Rats 2-Week Study

All rats survived to the end of the study (Table 2). Final mean body weights and body weight gains of males administered 50 mg/kg or greater and 200 mg/kg females were less than those of the vehicle controls. Irritation at the site of application occurred in all dosed groups except 12.5 mg/kg females. Differences in organ weights were not considered biologically significant (Table F1).

Table 2

Survival and Body Weights of Rats in the 2-Week Dermal Study of Pentaerythritol Triacrylate.

Crusts at the site of application were observed grossly in all dosed groups except 12.5 mg/kg females. Microscopically, lesions occurred at the site of application in all dosed groups (Table 3). In the 12.5 mg/kg group, epidermal hyperplasia, hyperkeratosis, and sebaceous gland hyperplasia were found in all treated rats and were characterized by increased layers of epidermal cells, thickened keratin layer, and prominence of sebaceous glands. More severe lesions occurred at doses of 25 mg/kg or greater and consisted of ulcer (focal full-thickness necrosis of the epidermis), degeneration (vacuolar change of epidermal cells), parakeratosis (retention of nuclei in keratin layer), mixed inflammatory infiltrate of the dermis (chronic active inflammation), and accumulation of neutrophils (suppurative inflammation) in the degenerative and parakeratotic areas of the superficial epidermis. These lesions were not seen in the vehicle controls and their severity generally increased with increasing dose.

Table 3

Incidences of Nonneoplastic Lesions of the Skin (Site of Application) in Rats in the 2-Week Dermal Study of Pentaerythritol Triacrylate.

Dose Selection Rationale: Lesions at the site of application occurred in all dose groups. Their severity generally increased with increasing dose and was moderate to marked in the higher dose groups. The incidences and severity of some of the changes (ulcers, degeneration, and suppurative inflammation) precluded use of 25 mg/kg or greater in a 3-month study; therefore, 0.75, 1.5, 3, 6, and 12 mg/kg were selected for use in the 3-month study in rats.

3-Month Study

All rats survived to the end of the study (Table 4). Final mean body weights and body weight gains of 12 mg/kg males were significantly less than those of the vehicle controls (Table 4 and Figure 1). Irritation at the site of application occurred in 12 mg/kg rats.

Table 4

Survival and Body Weights of Rats in the 3-Month Dermal Study of Pentaerythritol Triacrylate.

Figure 1

Growth Curves for Male and Female Rats Administered Pentaerythritol Triacrylate Dermally for 3 Months.

The hematology and clinical chemistry data for rats in the 3-month dermal study of pentaerythritol triacrylate are listed in Table E1. On day 23, an increase in segmented neutrophils occurred in 12 mg/kg males and females. At study termination, an increased neutrophil count occurred in 6 mg/kg males. The neutrophilia would be consistent with skin inflammation observed microscopically. No other hematology or clinical chemistry changes were considered to be toxicologically relevant.

Thymus weights of males administered 3 mg/kg or greater were significantly less than those of the vehicle controls (Table F2). There were no significant differences in sperm motility or vaginal cytology parameters between dosed groups and the vehicle controls (Tables G1 and G2).

No treatment-related lesions were observed grossly in rats except irritation at the site of application in the 12 mg/kg groups. Microscopically, the primary changes at the site of application consisted of epidermal hyperplasia, hyperkeratosis, epidermal degeneration and necrosis, and chronic active inflammation (Tables 5, D1, and D2). The incidence and severity of hyperplasia increased with increasing dose; most animals administered 3 mg/kg or greater were affected. Severity was minimal to mild and characterized by focally extensive to diffuse increased thickness of the epidermis, from the normal one to three cell layers thick to four to six layers thick. Hyperplasia was accompanied by minimal to mild increased thickness of the superficial keratin layer (hyperkeratosis). Minimal hyperkeratosis without accompanying hyperplasia was present in the 0.75 and 1.5 mg/kg groups. Degeneration was diagnosed in many animals treated with 1.5 mg/kg or greater.

Table 5

Incidences of Selected Nonneoplastic Lesions of the Skin (Site of Application) in Rats in the 3-Month Dermal Study of Pentaerythritol Triacrylate.

