Cancer weight of evidence for three lower acrylates: Conclusions and recommendations from an expert panel.

An international panel of experts was engaged to assess the cancer weight of evidence (WOE) for three lower acrylates: methyl acrylate, ethyl acrylate, and 2-ethylhexyl acrylate. The review was structured as a three-round, modified Delphi format, a systematic process for collecting independent and deliberative input from panel members, and it included procedural elements to reduce bias and groupthink. Based upon the available science, the panel concluded: (1) The MOA for point of contact tumors observed in rodent cancer bioassays that is best supported by available data involves increased cell replication by cytotoxicity and regenerative proliferation; (2) The WOE supports a cancer classification of "Not likely to be carcinogenic to humans" a conclusion that is more in line with an IARC classification of Group 3 rather than Group 2 B; (3) Quantitative cancer potency values based on rodent tumor data are not required for these chemicals; and (4) Human health risk assessment for these chemicals should instead rely on non-cancer, precursor endpoints observed at the point of contact (e.g., hyperplasia). The degree of consensus (consensus scores of 0.84-0.91 out of a maximum score of 1) and degree of confidence (7.7-8.7 out of a maximum score of 10) in the WOE conclusions is considered high.

Dermal carcinogenic activity of petroleum-derived middle distillate fuels.

In general, the carcinogenic potential of petroleum-derived materials is related to the polycyclic aromatic hydrocarbon (PAH) content. Thus it has been assumed that liquids which boil below the PAH distillation range (i.e., below approx. 370 degrees C (700 degrees F) would not be carcinogenic. Several early studies supported this conclusion but were of relatively short duration. Several recent and more rigorous studies have shown that repeated application of certain petroleum-derived materials boiling between approximately 177-370 degrees C (350-700 degrees F) (i.e., middle distillate fuels) can produce tumors in mouse skin. The current studies assessed the tumorigenic potential of a series of middle distillates which varied with respect to boiling range, composition, and source of blending stocks. All of the samples produced evidence of weak tumorigenic activity which was characterized by low tumor yields and long median latencies. However, the majority of the tumor yields were significantly different from the control. There were no apparent differences in response among the samples. Thus the various parameters examined did not substantially influence tumor outcome. In particular, there was no association of tumorigenic activity with aromatic carbon content; this finding, coupled with evidence that PAH levels were low, suggested that the tumorigenic responses were not PAH-dependent. In addition to the tumors, there was evidence of non-neoplastic dermal changes including hyperplasia. These may have contributed to the tumorigenic responses; however, the actual mechanism of tumor induction is unknown.

Alternative approaches in median lethality (LD50) and acute toxicity testing.

The LD50 test was introduced by Trevan in 1927 for biological standardization of dangerous drugs. Since then, the LD50 has gained wide acceptance as a measure of acute toxicity of all types of substances. Recently, however, the LD50 test has been criticized as an unnecessary waste of resources. Therefore, efforts have been made to reduce the number of animals used in such tests and to avoid using this test unless required by regulations. A review of the literature has shown that a relatively small number of animals per dose level (5) and a small number of dose levels (2 or 3) are usually sufficient to calculate an LD50 and slope using moving average methods. In addition, one sex should suffice since large sex differences are seldom encountered. When a formal LD50 is not required, one of several approximate methods may be used to estimate the lethal dose. Future approaches include in vitro cytotoxicity methods and computer-based structure-activity models. The in vitro methods are still in an early stage of development and will require extensive validation before they are accepted by the toxicology community. In conclusion, when LD50 tests are required, the most economical approach should be used, without undue concern for statistical precision.

Subchronic dermal toxicity study of triethanolamine in C3H/HeJ mice.

Triethanolamine (TEA) was applied to the skin of male and female C3H mice (15 per sex per dose group) three times weekly for 95 days (37 applications). TEA was administered at concentrations of 0 (acetone vehicle), 10, 33 and 100% (undiluted) in a volume of 50 microliters. The approximate daily doses of TEA were 0.14, 0.46 or 2.0 g/kg per male and 0.16, 0.54 or 2.3 g/kg per female, respectively. The animals were weighted weekly and observed for clinical signs including skin irritation. 10 mice per sex per dose group were designated for clinical chemistry and haematology at terminal killing. Complete autopsies were performed, and the liver, kidneys, brain, heart, spleen, thymus and testes were weighted. Histopathology was performed on tissues from control and high-dose mice and on target organs. Treatment-related effects were limited to a slight epidermal hyperplasia at the site of application at all TEA concentrations. The results indicate that TEA caused a mild local reaction at all concentrations tested, but did not cause systemic toxicity under these conditions.

