depression. Hypermagnesaemia has been shown to block neuromuscular transmission and diminish the responsiveness of the muscle fibers to direct stimulation. Therefore, the elevated plasma Mg2+ levels observed in phenol treated animals would be an important factor in the depression and weakening of motor functions also noted in rats. Plasma K+ levels were increased significantly and this elevation may be a sign of damage to liver tissue. In this study, plasma enzyme values ( LDH, GOT, GPT, and GLDH) were significantly elevated as in the " poisoning pattern" noted for organic solvents ( LDH> GOT> GPT> GLDH). This pattern indicates that the phenol concentration used in this study ( 100 mg/ m3) is sufficient in inducing liver damage in rats. Page 10 of 42 4.3 Prenatal Developmental Toxicity Adequacy of database for Prenatal Developmental Toxicity: The database for developmental toxicity is considered complete. Phenol was administered in two guideline studies in the rat and mouse at concentrations of 30, 60, or 120 mg/ kg/ day and 70, 140, or 280 mg/ kg/ day, respectively. There was no evidence of toxicity in these animals at concentrations below the highdose Fetal body weight was significantly reduced at 120 and 280 mg/ kg/ day in rat and mouse studies, respectively. Additionally, female mice experienced increased mortality, and clinical signs of CNS toxicity ( tremors, ataxia, lethargy) at the high dose ( 280 mg/ kg/ day). In a non guideline developmental study ( Kavlock, 1990), there were decreases in maternal body weight gain in maternal and offspring 870.3700 Prenatal Developmental Toxicity Study  Rat In a prenatal developmental toxicity study ( MRID # 43735402) 92 prenatally exposed mated female CD rats, 23 rats/ dose were administered phenol ( 99.9% purity) via gavage at dose levels of 0, 30, 60, or 120 mg/ kg/ day from gestation days ( GD) 6 to 15. Females were weighed daily during treatment and observed for clinical signs of toxicity. A total of 20 23 females/ group were confirmed to be pregnant at sacrifice on GD 20. The gravid uterus of each dam was weighed and the urine contents examined for implantation sites and fetus vitality ( live, dead, or reabsorbed). Each live fetus was weighed and examined for external, visceral, and skeletal malformations. There were no treatment related clinical signs, increases in mortality ( 100% survival), or decreases in body weight and body weight gain in rats dosed with phenol. The Maternal toxicity NOAEL is greater than or equal to 120 mg/ kg/ day ( highest dose tested) and the Maternal toxicity LOAEL is greater than 120 mg/ kg/ day ( not established). Treated animals exhibited no change in reproductive parameters when compared to controls; therefore, the Reproductive toxicity NOAEL is greater than or equal to 120 mg/ kg/ day ( highest dose tested) and the Reproductive toxicity LOAEL is greater than 120 mg/ kg/ day ( not established). There were no treatmentrelated effects on mean live fetal body weight/ litter in the low and mid dose treated groups. However, significant reductions from the control in mean fetal body weight/ litter was observed in the high dose ( 120 mg/ kg/ day) group. No evidence of teratogenicity was observed in the rats following administration of phenol and the Developmental toxicity NOAEL is 60 mg/ kg/ day. The Developmental toxicity LOAEL is 120 mg/ kg/ day, based on reduced fetal weight. This study is classified as Acceptable and satisfies the guideline requirements.

In an unpublished developmental toxicity study conducted according to GLP guidelines ( Argus Research Laboratories, 1997), pregnant Crl: CDRBR VAF/ Plus Sprague Dawley rats ( 25 per group) received phenol by oral gavage on GD 6 through 15. Animals were dosed 3 times/ day at a concentration of 0, 20, 40, or 120 mg phenol/ kg using a dosing volume of 10 mL/ kg. The corresponding daily doses were 0, 60, 120, and 360 mg/ kg/ day. It was noted that the test material was 90% phenol United States Pharmacopeia ( USP); the dosage calculations were adjusted for test material purity. The exposed dams were observed twice a day for viability and daily for clinical signs, abortions, and premature deliveries. In addition, the maternal body weights were recorded every day, and food consumption was also recorded periodically ( every 1 B 2 days). Rats were sacrificed on GD 20, and gross necropsy of the thoracic, abdominal, and pelvic viscera was performed. The number of corpora lutea in each ovary was recorded. The uterus of each rat was excised and examined for number and distribution of implantations, live and dead fetuses, and early and late resorption. Each fetus was weighed, sexed, and examined for gross external alterations. One half of the fetuses were examined for soft tissue alterations, and the rest were examined for skeletal alterations. Page 11 of 42 One high dose dam died on GD 11. This death was attributed to phenol treatment because it occurred only at the high dose, although there were no adverse clinical observations and no abnormal necropsy findings in this animal. Other high dose animals exhibited excess salivation and tachypnea ( rapid breathing). There were no other treatment related clinical observations and no treatment related necropsy findings. Dose dependent decreases in body weight of the exposed animals were observed. Statistically significant decreases from control in both maternal body weight ( 8%) and body weight gain ( 38% for GD 6 B 16) were observed at the high dose; although a statistically significant decrease in body weight gain ( 11%) was observed at the mid dose, the decrease at the mid dose ( relative to controls) in absolute maternal weight at the end of dosing ( 3%) was not statistically significant. Dose dependent decreases in food consumption were also observed during the dosing period. Fetal body weights in the high dose group were significantly lower than those of the controls, by 5 B 7%. The high dose group had a statistically significant decrease in ossification sites on the hindlimb metatarsals, but it is unlikely that this small change is biologically significant. The incidence of litters with incompletely ossified or unossified sternal centra was 0/ 23, 0/ 25, 3/ 23, and 3/ 24; this increase was not statistically significant. There were small, dose related increases in the number of litters with fetuses with " any alteration" and with " any variation" at 120 mg/ kg/ day and higher. However, neither of these changes was statistically significant, and the response was not clearly dose related. In addition, an increase in total variations is of questionable significance in the absence of any increase in individual variations. There were no other treatment related effects on uterine contents, malformations, or variations. The maternal NOAEL was 60 mg/ kg day, based on small decreases in maternal body weight gain at 120 mg/ kg day, and the developmental NOAEL was 120 mg/ kg day, based on decreased fetal body weight and delayed ossification at 360 mg/ kg day.

870.3700 Prenatal Developmental Toxicity  Mouse In a prenatal developmental toxicity study ( MRID # 43735401) 133 prenatally exposed mated female CD 1 mice, 31 36 animals/ dose were administered phenol ( 99.9% purity) by gavage at dose levels of 0, 70, 140, or 280 mg/ kg/ day from gestation days ( GD) 6 to 15. Females were weighed daily during treatment and observed for clinical signs of toxicity. Animals were sacrificed on GD 17. The gravid uterus of each dam was weighed and the uterine contents examined for implantation sites and fetus vitality ( live, dead, or reabsorbed). Each live fetus was weighed and examined for external, visceral, and skeletal malformations. There were no treatment related clinical signs or mortality observed in the maternal low dose group ( 70 mg/ kg/ day). At the 140 mg/ kg/ day dose, 1/ 31 animals died ( 3.2% mortality). Mild tremors were observed in mid dose females after dosing on GD 6 8; however, this effect was not observed on subsequent treatment days. A mortality rate of 14.3% ( 5/ 35 animals) was noted in dams of the 280 mg/ kg/ day dose group. Clinical signs of toxicity were observed in these mice following administration of phenol and included tremors, ataxia, lethargy, and irritability. The mortality in mid and high dose treated females was attributed to a dosing error. There was a significant treatment related decrease ( 10% at sacrifice, GD 17) in mean maternal body weight of the 280 mg/ kg/ day treated animals when compared to the control group. Mean maternal body weight gain was statistically reduced from the control in the 280 mg/ kg/ day dose group during the treatment period ( 31% decrease) and entire gestation period ( 28% decrease). Dams administered the phenol high dose exhibited a 66% decrease from control in absolute weight gain. The Maternal toxicity NOAEL is 140 mg/ kg/ day. The Maternal toxicity LOAEL is 280 mg/ kg/ day, based on increased mortality and clinical signs of CNS toxicity ( tremors, ataxia, and lethargy). There were no changes in reproductive parameters in treated animals when compared to controls. The Reproductive toxicity NOAEL is greater than or equal to 280 mg/ kg/ day ( highest dose tested). The Reproductive toxicity LOAEL is greater than 280 mg/ kg/ day ( not established). The low dose and mid dose groups ( 70 and 140 mg/ kg/ day) did not exhibit fetotoxicity or teratogenicity. At 280 mg/ kg/ day, an increase in cleft palate was observed in 8 of 214 fetuses ( 3 litters), although this increase was not statistically significant. Phenol was fetotoxic in both males and females of the Page 12 of 42 high dose ( 280 mg/ kg/ day) group with significant reductions ( 18%) from control in the mean fetal weight of treated animals. The Developmental toxicity NOAEL is 140 mg/ kg/ day. The Developmental toxicity LOAEL is 280 mg/ kg/ day, based on reduced fetal weight and an increase in the incidence of cleft palate. This study is classified as Unacceptable, although upgradable with the addition of on food consumption data, gross necropsy findings, complete developmental endpoints, and individual animal data.

Non Guideline Developmental Toxicity  Rat In a prenatal developmental ( gavage) toxicity study ( Narotsky and Kavlock 1995) phenol ( 99+% purity) was administered once daily from gestation days ( GD) 6 19 for a total exposure period of 14 days to groups of 17 prenatally exposed Fischer 344 rats/ dose at concentrations of 40 or 53.3 mg/ kg/ day. Dams were observed throughout pregnancy. Litters were examined postnatally and after final examination dams were sacrificed and uterine implantation sites were counted. Pups, 14 live litters/ dose, were maintained for 21 days. There were no treatment related effects in mortality ( 100 survival) of rats receiving phenol for 14 days. Clinical signs of toxicity following administration of both dose levels of phenol were observed and included altered respiration ( e. g., rales and dyspnea). Maternal weight loss was similar to control levels after two treatments but significantly reduced weight gains were observed after 4 and 10 treatments between GD 6 20. Maternal body weight gain was reduced 21 and 24% from controls in animals treated with 40.0 and 53.3 mg/ kg/ day phenol, respectively. The Maternal toxicity NOAEL is less than 40 mg/ kg/ day ( lowest dose tested). The Maternal toxicity LOAEL is 40 mg/ kg/ day, based on reduced body weight, body weight gain, and respiratory distress ( rales and dyspnea). One lowand two high dose animals experienced full resorption resulting in significantly reduced litter sizes and a marginally significant increase in prenatal loss in the high dose group. The three dams with resorbed litters had severe respiratory signs and an additional high dose female also had severe respiratory signs with excessive perinatal mortality in its litter. The high dose dam had markedly reduced pup weights on postnatal day ( PND) 1 resulting in a marginally significant reduction for the group. Kinked tails were observed in 2 of the 4 survivors in this litter. The Reproductive toxicity NOAEL is less than 40 mg/ kgday ( lowest dose tested). The Reproductive toxicity LOAEL is 40 mg/ kg/ day, based on increased incidence of resorption and significantly reduced litter size. The percentage loss of prenatal litters was 4, 13, and 22% for 0, 40, and 53.3 mg/ kg/ day, respectively. Developmental effects were evident only in litters with dams exhibiting severe respiratory signs, other females with similar respiratory effects successfully maintained apparently normal litters. The developmental effects of phenol were isolated to four litters: Three ( one from low dose group) were fully resorbed and the fourth exhibited high perinatal mortality. Postnatal loss was minimal with a small increase, less than 5%, at the high dose. Changes in pup weights were minimal and did not vary significantly from control levels. Kinked tails were also noted in one litter at 53.3 mg/ kg/ day. The Developmental toxicity NOAEL is less than 40 mg/ kg/ day ( lowest dose tested). The Developmental toxicity LOAEL is 40 mg/ kg/ day, based on increased incidence of perinatal loss.

