A REPORT OF ThE CERhR AmPhETAmINES ExPERT PANEL:
2.3 Genetic Toxicity
Appendix II
are.discussed.below..Thymus.and.spleen.were.also.examined.in.some.rats.from.lower.dose.groups.
All.rats.survived.in.the.13-week.study..Final.body.weights.were.significantly.reduced.in.both.sexes.
of.all.treated.groups..Final.mean.body.weights.in.the.188,.375,.and.750.ppm.groups.were.11,.18,.and.
38%.lower.in.males.and.15,.26,.and.32%.lower.in.females..Feed.intake.by.the.750.ppm.males.was.20%.
lower.than.controls..Hyperactivity.was.observed.in.all.dose.groups.and.severity.increased.according.
to.dose..According.to.study.authors,.organ.weight.changes.were.due.to.body.weight.reductions..
[The study authors’ statement is confirmed by dose-related reductions in absolute weight and increases in relative organ weights observed in many organs. The exceptions that appeared to be dose-related were an increase in absolute brain weight (≥94 ppm females) and decreases in relative (to body weight) liver (750 ppm males) and thymus (≥94 ppm males, ≥375 ppm females) weights.].A.histopathological.examination.was.conducted.in.controls.and.the.750.ppm.group..No.
treatment-related.lesions.were.observed.[data not shown]..
In.the.13-week.mouse.study,.8.males.of.the.500.ppm.group.died,.3.males.of.the.1000.ppm.group.died,.
and.7.females.and.6.males.from.the.2000.ppm.group.died..Mean.final.body.weights.of.all.treated.
groups.were.lower.than.controls..At.doses.≥250.ppm,.body.weights.were.18.–.30%.lower.than.controls.
in.males.and.13.–.19%.lower.than.controls.in.females..All.treated.mice.were.hyperactive.and.severity.
increased.according.to.dose..Fighting.was.observed.in.males.exposed.to.≥250.ppm..According.to.study.
authors,.increases.in.relative.organ.weights.were.due.to.reduced.body.weights..[Decreases in absolute organs weights were also consistent with reduced body weights.] A.histological.evaluation.was.
conducted.in.the.control.and.2000.ppm.groups,.males.in.the.1000.ppm.group,.and.all.mice.that.died.
during.the.study..No.treatment-related.lesions.were.observed.[data not shown].
Appendix II
Table 23. Results of In Vitro Genetic Toxicity Testing of d,l-Amphetamine Reference Concentration Testing with
Metabolic Activation
Species or Cell Type/
Strain
Endpoint Results
NTP.(24) 100.–.10,000..
µg/plate Yes
Salmonella.
typhimurium.
TA98,.TA100,.
TA1535,.and.
TA1537
Mutagenicity.
at.the.histidine.
operon
↑ mutagenicity.
in.TA98,.with.rat.
but.not.hamster.
S9.metabolic.
activation.
↔ in.all.other.
strains;.results.
judged.equivocal.
by.NTP FDA.(34) ≤.5000..
µg/plate Yes Mutagenicity ↔
NTP.(24) 50.–.1600..
µg/L Yes
Chinese.
hamster.ovary.
cells.
Sister.chromatid.
exchange ↔
NTP.(24) 300.–.1000..
µg/L Yes
Chinese.
hamster.ovary.
cells.
Chromosomal.
aberration ↔
↔ =.no.change,.↑ =.statistically.significant.increase.
Table 24. Results of In Vivo Genetic Toxicity Testing of d,l-Amphetamine
Reference Species Dose (route) Cell Tpe Endpoint Results FDA.(34) Mouse 2.5.–.10.mg/kg.bw.
(oral)
bone.marrow.
erythrocytes micronucleus ↔ Tariq.et.al..(73). Mouse 6.25.–.25.mg/kg.
bw.(2.oral.doses)
bone.marrow.
erythrocyte micronucleus ↑ at.≥12.5.
mg/kg.bw
↔.=..no.change,.↑.=.statistically.significant.increase.
2.4 Carcinogenicity 2.4.1 Human
No.data.were.identified.
2.4.2 Experimental animal
The.NTP.(24, 71).examined.the.carcinogenicity.of.d,l-amphetamine.sulfate.in.F344/N.rats.and.B6C3F1. mice.in.a.study.conducted.according.to.FDA.GLP.procedures..Rats.and.mice.were.randomly.assigned.
to.groups.and.administered.USP.grade.d,l-amphetamine.sulfate.(99%.purity).through.diet..Fifty.rats.
(7.–.8.weeks.old).and.mice.(8.–.9.weeks.old)/sex/group.were.given.diets.containing.0,.20,.or.100.ppm.
Appendix II
d,l-amphetamine.sulfate.for.103.weeks..Study.authors.estimated.doses.of.~0,.1,.and.5.mg/kg.bw/day.
in.rats..Doses.were.estimated.at.0,.4,.or.30.mg/kg.bw/day.in.male.mice.and.0,.3,.or.19.mg/kg.bw/day.
in.female.mice..However,.the.dose.estimate.in.high-dose.male.mice.may.have.been.affected.by.feed.
scatter..Dose.selection.was.based.upon.results.of.the.13-week.studies.described.in.Section.2.2.2..Drug.
stability.and.concentrations.in.feed.were.verified..Animals.were.observed.daily.and.weighed.before,.
during,.and.after.the.study..Following.sacrifice,.all.animals.were.necropsied.and.major.organs.from.
animals.in.all.dose.groups.were.fixed.in.10%.neutral.buffered.formalin.for.a.histological.analysis..
