Corneal epithelial cell viability following exposure to ophthalmic solutions containing preservatives and/or antihypertensive agents.

INTRODUCTION: This in-vitro study compared the toxicity of bimatoprost 0.01% containing benzalkonium chloride (BAK) 0.02% with other commercial BAK-free or BAK-containing prostaglandin analogs. METHODS: Six test solutions were evaluated: travoprost 0.004% with polyquaternium-1 0.001% (PQ), PQ, bimatoprost 0.01% with BAK 0.02%, latanoprost 0.005% with BAK 0.02%, tafluprost 0.0015% preservative free (PF), and BAK 0.02%. Phosphate-buffered saline (PBS) was the live control and 70% methanol was the dead control. Confluent human corneal epithelial cells were incubated with test solutions (diluted 1:5 or 1:10 with PBS) or control solutions for 10 or 25 min, after which cells were fluorescently labeled to distinguish live and dead cells. Data were expressed as a percentage of PBS live-cell fluorescence for automated readouts. Live and dead cells were manually counted for numeric analyses. RESULTS: For 1:5 and 1:10 dilutions using automated readout, cells exposed to bimatoprost with BAK, latanoprost with BAK, and BAK alone demonstrated significant reductions in the live cell signal compared with PBS, travoprost with PQ, and PQ alone (all P < 0.001). They also demonstrated significantly greater toxicity than tafluprost PF for 1:5 dilutions (all P < 0.001) and 1:10 dilutions (P ≤ 0.02), except for 1:10-diluted bimatoprost with BAK (P = 0.41). For 1:5 dilutions using manual cell count, cells exposed to bimatoprost with BAK demonstrated significant reductions in the percentage of live cells compared with PBS (P = 0.02). For 1:10 dilutions using manual cell count, cells exposed to bimatoprost with BAK, latanoprost with BAK, and BAK alone demonstrated significantly greater toxicity than PBS, travoprost with PQ, PQ alone, and tafluprost PF (all P ≤ 0.03). No significant differences were observed among PBS, travoprost with PQ, and PQ alone under any test conditions (P ≤ 0.63). CONCLUSION: This study demonstrated that BAKcontaining solutions, including bimatoprost 0.01% with BAK, were toxic to human corneal epithelial cells, whereas BAK-free solutions showed little to no evidence of toxicity.

Reduced in vivo ocular surface toxicity with polyquad-preserved travoprost versus benzalkonium-preserved travoprost or latanoprost ophthalmic solutions.

The study used a validated acute in vivo model to compare a new formulation of travoprost 0.004% ophthalmic solution(travoprost PQ), preserved with polyquaternium-1 (PQ), with commercially available formulations of benzalkonium-chloride(BAK)-preserved travoprost 0.004% ophthalmic solution(travoprost BAK) and BAK-preserved latanoprost 0.005%ophthalmic solution (latanoprost BAK). Adult male New Zealand albino rabbits (n = 36) were randomly divided into 6 groups. Phosphate-buffered saline (PBS), 0.001% PQ, 0.015% BAK, travoprost PQ, travoprost BAK or latanoprost BAK were applied onto rabbit eyes as 1 drop, for 15 times at 5-min intervals.The ocular surface reactions were investigated at hour 4 and day 1 using slitlamp examination; in vivo confocal microscopy (IVCM) for cornea, limbus and conjunctiva/conjunctiva-associated lymphoid tissue, conjunctival impression cytology and standard immunohistology in cryosections for detecting CD45+ infiltrating cells and MUC-5AC-labeled cells. PBS, PQ and travoprost PQ did not induce obvious irritation by clinical observation, changes in microstructures of the whole ocular surface as measured by IVCM analysis,inflammatory infiltration or cell damage as measured by impression cytology, altered levels of goblet cell counts or numerous CD45+ cells in the cornea. In contrast, all BAK-containing products induced diffuse conjunctival hyperemia and chemosis, abnormal changes in the ocular surface microstructure,significant total ocular surface toxicity scores,damaged epithelial cells, inflammatory cell infiltration and decreased goblet cell density. Travoprost PQ did not elicitocular surface toxicity when administered to rabbit eyes.These results suggest a greater safety advantage for the ocular surface of patients receiving chronic glaucoma treatment with PQ-preserved drugs.