Comparison of ketorolac tromethamine with other injectable nonsteroidal anti-inflammatory drugs for pain-on-injection and muscle damage in the rat.

The local tolerance of ketorolac tromethamine (Toradol, Syntex) was compared with that of four other injectable nonsteroidal anti-inflammatory drugs (NSAIDs) (diclofenac sodium, piroxicam, ketoprofen, and metamizol magnesium) in the rat paw-lick/muscle irritation assay as described previously. All drugs were tested at concentrations approved for clinical use. After subplantar (footpad) injection, ketorolac produced virtually no pain-on-injection as assessed by the number of paw-lick/lift responses during a 15 min observation period. The other NSAIDs produced slight to moderate paw-lick/lift responses. Redness and swelling at the injection site were less severe for ketorolac than for the other NSAIDs. After intramuscular (i.m.) injection, all of the NSAIDs produced some degree of muscle damage, as assessed histopathologically 24 h after injection. The lesions, consisting primarily of muscle degeneration, were less severe for ketorolac than for the other NSAIDs. Ketorolac and metamizol produced the smallest elevations in serum creatine kinase, as measured 2 h after i.m. dosing, not significantly different from isotonic saline. Overall, ketorolac was better tolerated in the assay than the other injectable NSAIDs, thereby suggesting the possibility of improved local tolerance on clinical use.

Feedback control of cholesterol biosynthesis in mice fed the liver carcinogens benzidine and 2-acetylaminofluorene.

Dietary feedback control (DFC) of hepatic cholesterol synthesis is absent or defective in hepatomas of trout, mouse, rat and man. DFC has also been shown to be defective in rats fed several liver carcinogens including 2-acetylaminofluorene (AAF). These studies have led to the hypothesis that loss of normal DFC is an early and consistent event in the development of liver cancer. To determine whether DFC was defective in "precancerous" mouse livers, we fed the liver carcinogens AAF (0.05% in chow) and benzidine (0.02% in drinking water) to male BALB/c and C57BL/6 mice for 3 or 6 weeks. We measured sterol synthesis as incorporation of 2-14C-acetate into digitonin-precipitable sterols by liver slices. DFC was tested by adding 2% cholesterol to the diet for 3 days prior to sacrifice. Benzidine (but not AAF) treatment enhanced sterol synthesis in C57BL/6, but not in BALB/c, mice. However DFC was normal in both strains with either carcinogen. The results indicate that defective DFC is not a consistent early finding in animals fed liver carcinogens. Since defective DFC has been a consistent finding in AAF-fed rats, additional research is needed to determine whether the same genetic factors which determine susceptibility to chemically-induced neoplasia are also important in influencing DFC in carcinogen-fed animals.

Comparison of teratogenic effects of aspirin and hydroxyurea in the Fischer 344 and Wistar strains.

The Fischer 344 rat is being used increasingly in toxicology studies. There have been few reports in which rats of this strain were used in teratology and reproduction studies, but comparison of teratologic data with other toxic and points and kinetic information would be greatly facilitated by using the same strain. Therefore, the embryotoxic effects of two positive teratogens, aspirin and hydroxyurea, were compared in Fischer rats and in the commonly used Wistar rats. Aspirin was administered in single oral doses of 500 and 625 mg/kg on d 10; hydroxyurea was injected ip at 500 mg/kg on d 11. Dams were sacrificed on d 20 and fetuses examined for skeletal and visceral defects. Male and female fetal weights and lengths were significantly reduced in treated groups in both strains. Both teratogens caused a significant increase in resorptions in Wistar and Fischer rats. A wide variety of skeletal alterations were induced by both teratogens in both strains. These included extra ribs, fused or missing ribs, extra thoracic and lumbar vertebrae, split vertebral centra, and missing vertebrae. Only a small number of Fischer fetuses exhibited visceral malformations such as hydrocephaly and cleft palate. The frequency of soft-tissue malformations, including cleft palate and severe cardiac anomalies, was much higher in Wistar fetuses.

