NDA 21-272
`
`I
`
`92
`
`\
`
`Pre- and Postnatal Developmental Toxicig Evaluation of UT-15 (Administered by
`Continuous Subcutaneous Infusion) in Rats
`
`Testing Facility:M
`
`[W
`
`Study Number: 65C-7019-600 (—~ No.)
`
`Study Dates: Initiation Date —— November 10, 1998
`Completion Date - May 23, 1999
`
`GLP Compliance: The study was conducted in compliance with GLP regulations.
`
`Animals: One hundred and fifty female Sprague-Dawley rats, 56 days old and weighing
`about 200-225 g, were obtained fromW
`After a week of quarantine, the females were mated with male Sprague-Dawley rats
`(previously received from the same supplier) from the («breeding colony. One hundred
`sperm-positive female rats [ranging in body weights from 211 to 269 g on gestational day
`(gd) 0], designated as F0 generation, were assigned to four groups of 25 rats each. (The
`day on which vaginal sperm or plugs were found was designated as gd 0).
`
`The animals were individually housed, except during mating periods, in solid bottom,
`polycarbonate cages with stainless steel wire lids and h cage litter. Certified
`Rodent Diet No. 5002 and tap water were available ad Iibitum.
`
`Dose Levels, Mode of Administration and Treatment Regimen: The target doses were 0,
`50, 150 and 450 ng/kg/min.
`
`Stock solutions (10 mg/ml) of UT415 (Lot No. UT15-98H01), formulated in vehicle
`(containing sodium citrate, citric acid and sodium chloride dissolved in sterile water for
`, injection) and adjusted to pH 7.4, were diluted (with vehicle) to achieve the desired final
`concentrations.
`
`All formulations and stock solutions were determined to be within 97—104% of target
`concentrations and were found to be stable at 25 and 40°C for four weeks.
`
`The test and vehicle solutions were administered by continuous subcutaneous infusion
`using a subcutaneously implanted . ’Qosmotic pump (M , which
`delivers —-_-fliour for a nominal duration of 28 days.
`
`On the morning of gd 5, each F0 female was anesthetized with isoflurane inhalation. The
`dorsal subscapular area was surgically prepared and an incision about 1.5 cm long was
`made. The osmotic pump, preloaded with appropriate dosing solution, was inserted into
`the subcutaneous pocket and the incision was closed with wound clips.
`
`

`

`NDA 21-272
`
`93
`
`The osmotic pump model used for the study requires about . ,__...—-sto reach steady
`state infusion rate once it is implanted. Therefore, the pump that was implanted on gd 5
`would reach the steady state by the morning of gd 6.
`
`On the morning of postnatal day 4, the animals were anesthetized and a new incision was
`made adjacent to the initial incision. The original osmotic pump was removed, inspected,
`and then replaced with a primedM
`r W second osmotic pump. The incision was
`closed with wound clips.
`
`The duration of exposure for F0 females was about 36 to 38 days, from gestational day 6
`to postnatal day 21 (gestational period = 21-23 days).
`
`It is stated that the top dose (450 ng/kg/min) was chosen to induce maternal toxicity or
`low levels of lethality (S 10%), and was based on the results of the Segment II study. The
`lower doses were assigned as fractions of the high dose.
`
`Observations and Measurements
`
`F0 Maternal Animals
`
`, Animals were checked for clinical signs of toxicity and mortality at least twice daily. The
`body weights were recorded on gd 0, 5, 9, 12, 15, 18 and 20, and on postnatal day (pnd)
`0, 4, 7, l4 and 21. Food consumption was recorded for gd 0-5, 5-9, 9-12, 12-15, 15-18,
`18-20, pnd 0-4, 4-7, 7-14 and 14-21.
`
`Beginning on gd 20, all females were examined twice daily for evidence of littering, or
`signs of dystocia. The F0 dams were allowed to rear their Fl young to pnd 2].
`
`F1 Progeny
`
`sexed and examined externally for
`All Fl pups/litter were counted, weighed,
`. malformations on the day of birth (designated pnd 0). Pups that were stillborn or died
`before pnd 4 were examined externally and viscerally, and any abnormal tissues or
`specimens were kept in buffered neutral 10% formalin. On pnd 4, the size of each litter
`was adjusted to eight (four per sex, if possible). Culled pups were decapitated and
`discarded. Litters with eight or fewer pups were not culled. Pups were counted, weighed
`individually, sexed, and examined externally on pnd 4, 7, 14 and 21 .
`
`Pups that died or were sacrificed moribund on pnd 5-21 were necropsied, and any
`abnormal tissues were preserved. Survival indices were calculated on pnd 0,4, 7, 14 and
`21.
`
`During the preweaning period (up to pnd 21), F1 pups were observed daily for
`developmental landmarks, including pinna detachment (pnd 1-4), incisor eruption (pnd 8-
`13) and eye opening (pnd 11-16).
`
`