Degeneration was a minimal focal change consisting of intraepidermal vacuolization, presumably due to intraor intercellular fluid accumulation. Vacuoles occasionally coalesced to form small vesicles that contained a few neutrophils. Epidermal necrosis was present in some males, although a dose-related response was not clear. Necrosis consisted of partial to full-thickness coagulative change of the epidermis and was likely a pathogenic sequela of degeneration. Intraepidermal infiltration of neutrophils (suppurative inflammation) often accompanied degeneration or necrosis of the epidermis. A mixed inflammatory cell infiltrate (chronic active inflammation) was present in the dermis of animals administered 1.5 mg/kg or greater, with dose-dependent increases in incidences and severity. Sebaceous glands at the site of application were slightly enlarged and prominent in animals with the other changes described above.

Mice 2-Week Study in B6C3F₁ Mice

All mice survived to the end of the study (Table 6). The final mean body weight and body weight gain of 25 mg/kg males were significantly greater than those of the vehicle controls, as was the mean body weight gain of 50 mg/kg males. Irritation at the site of application occurred in all dosed groups. Thymus weights of 50 mg/kg or greater males and of 200 mg/kg females were significantly less than those of the vehicle controls (Table F3).

Table 6

Survival and Body Weights of B6C3F₁ Mice in the 2-Week Dermal Study of Pentaerythritol Triacrylate.

Crusts were observed grossly at the site of application in all dosed groups. Microscopically, lesions were found at the site of application in all dosed groups (Table 7). Epidermal hyperplasia, hyperkeratosis, and sebaceous gland hyperplasia were found in most 12.5 mg/kg mice. These lesions were characterized by increased layers of epidermal cells, a thickened keratin layer, and prominence of sebaceous glands. More severe lesions that tended to occur at higher doses were ulcer (focal full-thickness necrosis of the epidermis), degeneration (vacuolar change of epidermal cells), parakeratosis (retention of nuclei in the keratin layer), dermal chronic active inflammation (mixed inflammatory cell infiltrate), and epidermal suppurative inflammation (accumulation of neutrophils) in the degenerative and parakeratotic areas of the superficial epidermis. These lesions were not seen in the vehicle controls and their severity generally increased with increasing dose.

Table 7

Incidences of Nonneoplastic Lesions of the Skin (Site of Application) in B6C3F₁ Mice in the 2-Week Dermal Study of Pentaerythritol Triacrylate.

Dose Selection Rationale: Lesions at the site of application occurred in all dose groups. Their severity generally increased with increasing dose and was moderate to marked in the higher dose groups. The incidences and severities of skin lesions, particularly ulcer, degeneration, and/or suppurative inflammation, that occurred in dosed mice precluded the use of doses of 25 mg/kg or greater in the 3-month study. Therefore, the doses selected for the 3-month study in B6C3F₁ mice were 0, 0.75, 1.5, 3, 6, and 12 mg/kg.

3-Month Study in B6C3F₁ Mice

One female vehicle control mouse was sacrificed during the first week of the study due to ataxia and one 1.5 mg/kg female died during week 8; all other mice sur vived to the end of the study (Table 8). There were no significant differences in final mean body weights or body weight gains between the dosed and vehicle control groups (Table 8 and Figure 2). Irritation at the site of application occurred in the 6 and 12 mg/kg male groups.

Table 8

Survival and Body Weights of B6C3F₁ Mice in the 3-Month Dermal Study of Pentaerythritol Triacrylate.

Figure 2

Growth Curves for Male and Female B6C3F₁ Mice Administered Pentaerythritol Triacrylate Dermally for 3 Months.

The hematology data for mice in the 3-month dermal study of pentaerythritol triacrylate are listed in Table E2. Similar to the rat study, an increase in segmented neutrophils occurred in the 6 mg/kg male mice and 12 mg/kg male and female mice and would be consistent with the skin inflammation observed microscopically. A minimal decrease (approximately 6% or less) in the hematocrit, hemoglobin concentration, and erythrocyte count occurred in 6 and 12 mg/kg mice and would be consistent with minimal erythropoietic suppression related to inflammation.