Feedback control of cholesterol biosynthesis in rats fed the liver carcinogens benzidine and 2-acetylaminofluorene.

Dietary feedback control (DFC) of hepatic cholesterol synthesis is absent in hepatomas of trout, mouse, rat and man. DFC has also been shown to be defective in rats fed several liver carcinogens, including 2-acetylaminofluorene (AAF). These studies have led to the hypothesis that loss of normal DFC is an early and necessary event in the development of liver cancer. To test this hypothesis, we fed the liver carcinogens benzidine (0.006% in drinking water) or AAF (0.05% in chow) to male Sprague-Dawley rats for either 3 or 6 weeks. We measured cholesterol synthesis as the incorporation of 2-14C-acetate into digitonin-precipitable sterols by liver slices. DFC was tested by adding 2% cholesterol to the diet for 3 days prior to sacrifice. DFC was defective in the AAF-fed rats but was normal in the benzidine-treated rats. These results are at variance with the previously stated hypothesis and, for the first time, suggest that defective DFC is not a consistent finding in the early stages of liver carcinogenesis. Additional research is necessary to explain why DFC is altered by exposure to some liver carcinogens such as AAF but unaffected by exposure to others such as benzidine.

Acute and subchronic toxicity studies with detirelix, a luteinizing hormone-releasing hormone antagonist, in the rat and monkey.

Acute (single dose), 2-week, and 3-month toxicology studies were conducted with detirelix, a luteinizing hormone-releasing hormone (LHRH) antagonist, in rats and cynomolgus monkeys. Acute studies were conducted by intravenous and subcutaneous injection. Subchronic studies were conducted by daily subcutaneous injection. Clinical signs after a single intravenous dose included lethargy, edema, cyanosis, pallor, and red ears in rats at greater than or equal to 0.3 mg/kg and lethargy and facial flushing in monkeys at greater than or equal to 0.5 mg/kg. In subchronic studies, detirelix at greater than or equal to 0.4 mg/kg/day (rats) and at greater than or equal to 0.2 mg/kg/day (monkeys) produced atrophy of the reproductive organs, inhibition of ovulation and spermatogenesis, decreased body weight gain in male rats and monkeys, and increased body weight gain in female rats. In the rat, morbidity and/or mortality occurred throughout the treatment phase at a subcutaneous dose of greater than or equal to 2.0 mg/kg/day. In both species, the time to recovery of normal reproductive organ morphology and function was directly related to dose. Exogenous testosterone decreased the severity of reproductive and body weight effects in male rats. In conclusion, the acute effects of detirelix were consistent with peripheral vasodilation. Subchronic effects were associated with inhibition of pituitary gonadotropic and gonadal hormone secretion.

Dermal oncogenicity bioassays of monofunctional and multifunctional acrylates and acrylate-based oligomers.

Several important components of photocurable coatings were studied for dermal tumorigenic activity by repeated application to the skin of mice. The substances tested were 2-ethylhexyl acrylate (EHA) and methylcarbamoyloxyethyl acrylate (MCEA) (monomers); neopentyl glycol diacrylate (NPGDA), esterdiol-204-diacrylate (EDDA), and pentaerythritol tri(tetra)acrylate (PETA) (cross-linkers); and three acrylated urethane oligomers. For each bioassay, 40 C3H/HeJ male mice were dosed 3 times weekly on the dorsal skin for their lifetime with the highest dose of the test agent that caused no local irritation or reduction in body weight gain. Two negative control groups received acetone (diluent) only. A positive control group received 0.2% methylcholanthrene (MC). NPGDA and EHA had significant tumorigenic activity with tumor yields of eight and six tumor-bearing mice (three and two malignancies), respectively. The MC group had 34 mice with carcinomas and 1 additional mouse with a papilloma. MCEA had no dermal tumorigenic activity but resulted in early mortality. No skin tumors in the treatment area were observed in the other groups. Additional studies will be necessary to elucidate possible relationships between structure and tumorigenic activity for the acrylates.

Aldicarb sulfoxide/aldicarb sulfone mixture in drinking water of rats: effects on growth and acetylcholinesterase activity.

