Mutation Research/Genetic Toxicology and Environmental

Home , Genotoxicity, Mutagen

Mutation Research 795 (2016) 7–30

Contents lists available at ScienceDirect

Mutation Research/Genetic Toxicology and

Environmental Mutagenesis

jo urnal homepage: www.elsevier.com/locate/gentox

Comm unity address: www.elsevier.com/locate/mutres

Updated recommended lists of genotoxic and non-genotoxic

chemicals for assessment of the performance of new or improved ଝ genotoxicity tests

a b c d e

David Kirkland , Peter Kasper , Hans-Jörg Martus , Lutz Müller , Jan van Benthem ,

f f,∗

Federica Madia , Raffaella Corvi

Kirkland Consulting, PO Box 79, Tadcaster LS24 0AS, England, UK

Federal Institute for Drugs and Medical Devices (BfArM), Kurt-Georg-Kiesinger Allee 3, D-53175 Bonn, Germany

Novartis Institutes for Biomedical Research, Preclinical Safety, CH-4002 Basel, Switzerland

F. Hoffmann-La Roche Ltd., Pharmaceutical Science and Roche Innovation Center Basel, CH-4070 Basel, Switzerland

RIVM-National Institute for Public Health and the Environment, Bilthoven, Netherlands

European Union Reference Laboratory for Alternatives to Animal Testing (EURL ECVAM), Systems Toxicology Unit, Institute for Health and Consumer

Protection (IHCP), European Commission—Joint Research Centre, TP 126, Via E. Fermi 2749, I-21027 Ispra, Varese, Italy

a r t i c l e i n f o a b s t r a c t

Article history: In 2008 we published recommendations on chemicals that would be appropriate to evaluate the sensitiv-

Received 16 September 2015

ity and speciﬁcity of new/modiﬁed mammalian cell genotoxicity tests, in particular to avoid misleading

Received in revised form 27 October 2015

positive results. In light of new data it is appropriate to update these lists of chemicals. An expert panel was

Accepted 28 October 2015

convened and has revised the recommended chemicals to ﬁt the following different sets of characteristics:

Available online 4 November 2015

•

Group 1: chemicals that should be detected as positive in in vitro mammalian cell genotoxicity tests.

Keywords:

Genotoxicity Chemicals in this group are all in vivo genotoxins at one or more endpoints, either due to DNA-reactive

or non DNA-reactive mechanisms. Many are known carcinogens with a mutagenic mode of action, but

In vitro test

Reliability a sub-class of probable aneugens has been introduced.

•

Improved tests Group 2: chemicals that should give negative results in in vitro mammalian cell genotoxicity tests. Chem-

EURL ECVAM icals in this group are usually negative in vivo and non-DNA-reactive. They are either non-carcinogenic

Reference chemicals

or rodent carcinogens with a non-mutagenic mode of action.

•

Group 3: chemicals that should give negative results in in vitro mammalian cell genotoxicity tests, but

have been reported to induce gene mutations in mouse lymphoma cells, chromosomal aberrations or

micronuclei, often at high concentrations or at high levels of cytotoxicity. Chemicals in this group are

generally negative in vivo and negative in the Ames test. They are either non-carcinogenic or rodent

carcinogens with an accepted non-mutagenic mode of action. This group contains comments as to any

conditions that can be identiﬁed under which misleading positive results are likely to occur.

This paper, therefore, updates these three recommended lists of chemicals and describes how these

should be used for any test evaluation program.

license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

1. Introduction

+/− In 2007, we published [1] the recommendations of a workshop,

Abbreviations: CA, chromosomal aberrations; MLA, mouse lymphoma tk

organised and funded by the European Reference Laboratory for

mutation assay; MN, micronuclei; UDS, unscheduled DNA synthesis; IARC, Inter-

national Agency for Research on Cancer; NTP, National Toxicology Program; RTG, Alternatives to Animal Testing (EURL ECVAM), in which ways to

relative total growth; E, equivocal; ip, intraperitoneal; HPRT, hypoxanthine-guanine

reduce the frequency of “misleading” or “irrelevant” positive results

phosphoribosyl transferase; −ve, negative; +ve, positive; MoA, mode of action.

ଝ (i.e. positive results found in vitro that are not predictive of in vivo

Disclaimer: This document represents the consensus of the authors’ views

genotoxic or carcinogenic activity), particularly in mammalian cell

expressed as individual scientists and does not necessarily represent the policies

tests (as highlighted in [2]), were discussed.

and procedures of their respective institutions.

∗

Corresponding author. Fax: +39 0332 789963.

E-mail address: [email protected] (R. Corvi).

http://dx.doi.org/10.1016/j.mrgentox.2015.10.006

8 D. Kirkland et al. / Mutation Research 795 (2016) 7–30

Several suggestions were identiﬁed for possible improve- and validation studies of in vitro and in vivo test methods, amongst

ments/modiﬁcations to existing tests, or new tests that showed which the validation of the in vivo comet assay, the micronucleus

potential. Such improvements or new assays need to show and comet assays in 3D reconstructed skin models, the Pig-a assay,

improved speciﬁcity (i.e. give fewer “misleading” positive results) the cell transformation assay in Bhas 42, and the micronucleus test

without compromising sensitivity (i.e. still detecting in vivo geno- in hen’s eggs (HET-MN) [24–28]. Moreover, as already mentioned,

toxins and DNA-reactive carcinogens). In recent years, several some projects that have used the lists have been carried out in order

experimental initiatives have identiﬁed that the reliability of to explore the ways to reduce misleading positive results in the

in vitro mammalian cell tests (i.e. improved speciﬁcity whilst retain- in vitro mammalian cell tests within the current testing practices,

ing high sensitivity) can be improved by using p53-competent and to identify the relevant parameters that need to be considered

human cells [3] and by choosing measures of cytotoxicity based on for the development of new or improved tests [3,4,29].

cell proliferation [4] rather than simple methods such as relative The extensive use of the EURL ECVAM lists of chemicals and

cell count or vital stains. the references to them for test method design, development and

However, if further improvements to in vitro tests are to be eval- implementation, prompted us to revise and update the lists in light

uated, or if new tests are to be developed, it is important to have a of new information.

reference set of chemicals to test where the outcomes (true posi- EURL ECVAM therefore kindly organised a meeting for the

tive, true negative or misleading positive) can be predicted on the authors in order to review and update these lists at a workshop

basis of results obtained under standard test conditions. In 2007, at EURL ECVAM, Ispra, Italy on 23rd October 2014. In reviewing

EURL ECVAM therefore convened an expert panel in order to iden- these lists it was important to question whether the chemicals pre-

tify recommended lists of chemicals, and these recommendations viously recommended were still justiﬁed in light of new data, or

were published in 2008 [5]. The lists covered the following 3 sets data previously not found. If the data did not support chemicals in

of chemicals: the respective lists, should they be deleted or allocated to another

group? If chemicals should be deleted, could replacements be iden-

•

Group 1: chemicals that should be detected as positive in in vitro tiﬁed? Therefore, as before, careful consideration was given to the

mammalian cell genotoxicity tests. Chemicals in this group are published evidence that would support the inclusion of each chem-

all in vivo genotoxins, either due to DNA-reactive or non DNA- ical in each of the lists, and in the tables provided here detailed

reactive mechanisms (e.g. induction of aneuploidy, inhibition of justiﬁcations and supporting references (not an exhaustive list,

topoisomerase). Most of them are also known carcinogens with but considered representative) are provided. Complete and con-

a mutagenic mode of action, but a sub-class of probable aneu- sistent data sets are still not available for all chemicals, although

gens has been introduced whose carcinogenic properties are not we have supplemented some of the Ames test data with new tests

clearly understood. in Salmonella typhimurium TA102 (see later). In the cases where

•

Group 2: chemicals that should, and routinely do, give negative there are still gaps and inconsistencies, our assessment is based on

results in in vitro mammalian cell genotoxicity tests. Chemicals in a weight of evidence approach. In order to develop weight of evi-

this group are usually negative in in vivo genotoxicity tests (when dence decisions, data of various kinds were taken as being more or

tested) and non-DNA-reactive. They are either non-carcinogenic less important. The information to build a weight of evidence that

or rodent carcinogens with an accepted non-mutagenic mode of a chemical is a DNA-reactive carcinogen or in vivo genotoxin, and

action. should be detected with a new or modiﬁed genotoxicity test, will

•

Group 3: chemicals that should give negative results in in vitro not necessarily be the same as that needed to decide a chemical is

mammalian cell genotoxicity tests, but have been reported to not DNA-reactive and should not be detected.

