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EC number: 700-242-3 | CAS number: 62037-80-3
The mouse has a pharmacokinetically distinct profile as compared to the rat and monkey. Therefore, the mouse is not the appropriate or preferred test species for human risk assessment with this substance. The substance, in deionized water, was administered orally by gavage to Crl:CD1(ICR) mice, at doses of 0.1, 0.5, and 5 mg/kg/day. There were no effects on reproduction (mating, fertility, or copulation indices, number of days between pairing and coitus, and gestation length). Test substance-related higher mean body weights, body weight gains, and food consumption were observed in the 5 mg/kg/day , but were not considered adverse. Increased liver weights and microscopic hepatocellular hypertrophy, consistent with peroxisome proliferation, were present in male and female F0 adults administered 0.5 or 5 mg/kg/day. Single cell necrosis of hepatocytes was also present in male mice in the 0.5 mg/kg/day dose group and in the males and females of the 5 mg/kg/day dose group. All other test substance-related microscopic changes in the livers of F0 male and female mice occurred at the 5 mg/kg/day dose level and included increased mitotic figures, lipofuscin pigment, and focal necrosis (females only). Microscopic renal tubular cell hypertrophy was present in F0 males given 0.5 and 5 mg/kg/day. An increase in mean absolute kidney weight was also present in body sexes given 5 mg/kg/day. No test substance-related effects were noted on postnatal survival. Lower mean offspring body weights and body weight gains were noted in the F1 males and females in the 5 mg/kg/day group during the pre-weaning period. Evaluation of the plasma levels of the test substance in dams and their offspring indicate that in mice, in utero exposure of fetuses to the test substance results in plasma concentrations that are less than those of the orally dosed dam, and that little or no exposure to nursing pups occurs during lactation. Also, the plasma kinetics of the test substance in juvenile mice directly dosed beginning at weaning appeared to be equivalent to those of adult mice. Based on these results, the no-observed-adverse-effect level (NOAEL) for reproductive toxicity was 5 mg/kg/day, as no effects on reproduction were observed at any of the doses levels tested. The NOAEL for systemic toxicity in F0 male mice was 0.1 mg/kg/day based on the low incidences of single cell necrosis observed in the liver at 0.5 mg/kg/day. The NOAEL for systemic toxicity in both maternal animals and their offspring was 0.5 mg/kg/day. In maternal animals, this NOAEL was based on microscopic changes in the liver at 5 mg/kg/day. In the offspring, the NOAEL was based on body weight decrements in the F1 males and females in the 5 mg/kg/day group during the pre-weaning period. However, as discussed previously, the mouse has a pharmacokinetically distinct profile as compared to the rat and monkey. Therefore, the mouse is not the appropriate test species for human risk assessment with this test substance.
Oral: NOAEL; OECD 414, rat. NOAEL for developmental toxicity was 10 mg/kg/day based on early deliveries and lower mean fetal weights at 100 mg/kg/day and higher. Reliability = 1.
The objective of the study was to determine the potential of the test substance to induce developmental toxicity after maternal exposure during the critical period of organogenesis, to characterize maternal toxicity at the exposure levels tested, and to determine a no-observed-adverse-effect level (NOAEL) for maternal and developmental toxicity. to 3 groups of 22 bred female Crl:CD(SD) rats once daily from gestation day 6 through 20. Dosage levels were 10, 100, and 1000 mg/kg/day. A concurrent control group of 22 bred females received the vehicle (deionized water) on a comparable regimen.The no-observed-adverse-effect level (NOAEL) for maternal and developmental toxicity was considered to be 10 mg/kg/day based on mortality and lower mean body weight gains and food consumption at 1000 mg/kg/day and early deliveries, microscopic findings in the liver (focal necrosis), and lower mean fetal weights at 100 and 1000 mg/kg/day. At 1000 mg/kg/day, there were additional test substance-related effects that were not considered adverse and consisted of higher kidney and liver weights and hepatocellular hypertrophy. To verify the findings of an apparent dose-related increase in dams that delivered litters just prior to scheduled euthanasia and caesarean section on gestation day 21, a study was conducted using the same experimental design, but was limited to a control group and a group dosed at 1000 mg/kg/day. Under the conditions of the follow-up study, test substance-related findings were observed that were consistent with and confirmed the findings of the original study. Findings included reductions in maternal body weight and food consumption, increased early deliveries, reduced foetal weight, increased maternal liver and kidney weight, microscopic hepatocellular hypertrophy and focal necrosis in the liver. These findings were consistent with those of the original study with the test material at this same dose level.