4.4 Reproductive Toxicity Adequacy of database for Reproductive Toxicity: The database for reproductive toxicity is considered complete. No additional studies are required at this time. In a two generational reproductive study in rats exposed to 200, 1000, or 5000 ppm phenol for 10 weeks/ generation, there were decreases in water and food consumption, body weight and body weight gain at the high dose ( potential reduced palatibility). Offspring toxic effects including decreases in body weight and litter survival were observed at 5000. This occurred concurrently with maternal toxicity ( decreased maternal body weight); believed to be secondary to the animals' aversion to the flavor of the phenol treated water and resulted in decreased maternal as well as offspring body Page 13 of 42 weight. In a non guideline reproductive study ( Bishop, et al. 1997) phenol was administered to mice at a concentration of 350 mg/ kg. There were no treatment related clinical signs or mortality observed in maternal, reproductive, and developmental parameters and the LOAEL was not established ( highest dose tested, 350 mg/ kg).

870.3800 Reproduction and Fertility Effects  Rat In a reproductive and fertility effects toxicity study ( Ryan, et al. 2001) phenol ( 100% purity) was administered to groups of 30 Sprague Dawley rats/ sex/ dose in drinking water at concentrations of 200, 1000, or 5000 ppm ( 14, 70, and 310 mg/ kg/ day for males and 20, 93, and 350 mg/ kg/ day for females, respectively, for both generations). The parental ( P1) generation was treated 10 weeks prior to mating, during mating ( 2 weeks), gestation, lactation, and until sacrifice. Females were allowed to go through natural parturition. The offspring of P1 was weaned on PND 22 and at least 1 rat/ sex/ litter was selected to receive phenol for 11 weeks prior to mating. These F1 pairs were treated in a similar regimen as the P1. The F2 generation was culled to 4 pups/ sex/ litter on PND 4, although phenol was not administered. After weaning, all of the F2 pups and F1 sires and dams were sacrificed. All animals were examined daily during gestation and weekly thereafter. At least 20 rats/ sex/ group in the P1 and F1 generations were subjected to a gross necropsy and tissue examination, while the F2 animals were euthanized and discarded without necropsy. There were 3 mortalities in the P1 generation male rats ( 1 control died spontaneously prior to cohabitation and 2 high dose died accidentally during bleeding and from a bladder infection during cohabitation), although these were not considered to be treatment related. Reduced palatability of the phenol treated drinking water was likely the cause of death in three high dose pups of the F1 generation. Three F1 dams, 1 each from the control, low dose, and high dose groups died spontaneously, but not considered treatment related. Minimal signs of clinical toxicity ( discolored or wet inguinal fur and redness around the nose or eyes) were observed in cases of mortality. These signs were observed in all groups; however, the incidence was higher in the phenol treated rats.

There were no treatment related effects observed for food and water consumption, body weight, and organ weight in the 200 and 1000 ppm dose groups. However, high dose animals treated with 5000 ppm phenol exhibited significant changes from control. Males exhibited a 23% decrease in water consumption in weeks 1 and 10, while females exhibited reductions of 32 and 37% in weeks 1 and 10, respectively. The decrease in water consumption at the high dose may have been a result of reduced palatability of the treated drinking water. Parental food consumption was significantly reduced 10 and 12% from control in males ( at week 1) and females ( at weeks 1 and 2), respectively. There were treatment related decreases from control in body weight and body weight gain in P1 generation rats treated with phenol. These reductions were concomitant with decreases in food and water consumption and observed in the high dose rats. Male body weights were 8% less than controls in week 10 while the female body weights were 7 and 10% less than controls at weeks 1 and 10, respectively. Body weight gain reductions were also significant decreased with reductions of 12 and 29% for male and female rats, respectively. Following treatment with 5000 ppm phenol, the absolute organ weight of male seminal vesicles was 12% less than control. Similarly, the absolute organ weights of adrenals, brain, ovaries, and spleen in the females were reduced 16, 5, 18, and 13 %, respectively. Animal final body weights were significantly reduced from controls in the high dose group with decreases of 9 and 12% for male and female rats, respectively.

In the dams there were no treatment related effects on body weight gain during the lactation phase of the study. However, throughout gestation and lactation the mean maternal body weight was significantly lower in the high dose animals compared to controls. Mean body weight gains were approximately 10, 12, and 11% less than controls for the pre mating, gestation, and lactation Page 14 of 42 periods, respectively. There were treatment related decreases from controls in F1 body weight in the 5000 ppm phenol treated rats. The statistically significant dose dependent decreases from controls were 5, 15, 18, 20, and 29% on PND 0, 4, 7, 14, and 21, respectively. These decreases occurred concomitantly with the maternal toxicity ( decrease in maternal body weight). The lower maternal body weight may have contributed to the lower birth weight of F1 generation as a result of the decreased food and water consumption during lactation and decreased palatability. The Maternal Toxicity NOAEL is 1000 ppm in the P1 generation. The Maternal Toxicity LOAEL is 5000 ppm, based on decreases in water and food consumption, body weight and body weight gain in the P1 generations. These effects are associated with flavor aversion to phenol in the drinking water.

There were no treatment related effects on reproductive performance in either generation ( P1 and F1). The estrus cycle, epididymal sperm count, motility, sperm morphology, testicular sperm count, and production rate were unaffected by phenol treatment in the P1 and F1 generations. However, testicular sperm count and production rate were significantly increased from control in the high dose group of the F1 generation. This was ascribed to lower testis weight rather than to a true alteration in sperm count or production, based on the lack of effects at the mid dose level. The Reproductive Toxicity NOAEL is greater than or equal to 5000 ppm in the P1 and F1 generations ( highest dose tested). The Reproductive Toxicity LOAEL is greater than 5000 ppm in the P1 and F1 generations ( not established).

The daily consumption of food and water was decreased from controls for the F1 generation highdose group. Food and water consumption declines were concomitant with reductions in body weight. Body weight gain was significantly decreased in the 5000 ppm phenol treated group at the end of the pre mating phase for males, whereas in females it was only reduced during gestation ( data not included). However, the percent of offspring alive after PND 0 were significantly decreased in the 5000 ppm treated groups with a 10% decrease on PND 4 for the P1 generation and decreases of 28 and 24% on PND 4 and 7 21, respectively, for the F1 generation. After culling, the percent survival was similar across all groups. The percent survival was similarly affected in the F2 generation; however, survival in the high dose group remained affected after culling and was reduced on PND 7 21. The decreased survival of the high dose treated group was likely a result of the maternal toxicity ( decreased body weight and water consumption) that occurred at this dose.

The F1 generation experienced significant decreases in absolute organ weights at all three dose levels. The absolute weight of the prostate in males was significantly less than the control in the low, mid, and high dose groups with decreases of 15, 17, and 16%, respectively. There were significant reductions at the mid and high dose treated males with absolute organ weight decreases of 10 and 16% for adrenals and 13 and 20% for spleen, respectively. The remaining organs experienced absolute organ weights significantly decreased from controls in the high dose group only. Final body weights of the F1 generation were significantly reduced from controls in the high dose group with decreases of 18 and 11% for male and female rats, respectively. The F2 generation pre weaning growth pattern was similar to the F1 generation with significant decreases of 7, 20, 20, 21, and 28% in litter body weight from control on PND ), 4, 7, 14, and 21, respectively. Similarly to F1 generation, the decreased maternal body weight ( F1 dams) and reduced palatability were likely significant factors that contributed to the decreased body weights in the high dose F2 generation. There were treatment related effects for both F1 and F2 generations with increases in litter mortality ( more so in the F2 generation) and reduced offspring body weights in the high dose group. This occurred concurrently with maternal toxicity ( decreased maternal body weight); believed to be secondary to the animals' aversion to the flavor of the phenol treated water and resulted in decreased maternal as well as offspring body weight. There were delays in vaginal patency of F1 females ( 38.3 days for treated females vs. 34.6 days for control females) and preputial separation of F1 males ( 47.8 days for treated males vs. 44 days for Page 15 of 42 control males) observed with decreases in pre and post weaning body weights in the high dose group. Therefore, the onset of puberty was delayed and attributed to decreased food and water consumption and reduced body weight. The Offspring Toxicity NOAEL is 1000 ppm. The Offspring Toxicity LOAEL is 5000 ppm based on decreases in body weight of F1 and F2 offspring, decreases in litter survival of P1 and F1 offspring, and delays in preputial separation in F1 males and vaginal patency in F1 females.

4.5 Chronic Toxicity Adequacy of database for Chronic Toxicity: The database for chronic phenol toxicity in rats and mice is presented in the NIH PB# 80 1759 study of the carcinogenicity database.

4.6 Carcinogenicity Adequacy of database for Carcinogenicity: The database for carcinogenicity is considered complete. No additional studies are required at this time. The two carcinogenicity studies performed by the National Cancer Institute produced no incidences of neoplasms in male and female mice or rats following administration of phenol, with the exception of a statistically significant increase in the occurrence of leukemia, lymphoma, or interstitial cell tumors in lowdose male rats. Due to the lack of significant tumors in high dose males, females, and mice, phenol was found to be non carcinogenic in the 2 year drinking water studies. Although phenoltreated rats and mice experienced a decrease in mean body weight and body weight gain, the reduction was not significantly different from the respective controls and there was no chronic toxicity at concentrations up to 5000 ppm. A 20 week dermal study exhibited effects of chronic irritation and hair growth inhibition with administration of 3 mg phenol ( in 200 uL acetone). A single papilloma was found 7 weeks into the study, but there was no evidence that it was significantly increased or treatment related. In a special, mechanistic study there was no evidence of tumor initiation or hepatocyte GSH depletion following administration of 100 mg/ kg/ day phenol.

870.4200 Carcinogenicity  Rat In a 24 month carcinogenicity study ( NIH PB# 80 1759), groups of 50 F344 rats/ sex/ dose were administered 0, 2500, or 5000 ppm phenol ( 98.47% purity, lot # 79380) in the drinking water for 103 weeks. There were treatment related increases in mortality in rats and survival rates, calculated over 105 weeks ( 103 weeks of study plus 2 week acclimation period), were 52, 44, and 60% ( males) and 76, 78, and 74% ( females) for controls, low dose, and high dose animals, respectively. A positive dose related trend in mortality in either sex was not significant according to the Tarone test. Food consumption was unaffected by treatment, but dose related decreases in water consumption were 80 ( 2500 ppm) and 90% ( 5000 ppm) of controls. Considering the test article was administered in the drinking water, reduced palatability may account for decreased consumption of water. There were no other treatment related clinical signs related to the consumption of phenol in drinking water. Male and female treated rats exhibited decreased mean body weight and body weight gain when compared to controls in both high and low dose groups. This deviation from the controls was observed for most of the study and tended to increase with time. Maximum differences in mean body weight between treated and control animals occurred at week 96 for male rats with decreases of 9 and 15% for low and high dose groups, respectively. Treated females exhibited a maximum weight difference at week 96; low and high dose animal weights were 7 and 9% lower than controls, respectively. The mean body weight changes observed in treated rats increased over time with greater deviations from the controls following administration of 5000 ppm ( high dose) of phenol. At study termination, body weight gains were Page 16 of 42 7 and 13% lower than control for treated males and 3 and 11% lower than control for treated females for low and high doses, respectively. The toxicity NOAEL is greater than or equal to 5000 ppm ( highest dose tested) for both male and female rats. The toxicity LOAEL is greater than 5000 ppm ( not established).