`

`NDA 21-272
`
`94
`
`At weaning on pnd 21, 20 F1 pups/sex/group were randomly selected from the maximum
`number of litters for generating the F2 animals. The selected animals were held for a
`minimum of 49 days until all selected F1 pups were at least 70 days old. Following this
`selection,
`the remaining offspring were examined for gross external abnormalities,
`euthanized and discarded.
`
`The selected pups were examined daily for clinical signs, and weighed weekly. Pups
`were assessed for vaginal opening (pnd 22-36), cleavage of the balanopreputial gland
`(preputial separation; pnd 35-44), and neurobehavioral development (auditory startle
`reflex on pnd 21-34, motor activity on pnd 35-45, and learning and memory on pnd 41-
`55). A11 F1 females were evaluated for estrous cyclicity during the last 14 days of the
`postwean holding period (just before mating).
`
`F1 males and females were then mated (1:1) for a period of 14 days. Females were
`examined daily during cohabitation for the presence of vaginal sperm or plug. Once
`vaginal sperms or plugs were found, the mating pairs were separated and individually
`caged. The F1 pregnant females were weighed on gd 0, 6, 9, 12, 15, 18 and 20, and on
`pnd 0 and 4, while the Fl males were weighed weekly.
`.
`
`Beginning on gd 20, all Fl pregnant females were monitored twice daily for parturition.
`
`F2 Progeny
`
`All F2 pups/litter were counted, sexed, weighed and examined grossly as soon as possible
`on the day of birth (pnd 0) and on pnd 4. Pups that were stillborn or died before pnd 4
`were examined externally, viscerally, and preserved in buffered neutral 10% formalin.
`Grossly malformed pups were sacrificed and examined. All F2 pups were decapitated and
`discarded on pnd 4.
`
`Necropsy ofF0 Females and F1 Parental Males and Females
`
`On pnd 21, all surviving F0 dams were necropsied and the thoracic and abdominal organs
`were examined grossly. The implantation sites were counted. Organs or tissues showing
`any abnormalities were preserved. Uteri from any F0 females that appeared nonpregnant
`were stained with 10% ammonium sulfide for confirmation of pregnancy status.
`
`On pnd 4 of F2 litter, Fl dams and nonpregnant F1 females were necropsied and
`examined as described above. Paired ovarian and uterine weights were recorded.
`
`At or after the pnd 4 date of their F2 litter, F1 males were necropsied and organs were
`examined grossly. Paired testes and epididymides weights were recorded.
`
`reproductive performance, gestational parameters and offspring
`for
`The indices
`parameters were calculated.
`
`for
`Quantitative continuous data were statistically analyzed using Bartlett’s test
`homogeneity of variances. If Bartlett’s test indicated lack of homogeneity of variances,
`
`
`
`'\
`
`

`

`NDA 21-272
`
`95
`
`then nonparametric statistical tests (Kruskal-Wallis test followed by Mann-Whitney U
`test for pairwise comparisons; Jonckheere’s test to identify dose-response trends) were
`employed. If Bartlett’s test indicated homogeneous variances, then parametric statistical
`tests [appropriate General Linear Models (GLM) procedures for the Analyses of Variance
`(ANOVA)] were used. Prior to GLM analysis, an arcsine-square root transformation was
`performed on all litter-derived percentage data to allow use of parametric methods All
`indices were analyzed by Chi--Square test and by the Cochran--Armitage test for linear
`' trend on proportions. When ChiSquare revealed significant differences among groups,
`then a Fisher’s Exact Probability test was used for pairwise comparisons A test for
`statistical outliers was performed on parental body weights and F0 maternal feed
`consumption.
`
`Results
`
`F0 Maternal Animals
`
`No F0 females died during the study.
`
`There were more incidences of swelling at the implantation site noted in drug treated
`animals (not dose-related) than in controls during gestation and lactation periods.
`
`Mean maternal body weights were similar across control and treatment groups during the
`gestation period except on gd 9, when the mean body weight of the high dose group was
`significantly lower than the control value. Dose.related reductions in food consumption
`were noted in treated groups on gd 5-9, 5-20 (gestational treatment period) and 0~20
`(entire gestational period).
`
`During the lactation period, there were no statistically significant body weight differences
`among groups with the exception that the mean body weights of the. mid and high dose
`groups were significantly lower than control on pnd 7. The food consumption was
`reduced at all dose levels (not dose-related) on pnd 4-7, corresponding to the time of
`implantation of the second osmotic pump on pnd 4. No treatment-related effects were
`observed for food consumption prior to or after pnd 4-7.
`
`F0 reproductive and lactation indices are presented in Table 23. The fertility and
`gestation indices, the length of gestation period, the number of implantation sites per
`litter or the percent postimplantation loss per litter were similar across control and treated
`groups. There were no treatment-related effects on the number of live, dead or total pups
`at birth, stillbirth or livebirth indices, and lactation index.
`
`The necropsy of the F0 females showed no treatment-related findings except for “serum
`pockets” surrounding the pump, the incidence of this observation being higher in treated
`groups (3 to 6 animals/group; not dose related) than in the control group (2 animals).
`
`

`

`NDA 21-272
`
`F1 Progeny
`
`96
`
`F1 litter size and pup body weights during lactation are presented in Table 24. There were
`no treatment-related effects on litter size, survival indices, pup body weights and sex ratio
`(percent male pups per litter) of pups.
`
`There were no treatment-related necropsy findings in pups that died or were sacrificed
`moribund on pnd 0 through 21.
`
`F0 maternal treatment had no effect on eye opening, vaginal patency and preputial
`separation in F1 offspring. Pinna detachment was significantly delayed at low dose (by
`0.26 days compared to control), but unaffected at mid and high doses. lncisor eruption
`was significantly accelerated (by 0.47 days) at the high dose. Dose-related effects of
`treatment were not seen in auditory startle behavior, motor activity, and learning and
`memory assessments.
`
`Fl male and female body weights during prebreed, mating and holding periods until
`sacrifice were unaffected by the F0 maternal treatment with UT-lS. There were no dose-
`related clinical signs in F1 animals; the estrous cyclicity, evaluated during the final 14
`days of prebreed period, was similar across all groups.
`
`Fl female and male reproductive indices are presented in Table 25. The mating index for
`the high dose group (73.7%) was significantly lower than that for the control group
`(95%). No significant differences from control were noted for this parameter at lower
`doses. Although not statistically significant, dose-related decreases in fertility and
`pregnancy indices were seen in treated groups (for both indices, control 100%, low dose
`94.7%, mid dose 90.0% and high dose 85.7%). There were no treatment-related effects
`on gestational
`length, number of implantation sites per litter, or the percent post-
`irnplantation loss per litter.
`
`Fl maternal body weights during pregnancy and lactation were unaffected by treatment.
`
`There were no unscheduled deaths among Fl males. One Fl female at 450 ng/kg/min
`was euthanized on day 36 during the prebreed period because of an inguinal mass, and
`one Fl female at 50 ng/kg/min was euthanized moribund on pnd 0. No treatment-related
`findings were seen on necropsy. Absolute or relative reproductive organ weights were
`unaffected by drug treatment.
`
`F2 Progeny
`
`F0 treatment had no effects on the numbers of live, dead or total F2 pups, live or still-
`birth indices (Table 25), average number of pups per litter, average pup body weights per
`litter, and percent male pups per litter on pnd 0 and 4 (Table 26).
`
`There were no treatment-related findings at the F2 necropsy on pnd 0 through 4.
`
`
`
`