There were no biologically significant differences in organ weights between dosed and vehicle control groups (Table F4). The number of spermatid heads per testis in 3 mg/kg males and the numbers of spermatid heads per gram cauda in 6 and 12 mg/kg males were significantly greater than those in the vehicle controls (Table G3); however, these differences were not considered to be biologically significant. There were no significant differences in vaginal cytology parameters between the dosed and vehicle control groups (Table G4).

No lesions were observed grossly in mice except irritation at the site of application in 6 and 12 mg/kg males. Similar to the effects in rats, the primary microscopic changes at the site of application were epidermal hyperplasia, degeneration, and necrosis; dermal chronic active inflammation, and sebaceous gland hyperplasia (Tables 9, D3, and D4). The incidences of epidermal hyperplasia increased with increasing dose, and the severity was generally greater in the 6 and 12 mg/kg groups; most mice administered 3 mg/kg or greater were affected, with lower incidences in lower dose groups. Severity was minimal to moderate and characterized by focally extensive to diffuse increased thickness of the epidermis, from the normal one to three cell layers thick to four to eight layers thickness. Hyperplasia was accompanied by minimal to mild increased thickness of the superficial keratin layer (hyperkeratosis).

Table 9

Incidences of Selected Nonneoplastic Lesions of the Skin (Site of Application) in B6C3F₁ Mice in the 3-Month Dermal Study of Pentaerythritol Triacrylate.

Degeneration was diagnosed in several male mice exposed to 1.5 mg/kg or greater, but there was no clear dose-related response trend. Degeneration was a minimal, focal change consisting of intraepidermal vacuolization, presumably due to intra- or intercellular fluid accumulation. Epidermal necrosis was diagnosed with increasing incidence and severity in male mice at doses of 1.5 mg/kg and greater. Necrosis consisted of partial to full thickness coagulative change of the epidermis and was likely a pathogenic sequela of degeneration. A mixed inflammatory cell infiltrate (chronic active inflammation) was present in the dermis of most animals administered 1.5 mg/kg or greater, with dose-dependent increases in incidences and severity. Slight superficial dermal fibrosis occurred in some 6 and 12 mg/kg mice. Sebaceous glands at the site of application were slightly enlarged and prominent in mice with the other changes described above, an effect that was likely the result of dermal irritation.

Dose Selection Rationale: There were no effects on survival or body weight of B6C3F₁ mice treated with pentaerythritol triacrylate in the 3-month study. In selecting doses for a 2-year dermal administration bioassay based on findings in 3-months studies, dose levels containing any severity of ulceration and necrosis or other changes (e.g. inflammation or hyperplasia) at the skin site of application that are moderate to marked are generally avoided. Therefore, because of the presence of some of these lesions, 6 and 12 mg/kg would not have been selected for use in a 2-year bioassay for the males. The severity of lesions likely would have precluded use of 12 mg/kg in females. The incidence and/or severity of lesions was generally lower in females administered 6 mg/kg or less and males administered 3 mg/kg or less. Because doses were being selected for a different strain (Tg.AC) and for a different duration (6 months), five dose groups were used to allow a margin of error, ranging from doses that produced minimal or no lesions to 12 mg/kg.

6-Month Study in Tg.AC Hemizygous Mice

Estimates of 6-month survival probabilities for male and female Tg.AC hemizygous mice are shown in Table 10 and in the Kaplan-Meier survival curves (Figure 3). Survival of all dosed groups of mice was similar to that of the vehicle controls.

Table 10

Survival of Tg.AC Hemizygous Mice in the 6-Month Dermal Study of Pentaerythritol Triacrylate.

Figure 3

Kaplan-Meier Survival Curves for Male and Female Tg.AC Hemizygous Mice Administered Pentaerythritol Triacrylate Dermally for 6 Months.