Polyoxyethylene hydrogenated castor oil modulates benzalkonium chloride toxicity: comparison of acute corneal barrier dysfunction induced by travoprost Z and travoprost.

PURPOSE: To determine the element that modulates benzalkonium chloride (BAC) toxicity by using a new electrophysiological method to evaluate acute corneal barrier dysfunction induced by travoprost Z with sofZia (Travatan Z(®)), travoprost with 0.015% BAC (Travatan(®)), and its additives. METHODS: Corneal transepithelial electrical resistance (TER) was measured in live white Japanese rabbits by 2 Ag/AgCl electrodes placed in the anterior aqueous chamber and on the cornea. We evaluated corneal TER changes after a 60-s exposure to travoprost Z, travoprost, and 0.015% BAC. Similarly, TER changes were evaluated after corneas were exposed for 60 s to the travoprost additives ethylenediaminetetraacetic acid disodium salt, boric acid, mannitol, trometamol, and polyoxyethylene hydrogenated castor oil 40 (HCO-40) with or without BAC. Corneal damage was examined after exposure to BAC with or without travoprost additives using scanning electron microscopy (SEM) and a cytotoxicity assay. RESULTS: Although no decreases of TER were noted after exposure to travoprost Z with sofZia and travoprost with 0.015% BAC, a significant decrease of corneal TER was observed after 0.015% BAC exposure. With the exception of BAC, no corneal TER decreases were observed for any travoprost additives. After corneal exposure to travoprost additives with BAC, HCO-40 was able to prevent the BAC-induced TER decrease. SEM observations and the cytotoxicity assay confirmed that there was a remarkable improvement of BAC-induced corneal epithelial toxicity after addition of HCO-40 to the BAC. CONCLUSIONS: Travoprost Z with sofZia and travoprost with BAC do not induce acute corneal barrier dysfunction. HCO-40 provides protection against BAC-induced corneal toxicity.

Effects of glaucoma medications and preservatives on cultured human trabecular meshwork and non-pigmented ciliary epithelial cell lines.

AIMS: We investigated the potential cytotoxicity of various topical ophthalmic glaucoma formulations containing different preservatives in cultured human trabecular meshwork (TM) and non-pigmented ciliary epithelial (NPCE) cell lines. METHODS: We tested 0.004% travoprost preserved with either 0.015% benzalkonium chloride (BAK), sofZia or 0.001% Polyquad (PQ); and 0.005% latanoprost preserved with 0.020% BAK. We also tested a range of BAK concentrations in balanced salt solution (BSS). TM cells were treated for 10 min at 37°C with solutions diluted 1:10 to mimic the reduced penetration of topical preparations to the anterior chamber. Viability was determined by the uptake of the fluorescent vital dye calcein-AM (n = 6). RESULTS: BAK solutions (diluted 1:10) demonstrated a dose-dependent reduction in cell viability in both cell types (TM and NPCE). With a 1:10 dilution of 0.020% BAK, there were significantly more living NPCE cells (89 ± 6%) than TM cells (57 ± 6%; p < 0.001). In TM cells, travoprost + BAK had statistically fewer live cells (83 ± 5%) than both travoprost + sofZia (97 ± 5%) and travoprost + PQ (97 ± 6%; p < 0.05). Compared with BSS-treated NPCE cells, travoprost had statistically fewer live cells (p < 0.05) when preserved with BAK (85 ± 16%), sofZia (91 ± 6%) or PQ (94 ± 2%). CONCLUSIONS: These results demonstrate that substitution of BAK from topical ophthalmic drugs results in greater viability of cultured TM cells, the cells involved in the conventional outflow pathway. Cultured NPCE, responsible for aqueous inflow, appear more resilient to BAK.

In vitro comparative toxicology of polyquad-preserved and benzalkonium chloride-preserved travoprost/timolol fixed combination and latanoprost/timolol fixed combination.