Female Crl:CD(SD) rats were exposed to the test substance via gavage once daily from gestation day 6 through 20 at doses of 10, 100, and 1000 mg/kg/day . The no-observed-adverse-effect level (NOAEL) for maternal and developmental toxicity was considered to be 10 mg/kg/day based on mortality and lower mean body weight gains and food consumption at 1000 mg/kg/day and early deliveries, microscopic findings in the liver (focal necrosis), and lower mean fetal weights at 100 and 1000 mg/kg/day. At 1000 mg/kg/day, there were additional test substance-related effects that were not considered adverse and consisted of higher kidney and liver weights and hepatocellular hypertrophy.
Justification for selection of Effect on developmental toxicity: via oral route: OECD Guideline, GLP study
The substance has been evaluated in a battery of acute, repeated-dose, and genotoxicity studies. The test substance has low acute toxicity in laboratory animals, and the results of a battery ofin vitroandin vivogenotoxicity studies demonstrate that the substance is not genotoxic. The sentinel effects in these studies, liver toxicity, was consistent with the known effects of PPARαagonists on the rodent liver. Effects on offspring were mostly limited to body weight decrements at maternally toxic doses. Therefore, the substance was not uniquely toxic to the offspring. The substance was not embryolethal or teratogenic in rats and produced no effects on fertility or prenatal development in mice at any of the doses tested, including the maternally toxic doses. In addition, no target organ weight or pathological changes were produced in reproductive or endocrine organs of rats or mice in repeated dose oral studies. Based on these considerations, the substance does not need to be classified for reproductive/developmental toxicity according to EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008. Detailed justification for non classification is provided in the following paragraphs.
Based on the results of biochemical studies (liver β-oxidation) as well as microscopic changes observed in the liver of rodents, the substance is a member of a diverse class of compounds known as peroxisome proliferators. Compounds in this class activate peroxisome proliferator alpha (PPARα). PPARα is a nuclear receptor involved in lipid homeostasis, as well as other physiologic processes. In rodents, peroxisome proliferators characteristically produce effects in the liver including hepatocellular hypertrophy and cell proliferation, as well as liver cell injury. It is well established that compounds in this class produce liver toxicity in rodents and that humans are relative non-responders to these liver effects (Cunningham et al., 2010). As with other peroxisome proliferators, the most sensitive target organ effects following exposure to the substance in rodents occur in the liver and include increased liver weight, microscopic hepatocellular hypertrophy, and liver necrosis.
Developmental and Reproductive Toxicity Studies with the substance
The developmental and reproductive toxicity of the substance has been evaluated in a developmental toxicity study in rats (OECD Guideline 414; OPPTS Guideline 870.3700) and a modified one-generation reproduction toxicity study in mice (OECD Guideline 421; U.S. EPA OPPTS Guideline 870.3550). Consistent with previous toxicity studies with this material, liver toxicity, including liver necrosis was the sentinel effect observed in maternal animals in these studies, with effects on offspring occurring only at maternally toxic doses. Pathological evaluation of reproductive and endocrine organs has also been conducted as part of the 90-day oral studies in rats and mice (DuPont-17751-1026; DuPont-18405-1307). Dose levels in these studies were the same as those used in the respective developmental and one-generation reproductive studies in rats and mice. No test substance-related effects on organ weights or histopathology of endocrine or reproductive organs were observed at any of the doses tested. The result of the developmental and reproductive toxicity studies with the substance are discussed in more detail below.