`

`NDA 21-272
`
`Table 23.
`
`1'
`
`97
`
`Summary and Statistiwl Analysis of the F0 Reproductive and Lactational indexes for the F1
`Litters
`(page 1 of 3)
`
`-
`UT-15 {mag/gin, subcutaneous)
`0 450 50 150
`
`
`
`
`
`-
`
`No. of Sperm Positive Females
`.
`
`No. of Pregnant Females
`
`25
`
`25
`
`23
`25
`Fertility Index (no. pregnant females/no. sperm positive females)
`100.0
`92.0
`25a
`23
`
`No.0fFemaleswithUvethters(pndO)
`
`-
`
`25
`
`25
`
`100.0
`25
`
`25
`
`25
`
`100.0
`25
`
`Gestationd Index (no. females with live litters/no. females pregnant)
`100.0
`100.0
`100.0
`100.0
`
`
`Gatatimai Length (days)b
`
`22.0 ,
`1 0.1
`N=24
`
`No. Implantation Sites per Litter"
`
`14.33
`1 0.35
`N=24
`Percent Postimplamation Loss per Litter”
`
`21.9
`1 0.1
`11:23
`
`15.04
`10.40
`N=23
`
`22.0
`1 0.1
`N=25
`
`15.32
`1 0.34
`N=25
`
`22.1
`1 0.1
`N=25
`
`14.76
`1 0.36
`N=25
`
`5.21
`5.14
`437
`9.35
`11.24
`11.63
`11.39
`11.75
`N=25
`N=25
`N=23
`N=24
`
`
`No. of Live Littersn
`
`Postnatal Dayo
`Postnatal Day 4
`Postnatal Day 7
`_ Postnatal Day 14
`Postnatal Day 21
`
`24
`24
`24
`24
`24
`
`23
`23
`23
`23
`23
`
`25
`25
`25
`25
`25
`
`25
`25
`25
`25
`25
`
`NumberofljvePupsoni’ostl'latalDayOb
`142
`14.6
`14.6
`13.5
`1 0.4
`1 0.4
`1 0.3
`1 0.4
`
`11:24
`11:23
`N=25
`N=25
`.
`
`(continued)
`
`E
`
`E
`E
`E
`
`E E
`
`E
`
`E
`E
`
`E E
`
`E
`
`E
`E
`E
`
`E
`E
`
`E
`
`
`
`E
`
`E
`E
`E
`
`
`
`‘
`
`
`
`
`
`.
`
`

`

`NDA 21-272
`
`98
`
`Table 23. (continued)
`
`!
`
`Summary ands-919ml Amcysis of the so Reproductive and Ladafiond Indexes for the F1
`Litters
`(P89920f3)
`
`
`01.15 (9mm. subcutaneous)
`
`0
`50
`150
`450
`
`NumbetofDeadPupsonPocmatalDayob
`t
`—
`.
`0.0
`1 0.0
`N=24
`TotalNumbarofopsmPoshaleayOb
`13.5
`1 0.4
`14:24
`
`0.1
`1 0.1
`N=23
`
`14.7
`1 0.4
`N=23
`
`smmundex(no.0eadonpndonom1m.onpnc10)b
`0.3
`1 0.3
`=24
`
`0.9
`1 0.6
`N=23
`
`WeBilthhdex(m.livempnd0nolalno.onpndO)b
`99.7
`1 0.3
`N=24
`
`99.2
`1 0.0
`14:23
`
`atDaySurvlvalIndex(no.survivirrguiayslno.liveonpndO)b
`99.7
`99.4
`1 0.3
`1 0.4
`N=24
`14:23
`
`7DoySu~wa11nm1no.su~m7doys/no.0veonpnd4)b
`9
`100.0
`100.0
`1 0.0
`1 0.0
`11:24
`N=23
`
`_
`0.1
`1 0.1
`N=25
`
`14.7
`1 0.4
`N=25
`
`0.6
`1 0.6
`N=25
`
`99.4
`1 0.6
`N225
`
`99.5
`1 0.3
`11:25
`
`100.0
`1 0.0
`11:25
`
`14DaySuvlvallndex(no.smiving14days/no.liveonpnd7)b
`~
`9
`99.5
`100.0
`1 0.5
`1 0.0
`01:24
`N=23
`21 Day Swivel Index (no. surviving 21 dayslno. We on pnd 14)b
`s
`100.0
`100.0
`1 0.0
`1 0.0
`14:24
`N=23
`
`100.0
`1 0.0
`N45
`
`,
`
`99.5
`1 05
`14:25
`
`0.0
`1 0.0
`N=25
`‘
`
`14.2
`1 0.4
`N=25
`
`'
`
`0.3
`1 0.3
`N=25
`
`99.8
`1 0.3
`«=25
`
`99.7
`1 0.3
`N=25
`
`100.0
`1 0.0
`N=25
`
`100.0
`1 0.0
`N=25
`
`100.0
`1 0.0
`N=25
`
`Lactafional index (no. surviving 21 dayslno. five on pnd 4)b
`100.0
`99.5
`a!
`99.5
`100.0
`1 0.0
`1 0.5
`1 0.5
`1 0.0
`01:25
`11-25
`71:24
`N=23
`
`
`(continued)
`
`\
`
`‘
`
`a
`
`§
`i
`
`
`
`
`
`

