John L. O'Donoghue

The relative neurotoxicity of methyl-n-butyl ketone, n-hexane and their metabolites.

Methyl n-butyl ketone (MnBK) and n-hexane produce a polyneuropathy in experimental animals. Metabolic studies have revealed that both of these compounds are metabolized to similar metabolites; that is 2-hexanol, 5-hydroxy-2-hexanone, 2,5-hexanediol, and 2,5-hexanedione. The latter two metabolites have been shown to be neurotoxins and the former two may be neurotoxic by virtue of the fact that both are metabolized to 2,5-hexanedione. The relative neurotoxicity of MnBK, n-hexane, and their metabolites was compared by administering equimolar doses of each compound by gavage to groups of male rats, 5 days/week over a 90-day period. A control group was given distilled water. The endpoint used to determine relative neurotoxicity was the time (days) required to produce clinical evidence of severe hindlimb weakness or paralysis. All test compounds produced both clinical and histologic evidence of neuropathy. Morphologically, this nerve damage was identical to that previously described following MnBK, n-hexane, and 2,5-hexanedione administration. The relative neurotoxicity of the test compounds in decreasing order of potency was: 2,5-hexanedione, 5-hydroxy-2-hexanone, 2,5-hexanediol, methyl-n-butyl ketone, 2-hexanol, and n-hexane. The neurotoxic potency was directly related to the amount of 2,5-hexanedione produced by each compound as determined by measuring the area under the serum concentration-time curve of 2,5-hexanedione. Additionally, atrophy of testicular germinal epithelium similar to that previously reported for 2,5-hexanedione occurred in the animals receiving 2,5-hexanedione, 2,5-hexanediol, 5-hydroxy-2-hexanone, MnBK, and 2-hexanol. n-Hexane administration had a lesser effect on the germinal epithelium than did the other compounds. Effects on body weight response and feed consumption paralleled the neurotoxic potency of each compound.

Screening for Neurotoxicity Using a Neurologically Based Examination and Neuropathology

Testing chemicals for toxicity by screening methods is an integral part of providing high-quality products and processes. Screening tests must be sensitive, reproducible, readily performed by a trained staff, generally acceptable across laboratories, costeffective, timely, and predictive of significant hazards. The screening process begins with routine acute and subacute tests commonly in use in toxicology laboratories worldwide. When these tests indicate a neurotoxicologic effect or when structure-activity analysis suggests a concern, more specific tests, including a neurologically based functional observational battery (FOB) and neuropathology, are indicated. The FOB provides a highly structured framework for collection of clinical signs. When teamed together, the FOB and neuropathology examinations are complementary tests that provide a clinicopathologic description of a neurotoxicant that can be used for risk assessment.

Studies on the respiratory uptake and excretion and the skin absorption of methyl n-butyl ketone in humans and dogs

Abstract Methyl n-butyl ketone (MnBK) has produced peripheral neuropathy in experimental animals and is implicated in an occupationally produced neuropathy. Since occupational exposure to MnBK is by inhalation or skin contact, both the absorption and elimination of MnBK vapor and its absorption through skin were investigated. Studies were carried out first with male beagle dogs and subsequently with human volunteers. Humans exposed for 7.5 hours to 10 or 50 ppm or for 4 hr to 100 ppm of MnBK vapor absorbed between 75 and 92% of the inhaled vapor. Unchanged MnBK was not eliminated extensively in the postexposure breath or in urine. 2,5-Hexanedione, a metabolite of MnBK known to be neurotoxic in rats, was found in the serum of humans exposed to either 50 or 100 ppm of MnBK. The absorption and elimination of MnBK in dogs was similar to that observed in humans. The skin absorption of [1-14C]MnBK or a 9 1 ( v v ) mixture of methyl ethyl ketone (MEK) [1- 14 C]MnBK was determined by excretion analysis. Two volunteers exposed by skin contact to [1-14C]MnBK absorbed 4.8 μg min−1 cm−2 and 8.0 μg min−1 cm−2, respectively. Skin exposure to MEK [1- 14 C]MnBK resulted in the respective absorption of 4.2 and 5.6 μg min−1 cm−2 by two individuals. Two volunteers given an oral dose of [1-14C]MnBK (2 μCi; 0.1 mg/kg) excreted 49.9 and 29.0% of the dose, respectively, as respiratory 14CO2 within 3 to 5 days and 27.6 and 25.0% of the dose, respectively, in urine within 8 days. Both [1-14C]MnBK and MEK [1- 14 C]MnBK were absorbed through the skin of dogs. These findings show that MnBK is readily absorbed by the lungs, the gastrointestinal tract, and through the skin, is not eliminated extensively unchanged in breath or urine, and is metabolized to CO2 and 2,5-hexanedione. Radioactivity derived from [1-14C]MnBK was excreted slowly by man, suggesting that repeated daily exposure to high concentrations of MnBK may lead to a prolonged exposure to neurotoxic metabolites.