Developmental Toxicity Study in Rats
In a developmental toxicity study in rats, the substance in deionized water was administered orally by gavage to 3 groups of 22 bred female Crl:CD(SD) rats once daily from gestation day 6 through 20. Dosage levels were 0, 10, 100, and 1000 mg/kg/day. The dosing protocol and end points evaluated were in accordance with OECD Guideline 414 and OPPTS Guideline 870.3700.
Maternal toxicity was present in the 100 and 1000 mg/kg/day dose groups. At 1000 mg/kg/day, test substance-related death occurred in one dam. Test substance-related mortality was also seen in 3 of 20 female rats after 8 - 37 days of treatment with 1000 mg/kg/day in a previous 90-day study with the substance (DuPont-17751-1026), indicating that 1000 mg/kg/day represents an excessively toxic dose level in female rats. In the current developmental toxicity study, liver changes including increased liver weights (34% above control), microscopic hepatocellular hypertrophy (in 19/22 dams) and focal liver necrosis (in 5/22 dams) were also observed in dams at this dose level of 1000 mg/kg/day. Increased liver weight (12.1% above control) and focal liver necrosis (in 2/22 dams) were also present in dams in the 100 mg/kg/day group. The liver changes observed in dams in the 100 and 1000 mg/kg/day dose groups were consistent with the known PPARα activity of the test material and were similar to those seen in the previous 90-day study in rats with the substance. Although clinical chemistry parameters were not evaluated in this study, in the previous 90-day study, changes in some serum lipid and protein parameters (decreased cholesterol and globulin) that are also consistent with a PPARα agonist were observed at 100 and/or 1000 mg/kg/day.
Four of 22 pregnant females in the 100 mg/kg/day group and 9 of 21 females in the 1000 mg/kg/day group delivered early in the morning on gestation day 21. When allowed to deliver naturally, Sprague-Dawley rats normally deliver on gestation day 22 but may range from day 21-23 post coitus (Holsen et al., 2006). The historical mean gestation length in rats (from reproduction studies where natural delivery is allowed to occur) for the conducting laboratory is 21.9 days (n = 97 studies) with a range of 21.5 to 22.3 days. Therefore, the early morning deliveries (prior to scheduled Caesarian section on day 21) in some animals in the 100 and 1000 mg/kg/day groups were considered to be a test substance-related effect manifest as delivery a few hours prior to the more typical delivery time. The significance of this finding is uncertain, as parturition in dams delivering early was otherwise normal, all their offspring were delivered alive, and there was no evidence of increased resorptions compared to those dams sacrificed at the scheduled Caesarian section. Also, while mean fetal weights in the 1000 mg/kg/day group were decreased relative to controls (discussed below), the body weights of fetuses that were delivered early were not different from those of offspring at this same dose level that were delivered by scheduled Caesarian section. The timing of parturition in rats–unlike humans–is luteal-dependant, with progesterone withdrawal as an important initiator. In humans, the best evidence suggests maternal serum progesterone does not play a dominant role in human parturition, and that parturition in humans has significantly different regulators and mediators than rats (Mitchell and Taggert, 2009). Therefore, the slight effects (in hours) on the timing of parturition in rats at these high dose levels is unlikely to be of relevance to humans.
The maternal toxicity noted at 1000 mg/kg/day was associated with decreases in fetal weight (approximately 28% below controls). Fetal weights were also slightly decreased (8.8%) in the 100 mg/kg/day group. The only fetal malformations observed in the study occurred in 2 fetuses from 2 different litters in the 10 mg/kg/day group, and no malformations were observed in the other groups, including the 1000 mg/kg/day group. A higher mean litter proportion of the skeletal variation, 14th rudimentary rib, was observed in the 1000 mg/kg/day group, but this finding was not considered adverse, as 14th rudimentary ribs are resorbed during postnatal development (Holson et al, 2006; Wickramaratne, 1988). No test substance-related developmental variations were observed in the 10 and 100 mg/kg/day groups.