`

`NDA 21-272
`
`Table 23. (continued)
`
`99
`
`Summary and Statistics! Analysis of the F0 Reproductive and Lactafional Indexes for the F1
`Litters
`(page 3 of 3)
`
`
`mandamm.butwasidudedodyformmnyandgestafionalhdexabecauseshe
`wasnotweighedonhercotredposmatal dayOandmemforealsuwequemdatadufinglaaafionwere
`uncrowdedonheeomctdays.
`,
`_
`asmemanzs.E.M.:pnd=posmatalday. Nlhdexesareheavetagepeteentpermter.
`Wmtmmpnwdmmamwamnuwmmmmem
`mmwfiamhmummfiflnmbmnmmbmmdmm
`employed.
`
`

`

`NDA 21-272
`
`Table 24.
`
`100
`
`SumryandStafisficalAnalysis of(0ieF11L;tftzSizeand Pup BodyWeights During Lactation
`P899
`
`m U
`
`T~15 (Min, subwtaneous} '
`
`
` 0 50 150 450
`
`
`
`No. of Live Linens;
`PM Day 0
`Postnatal Day 4
`Postnatal Day 7
`Postnatal Day 14
`Pounatal Day 21
`
`24
`24
`24
`24
`24
`
`~
`
`23
`23
`23
`23
`23
`
`Average Number of Pups per me: (pnd 0)3
`*
`13.5
`:04
`N=24
`
`Avenue NunberdeSpeerarmna
`13.5
`:04
`N324
`
`Average M.u1'lberofPu.'.>sperLitternd7)a
`8
`8.0
`109
`N=24
`
`AvaugeNumberofPLps puma“! 14)a
`
`14.6
`:03
`N=23
`
`14.5
`103
`N=23
`
`.
`
`8.0
`100
`N:23
`
`25
`25
`25
`25
`25
`
`14.6
`10A
`N=
`
`14.5
`:04
`N=25
`
`8.0
`:00
`N=25
`
`25
`25
`25
`25
`25
`
`14.2
`10A
`N45
`
`14.1
`10A
`N825
`
`8.0
`:00
`N=25
`
`#
`
`.
`
`an
`:09
`N=24
`
`an
`100
`N=23
`
`an
`:on
`N=25
`
`an
`100
`N325
`
`Average NumberofPups perUfleflpnd 21)a
`8.0
`8.0
`8.0
`#
`8.0
`ion
`:00
`:00
`100
`N=25
`N=25
`N=23
`N=24
`
`
`Mmmnmmwwmmwwamwwmma
`6.58
`1 0.09
`N824
`
`6.44
`1 0.10
`N=23
`
`6.55
`1 0.13
`N=25
`
`8.62
`1 0.11
`N=25
`
`‘
`AvaagelflaieBodyWeighttgwerLMeqpndO)‘
`6.80
`6.76
`6.62
`6.78
`gun,
`30M
`gnu
`gum
`
`N324 N825 N823 N=25
`
`
`
`(continued)
`
`

`

`NDA 21-272
`
`Table 24. (continued)
`
`101
`
`Summaryand Stafisfioalwysisofmefi LitterSizeandPupBodyWeights Outing Lactation
`(Page 2 0“)
`
`WU
`
`T-15
`‘
`subwtaneous
`0
`50
`1 50
`450
`
`
`Average Femalc Body Weight (g) per Litter (pnd 0)3
`~
`6.41
`6.28
`1 0.09
`1 0.10
`N=24
`N=23
`
`6.39
`1 0.12
`~=25
`
`6.46 1
`1 0.11
`~=25
`
`AmgePwBodvWeUMmpetLMertpndo‘
`11.16
`1 0.21
`N=24
`
`10.71
`1 0.18
`11:23
`
`Average Mal. Body Weight (g) per Litter (pod 4P
`11.42
`10.94
`10.20
`1020
`N=24
`N=23
`
`Avuage Female Body Weight (g) per Litter (pnd 4)8
`10.93
`10.52
`1 0.22
`10.18
`N=24
`N=23
`
`Avemge Pup BodyWeigl1t(g)perLflter(pnd 7F
`17.05
`1 0.26
`N=24
`
`1721
`1 027
`N=23
`
`Average Inch BodyWenght‘ (g) perLittef (pod 7P
`1 8.19
`17.68
`1 026
`1 0.30
`N=24
`NflS
`
`Average Female Body Weight (g) per Liter (pnd 7P
`17.54
`16.74
`10.28
`10.26
`N=24
`11:23
`
`10.78
`z 02
`N=25
`
`11.01
`10.25
`N=25
`
`10.61
`1 0.21
`N=25
`
`17.46
`1 029
`NflS
`
`17.82
`1 0.33
`N=25
`
`1 7.17
`10.28
`N=25
`
`10.87
`1 0.24
`N=25
`
`11.16
`1 025
`N=25
`
`10.60
`1 0.24
`N=25
`
`17.55
`1 0.28
`N=25
`
`1 7.94
`1 0.30
`N=25
`
`17.18
`10.28
`N=25
`
`AveragePupBodyWeight(9)peruuec(pnd 1403
`36.98
`1 0.46
`N=24
`
`35.62
`1 0.59
`N823
`
`36.44
`1 0.45
`N=25
`
`36.46
`1 0.45
`N=25
`
`Average Male Body Weoght' (a) per Utter‘ (pm we
`36.97
`37.19
`37.45
`36.33
`1 0.49
`1 0.50
`1 0.47
`1 0.62
`
`N=24 N=25 N=23 N=25
`
`
`
`(continued)
`
`
`
`
`