induce gene mutations in mouse lymphoma cells, chromosomal Tables 1–3 summarise suggested reference substances for the

aberrations or micronuclei, often at high concentrations or at three groups. Classiﬁcation is mainly based on in vivo genotoxi-

high levels of cytotoxicity. Chemicals in this group are generally city and DNA reactivity, while carcinogenicity data are used as a

negative in in vivo genotoxicity studies (when tested) and nega- supplementary criterion. The deﬁnition of “DNA reactivity” is pri-

tive in the Ames test. They are either non-carcinogenic or rodent marily based on results from bacterial mutagenicity tests (“Ames

carcinogens with an accepted non-mutagenic mode of action. test”), i.e. Ames-test-positive indicates DNA reactivity while Ames-

test-negative indicates the absence of it. However, aneugens have

Since its publication the EURL ECVAM recommended lists of now been included which, although not DNA reactive per se, are

genotoxic and non-genotoxic chemicals have become a major ref- genotoxic and their inclusion is justiﬁed by the speciﬁc informa-

erence for test developers in the area of genotoxicity. The whole tion given in the tables. The tables contain further information

lists, or part of them, have been used in the evaluation of a large about the chemicals, including information on the requirement for

number, if not most, of the newly developed genotoxicity assays metabolic activation, or the mode of action if known. Within the

[6–13]. tables, subgroups of chemicals are identiﬁed based on chemical

The lists have also been employed for the selection of chemicals classes or a speciﬁc pattern of results in genotoxicity tests. It is

used to assess whether modiﬁcations to existing protocols led to recommended that examples from each subgroup are included for

improvements of the assays in terms of performance, and in par- any test evaluation program. However, some scientists may have

ticular in relation to an enhanced speciﬁcity [14]. Amongst the new their own “favourite” chemicals for inclusion in such an evaluation

and improved assays, many are high-throughput screening meth- program, and the lists given here are not meant to be exhaustive.

ods [15–19]. In certain cases existing tests have been miniaturised

or automated and the new versions of these traditional tests needed

in vivo

to undergo validation [20–23]. 2. Considerations for updating group 1— genotoxins

The recommended chemical lists have likewise served to sup- and DNA-reactive, mutagenic carcinogens that should be

in vitro

port the chemical selection of several international inter-laboratory detected as positive in mammalian cell tests (“true

positives”)

1 p-Chloroaniline was included as a group 1 chemical in the previ-

In this manuscript, substances with a mutagenic mode of action refer to

ous publication [5] although it was non-carcinogenic according to

those substances that are positive in the Ames test, while substances with a non-

mutagenic mode of action are negative in the Ames test. the Carcinogenicity Potency Database [30], and gave mixed (posi-

D. Kirkland et al. / Mutation Research 795 (2016) 7–30 9 forms 1B1,

forms

7 (N

1A1,

mutagen

(CYP2B6) (CYP (CYP1B1),

metabolic metabolic metabolic

hydrolase),

gene clastogen

adducts adducts

alkylation) 6 Requires Requires Comments alkylation) Strong Strong Requires (O bulky epoxide activation activation activation bulky

. . . with in oral

skin sites rats, lung female

. [87] [108] [113]

in in

hamsters mice small after after

ﬁndings IARC after

and Skin

skin

rats),

[78] carcinogen carcinogen carcinogen ,

by carcinogen thyroid

tumours in

1 2A 2A 2A multiple rats. mice male mice mice, following

mice

[30]

tested human

at and tumours

tumours in in in in in

system,

application application application

to and

mice (not Group Group Group Group

subcutaneous gerbils classiﬁed

rats

dermal Stomach mice Carcinogenicity Tumours Nervous Haematopoietic [30,104] Vascular administration tumours tumours IARC dermal dermal IARC IARC and intestine oesophageal tumours and tumours carcinogenicity. in Not systemically tumours IARC male regard , , HPRT CA cell

, MN [51,76] MN

HPRT

, , [73–75]

CA endpoints UDS mutations

, [76] MN

[86] variable ,

[37,111]

and [73]

concentrations [85,97,99–101] [99,100]

[76] metabolism

HPRT , low

but , [37] [86,102,103] UDS

[37,96,97]

, mammalian

[76,77] at multiple MLA MN

low

[84] and

CA UDS

MLA at for for for needs

MLA

vitro vitro

In for but +ve and +ve [85] +ve [73,74,98] and +ve [37,73,76,86,99,100,107] +ve [73,74,112] [86,102] mutations in MLA concentrations responses tests mutations and

CA and

, [70] gene

trial comets marrow

than

, [94,95] [110] ve and

[71] [71,83]

MN MN

and

mutations −

, , gene

stomach

comet

bone

, in UDS transgenic [62]

JaCVAM

for

, ,

[71] [71] [71,83] [60,61] [82] [90] [62,68,109]

more tissues

marrow the genotoxicity

CA CA CA MN MN

tests.

mutations but in

transgenic comets blood , for for for results for for

vivo many bone

tests in mutations In +ve [50,62–70] [70,72] +ve [51,62,68,70] and +ve [63,91–93] and [24] +ve +ve mutations in mutations liver and +ve gene genotoxicity

cell

proﬁle

a mammalian

test

[36,59] [36,79–81] [36,79,80,105,106] [36,45,79,106] vitro

+ve +ve Genotoxicity Ames [31,36,45,79,88–89] +ve +ve +ve in

positive

detected

(57-97-6) be

genotoxins/carcinogens hydrocarbons

should

vivo

which

aromatic

alkylators 7 N

]pyrene a and number) genotoxins

6 1

Polycyclic

Ames-positive

vivo I. Chemical (CAS Cyclophosphamide (6055-19-2) (i) (50-32-8) Benzo[ (ii) MMS (66-27-3) 7,12-Dimethylbenzanthracene ENU (759-73-9)

In Table

10 D. Kirkland et al. / Mutation Research 795 (2016) 7–30 [123]

S9) highly

Forms

with with

not CYP1A2 requires after

liver

by and

guanine

rat

amine amine

- 6 guanine agent

in by [161] [161] activation on

amine, O acetylated.

metabolic metabolic [123]

genotoxicity, genotoxicity,

then

adduct -methyl

7 Hydroxylated requires requires CYP2E1(which expressed N Alkylating Heterocyclic Comments Aromatic Heterocyclic potent potent produces adducts activation C8 activation activation metabolic and . .

. in and

with [30]

and and [30]

[30]

. in dermal

. rats rats rats ﬁndings IARC gland kidney, oral

mice

rats

[30] carcinogen carcinogen carcinogen carcinogen

in in lung, tumours

after

and

and 2B 2A 2A 2B multiple [129] male

prostate after

gland vascular

also human in

[30] in

system,

mutations

rats

haematopoietic and

and kidney bladder mammary but

tumours tumours

Group Group Group Group

tumours GI classiﬁed

Tumours carcinogen application liver/bladder mice, tumours, also organs, Carcinogenicity Liver Liver, Liver Mainly Not testes Induces administration mice, mice, nervous carcinogenicity. IARC IARC mammary IARC regard IARC tumours tumours skin , and [86]

and

both and , cell

UDS for +ve UDS UDS

MN CA variable [157] have [76]

CA [121] [171] , ,

, for and and −

[37,96] and and

CA but

UDS , [37] [37]

, [73] [154–156] [73,156,169] but

MN but

for ,

, , MLA

, mammalian

MLA MLA MN MN MN [148,160]

mutations mutations mutations mutations reported

[157] [158,159]

for for for for for

[73,170]

vitro

ve In +ve [107,112,119] [86,102,120] [99,121,122] +ve [131,132] [102,133] +ve [76,138] [100,139,140] +ve +ve CA UDS HPRT results − tests HPRT responses HPRT been HPRT in

, CA in

[32]

for ,

ve for +ve MN

− ve and , [153]

ve kidney

multiple and −

− UDS

comets comets and ,

but both in CA

for

, comet bone liver but [63,116]

generally [116,127]

endpoints

both and and

, [94,95]

mutations

[117] but

[94,95,127]

in in

[152]

, marrow [94,114]

mutations [71] [71,166,167]

but − [151] MN

but

[32] , results UDS

[151]

genotoxicity many UDS gene transgenic MN [71]

[24] [168] UDS transgenic

and

bone marrow , , mutations

[24]

for +ve comets brain [62,148,152] for for for for for

CA in

vivo

ve mutations tests for comets MN hepatocytes In +ve [117] [118] +ve [71,128,129] [24,130] and +ve [71,83] [93,116,137] +ve +ve [165] and transgenic marrow gene results including liver − mutations bone CA and tissues

proﬁle

a test

[36,45,79,114,115] [36,79,124–126] [36,79,89,134–136] [141–150] [149,150,162–164]

+ve +ve +ve Genotoxicity +ve Ames +ve ) [4,5-b]pyridine

amines

(2-amino-1-methyl-6-

Continued ( number)