The conclusions drawn from this OECD 414 developmental toxicity study are that the top dose of 1000 mg/kg/day was extremely toxic to the dams, and included lethality and liver toxicity. The mid-dose of 100 mg/kg/day also produced maternal toxicity based on low incidences of liver necrosis. Despite this maternal toxicity, the only developmental toxicity observed was a small decrease in gestation length, but with no evidence of embryolethality or malformations in the embryos. Changes at 100 mg/kg/day were limited to a very slight decrease (less than 10%) in fetal body weight.
Oral (Gavage) Reproduction/Developmental Toxicity Screening Study with the substance in Mice
A modified one-generation reproduction toxicity study was conducted in mice with the test substance (DuPont-18405-1037), but at lower dose levels compared to those used in studies with rats. In mice, as in rats, the most sensitive target organ toxicity following administration of the test substance occurs in the liver, with changes typical of a PPARα agonist. However,both male and female mice show greater sensitivity to these effects compared with the rat. Thus, in a 28-day oral study, changes in female mice given 30 mg/kg/day included increased liver weight (to double the control group mean); 2- to 3-fold elevations in some liver enzymes; microscopic hepatocellular hypertrophy and single cell liver necrosis; and a 9-fold elevation in hepatic β-oxidation activity–an indicator of PPARα activation. Less severe liver changes (a 34% increase in liver weight, minimal to mild hepatocellular hypertrophy, and a 6-fold elevation in hepatic β –oxidation) were noted in females in the 3 mg/kg/day dose group, and no effects were observed at 0.1 mg/kg/day. In contrast, no liver effects were produced in female rats administered 30 mg/kg/day for 28-days. These differences between rats and mice are likely due in large part to pharmacokinetic differences between the species. The elimination half-life of the substance is approximately 10 hours in female mice compared to about 1 hour in female rats, and plasma levels in mice following repeated dosing are approximately 10-fold higher compared with rats. In this regard, rats are more kinetically similar to primates (half-life in female monkeys about 2 hours).
In the modified one-generation reproduction toxicity study in mice (DuPont-18405-1037), the substance in deionized water was administered orally by gavage to groups of 25 male and female Crl:CD1(ICR) mice at dosage levels of 0, 0.1, 0.5, and 5 mg/kg/day. The study was conducted in accordance with OECD Guideline 421 and U.S. EPA OPPTS Guideline 870.3550, but also included a number of enhancements that provided a more comprehensive assessment of fertility and developmental endpoints. These included the following:
No test substance-related effects were observed on F0 reproductive performance (mating, fertility, or copulation indices, and mean days between pairing and coitus), mean gestation length, the process of parturition, mean numbers of implantation sites, or unaccounted-for sites. In addition, there were no effects on the anatomic pathology of reproductive organs at any of the dose levels tested. In dams, systemic toxicity was observed at 5 mg/kg/day (at 0.5 and 5 mg/kg/day in males) and was primarily associated with liver effects. Liver weights in the 5 mg/kg/day female group were markedly increased (up to 2-fold compared to controls in dams), and microscopic changes in the liver at this dose level included hepatocellular hypertrophy (in all dams), single cell liver necrosis (in 21/24 dams) and focal coagulative necrosis (in 5/24 dams). Similar changes were present in males at 0.5 and 5 mg/kg/day. As noted previously, similar liver effects were produced in 28-day, as well as 90-day, studies in mice with the substance (DuPont-18405-1307). In those studies, liver toxicity was further confirmed by increases in liver-related clinical chemistry parameters including alanine amino transferase, sorbitol dehydrogenase, and alkaline phosphatase. These liver changes are consistent with those observed in rodents with other peroxisome proliferators, and as noted previously, are of questionable relevance to humans.