`

`NDA 21-272
`
`102
`
`Table 24 (continued)
`
`SumneryandStatisbeathalysisofmeF1LfltasueandPupBodyWeightsthgLachhon
`(page13 of 4)
`
`UT-1sgng591min: subwtaneous)
`
`0
`50
`150
`450
`
`Average Female Body Weight (g) per Utter (pm! 14)8
`36.51
`34.92
`10.47
`1053
`N=24
`N=23
`
`3577
`+0.44
`N=25
`
`AveragePupBodyWeiahtwwerUmrmndflF
`60.52
`5; 0.82
`N=24
`
`57.26
`g 0.84
`N=23
`
`59.15
`+ 0.88
`N=25
`
`'
`
`Average Male BodyWeight (g) per Uttar (pnd 21)a
`81.54
`58.70
`10.88
`1 0.96
`N=24
`N=23
`
`60.58
`+ 1.05
`N=25
`
`Average Penal. Body Weight (g) per Litter (pnd 21)a
`
`35.98
`+ 0.44
`N825
`
`5948
`+ 0.77
`N=25
`
`60.93
`1 0.91
`NflS
`
`58.20
`57.89
`55.81 “
`59.52 3
`1 0.74
`10.81
`1 0.77
`10.81
`N824
`N=23
`Nfls
`N=25
`
`
`Pemt Male Pups per Utter (pnd 0)8
`'
`
`Percent Male Pups per Utter (pnd 4)8
`
`Percent Male Pups per Utter (pnd 7P
`8
`
`46.7
`1 2.3
`N=24
`
`47.0
`1 2.3
`N=24
`
`49.5
`1 0.9
`N=24
`
`48.6
`:I; 2.5
`N=23
`
`48.3
`+ 2.4
`N=23
`
`50.0
`1 0.0
`N=23
`
`Percent Male Pups per Litter (and 14)a
`8
`
`49.8
`1 1.0
`N=24
`
`‘
`
`50.0
`+ 0.0
`N=23
`
`44.8
`+ 2.7
`N=25
`
`45.6
`+ 2.7
`N=25
`
`48.0
`+ 1.6
`N=25
`
`48.0
`+ 1.6
`03-225
`
`‘
`
`47.5
`+ 27
`N=25
`
`46.8
`+ 2.8
`N=25
`
`49.0
`+ 1.2
`N=25
`
`49.0
`+ 1 .2
`N=25
`
`PawntMalePupsperLittedpndzna
`I
`
`49.0
`48.3
`50.0
`49.8
`+ 1.2
`1 1.6
`+ 0.0
`1 1.0
`
`
`
`N=23 N=25N=24 N=25
`
`(continued)
`
`

`

`NDA 21-272
`
`'
`
`103
`
`Table 24. (continued)
`
`Summary and Statistical Arafysis of the F1 Litwr Size and Pup Body Weights During Lactation
`(page 4 0“)
`
`
`'
`“Reported as the mean 1; s.E.M.; pndaposmaal day.
`’Ba‘fien'smforhomogmityofvadanceswassiwiflml(p<0.001)oreouidno!bedmebeausemete
`waszerovaflamehoneormore m,hereforemnpammeficstafisfiealprocodumwere
`empioyed.
`3942.05; ANOVA Test.
`p<0.01; Dunnett's Test.
`
`APPEARS THIS WAY
`053%" GREGIML
`
`

`

`NDA 21-272
`
`-
`
`104
`
`Table 25.
`
`Summaryend Statistical Analysisoflhefi Reptoductlveand Lactational Indexes forthe F2
`Utlets
`(Pennants)
`
`
`UT-15 (M. subcutaneous)
`
` 0 50 150 450
`
`
`
`
`No. Animals Started on Study .
`Males
`Females
`
`.
`
`No. Parades Paired
`
`No. of Females that Mated
`
`20
`20
`
`20
`
`19 2 ‘P
`
`20
`20
`
`20
`
`19
`
`20
`20
`
`20
`
`20
`
`20
`20
`
`19a
`
`14
`
`Mating Index (no. females that matedlno. females paired)
`95.0
`
`95.0
`
`100.0
`
`73.7
`
`No. of Pregnant Female:
`
`19
`18
`Fettility Index (no. ptegnantfemaleslno. femalesthatmabd)
`100.0
`94.7
`
`No.6fFemaleswithiveUttersmnd0)
`
`19
`
`1 8
`
`18
`
`90.0
`
`18
`
`12
`
`'
`85.7
`
`12
`
`Gestational Index (no. females with live litters/no. females ptegnant)
`100.0
`,
`100.0
`
`100.0
`
`100.0
`
`No. Males Paired
`
`No. of Males that Mater!
`
`20
`
`19 £ '9
`
`20
`
`19
`
`20
`
`20
`
`19
`
`14
`
`Mating lndex(nomalesthatmatedlm.malespaired)
`95.0
`
`95.0
`
`100.0
`
`73.7
`
`No. Males Sirlng Litters
`
`19
`
`18
`
`18
`
`12
`
`Fertility lndex(no.malessi'inglitterslno.maleslhatmated)
`100.0
`94.7
`
`90.0
`
`85.7
`
`Pregnancy lndex (no. pregnant femaleslno. males that mated)
`100.0 85.7 94.7 90.0
`
`
`
`
`
`(continued)
`
`

