3
Health Effects: Toxicity Studies

The Army’s toxicity review of iodotrifluoromethane (CF3I) (McCain and Macko 1999) and its update (Chaney 2002) covered health-effects data on the acute, subacute, subchronic,1 reproductive and developmental toxicity, and on genotoxicity and carcinogenicity. In this chapter, the subcommittee reviews the toxicity data on CF3I and indicates whether it agrees with the Army’s identification of relevant studies and its interpretation of the data. Chapter 4 deals with the cardiac-sensitization potential of CF3I, and Chapter 5, with the Army’s consideration of a physiologically based pharmacokinetic model to determine concentration and effects.

ANIMAL STUDIES

Acute Exposure

In an acute inhalation study, groups of 30 young adult male Fischer 344 rats were given a single 4-h nose-only exposure to CF3I at 0.0%, 0.5%, or 1.0% (0, 5,000, or 10,000 ppm). Ten rats in each exposure group were sacrificed immediately after exposure and on days 3 and 14 after exposure (Kinkead et al. 1994; Dodd et al. 1997). No deaths or clinical signs of toxicity were observed immediately after exposure or during the 3- or 14-

1  

As used by the subcommittee, acute means continuous exposure for up to 24 h, subacute means repeated exposures for at least 1 month; and subchronic means repeated exposures for at least 1 month but no longer than 3 months. In the evaluation of the toxicity of CF3I, rats are the preferred animal model for acute, subacute, and subchronic tests (Dodd et al. 2000).



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Iodotrifluoromethane: Toxicity Review 3 Health Effects: Toxicity Studies The Army’s toxicity review of iodotrifluoromethane (CF3I) (McCain and Macko 1999) and its update (Chaney 2002) covered health-effects data on the acute, subacute, subchronic,1 reproductive and developmental toxicity, and on genotoxicity and carcinogenicity. In this chapter, the subcommittee reviews the toxicity data on CF3I and indicates whether it agrees with the Army’s identification of relevant studies and its interpretation of the data. Chapter 4 deals with the cardiac-sensitization potential of CF3I, and Chapter 5, with the Army’s consideration of a physiologically based pharmacokinetic model to determine concentration and effects. ANIMAL STUDIES Acute Exposure In an acute inhalation study, groups of 30 young adult male Fischer 344 rats were given a single 4-h nose-only exposure to CF3I at 0.0%, 0.5%, or 1.0% (0, 5,000, or 10,000 ppm). Ten rats in each exposure group were sacrificed immediately after exposure and on days 3 and 14 after exposure (Kinkead et al. 1994; Dodd et al. 1997). No deaths or clinical signs of toxicity were observed immediately after exposure or during the 3- or 14- 1   As used by the subcommittee, acute means continuous exposure for up to 24 h, subacute means repeated exposures for at least 1 month; and subchronic means repeated exposures for at least 1 month but no longer than 3 months. In the evaluation of the toxicity of CF3I, rats are the preferred animal model for acute, subacute, and subchronic tests (Dodd et al. 2000).

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Iodotrifluoromethane: Toxicity Review day observation period, and there were no biologically significant effects on body weight. Although there were some statistically significant variations in hematologic and clinical-chemistry measures examined, including thyroxine (T4) and T4-binding globulin, all were within historical and biologic limits and were considered unrelated to treatment. Groups of five male and five female Sprague-Dawley rats were given a single whole-body inhalation exposure to CF3I at 0.0%, 10.0%, 12.8%, 20.0%, or 32.0% (0, 100,000, 128,000, 200,000, or 320,000 ppm) for up to 4 h (Ledbetter 1994). All rats exposed to 32.0% test material died within 20 min of the start of exposure. However, hydrogen fluoride at 7 ppm had contaminated the test gas, and that necessitated the installation of a potassium hydroxide scrubber for the later 20.0% exposure. All male and female rats exposed to 20.0% CF3I died within 20 min of the start of exposure. A new sample of test material was used for the 10.0% and 12.8% exposures. Within 30 min of the start of exposure, all male and female rats exposed to 10.0% CF3I became unconscious or semiconscious, and they had limb twitching for the remainder of exposure. After cessation of exposure, the rats awakened after about 3 min. Male and female rats exposed to 12.8% CF3I appeared to enter a deep sleep and remained there until the end of exposure. All male and female rats exposed to 10.0% or 12.8% CF3I survived the 2-wk observation period, and no other clinical signs of toxicity were noted. At necropsy, the rats exposed to 32.0% test material had dark red and puffy lungs that were consistent with hydrogen fluoride exposure. Rats exposed at 20.0% had puffy lungs that were much less red than the 32.0% animals. The lungs of two male rats exposed to 12.8% CF3I had slight redness or red foci, but no other treatment-related effects were noted in the remaining rats. Given the animal responses seen at 12.8%, the responses seen at 20.0% and 32.0% may have been indicative of CF3I toxicity with little contribution from HF. Ledbetter (1994) also conducted a nose-only 15-min exposure to 24.2% or 28.8% CF3I with groups of five male and five female Sprague-Dawley rats. All the female rats and two male rats died during exposure to 28.8% CF3I; no female rats and one male rat died during exposure to 24.2% CF3I. All surviving rats were shaky when removed from the exposure chamber, but they recovered within minutes. On the basis of the results, the authors calculated a 15-min CF3I LC50 (the concentration of a substance that is estimated to be lethal to 50% of the test animals) of 27.4%. Typically, three concentrations are used for the determination of LC50. However, the authors reasoned, and the subcommittee concurs, that the steepness of the LC50 curve between the two exposure concentrations made it unnecessary