A 1:1 mixture of aldicarb sulfoxide/aldicarb sulfone was administered to young adult Wistar rats via the drinking water at nominal concentrations of 19.2, 4.8, 1.2., 0.3, 0.075, or 0 ppm for 29 d. Blood was collected after 8, 15, and 29 d of treatment for plasma and erythrocyte cholinesterase determinations, and brain cholinesterase was determined at sacrifice. Body weight, food intake, and water consumption were measured weekly. Body weight gain and water consumption were reduced at 7, 14, 21, and 29 d in male and female rats at 19.2 ppm. Food consumption was reduced in males at 7, 14, 21, and 29 d but was reduced in females only on d 7. Both plasma and erythrocyte cholinesterase activity were reduced after 8, 15, and 29 d in male and female rats at 19.2 ppm. Males at 4.8 ppm showed reductions in plasma activity only after d 8 and in erythrocyte activity only after d 29. Female rats at 19.2 ppm also displayed depressions in brain cholinesterase activity not observed in similarly treated males. Since the only effects noted at 4.8 ppm were reductions in plasma and red blood cell cholinesterase activity in males only and at only one of three sampling periods, these two instances are not believed to be of any biological significance. The data suggest that 4.8 ppm in drinking water is a no observable ill-effect level for exposure of rats to aldicarb residues based on the parameters measured in this study.

Dermal oncogenicity studies on various ethyleneamines in male C3H mice.

The dermal oncogenic potential of diethylenetriamine, high purity and commercial grades (DETA-HP and DE-TA-C), triethylenetetramine (TETA), tetraethylenepentamine (TEPA), and polyamine HPA No. 2 was assessed by applying 25-microliter aliquots of aqueous solutions to the skin of groups of 50 male C3H/HeJ mice. The concentrations applied were 5.0, 5.0, 5.0, 25.0, and 10.0% by volume for DETA-HP, DETA-C, TETA, TEPA, and HPA No. 2, respectively. Applications were made thrice weekly until the death of the animals. A negative control group received deionized water (solvent) only. All animals were individually housed. No treatment-related skin tumors were observed, nor was there evidence of increased incidence of any internal tumor. Twenty TEPA-treated mice had hyperkeratosis and 13 had necrosis of the epidermis, both indicative of skin irritation. Such lesions were absent or occurred very infrequently in the other groups of mice. The mean survival times were 587, 662, 627, 591, 601, and 626 days for the DETA-HP, DETA-C, TETA, TEPA, HPA No. 2, and water control groups, respectively. In no case was the mortality rate significantly different from that of the controls. The results indicate that none of these compounds was oncogenic under the conditions of these studies.

Evaluation of the teratogenicity of ethylenediamine dihydrochloride in Fischer 344 rats by conventional and pair-feeding studies.

Ethylenediamine dihydrochloride (EDA.2HCl) was fed to groups of 20 (40 controls) timed-pregnant rats on Gestation Days 6 through 15 at 1.0, 0.25, 0.05, or 0 g/kg/day. The day of discovery of a vaginal plug was considered gestation day 0. On Gestation Day 21, the fetuses were delivered by cesarean section, and the standard endpoints for teratogenicity were evaluated. At 1.0 and 0.25 g/kg/day, reductions in maternal diet consumption and weight gain were observed during the exposure period. At 1.0 g/kg/day, fetal weight and crown-rump length were significantly reduced and the percentage of litters with resorptions, with skeletal variants, and with missing or shortened innominate arteries was increased. To circumvent the possible problem of palatability of the diet and/or the effects of reduced food intake, a probe study was performed in which 1.0 or 0 g/kg/day was given to pregnant rats by gavage on Gestation Days 6 through 15. However, food intake was also substantially reduced with gavage dosing of the test substance. To determine whether the arterial defects were the result of reduced food intake, a pair-feeding study was performed in which EDA.2HCl was fed on Gestation Days 6 through 15 at 1.0 g/kg/day. A pair-fed control group received the same amount of diet consumed by the EDA.2HCl-treated rats. An untreated control group was fed ad libitum. All groups contained 20 pregnant females. Maternal weight gain, fetal weight and length, and the length of the innominate artery were all reduced in the EDA.2HCl-treated group compared to both control groups. Two fetuses each in the EDA.2HCl-treated and pair-fed control groups had missing innominate arteries versus none in the untreated controls. The four affected fetuses were from four different litters. Ingestion of EDA.2HCl resulted in reduced maternal weight gain, fetal size, and length of the innominate artery, but the missing innominate artery was not a result of EDA.2HCl treatment. Therefore, there was no evidence of teratogenicity in Fischer 344 rats from EDA.2HCl ingestion during organogenesis.

