Robert M. Diener

The Toxicity Profile of Hydrolyzed Aqueous Olive Pulp Extract

The toxicity profile of HIDROX™ (Hydrolyzed Aqueous Olive Pulp Extract; OPE) was characterized in a series of toxicology studies. A limit dosage of 2000 mg/kg produced no toxicity in mice (acute oral NOAEL: 2000 mg/kg). In rats, an acute oral NOAEL of 2000 mg/kg was established, based on reductions in weight gains in both sexes at 5000 mg/kg. Reduced gains in female rats at 1500 and 2000 mg/kg were not significantly different from control values. Daily oral dosages of 1000, 1500 and 2000 mg/kg/day for 90 days produced small decreases in body weight gains at 2000 mg/kg/day in the male rats and in all groups of female rats. Feed consumption was comparable to controls. There were no adverse clinical, hematologic, biochemical, organ weight or gross necropsy effects. Focal, minimal or mild hyperplasia of the mucosal squamous epithelium of the limiting ridge of the forestomach occurred in some rats at 2000 mg/kg/day; this change was attributed to local irritation by repeated intubation of large volumes of viscous, granular dosing suspension. A NOAEL of 2000 mg/kg/day was established for the 90‐day study, based on the lack of significant adverse effects. Toxicokinetic data indicated that hydroxytyrosol (HT, the major component of OPE) was rapidly absorbed. Mean concentrations were measurable through 1 to 4 hours (tlast) at 1000 and 1500 mg/kg/day and through 8 hours at 2000 mg/kg/day. Dosages of OPE ranging from 500 to 2000 mg/kg/day did not adversely affect any of the mating, fertility, delivery or litter parameters investigated in an oral rat dosage‐range reproduction study. Adverse effects were also absent in a rat developmental toxicity study in which pregnant dams were treated with 1000, 1500 or 2000 mg/kg/day on days 6 through 20 of gestation. Plasma levels for pregnant and lactating rats were comparable to non‐pregnant rats; minimal levels crossed the placenta. Quantifiable levels were not identified in maternal milk or plasma from nursing pups. A bacterial reverse mutation and a CHO chromosome aberration assay revealed evidence of mutagenic activity at high dosages with S9 metabolic activation. However, three rat micronucleus evaluations performed after single and repeated (28‐day) dosages of up to 2000 mg/kg/day and dosages of 5000 mg/kg/day for 29 days resulted in negative findings; therefore, OPE was not considered to be mutagenic in this in vivo assay.

Evaluation of the Developmental Toxicity of Linalool in Rats

The developmental toxicity of linalool, a widely used fragrance ingredient, was evaluated in presumed pregnant Sprague-Dawley rats (25/group). Oral dosages of 0, 250, 500, or 1000 mg/kg/day linalool were administered by gavage on gestational days 7 to 17. The presence of spermatozoa and/or a copulatory plug in situ was designated as gestational day 0. Rats were observed for viability, clinical signs, body weights, and feed consumption. Caesarean sectioning and necropsy occurred on gestational day 21. Uteri were examined for number and distribution of implantations, live and dead fetuses, and early and late resorptions. Numbers of corpora lutea were also recorded. Fetuses were weighed and examined for gender, gross external changes, and soft tissue or skeletal alterations. There were no maternal deaths, clinical signs, or gross lesions that were considered related to linalool. During the dosage period, mean relative feed consumption was significantly reduced by 7% and mean body weight gains were reduced by 11% at 1000 mg/kg/day. During the postdosage period, feed consumption values at 1000 mg/kg/day were significantly higher than vehicle control values, which corresponded to the increase in body weight gains during this period. Caesarean section and litter parameters, as well as fetal alterations, were not affected by linalool at any of the three dosages tested. On the basis of these data, the maternal no observed adverse effect level (NOAEL) of linalool is 500 mg/kg/day, whereas the developmental NOAEL is ≥ 1000 mg/kg/day. It is concluded that linalool is not a developmental toxicant in rats at maternal doses of up to 1000 mg/kg/day.

Developmental toxicity studies of four fragrances in rats.