Aromatic 1

(2-amino-3methylimidazo[4,5-f]quinoline)

(76180-96-6) (iii) Chemical (CAS 2,4-Diaminotoluene (95-80-7) IQ PhIP.HCl (105650-23-5) phenylimidazo 2-Acetylaminoﬂuorene (53-96-3) Dimethylnitrosamine (62-75-9) Table

D. Kirkland et al. / Mutation Research 795 (2016) 7–30 11 has

damage and

compared forms

inhibitor

also

Forms agent

rats CYP3A4, highly

but agent, in

by activity

not

DNA–DNA oxidative adducts

humans. adducts

adducts

No Activated Comments Topoisomerase Alkylating with Cross-linking akylating expressed crosslinks, Induces which DNA various induces .

but

in ,

[78] (high on and

of for

base)

mice

the

rats 1 [30] mice

effects breaks animals

tongue

rats

of according

[209] ﬁndings

gene [113] in

based

carcinogen intestine

myeloid (free carcinogen in

the the a

carcinogen

the humans

translocations

CPDB

recently Group

of 1 2B 2A 2B evidence

evidence rats,

+ve

in cavity of

large

as and acute

in in

but gene, [30]

tumours adeno- genotoxic

tumours

injection

and adenomas Group Group Group Group

leukaemia limited of . . MLL

CPDB 11q23

non-carcinogenic inducing and Carcinogenicity Liver [30] Non-carcinogenic according [51] Lung Inadequate Induces Induces [237] involved leukaemia IARC peritoneal IARC mas/adenocarcinomas distinct classiﬁed considered lung, sarcomas/ﬁbrosarcomas IARC at haematopoiesis) localization site humans to splenic carcinomas carcinogenicity within the tumours frequency and IARC UDS

MLA cell MN

UDS

, MLA for

, MN UDS

and

results, ,

and HPRT

, [208] and and

+S9,

MLA

, [51,76] HPRT

, HPRT [183]

, low

, low mutations

[194] [99,194] [76,178] at [132,219] [76]

at [204,205]

MLA mammalian

for

CA CA CA CA

CA HPRT Comet,

mutations

, , [86,103,236]

for for in for for +ve

vitro

In variable mutations mutations but +ve [107] [100,179] [73,180] +ve [51,192,193] [85,140,194–197] [198] +ve [200] [206,74,75,207] +ve [73–75,112,220,221] [96,221–224] [73,198] +ve [119,221,234] [37,111,221,235] concentrations concentrations UDS HPRT tests mutations , , , in [71] MN

and and , and

and

MN for

for

, [94] ve for CA ve

[24] mutations [24,130]

− transgenic −

[32,188–190]

marrow

[172] and UDS [51]

mutations mutations mutations

[232,233] [51,214,215] [228,229] but

, liver

but

and [181,182]

MN ,

, [177] in genotoxicity MN comets CA MN CA CA

bone [51,68,70,116,216]

[71]

transgenic in for for results for for for for

vivo

mutations liver tests In +ve [173–176] +ve +ve [78,188] [71,191] +ve [200,203] +ve and [217,218] +ve [230,231] equivocal MN transgenic comets comets transgenic blood transgenic +ve

proﬁle results

[199–201] a

+ve

test +ve

clear [30,79] [36,51] [81,89,184–187] [45,79,89,225–227]

+ve +ve Genotoxicity +ve Ames and [202] [88,89,186,210–213] Weak +ve +ve salt

HCl

and

base

-oxide N ) C 20265-96-7)

Continued ( number)

Others 1

-Chloroaniline—free (106-47-8; Chemical (CAS (iv) Aﬂatoxin (50-07-7) (1162-65-8) p Mitomycin Etoposide (33419-42-0) Cisplatin (15663-27-1) (56-57-5) 4-Nitroquinoline- Table

12 D. Kirkland et al. / Mutation Research 795 (2016) 7–30 likely

analogue analogue analogue

MoA, repair

including

carcinogen carcinogen, possible

MoA, aneuploidy action.

multiple of aneugen aneugen aneugen Nucleoside nucleoside nucleoside oxidant?

Likely MoA: MoA: MoA: MoA: MoA: Inorganic Inorganic possible inhibitor? MoA: including multiple MoA: aneuploidy mode

MoA,

. in

is ip in

[292]

humans; . [201]

ve cell

. to −

rats after lung,

ﬁndings Comments

tumours not not not not to [30]

dosing in

mice

[30] animals

carcinogen carcinogen but

and testicular

, 1 3 3 3 1 in [78] ve rats mice

“sufﬁcient”

oral −

lymphoreticular

in in and mice

liver squamous

[290]

and and after

. Group Group Group Group Group

and

carcinogenicity

Carcinogenicity Kidney, mice carcinomas Haematopoietic, Carcinogenicity Carcinogenicity Carcinogenicity IARC Vaginal Carcinogenicity Lung [291] Carcinogenicity [306] IARC IARC established established established established experimental tumours haematopoietic tumours IARC IARC dosing prostate rats considered its

in at

, , , , and

for for

cell +ve

, MLA , MLA

MN MLA but

[250] ve

[253] test ,

[250] +ve classiﬁable and [224] cells

− low

, before CA

[73,253]

[289] , and

, ,

CA CA

and MN at –S9

ve [296–300] not a a but

TK6 as

HPRT

h CA [224] [38] −

MLA

[37]

[37] but in in

and 3 in [248,249] [276] [76]

CA [254]

to to 24

in or

and and

mammalian

MN MN MN MN CA CA structural MLA

MLA

and g/ml

mutations re-evaluation [NTP] with [275]

group [73] [301–303] for for for for for in for for ␮ for

vitro

In re-evaluated polyploidy MLA MN concentrations, MLA treatment equivocal +ve [73,75,245,246] [76,247] +ve [73,75,220,221,252] +ve +ve [119,207,262–264] [265] +ve [73–75,98,112,272–274] +ve [278,279] +ve [284–286] [75,220] +ve [75,220,289] +ve [304,305] MLA gene <10 mutations numerical mutations tests after polyploidy according “uninterpretable” MN according TA100.

and carcinogen;

in is

liver TA98

for

in for

bone

[24] MN

and human [251] for and

marrow marrow

, in and comets

comets

-ve −

[269]

, and

liver colon and

[24] for

lung MN liver mutagen

for

bone

bone bone

CA TA1538, but

, ve in [282,283] [294,295] and in in and possible for

mutations and

and [255] and

[51,277] for

liver liver

−

MutaMouse =

blood [24] [260,261]

in in [24] genotoxicity CA CA MN MN MN MN MN MN of

+ve

+ve TA97a,

and

stomach, [278] ,

blood for for for for for for for for

vivo liver stomach

group stomach tests in comets In +ve [242–244] +ve [62,68,77] +ve [77,256,257] [258] Strong [251] +ve [77,267–270] [271] +ve +ve +ve [92,116,283,288] +ve kidney in and marrow transgenic marrow, duodenum mice comets transplacental liver,

TA97

, carcinogen; of and

coli TA1537 ,

[51] [276]

strains strains

and

to in

proﬁle

Ames

TA102 human

reports

in TA102 +ve a in TA1535,

[238,239] TA102

are

standard standard

but in test [287] and

, , +ve

results [31] [36] [259] [186,266] according in in [281,293]

+ve

probable strains

there

coli

ve ve ve ve ve ve ve ve ve =

E: − − Genotoxicity − E. both Ames [51,240,241] [280] [281] [186] [280] but − − − − − − equivocal

group

typhimurium

negative S.

means

carcinogen;

carcinogens

column human

this

and/or

group

) strains”

sulfate

chloride genotoxins aneugens

arabinoside

arsenite

Continued ( number)

vivo

classiﬁcation:

“Standard a (10108-64-2) II. Chemical (CAS (7784-46-5) (143-67-9) Cadmium (147-94-4) Probable Colchicine (64-86-8) Cytosine 5-Fluorouracil (51-21-8) Sodium Hydroquinone (123-31-9) Vinblastine Non-aneugens Azidothymidine (30516-87-1) Taxol (33069-62-4) Table IARC

D. Kirkland et al. / Mutation Research 795 (2016) 7–30 13 on

used

[307] available

[316]

MN protocols

available

data

vivo but

, website in

details

different ve g/kg.