Dermal oncogenicity bioassays of di-sec-butoxyacetophenone and diethoxyacetophenone.

Groups of 40 male C3H/HeJ mice were treated 3 times per week for their lifetime on the skin of the back with undiluted di-sec-butoxyacetophenone (DBAP), or with diethoxyacetophenone (DEAP), undiluted and as a 50% dilution in acetone. Approximate doses per application were 22.4 mg for DBAP, and 25.0 or 11.3 mg for DEAP. Two negative control groups received acetone only. Both DBAP and DEAP (undiluted) had weak tumorigenic activity. In the DBAP group, one mouse developed a squamous-cell papilloma, whereas one mouse in the high-dose DEAP group had a squamous-cell carcinoma, both tumors appearing in the treatment area. No skin tumors were observed in the other groups. Both DEAP-treated groups also had substantial incidences of hyperkeratosis, epidermal hyperplasia, and dermatitis. No significant reduction in survival was observed in the test groups. The occurrence of the skin tumors in the DBAP- and DEAP-treated groups is considered to be treatment-related and suggestive of oncogenic potential, because of the extremely low historical control incidence of skin tumors in similar studies.

Dermal oncogenicity studies on ethylenediamine in male C3H mice.

The dermal oncogenic potential of ethylenediamine (EDA) was assessed by applying 25 microliter of a 1% solution in deionized water to the skin of 50 male C3H/HeJ mice. This was the highest concentration not producing irritation or weight changes in a preliminary 2-week study. Two EDA samples (Nos. 1 and 2) from different production sources were tested. Applications were made thrice weekly until the death of the animals. A negative control group received deionized water only. This group and the EDA-treated groups were individually housed. A fourth group of 40 mice, housed 5 per cage, received 0.1% 3-methylcholanthrene (MC) in acetone as a positive control substance. A fifth group of 40 mice, housed 5 per cage, also received deionized water to determine the effect of group housing on survival. No skin tumors were observed in the EDA-treated groups. In the positive control group, however, 39 animals (98%) had skin tumors including 37 (92%) with confirmed squamous cell carcinomas. Eleven mice (22%) which received EDA No. 1 had dermal fibrosis indicative of probable skin irritation in this group; there was no such lesion in the controls. The mean survival times were 639, 626, and 598 days for the EDA No. 1, water control, and EDA No. 2 groups, respectively. The survival time of the EDA No. 2 group was significantly reduced compared to the individually housed water controls by one of two statistical tests. Irrespective of this difference, the study is considered to be a valid assessment of the oncogenic potential of EDA No. 2 because the magnitude of the difference in mean survival time was small.(ABSTRACT TRUNCATED AT 250 WORDS)

Three-generation reproduction and dominant lethal mutagenesis studies of ethylene glycol in the rat.

To assess the possible effects of ethylene glycol (EG) on reproductive performance and mutagenesis, three-generation reproduction and dominant lethal mutagenesis studies were performed in the Fischer 344 rat. EG was included in the diet at approximate dosages of 1.0, 0.2, and 0.04 g/kg/day during three generations of reproduction. Each generation was bred once. In a dominant lethal mutagenesis study, the F2 males from the reproduction study were bred to three consecutive lots of untreated females at weekly intervals. Concomitantly, another group of untreated F2 males that received a single ip injection of 0.50 mg/kg triethylenemelamine (TEM) were bred similarly to serve as a positive control group. No evidence of reduced fertility or increased fetal death was observed in any of the groups receiving EG. Dominant lethal effects in the TEM group confirmed the susceptibility of the rats to a known mutagen. In conclusion, there were no reproductive or dominant lethal effects associated with the inclusion of as much as 1.0 g/kg/day of EG in the diet.

Dermal oncogenicity studies on two methoxysilanes and two ethoxysilanes in male C3H mice.

