Commission Directive 2000/32/EC of 19 May 2000 adapting to technical progress for the 26th time Council Directive 67/548/EEC on the approximation of the laws, regulations and administrative provisions relating to the classification, packaging and labelling of dangerous substances (Text with EEA relevance.)
Date: 2000-05-19
Institutions: European Commission
Subjects: approximation of laws, classification, dangerous substance, labelling, packaging

8.6.2000

EN

Official Journal of the European Communities

L 136/1

I

(Acts whose publication is obligatory)

COMMISSION DIRECTIVE 2000/32/EC

of 19 May 2000

adapting to technical progress
the 26th time Council Directive 67/548/EEC on the
approximation of the laws, regulations and administrative provisions relating to the classification,
packaging and labelling of dangerous substances (*)

for

(Text with EEA relevance)

THE COMMISSION OF THE EUROPEAN COMMUNITIES,

Having regard to the Treaty establishing the European
Community,

Having regard to Council Directive 67/548/EEC of 27 June
the laws, regulations and
1967 on the approximation of
classification,
administrative
packaging and labelling of dangerous substances (1), as last
amended by European Parliament and Council Directive
1999/33/EC (2), and in particular Article 28 thereof,

provisions

relating

the

to

(4) Annex IX to Directive 67/548/EEC contains the provisions
relating to child-proof fastenings. Those provisions should
be adapted and updated. It is necessary to extend the scope
of the use of child-proof fastenings.

(5) The measures provided for

in this Directive are in
accordance with the opinion of
the Committee on the
Adaptation to Technical Progress of the Directives for the
Elimination of Technical Barriers to Trade in Dangerous
Substances and Preparations,

Whereas:

HAS ADOPTED THIS DIRECTIVE:

together with particulars of

(1) Annex I
to Directive 67/548/EEC contains a list of
the
dangerous
substances,
classification and labelling of each substance. Present
scientific and technical knowledge has shown that the list
of dangerous substances in that Annex should be adapted.
require
Certain language
corrections in specific sections of
the foreword and of
Table A to Annex I.

the Directive

versions of

(2) Annex III

to Directive 67/548/EEC contains a list of
phrases indicating the nature of special risks attributed to
substances and preparations. Annex IV to
dangerous
the phrases
Directive 67/548/EEC contains a list of
indicating
dangerous
to Directive
substances
67/548/EEC contains a guide to the classification and
labelling of dangerous substances and preparations. Certain
language versions of the Directive require corrections in
specific sections of Annexes III, IV and VI.

the
and preparations. Annex VI

concerning

advice

safety

(3) Annex V to Directive 67/548/EEC lays down the methods
for the determination of the physico-chemical properties,
toxicity and ecotoxicity of substances and preparations. It
is necessary to adapt that Annex to technical progress.

(*) Adopted after the 27th adaptation.
(1) OJ 196, 16.8.1967, p. 1.
(2) OJ L 199, 30.7.1999, p. 57.

Article 1

Directive 67/548/EEC is hereby amended as follows:

1. Annex I is amended as follows:

(a) Note Q in Annex 1A to this Directive replaces the

corresponding note in the Foreword.

(b) The rows in Annex 1B to this Directive replace the

corresponding rows in Table A.

(c) The entries in Annex 1C to this Directive replace the

corresponding entries.

(d) The entries in Annex 1D to this Directive are inserted.

2. The risk phrase in Annex 2 to this Directive replaces the

corresponding phrase in Annex III.

3. Annex IV is amended as follows:

(a) The safety phrases in Annex 3A to this Directive

replace the corresponding phrases in Annex IV.

L 136/2

EN

Official Journal of the European Communities

8.6.2000

(b) The combined safety phrases in Annex 3B to this
in

corresponding

phrases

replace

the

Directive
Annex IV.

4. Part B of Annex V is amended as follows:

(a) The text

in Annex 4A to this Directive replaces

Chapter B.10.

(b) The text

in Annex 4B to this Directive replaces

Chapter B.11.

(c) The text

in Annex 4C to this Directive replaces

Chapter B.12.

(d) The text

in Annex 4D to this Directive replaces

Chapters B.13 and B.14.

Article 2

1. Member States shall bring into force the laws, regulations
and administrative provisions necessary to comply with this
Directive by 1 June 2001 at the latest. They shall forthwith
inform the Commission thereof.

those provisions,

When Member States adopt
they shall
contain a reference to this Directive or be accompanied by
such a reference on the occasion of their official publication.
Member States shall determine how such reference is to be
made.