Comments − No [315] 2 5 FDA

and to

24 [30] [309] [30] [309] [30] [317]

rats

ﬁndings

in mice mice mice mice mice mice were

[316]

According and and and and and and

. these

studies rats rats rats rats rats rats

[315]

data in in in in in carcinogen in

ve ve ve ve ve ve ve − FDA Carcinogenicity − − − − − − month mice , ), ) ), b &

at b b )

[37] S9, for 56%

V79 [51] and − 38

+ve [317] very ,

& to

, tests.

cells as

how

+ g/ml g/ml +ve

[308]

to tests g/ml

, g/ml 25

up up ␮ ␮ 75–80%

17 g/ml g/ml ␮

␮

rat

[315] V79

[316] and ␮ ␮

biological cell or clear MLA

in HPRT

− reduced MLA [307] concs

500 2160 2120

than and 5000

[250] ve

signiﬁcant

not data for +S9 ve but HPRT

“uninterpretable”

MI to to to 1500 5000

reduced

−

and − h

inducing

genotoxicity questionable) to to

re-evaluated [309]

g/ml

+ve lymphocytes control,

“uninterpretable” dose-response, (up (up (up FDA

mg/ml up

[309]

) and

[37] plasma

was ␮ higher

lymphocytes MLA, b

)

cells, was scored up [51] up

cell

b to but

no 10

lymphocytes ,

CA mammalian survival

CA MLA CA MLA CA CA, MLA MLA

g/ml g/ml

V79 S9, 2120 cells

human g/ml concs

␮ ␮ ,

[51]

(statistically −

for for for for for for for for

respectively,

negative up in ␮ to

re-evaluated

at (hum (human in

vitro

ve ve ve ve ve ve ve ve − − − MLA questionable therapeuric In − [250] and − [250] − − − and CA SCE concentrations many CA low 64% “uninterpretable” but CHO/HPRT 506 and CA CA (up re-evaluated cells lymphocytes, toxicity, only 5000 1000 signiﬁcance respectively according relative mammalian

vitro

in or

iv, po) 2x

po x bone [317] MN,

tests

MN died), 250 h ) 1

oral

top

b [50] FDA

[309]

results

TK 48 dosing; and for single single scored,

dosed

toxicity

bone to

CA UDS good

harvest 125,

or bone (3/12 cytogenetics cytogenetics cytogenetics

dose,

mg/kg rat and at marrow oral

marrow

24 mg/kg [307] and were

apart,

liver

in with

mg/kg mg/kg

sampling

vivo vivo vivo

2nd tox mg/kg

MTD h

exposure

40 12.5

negative

h 12,

marrow cells sampled CA

sampling) after

bone MN in rat MN in CA in MN mouse

[309] indicate bone

genotoxicity

24 to to 100 900 h According mice

48 150

PCE/animal . ,

was

after

for for for for for for for for for dose; rat give

mg/kg to to

other

up up

to bone

h vivo

2 24,

ve ve ve ve ve ve ve ve ve (up up no studies − marrow oral marrow In − − − − [311] − [315] [316] − rodents systemic orally, 8, in (mouse (2) 1000 (up dose; 24, − dose studies marrow times but 500 24 (1) − should

that

) 4

and in [36]

[311]

and coli Table data

( b

b E. coli

strains strains strains strains strains strains carcinogens) strains

coli [49] in E.

proﬁle

E.coli E.

TA102

a and in in

in [317]

and TA102

standard standard standard standard standard standard standard

test [310] and and

genotoxicity

coli

in in in in in in in also

E. coli

ve ve ve ve ve ve ve non-genotoxic

vivo − − − − − − Genotoxicity Ames and [309] [36] [309] [32,36] [312,313] [314] E. in −

(including

negative

with

chemicals

number)

trihydrate

(CAS

diclofenac

(7177-48-2) (69-65-8) (124937-51-5) trimethyl-ammonium chloride (999-81-5) (68291-97-4) (15307-79-6) (122852-42-0) Non-carcinogens

I. Chemical D-mannitol Tolterodine (2-chloroethyl) Sodium Zonisamide Ampicillin Alosetron Non-DNA-reactive Table

14 D. Kirkland et al. / Mutation Research 795 (2016) 7–30 Comments

and

to due

[51] [51] [30] [30] liver;

kidney: rats liver

[51] tumours adducts sales

ﬁndings in apply

rat

mice mice mice mice mice carcinogen carcinogen carcinogen

breaks

to mouse in

that hormonal liver DNA

proliferation. [333]

3 2B 2B 2B

mouse

may

prolactin and and and and and in

sulphate

deﬁciency.

to due tubules

of due

and strand

liver and rats rats rats rats rats

due carcinogen [51]

Group Group Group Group induce

glubulin

in in in in in renal possible

not

␮

choline

is ve ve ve ve ve 2

secretion. IARC ephedrine and effects restrictions Carcinogenicity − − − It − − [30,51] Thyroid [325] Liver Tumours Tumours IARC promotion IARC does but mouse mice to peroxisome ␣ IARC , ,

, , , , , g/ml 50% [37] for and

MLA

cell

␮

g/ml [250]

[51] [250]

␮ >90%

90% 90% 90%

for

[250] [250] [250] [250] [250] but but and

CA as , , 30% 500 trials

to to to

[51]

[98] to to to

1600 but but re-evaluated as as as as

up up up and [51] [51]

+ve

, , , [319] CA [331,332]

MLA

up to up up [51] [138]

h-S9

for but

mammalian [37] [51] [51] [37] [321]

6/7 CA MLA CA MLA CA MLA CA MLA MLA CA MLA CA MN

g/ml 24 re-evaluation

␮

for for for for for for for for for for for for for for − at

re-evaluated

[51]

vitro “uninterpretable” the

ve ve ve ve ve ve ve ve ve ve ve ve ve ve but toxicity re-evaluated re-evaluated toxicity in − [51] − − [51] “Uninterpretable” and − − − − − 5000 CA toxicity toxicity toxicity, − In − − − − − re-evaluated re-evaluated as MN tests “uninterpretable” toxicity “uninterpretable” “uninterpretable” “uninterpretable” “uninterpretable”

, tests

[51]

[324]

[71]

CA MN

, transgenic liver

and [51] [330]

[51] in

and

MN CA MN MN

genotoxicity

[94] for for for for

data data data data data

vivo

ve ve ve ve No No No − In No No − − − mutations UDS ) ) ) 4 4 4

[36] [36] [36]

[320] TA102

Table Table Table ( ( ( coli

in [318] [322,323] [329]

coli strains strains strains strains strains strains strains strains strains

proﬁle coli coli coli

in also

data in E. E. E.

TA102 TA102 TA102

a and in in in and in in in

) and

standard standard standard standard standard standard standard standard standard 4

test

and and and in in in in in in in in in also also also

ve ve ve ve ve ve ve ve ve Table − − − − − ( Genotoxicity Ames [36] [36,44] and and [36,280] [51] [51,326] [327,328] [36] and − − − − genotoxicity

vivo

with

carcinogens

thiourea

) number)

HCl

(CAS

chloride

Continued (

2 -dicyclohexyl

Non-genotoxic

Non-carcinogens (1212-29-9) (61-82-5) (109-69-3) sulphate/hydrochloride (134-72-5/134-71-4) (834-28-6) (111-42-2) (67-72-1) stearate (114-07-8/643-22-1) phthalate (117-81-7)

N , -Butyl II. III. Amitrole Chemical N Ephedrine Phenformin n Diethanolamine Hexachloroethane Erythromycin/erythromycin Di-(2-ethylhexyl) Table

D. Kirkland et al. / Mutation Research 795 (2016) 7–30 15 Comments b b b

) b and in mice from

[51]

and

in to

strain- [309] [309] [309]

(poor rats female ﬁndings

due

-globulin tumours relevance relevance relevance

class-

MoA MoA MoA

+ve for

due strain

speciﬁc

␮

in in

, carcinogen 2 mice mice mice

ureteral [344] proliferation resulting

3 3 3 3

[51]

bladder

␣

but

[51]

[51] to kidney formation clinical clinical clinical

and and and

and

effects

rats,

and

and mice rats

inﬂammation mineralisation

hepato-carcinogen tubule tumours

rat tumours tumours rats

. Group Group Group Group

due in in to to high-dose

rats rats rats

F344

ve ve nephropathy. IARC in rodent-speciﬁc speciﬁc tumourigenicity. IARC − and Carcinogenicity Male [51] Bladder [339] Liver due Renal Weak +ve Non-genotoxic +ve Non-genotoxic, Kidney due − [309] (leiomyosarcoma, Liver [51] +ve Non-genotoxic tumours peroxisome without without without hyperplasia carcinomas calculus mice, IARC F344 kidney clearance). IARC bioaccumulation ) b ) ) ), for for for for and to b b b

for

) b ve ve ve ve to to to to up conc

MLA

− − − − -ve

g/ml g/ml g/ml , , , , , tests

up up up up ␮ ␮ ␮

but

, and

cells, re-evaluated re-evaluated re-evaluated re-evaluated re-evaluated

cell

higher

[250] [250] [250] [250] [250] [309]

technically

solubility 5000 4000 5000 [37] )

at cells b but but but but but

, , , , , of to to to (CHO TA100.

but

‘uninterpretable’ [51,319]

, UDS

MLA [309] [309] and

CHL g/ml

[37] [37] [37] (up (up [51] (up [37]

) ) ) and

␮

lymphocytes lymphocytes lymphocytes lymphocytes limit b b b

[51] in

cells

cytotox

mammalian CA MLA MLA MLA CA MLA MLA MLA MLA MLA MLA,

g/ml g/ml g/ml TA98 2500

mutations CHO g/ml;

␮ ␮ ␮

g/ml; for for for for for for for for for for for ␮ to

hepatocyte [51] in [51] (human (human [51] (human [51] (human ␮

vitro ”uninterpretable” “uninterpretable” ”uninterpretable” “uninterpretable” “uninterpretable”

ve ve ve ve ve ve ve ve ve ve ve − (up In − [250] − − − − [344] − − − − − [309] CA CA CA CA 40 CA rat as as as as as and CA and CA CA 5000 1000 5000 200 re-evaluated CA HPRT compromised TA1538,

humans.