Four fragrances, 6-acetyl-1,1,2,4,4,7-hexamethyltetraline (AHTN), 1,3,4,6,7,8-hexahydro-4,6,6,7,8,8-hexamethylcyclopenta-gamma-2-ben zopyran (HHCB), musk ketone and musk xylene were tested for developmental toxicity in Sprague-Dawley rats (25/group, 3 groups/fragrance, 2 fragrances/corn oil control). Dosages tested were HHCB: 50, 150, 500 mg/kg per day; AHTN: 5, 15, 50 mg/kg per day; musk ketone: 15, 45, 150 mg/kg per day; musk xylene: 20, 60, 200 mg/kg per day. All dosages tested exceeded multiples of the estimated maximal daily human dermal exposure. Treatment (gavage, 5 ml/kg) occurred on GDs 7-17 and Caesarean-sectioning on GD 20. Based on the results of these studies, none of the four fragrances tested were more toxic in the conceptuses than in the dams. Maternal NOAELs were 50, 5, 15 and 20 mg/kg per day for HHCB, AHTN, musk ketone and musk xylene, respectively (150, 50, 45 and 60 mg/kg per day caused clinical signs and reduced weight gain and feed consumption). Developmental NOAELs were 150, 50, 45 and 200 mg/kg per day for HHCB, AHTN, musk ketone and musk xylene, respectively. No adverse effects on embryo-fetal viability, growth or morphology occurred at the highest dosages of AHTN (50 mg/kg per day) or musk xylene (200 mg/kg per day). Developmental toxicity occurred at the high-dosages of HHCB (axial skeletal malformations at 500 mg/kg per day) and musk ketone (increased postimplantation loss and reduced fetal body weight at 150 mg/kg per day). The results of this study indicate that under conditions of normal use, the tested fragrances do not pose a risk to human conceptuses.

Oral (drinking water) developmental toxicity studies of bromodichloromethane (BDCM) in rats and rabbits.

Crl:CD(SD)IGS BR VAF/Plus (Crl SD) rats and Hra(NZW) SPF rabbits were tested for potential developmental toxicity from bromodichloromethane (BDCM) provided continuously in the drinking water during gestation (gestation days [GDs] 6 to 21 in rats and GDs 6 to 29 in rabbits). Concentrations of 0, 50, 150, 450, or 900 ppm of BDCM were used for rats; 0, 15, 150, 450, or 900 ppm were used for rabbits (in dose range-finding studies, 1350 ppm was excessively maternotoxic to both species). Investigated maternal parameters included viability, clinical signs, water and feed consumption, and body weights. Maternal gross lesions, gravid uterine weights, abnormal placentas, and numbers of corpora lutea, implantation sites, live and dead fetuses, and early and late resorptions were observed at time of Caesarean sectioning (GD 21 in rats; GD 29 in rabbits). Body weights, sex ratios, and morphological abnormalities (external, soft tissue, and skeletal) were noted in the fetuses. Mean consumed doses of BDCM were calculated to be 0, 2.2, 18.4, 45.0, or 82.0 mg/kg/day for the rats, and 0, 1.4, 13.4, 35.6, or 55.3 mg/kg/day for the rabbits (approximate human intake is 0.8 microg/kg/day [0.0008 mg/kg/day] in adults). In pregnant rats, toxicologically important, statistically significant effects included reduced absolute (g/day) and relative (g/kg/day) water consumption values at > or =50 ppm (2.2 mg/kg/day) and reduced body weight gains (also when corrected for gravid uterine weight) and absolute (g/day) and relative (g/kg/day) feed consumption values at >450 ppm (45.0 mg/kg/day). These parameters were also significantly reduced at > or =450 ppm (35.6 mg/kg/day) in pregnant rabbits (significant weight loss occurred in the rabbits at 900 ppm, i.e., 55.3 mg/kg/day). Thus, the maternal no-observable-adverse-effect level (NOAEL) for BDCM was 150 ppm, i.e., 18.4 and 13.4 mg/kg/day in rats and rabbits, respectively. No adverse effects on embryofetal viability, growth, sex ratio, gross external, soft tissue, or skeletal morphology occurred at 900 ppm in rats or rabbits. Minimal delays in the ossification of forepaw phalanges and hindpaw metatarsals and phalanges occurred in rat fetuses at 900 ppm; delays were considered marginal, reversible, and associated with severely reduced maternal weight gain. Therefore, the developmental NOAEL for rats was 450 ppm (45.0 mg/kg/day), whereas in rabbits it was 900 ppm (55.3 mg/kg/day). These NOAELs are 56,250 and 69,120 times the human adult exposure level of 0.0008 mg/kg/day, respectively. Based on the results of these studies, BDCM should not be identified as a risk to development of human conceptuses.