PURPOSE: To compare, in vitro, the cytotoxicity profile of a new formulation of travoprost 0.004%/timolol 0.5% fixed combination ophthalmic solution preserved with polyquaternium-1 0.001% (travoprost/timolol PQ) instead of benzalkonium chloride (BAK) with (1) commercially available travoprost 0.004%/timolol 0.5% fixed combination ophthalmic solution (travoprost/timolol BAK), (2) commercially available latanoprost 0.005%/timolol 0.5% fixed combination ophthalmic solution (latanoprost/timolol BAK), and (3) their associated BAK concentrations. METHODS: Compounds tested on Wong-Kilbourne-derived human conjunctival epithelial cells: (1) phosphate-buffered saline, (2) polyquaternium-1 0.001% (Polyquad(®), PQ), (3) travoprost/timolol PQ, (4) travoprost/timolol BAK with 0.015% BAK (DuoTrav(®)), (5) BAK 0.015%, (6) latanoprost/timolol BAK with 0.020% BAK (Xalacom(®)), and (7) BAK 0.020%. Toxicological assays were used to assess cell viability [neutral red (NR), Alamar blue (AB)], apoptosis (YO-PRO-1, Hoechst 33342), and oxidative stress (H(2)DCF-DA, hydroethidine). The apoptosis and oxidative stress assays were each reported according to cell viability as observed with NR and AB (totaling 10 analyses per treatment). RESULTS: The NR and AB assays demonstrated that cells incubated with travoprost/timolol PQ had significantly better viability than cells incubated with latanoprost/timolol BAK, travoprost/timolol BAK, BAK 0.015%, and BAK 0.020% (P<0.0001 for all). As assessed with YO-PRO-1 and Hoechst 33342 relative to cell viability determined with NR or AB, travoprost/timolol PQ produced significantly less apoptosis than travoprost/timolol BAK and latanoprost/timolol BAK and their respective BAK concentrations alone (P<0.0001 for all). Also, travoprost/timolol BAK induced less apoptosis than latanoprost/timolol BAK (P<0.0001). As assessed with H(2)DCF-DA as a ratio to NR or AB, all of the compounds without BAK (phosphate-buffered saline, PQ 0.001%, and travoprost/timolol PQ) and travoprost/timolol BAK produced significantly less reactive oxygen species than latanoprost/timolol BAK (P<0.0001 for all). As assessed with hydroethidine as a ratio to NR or AB, travoprost/timolol PQ produced significantly fewer superoxide anions than latanoprost/timolol BAK (P<0.0001). In contrast, release of superoxide anions (hydroethidine method) after incubation with travoprost/timolol BAK was not significantly different from incubation with latanoprost/timolol BAK or travoprost/timolol PQ. CONCLUSION: Travoprost/timolol PQ may be better for ocular surface health than either BAK preserved formulations of latanoprost/timolol or travoprost/timolol.

In vitro toxicity of topical ocular prostaglandin analogs and preservatives on corneal epithelial cells.

PURPOSE: To determine the effect of 4 formulations of commercially available prostaglandin analogs (PGAs) on human corneal epithelial cells in vitro. METHODS: The test solutions (PGAs) examined were tafluprost 0.005% with 0.010% benzalkonium chloride (BAK), travoprost 0.004% with 0.015% BAK, travoprost 0.004% with sofZia, and latanoprost 0.005% with 0.020% BAK. Also tested independently were the 4 respective BAK or sofZia concentrations related to each PGA. Balanced salt solution (BSS) was used as the live control, and a fixative solution containing 70% methanol and 0.2% saponin was used as the dead control. Immortalized human corneal epithelial cells were exposed to test or control solution for 25 min at 37 degrees C and 5% CO(2). A live/dead assay was used to measure the toxicity of the PGAs. RESULTS: The percentage of live cells in the PGA groups ranged from 2% to 72% of the BSS group (live control). The PGA with the highest relative live cell percentage, at 72% of the live control, was travoprost with sofZia. The next highest PGA, exhibiting 14% live cells, was the formulation of travoprost containing BAK. The other 2 PGAs, tafluprost and latanoprost, had few surviving cells, with 3% and 2% live cells, respectively. The BAK concentrations exhibited 4%, 3%, and 3% for the 0.01%, 0.015%, and 0.02% concentrations, respectively. The stand-alone sofZia cell survival was 68% of the live control. CONCLUSIONS: All 4 PGA formulations tested demonstrated significantly more toxicity in human corneal epithelial cells than the live control, but there were significant differences among the PGAs. Travoprost with sofZia exhibited the least toxicity, followed by travoprost with BAK, and then tafluprost and latanoprost. The stand-alone preservative systems were also tested and showed similar survival percentages to each respective PGA. The true clinical implications of these findings require further investigation.