Included.among.organs.analyzed.were.mammary.gland,.prostate,.testes,.seminal.vesicles,.ovaries,.
and.uterus..Survival.data.were.analyzed.using.Kaplan.Meier,.Cox,.or.Tarone.life.table.test.methods..
Continuous.data.were.analyzed.using.the.Dunn,.Shirley,.or.Jonckheere.test..Tumor.incidence.data.
were.assessed.using.logistic.regression.analysis,.Cox.life.table.test,.Fisher.Exact.test,.or.Cochran-Armitage.trend.test.
No.difference.in.survival.was.observed.in.the.rat.carcinogenicity.study..Body.weights.were.10.–.34%.
lower.in.female.rats.exposed.to.20.or.100.ppm.and.male.rats.exposed.to.100.ppm..[Compared to control animals, CERHR calculated body weight reductions of 11% in 20 ppm males, 10%
in 20 ppm females, and 20% in 100 ppm females, based on body weights presented in Table F1 and F2 of the NTP report. The Panel notes that body weight reductions >10% indicate that maximum tolerated dose was exceeded.].Feed.intake.of.the.100.ppm.females.was.84%.of.
control.levels..An.increase.in.testicular.interstitial.cell.adenomas.in.male.rats.was.not.considered.to.be.
treatment-related.by.study.authors.due.to.the.common.occurrence.of.that.tumor..[The results section listed the interstitial cell adenoma incidence as 34/50 in controls, 43/50 in the 20 ppm group, and 48/50 in the 100 ppm group. However, Table A3 of the NTP reported listed the incidence as 43/50 in controls, 43/50 in the 20 ppm group, and 48/50 in the 100 ppm group.] Thyroid.
follicular.cell.adenomas.were.not.dose-related.and.hyperplasia.was.not.observed.prior.to.development.
of.adenoma..An.increase.in.cataracts.and.retinal.atrophy.in.females.was.believed.to.be.due.to.excessive.
light.exposure.resulting.from.cage.placement..Because.amphetamines.can.cause.hyperthermia,.pupil.
dilation,.and.increased.activity,.it.was.also.postulated.by.study.authors.that.the.drug.could.have.
indirectly.contributed.to.the.formation.of.eye.lesions..An.increase.in.bone.myelofibrosis.was.thought.
to.be.due.to.loss.of.adipose.tissue.secondary.to.weight.loss..The.study.authors.concluded.that.none.
of.these.lesions.were.directly.caused.by.drug.exposure..Dose-related.reductions.were.observed.for.
neoplasms.in.adrenal.gland,.pituitary.gland,.mammary.gland,.and.uterus..
In.the.mouse.study,.there.were.no.differences.in.survival.of.treated.groups..Mean.body.weights.of.
males.were.reduced.by.10.–.36%.in.the.100.ppm.group.and.10.–.19%.in.the.20.ppm.group..Mean.female.
body.weights.were.reduced.by.10.–.34%.in.the.100.ppm.group.and.10.–.19%.in.the.20.ppm.group..
Final.body.weights.in.the.100.ppm.groups.were.60.–.70%.of.control.values..[Based on information presented in NTP Tables F3 and F4, CERHR estimated that compared to controls, body weights of animals in the 100 ppm group were reduced by ~25%; this suggests that maximum tolerated dose was exceeded.].Average.feed.consumption.was.not.reduced.in.treatment.groups,.but.the.authors.
stated.that.the.values.were.not.corrected.for.scatter,.which.may.have.led.to.overestimation.in.males..
Seventeen.females.in.the.100.ppm.group.were.mistakenly.bypassed.during.sacrifice.at.the.end.of.the.
study..They.received.water.but.no.feed.for.5.days,.at.which.time.they.were.killed,.necropsied,.and.
apparently.included.in.histological.analyses..A.slight.increase.in.thyroid.follicular.cell.neoplasms.in.
Appendix II
cell.hyperplasia,.a.lack.of.increase.in.follicular.cell.adenomas.in.female.mice,.and.a.reduced.incidence.
of. follicular. cell. hyperplasia. in. female. mice..Treated. mice. displayed. dose-related. reductions. in.
neoplasms.of.the.liver,.pituitary,.lung,.and.harderian.gland..Ovarian.atrophy.was.increased.in.females.
of.the.100.ppm.group,.but.it.was.not.stated.if.the.effect.was.statistically.significant..The.incidences.
of.ovarian.atrophy.were.14/49.in.the.control.group,.12/48.in.the.20.ppm.group,.and.25/46.in.the.100.
ppm.group.[P = 0.0054 chi-square performed by CERHR]..Study.authors.postulated.that.ovarian.
atrophy.may.have.been.due.to.reduced.body.weight.rather.than.a.direct.drug.effect.
The.study.authors.concluded.that.there.was.no.evidence.of.carcinogenic.activity.of.d,l-amphetamine.
sulfate.in.male.or.female.F344/N.rats.or.B6C3F1.mice..
In.a.review.of.the.NTP.studies,.the.FDA.noted.that.the.high.dose.in.rats.(5.mg/kg.bw.=.30.mg/m2).is.
equivalent.to.the.maximum.advised.dose.for.children,.“30.mg/25.kg.´.25.=.30.mg/m2,”.and.that.the.
high.doses.in.male.(30.mg/kg.bw.=.90.mg/m2).and.female.(19.mg/kg.bw.=.57.mg/m2).mice..are.only.
3.and.2.times.the.maximum.advised.children.’s.dose.(34)..
2.5. Potentially susceptible populations