Various neoplasms were observed in both control and treated rats. The low dose male rats exhibited increased tumor occurrence over controls. Pheochromocytomas of the adrenal medulla were found in 44% of low dose males; significantly higher ( p= 0.046) than the 26% in controls and 18% in high dose males. The incidence of either leukemia or lymphomas in high and lowdose males were higher than controls, but were only significantly higher ( p= 0.008) in low dose treated. The incidence of interstitial cell tumors in the testis of males is also significantly higher ( p= 0.05) in low dose males than control; found in 49 of the 50 animals. Results of histopathologic examinations suggest phenol may have increased the incidence of pheochromocytoma, leukemia or lymphoma in low dose male rats. Females did not exhibit increased incidences of tumors at any time in this study. No significant tumors could be clearly associated with the administration of phenol due to the lack of significant tumors in high dose males and absence of any significant neoplasms in females as well as the high spontaneous tumor rate observed in matched controls. Phenol was not carcinogenic for either male or female F344 rats in this 2 year carcinogenicity drinking water study.

870.4200 Carcinogenicity  Mouse In a 24 month carcinogenicity study ( NIH PB# 80 1759), groups of 50 B6C3F1 mice/ sex/ dose received 0, 2500, or 5000 ppm phenol ( 98.47% purity, lot # 79380; purity not reported, lots # A4X and B4A) in drinking water for 103 weeks. There was a treatment related increase in mortality in mice and the rate of survival was calculated for the entire study of 103 weeks and included a 2 week acclimation period at the beginning of study for a total of 105 weeks. Survival rates were 84, 90, and 96% ( males) and 82, 80, and 84% ( females) for controls, low dose, and high dose, respectively. Other than a reduced tendency to fight observed among treated male mice at the beginning of week 80 there were no treatment related clinical signs from the consumption of phenol in drinking water. Food consumption was unaffected by treatment while water consumption of treated animals was 75% ( 2500 ppm) and 50 60% ( 5000 ppm) of controls. Considering phenol was administered in the drinking water, reduced palatability may account for decreased consumption of water. Male and female treated mice exhibited decreased mean body weights when compared to controls in both high and low dose groups. This deviation from the controls was observed for most of the study and tended to increase with time. Body weights varied between treated and control animals as early as 2 weeks ( approximately 1 g variation) into the study although maximum differences were not observed until week 100 for males, with decreases from control of 13 and 18% for low and high dose treated mice, respectively. The maximum variation for females between treated and control animals occurred at week 85 in which decreases of 8 and 12% from control were observed in low and high dose groups, respectively. At the conclusion of the study, body weights for treated males were 3 ( low dose) and 9% ( highdose less than control while treated females were 6 and 10% less than control at the low and high doses, respectively. The toxicity NOAEL is greater than or equal to 5000 ppm ( highest dose tested) for male and female mice. The toxicity LOAEL is greater than 5000 ppm ( not established).

Neoplasms observed in treated animals were of the usual number and type found in mice. Uterine endometrial stromal polyps were increased in 5 of 48 high dose female mice, although this was not significantly different from similar historical control mice. Any other neoplasms noted were occurrences normally associated with aged B6C3F 1 mice and were not treatment related. Results of the histopathologic examinations suggest phenol was not toxic or carcinogenic to B6C3F 1 mice under the conditions of this 2 year drinking water carcinogenic bioassay. No tumor at any site in Page 17 of 42 the mice could be clearly associated with the administration of phenol in this study. Non Guideline  Mechanistic Study  Mouse In a mechanistic study ( Stenius, et al. 1989) phenol (> 99.57% purity) was administered to 10 partially ( 70%) hepatectomized male Sprague Dawley rats by gavage 5 days/ week for 7 weeks at a concentration of 100 mg/ kg/ day. Animals were sacrificed, by decapitation, 1 week after last treatment. Sections of the rat liver were prepared and stained to measure for induction of   glutamyltranspeptidase ( GGT) positive enzyme altered foci as an indicator of tumor initiation. Additional studies involved single oral administrations of phenol to measure inductions of hepatic ornithine decarboxylase ( ODC), glutathione ( GSH) depletion, and in vivo lipid peroxidation. Phenol did not increase the number or volume of foci and was found to have no tumor initiating properties within the confines of this study. Lipid peroxidation was not induced following administration of phenol as measured by malondialdehyde ( MDA) in the urine. There were small and inconsistent effects observed in hepatic ODC in which there was an increase at the mid dose, but a decrease at the high dose. Observed measurements of hepatic ODC were 18.8, 32.3, and 11.4 pmol/ mg/ h for the phenol doses of 0, 50, and 100 mg/ kg/ day, respectively. Phenol did not induce GSH depletion in hepatocytes. Phenol is not a potent stimulator of foci development and can be regarded as a negative control in this study because it is not susceptible to oxidationreduction reactions. The Subchronic toxicity NOAEL is greater than or equal to 100 mg/ kg/ day ( highest dose tested). The Subchronic toxicity LOAEL is greater than 100 mg/ kg/ day ( not established).

4.7 Mutagenicity Mutagenicity studies were from open literature studies, with the exception of one gene mutation toxicology study ( Malcolm, et al. 1985; Acceptable Guideline). The results of these studies indicate that phenol was not mutagenic in Salmonella typhimurium or Drosophila melanogaster and did not induce micronuclei or bone marrow chromosomal aberrations in mice. However, mutagenic effects were observed in Chinese Hamster Ovary cells and spermatocytes in mice and HL60 cells. The genotoxic potential of phenol appears to depend on the competing processes of activation to a genotoxic form and metabolic inactivation ( e. g., via conjugation). Phenol tended to be negative in bacterial gene mutation assays but was positive or equivocal in mammalian cell gene mutation assays. Phenol tended to induce micronuclei in mice when administered intraperitoneally but was negative ( or positive only at very high doses) when administered orally. This difference is likely due to the first pass conjugation and inactivation of orally administered phenol. Phenol was also positive in in vitro micronucleus tests with human lymphocytes and CHO cells. Results from DNA damage assays are inconsistent, but they tend to show that phenol can cause sister chromatid exchanges or cell transformation if it is not metabolically inactivated. Overall, phenol did not exhibit strong mutagenic effects.

Bacterial Reverse Mutation Test in Salmonella typhimurium Mutagenicity Assay; OPPTS 870.5100 [ § 84 2]. In a microbial reverse gene mutation assay ( Florin, et al. 1980) Salmonella typimurium strains TA 98, TA 100, TA 1535, and TA 1537 were exposed to one dose of phenol ( purity not reported) at a concentration of 3 umol/ plate in the presence and absence of metabolic activation ( derived from aroclor or methylcholanthrene induced rats). The mutagenic response was verified with positive controls, N methyl N' nitro N nitrosoguanisine for S9 and 2 aminoanthracene for S9+ experiments. Positive controls elicited the appropriate responses in the corresponding assays; however, there was no evidence that phenol induced a mutagenic effect in any strain with or without metabolic.

Bacterial Reverse Mutation Test in Salmonella typhimurium Mutagenicity Assay; OPPTS 870.5100 [ § 84 2]. In a bacterial reverse mutation assay ( Haworth, et al. 1983) Salmonella Page 18 of 42 typhimurium strains TA 98, TA 100, TA 1535, and TA 1537 were exposed to phenol ( 99.5% purity) at concentrations of 0, 33, 100, 333, 1000, 2500, or 3333

g/ plate in the presence and absence of metabolic activation from aroclor induced rats and hamsters. Concurrent positive controls included 2 aminoanthracene ( 2 AA) for S9+ and for S9 4 nitro o phenylenediamine ( NOPD), sodium azide ( SA), and 9 aminoacridine ( 9 AAD). There was no evidence of induced mutant colonies over background. Positive controls produced appropriate responses in corresponding strains of the bacterial reverse mutagenesis test. S. typhimurium did not show mutagenic activity in the presence or absence of metabolic activation following administration of phenol.

Bacterial Reverse Mutation Test in Salmonella typhimurium Mutagenicity Assay; OPPTS 870.5100 [ § 84 2]. In a bacterial reverse mutation assay ( Pool and Lin, 1982) histidine requiring mutants of Salmonella typhimurium bacterial strains, TA 98, TA 100, TA 1535, TA 1537, and TA 1538, were exposed to phenol ( 98% or analytical grade purity) at concentrations of 0.5, 5, 50, 500, or 5000 µ g/ plate. Phenol was administered to 4 plates/ dose in the presence and absence of metabolic activation derived from aroclor induced Sprague Dawley rats. Positive controls, 2 µ g/ plate sodium azide ( TA 100, TA 1535), 3 µ g/ plate 2 Nitrofluorene ( TA 98, TA 1538), and 60 µ g/ plate 9 aminoacridine ( TA 1537) were used in the absence of metabolic activation while 5 µ g/ plate 2 aminoanthracene served for all experiments performed in the presence of metabolic activation. A treatment related toxicity effect was observed at the highest dose, 5000 µ g/ plate, as was indicated by the thinning of the bacterial background lawn and a reduced number of spontaneous revertants ( 11 100% decreases in mutant colonies in presence and absence of S9). For the remaining test doses, phenol did not significantly induce mutant colonies over background. The number of histidine revertants scored in the presence of phenol never more than slightly exceeded the number of spontaneously arising revertants. A small, dose related increase in mutant colonies was observed in the TA 100  S9 experiment with the non cytotoxic doses of phenol ( 11 14% increase for phenol concentrations ranging from 0.5 to 500 µ g/ plate); however, this increase never reached a doubling of the number of spontaneous revertants and was not significant. There was no evidence of mutagenic activity following administration of phenol to 5 bacterial strains of S. typhimurium in the presence or absence of metabolic activation.

Bacterial Reverse Mutation Test in Salmonella typhimurium Mutagenicity Assay; OPPTS 870.5100 [ § 84 2]. In a bacterial reverse mutation assay ( Gocke, et al. 1981) histidine requiring mutants of S. typhimurium bacterial strains, TA 98, TA 100, TA 1535, TA 1537, and TA 1538, were exposed to phenol ( purity not reported) at concentrations of approximately 9, 12, 54, 78, or 90 µ moles/ plate in the presence and absence of metabolic activation from aroclor induced rats or hamsters. The numbers of his+ revertants observed with phenol treatment indicated no evidence of induced mutant colonies over background following administration of phenol in the absence of metabolic activation. However, in the presence of metabolic activation, mutant colonies were increased with His+ revertants/ plate observed at the maximum level at the mid dose ( 54 µ mole/ plate) and individual increases of 14, 21, 143, 121, and 57% over control were measured at the concentrations of 9, 12, 54, 78, and 90 µ mole/ plate, respectively. The higher concentrations, 78 and 90 µ mole/ plate, exhibited lower amounts of his+ revertants than 54 µ mole/ plate, but were still well above control levels. Phenol was not mutagenic in the bacterial strains in the absence of metabolic activation, but mutagenic effects were measured by a significant increase over control in histidine revertants in the presence of metabolic activation; predominantly in the Ames tester strains of S. typhimurium that are sensitive for frameshift mutatgens ( ie., TA 98).

Sex Linked Recessive Lethal Test in Drosophila melanogaster Mutagenicity Assay; OPPTS 870.5275 [ § 84 2]. In an in vivo microbial gene mutation assay ( Gocke, et al. 1981) Berlin K ( wild type) and Basc strains of Drosophila melanogaster were exposed to phenol ( purity not reported) via an adult feeding method at a concentration of 50 mM ( one dose close to the LD 50). Approximately 1200 X chromosomes were tested per experiment in each of 3 successive broods Page 19 of 42 ( 3, 3, and 4 days, respectively) with 3543, 3458, and 2139 chromosomes tested for sex linked recessive lethals in Broods 1, 2, and 3, respectively. There were no significant increases in recessive lethals observed following administration of phenol to Broods 1, 2, and 3 with only 17 ( 0.48%), 6 ( 0.17%), and 7 ( 0.33%) sex linked recessive lethals, respectively, measured in the chromosomes tested. After feeding phenol to adult flies, the frequency of recessive lethals was increased, but not to significant levels. Phenol was not mutagenic within the confines of this study.