In vitro effects of preservative-free tafluprost and preserved latanoprost, travoprost, and bimatoprost in a conjunctival epithelial cell line.

PURPOSE: This study compared the toxicity profiles of three antiglaucoma prostaglandin F2alpha analogs, latanoprost, travoprost, and bimatoprost which contain benzalkonium chloride (BAK), with tafluprost, a new preservative-free prostaglandin analog. METHODS: IOBA-NHC cells were exposed to BAK-containing prostanoid solutions, their respective BAK concentrations, and preservative-free tafluprost solution for 30 min. Membrane integrity, apoptosis, oxidative stress, and cells morphology were evaluated. RESULTS: Preservative-free tafluprost resulted in significantly higher membrane integrity and lower pro-apoptotic and pro-oxidative effects than preservative-containing prostaglandin analog preparations. CONCLUSIONS: These results suggest that tafluprost, a new preservative-free prostaglandin analog, has very low or no pro-apoptotic, pro-necrotic, or pro-oxidative effects in vitro compared to preservative-containing formulations.

In vitro studies of antiglaucomatous prostaglandin analogues: travoprost with and without benzalkonium chloride and preserved latanoprost.

PURPOSE: With use of the Wong-Kilbourne derivative Chang conjunctival cell line, this study compared in vitro the ocular toxicity of three topical intraocular pressure (IOP)-lowering agents: travoprost 0.004% containing 0.015% benzalkonium chloride (BAK), travoprost Z 0.004%, a new formulation without BAK, and latanoprost 0.005% containing 0.02% BAK. METHODS: Neutral red, Alamar blue, YOPRO-1, and annexin V/7-AAD assays were used to evaluate the effects of the IOP-lowering agents and BAK on cellular viability, membrane integrity, and apoptosis in the conjunctival cell line using microtitration fluorometric analysis and flow cytometry. All assessments were performed in a masked manner. RESULTS: Assessment of cell viability and membrane integrity revealed a significant effect by latanoprost with BAK or BAK alone but no effect by travoprost Z without BAK or buffer alone (P < 0.0001). Latanoprost with BAK, travoprost with BAK, and BAK alone were cytotoxic in Chang conjunctival cells, whereas no cytotoxicity was observed in cells exposed to travoprost Z without BAK or in cells treated with buffer (P < 0.0001). No increase in apoptosis or necrosis was observed in cells treated with control or travoprost Z without BAK compared with BAK, travoprost with BAK, and latanoprost with BAK (P < 0.0001). CONCLUSIONS: Latanoprost with BAK, travoprost with BAK, and BAK alone have significant cytotoxic effects on human conjunctiva-derived cells and are associated with apoptosis. These effects likely result from BAK used as a preservative. IOP-lowering agents with alternative preservatives instead of BAK will most likely have fewer ocular surface adverse effects than agents containing BAK.

Cloprostenol, a prostaglandin F(2alpha) analog, induces hypoxia in rat placenta: BOLD contrast MRI.

Blood oxygen level dependent (BOLD) contrast was used to monitor hypoxia induced by cloprostenol, a prostaglandin F(2alpha) (PGF(2alpha)) analog, in the rat embryo-placental unit (EPU). It is shown that administration of cloprostenol (0.025 mg/rat) at mid-gestation (day 16) reduced EPU oxygenation, as detected by BOLD contrast MRI, in correlation with induction of vascular endothelial growth factor (VEGF) gene (Vegfa) expression in the corresponding placenta (r = 0.56, p = 0.03). Elevated VEGF mRNA expression in response to cloprostenol treatment was also observed at early gestation (day 9) in the forming placenta (p = 0.04) and uterus (p = 0.03). Cloprostenol increased the expression levels of endothelin-1 (ET-1) gene (Edn1) (p = 0.03) and its corresponding peptide (p = 0.02) in the forming placenta, as well as the expression of the endothelin receptor type A (ETA) gene (Ednra) in both the forming placenta (p = 0.009) and the uterus (p = 0.01). The levels of the endothelin receptor type B (ETB) gene (Ednrb) were not affected in response to cloprostenol, but a significant elevation in the expression level of this receptor was observed in the uterus at mid- and late gestation (day 22) (p = 0.04 and 0.01 respectively), suggesting a role for ETB in the vasodilatory status of the pregnant uterus. It is suggested that PGF(2alpha) induces uteroplacental vasoconstriction in the rat, and that ET-1 may take part in mediating this effect, probably via activation of ETA receptor. The uteroplacental vasoconstriction induces hypoxia, as manifested by significant changes in BOLD MRI and by upregulation of VEGF.