Body Weights and Clinical Findings

With the exception of the 3 mg/kg group, body weights of male mice were less than those of the vehicle controls during the last 3 to 6 weeks of the study (Figure 4 and Table 11). Females administered 3 mg/kg had generally reduced body weights during the last month of the study (Figure 4 and Table 12). Treatment-related clinical findings included masses on the torso of males and females administered 3 mg/kg or greater; a single 0.75 mg/kg male and two 1.5 mg/kg males were also affected. Papilloma occurred at the site of application in the 3 mg/kg or greater male and female groups (Table 13).

Figure 4

Growth Curves for Male and Female Tg.AC Hemizygous Mice Administered Pentaerythritol Triacrylate Dermally for 6 Months.

Table 11

Mean Body Weights and Survival of Male Tg.AC Hemizygous Mice in the 6-Month Dermal Study of Pentaerythritol Triacrylate.

Table 12

Mean Body Weights and Survival of Female Tg.AC Hemizygous Mice in the 6-Month Dermal Study of Pentaerythritol Triacrylate.

Table 13

Skin Papilloma Formation at the Site of Application in Tg.AC Hemizygous Mice in the 6-Month Dermal Study of Pentaerythritol Triacrylate.

Papillomas were observed earlier in the 6 and 12 mg/kg groups than in the 3 mg/kg groups. One vehicle control male, two 1.5 mg/kg males, and one 1.5 mg/kg female also had in-life papillomas.

Pathology and Statistical Analyses

This section describes the statistically significant or biologically noteworthy changes in the incidences of neoplasms and nonneoplastic lesions of the skin, liver, spleen, mandibular lymph node, kidney, thymus, and epididymis. Summaries of the incidences of neoplasms and nonneoplastic lesions, individual animal tumor diagnoses, and statistical analyses of primary neoplasms that occurred with an incidence of at least 5% in at least one animal group are presented in Appendix A for male mice and Appendix B for female mice.

Skin: Histologic evaluation revealed that squamous cell papillomas at the site of application were present at the end of the study in all 3 and 6 mg/kg males, most females administered 3 or 6 mg/kg, and most 12 mg/kg males and females (Tables 14, A3, and B3). Two 3 mg/kg and three 12 mg/kg males and one 12 mg/kg female had squamous cell carcinomas at the site of application. Significantly increased incidences of nonneoplastic lesions occurred at the site of application, including hyperkeratosis in 3 mg/kg or greater males and females, chronic active inflammation in 6 and 12 mg/kg males and females and 3 mg/kg females, and epidermal hyperplasia in males and females administered 3 mg/kg or greater; the incidence of epidermal hyperplasia was also significantly increased in 1.5 mg/kg females. The severity of epidermal hyperplasia increased with increasing dose.

Table 14

Incidences of Neoplasms and Nonneoplastic Lesions of the Skin (Site of Application) in Tg.AC Hemizygous Mice in the 6-Month Dermal Study of Pentaerythritol Triacrylate.

Other Organs: Males administered 0.75, 3, or 6 mg/kg had significantly increased incidences of chronic active inflammation of the liver (Table 15). The incidences of hematopoietic cell proliferation of the liver in 12 mg/kg females and of the spleen in 6 and 12 mg/kg males and females were significantly increased. Females administered 12 mg/kg had significantly increased incidences of hematopoietic cell proliferation of the mandibular lymph node and thymocyte necrosis.

Table 15

Incidences of Selected Nonneoplastic Lesions in Tg.AC Hemizygous Mice in the 6-Month Dermal Study of Pentaerythritol Triacrylate.

There was a change that occurred in several animals administered 6 or 12 mg/kg. This change was observed in one or more organs and was characterized by somewhat variable morphology and uncertain biological behavior. Florid lesions, diagnosed as myelodysplasia, were identified in five 12 mg/kg males, while milder lesions, diagnosed as cell, infiltration, nonspecified site, were identified in several animals. The change was characterized predominantly by myeloid infiltration/proliferation that tended to be perivascular in the liver and lungs. Infiltrating cells were predominantly mature and immature granulocytes (eosinophils and neutrophils), with lesser numbers of admixed mononuclear cells. In severely affected livers, there was bridging between portal tracts and accumulations of brightly eosinophilic crystalline material in the lumen of bile ductules. The change was commonly observed in the mediastinal, mandibular, axillary, and mesenteric lymph nodes, and often included a pronounced plasma cell population in the medullary sinuses. The epididymis and spleen were also often involved.