Sex Linked Recessive Lethal Test in Drosophila melanogaster Mutagenicity Assay; OPPTS 870.5275 [ § 84 2]. In an in vivo microbial gene mutation assay ( Woodruff, 1985) the Basc strain of male Drosophila melanogaster was exposed to phenol ( 99.9% purity) in an adult feeding study at concentrations of 0 or 2000 ppm and in an adult injection study at concentrations of 0 or 5250 ppm. In the feeding assay, males were treated with phenol at 0 and 200 ppm in 5761 and 6641 tests, respectively, for 3 days in glass shell vials containing a glass fiber disc soaked with 0.2 to 0.5 mL of solution. The test solution, 0.2 to 0.3

L, was injected in 6357 and 5887 tests at the 0 and 5250 ppm doses of phenol, respectively, in the injection assay. Cytotoxic effects were observed in the high dose groups with a 30% mortality at 2000 ppm in the feeding study and 6% at 5250 ppm in the injection study. Sterility was also observed in 8% of the tests at 5250 ppm phenol ( injection assay). Sex linked recessive lethal mutations in the feeding study were 7 ( 0.12%) and 11 ( 0.17%) at the 0 and 2000 ppm doses, respectively. In the injection study, 5 ( 0.08%) and 6 ( 0.10%) recessive lethal mutations were observed at 0 and 5250 ppm, respectively. One cluster of 32 lethals and one cluster of 86 lethals were observed in the treated feeding experiment. The sex linked recessive lethal mutations in treated flies were not significantly different from those found in controls. Phenol was not mutagenic in the Basc strain of male D. melanogaster at the concentrations used in this study.

In Vitro Mammalian Cell Gene Mutation Test in Chinese Hamster Ovary ( CHO) Cells; OPPTS 870.5300 [ § 84 2]. In an in vitro mammalian gene mutation assay ( Paschin and Bahitova, 1982) the induction of point mutations in mammalian somatic cells at the hypoxanthine guanine phosphoriboxyl transferase ( HGPRT) locus of V79 was measured in CHO cells exposed to individual phenol (" pure" grade) at concentrations of 0, 25, 50, 100, 250, or 500 µ g/ mL or a mixture of 12 µ g/ mL benzo[ a] pyrene and phenol at concentrations of 0, 100, 250, or 500 µ g/ mL. The selective agent, 8 azaguanine ( AG), was added to determine resistant ( AGr) mutants. For each dose 10 12 petri dishes were used and experiments were performed in triplicate. There were no treatment related effects on cell survival at individual phenol concentrations up to and including 250 µ g/ mL. At the 500 µ g/ mL dose, cell survival was approximately 55% of control. The combination of various doses of phenol with a constant concentration of benzo[ a] pyrene ( 12 µ g/ mL) resulted in decreased cell survival at all three doses of phenol; 100 ( 45%  ) , 250 ( 83%  ) , and 500 µ g/ mL ( 99%  ) . At phenol concentrations of 25, 50, 100, 250, and 500 µ g/ mL, there were dose dependent increases of 1.2, 1.2, 1.7, 2.5, and 4.3 fold, respectively, in the number of revertant colonies. Statistically significant increases of 52 and 72% were observed in the frequency of AGr ( selective agent, 8 azaguanine added to determine resistant mutants) mutants over spontaneous levels at 250 and 500 µ g/ mL, respectively. The combination of phenol with benzo[ a] pyrene at a concentration of 12 µ g/ mL, exhibited an increase in frequency of 8 azaguanine resistant colonies with increasing concentrations of phenol. The results were the same as the sum of the separate effects of phenol and benzo[ a] pyrene; indicating that phenol did not block or activate the mutagenicity of benzo[ a] pyrene. Evidence of mutagenicity was exhibited by the statistically significant increase in the frequency of 8 azaguanine resistant mutants over the spontaneous level at phenol concentrations of 250 and 500 µ g/ mL. Additive results were observed in the combination experiment with 12 µ g/ mL benzo[ a] pyrene at the 250 and 500 µ g/ mL doses of phenol.

In Vitro Mammalian Chromosome Aberration Test in HL60 Cell Cultures; OPPTS 870.5375 [ § 84 2]. In an in vitro mammalian chromosome aberration assay ( Kolachana, et al 1993) the formation of active oxygen in complex biological systems was measured in preincubated HL60 Page 20 of 42 cell cultures exposed to phenol ( distilled high purity) at a concentration of 100 µ M for 30 minutes. DNA was extracted to measure the levels of 8 hydroxy 2' deoxyguanosine ( 8OHdGua), which is a product of oxidative damage. 8OHdGua has been shown to be mutagenic and was used as a marker for oxidative DNA damage. A statistically significant, 3.5 fold increase of 8OHdGua levels in DNA was observed in HL60 cells; from the control level of 0.080 to 0.270 pmol 8OHdGua/ µ g after 30 minutes of incubation. There was little or no cytotoxicity ( not reported) in the cells for at least 6 hours of exposure to phenol; indicating the 8OHdGua formation in these cells does not occur after cell death. Therefore, phenol induced rapid 8OHdGua formation in HL60 cells ( increasing the steady state level of 8 hydroxy 2' deoxyguanosine in DNA) that returned to normal levels following further incubation, presumably due to the rapid repair of DNA damage. However, the increase in this oxidative damage product indicated the genotoxic effects resulting from active oxygen.

Mammalian Spermatogonial Chromosomal Aberration Test in Rats; OPPTS 870.5380 [ § 84 2]. In a mammalian spermatogonial chromosomal aberration assay ( Bulsiewicz, 1977) male and female mice of the Porton strain were exposed to phenol ( purity not reported) in drinking water at concentrations of 0.08, 0.8, or 8.0 mg/ L/ day for 30 days. The parent ( P) generation was initiated when 6 males/ dose were mated with 6 females/ dose. After the 30 day treatment, the males were anesthetized and testes were prepared for analysis. Females continued to receive phenol throughout pregnancy and bringing up of newborns. Two mice, 1 male and 1 female, from each of six families were mated for F2 generation and this process was repeated for a total of 5 generations. The testes of 6 males from each generation were prepared and stained to observe 20 metaphases from spermatogonia and 120 150 from primary spermatocytes for chromosomal anomalies. Treatment related effects on mortality were observed at the high dose in which 8 of F3, 16 of F4, and 22 mice of F5 generations died with exposure to 8 mg/ L phenol per day. Numerous associations and pulverization of the chromosomes made their analysis impossible in many cases. Pulverization was observed at the 8 mg/ L dose. Chromosomal aberrations were more frequent in this group with aberrations observed in 537 of the 660 plates analyzed.

There was evidence of chromosomal aberrations in spermatogonia and primary spermatocytes in all generations for the three doses of phenol, and included breaks and fractures of chromatids and chromosomes, small chromosomes, ring chromosomes, centric fusions, acentric fragments, aneuploidy, and polyploidy for spermatogonia and chromatid breaks, univalents, multivalents, aneuploidy, and polyploidy for primary spermatocytes. The induction of these aberrations were dose dependent and more apparent in later generations. Phenol produced dose dependent changes in the reproductive cells of mice that increased in intensity in successive generations. Aberrations were observed in each dose group; 0.08 mg/ L/ day ( 27% spermatogonia, 5% primary spermatocytes), 0.8 mg/ L/ day ( 52% spermatogonia, 22% diakinetic metaphasal plates of spermatozoa), and 8 mg/ L/ day ( 81% spermatogonia, 24% primary spermatocytes). Phenol in low concentrations produced only minor changes in chromosomes that are probably eliminated by regulative processes in the body. Higher doses of phenol produced qualitative and quantitative changes such as large numbers of spermatogonial metaphases and total absence of spermatocytes, spermatids, and spermatozoa in testes preparation of treated mice. These effects and the excessive number of proliferating spermatogonia indicate potential carcinogenicity of phenol at higher concentrations. Phenol was mutagenic in the Porton strain mice due to the evidence of a concentration related ( 0.08 8.0 mg/ L/ day) positive response of spermatogonial chromosome aberrations following 30 days of exposure to phenol in water.

Mammalian Bone Marrow Chromosome Aberration Test in Mice; OPPTS 870.5385 [ § 84 2]. In a mammalian bone marrow chromosome aberration assay ( Kolachana, et al. 1993) the formation of active oxygen in complex biological systems was measured in B6C3F 1 male mice exposed to phenol ( distilled high purity), 3 animals/ dose, administered via intraperitoneal injection at a concentration of 75 mg/ kg/ day individually and combined in separate experiments with 75 mg/ kg/ day of hydroquinone or 75 mg/ kg/ day of catechol. Animals were sacrificed 1 hour after treatment and bone marrow cells were isolated and harvested from mice femurs. DNA was Page 21 of 42 extracted from bone marrow cells to measure levels of 8 hydroxy 2' deoxyguanosine ( 8OHdGua); a product of oxidative damage that has been shown to be mutagenic. 8OHdGua was used as a marker for oxidative damage resulting from phenol exposure. Phenol, hydroquinone, and catechol administered individually did not have significant effects on the steady state level of 8OHdGua. With a range of 0.045 0.053 pmol 8OHdGua/ µ g DNA, the phenol ( 11% increase), hydroquinone ( 7% increase), and catechol ( 26% increase) treated cells were not statistically different from the control. The combined experiments with either hydroquinone or catechol induced a 2 fold increase in 8OHdGua levels over control values. The phenol and hydroquinone experiment increased the indicator level to 0.104 pmol 8OHdGua/ µ g DNA; a 226% increase over control and 49 and 47% increases over individual phenol and hydroquinone levels, respectively. Combined administrations of phenol and catechol induced a 172% increase over control for the 8OHdGua level and was 65 and 73% greater than individual phenol and catechol levels, respectively. The levels in combined experiments were significantly greater than their individual counterparts. Individual treatment with phenol, hydroquinone, or catechol did not significantly increase 8 hydroxy 2' deoxyguanosine levels in mouse bone marrow cells compared to the control. The phenol and hydroquinone mixture was the most effective combination although administration of all combinations of the test articles significantly increased the 8OHdGua levels via oxidative DNA damage. The phenolic metabolites of benzene used in this study, ( phenol, hydroquinone, and catechol), increased the steady state levels of 8OHdGua in the bone marrow of B6C3F 1 mice in vivo; indicating that activation of these compounds produces active oxygen capable of causing oxidative DNA damage.

Mammalian Erythrocyte Micronucleus Test in Mice; OPPTS 870.5395 [ § 84 2]. In a bone marrow erythrocyte micronucleus assay ( Barale, et al. 1990) Swiss CD 1 male mice bone marrow cells were exposed to individual and combined administrations of phenol and hydroquinone. Groups of 3 mice/ dose received an intraperitoneal injection of phenol at a concentration of 40, 80, or 160 mg/ kg body weight ( bw) or hydroquinone at 40, 60, or 80 mg/ kg bw. The mixture experiments involved all possible combinations of phenol and hydroquinone. Phenol at a concentration of 40 mg/ kg was co administered with each of the 3 hydroquinone concentrations in 3 separate experiments and this process was repeated for the 60 and 80 mg/ kg doses of phenol. Animals were sacrificed 18 hours after treatment and bone marrow cells were harvested to score PCEs ( at least 3000 per animal) and normochromatic erythrocytes ( NCEs) for each animal. There were no clinical signs of toxicity observed following treatment with phenol or hydroquinone individually. In the combination experiment the myelotoxicity ( expressed as the increase of NCE/ PCE ratio and compared to control ratio) increased as the concentration of hydroquinone increased. This ratio exhibited very little change with increasing concentrations of phenol at each hydroquinone dose level. At 40 mg/ kg hydroquinone the ratio range for 40 160 mg/ kg of phenol was 1.03 1.04 fold greater than the control. The 60 and 80 mg/ kg of hydroquinone exhibited NCE/ PCE ratios for the phenol range that were 1.23 1.25 fold and 1.23 1.45 fold greater, respectively, than the control ratio. Only at the high dose of hydroquinone ( 80 mg/ kg) was a dose related effect observed with increasing concentrations of phenol.