2. Member States shall communicate to the Commission the
main provisions of national law which they adopt in the field
covered by this Directive and a correlation table between this
Directive and the national provisions adopted.

(e) The text in Annex 4E to this Directive replaces Chapter

Article 3

B.17.

(f) The text in Annex 4F to this Directive replaces Chapter
B.23. The title of Chapter B.23 in the explanatory note
is changed accordingly.

This Directive shall enter
into force on the third day
following its publication in the Official Journal of the European
Communities.

(g) The text in Annex 4G to this Directive is added.

Article 4

5. The fourth indent of the general introduction to Part C of

Annex V is deleted.

This Directive is addressed to the Member States.

6. The texts

in Annex 5 to this Directive replace the

corresponding texts in Annex VI.

7. Annex IX is amended as set out

in Annex 6 to this

Directive.

Done at Brussels, 19 May 2000.

For the Commission

Margot WALLSTR(cid:214)M

Member of the Commission

8.6.2000

EN

Official Journal of the European Communities

L 136/3

ANNEX 1A

FOREWORD TO ANNEX I

Explanation of the notes relating to the identification, classification and labelling of substances

DA:

Note Q:

Klassificeringen som kr(cid:230)ftfremkaldende kan udelades for fibre, som opfylder en af fłlgende betingelser:

(cid:151) en kortvarig biopersistensprłve ved inhalation har vist, at fibre, der er l(cid:230)ngere end 20 (cid:181)m, har en v(cid:230)gtet halve-

ringstid p(cid:229) mindre end 10 dage

(cid:151) en kortvarig biopersistensprłve ved intratrakeal instillation har vist, at fibre, der er l(cid:230)ngere end 20 (cid:181)m, har en

v(cid:230)gtet halveringstid p(cid:229) mindre end 40 dage

(cid:151) en egnet intra-peritoneal prłve ikke har vist kr(cid:230)ftfremkaldende virkning, eller

(cid:151) en egnet langvarig inhalationsprłve ikke har vist relevante sygdomsfremkaldende virkninger eller neoplastiske

forandringer.

SV:

Note Q:

˜mnet beh(cid:246)ver inte klassificeras som cancerframkallande om det kan visas att det uppfyller ett av f(cid:246)ljande villkor:

(cid:151) ett korttidstest f(cid:246)r att best(cid:228)mma den biologiska best(cid:228)ndigheten vid inhalation har visat att fibrer l(cid:228)ngre (cid:228)n 20 (cid:181)m

har en viktad halveringstid p(cid:229) mindre (cid:228)n 10 dagar

(cid:151) ett korttidstest f(cid:246)r att best(cid:228)mma den biologiska best(cid:228)ndigheten vid intratrakeal instillation har visat att fibrer l(cid:228)ngre

(cid:228)n 20 (cid:181)m har en viktad halveringstid p(cid:229) mindre (cid:228)n 40 dagar

(cid:151) ett l(cid:228)mpligt intraperitonealt test har inte givit bel(cid:228)gg f(cid:246)r f(cid:246)rh(cid:246)jd cancerogenitet

(cid:151) fr(cid:229)nvaro av relevant patogenitet eller neoplastiska f(cid:246)r(cid:228)ndringar i ett l(cid:228)mpligt l(cid:229)ngtids inhalationstest.

(Does not concern the ES version)

(Does not concern the DE version)

(Does not concern the EL version)

(Does not concern the EN version)

(Does not concern the FR version)

(Does not concern the IT version)

(Does not concern the NL version)

(Does not concern the PT version)

(Does not concern the FI version)

ANNEX 1B

TABLE A

Z

(cid:145)18

(cid:145)64

Symb.

Ar

Gd

ES

Arg(cid:243)n

DA

Argon

DE

Argon

EL

Aqc(cid:252)

EN

Argon

FI

Argon

FR

Argon

IT

Argon

NL

Argon

PT

SV

`rgon

Argon(cid:146)

Gadolinio

Gadolinium

Gadolinium

Cadok(cid:223)mio

Gadolinium

Gadolinium

Gadolinium

Gadolinio

Gadolinium

Gadol(cid:237)nio

Gadolinium(cid:146)