The dermal oncogenic potential of beta-(3,4-epoxycyclohexyl)ethyltrimethoxysilane (EEMS), gamma-glycidoxypropyltrimethoxysilane (GPMS), beta-(3,4-epoxycyclohexyl)ethyltriethoxysilane (EEES), and gamma-glycidoxypropyltriethoxysilane (GPES) was assessed by applying 25-microliters aliquots of acetone solutions to the skin of 40 male C3H/HeJ mice. The concentrations applied were 100, 25, 10, and 10% by volume for EEMS, GPMS, EEES, and GPES, respectively. Applications were made thrice weekly until the death of the animals. A negative control group received acetone (solvent) only. No treatment-related skin tumors were observed, nor was there evidence of increased incidence of any internal tumor in the groups that received GPMS, EEES, or GPES. In the group treated with EEMS, four mice were observed with squamous cell carcinomas of the treated skin and two mice had subcutaneous sarcomas outside of the treated area. No skin tumors were observed in the group treated with acetone, but two mice had subcutaneous sarcomas outside of the treated area. The mean survival times were 529, 482, 545, 492, and 502 days for the EEMS, GPMS, EEES, GPES, and acetone control groups, respectively. In no case was the mortality rate significantly different from that of the controls. The results indicate that only EEMS was oncogenic under the conditions of these studies.

False positive result for a peptide drug in the gene conversion assay with Saccharomyces cerevisiae strain D7.

A battery of mutagenicity tests was performed with nafarelin, an agonist analogue of luteinizing hormone releasing hormone (LHRH) containing tryptophan (Trp) and histidine (His). Included were the Ames assay and the gene conversion assay with yeast strain D7. Both tests were negative without S9 activation, and the Ames test was negative with S9, but the yeast test was positive with S9 activation. Since the yeast test is based on conversion of cells to Trp independence, release of Trp by metabolism of the drug could account for the positive result. The test was repeated using Trp instead of the drug. The result was positive even at the lowest Trp concentration. In another experiment with the drug, amino acid analysis of the incubation mixture revealed the presence of Trp but no detectable His. Since the Ames test is based on mutation to His-independent cells, these data are completely consistent with the negative result in the Ames test and the false positive result in the yeast test. These data suggest the need for caution in interpreting the results from mutagenicity assays with peptide drugs.

Dermal oncogenicity bioassays of acrylic acid, ethyl acrylate, and butyl acrylate.

Male C3H/HeJ mice (40 per group) were treated with 25-microliter applications of undiluted ethyl acrylate, 1% acrylic acid, or 1% butyl acrylate on the dorsal skin 3 times weekly for their lifetime. A negative control group received acetone (diluent) only, and a positive control group received 0.1% 3-methylcholanthrene (MC). No epidermal tumors were observed in the animals that received any of the three test substances. In the positive control group, 39 animals had skin tumors, including 33 with confirmed squamous-cell carcinomas. Nonneoplastic skin changes such as dermatitis, dermal fibrosis, epidermal necrosis, and hyperkeratosis were observed in several mice that received ethyl acrylate. No statistically significant effects on survival were seen. Therefore, there was no evidence for local carcinogenic activity of acrylic acid, ethyl acrylate, or butyl acrylate under the conditions of these studies.

Subchronic and reproductive toxicology studies on acrylic acid in the drinking water of the rat.

In the subchronic study acrylic acid was incorporated in the drinking water of Fischer 344 rats (15 per group) for three months at dosage levels of 0.75, 0.25, 0.083 or 0 g/kg/day. No deaths occurred during the study but several treatment-related effects were observed. At 0.75 g/kg/day dosage level, body weight gain was reduced as were food and water consumption. Changes in organ weights and clinical chemistry parameters were observed along with increases in urine specific gravity and protein concentration. Similar, though less profound, changes occurred at 0.25 g/kg/day. At 0.083 g/kg/day, the only effect was a reduction of water consumption by male rats. There were no significant treatment-related histopathologic changes. Many of the effects observed may have been the result of decreased water and food consumption rather than specific toxic effects of acrylic acid. In the reproduction study, the same dosages of acrylic acid were given to groups of 10 males and 20 females for 90 days after which the animals were mated. Treatment was continued throughout gestation and lactation. Treatment-related effects included decreased body weight gain, and reduced food and water consumption in F0 rats at the 2 highest dosage levels. Organ weight changes occurred in both F0 and F1 animals and reduced body weight gain was seen in the F1 pups at the highest level. No statistically significant changes in reproductive indices were observed, perhaps because of an atypical control group.