The administration of phenol at concentrations 40 160 mg/ kg bw did not result in micronuclei induction in the mouse bone marrow cells; therefore, phenol was not genotoxic in this assay. A statistically significant increase in MNPCEs was observed for hydroquinone although it was considered weakly toxic. The increase in MNPCEs was observed as a function of dose for hydroquinone with 1.6, 2.1, and 4.9 fold increases over control for the 40, 60, and 80 mg/ kg doses, respectively. Combined treatment of the test articles increased the number of MNPCEs compared to the control. Co administration of phenol and hydroquinone resulted in greater than a 2 fold increase in the amount of MNPCEs observed with hydroquinone alone. Greater than additive effects were apparent with a 3.4 fold increase at the lowest concentration ( phenol 40 mg/ kg, hydroquinone 40 mg/ kg) increasing as the doses were raised. There was an 11 fold increase over control MNPCEs at the maximum concentration ( phenol 160 mg/ kg, hydroquinone 80 mg/ kg). Phenol was not toxic and hydroquinone was weakly toxic to mouse bone marrow cells; however, a mixture of the two induced a dramatic and significant decrease in bone marrow Page 22 of 42 cellularity. A powerful synergism between phenol and hydroquinone was observed in the induction of genotoxicity and somewhat in the myelotoxicity of the mouse bone marrow cells.

Mammalian Erythrocyte Micronucleus Test in Mice; OPPTS 870.5395 [ § 84 2]. In a mammalian erythrocyte micronucleus assay ( Chen and Eastmond, 1995) CD 1 male mice, in groups of 5/ dose were exposed to intraperitoneal injections of phenol ( 99% purity) at concentrations of 0, 50, 75, 100, or 160 mg/ kg/ day or combination injections of hydroquinone ( 60 mg/ kg) and 0, 50, 75, 100, or 160 mg/ kg/ day for 3 consecutive days at 24 hour intervals. Twenty four hours following the final dose, the femoral bone marrow cells were harvested from male mice and slides were prepared for scoring of MNPCEs per 2000 PCEs. The ratio of PCEs to normochromatic erythrocytes ( NCEs) was determined for each animal on basis of the number of mature cells ( NCEs) encountered while accumulating 200 PCEs and was an indirect measure of myelotoxicity in mouse bone marrow cells. There was no depression in the PCE/ NCE ratio with the individual administrations of phenol and hydroquinone ( with 0 mg/ kg phenol), while the mixture exhibited significant decreases ( 42 87% reduction) in the ratio for all concentrations of phenol. The depression in bone marrow erythropoiesis from the control ratio was dose related in the hydroquinone ( constant 60 mg/ kg) and phenol ( increasing concentration 50 160 mg/ kg/ day) mixture and exhibited a decrease in PCE: NCE ratios from 1.17 for control to 0.68 ( 50 mg/ kg/ day), 0.36 ( 75 mg/ kg/ day), 0.26 ( 100 mg/ kg/ day), and 0.15 ( 160 mg/ kg/ day) for the doses of phenol. Individually, phenol was weakly and hydroquinone was moderately toxic to mouse bone marrow cells. The frequencies of MN ranged from 2 16 MN/ 2000 PCE following treatment with the individual chemicals. Concentrations of phenol up to 100 mg/ kg/ day increased MN only 5 MNPCEs/ 2000 PCEs; slightly above controls ( approximately 1 MNPCEs/ 2000 PCEs). The maximum dose of phenol ( 160 mg/ kg/ day) increased MN to 10 per 2000 PCEs. The hydroquinone experiment increased frequencies of MN more so than phenol. A hydroquinone concentration of 60 mg/ kg/ day bw increased MNPCEs to approximately 16 MN/ 2000 PCEs.

When mice were administered a mixture of various doses of phenol ( 50, 75, 100, or 160 mg/ kg/ day) and a constant hydroquinone ( 60 mg/ kg/ day) concentration a pronounced increase in frequency of MN was observed. Dose related increases in MN frequencies from 18 80 MN were noted with increasing phenol concentrations ( when combined with hydroquinone). Statistically significant increases in MN were observed at the two highest doses of phenol ( 100 and 160 mg/ kg/ day). The high dose of the mixture ( Phenol 160 mg/ kg/ day, Hydroquinone 60 mg/ kg/ day) was approximately 4 fold greater than the expected additive MN frequencies of individual doses of phenol and hydroquinone. There was no myelotoxicity observed in individual treatments of phenol and hydroquinone as measured by the ratio of PCEs to NCEs. Significant decreases in the PCE/ NCE ratio with the mixture indicated myelotoxic effects associated with treatment. There were no signs of genotoxicity with phenol ( 50 160 mg/ kg/ day) alone and minimal increases in MN with hydroquinone alone during the study. However, the combined administration of phenol and hydroquinone to CD 1 male mice resulted in a statistically significant increase in the frequency of MN. There was a synergism in myelotoxicity and genotoxicity for combined administrations of phenol and hydroquinone to male mice bone marrow erythroblast chromosomes. Phenol was genotoxic when combined with hydroquinone.

Mammalian Erythrocyte Micronucleus Test in Mice; OPPTS 870.5395 [ § 84 2]. In a mammalian erythrocyte micronucleus assay ( Gocke, et al. 1981) NMRI mice in groups of four/ dose ( 2 males, 2 females) were exposed to intraperitoneal phenol ( purity not reported) at concentrations of 47, 94, or 188 mg/ kg. Doses were administered twice, once at 0 hours and again at 24 hours. Animals were sacrificed and bone marrow cell smears were prepared 6 hours after last treatment. For each animal, 1000 polychromatic erythrocytes were scored. There were no treatment related effects on mortality ( 100% survival) in mice receiving treatment. Variations in micronuclei polychromatic erythrocytes ( MNPCEs) were observed with increasing doses of phenol. A 30% decrease from control levels of MNPCEs was observed with the low dose ( 47 mg/ kg); however there were increases with the higher doses of phenol. The 94 and 188 mg/ kg concentrations of phenol resulted in 2.4 and 1.6 fold increases, respectively, in the number of MNPCEs observed in Page 23 of 42 controls. There were no significant increases in the frequency of micronucleated polychromatic erythrocytes in mouse bone marrow cells at the concentrations of phenol used in this study. Phenol was not mutagenic in NMRI mouse bone marrow cells.

4.8 Neurotoxicity Adequacy of database for Neurotoxicity: Upon review of the toxicity profile, the ADTC committee noted neurotoxic signs in acute dermal toxicity studies ( convulsions, Brown, et al.; tremors, OTS 0515567; stimulation of motor nerve endings or spinal motor centers, Conning) and a 15 day inhalation study ( impaired balance, involuntary muscle twitches, jerkiness, and increased sluggishness). The IRIS Toxicological profile reports numerous nervous system effects have been observed following phenol dosing in animals. Studies performed by ClinTrials BioResearch presented one animal with tremors that later died ( apparently of dehydration) following dosing in drinking water ( 1998). Additionally, several gavage studies have shown rats and mice with phenol related tremors ( 43735402; Dow Chemical Co., 1994; Moser et al., 1995). However, in a specialized 13 week neurotoxicity study in male and female rats that included a Functional Observational Battery ( FOB) and a detailed neurohistopathology evaluation ( ClinTrials BioResearch, 1998), nervous system effects were observed as tremors in one animal and decreased motor activity in females. A short term gavage screening study ( Moser et al., 1995) found that the only effect in an FOB was a marginal decrease in motor activity and increased rearing post exposure. Part of this could be attributed to the dehydration observed in the study, but it was also concluded that phenol contributed to the decreased motor activity.

4.9 Metabolism and Pharmacokinetics Adequacy of database for Metabolism and Pharmacokinetics: The database for metabolism is considered to be complete. No additional studies are required at this time. Phenol is essentially completely metabolized in 24 hours. The portal of entry metabolism for the inhalation and oral routes appears to be extensive and involves sulfate and glucuronide conjugation and, to a lesser extent, oxidation. Phenol was predominantly conjugated with sulfate and lower amounts of glucuronic acid and the metabolites were rapidly excreted in the urine. The major metabolites found include phenyl sulfate, quinol sulfate, phenyl glucuronide, and quinol glucuronide.

870.7485 Metabolism and Pharmacokinetics  Rat In a metabolism and pharmacokinetics study ( Capel, et al. 1971) 3 female Wistar Albino rats were exposed to phenol ( purity not reported) by stomach syringe or tube at a concentration of 25 mg/ kg ( 5 14C µ Ci/ animal). Animals were housed in metabolism cages that collected urine in receptacles containing a few mL of saturated aqueous HgCl 2 solution to prevent bacterial breakdown of conjugates. After 24 hours, radiochromatogram scans were performed on urine of treated animals. An average of 95% of 14C phenol ( 25 mg/ kg) was excreted in the urine in 24 hours. Four metabolites, the sulfates and glucuronides of phenol and quinol, were found in the urine of rats after an oral dose of phenol. The phenyl sulfate and quinol sulfate metabolites represented 54 and 1%, respectively of the metabolites recovered in the urine. The remaining phenol metabolites recovered from the urine were phenyl glucuronide ( 42%) and quinol glucuronide ( 2%).

In a metabolism and pharmacokinetics study ( Hughes and Hall 1995) female Fischer 344 rats were exposed to [ U 14C] phenol (> 98% radiochemical purity, specific activity 100 µ Ci/ µ mol) via oral, dermal, intratracheal, and intravenous administrations at a concentration of 63.5 nmol ( 3 µ Ci/ animal, approximately 350 nmol/ kg). Animals were housed in metabolism cages. Urine was collected at 4, 8, 12, 24, 48, and 72 hours and feces at 24, 48, and 72 hours. Excretion samples Page 24 of 42 were analyzed for radioactivity. Animals from the oral, iv, and it experiments were sacrificed 72 hours after treatment; organs, muscle tissue, epididymal fats, and contents of gastrointestinal organs were examined. Treated skin was removed from animals of dermal exposure experiments then sacrificed and handled in a similar manner as previously described for other animals.

The sum of radioactivity detected in excreta and tissues is reported as disposition results expressed as a percentage of the recovered dose. The total mean recovered radioactivity by all four routes was 90.5% (+/ 10.2). Phenol was absorbed well and eliminated primarily in urine after oral, dermal, iv, and it administration. Urine provided 75 95% of recovered dose within 72 hours after treatment via all routes. Similar excretion profiles ( 90% of recovered dose) of radioactivity were observed among iv, oral, and it routes. Dermal administration exhibited the lowest total amount of radioactivity ( 75% of recovered dose). Excretion was extensive within 4 hours for oral, it, and iv administrations with  70 85% of recovered dose found in urine. Essentially complete recovery was established after 12 hours. Since iv is considered a standard for 100% absorption, it was concluded that oral and it routes resulted in phenol absorption that is nearly complete. At 4 hours, dermal exposure resulted in only 40% of recovered phenol in urine. This elimination was 70% by 12 hours and essentially complete at 24 hours. Skin absorption was 15 20% lower than other routes and may have been due to anatomical difference between the skin and gastrointestinal tract and/ or lung. The stratum corneum acts as a barrier and may have slowed the absorption of phenol through the skin; however, once phenol was absorbed it was rapidly eliminated in the urine, similar to the other experiments.