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Iodotrifluoromethane: Toxicity Review to sacrifice additional test animals for the small amount of information that would be gained. The acute exposure studies discussed by the Army in its review of the toxicity of CF3I and discussed above are summarized by the subcommittee in Table 3-1. The subcommittee finds that the Army’s interpretation of the acute toxicity studies of CF3I was appropriate, and no further acute toxicity testing is recommended. The Army’s review of CF3I also considered overall toxicity. Ledbetter (1994) reported that for acute exposures in rats, the 15-min LC50 was 27.4% (274,000 ppm). That is about 100 times higher than the exposure that causes cardiac arrhythmias in dogs (see Chapter 4). Therefore, the subcommittee finds that noncardiac acute-toxicity end points would not pose a problem at projected exposures of 0.2% for up to 5 min and 0.4% for less than 1 min. Subacute and Subchronic Exposure In a 2-wk range-finding study, groups of five male Fischer 344 rats were exposed to 0.0%, 3.0%, 6.0%, or 12.0% CF3I nose-only for 2 h/day, 5 days/wk (see Table 3-2) (Dodd et al. 1997; Kinkead et al. 1995). Rats in the 6.0% and 12.0% exposure groups were lethargic after treatment, but, none died during the study. The body weight of male rats exposed to 12.0% TABLE 3-1 Summary of Acute Rat Inhalation Exposure Studies Animal CF3I Exposure Results Reference 30 male Fischer 344 rats Single 4-h, nose only; 0.0%, 0.5%, or 1.0% No deaths during the 3- or 14-day observation period; slight decreases in thyroxine and thyroxine-binding globulin Kinkead et al. 1994; Dodd et al. 1997 5 male and 5 female Sprague-Dawley rats Single 4-h, whole body, 0.0%, 10.0%, 12.0%, or 20.0% at 20.0%, death; at 10.0% and 12.0%, narcosis, no deaths Ledbetter 1994 5 male and 5 female Sprague-Dawley rats 15-min, nose only; 24.2%or 28.8% LC50, 27.4% Ledbetter 1994