Comparison of the relative toxicity of travoprost 0.004% without benzalkonium chloride and latanoprost 0.005% in an immortalized human cornea epithelial cell culture system.

The purpose of this study was to compare the relative toxicity of a new topical ophthalmic glaucoma medication, travoprost 0.004% without benzalkonium chloride (BAK), with that of commercially available latanoprost 0.005% (preserved with 0.02% BAK) in immortalized human corneal epithelial cells (HCEs). Tissue culture plates (96 well) containing HCEs were divided into 6 groups. Two groups served as negative controls (70% methanol and gentamicin). Another 2 groups--1 in corneal epithelial culture media and the other in a hydroxypropyl (HP)-Guargellable lubricant eyedrop--served as live controls. The travoprost 0.004% without BAK and latanoprost 0.005% groups were exposed to 100 microL of the undiluted solutions. Cells were incubated for 25 min at 37 degree C. A live/dead assay was used to measure the effects of travoprost without BAK and of latanoprost on HCEs compared to 70% methanol and culture medium. Between the 2 glaucoma medications tested, travoprost 0.004% preserved without BAK showed significantly less toxicity on HCEs than did latanoprost 0.005%. This difference may have ramifications in terms of tolerability for patients who use these topical glaucoma drugs on a long-term basis.

In vitro study of inflammatory potential and toxicity profile of latanoprost, travoprost, and bimatoprost in conjunctiva-derived epithelial cells.

PURPOSE: Conjunctiva-derived epithelial cells were used to investigate, in vitro, the expression of various inflammation-associated markers known to be overexpressed in patients with glaucoma after contact with the three major commercially available eye drops containing prostaglandin analogues. The impact on cellular viability and apoptosis in the same cell line was evaluated, to address the possible proinflammatory and/or toxic origin of the most frequent clinical impairments induced by prostanoids (i.e., conjunctival hyperemia). METHODS: Conjunctiva-derived cells were treated in vitro with the commercial solutions of latanoprost, travoprost, bimatoprost, prostaglandin (PG)F2alpha, tumor necrosis factor (TNF)-alpha, and different concentrations of benzalkonium chloride (BAC). Expressions of three inflammation- and immune-related markers, intercellular adhesion molecule (ICAM)-1, platelet-endothelial cell adhesion molecule (PECAM)-1 and HLA DR, were evaluated with flow cytometry after 24 to 72 hours of contact at low, subtoxic concentrations. Toxicological tests were also performed with cold-light cytofluorometry, in which cellular viability and apoptosis were evaluated with the neutral red and Hoechst/propidium iodide tests, respectively. RESULTS: TNFalpha induced or stimulated expression of the three inflammatory markers, whereas the PGF2alpha, latanoprost, travoprost, and bimatoprost solutions did not induce an increase in these markers and even produced a marked reduction of ICAM-1 and PECAM-1 expression in those solutions most concentrated in BAC, thus suggesting a toxic phenomenon in cellular membranes induced by the preservative rather than the medication itself. Cytotoxic assays confirmed this hypothesis and showed significant toxicity with prostaglandin analogues after prolonged contact, proportional to the concentration of BAC in the solution and similar to that of the corresponding concentration of BAC alone, bimatoprost having both the least concentration of BAC and the least cytotoxic in these experimental conditions. CONCLUSIONS: The comparison of latanoprost, travoprost, and bimatoprost, in their commercial formulations, showed that none of them appeared to induce direct stimulation of the inflammatory pathways involving adhesion molecules or class II antigens, although these markers have been found ex vivo in conjunctival specimens from patients treated with prostaglandins. In fact, their toxicity was mild and seemed to be primarily related to the concentration of BAC, their common preservative, which may be the major factor responsible for long-term ocular surface reactions in patients receiving topical prostaglandins, but most likely is not a factor in early and transient conjunctival hyperemia.

The comparative cardiovascular, pulmonary, ocular blood flow, and ocular hypotensive effects of topical travoprost, bimatoprost, brimonidine, and betaxolol.