TA97a,

or to

or po

x po; UDS ) [309] MN, MN,

[335]

tests

1 after )

oral

mice x po) b for [50]

1x TA97

oral

x CA,

1 and

or comet 1 2x cells CA

carcinogenicity

rat and

cytogenetics cytogenetics cytogenetics cytogenetics

CD-1 blood

mg/kg marrow marrow for

: in

its

mg/kg

[344] single

[338] [51,340] [342] in in and sampling

mg/kg [71]

marrow sampling to vivo vivo vivo vivo

h mg/kg mg/kg

marrow

kidney

h TA1537 400 2000

UDS [51]

bone bone transgenic MN MN MN MN in in MN in MN in

as [309]

genotoxicity

liver

48 to to 400 1000 500 mg/kg

bone

for

for for for for for for for for for for for

bone

48, to to to days

vivo

up up

24,

rat

ve ve ve ve ve ve ve ve ve ve ve

mutations up up 90 studies hepatocytes; 2) In − − − − [343] − − [309] − [309] − − − − dose; 6, comet mouse (mouse (mouse (rat 2 1000 dose: 24, up 1) PCE, TA1535,

classiﬁable

strains not

and

[345]

group [337]

[334] [341] b b

b b [346] coli

TA102

typhimurium

strains strains strains strains strains strains strains strains strains strains strains

E. coli coli TA102 in

proﬁle coli

TA102 E. E. E.coli E.coli in

TA102 TA102 E.

a in and in in in in

in in in [344]

carcinogen; and

standard standard standard standard standard standard standard standard standard standard standard

test and and and and and

Salmonella

coli and and and in in in in in in in in in in in

ve ve ve ve ve ve ve ve ve ve ve human − − − − − − Genotoxicity Ames [36] [336] [36,44,240] [287,320] [320] [36] [309] [309] [44,51] [309] [36] [309] in − − − − −

means

docs.

possible

column =

BfArM this

group

) number)

internal

strains”

alcohol (CAS

from

carbamate

Continued (

classiﬁcation:

(75-65-0) (5989-27-5) (91374-21-9) (287714-41-4) (107753-78-6) (598-55-0) phosphate (78-42-2) (108-78-1) (110-86-1) (688046-61-9) (97240-79-4) “Standard Details a Chemical D-Limonene Ropinirole Rosuvastatin Tert-butyl Zaﬁrlukast Tris(2-ethylhexyl) Melamine Methyl Pyridine Pyriofenone Topiramate b Table IARC

16 D. Kirkland et al. / Mutation Research 795 (2016) 7–30 cells

for for for

mutations

or SHE

in deemed deemed deemed

cells,

cell at at in at cytotoxic cytotoxic cytotoxic

induce

for

system(s) system(s) system(s) +ve +ve +ve +ve

be be be be –ve

test test test

May May May Comments May [361] precipitating concentrations excessively excessively excessively transformation the the the also concentrations concentrations p53-deﬁcient concentrations reported

been

have

but [30] [30] [51]

not not

; , ﬁndings tumours

mice mice mice tests,

[360] [360] 3 3 3 .

and and and

rats. rats thyroid

[51]

rats rats rats mice rats; mice in in

Group Group Group

in in in in in in

genotoxicity mice

ve ve ve ve ve ve tested IARC Carcinogenicity − IARC − − − − − tested possible IARC in cell

in ,

be mammalian

CA in cell but , ,

CA and and ,

but +ve

for

in cell MN , HPRT

, but g/ml

for for human may , [76] [250]

vitro

␮

, − hamster CHO toxic

and at ve

both

MN [250] [250]

for but

high and − after

in and

, in at

with ,

human human human human in [51] − +ve [350]

that

at for both in

4000

[250] hamster hamster hamster

MLA MLA and

CA as as as

[353,354] [51,319]

, [37] [51] concentrations

[38] in 2.4

p53-deﬁcient MN

[321]

[362] [349] at +S9 for for −

[3] for

[319] at

generally

and for

mammalian MN MLA

toxicity

above [3] 5–8 MLA CA CA MLA CA mM) high

MLA results

both

but +ve +ve

[3] mutation +ve

ve but

, p53-deﬁcient [3] [3] at in

cells for for

p53-competent in for for for for for types

− for

vitro very

ve ve In HPRT (30–40 hamster +ve [350] Weak − +ve [51,358,359] Weak +ve p53-deficient p53-deficient p53-competent p53-competent MN p53-deficient concentrations, -ve both re-evaluation types mutations p53-competent re-evaluated cells cells cells +ve weak cell 70-90% but at cytotoxic tests − +ve re-evaluated re-evaluated CHL cells and concentrations “uninterpretable” “uninterpretable” p53-competent for concentrations, precipitate cells, negative cytotoxicity.

give

levels

should

and high

at trial [51] that and in

[348] [362]

in liver

CA CA

mice

liver [24] others dosing

comets and [352] in [357,358] and

JaCVAM

and in for and trial

MN MN MN MN comets MN genotoxicity

ve for for for for for for using −

vivo concentrations

ve ve ve ve ve ve poisons − − − tests In − and − − [24] stomach stomach rats JaCVAM

high

at vivo

metabolic

often

) 4

[51]

coli TA102

Table TA102

[347] [351] ( E.

TA102

genotoxicity in strains strains strains strains

strains Salmonella

carcinogens), micronuclei,

proﬁle

for also

or also

TA102 TA102

TA102 and

and in in in

) ) including and

standard standard multiple standard standard standard 4 4

and and in in in in in in also TA104

equivocal

ve ve ve ve ve ve Table Table aberrations − − − Genotoxicity − ( Ames test [36] [36] and [355,356] [44,51] [51,362] and strains, ( − − or non-genotoxic

negative

(including

are chromosomal

that

cells, maleate

chemicals

acid

lymphoma number) alcohol

(100-51-6) (113-92-8) (CAS (536-33-4) (118-92-3) (94-96-2) (resorcinol) (108-46-3) Non-carcinogens

-Anthranilic mouse

I. Chemical Chlorpheniramine Ethionamide o 2-Ethyl-1,3-hexanediol Benzyl 1,3-Dihydroxybenzene Table Non-DNA-reactive in

D. Kirkland et al. / Mutation Research 795 (2016) 7–30 17 this

at for for AG

cells in

dosing

[378]

+ve TA98 ip

to –ve TA1538

through

that deemed deemed be in

adducts Hoechst oxidative but

after

+ve detoxiﬁcation

at at

cytotoxic cytotoxic

cytotoxicity,

to of

tests +ve

system system DNA +ve +ve

liver p53-deﬁcient all

to likely according

[381]

, be be for

induce in in

test test

ve − May Reported mouse and induction concentrations concentrations Comments [373] Likely May and overwhelm excessively excessively according and stress strain concentrations the the study

tested by [30] [30] ve

−

not ﬁndings

, mice mice mice

[30,51]