`

`NDA 21-272
`
`Table 25 (continued)
`
`105
`
`SummatymdStatistkal/Analysiscfthe!’ RewodudivemdLactationalhdexesforfier
`Linens
`(113902013)
`
`W U
`
`T-15 gum subwtanwus)
`
` 0 50 150 450
`
`
`
`
`Days mt! Span-n Positive (daysP-c
`
`Gestational Length Mays)”-d
`
`N0. of Live Litters:
`
`9mm Day 0
`. P051313! Day 4
`N0. Implmtafion 511.5 per Lamb
`
`3.7
`+ 0.5
`N=19
`
`213
`+ 0.1
`11:19
`
`19
`19
`
`14.54
`10.01
`11:19
`
`Percent Posfimplanmtion Loss per Litterb
`
`729
`11.51
`N=19
`
`NunberovaePupsonPosmaalDeyOb
`13.8
`1 0.7
`~=19
`
`NummneadPupsoanuDayob
`t
`.
`0.3
`t 0.1
`N=19
`
`TotalNumberofPupsonPoshatalDayOb
`‘
`14.1
`1 0,3
`14:19
`
`3.4
`_+_ 0.0
`N=17
`
`22.4
`+ 0.1
`N=16
`
`15
`17e
`
`1672
`10.53
`11:18
`
`3.75
`+2.00
`N-1a
`
`152
`+ 0.7
`~=1a
`
`0.4
`t. 0.3
`N=18
`
`15.6
`1 0.7
`=18
`
`’
`
`4.0
`1 05
`N=19
`
`3.7
`+ o.0 _
`N=12
`
`_
`
`_
`
`22.1
`3 0.1
`N=17
`
`15
`18
`
`17.00
`+051
`N=18
`
`7.36
`11.70
`N=18
`
`15.0
`+ 05
`N-18
`
`02
`+ o 1
`N=18
`
`159
`+ 06
`N=18
`
`222
`1» 02
`N=10
`
`12
`12
`
`17.17
`31.11
`N=12
`
`11.90
`32.81
`14:12
`
`15.0
`g 1.0
`10:12
`
`0.4
`+ 0.3
`N=12_
`
`1s.4
`+ 1.0
`N=12
`
`31mm» Index1no.ueadonpnd0/to1a1no.onm0)b
`2.6
`1.0
`2.1
`1.6
`_+_ 1.5
`+ 0.5
`1 1.0
`10.5
`
`N=19 N=12 . 11-15 N=18
`
`
`
`(confined)
`
`
`
`
`
`
`
`

`

`NDA 21-272
`
`106
`
`Table 25 (continued)
`
`Summary and Statistical Anaiysisanhe F1 Reproductive and Laciafional indexes forthe F2
`Litters
`(page3of3)
`
`W U
`
`min subcumneous
`150
`
`‘50
`
`0
`
`T-15
`50
`
`Liam Il'u'lex(I'm.Iivem'lpndwhotalnva.mum!(Iv)b
`A
`98.4
`1 0.6
`N=19
`
`97.9
`1 1.6
`N318
`
`99.0
`1 0.5
`N318
`
`97.4
`:I_- 1.6
`N312
`
`ADaySquallndex(m.mivm4dayslm.flveon pndO)b
`95.5
`99.1
`99.0
`96.3
`3; 2.8
`i 0.5
`3 0.5
`g 1.5
`
`N319 "‘12 N=17e "‘18
`
`
`
`3Femala32‘waseufimizedonshdyday36ducbamshmelenhguhdareamatwasdispladng
`mmwmmmm
`bRepubdastnzSfiM; pnd=pomtalday. Mitexesaremeaverageperwupufiuer.
`coaysmemanewmmqubrmambsmmmspmmdmh
`
`dGWWcouidnotbeawamdforfenueshatwerepremantbutformidtspermwere.
`mmmmevagmm.
`°Fum256wasummmdmbundmposmday0anaddwmmrmm Herpupswerealso
`mtwmpmudayowmmmbmbmmedmmwmfimofme4day
`
`W'nestforhanogeneflyofvadanceswassignificanwnonorcouldmtbedonebmusemere
`waszuovarimcehmeumwwps.mmemnpammemcshfisficalprocedumswere
`employed.
`'
`.05;CI+SquesL
`p<0.05;CoumAnnltageTw.
`
`APPEARS nus WAY
`on ommm
`
`

`

`NDA 21-272
`
`107
`
`Table 26.
`
`4
`
`i.
`
`SmmyamsufisficalwyfidequuerSize. F2 Pup'BodyWeightsandPercem F2
`Malestthactafion (page1 012)
`
`W U
`
`«
`
`0
`
`T-15 129159329 subwhneouc)
`50
`150
`
`450
`
`i4
`
`
`
`No. of Uve Litters:
`
`P08013181 Day 0
`P052313! Day 4
`
`19
`19
`
`AmuseNuWofPups perUttedpnd 0P
`‘
`13.8
`3; 0.7
`N=19
`
`_
`
`18
`173
`
`‘
`
`152
`3 0.7
`N318
`
`18
`18
`
`‘
`15.8
`:0; 0.5
`N=18
`
`12
`12
`
`15.0
`1 1.0
`N=12
`
`.
`AverageNunberofPupsperUflarcpnde
`14.3
`15.6
`14.7
`13.6
`1 1.0
`+ 0 5
`1 0.7
`'1: 0.7
`N=12
`N219
`N=17a
`"=19
`
`
`Average Pup Body Weight (g) per Utter (pnd 0P
`6.65
`30.11
`11:19
`
`6.37
`30.15
`N=18
`
`6.39
`10.14
`N=18
`
`Avatage um Body Weight (9) per Lfltedpnd 0)b
`6.79
`10.13
`11:19
`
`6.52
`10.15
`N=18
`
`.
`
`AmagethBodyWeightu)peruner(pndO)b
`6.53
`6.12
`10.10
`10.14
`=19
`N=17°
`AveragePupBodyWeinmmperuuevmndaP
`10.12
`z 0.32
`11:19
`
`9.80
`1 0.39
`11:179
`
`Average m1: Body Weight (g) perUneramd 4)!)
`10.26
`10.00
`30.35
`30.40 -
`N=19
`N=17a
`
`6.54
`10.14
`11:19
`
`'
`6.25
`£0.14
`N=16
`
`9.33
`i 0.35
`N=1a
`
`10.12
`10.35
`N=18
`
`6.35
`30.17
`N=12
`
`6.54
`3020
`N212
`
`6.13
`$0.14
`=12
`
`9.42
`10.44
`N=12
`
`9.71
`10.44
`N=12
`
`Average roman. Body Weight (9);»! uuer (pnd 4)b
`9.10
`9.66
`9.98
`934
`3; 0.42
`g 034
`~
`1 0.30
`1 028
`
`=19 11:12 N=16° N=18
`
`
`
`(continued)
`
`