OBJECTIVE: This study evaluated systemic and ocular acute safety and intraocular pressure (IOP)-lowering efficacy of travoprost 0.004% and bimatoprost 0.03%, compared to brimonidine 0.2% and betaxolol 0.25% in healthy subjects. PATIENTS AND METHOD: Nineteen (19) young men, ages between 24 and 42, were enrolled in a single-center, institutional randomized, double-masked, crossover clinical trial. Baseline IOP, heart rate, blood pressure, and respiratory rate were recorded at hour 0. At minute 30, heart rate, blood pressure, respiratory rate, and spirometry were measured. At hour 1, color Doppler imaging of retrobulbar vessels was performed. At hour 2, heart rate, blood pressure, and respiratory rate were measured; spirometry and a 15-minute treadmill test were performed. The same protocol was applied after one drop of a study medication was instilled into each eye on four subsequent visits at 5-day intervals. RESULTS: Travoprost and bimatoprost did not cause significant reductions in systolic blood pressure during exercise and recovery. The mean respiratory rate and forced expiratory volume in 1 second were not significantly altered by any study medication. Travoprost reduced the resistive index and increased blood velocities in the ophthalmic artery and its branches. Bimatoprost caused a significant increase in end diastolic velocity of the ophthalmic artery. At hour 6, all medications reduced IOP significantly (p < 0.05). The most frequent ocular side effect of travoprost and bimatoprost was conjunctival hyperemia. CONCLUSION: Travoprost and bimatoprost were found to be systemically safe and caused an increase in blood-flow velocities of the retrobulbar vessels after a single-dose application. Their ocular hypotensive effect was comparable to that of brimonidine and greater than that of betaxolol in healthy subjects.

Endotoxin-induced abortion in early pregnant gilts and its prevention by flunixin meglumine.

The objective of the study was to examine the effect of endotoxin on early pregnancy in gilts and to test the potential of flunixin meglumine (FM), a cyclooxygenase inhibitor, to counteract abortifacient action of the endotoxin. Ten gilts at 30 days gestation were used in the experiment. Eight were injected with lipopolysaccharide (LPS) of Salmonella typhimurium, while 2 were treated with 500 micrograms cloprostenol (CP). Six of the LPS-injected gilts were treated with a total of 4 mg/kg body weight FM in 2 different dose regimens. Clinical observations were recorded and plasma levels of 15-keto-13, 14-dihydro-PGF2 alpha, progesterone and estrone sulfate (ES) were determined with radioimmunoassay. LPS induced typical signs of endotoxemia and a monophasic fever in all LPS-treated gilts. No antipyretic effect of FM was observed. The CP-treated gilts aborted within 34 h as did the gilts treated by LPS only. Of the 6 LPS + FM-treated gilts, 1 aborted within 34 h, while 5 maintained gestation. These were aborted about a week later by CP and the aborted fetuses anatomically examined. Two of the litters were lost (devoured by the dams), 2 showed no signs of earlier death and 1 showed extensive fetal death. The PGF2 alpha metabolite concentrations increased at least 10 fold immediately after the LPS injection. Progesterone plasma concentration decreased rapidly. A 5-10 fold increase in the plasma metabolite levels accompanied all abortions. CP caused no immediate change in the PGF2 alpha metabolite levels, but the abortion-related response was similar to that in LPS-injected gilts. In the FM-treated gilts, the LPS-induced PGF2 alpha metabolite response was rudimentary and the progesterone decrease temporary in nonaborting gilts. The elevated concentrations of ES decreased within 48 h in gilts aborting at 30 days gestation, while in nonaborting gilts a slow, graduate decrease of ES occurred within 3-5 days of the LPS injection. These results indicate that FM apparently suppressed LPS-induced prostaglandin synthesis and thus prevented luteolysis and abortion in early pregnant gilts.

[The fertility performance (litter characteristics) of laboratory mice over 6 generations after the administration of zinc metallibure as suisynchronous premix and cloprostenol as estrophane]. / Untersuchungen zu Fruchtbarkeitsleistungen (Wurfmerkmale) von Labormäusen im Verlaufe von 6 Generationen nach Verabreichung von Zink-Metallibur als Suisynchron-Prämix und Cloprostenol als Oestrophan.

Suisynchron premix and oestrophane were tested through 6 generations of laboratory mice for their action on fertility (litter parameters). No fertility-reducing effect was recordable.