[51] 3 3

and and and

[51]

mice

considered

rats rats rats mice

Group Group

in in in in and

rats

ve ve ve ve rats inhalation Carcinogenicity Overall IARC − − − − IARC in or to

cell as , CA

and and and ,

(may for

cell S9 CA

and MLA g/ml

[37]

due

for

RTG − toxicity

␮

hamster

under

MLA [250] generally h +ve for [51]

and (not

human 3

human human human +ve

MLA

reported both with

<20% but S9

hamster hamster hamster 1500 in

+S9

mutations in

mechanism), [3] CA,

[37] − MN

in in in

at and

[376,377] [51,76,376]

probably ve

also re-evaluated mM

− for for −

cells,

mammalian MN CA MN CA MN +ve

cells [3] MLA MLA +S9

HPRT

p53-competent

recovery), +S9) p53-deﬁcient

but but

[3]

but

, , +ve inconclusive +ve but cytotoxicity, [3] in [3]

in for for for for for

in , , , for for CA

for

oxidative vitro 1.6-1.9

ve ve/inconclusive ve ve S9, ve ve ve In hamster cells − MN +ve [253,363,371,372] +ve − [37] [321] +ve [51,383] [99] [37] − [235] − tested − p53-competent p53-competent p53-competent conditions at p53-deficient (+17 human “uninterpretable” -ve cells, types − − inconclusive cells cells − p53-deficient p53-deficient weakly high tests be ip

for [370] ip in

only

[24] [24] -ve

3 CA

[51] and

doses ve , and and mice

mice and −

dosed

UDS

, in for dose

trial trial

results: for 50

[51] after

ip [367] for

orally [51] male +ve LD

[375] [382] and

daily MN mutations

−

− stomach stomach -ve +ve in

, mice

MN, MN MN MN in in dosing JaCVAM JaCVAM genotoxicity

dosed but -ve in +ve and

dosing

, , 1 ip in in [71]

if for for for for

ip intermediate vivo

ve ve ve ve ve tests − liver liver − doses In Conﬂicting [365] [366] [352] [368,369] − − − after comets weak comets an transgenic after liver by ) 4

in [44]

ve [45]

ve Table Hoechst −

(

−

and coli [364] to strains strains strains

strains

also E.

proﬁle ,

and

in uvrA

TA102

TA102 [381]

and

standard multiple standard standard [51,341,374] standard

WP2 according

in in in in in also

study

coli coli

ve ve ve ve ve − Genotoxicity − including AG Ames test [36,336,363] and [51,378–380] [36,44] E. E. TA104 − − − ) racemic

(89-78-1)

number) given

Continued

(

-Menthol (CAS (97-53-0) menthol (15356-70-4) Also (100-02-7) (127-69-5) (78-84-2) l , -Nitrophenol Chemical Eugenol p Sulﬁsoxazole Isobutyraldehyde d Table

18 D. Kirkland et al. / Mutation Research 795 (2016) 7–30 for for

through at at

deemed deemed

oxidative

+ve +ve +ve cytotoxic cytotoxic

be be be

system(s) system(s)

to to to

test test

Likely induction concentrations concentrations Comments Likely Likely excessively excessively stress the the

[51] [51] [30]

tumours [51]

, ﬁndings deposits

ionic mice mice mice

bladder carcinogen

[30] tumours to

, mice

3 2B

and and and and

[30] due and

mice

mouse

rats rats rats cytotoxicity/chronic

Group Group

in in rats in to and

and

ve ve ve ve irritation. IARC tumours rats imbalance microcrystalline Carcinogenicity − − IARC − − Forestomach Rat due tumours , ve , in cell MN ,

−

and and

, , +ve cell [37]

ve but both with low +S9 for where

, − [111] at

scored

[250] but in [76] at

[51,349] , CA [353]

hamster MLA

[319] human human human human +ve

[37] be only low ,

hamster hamster MN CA cytotoxicity but MLA for

mutations

for [37]

CA g/ml MLA

, at

RTG in TA100.

of but in ␮ [51]

[76]

>10 for for

p53-deﬁcient

re-evaluation

for associated generally +ve could

and

mammalian at MN MLA and 30%

in CA, CA MLA HPRT

+ve

+ve +ve [3]

8000 but

+ve , ,

levels [3] [3] [3]

for for

cells for upon for for for

+ve

vitro p53-deﬁcient

TA98

ve ve

In re-evaluated inconclusive few Strong +ve Equivocal [250] +ve +ve [37,393,394] [97,101] [131] − probably p53-competent p53-competent − types hamster for p53-competent cells cells cells +ve (toxicity?) +ve high around tests cells, p53-deﬁcient “uninterpretable” p53-competent concentrations but clear p53-deﬁcient CA concentrations in TA1538,

humans.

in TA97a, in

ip ,

+ve too ip

and

or assay ve

using

[24]

since −

[51] dosing

and for

for [388] after

and

[396] MN

at for

JaCVAM

ip using

[51]

[386] TA97 ve CA trial MN

saccharin

and injection

results,

CA only

for &

comet +ve ,

− ,

[51] or −

mice

carcinogenicity for

, MN group

and for

MN +ve in doses

[71] oral

liver stomach

was

ve its

[391] scored

ve for

per

− ve after MN transgenic

in in

JaCVAM − genotoxicity

[392] E control,

mOsmol/kg

alkaline −

splenocytes marrow TA1537 after ,

weak in

[24]

for or for cells

, in

borderline

in +ve

vivo

mice dosing 600

ve ve ve mechanism Limited Conﬂicting − dosing tests few animals liver in In − +ve [390] [32] Mainly (81-07-2) comets at but injection trial mutations ip no mice CA near-toxic bone comets −

TA1535,

classiﬁable

humans)

strains not

= )

4 3 (for

[51] [36] data

strains TA102

and Table

group ( [395]

in [387] TA102

strains strains strains strains strains

typhimurium in

proﬁle irrelevant coli

also

TA102

E. standard TA102 TA102

by also

genotoxicity

and in in

in in

) ) E standard standard standard [384,385] standard standard ve 4 4

carcinogen; and

− and

vivo in in in or in in also also ,

Salmonella a

in ve ve ve ve ve ve

Table Table − − − Genotoxicity − − ( Ames test [51] and [280] [31] [36,389] and ( TA104 − human

means carcinogenic

or limited

possible

column =

this

with in

carcinogens

group

chloride )

strains”

[AKA

saccharin

number)

Continued ( acrylate

Non-genotoxic classiﬁcation:

Non-carcinogens (124-64-1) (CAS butylhydroquinone (1948-33-0) (128-44-9) disulﬁram] (97-77-8) (140-88-5) disulﬁde phosphonium (57-13-6)

“Standard a III. Chemical Tertiary- Tetraethylthiuram Ethyl Tetrakis(hydroxymethyl) II. Urea Sodium Table IARC

D. Kirkland et al. / Mutation Research 795 (2016) 7–30 19

tive and negative) results in in vivo genotoxicity tests. When both that ﬂuometuron may not be a good candidate for a “true nega-

the free base and the hydrochloride salt of the chemical are con- tive” group.

•

sidered together (and both should behave the same in aqueous Cyclohexanone is negative in standard Ames strains [36] but not

biological systems) there are positive results in the in vivo MN test, tested in TA102 or E. coli. There are both negative and positive

and it is considered to be carcinogenic based on a more rigorous NTP conclusions for mutation induction in the mouse lymphoma tk

study. p-Chloroaniline (free base and HCl salt) has therefore been mutation assay [37,38] and reports of induction of chromosomal

retained in group 1 and the supporting data have been updated. aberrations (CA) both in vitro [39–41] and in vivo [42]. Whilst

By contrast, chloramphenicol, which was also included as a most of the CA studies are old, and results may be questionable,

group 1 chemical in the previous publication [5], is negative in the uncertainties with this data set suggest that this is not a good

the Ames test [31], although neither TA102 nor an Escherichia coli candidate for the “true negative” group.

•

strain were used. It is also reported negative for micronuclei in sev- Progesterone is positive for induction of MN in vitro via an aneu-

eral bone marrow studies in mice according to Morita et al. [32]. It genic mode of action [43] and therefore is not appropriate for the

is not considered a strong candidate for inclusion in group1 and “true negative” group.

•

chloramphenicol has therefore been deleted from this group. Trisodium EDTA trihydrate is negative in the Ames test strains

It is not the intention that all group 1 chemicals should be tested [44,45] and in most in vitro genotoxicity tests, but a few posi-

in order to evaluate a new or modiﬁed mammalian cell test system, tive results for EDTA and its salts have been observed, depending

but rather that a choice of chemicals belonging to different struc- on assay type and cell type [46]. Moreover, in the bone mar-

tural classes, and acting in different ways, should be available. To row micronucleus assay acute doses of EDTA disodium salt

increase the options, we decided to add some chemicals to this (5–20 mg/kg body weight) induced a dose-dependent increase

list and therefore we included 5-ﬂuorouracil, cytosine arabinoside in the incidence of micronucleated polychromatic erythrocytes

(nucleoside analogues), colchicine, and vinblastine (aneugens). We at 24-h sampling [47]. It is therefore considered that it may not

also added mitomycin C and 4-nitroquinoline-N-oxide as these are be a good candidate for the “true negative” group.

recommended positive control chemicals in OECD Test Guidelines

for mammalian cell genotoxicity tests.