In F1 offspring, the mean numbers of pups born, live litter size, percentage of males at birth, postnatal survival, the general physical condition, and post weaning survival were unaffected by test substance administration at all dosage levels. Effects on offspring occurred only at the high dose (5 mg/kg/day) which also produced maternal toxicity. There were no effects on pup body weights at birth, but lower mean body weights and body weight gains were observed in F1 males and females in the 5 mg/kg/day group during the pre-weaning period. This resulted in slight delays (within laboratory historical control range) in the attainment of balanopreputial separation and vaginal patency of 2.6 days and 3.4 days respectively, at which time the body weight of treated pups were similar to those of controls at attainment (no statistically significant differences in body weight at attainment). Therefore, these delays were attributed to the effects on mean body weight observed in this group during the pre-weaning period and were not considered to be a direct effect of test substance administration (Carney et al., 2004; Ashby and Lefevre, 2000).
The no-observed-adverse-effect level (NOAEL) for effects on fertility was greater than 5 mg/kg/day, the highest dose tested, as no effects on reproduction were observed at any of the doses levels tested. The NOAEL for systemic toxicity was 0.5 mg/kg/day in dams (0.1 mg/kg/day in males) based on liver toxicity at higher doses. No embryolethality, malformation, or effects on offspring survival were observed at any dose levels tested. Primary effects on offspring were limited to decreased body weight gain during the preweaning period and a small and not biologically significant delay in sexual maturation of the pups at the maternally toxic dose of 5 mg/kg/day. Thus the NOAEL for offspring was also 0.5 mg/kg/day.
The substance has been evaluated in a developmental toxicity study in rats and an enhanced one-generation reproduction study in mice. The sentinel effects in these studies, liver toxicity, was consistent with the known effects of PPARα agonists on the rodent liver. Effects on offspring were mostly limited to body weight decrements at maternally toxic doses. Therefore, the substance was not uniquely toxic to the offspring. The substance was not embryolethal or teratogenic in rats and produced no effects on fertility or prenatal development in mice at any of the doses tested, including the maternally toxic doses. In addition, no target organ weight or pathological changes were produced in reproductive or endocrine organs of rats or mice in repeated dose oral studies. Based on these considerations, the substance does not need to be classified for reproductive/developmental toxicity according to EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.
Ashby, J.; Lefevre, P.A. The peripubertal male rat assay as an alternative to the Hershberger castrated male rat assay for the detection of anti-androgens, oestrogens and metabolic modulators. Journal of Applied Toxicology2000, 20, 35-47.
Carney, E.W.; Zablotny, T.L.; Marty, M.S.; Crissman, J.W.; Anderson, P.; Woolhiser, M.; Holsapple, M. The effects of feed restriction during in utero and postnatal development in rats. Toxicological Sciences2004,82, 237-249.
Cunningham, M.L., Collins, B.J., Hejtmancik, M.R., Herbert, R.A., Travlos, G.S., Vallant, M.K., Stout, M.D. Effects of the PPARα agonist and widely used antihyperlipidemic drug gemfibrozil on hepatic toxicity and lipid metabolism. 2010, PPAR Research, Volume 2010, Article ID 681963
DuPont-17751-1026. A 90-day oral (gavage) toxicity study of H-28548 in rats with a
28-day recovery.2009, Unpublished data.
DuPont-18405-1307. H-28548: Subchronic Toxicity 90-Day Gavage Study in Mice.2010, Unpublished data.
DuPont-18405-1037. An Oral (Gavage) Reproduction/Developmental Toxicity Screening Study of H-28548 in Mice.2010, Unpublished data.
Holson, J.F.; Nemec, M.D.; Stump, D.G.; Kaufman, L.E.; Lindstrom, P.; Varsho, B.J. Significance, Reliability, and Interpretation of Developmental and Reproductive Toxicity
Study Findings. In: Developmental and Reproductive Toxicology - A Practical Approach;Hood, R.D., Ed. CRC Press: Boca Raton, FL,2006; p. 381.
Mitchell, B.F. and Taggart, M.J. Are animal models relevant to key aspects of human parturition? Am J Physiol Regul Integr Comp Physiol, 2009, 297: R525–R545.
Wickramaratne, G.A. The post-natal fate of supernumerary ribs in rat teratogenicity studies. Journal of Applied Toxicology1998, 3(2), 91-94.
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