The recovered phenol found in the urine of treated rats 8 hours after exposure was chiefly in the form of two metabolites. Phenyl sulfate was recovered in the oral ( 63.4%), dermal ( 48.4%), it ( 68.7%), and iv ( 72.6%) routes of administration. The second metabolite, phenyl glucuronide, was also present after oral ( 26.8%), dermal ( 16.2%), it ( 19.4%), and iv ( 14.3) administrations of treated animals. Tissue examination of rats following phenol treatment indicated distribution throughout the bodies of the animals, and was found in every tissue sampled. The greatest concentration of radioactivity was found in the contents of the large intestine 72 hours after exposure, regardless of administration route. Absorption of phenol was extensive following iv, oral, and it administration while dermal absorption was slightly lower by comparison. Phenol was predominantly conjugated with sulfate and lower amounts of glucuronic acid. These metabolites were rapidly excreted in the urine. Phenol was distributed throughout the body in low amounts and appeared to concentrate in liver, lung, and kidney. Overall, the exposure of phenol to rats by oral, dermal, intratracheal, and intravenous routes resulted in rapid absorption, conjugation, and elimination in urine.

870.7485 Metabolism and Pharmacokinetics  Mouse In a metabolism and pharmacokinetics study ( Capel, et al. 1971) 3 groups of 10 female ICI mice were exposed to phenol ( purity not reported) by stomach syringe or tube at a concentration of 25 mg/ kg ( 2.5 14C µ Ci/ animal). Animals were housed in metabolism cages that collected urine in receptacles containing a few mL of saturated aqueous HgCl 2 solution to prevent bacterial breakdown of conjugates. The urine of the 10 mice were pooled for each group. After 24 hours, radiochromatogram scans were performed on urine of treated animals. An average of 66% of 14C phenol ( 25 mg/ kg) was excreted in the urine in 24 hours. Four metabolites, the sulfates and glucuronides of phenol and quinol, were found in the urine of mice after an oral dose of phenol. The phenyl sulfate and quinol sulfate metabolites represented 46 and 5%, respectively, of the metabolites recovered in the urine. The remaining phenol metabolites recovered from the urine were phenyl glucuronide ( 35%) and quinol glucuronide ( 15%).

4.10 Dermal Penetration Page 25 of 42 Adequacy of database for Dermal Penetration: Dermal penetration studies were available from the open literature and IRIS Toxicological profile for phenol. Phenol is readily absorbed by the inhalation, oral, and dermal routes. Once absorbed, phenol is widely distributed in the body, although the levels in the lung, liver, and kidney are often reported as being higher than in other tissues ( on a per gram tissue basis). Elimination from the body is rapid, primarily as sulfate and glucuronide conjugates in the urine, regardless of the route of administration. Phenol does not appear to accumulate significantly in the body.

870.7600 Dermal Penetration  Mouse In a dermal penetration study ( Behl and Linn, 1983) skin of the hairless male Skh hr  1 strain of mice was exposed to phenol (> 98% purity) in several experiments. 1) Abdominal and dorsal skin patches were excised from mice aged 36, 92, 124, 340, 381, and 441 days to measure the permeability coefficient of phenol in various aged mice. 2) Skin was immersed in saline for 50 hours to determine hydration influences which were measured by carrying out four permeation experiments in succession on the same piece of skin, with saline rinsing between experiments. 3) Mice were sacrificed ( cervical dislocation) and the abdominal and dorsal skin surfaces were stripped 5, 10, or 25 times with cellophane tape. The epidermis was removed from the dermal sections to begin permeation process. 4) Permeation of phenol was examined in pieces of skin from 5 different dorsal locations of 6 mice at concentrations of 0.5, 1.0, 2.0, 4.0, or 6.0% ( w/ v). These phenol concentrations were rotated and varied between mice. The permeation experiment was initiated within 30 minutes of skin exposure to phenol solution and completed within an additional 2 hours.

The phenol permeability coefficient through hairless mouse skin underwent no appreciable change with age and the overall average range from 18.3 to 25.4 P X 103 cm/ hr for mice aged 36 to 381 days. The 441 day old mice exhibited a slightly smaller permeability coefficient of 12.5 P X 103 cm/ hr; however, it was concluded that this decline was due more to animal variability than age. Additionally, the permeability coefficients for phenol were normalized to animal mass ( perm coefficient/ body weight) and age patterns were reexamined. Normalized permeability coefficients were approximately 105, 79, 58, 50, 56, and 32 normalized P X 103 cm/ hr/ g for mice aged 36, 92, 124, 340, 381, and 441 days, respectively. The mass weighted values decreased appreciably over the initial year of age, in each case the fall in magnitude was twofold. Permeability coefficients through abdominal and dorsal skin of various aged mice showed no significant difference in values of the two sites. Within the confines of this study there was no factorable site dependency to the permeation of phenol. This was further supported by the hydration study in which permeability coefficients of both dorsal and abdominal skin remained unchanged, within the range of 21 24 P X 103 cm/ hr during the full 50 hours of hydration. Phenol permeability coefficients for fresh and extensively hydrated skins appeared to be essentially the same. Stripping increased permeability of abdominal and dorsal skins to phenol. Abdominal permeability coefficients increased with 28.4, 210, 354, and 318 P X 103 cm/ hr for 0, 5, 10, and 25 strippings, respectively. Similar results were observed in the 0, 5, 10, and 25 strippings of dorsal skin with 22.4, 120, 277, and 275 P X 103 cm/ hr, respectively. The permeability coefficients obtained tended to level off at 10 strippings. Abdominal permeability for partially and fully stripped skins invariably exceed those of dorsal surfaces even though there is no apparent difference for the intact skins. Abdominal stratum corneum may be more easily removed and the permeability of remaining abdominal strata may be intrinsically greater than that of the dorsal skin. Phenol exhibited dose related increases in skin permeability for all 6 mice of permeation study. Regardless of location of excised dorsal skin, the permeability increased as concentration of phenol increased. The overall mean permeability coefficients were 20.3, 22.7, 30.7, 46.6, 165.4, and 202.7 P X 103 cm/ hr for 0, 0.5, 1.0, 2.0, 4.0, and 6.0 % ( w/ v).

Phenol chemically alters the mouse skin stratum corneum, leading to increased mass transfer rates; a phenomenon also observed in isolated human epidermis with the threshold concentration Page 26 of 42 of 1.5% being virtually the same for both mouse and human tissue. Altering the stratum corneum decreased the partitioning dependency of the skin permeation process. Significant effects on the lipid medium were exhibited by a 6% ( w/ v) concentration of phenol. Phenol administered at a concentration  5% is doubly dangerous because the permeability coefficient is 10 fold enhanced. Therefore, an absorption rate of 5% compared with a 1% solution is not just 5 times but 50 times greater. It was observed that age and hydration have little effect on the permeability of phenol through human and mouse skin and the effects on the skin were a result of destroyed barrier integrity. Permeation and chemical denaturation of skin by phenol were found to be similar between mouse and human skin tissue; indicating that the hairless mouse is an adequate model for chemical permeability in humans. The results suggest that the stratum corneum is proportionally impaired as the phenol concentration is increased.

5.0 Toxicity Endpoint Selection 5.1 See Section 7.1, Summary of Toxicological Doses and Endpoint Selection, Table 3.

5.2 Dermal Absorption Dermal Absorption Factor: 50%

From the available data, dermal absorption percentages of 20 50% have been observed from in vivo and in vitro studies. The ADTC selected the 50% dermal absorption value for phenol for use as a conservative value in risk assessments. This value also takes into account the irritant properties of phenol which may increase its dermal absorption. Phenol is not expected to result in chronic dermal exposure due to its volatile nature and short half life. Therefore, the ADTC did not select a long term dermal endpoint for phenol, and this risk assessment is not required.

5.3 Classification of Carcinogenic Potential Phenol is characterized as a Group D, not classifiable as to human carcinogenicity ( This group is used for agents with inadequate human and animal evidence of carcinogenicity or for which no data are available). The updated toxicological review in the EPA IRIS database ( USEPA, 2002) provides a summary of the weight of the evidence with respect to the carcinogenic potential of phenol, and this is reproduced in part below.

Chronic drinking water bioassays of phenol have been conducted in rats and mice ( NIH PB# 80 1759). In these studies, NCI concluded that phenol was not carcinogenic in male or female F344rats or B6C3F1 mice. However, the report also noted that leukemia and lymphoma were significantly increased in low dose male rats, although there was no significant increase at the high dose. The increases in leukemia are of particular interest in light of the leukemogenic effects of benzene ( for which phenol is a metabolite) in humans. ( In experimental animals, benzene has not been shown to induce leukemia, although increases in lymphoma have been observed [ e. g., NTP, 1986].).

In contrast with the negative results for oral carcinogenicity, dermally administered phenol has been consistently observed to be a promoter. Several authors ( Salaman and Glendenning, 1957; Boutwell and Bosch, 1959; Wynder and Hoffmann, 1961) observed that dermally applied phenol promoted DMBA initiated skin tumors. These studies have generally reported significant skin ulceration at all phenol doses tested. The exception is Wynder and Hoffman ( 1961), who reported that 5% phenol promoted DMBA initiated tumors in mice in the absence of any toxic reactions. When the same phenol dose was administered in different volumes, higher promotion activity was exhibited by the more concentrated solution, which also produced severe skin Page 27 of 42 ulceration, suggesting that some of the promotion activity may have been related to the rapid cell division of repair of skin damage ( Salaman and Glendenning, 1957).

A more recent study of the tumor promoting ability of phenol was conducted by Spalding et al. ( 1993) in which a transgenic mouse line possessing the properties of genetically initiated skin and sensitive to TPA ( a well described promotor of skin papillomas in two stage models) was treated topically 2 times/ week for up to 20 weeks with several chemicals including phenol. Papillomas were induced with the chemicals benzoyl peroxide, TPA, and 2 butanol peroxide. Three mg of phenol administered for 20 weeks resulted in a single papilloma in one of five male mice at 7 weeks and persisted over the 20 week treatment period. This response was not statistically significant compared to controls.

A mechanistic study conducted by Stenius et al. ( 1989) was designed to assess the toxicity and carcinogenicity of phenol when administered to partially hepatectomized male SD rats. Phenol was administered by gavage 5 days/ week for 7 weeks at a concentration of 100 mg/ kg/ day. Animals were sacrificed, by decapitation, 1 week after last treatment. Sections of the rat liver were prepared and stained to measure for induction of   glutamyltranspeptidase ( GGT) positive enzyme altered foci as an indicator of tumor initiation. Additional studies involved single oral administrations of phenol to measure inductions of hepatic ornithine decarboxylase ( ODC), glutathione ( GSH) depletion, and in vivo lipid peroxidation.

Phenol did not increase the number or volume of foci and was found to have no tumor initiating properties within the confines of this study. Lipid peroxidation was not induced following administration of phenol as measured by malondialdehyde ( MDA) in the urine. There were small and inconsistent effects observed in hepatic ornithine decarboxylase ( ODC) in which there was an increase at the mid dose, but a decrease at the high dose. Observed measurements of hepatic ODC were 18.8, 32.3, and 11.4 pmol/ mg/ h for the phenol doses of 0, 50, and 100 mg/ kg/ day, respectively. Phenol did not induce GSH depletion in hepatocytes.

6.0 FQPA Considerations The ADTC concluded that the available toxicity database for phenol was adequate for hazard characterization for the registered uses. In addition to several reports from the scientific literature, there is a recent ( 2002) updated Toxicological Profile in the IRIS database for phenol.

6.3 Developmental Toxicity Study Conclusions The toxicity profile for phenol presented several developmental and reproductive toxicity studies found in the open literature. These studies are also referred to in the IRIS update. In addition, the National Toxicology Program conducted developmental toxicity studies in both rats and mice. The ADTC considered the NTP studies as well as the published report by Ryan et al. ( 2001) to be acceptable for regulatory purposes. Published reports by Kavlock ( 1990) and Bishop ( 1997) were considered unacceptable for purposes of determining the developmental toxicity of phenol, based on study design.