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Iodotrifluoromethane: Toxicity Review TABLE 3-2 Summary of Subacute and Subchronic Rat Inhalation Exposure Studies Animal CF3I Exposure Results Reference 5 male Fischer 344 rats 2-wk, 5-day/wk, 2-h/day, nose only; 0.0%, 3.0%, 6.0% or 12.0% No deaths; at 6.0% and 12.0%, WBC count decreased 20% Kinkead et al. 1995; Dodd et al. 1997 15 male and 15 female Fischer 344 rats 13-wk, 5-day/wk, 2-h/day, whole body; 0.0%, 2.0%, 4.0%, or 8.0% 8 deaths, not attributed to treatment; at 4.0% or 8.0%, dose-related increase found for micronucleated RBCs in male and female rats at 4 wk, T3 decreased up to 50% in male and female rats at 4 or 13 wk; at 8.0%, rhinitis in male and female rats at 4 wk but not at 13 wk; 8.0%, necrosis of nasal turbinates in male rats (56%) and female rats (40%); 8.0%, mild increase in thyroid follicular colloid in male and female rats at 13 wk Dodd et al. 1997 CF3I was statistically decreased on study days 7 and 14, and the body weight of male rats exposed to 6.0%, on study day 14. In addition, the white-blood-cell (WBC) count of rats in the 6.0% and 12.0% exposure groups was decreased by about 20%, whereas the serum thyroglobulin and reverse triiodothyronine (rT3) levels were statistically increased but still within acceptable biologic limits. No treatment-related effects were found on histologic examination of the thyroid or parathyroid glands. Dodd et al. (1997) exposed groups of 15 male and 15 female Fischer 344 rats to 0.0%, 2.0%, 4.0%, or 8.0% CF3I for 2 h/day, 5 days/wk for up to 13 wk (see Table 3-2). Five male and five female rats in each group were sacrificed after 30 days of treatment, and the remainder after 13 wk of exposure. Six male rats in the 2.0% group died after the ninth exposure, and one died following the 13th exposure. One male rat in the 8.0% exposure group died after the 10th exposure. All deaths were attributed to the animal-restraint system, not to treatment. During exposure, all rats in the 8.0% group were highly active, the 4.0% group was moderately active, and the 2.0% group was slightly active compared with the control rats. The body weights of male and female rats in the 8.0% group decreased slightly during the first 3 wk of treatment and did not return to their initial weight

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Iodotrifluoromethane: Toxicity Review until after day 28 of the study. Body weights of rats in the 4.0% exposure group were also statistically (p < 0.01) decreased relative to controls from study day 14 to the end. No significant treatment-related effects were found on the body weight of male and female rats exposed to 2.0% CF3I. After 4 wk of exposure, the mean hemoglobin, red-blood-cell (RBC) count, and lymphocyte percentage were statistically (p < 0.01) decreased by 6-23% in male rats exposed to 8.0% CF3I. By 13 wk of treatment, those measures returned to normal (Dodd et al. 1997). A dose-related increase in micronucleated RBCs was found in male and female rats exposed to 4.0% or 8.0% CF3I for 4 wk, and in male and female rats exposed to CF3I at higher than 2.0% for 13 wk. In addition, the ratio of polychromatic to normochromatic RBCs was decreased in a dose-related manner in male and female rats exposed to 4.0% or 8.0% CF3I for 4 or 13 wk. No biologically significant treatment-related effects were found in clinical-chemistry measures investigated (Dodd et al. 1997). However, the triiodothyronine (T3) of male and female rats exposed to 8.0% CF3I was decreased by up to 50% after 4 or 13 wk of treatment, and dose-related biologically significant decreases in T3 were found in male and female rats exposed to 2.0% or 4.0%. With the decrease in T3 in those groups, thyroid-stimulating hormone (TSH) was increased by up to a factor of 2 in a dose-related manner in male and female rats exposed for 4 or 13 wk to 2.0%, 4.0%, or 8.0% CF3I. In addition, rT3 was increased in a dose-related manner after 4 or 13 wk of exposure in all CF3I groups, although no biologically significant effects in T4 were found. On necropsy, a mild increase in thyroid follicular colloid was found in all CF3I groups, although no biologically significant effects were found in the absolute or relative organ weights after 4 or 13 wk of treatment. Treatment-related microscopic effects, such as rhinitis, were found after 4 wk of treatment in male and female rats exposed to CF3I at greater than 4.0% but not after 13 wk of exposure. The subcommittee finds that all subacute and subchronic studies summarized in Table 3-2 and reviewed in the Army’s 2002 update appear to be appropriate, and no further testing is recommended. Genotoxicity The Army’s 1999 review of the toxicity of CF3I evaluated several genotoxicity studies (McCain and Macko 1999). The Salmonella typhimurium histidine reversion (Ames) assay was conducted with CF3I at 1,060, 2,775, 10,586, 23,230, and 85,908 ppm (0.11%, 0.28%, 1.1%, 2.3%, and