`

`NDA 21-272
`
`Table 26 (continued)
`
`108
`
`SummaryandStatlsficaiAnalysisafmeFzLitterSize,F2PupBodyWeightsaMPercentF2
`MalesDuringLacufion (pageZon)
`
`W
`
`min subcuhneous
`150
`
`450
`
`0
`
`UT~15
`50
`
`Patent Mane Pups per Litter (pnd up
`
`48.8
`- i 2.6
`N=19
`
`543
`1 43
`N318
`
`47.9
`1 2-7
`N=18
`
`52.4
`it 3-6
`N=12
`
`Percent Male Pups per Laser (pnd 4»
`
`53.2
`47.7
`55.5
`49.5
`12.6
`14.4
`1 2.6
`t 3.8
`
`N=19
`".473
`N=18
`N312
`
`“szsswawummdmmmposmamdayomerdelwemgwm. Herpupswerealso
`W on poshahl day 0.
`humorous as the mean 3; 3.514.;W day.
`cOm Mar had male pups only.
`
`
`
`APPEARS ““8 WAY
`0N ORIGINAL
`
`

`

`NDA 21-272
`
`109
`
`SUNIMARY OF GENETIC TOXICITY STUDIES
`
`Bacterial Reverse Mutation Assay
`
`[This assay‘uses tester strains of Salmonella typhimurium and Escherichia coli that
`contain mutations causing them to require amino acids histidine and tryptophan,
`respectively, in order to grow. The principle of this bacterial reverse mutation test is that
`it detects chemicals that induce mutations which revert mutations already present in the
`tester strains, thus restoring the functional capability of the bacteria to synthesize the
`essential amino acid. The revertant bacteria are detected by their ability to grow in the
`absence of the amino acid required by the parent tester strain]
`
`.. M
`.
`W:
`.
`
`._..———-———-
`
`W
`
`Study Number: AA20YG.502.BTL _A
`UT~15 (Sponsor’s project No.)
`
`, Study Dates: September 20 to October 21, 1999
`
`GLP Compliance: The study was conducted in compliance with GLP regulations.
`
`Lot No. of the Test Comp_ound: UTl 5M1X-99G001
`
`Concentrations Tested: 0, 100, 333, 1000, 3333 and 5000 ug/platc. (The doses were
`selected based on the results of a preliminary assay, in which doses up to 5000 ug per
`plate did not produce any precipitation or significant cytotoxicity.)
`
`Vehicle: Sodium citrate formulation - pH 7.0 [prepared by mixing sodium citrate,‘citric
`acid and sodium chloride in sterile water for injection (sponsor-specified vehicle)]
`
`Tester Strains: Salmonella typhimurium strains TA98, TA100, TA1535, TA1537 and
`Escherichia coli WP2 uvrA.
`
`Metabolic Activation System: Aroclor 1254-induced rat liver S—9 fraction in a cofactor
`pool containing g1ucose~6-phosphate, B-nicotinamide adenine dinucleotide phosphate and
`magnesium and potassium chlorides in phosphate buffer (pH 7.4)
`
`Positive Control Compgunds Not Regm' g 8-9 Activation: 2-nitrofluorene (TA98),
`sodium azide (TA100 and TA1535), 9-aminoacridine (TA1537) and methyl methane—
`sulfonate (WP2 uvrA)
`
`Positive Control Compound Reguiring S—9 Activation : 2-aminoantracene (all strains — to
`test the activity of 8-9 mix)
`
`

`

`NDA 21-272
`
`110
`
`Test Procedure: The test article was exposed to the test system using the plate
`incorporation assay procedure. The vehicle or solutions of test article or reference
`compounds, S-9 or sham mix and cultures of tester strains were added into respective
`tubes containing molten minimal top agar at 45 i 2° C . Afier mixing, the mixture was
`overlaid onto the surface of Vogel-Bonner minimal agar plates. When the overlay had
`solidified, the plates were inverted and incubated for about 48 to 72 hours at 37 1 2° C.
`Revertant colonies were counted manually or by an automated colony counter. For each
`replicate plating, the mean and standard deviation of the number of revertants per plate
`were calculated. (All concentrations of test article and vehicle and positive controls were
`plated in triplicate.)
`
`The growth of the background lawn was evaluated for evidence of test article toxicity
`using a dissecting microscope. The precipitate was evaluated by visual examination. Test
`article toxicity and the degree of precipitation were scored relative to the vehicle control
`plates.
`
`A test compound is considered to be positive for mutagenic effect if it produces a dose-
`related increase in the mean revertants per plate of at least one tester strain with a
`minimum of two increasing concentrations of test article.
`[The increase in mean
`revertants at the peak of the dose response should be equal to or greater than two (for
`strains TA98, TA100 and WPZuvrA) or three (for strains TA1535 and TA1537) times the
`mean vehicle control value.)
`
`A dose level is considered toxic if one or both of the following criteria are met: 1. greater
`than 50% reduction in the mean number of revertants per plate as compared to the mean
`vehicle control value, and 2. a reduction in the background lawn.
`
`An assay is considered to be valid if the following criteria are met:
`
`1. each tester strain must have appropriate genotypic characterics. 2. all cultures must
`demonstrate the characteristic mean number of spontaneous revertants in the vehicle
`control as follows: TA98, 10-50; TAIOO, 80-240; TA1535, 5—45; TA1537, 3-21;
`WP2uvrA, 10-60. 3. tester strain culture titers must be greater than or equal to 0.3x109
`cells/ml.
`4. each positive control must have a three-fold increase in the number of
`revertants over the respective vehicle control value, and 5. a minimum of three non-toxic
`levels are required for evaluation.
`
`Results: The results are summarized in Table 27. The test compound, under the
`conditions of the study, did not produce any significant dose-related increase in revertant
`colonies with any tester strains with or without metabolic activation. Respective positive
`control compounds produced significant increases in mutant frequencies, demonstrating
`the sensitivity of the tester strains as well as the activity of the 8-9 mix.
`
`No precipitation was observed at any concentrations in this study. Evidence of toxicity
`was observed at 5000 ug/plate with tester strains TA100, TA1535 and TA1537 in the
`absence of metabolic activation.
`
`‘r'.'*-—v .1. 7.--. .... ~_.V . -__
`
`.___.._._...- ~
`
`