Contact Hypersensitivity Studies in BALB/c Mice

These studies are described in Appendix K. There were no deaths, body weight changes, or clinical findings related to pentaerythritol triacrylate treatment in dosed mice. Results of the irritancy studies of the approximately 10% and 45% pentaerythritol triacrylate mixtures indicated that the maximal nonirritating and minimal irritating doses were 0.1% and 0.25% pentaerythritol triacrylate, respectively, for each mixture (Figures K4 and K5).

The mouse ear swelling test did not indicate the approximately 10% pentaerythritol triacrylate mixture as a contact sensitizer in female BALB/c mice at the doses tested 24 or 48 hours after dosing (Figure K6). Testing of the approximately 45% pentaerythritol triacrylate mixture with the ear swelling test paradigm indicated pentaerythritol triacrylate as a contact sensitizer at 0.1%, with significant increases (compared to background controls) in percent ear swelling noted 24 and 48 hours postchallenge (Figure K7). In the first local lymph node assay using the approximately 10% pentaerythritol triacrylate mixture, a significant increase in lymph node cell proliferation occurred in all dosed groups of mice compared to the vehicle controls; in the second assay using lower sensitizing doses, a significant response occurred only in the 0.05% group (Figure K8). Local lymph node assay of the approximately 45% pentaerythritol triacrylate mixture yielded a positive response only with a sensitizing concentration of 0.25% (Figure K9).

In summary, for both the approximately 10% and 45% pentaerythritol triacrylate mixtures, the maximal nonirritating concentration was determined to be 0.1%, and the minimal irritating concentration to be 0.25%. The mouse ear swelling test yielded negative results for pentaerythritol triacrylate as a potential contact sensitizer when the approximately 10% pentaerythritol triacrylate mixture was used and positive results when the approximately 45% pentaerythritol triacrylate mixture was used. Positive responses were seen in local lymph node assays at concentrations of 0.05%, 0.1%, and 0.25% pentaerythritol triacrylate when the approximately 10% pentaerythritol triacrylate mixture was used and at a concentration of 0.25% pentaerythritol triacrylate when the approximately 45% pentaerythritol triacrylate mixture was used. These data indicate the potential of pentaerythritol triacrylate as a weak contact sensitizer.

Genetic Toxicology

Pentaerythritol triacrylate (33 to 10,000 µg/plate) was not mutagenic in Salmonella typhimurium strain TA98, TA100, TA1535, or TA1537 with or without Aroclor-induced rat or hamster S9 enzymes (Zeiger et al., 1987; Table C1). No increase in the frequency of micronucleated normochromatic erythrocytes (NCEs) was observed in peripheral blood samples from male or female B6C3F₁ mice treated dermally with pentaerythritol triacrylate for 3 months. In contrast, treatment of Tg.AC hemizygous female mice administered pentaerythritol triacrylate dermally for 6 months induced a significant increase in micronucleated NCEs; the trend test analysis was highly significant (P#0.001), and the two highest dose groups showed mean values significantly elevated over the vehicle control frequency. In Tg.AC hemizygous male mice treated with pentaerythritol triacrylate for 6 months, a small increase in micronucleated NCEs was detected; however, the response was judged to be equivocal due to a positive trend test (P=0.001) without any one dose group being significantly elevated over the vehicle control frequency. In both micronucleus studies, the dose range tested was 0.75 to 12 mg/kg. The NCE/polychromatic erythrocyte (PCE) ratios in peripheral blood were not significantly altered by chemical treatment in the 3-month studies, indicating an absence of induced bone marrow toxicity in this group of animals. However, the NCE/PCE ratios in peripheral blood of Tg.AC mice treated for 6 months were significantly altered in the 6 and 12 mg/kg groups. Male and female mice in these dosed groups had markedly elevated levels of immature PCEs in their blood, implying a stimulation of erythropoiesis, perhaps in response to pentaerythritol triacrylate-induced toxicity.