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Iodotrifluoromethane: Toxicity Review 8.6%) (Mitchell 1995a). For the mouse lymphoma forward-mutation assay with L5178-Y cells, five concentrations of CF3I, from 8.0% to 51.0%, were tested (Mitchell 1995c) with and without metabolic activation with S-9. In the in vivo mouse bone-marrow RBC micronucleus test, mice were exposed to CF3I at 2.5%, 5.0%, or 7.5% (Mitchell 1995b). The Army concluded that two of the five strains of Salmonella (TA 1537 and TA 98) tested with and without S-9 metabolic activation were weakly positive for inducing frame-shift and base-pair mutations, two (TA 1535 and TA 100) were strongly positive, and in the micronucleus test the two highest concentrations were positive for structural chromosomal aberrations in both sexes. The mouse lymphoma assay was negative for gene mutations. All in vitro studies were conducted in exposure chambers in which the agent was in direct contact with the media of the cells, so solubility in all likelihood was not an issue. CF3I was also positive in both sexes in a micronucleus study conducted in male and female Fischer 344 rats after 4 or 13 wk of exposure via inhalation (nose only) to 2.0%, 4.0%, or 8.0% CF3I vapor for 2 h/day, 5 days/wk. The two highest concentrations were positive at the end of 4 wk, and all concentrations were positive after 13 wk of exposure (Kinkead et al. 1996; Dodd et al. 1997). In the Army’s 2002 update (Chaney 2002), the only new genotoxicity study reviewed was that of Dodd et al. (1998, 1999). It was a micronucleus study conducted as a component of a reproductive investigation in male and female Sprague-Dawley rats. Animals were exposed in whole-body inhalation chambers to 0.2%, 0.7%, and 2.0% CF3I for 7 or 12 wk. Dodd et al. and the Army concluded that CF3I did not induce an increase in the number of micronuclei in rats of either sex. Table 3-3 summarizes the available genotoxicity data on CF3I. The conclusions reached by the Army regarding most of the genotoxicity data seem appropriate. However, the subcommittee finds that the Dodd et al. study (1998, 1999) cited in the 2002 update with regard to the micronucleus test, although negative, has a weakness. Specifically, the doses used in the study were below those used in earlier studies; for example, the highest dose used by Dodd et al. was 2.0%. The highest doses used in the two previous studies, in which positive results were found, were 7.5% and 8.0% in the mouse and rat, respectively. It is also notable that in the Dodd et al. study the ratio of polychromatic to normochromatic RBCs was the same in all groups, including controls. That indicates that the dose used by Dodd et al. could have been higher. The subcommittee finds that the Dodd et al. study should not be viewed as having equal weight with the other micronucleus studies.

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Iodotrifluoromethane: Toxicity Review TABLE 3-3 Summary of Genotoxicity Studies Organism End Point Dose, % Results Reference Salmonella typhimurium TA-1537 with/ without activation Gene mutations 0.11, 0.28, 1.1, 2.3, 8.6 Weakly positive Mitchell 1995a Salmonella typhimurium TA-98 with/without activation Gene mutations 0.11, 0.28, 1.1, 2.3, 8.6 Weakly positive Mitchell 1995a Salmonella typhimurium TA-1535 with/ without activation Gene mutations 0.11, 0.28, 1.1, 2.3, 8.6 Strongly positive Mitchell 1995a Salmonella typhimurium TA-100 with/ without activation Gene mutations 0.11, 0.28, 1.1, 2.3, 8.6 Strongly positive Mitchell 1995a Mouse lymphoma L5178-Y Gene mutations and chromosomal aberrations 8.0, 17.7, 30.6, 42.6, 45.4, 49.7, 51.8 Negative Mitchell 1995c Mouse (male and female) Micronuclei 2.5, 5.0, 7.4 Positive in male and female Mitchell 1995b Fischer 344 rat (male and female) Micronuclei 2.0, 4.0, 8.0 Positive in male and female Kinkead et al. 1996 Sprague-Dawley rat (male and female) Micronuclei 0.2, 0.7, 2.0 Negative in male and female Dodd et al. 1998, 1999 The differences in the data on the micronucleus tests, although possibly explained by dose, are of concern and cannot be dismissed. The micronucleus test detects chromosomal aberrations. The mouse lymphoma assay can detect both gene and chromosomal aberrations, but this study was negative for CF3I. Given the varied genotoxicity results, the subcommittee suggests that it would be prudent to verify the micronucleus results in a mouse or rat bone marrow chromosomal-aberration study. We offer this