Soaps and Detergents: Alternatives to Animal Eye Irritation Tests

An extensive computer search was conducted, and a comprehensive overview of the current status of alternatives to animal eye irritation tests was obtained. A search of Medline and Toxline databases (1988 to present) was supplemented with references from sources regarding in vitro eye irritation. Particular attention was paid to soap and detergent products and related ingredients. Eighty-five references are included in the review; the in vitro assays are categorized, and their predictive values for assessing acute ocular irritation are evaluated and compared with the Draize rabbit eye irritation assay and with each other. The present review shows that the increased activity of scientists from academia, industry, and regulatory agencies has resulted in substantial progress in developing alternative in vitro procedures and that a number of large, interlaboratory evaluations and international workshops have assisted in the selection process. However, none of these methodologies has obtained acceptance for regulatory classification purposes. Conclusions drawn from this review include that (a) no single in vitro assay is considered capable of replacing the Draize eye irritation test; (b) the chorioallantoic membrane vascular assay (CAMVA) or the hen egg test-chorio-allantoic membrane test (HET-CAM), the chicken or bovine enucleated eye test, the neutral red and plasminogen activation assays for cytotoxicity, and the silicon microphysiometer appear to have the greatest potential as screening tools for eye irritation; and (c) choosing a specific assay or series of assays will depend on the type of agent tested and the impact of false-negative or false-positive results. New assays will continue to be developed and should be included in future evaluations, when sufficient data are available.

Oral and dermal developmental toxicity studies of phenylethyl alcohol in rats.

Phenylethyl alcohol (PEA) was tested for developmental toxicity. Pregnant rats were fed 0, 83, 266, or 799 mg/kg/d PEA on gestation days (GDs) 6 to 15; only minimal, nonsignificant effects were observed. In dermal studies, PEA (neat) was applied to the skin on GDs 6 to 15 at dosages of 0, 140, 430, or 1400 mg/kg/d and at 0, 70, 140, 280, 430, or 700 mg/kg/d in a corroborative study. Observations included maternal and embryo-fetal toxicity/abnormalities at 1400 mg/kg/d, increased incidences of rudimentary cervical ribs at ≥430 mg/kg/d, and reduced fetal body weights at ≥140 mg/kg/d. Dermal maternal and developmental no-observed-adverse-effect levels are 70 mg/kg/d, based on dermal irritation and reductions (nonsignificant) in fetal body weights. Human exposure from fragrances is 0.02 mg/kg/d, resulting in a margin of safety >2600, when marked differences in dermal absorption between rats and humans are considered. Under normal fragrance use conditions, PEA is not a developmental toxicity hazard for humans.

Safety Evaluation of ABELCET, an Amphotericin B Lipid Complex (ABLC): Toxicity Studies in Rats:

ABELCET (ABLC) is a widely used amphotericin B lipid complex formulation that is approved for use in the treatment of invasive fungal infections in patients who are refractory or intolerant of conventional amphotericin B (AmB). The safety profile of ABLC has been characterized in two acute and two repeat-dose toxicity studies in rats. The acute toxicity studies indicated that single intravenous doses of ABLC are at least 20 times less toxic than conventional amphotericin B doses without the lipid formulation, Fungizone. Intravenous doses of 0, 1, 3, or 10 mg/kg/day to groups of rats (10 to 15 rats/sex/group) for 31 days elicited no mortality or overt clinical signs of toxicity, whereas alternate intravenous/intraperitoneal doses (three each per week) for 6 months, produced one death in the control group, one in the intermediate-dose group, and two in the high-dose group. Clinical signs (predominantly piloerection and hunched posture at 10 mg/kg/day) were attributed to granulomatous inflammatory lesions in the abdominal wall, mesentery, and omentum, which were produced by the intraperitoneal injections of ABLC. Feed consumption and body weight gains decreased in high-dose male rats in the one-month study and were significantly lower in male rats at 3 and 10 mg/kg/day in the 6-month study. In contrast, water consumption increased in male and female rats in both studies. Trends of minimal to moderate, dose-related increases in relative kidney, liver and spleen weights, and histological evidence of hypertrophy and hyperplasia of reticuloendothelial cells in the liver and spleen and mild, dose-related impairment of renal function occurred in both the 1- and 6-month studies. Examination of high-dose rats following a recovery period of 28 days after completion of 31 days of dosing suggested that treatment-related changes were reversible. The observed changes for ABLC are similar to those for other amphotericin B lipid formulations, such as AmBisome (LAmB), except for the hepatoxicity, which was observed for LAmB, but not for ABLC.