Whereas some early data did not include tests on S. typhimurium

Group 1, therefore now consists of a total of 25 chemicals

TA102 or E. coli WP2 strains, we have now searched for addi-

and they are detailed in Table 1, together with the reasons for

tional data in these strains. Such data were not available for 5 of

their selection. We have chosen chemicals that represent differ-

the chemicals we wished to retain within group 2, and so new

ent classes and exhibit different modes of action. The focus is on

tests in TA102 have been performed at the Novartis Institutes

chemicals that are considered DNA-reactive carcinogens and in vivo

for Biomedical Research (Switzerland) using standard plate incor-

genotoxins. Furthermore, we have included in vivo genotoxins such

poration methodology. A description of the methods is given in

as aneugens and clastogens (e.g. topoisomerase inhibitors, nucle-

Appendix 1. The data from these studies are summarised in Table 4.

oside analogues) that may not be carcinogenic, and are negative

Some new substances have been added to group 2, mainly from the

or equivocal in the Ames test, but which we would expect in vitro

pharmaceutical area, that are commercially available and for which

mammalian cell tests to detect as positive. The chemicals in Table 1

robust testing data have been reviewed in regulatory submissions

are arranged into 2 sections (Ames-positive and Ames-negative or

to the German Federal Institute for Drugs and Medical Devices

equivocal) and several subgroups. They are not listed in any order

(BfArM). Thus, only chemicals that have been shown to be nega-

of priority, but for any test evaluation program it is recommended

tive in Ames tests including the standard strains plus either TA102

that examples from each subgroup are included. It should be noted

or E. coli are included in group 2. For the remainder, the references

that the mode of action for tumour induction might not be the same

supporting the various notes in Table 2 have been updated.

as that leading to genotoxic responses.

Group 2 therefore consists of 27 non-DNA-reactive (Ames test

negative) chemicals and they are detailed in Table 2. The chem-

icals in Table 2 are arranged into 3 subgroups. The 8 chemicals

3. Considerations for updating group 2—Non-DNA-reactive

in subgroup I are the only ones that had clearly negative in vivo

chemicals (including non-genotoxic carcinogens) that

genotoxicity data as well as being negative in vitro and non-

should, and routinely do give negative results in in vitro

carcinogenic. There are a large number of non-carcinogens that are

mammalian cell genotoxicity tests (“true negatives”)

non-genotoxic in vitro, but for which no published in vivo geno-

toxicity data could be found. However, these are included in this

After searching for new or previously missed data, the authors

group (subgroup II) because the existing data suggest they should

felt that this group of chemicals also needed revision. Several chem-

be negative in any modiﬁed or new in vitro genotoxicity test sys-

icals have been deleted from the previous group 2 list [5] for the

tems. Subgroup III consists of non-genotoxic carcinogens for which

following reasons:

the “non-genotoxic” status is supported by negative in vitro and

• in vivo genotoxicity data.

Phenanthrene—whilst there is reasonable evidence of lack of

mutagenic/clastogenic effects in mammalian cells, phenanthrene

may be a bacterial mutagen. Oesch et al. [33] found a positive 4. Considerations for updating group 3— Non-DNA-reactive

response in TA100 at 12 ␮g/plate and above in the presence of chemicals (including non-genotoxic carcinogens),

high levels (probably around 30%) of Aroclor-induced rat liver S9. metabolic poisons and others that should give negative

in vitro

Also, there are no in vivo data in standard systems,and the “non- results in mammalian cell genotoxicity tests, but

carcinogen” label is based on limited data; only mice have been have been reported to induce mutations in mouse

studied using dermal, intraperitoneal or subcutaneous adminis- lymphoma cells, chromosomal aberrations or micronuclei,

tration [34], studies were all shorter, and inmany cases group often at high concentrations or at high levels of cytotoxicity

sizes were smaller, than normal. Thus, this is probably not a good (“misleading” or “irrelevant” positives)

candidate for a “true negative” group.

•

Fluometuron—according to a review by EFSA [35], a soil metabo- The authors deemed it most important to focus time and effort

lite of ﬂuometuron, desmethyl ﬂuometuron, is clastogenic in vitro on the group 3 chemicals. In light of new data, particularly from

and also induces tumours in mice. It was therefore considered Fowler et al. [3], several chemicals that were previously considered

20 D. Kirkland et al. / Mutation Research 795 (2016) 7–30

Table 4

Summary of TA102 revertant counts for new tests performed.

Chemical S9 Revertant counts (mean of 2 or 3 plates) at concentrations of chemical (␮g/plate)

0 15 50 150 500 1500 5000 POS

−

Ephedrine hydrochloride 540 514 549 548 562 542 514 BGR 2326

+ 722 585 612 662 543 562 593 BGR 3437

−

Hexachloroethane 540 522 509 561 590 500 BGR 551 BGR 2326

+ 722 661 650 746 736 622 BGR 595 BGR 3434

−

Ampicillin trihydrate 540 451 558 559 551 489 385 BGR 2326

+ 722 653 719 625 561 574 452 BGR 3434

2-Ethyl-1,3-hexanediol − 531 551 516 576 513 556 462 BGR 2850

+ 784 756 720 769 773 762 697 2964

−

Chlorpheniramine maleate 508 477 472 492 406 280 BGR BGO 2627

+ 624 633 623 596 646 513 BGO 1988

−

d,l-Menthol 531 562 624 666 514 BGO TNG 2650

+ 784 766 803 737 731 BGO TNG 2964

−

Ethionamide 531 563 554 567 542 511 237 BGR 2650

+ 784 664 760 774 645 600 396 BGR 2964

−

Ethyl acrylate 531 577 558 531 473 438 479 2650

+ 784 677 622 652 570 581 516 2964

−

Tetraethylthiuram disulﬁde 531 566 560 535 481 BGO TNB 2650

+ 784 777 773 713 592 475 70 BGR 2964

Chemical S9 Revertant counts (mean of 2 or 3 plates) at concentrations of chemical (␮g/plate)

0 3.13 6.25 12.5 25 50 POS

Erythromycin − 531 521 448 545 PPC PPC 2698

+ 734 681 637 688 PPC PPC 2062

Chemical S9 Revertant counts (mean of 2 or 3 plates) at concentrations of chemical (␮g/plate)

0 0.5 1.5 5 15 50 POS

Curcumin − 473 537 501 530 452 BGR BGO 2985

+ 692 720 684 720 728 581 BGR 2034

Tetrakis(hydroxymethyl) phosphonium chloride − 473 594 613 547 617 621 BGR 2985

+ 692 737 599 542 600 585 BGR 2034

Chemical S9 Revertant counts (mean of 2 or 3 plates) at concentrations of chemical (␮g/plate)

0 12.8 42.7 128 427 1282 4274 POS

Phenformin hydrochloride − 540 487 500 516 529 441 BGR BGO 2326

+ 722 608 702 692 646 626 BGR 348 BGR 3434

POS = positive control, as described in Appendix 1; BGR = reduced background growth, BGO = no background growth, TNB = precipitation and no background growth,

TNG = absolutely no growth (no colonies, no background growth); PPC = precipitate.

to be “misleading” positives [5], had been found not to be geno- tocytes of mice [52], and positive for comets in stomach and colon

toxic either in p53-defective hamster cells or in p53-competent of mice [53].

•

human cells when following current protocols. Some were non- Sodium xylenesulfonate—since the only negative carcinogenic-

toxic up to the maximum concentration that could be tested whilst ity data are in rats (not tested in mice), and there are no in vivo

others induced toxicity but were still not genotoxic. Therefore, the genotoxicity data, so categorisation as a non-carcinogen may not

previously reported positive results in mammalian cells were rare be robust, and there are no data to conﬁrm if it is non-genotoxic

events, not easily reproduced, and the conditions under which the in vivo.

•

positive results might be found have not been identiﬁed. We con- Curcumin—reported positive in many mammalian cell tests,

sidered whether to delete these chemicals from group 3, since by possibly as a result of apoptosis [54], but also positive in p53-

now the majority of mammalian cell tests with these chemicals competent human cells [3]. It may therefore not be useful to

have given negative results. However, since some positive results determine whether new or modiﬁed tests show acceptable speci-

have been published, we decided to keep some of them in group 3. ﬁcity.

Details are given in Table 3.

Some chemicals that were previously included in group 3 [5]

These chemicals have therefore been deleted from the previous

are now known to be positive, either in the Ames test (therefore

group 3 list.

must be considered to be potentially DNA-reactive) or in vivo, or

The following chemicals were considered for inclusion in group

there are other reasons to question the reported positive results

3, but were eventually not included for the reasons given below:

in mammalian cells, or the data on absence of in vivo genotoxic or

carcinogenic activity are unclear. These chemicals are: •

Benzoate sodium, since the only negative carcinogenicity data are

in rats (not tested in mice), categorisation as a non-carcinogen

may not be robust.