`

`NDA 21-272
`
`11]
`
`Historical negative and positive control data are presented in Table 28.
`
`The present study is considered to be a valid study since all criteria for an acceptable
`study are met.
`'
`
`Table 27
`
`Salmonella/E. e011 Mutagonicicy Assay
`Sumaty of Results
`
`'
`‘
`: {IT-15
`Test Article Id
`
`
`MAAZOYG 502.311..Study Number Experiment No : 31
`
`Average Revertants Per Plate 1' Standard Deviation
`Liver Microsones: None
`
`Dose (pg)
`
`mes
`
`moo
`
`nusas
`
`111537
`
`wrz uvrA
`
`0.0
`100
`333
`1000
`3333
`5000
`P03
`
`17 i
`17 i
`21 t
`19 i
`16 i
`10 it
`257 2‘:
`
`10
`104 :l:
`3
`97 t 17
`2
`83 i
`5
`3
`h 105 :t
`6
`S
`77 d:
`24
`1
`46 i
`8
`36. #19 i 58
`
`2
`1!; :l:
`S
`13 t
`3
`14 1':
`1
`10 1
`2
`12 i
`3
`lo 1*
`163 i 26
`
`1
`5 :t
`3
`6 it
`2
`5 t
`2
`b t
`2
`4 1
`1
`2 I
`730 1' 171
`
`14 d:
`13 1’
`18 i
`14 i
`6 i
`9 I!
`72 :l:
`
`2
`3
`2
`3
`3
`4
`7
`
`Liver Hicrosomes: Rat liver 59
`
`Dose (pg)
`
`TA98
`
`TA100
`
`IA1535
`
`TA1537 W2 uvrA
`
`2
`13:
`51 2
`9t _2
`29
`115.";
`3
`21.1
`00
`6
`14 2':
`5 i
`It
`10 :l:
`2
`8
`121; t
`3
`29 i
`100
`2
`14 i
`h i
`2
`13 1'
`3
`19
`87 :t
`1
`22 1
`333
`1
`12 i
`7 i
`3
`12 1
`lo
`2
`1015 i
`l;
`22 i
`1000
`2
`15 i
`4 i
`3
`12 1
`2
`86 t 12
`2
`' 21 it
`3333
`6
`13 i
`4 2!:
`3
`10 i
`S
`78 i 20
`I:
`21 i
`5000
`312 :l: 1&1
`31 1'
`69 i
`491 1 47
`355 i 115
`Po:
`9
`13
`......................................................................
`
`0.0 I Vehicle plating Inquot 02 500 31.
`En - 20:11.1“ Control cone-nuance: u ”can“ In Materials and Methods 'ueuun.
`
`{\ij iii-€33
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`NDA 21-272
`
`112
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`\. ~
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`‘
`
`Table 28.
`
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`
`
`
` SD-rmdud dcv'nfion; Minsminimmn value; Max-Winn value; Neg-negative control
`
`E
`x
`
`limited to deionized water, dimethyl sulfnxide, ethanol and acetone);
`(Including but not
`Pas-positive control
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`

`

`NDA 21-272
`
`113
`
`'
`
`Mouse Lymphoma L5178Y Cell Assay '
`
`1
`
`[The objective of this assay was to evaluate the ability of UT-15 to induce forward
`mutations at the thymidine kinase locus in the mouse lymphoma L5178Y cell line. These
`cells are heterozygous for the enzyme thymidine kinase ('TK+/'). Chemical-induced
`mutations at the TK locus result in the loss of thymidine kinase activity with the
`formation of a homozygous strain (TK’I'). Both TK+" and TK’/' strains can grow in normal
`medium, but the incorporation of 5—trifluorothymidine (TFT) into medium results in cyto-
`toxicity to the TIC" cells, with growth and replication occurring only in mutant TK‘I‘
`cells. Thus, resistance to TFT indicates mutation, induced by the test compound, at the
`TK locus. In this assay system, both gene mutations (large colonies) as well as
`chromosome aberrations (small colonies) can be detected]
`
`W4 M
`
`I'M—’-
`
`Study Number: 19159-0-431 ICH ( om
`
`Study Dates: January 22 to March 11, 1998
`
`GLP Compliance: The study was conducted in compliance with GLP regulations.
`
`Lot No. of the Test Compgund: LRX-98A01
`
`Concentrations Tested: 1. without metabolic activation — 15.7, 31.3, 62.5, 125, 250, 300,
`
`400, 500 and 600 ug/ml for the first trial (4-hour treatment period) and 15.7, 25.0, 31.3,
`37.5, 50, 62.5, 75, 87.5, 100, 125 and 150 u