In the NTP rat developmental toxicity study ( MRID # 43735402), phenol, in distilled water, was administered to groups of 23 rats/ dose via gavage at dose levels of 0, 30, 60, or 120 mg/ kg/ day from gestation days ( GD) 6 to 15. No significant maternal toxicity was observed up to the highdose of 120 mg/ kg/ day tested in this study. In offspring, the only effect noted was a significant decrease in mean fetal body weight, but no teratogenic effects were observed. Therefore, the maternal toxicity LOAEL is greater than 120 mg/ kg/ day and the Developmental toxicity LOAEL is 120 mg/ kg/ day. Page 28 of 42 In the NTP mouse developmental toxicity study ( MRID # 43735401), phenol was administered in distilled water to groups of 31 36 mice/ dose by gavage at dose levels of 0, 70, 140, or 280 mg/ kg/ day from gestation days ( GD) 6 to 15. Clinical signs of toxicity were observed in maternal animals at a dose of 280 mg/ kg/ day, which included signs of neurotoxicity ( tremors, ataxia, lethargy). In addition, maternal body weight was decreased during the treatment period ( 31% decrease) and entire gestation period ( 28% decrease). Offspring in the 280 mg/ kg/ day dose group experienced an increase in cleft palate in 8 of 214 fetuses ( 3 litters) but, this increase was not statistically significant. The fetuses, male and female combined, exhibited reduced mean fetal weights. The high dose ( 280 mg/ kg/ day) was fetotoxic due to significant reductions ( 18%) from controls in mean fetal weight in treated animals. The Maternal and Developmental toxicity NOAEL was 140 mg/ kg/ day, and the Maternal and Developmental toxicity LOAEL was 280 mg/ kg/ day in this study.

6.4 Reproductive Toxicity Study Conclusions In a two generation reproduction toxicity study conducted by Ryan et al ( 2001), phenol ( 100% purity) was administered to groups of 30 Sprague Dawley rats/ sex/ dose in drinking water at concentrations of 200, 1000, or 5000 ppm ( 14, 70, and 310 mg/ kg/ day for males and 20, 93, and 350 mg/ kg/ day for females, respectively) for both generations. There were treatment related decreases from control in body weight and body weight gain in P1 generation rats treated with phenol. These reductions were concomitant with decreases in food and water consumption and observed in the high dose rats. There were also treatment related decreases from controls in F1 body weight in the 5000 ppm phenol treated rats. The lower maternal body weight may have contributed to the lower birth weight of F1 generation as a result of the decreased food and water consumption during lactation and decreased palatability. The Maternal Toxicity NOAEL is 1000 ppm in the P1 and F1 generations. The Maternal Toxicity LOAEL is 5000 ppm based on decreases in water and food consumption, body weight ( average 11% decrease during pre mating weeks 1 and 10, gestation, and lactation) and body weight gain ( average 18% decrease during pre mating weeks 1 through 10) in the P1 and F1 generations. These effects are associated with flavor aversion to phenol in the drinking water.

There were no treatment related effects on reproductive performance in either the P1 or F1 generation. The estrus cycle, epididymal sperm count, motility, sperm morphology, testicular sperm count, and production rate were unaffected by phenol treatment in the P1 and F1 generations. However, the percent of offspring alive after PND 0 was significantly decreased in the 5000 ppm groups with a 10% decrease on PND 4 for the P1 generation and decreases of 28 and 24% on PND 4 and 7 21, respectively, for the F1 generation. The Reproductive Toxicity NOAEL is greater than or equal to 5,000 ppm in the P1 and F1 generations ( highest dose tested). The Reproductive Toxicity LOAEL is greater than 5,000 ppm in the P1 and F1 generations ( not established).

There were treatment related effects for both F1 and F2 generations with increases in litter mortality ( more so in the F2 generation) and reduced offspring body weights in the high dose group. This occurred concurrently with maternal toxicity ( decreased maternal body weight); believed to be secondary to the animals' aversion to the flavor of the phenol treated water and resulted in decreased maternal as well as offspring body weight. There were delays in vaginal patency of F1 females ( 38.3 days for treated females vs. 34.6 days for control females) and preputial separation of F1 males ( 47.8 days for treated males vs. 44 days for control males) observed with decreases in pre and post weaning body weights in the high dose group. Therefore, the onset of puberty was delayed and attributed to decreased food and water consumption and reduced body weight. The Offspring Toxicity NOAEL is 1000 ppm. The Offspring Toxicity LOAEL is 5000 ppm based on decreases in body weight of F1 and F2 offspring ( 5 7% on PND 0; 15 30% on PND 4 21), decreases in litter survival of P1 and F1 offspring ( P1 generation: 90% treated vs. 99% control on PND 4; 96% treated vs. 100% control Page 29 of 42 animals on PND 7 21 and F1 generation: 67% treated vs. 93% control on PND 4; 74% treated vs. 98% control on PND 7 21), delays in preputial separation in F1 males ( 47.8 days for treated males vs. 44 days for control males), and delays in vaginal patency in F1 females ( 38.3 days for treated females vs. 34.6 days for control females).

6.5 Information from Literature Sources Information describing hazards of phenol were obtained mainly from published scientific literature on the toxicity of this compound, including technical reports from the National Toxicology Program ( ntp server. niehs. nih. gov) and the USEPA's IRIS website ( epa. gov/ iris) . Product chemistry data were obtained from the Handbook of Physics and Chemistry, 64th edition ( CRC Press), 1983.

6.6 Pre and/ or Postnatal Toxicity A. Determination of Susceptibility The ADTC concluded that there is no evidence for susceptibility of phenol from the available data on developmental and reproductive toxicity.

B. Degree of Concern Analysis and Residual Uncertainties The ADTC concluded that there are low concerns and no residual uncertainties for pre and/ or postnatal toxicity with phenol for any of the available studies. Conservative NOAELs were established for all developmental and offspring effects. The developmental and reproductive toxicity studies conducted with phenol provide adequate information on the dose response relationships for developmental and reproductive toxicity and are considered adequate studies for regulatory purposes.

C. Proposed Hazard based Special FQPA Safety Factor( s):

The hazard based default special FQPA safety factor ( 1X) can be removed when assessing dietary risks resulting from the uses of phenol.

6.7 Recommendation for a Developmental Neurotoxicity Study The ADTC did not recommend a developmental neurotoxicity study.

7.0 Summary of Toxicological Doses and Endpoints for Phenol for Use in Human Risk Assessment1 7.1 Summary Table of Toxicological Dose and Endpoint Selection ( Table 3) Page 30 of 42 Exposure Scenario Dose ( mg/ kg/ day) used in risk assessment UF Special FQPA SF and Level of Concern for Risk Assessment Study and Toxicological Effects Acute Dietary ( gen population) This risk assessment is not required.

Acute Dietary ( females 13 49) This risk assessment is not required.

Chronic Dietary ( all populations) NOAEL= 60 UF = 100 Chronic RfD = 0.6 mg/ kg/ day 100X Developmental toxicity study in rats ( NTP, 1983) Based on significant reductions from the control in mean fetal body weight/ litter at 120 mg/ kg/ day.

Incidental Oral Short Term ( 1  30 Days)

Residential Only NOAEL= 60 mg/ kg/ day MOE = 100 Developmental toxicity study in rats ( NTP, 1983) Based on significant reductions from the control in mean fetal body weight/ litter at 120 mg/ kg/ day.

Incidental Oral Intermediate Term ( 1  6 Months)

Residential Only NOAEL= 60 mg/ kg/ day MOE = 100 Developmental toxicity study in rats ( NTP, 1983) Based on significant reductions from the control in mean fetal body weight/ litter at 120 mg/ kg/ day.

Dermal1 Short and intermediateterm NOAEL = 60 mg/ kg/ day MOE = 100 Developmental toxicity study in rats ( NTP, 1983). Based on significant reductions from the control in mean fetal body weight/ litter at 120 mg/ kg/ day.

Inhalation2 ( All durations) LOAEL = 0.1 mg/ L MOE = 300 ( ST, IT)

MOE = 1000 ( LT) Dalin and Kristofferson: Physiological Effects of a Sub lethal Concentration of Inhaled Phenol on the Rat. Ann. Zool. Fennici 11: 193 199, 1974 LOAEL of 0.1 mg/ L, based on alterations in sliding angle from tilting plane test, and significant increases in liver enzymes Cancer Data inadequate for assessment of human carcinogenic potential ( USEPA, 2002)

1a dermal absorption factor of 50% should be used since an oral endpoint was selected.

7.2 Rationale for Toxicological Dose and Endpoint Selection 8.0 Toxicity Profile Tables Page 31 of 42 8.1 Acute Toxicity Profile Table  ( See Section 4.1, Acute Toxicity, Table 2).

8.2 Subchronic, Chronic and Other Toxicity Profiles Table ( Table 4)

Guideline Number/ Study Type/ Test Substance (% a. i.) MRID Number ( Year)/ Citation/ Classification/ Doses Results Non guideline  Subchronic ( Oral) Range Finding Study ( Rat) Phenol purity not reported NIH PB#  80 1759 Acceptable  Non guideline 0, 100, 300, 1000, 3000, and 10000 ppm NOAEL = 3000 ppm LOAEL = 10,000 ppm, based on significant decreases in mean body weight gain.

Considering the test article was administered in the drinking water, palatability at the high dose may have affected water consumption..

Non guideline  Subchronic ( Oral) Range Finding Study ( Mouse) Phenol purity not reported NIH PB#  80 1759 Acceptable  Non guideline 0, 100, 300, 1000, 3000, and 10000 ppm NOAEL = 3000 ppm LOAEL = 10,000 ppm, based on significant decreases in mean body weight gain.

Considering the test article was administered in the drinking water, palatability at the high dose may have affected water consumption..

Special Study  Nonguideline Two Week Inhalation Study ( Rat) Phenol purity not reported Dalin and Kristoffersson. 1984 Acceptable  Non guideline 600 +/ 10 L/ hr in an inhalation chamber with a phenol concentration of 100 mg/ m3 CNS effects measured by the " tilting plane" method showed significant decreases in the value of the sliding angle after exposure to phenol. The mean sliding angle was 71.2 ± 2.4 degrees prior to phenol administration and following treatment there was a significant decrease ( 6%) to 66.8 ± 1.7 degrees. Rats had elevated plasma Mg2+ levels ( Hypermagnesaemia) and exhibited toxic effects in the central nervous system ( CNS).