• •

Phthalic anhydride—would be likely to form phthalic acid in Benzoin, since positive results have now been found for induction

aqueous media, possibly leading to pH effects which could con- of UDS in rat hepatocytes [55] and for induction of hprt mutations

found the results [48]. [56,57]. Such results are not typical of chemicals that may give

•

Propyl gallate—reported positive in TA102 in the presence of S9 “misleading positive” results in vitro.

•

[49] and for induction of MN and CA in vivo [50]. Methyl methacrylate, since weak positive in vivo MN results have

•

2,4-Dichlorophenol—equivocal or positive in the Ames test with been reported in 2 separate studies using ip dosing [51]and clear

hamster S9 [51,36], positive for CA in bone marrow and sperma- positive MN results have been reported after 1-day inhalation

D. Kirkland et al. / Mutation Research 795 (2016) 7–30 21

exposure [58]. Thus, it is not possible to categorise this chemical aware of quality and should try to obtain certiﬁcates of analysis for

as being clearly negative for in vivo genotoxicity. the test compounds including details of purity and impurities. For a

•

Diphenhydramine HCl, since, although there is no evidence of good preparation of test chemical solutions, care should be taken in

carcinogenic activity for male or female mice, there is equivocal the suitable choice of the solvent/vehicle. Moreover, careful han-

evidence of carcinogenic activity in male (marginally increased dling of chemicals is always necessary, especially when handling

incidences of uncommon astrocytomas or gliomas and of alveo- IARC group 1 chemicals or chemicals that are coded.

lar/bronchiolar neoplasms) and female rats (marginal increase in

the incidence of pituitary gland adenomas) [51]. In the absence

6. Conclusions

of any in vivo genotoxicity data it is difﬁcult to categorise this

substance as clearly non-carcinogenic and non-genotoxic in vivo.

After careful consideration of the published literature, and in

particular new data published since 2008, the authors have updated

We discussed the possibility of deleting ethyl acrylate from the lists of chemicals that can be used in the evaluation of modiﬁed

group 3. There are some reports of induction of MN in vivo, but or new mammalian cell genotoxicity assays. These lists basically

there are more robust studies showing negative results (see Table 3 arrange the chemicals according to whether positive results should

for references), and so the weight of evidence indicates lack of true be expected in vitro or whether negative results should be expected,

genotoxic activity in vivo. It was also negative in a new test in TA102 and the latter includes chemicals currently suspected of giving

(Table 4). We therefore decided to retain ethyl acrylate in group 3. “misleading” or “irrelevant” positive results in existing assays. It

To compensate for the deletions, a number of new chemicals should be noted that the chemicals that have been deleted since

have been added. Details are given in Table 3. In the revisions the 2008 publication are not necessarily considered to be “wrong”.

and additions to group 3 we checked carefully that the negative However, they are considered to be less robust candidates for the

Ames results were robust. Whereas some early data did not include groups we have presented here, and any data obtained with them

tests on S. typhimurium TA102 or E. coli WP2 strains, we have now should be viewed with some caution. It is hoped these lists may

searched for additional data in these strains. As data were not avail- continue to provide useful reference chemicals for those scientists

able for 8 of the chemicals we wished to retain within or add to seeking to modify existing assays or introduce new assays.

group 3, new tests in TA102 have been performed at the Novar-

tis Institutes for Biomedical Research (Switzerland) using standard

Conﬂict of interests

plate incorporation methodology. A description of the method is

given in Appendix 1. The data from these studies are summarised None.

in Table 4. Thus, only chemicals that have been shown to be nega-

tive in Ames tests, including the standard strains plus either TA102

Funding sources

or E. coli, are included in group 3.

Group 3 now consists of 17 non-DNA-reactive (Ames test nega-

None.

tive) chemicals and they are detailed in Table 3, together with the

reasons for their selection. The chemicals in this group have been

selected primarily because most are negative for in vivo genotoxi- Acknowledgements

city. Where possible, Table 3 includes comments on the conditions

(cytotoxicity, apoptosis, oxidative stress or cell type) under which The authors would like to thank EURL ECVAM for organising and

positive results in mammalian cells in vitro might occur. It is sug- funding the workshop that enabled this project to be completed.

gested to include such chemicals into a validation study for a new We are also indebted to Wilfried Frieauff and Petra Kimlickova from

or modiﬁed in vitro mammalian cell genotoxicity test to identify the Novartis Institutes for BioMedical Research, Basel, Switzerland,

whether negative results are obtained or to establish the conditions for kindly performing the additional bacterial mutation tests in

under which positive results are found. The chemicals in Table 3 are TA102.

arranged into 3 subgroups. Those in sub-group I are negative for

in vivo tests and should be given priority. However, for the chem-

Appendix 1.

icals in subgroup II, no adequate published in vivo genotoxicity

data could be found. Only two chemicals (sodium saccharin, ethyl

Methodology for new tests in S. typhimurium TA102

acrylate) are included in subgroup III being mainly negative for

genotoxicity in vivo but with tumours induced via a non-genotoxic

Tests were kindly performed by the Novartis Institutes for

mechanism.

BioMedical Research, Preclinical Safety, Basel, Switzerland.

5. Note on concordance Bacteria

Strain TA102 was received from Hoffmann—LaRoche Ltd., Basel,

It should be noted that “concordance” is a measure fre- Switzerland. It is maintained and the genotype of each freshly

quently used when evaluating the performance of a test system. frozen batch was regularly checked as described by Maron and

In this case it is the level of agreement between the results Ames [397]. The strain is kept as frozen aliquots in 8 % DMSO at

◦ ◦

− −

in the in vitro tests relative to the expected in vivo test out- 70 C to 90 C. Overnight cultures were prepared in nutrient

◦

comes for genotoxicity or carcinogenicity. Concordance relies on broth shaking ca. 15 h at 30–39 C at 40–70% humidity.

having relatively equal numbers of carcinogens/genotoxins and The overnight cultures used for testing were checked to ensure

non-carcinogens/non-genotoxins. Many previous collaborative or that revertant numbers on negative control plates fell within his-

validation trials have contained large numbers of carcinogens but torical control ranges, and positive control chemicals induced at

few non-carcinogens. In Tables 1 and 2 there are similar numbers least a 1.5-fold increase in revertants. In addition, the overnight

of carcinogens/genotoxins and non-carcinogens/non-genotoxins, cultures were assessed for the following: (i) presence of the plas-

providing a good balance for concordance calculations. It should mid pKM101, i.e. ampicillin resistance, (ii) presence of the plasmid

also be noted that whilst we believe all of the chemicals listed pAQ1, i.e. tetracycline resistance, (iii) number of living cells. The

in these tables are commercially available, laboratories should be OD600 of the culture was also measured.

22 D. Kirkland et al. / Mutation Research 795 (2016) 7–30

Chemicals growth. Colony counting was done with an image analyzer (Sor-

The chemicals that needed to be tested in TA102, because no cerer; Perceptive Instruments, UK). The data were recorded and

published data in either TA102 or E. coli WP2 strains could be found, printed by a computer system (Gendata program).

were as follows:

Data analysis

•

Ampicillin trihydrate (CAS No. 7177-48-2), 100.2% pure The results of a test were considered valid if the negative control

•

Ephedrine hydrochloride (CAS No. 50-98-6), 99% pure data were within the range of the historical control data obtained

•

Erythromycin (CAS No. 114-07-8) in the testing laboratory, the positive controls induced at least a

•

Phenformin hydrochloride (CAS No. 834-28-6), 99.0% pure 1.5-fold increase in revertants, and the sterility tests of the test

•

Hexachloroethane (CAS No. 67-72-1), 99% pure chemical solutions, vehicle, phosphate buffer and S9-mix used all

•

2-Ethyl-1,3-hexanediol (CAS No. 94-96-3), 96.5% pure provided acceptable results.

•

Chlorpheniramine maleate (CAS No. 113-92-8), 99.0% pure A test chemical was judged to be mutagenic if it produced, in

•

Curcumin (CAS No. 458-37-7), 76.0% pure at least one concentration, an increase by a factor of 1.5 above the

• d l

, -Menthol (CAS No. 15356-70-4), 98.5% pure negative control level.

•

Ethionamide (CAS No. 536-33-4), 98.0% pure

•

Ethyl acrylate (CAS No. 140-88-5), 98.5% pure Results

•

Tetraethylthiuram disulﬁde (CAS No. 97-77-8), 97.0% pure The results are summarised in Table 4. Revertant counts on all

•

Tetrakis (hydroxymethyl) phosphonium chloride (CAS No. 124- solvent control plates were within the normal range and positive

64-1), 77.8% pure. control chemicals induced acceptable mutagenic responses. It can

be seen that all 13 test chemicals gave no signiﬁcant increases in

All were obtained from Sigma–Aldrich Chemie GmbH, Kappel- TA102 revertants either in the absence or presence of S9.

weg 1, 91625 Schnelldorf, Germany and were dissolved in DMSO,

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