Mutagenicity studies on ketone solvents: methyl ethyl ketone, methyl isobutyl ketone, and isophorone

3 ketone solvents (methyl ethyl ketone (MEK), methyl isobutyl ketone (MiBK), and isophorone) were tested for potential genotoxicity. The assays of MEK and MiBK included the Salmonella/microsome (Ames) assay, L5178Y/TK+/- mouse lymphoma (ML) assay, BALB/3T3 cell transformation (CT) assay, unscheduled DNA synthesis (UDS) assay, and micronucleus (MN) assay. Only the ML, UDS, and MN assays were conducted on samples of isophorone. No genotoxicity was found for MEK or isophorone. The presence of a marginal response only at the highest, cytotoxic concentration tested in the ML assay, the lack of reproducibility in the CT assay, and clearly negative results in the Ames assay, UDS and MN assays, suggest that MiBK is unlikely to be genotoxic in mammalian systems.

Mortality study of employees engaged in the manufacture and use of hydroquinone

Mortality in a 1942–1990 cohort of 858 men and 21 women employed in the manufacture and use of hydroquinone was evaluated through 1991. Average exposure concentrations, 1949–1990, ranged from 0.1 to 6.0 mg/m3 for hydroquinone dust and from less than 0.1 to 0.3 for quinone vapor (estimated 8-h time-weighted averages). Compared with general population and occupational referents, there were statistically significant deficits in total mortality and deaths due to cancer. No significant excesses were observed for such hypothesized causes as kidney cancer [2 observed vs 1.3 expected (both control groups), P ∼ 0.39], liver cancer (0 vs 0.8, 1.3), and leukemia (0 vs 2.3, 2.7). Dose-response analyses of selected causes of death, including renal carcinoma, demonstrated no statistically significant heterogeneities or linear trends according to estimated career hydroquinone exposure (mg/m3-years) or time from first exposure.

Comparative neurotoxicity and metabolism of ethyl n-butyl ketone and methyl n-butyl ketone in rats

Abstract The six-carbon compounds methyl n -butyl ketone (MnBK) and n -hexane are metabolized by oxidation of their subterminal carbons through a series of steps leading to the production of 2,5-hexanedione (2,5Hxdn). Each oxidative step increases the neurotoxic potential so that 2,5Hxdn is the most active compound in the metabolic series. The most likely seven-carbon compound commercially available that would produce a metabolic analog to 2,5Hxdn is ethyl n -butyl ketone (EBK). A study was conducted to determine the neurotoxicity of EBK and to identify its major metabolites. Rats were exposed to 700 ppm MnBK by inhalation for 11 weeks or EBK for 24 weeks, twice the exposure period needed to produce a severe, clinically evident neuropathy with MnBK. No clinical or neuropathological evidence of neurotoxicity was evident after EBK exposure. These findings appear to be due to the low serum concentrations of 2,5-heptanedione (6.8 ± 4.0 μg/ml) formed from EBK in comparison to the much higher concentrations of 2,5Hxdn (133.2 ± 36.7 μg/ml) formed from MnBK. This argument is strengthened by the fact that in a companion study, 2,5-heptanedione administered to rats 5 days per week by gavage in 1000 and 2000 mg/kg/day doses was found to produce the same type of neurotoxicity as seen with 2,5-Hxdn.

Measurement of Cell Proliferation in the Kidneys of Fischer 344 and Sprague-Dawley Rats after Gavage Administration of Hydroquinone

Oral administration of hydroquinone (HQ) over 2 years to male Fischer 344 (F344) rats results in a dose-related nephropathy and an increase in the incidence of renal tubule adenomas. Female F344 rats, B6C3F1 mice, and Sprague-Dawley (SD) rats are resistant to the chronic renal toxicity of HQ, and nephrotoxicity was not seen in dogs or humans following subchronic exposure. To better characterize the early development of renal toxicity in rats, cell proliferation was quantitated within the proximal (P1, P2, and P3) and distal tubule segments of the kidney in rats given 0, 2.5, 25, or 50 mg/kg HQ by gavage. Male and female F344 rats were treated for 1, 3, or 6 weeks, and male SD rats were treated for 6 weeks. Cell proliferation was quantitated by incorporation of bromodeoxyuridine, detected immunohistochemically, into newly synthesized DNA. At 6 weeks, renal cell proliferation was increased over vehicle-controls in male F344 rats dosed at 50 mg/kg. Significant elevations (p < 0.001) occurred in the P1 segments (87%) and in the P2 segments (50%) but the elevation in the P3 segment (34%) was not statistically significant. Urinalyses revealed increases in the rate of excretion of enzymes indicative of proximal tubular damage. Histopathologic evaluation of the kidneys was consistent with a dose-related tubular degeneration in the male F344 rat. No chemical-related effects were observed in the kidneys of female F344 and male SD rats. These data parallel the findings of sex- and strain-specific kidney adenomas in the 2-year bioassays, and suggest that chemically induced cell proliferation secondary to toxicity may be important in the pathogenesis of benign renal tumors in male F344 rats treated with HQ.