The Pharmacokinetics of Phenylethyl Alcohol (PEA) Safety Evaluation Comparisons in Rats, Rabbits, and Humans

The present studies were conducted to compare the dermal absorption, plasma pharmacokinetics, and excretion of phenylethyl alcohol (PEA) by pregnant and nonpregnant rats, rabbits, and humans. The PEA is a natural fragrance material that is widely used in perfumes, soaps, and lotions and is a major ingredient of natural rose oil. Following dermal (430, 700, or 1400 mg/kg body weight [bw]), gavage (430 mg/kg bw), or dietary (430 mg/kg bw) administration of PEA to rats, plasma concentrations of PEA were found to be low regardless of the route of administration. The plasma concentrations of phenylacetic acid (PAA, the major metabolite of PEA) greatly exceeded the concentrations of PEA and were highest after gavage, followed by dermal then dietary administration. Absorption, distribution, metabolism, and excretion were compared following topical application of 14C-labeled PEA to rats, rabbits, and humans (specific activities of dosing solutions: 58-580, 164, and 50 µCi/mL, respectively). In rabbits, the plasma concentration–time profile for PAA was markedly prolonged compared to rats or humans. In humans, only 7.6% of the applied dose of PEA was absorbed, versus 77% in rats and 50% in rabbits. Based on a human dermal systemic exposure of 0.3 mg/kg per day from the use of multiple consumer personal care products containing PEA, a rat dermal no observed adverse effect level of 70 mg/kg per day, and the percentage of dose absorbed in humans, the margin of safety exceeds 2600 concluding that, under normal fragrance use conditions, PEA is not a developmental toxicity hazard for humans.

Evaluation of the Developmental Toxicity of Dihydromyrcenol in Rats

Dihydromyrcenol, a widely used fragrance ingredient, was evaluated for developmental toxicity in pregnant Sprague-Dawley rats (25/group). Oral dosages of 0, 250, 500, or 1000 mg/kg/d in corn oil were administered on gestational days 7 to 17. Rats were observed for viability, clinical signs, body weights, and feed consumption. Caesarean sectioning and necropsy occurred on gestational day 21. Fetuses were weighed and examined for sex, gross external changes, and soft tissue or skeletal alterations. All rats survived until scheduled termination. No clinical signs were attributed to dihydromyrcenol. There were no gross tissue changes at necropsy. The 1000-mg/kg/d dosage group had reduced mean maternal body weight gains of 5% compared with controls, whereas absolute and relative feed consumption were significantly reduced during the dosage period. This threshold systemic maternal toxicity was associated with threshold developmental toxicity in the 1000-mg/kg/d dosage group. Fetal effects included a minimal ∼3% reduction in fetal body weight; reversible variations in ossification, including retarded ossification of the metatarsal bones in the hindpaws; and an increase in supernumerary thoracic ribs with associated increases or decreases in thoracic and lumbar vertebrae, respectively. Based on these data, maternal and developmental no observable effect levels of 500 mg/kg/d and maternal and developmental no observable adverse effect levels of 1000 mg/kg/d were established for dihydromyrcenol. It was concluded that dihydromyrcenol is not a selective developmental toxicant in rats under the conditions of this study and that a margin of safety of 25 000 exists between reversible developmental delays in rats and the estimated daily human exposure level of 0.02 mg/kg/d.

Evaluation of the Developmental Toxicity of Methyl Dihydrojasmonate (MDJ) in Rats

Methyl dihydrojasmonate (MDJ) is a widely used fragrance ingredient. MDJ was evaluated for developmental toxicity in presumed pregnant Sprague-Dawley rats (25/group) at oral dosages of 0, 40, 80 or 120 mg/kg/day in corn oil administered on gestational days 7–20. Dams were observed for viability, clinical signs, body weights, and feed consumption. Caesarean-sectioning and necropsy occurred on gestational day 21. Fetuses were weighed and examined for gender, gross external changes, and soft tissue or skeletal alterations. No maternal or fetal deaths occurred. MDJ-related maternal clinical signs included an increased incidence of sparse hair coat and ungroomed appearance at 120 mg/kg/day. Two dams in this group also had tan areas in the liver and a pale spleen. The 120 mg/kg/day dosage also caused reduced mean maternal body weight gains and body weights during the dosage period and reduced absolute and relative maternal feed consumption for the entire dosage period. No Caesarean-sectioning or litter parameters were affected by dosages of MDJ as high as 120 mg/kg/day, although at the highest dosage a tendency toward slightly reduced, but not statistically significant, fetal mean body weight was observed. No fetal gross external, soft tissue or skeletal changes were attributable to dosages of MDJ as high as 120 mg/kg/day. Based on these data, maternal No-Observable-Adverse-Effect-Level (NOAEL) of 80 and developmental NOAEL of equal to or greater than 120 mg/kg/day were established for MDJ. It is concluded that MDJ is not a developmental toxicant in rats under the conditions of this study.