870.3700a ( § 83 3) Developmental Rat Phenol purity 99.9% 43735402 Acceptable  Non guideline 0, 30, 60 or 120 mg/ kg/ day Maternal Toxicity NOAEL

120 mg/ kg/ day ( highest dose tested) LOAEL > 120 mg/ kg/ day ( not established) Reproductive Toxicity NOAEL

120 mg/ kg/ day ( highest dose tested) LOAEL > 120 mg/ kg/ day ( not established) Developmental Toxicity NOAEL = 60 mg/ kg/ day LOAEL = 120 mg/ kg/ day, based on reduced fetal weight Guideline Number/ Study Type/ Test Substance (% a. i.) MRID Number ( Year)/ Citation/ Classification/ Doses Results Page 32 of 42 870.3700a ( § 83 3) Developmental Rat Phenol purity 90% Argus, 1997 Acceptable  Guideline 3 time daily with 0, 20, 40, or 120 mg/ kg/ dose ( 0, 60, 120, or 360 mg/ kg/ day) Maternal Toxicity NOAEL = 60 mg/ kg/ day LOAEL = 120 mg/ kg/ day, based on small decreases in maternal body weight Developmental Toxicity NOAEL = 120 mg/ kg/ day LOAEL = 360 mg/ kg/ day, based on decreased fetal body weight and delayed ossification 870.3700a ( § 83 3) Developmental Mouse Phenol purity 99.9% 34735401 Unacceptable ( Upgradable) 0, 70, 140, or 280 mg/ kg/ day Maternal Toxicity NOAEL = 140 mg/ kg/ day LOAEL = 280 mg/ kg/ day, based on increased mortality, significant reductions in body weight, and clinical signs of CNS toxicity ( tremors, lethargy, and ataxia) Reproductive Toxicity NOAEL

280 mg/ kg/ day ( highest dose tested) LOAEL > 280 mg/ kg/ day ( not established) Developmental Toxicity NOAEL = 140 mg/ kg/ day LOAEL = 280 mg/ kg/ day, based on reduced fetal weight and an apparent increase in te incidence of cleft palate Non guideline  Developmental Toxicity  Rat Phenol purity > 99% Narotsky and Kavlock. 1995 Acceptable  Non guideline 40, and 53.3 mg/ kg/ day Maternal Toxicity NOAEL > 40 mg/ kg/ day ( lowest dose tested) LOAEL = 40 mg/ kg/ day, based on reduced body weight gains and severe respiratory signs Reproductive Toxicity NOAEL = 40 mg/ kg/ day LOAEL = 53.3 mg/ kg/ day, based on significantly reduced litter sizes, full resorption, and prenatal loss Developmental Toxicity NOAEL

53.3 mg/ kg/ day ( highest dose tested) LOAEL > 53.3 mg/ kg/ day ( not established) Guideline Number/ Study Type/ Test Substance (% a. i.) MRID Number ( Year)/ Citation/ Classification/ Doses Results Page 33 of 42 870.3800 ( § 83 4) Reproduction  Rat Phenol purity 100% Ryan, et al. 2001 Acceptable Guideline 0, 200, 1000, and 5000 ppm Males: 0, 14, 70, and 310 mg/ kg/ day Females: 0, 20, 93, and 350 mg/ kg/ day Maternal Toxicity NOAEL = 1,000 ppm in the P1 and F1 generations LOAEL = 5,000 ppm based on decreases in water and food consumption, body weight ( average 11% decrease during premating weeks 1 and 10, gestation and lactation) and body weight gain ( average 18% decrease during pre mating weeks 1 through 10) in the P1 and F1 generations. These effects are associated with flavor aversion to phenol in the drinking water Reproductive Toxicity NOAEL

5,000 ppm in the P1 and F1 generations ( highest dose tested) LOAEL > 5,000 ppm in the P1 and F1 generations ( not established) Offspring Toxicity NOAEL = 1,000 ppm LOAEL = 5,000 ppm based on decreases in body weight of F1 and F2 offspring ( 5 7% on PND 0; 15 30% on PND 4 21), decreases in litter survival of P1 and F1 offspring ( P1 generation: 90% in treated animals vs. 99% in control animals on PND 4; 96% in treated animals vs. 100% in control animals on PND 7 21. F1 generation: 67% in treated animals vs. 93% in control animals on PND 4; 74% in treated animals vs. 98% in control animals on PND 7 21) and delays in preputial separation in F1 males ( 47.8 days for treated males vs. 44 days for control males) and vaginal patency in F1 females ( 38.3 days for treated females vs. 34.6 days for control females) Guideline Number/ Study Type/ Test Substance (% a. i.) MRID Number ( Year)/ Citation/ Classification/ Doses Results Page 34 of 42 870.4200a ( § 83 2) Carcinogenicity  Rat Phenol purity 98.47% NIH PB# 80 1759 Acceptable  Non guideline 0, 2500, or 5000 ppm Male: 0, 322, or 645 mg/ kg/ day Female: 0, 360, or 721 mg/ kg/ day NOAEL

5000 ppm ( highest dose tested) for both male and female rats LOAEL > 5000 ppm ( not established)

Various neoplasms were observed in both control and treated rats. The low dose male rats exhibited increased tumor occurrence over controls. Pheochromocytomas of the adrenal medulla were found in 44% of low dose males; significantly higher ( p= 0.046) than the 26% in controls and 18% in high dose males. The incidence of either leukemia or lymphomas in high dose and low dose males were higher than controls, but were only significantly higher ( p= 0.008) in low dose treated. The incidence of interstitial cell tumors in the testis of males is also significantly higher ( p= 0.05) in low dose males than control; found in 49 of the 50 animals. Results of histopathologic examinations suggest phenol may have increased the incidence of pheochromocytoma, leukemia or lymphoma in low dose male rats. Females did not exhibit increased incidences of tumors at any time in this study.

870.4200b ( § 83 2) Carcinogenicity  Mouse Phenol purity 98.47% NIH PB# 80 1759 Acceptable  Non guideline 0, 2500, or 5000 ppm Male: 0, 590, or 1180 mg/ kg/ day Female: 0, 602, or 1204 mg/ kg/ day NOAEL

5000 ppm ( highest dose tested) for male and female mice LOAEL > 5000 ppm ( not established)

Neoplasms observed in treated animals were of the usual number and type found in mice. Uterine endometrial stromal polyps were increased in 5 of 48 high dose female mice, although this was not significantly different from similar historical control mice. Any other neoplasms noted were occurrences normally associated with aged B6C3F1 mice and were not treatment related.

Results of the histopathologic examinations suggest phenol was not toxic or carcinogenic to B6C3F1 mice under the conditions of this 2 year drinking water carcinogenic bioassay; no tumor at any site in the mice could be clearly associated with the administration of phenol in this study. Guideline Number/ Study Type/ Test Substance (% a. i.) MRID Number ( Year)/ Citation/ Classification/ Doses Results Page 35 of 42 Non guideline  Mechanistic Study Phenol purity 99.5% Stenius, et al. 1989 Acceptable  Non guideline 0 or 100 mg/ kg/ day 5 days/ week for 7 weeks NOAEL

100 mg/ kg ( highest dose tested) LOAEL > 100 mg/ kg ( not established)

Phenol is not a potent stimulator of foci development and can be regarded at a negative control in this study because it should not be susceptible to oxidation reduction reactions.

870.5100 ( § 84 2) Bacterial reverse mutation test Phenol purity not reported Florin, et al. 1980 Acceptable  Non guideline 2.3  2343  g/ plate 3  mol/ plate Negative There was no evidence of induced mutant colonies over background. Positive controls produced the appropriate responses in the corresponding strains of the bacterial reverse mutagenesis test. S. typhimurium did not show mutagenic activity in the presence or absence of metabolic activation when phenol was administered at 3  mol/ plate.

870.5100 ( § 84 2) Bacterial reverse mutation test Phenol purity 99.9% Haworth, et al. 1983 Acceptable  Non guideline 0, 33, 100, 333, 1000, 2500, and 3333  g/ plate Negative There was no evidence of induced mutant colonies over background. Positive controls produced appropriate responses in corresponding strains of the bacterial reverse mutagenesis test. S. typhimurium did not show mutagenic activity in the presence or absence of metabolic activation following administration of phenol.

870.5100 ( § 84 2) Bacterial reverse mutation test Phenol purity 98% Pool and Lin. 1982 Acceptable  Non guideline 0.5, 5, 50, 500, and 5000  g/ plate Negative The most abundant phenolic compounds found in smokehouse smoke condensates were not mutagenic in the Salmonella Typhimurium assay. There was no evidence of mutagenic activity following administration of phenol to 5 bacterial strains of S. typhimurium in the presence or absence of metabolic activation.

870.5100 ( § 84 2) Bacterial reverse mutation test Phenol purity not reported Gocke, et al. 1981 Acceptable  Non guideline 0  3000  g/ plate Negative Phenol was not mutagenic in the bacterial strains in the absence of metabolic activation. Phenol showed mutagenic effects, in the presence of metabolic activation, predominantly in the Ames tester strains of S. typhimurium that are sensitive for frameshift mutagens ( ie., TA 98). Guideline Number/ Study Type/ Test Substance (% a. i.) MRID Number ( Year)/ Citation/ Classification/ Doses Results Page 36 of 42 870.5275 ( § 82 4) Sex linked recessive lethal test in Drosophila melanogaster Phenol purity not reported Gocke, et al. 1981 Acceptable  Non guideline 50 mM Negative Phenol was administered to D. melanogaster and 3543, 3458, and 2139 chromosomes were tested for sex linked recessive lethals in Broods 1, 2, and 3, respectively. There were no significant increases in recessive lethals observed following administration of phenol to Broods 1, 2, and 3 and only 17 ( 0.48%), 6 ( 0.17%), and 7 ( 0.33%) sex linked recessive lethals, respectively, were measured in the chromosomes tested.

After feeding phenol to adult flies, the frequency of recessive lethals was increased, but not to significant levels. Phenol was not mutagenic within the confines of this study.

870.5275 ( § 82 4) Sex linked recessive lethal test in Drosophila melanogaster Phenol purity 99.9% Woodruff, et al. 1985 Acceptable  Non guideline Feeding: 0 and 2000 ppm Injection: 0 and 5250 ppm Negative Phenol was tested up to cytotoxic concentrations 2000 and 5250 ppm in feeding and injection studies, respectively. The number of sex linked recessive lethal mutations in the feeding study were 7 and 11 at the 0 and 2000 ppm doses, respectively. The injection assay resulted in 5 ( 0 ppm) and 6 ( 5250 ppm) sex linked recessive lethal mutations, while the feeding study exhibited 0.12 ( 0 ppm) and 0.17 ( 2000 ppm) lethals. These sex linked recessive lethal mutations were not significantly different from those found in controls. One cluster of 32 lethals and one cluster of 86 lethals were observed in the treated feeding experiment. Phenol was negative in inducing sex linked recessive lethal mutation in D. melanogaster. Guideline Number/ Study Type/ Test Substance (% a. i.) MRID Number ( Year)/ Citation/ Classification/ Doses Results Page 37 of 42 870.5300 ( § 84 2) In Vitro mammalian call gene mutation test Phenol purity not reported Pashin and Bahitova. 1982 Acceptable  Non guideline Phenol: 0, 25, 50, 100, 250, and 500  g/ mL Benzo[ a] pyrene and Phenol mix: 100, 250, 500  g/ mL. The mixture includes the addition of sodium Phenobarbital metabolic activation. Positive At doses of 25, 50, 100, 250, and 500  g/ mL phenol, there was a dose dependent increase in the number of revertant colonies ( 1.2, 1.2, 1.7, 2.5, and 4.3 fold increases, respectively). Statistically significant increases were observed in the frequency of AGr ( selective agent, 8 azaguanine added to determine resistant mutants) mutants over spontaneous levels with 52 and 72% increases at 250 and 500  g/ mL, respectively. The combination of phenol with benzo[ a] pyrene at a concentration of 12  g/ mL, exhibited an increase in frequency of 8 azaguanine resistant colonies with increasing concentrations of phenol. The results were the same as the sum of the separate effects of phenol and benzo[ a] pyrene and the researchers concluded that phenol did not block or activate the mutagenicity of benzo[ a] pyrene.

870.5375 ( § 84 2) In Vitro mammalian chromosome aberration test Phenol purity not reported ( high commercial grade) Kolachana, et al. 1993 Acceptable  Non guideline HL60 cells exposed for 30 minutes to 100  M phenol Positive Phenol increased the steady state level of 8 hydroxy 2' deoxyguanosine in DNA of human leukemia HL60 cells. The increase in this oxidative damage product was 3.5 fold and indicates genotoxic effects resulting from active oxygen following exposure to phenol.

870.5380 ( § 84 2) Mammalian spermatogonial chromosomal aberration test  Rat Phenol purity not reported Bulsiewicz. 1977 Acceptable  Non guideline 0.08, 0.8, or 8.0 mg/ L/ day for 30 days for 5 generations Positive There was evidence of a concentration related ( 0.08 8.0 mg/ L) positive response of spermatogonial chromosome aberrations in Porton strain male mice following 30 days of exposure to phenol in water.