A 14-Week Vapor Inhalation Toxicity Study of Methyl Isobutyl Ketone

In a 2-week probe study male and female Fischer-344 rats and B6C3F1 mice were exposed 6 hr/day to 2000, 500, 100, or 0 ppm methyl isobutyl ketone (MIBK). At 2000 ppm there was a slight increase in male rat liver weight (absolute and relative). The only changes observed histologically were increases in regenerative tubular epithelia and hyalin droplets in kidneys of male rats exposed to 2000 or 500 ppm. Exposure levels for a subchronic study were 0, 50, 250, or 1000 ppm methyl isobutyl ketone vapors 6 hr/day, 5 days per week, for 14 weeks. The 14 weeks of exposure had no adverse effect on the clinical health or growth of rats or mice. Male rats and male mice exposed to 1000 ppm MIBK had a slight but statistically significant increase in liver weight and the liver weight/body weight ratio. Liver weight was also increased slightly in male mice exposed to 250 ppm. No gross or microscopic hepatic lesions related to MIBK exposure were observed. Furthermore, the only microscopic change observed was an increase in the incidence and extent of hyalin droplets within proximal tubular cells of the kidneys of male rats exposed to 250 and 1000 ppm of MIBK. The relevance of the male rat kidney tubular effect to humans is not known. In conclusion, other than the male rat kidney effect, exposure of male and female rats and mice to MIBK at levels up to 1000 ppm for 14 weeks was without significant toxicological effect.

Hydroquinone: Genotoxicity and Prevention of Genotoxicity Following Ingestion

Plant-derived polyphenolics and other chemicals with antioxidant properties have been reported to inhibit the expression of genotoxic activity by pro-oxidant chemicals (Sai et al., 1992, 1994; Teel and Castonguay, 1992). In vitro and in vivo studies with ionizing radiation suggest that hydroquinone (HQ) may have similar protective effects (Babaev et al., 1994). The present study was conducted to determine whether HQ is capable of inhibiting the induction of micronuclei in the bone marrow of mice following exposure to an oxidant, potassium bromate or KBrO3 (Nakajima et al., 1989; Sai et al., 1992, 1994). To be able to interpret the results of this work, it was also necessary to determine whether HQ is itself genotoxic when fed in the diet. HQ diets (0.8%) fed to mice for 6 days reduced the background incidence of micronuclei compared with the basal diet. KBrO3 dosed ip (12.5-100 mg/kg) produced a dose-dependent increase in micronuclei as reported by others. Mice fed 0.8% HQ diets 6 days, and then dosed intraperitoneally with KBrO3, showed a 36% reduction in micronuclei across the range of KBrO3 dose levels. This effect was associated with a reduction in the background micronucleus response as well as a reduction in response to KBrO3. Statistical significance (P < or = 0.05), observed at a dose of 25 mg/kg KBrO3 in the mice fed the control diet, was abolished in the group fed 0.8% HQ. When mice were given 50 mg HQ/kg by oral gavage and then given 50 mg KBrO3/kg ip 20 min later, the micronucleus response induced by KBrO3, was lower in animals given HQ. The results of this study demonstrate that large doses of HQ may be given orally without induction of micronuclei or bone marrow depression, that HQ reduces the background micronucleus response in animals fed a basal diet, and that the HQ reduces the micronucleus response to KBrO3 as well as background incidence of micronuclei in KBrO3-dosed animals. The protective effect of HQ may be due to enzyme induction or a direct antioxidant effect of HQ against oxidants commonly present in the diet.

Lack of nephrotoxicity and renal cell proliferation following subchronic dermal application of a hydroquinone cream.

Hydroquinone (HQ) is used in over-the-counter formulations of skin-lightening creams sold in the United States and European Union. HQ was introduced into these formulations to provide a safe and effective alternative to mercury and other less effective ingredients. Recent studies involving subchronic oral exposure of male F344 rats to HQ have shown nephrotoxicity and renal tubule cell proliferation (English et al., 1994), while chronic exposures of male F344 rats were reported to cause renal cell adenomas (NTP, 1989). Previous subchronic dermal toxicity studies (CTFA, 1986; NTP, 1989) with HQ failed to detect nephrotoxicity; however, these studies were not specifically designed to assess renal structure and function. More sensitive endpoints were used in the present subchronic study to address concerns over potential toxicity from repeated dermal exposure to HQ. Male and female F344 rats were given topical applications with 0, 2.0, 3.5, or 5.0% HQ in an oil-in-water emulsion cream for 13 wk (5 days/wk). Body weights, feed consumption and water consumption were monitored, and animals were observed for clinical signs of toxicity and dermal irritation. Blood taken at termination was analysed for haematological and clinical chemistry effects. Erythema, which abated when exposure stopped, was the only dermatological effect seen at the HQ-cream application sites. Cell proliferation in the kidneys was evaluated after 3, 6 and 13 wk of treatment using bromodeoxyuridine (BrdU) labelling, but no changes indicative of sustained cell proliferation were seen. The renal histopathological lesions noted after oral exposure to HQ were not present after dermal exposure. Thus, topical exposure to HQ does not result in the renal toxicity observed in previous studies with F344 rats given HQ orally.