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Iodotrifluoromethane: Toxicity Review recommendation because positive results have been found in two species in previous micronucleus assays, and there is a potential for chronic but intermittent exposure to CF3I. Chronic exposure to a mutagen or clastogen may facilitate carcinogenesis. The bone marrow chromosomal-aberration study would focus on structural aberrations as opposed to micronuclei. If such a study demonstrates positive results, it would be appropriate to conclude that CF3I is a clastogen. Carcinogenicity No published studies of the carcinogenicity of CF3I in animals were found by the Army for its toxicity reviews or by the subcommittee. In its 2002 update review of CF3I, the Army cited a study by Koski et al. (1997) that used free-radical modeling as a predictor of carcinogenicity; CF3I was determined to be a potent toxicant and an expected carcinogen. The information from the Ames assays and the micronucleus tests suggests that CF3I is a potential mutagen and clastogen in humans. The subcommittee’s concern with respect to mutagenicity is that chemicals that exhibit such activity may be determined to be carcinogenic. Therefore, the subcommittee recommends short-term testing for carcinogenicity. Studies of in vitro cellular transformation, such as the Syrian hamster embryo cell culture (LeBoeuf et al. 1999), and possibly transgenic-animal studies in one or more of three models—P53+/ hemizygous knockout mouse (Pritchard et al. 2003), Tg.AC (Pritchard et al. 2003), or rasH2 (Pritchard et al. 2003)—should be considered. The subcommittee finds that if any of the recommended short-term carcinogenicity tests are positive, the Army must consider whether, given its proposed use and exposure scenarios, a 2-year, in vivo, inhalation bioassay for carcinogenicity should be conducted. Reproductive and Developmental Toxicity Only one reproductive study was identified by the subcommittee in its review of the available literature. This study was reviewed by the Army in its 1999 and 2002 reports. The study, “Reproductive Toxicity Screen of Trifluoroiodomethane in Sprague-Dawley Rats,” was conducted by Dodd et al. (1998). Male and female rats were exposed in whole-body inhalation chamber to CF3I at 0.2%, 0.7%, or 2.0%. Index of effects on fertility, pregnancy, lactation, and pup development were evaluated, and there were no indications of adverse effects for any of the reproductive or developmen-

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Iodotrifluoromethane: Toxicity Review tal indices. The Army’s conclusion that CF3I is not a reproductive toxicant in Sprague-Dawley rats seems appropriate. There is evidence from another study that CF3I may adversely affect the testes. In a subchronic (13-wk) inhalation study, Fischer 344 rats were exposed nose-only to 0.0%, 2.0%, 4.0%, or 8.0% CF3I (Dodd et al. 1997). The authors noted mild atrophy and degeneration of the testes and a relative decrease in testicular weight at 8.0% in male rats at 13-wk. The study used higher doses than, and a different rat strain from, the reproductive-toxicity study. The animals were not mated in the former study (Dodd et al. 1997), so there was no opportunity to determine the possible influence of the testicular effects on reproduction. The authors stated that CF3I appeared to produce an indirect effect on the testes, although they did not suggest a mechanism by which they thought the alterations occurred. They did note that they believed the exposure design was partly responsible—that is, heat stress associated with nose only exposure in the treated animals. In the Dodd et al. (1998) reproductive study, no macro or micro adverse testicular effects were reported in the 16 male rats. Support for heat stress as the basis for atrophy and degeneration of the testes is provided by two 28-day subacute studies of HFC-143a (Malley 1993), in which animals were exposed nose only at 2,000, 10,000, or 39,000 ppm or whole body at concentrations of 2,000, 10,000, or 40,000 ppm. Those exposed nose only had morphologic changes in the testes and decreased body weight. Elevated temperatures occurred during the exposure period. In the animals exposed whole body, there were no significant differences in body weight, testicular weight, or evidence of testicular changes. In another study (Malley 1993), rats were exposed whole body to 2,000, 10,000, or 40,000 ppm of HCF-143a five times per week for 90 days. No evidence of testicular atrophy or degeneration was found under these conditions. Because there is evidence that testicular changes can be associated with heat stress from nose-only inhalation exposure (Lee et al. 1993; Malley 1993; Rothenberg et al. 2000), the subcommittee concludes that the effects seen in the study by Dodd et al. (1997) were most likely due to heat stress and not CF3I exposure. A search of the published literature by the subcommittee failed to identify any other reproductive or developmental toxicity studies for CF3I. Therefore, given the lack of reproductive or development toxicity from exposure to CF3I and the availability of developmental toxicity studies conducted for several other halocarbons that failed to demonstrate any adverse effects, no additional testing of CF3I for reproductive or developmental effects is recommended by the subcommittee.

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Iodotrifluoromethane: Toxicity Review HUMAN STUDIES No published human studies of the toxicity of CF3I were found by the Army or by the subcommittee. There is minimal human experience with CF3I (see Chapters 4 and 6), but no human health studies were identified.