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Index No

Chemical name

006-011-00-7

carbaryl (ISO)
1-naphthyl methylcarbamate

006-013-00-8 metam-sodium (ISO)

sodium methyldithiocarbamate

ANNEX 1C

Notes
related to
substances

EC No

CAS No

Classification

Labelling

Concentration limits

Notes
related to
preparations

200-555-0

63-25-2

Carc. Cat. 3; R40
Xn; R22
N; R50

Xn; N
R: 22-40-50
S: (2-)22-24-36/37-46-61

205-293-0

137-42-8 Xn; R22

R31
C; R34
R43
N; R50-53

C; N
R: 22-31-34-43-50/53
S: (1/2-)26-36/37/39-45-60-61

006-015-00-9

diuron (ISO)
3-(3,4-dichlorophenyl)-1,1-dimethylurea

206-354-4

330-54-1

006-016-00-4

propoxur (ISO)
2-isopropoxyphenyl N-methylcarbamate
2-isopropoxyphenyl methylcarbamate

204-043-8

114-26-1

006-017-00-X aldicarb (ISO)

204-123-2

116-06-3

2-methyl-2-(methylthio)propanal-O-
(N-methylcarbamoyl)oxime

006-018-00-5

aminocarb (ISO)
4-dimethylamino-3-tolyl methylcarbamate

217-990-7

2032-59-9

Carc. Cat. 3; R40
Muta. Cat. 3; R40
Xn; R22-48/22
N; R50-53

Xn; N
R: 22-40-48/22-50/53
S: (2-)13-22-23-37-46-60-61

T; R25
N; R50-53

T+; R26/28
T; R24
N; R50-53

T; R24/25
N; R50-53

T; N
R: 25-50/53
S: (1/2-)37-45-60-61

T+; N
R: 24-26/28-50/53
S: (1/2-)22-36/37-45-60-61

T; N
R: 24/25-50/53
S: (1/2-)28-36/37-45-60-61

006-019-00-0

di-allate (ISO)
S-(2,3-dichloroallyl)-N,N-
diisopropylthiocarbamate

218-961-1

2303-16-4

Carc. Cat. 3; R40
Xn; R22
N; R50-53

Xn; N
R: 22-40-50/53
S: (2-)25-36/37-60-61

006-020-00-6

barban (ISO)
4-chlorbut-2-ynyl N-(3-chlorphenyl)carbamate

202-930-4

101-27-9 Xn; R22

006-023-00-2 mercaptodimethur (ISO)

217-991-2

2032-65-7

R43
N; R50-53

T; R25
N; R50-53

Xn; N
R: 22-43-50/53
S: (2-)24-36/37-60-61

T; N
R: 25-50/53
S: (1/2-)22-37-45-60-61

methiocarb
3,5-dimethyl-4-methylthiophenyl
N-methylcarbamate

006-024-00-8

proxan-sodium (ISO)
sodium O-isopropyldithiocarbamate

205-443-5

140-93-2 Xn; R22
Xi; R38
N; R51-53

Xn; N
R: 22-38-51/53
S: (2-)13-61

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Index No

Chemical name

Notes
related to
substances

EC No

CAS No

Classification

Labelling

Concentration limits

Notes
related to
preparations

006-026-00-9

carbofuran (ISO)
2,3-dihydro-2,2-dimethylbenzofuran-7-yl
N-methylcarbamate

216-353-0

1563-66-2

T+; R26/28
N; R50-53

006-028-00-X dinobuton (ISO)

213-546-1

973-21-7

2-(1-methylpropyl)-4,6-dinitrophenyl isopropyl
carbonate

006-029-00-5

dioxacarb (ISO)
2-(1,3-dioxolan-2-yl)phenyl
N-methylcarbamate

230-253-4

6988-21-2

T; R25
N; R50-53

T; R25
N; R51-53

006-033-00-7 metoxuron (ISO)

243-433-2

19937-59-8 N; R50-53

3-(3-chloro-4-methoxyphenyl)-1,1-
dimethylurea

006-034-00-2

pebulate (ISO)
N-butyl-N-ethyl-S-propylthiocarbamate

006-035-00-8

pirimicarb (ISO)
5,6-dimethyl-2-dimethylamino-pyrimidin-
4-yl-N,N-dimethylcarbamate

006-037-00-9

promecarb (ISO)
3-isopropyl-5-methylphenyl
N-methylcarbamate

214-215-4

1114-71-2 Xn; R22

245-430-1

23103-98-2

220-113-0

2631-37-0

N; R51-53

T; R25
N; R50-53

T; R25
N; R50-53

T+; N
R: 26/28-50/53
S: (1/2-)36/37-45-60-61

T; N
R: 25-50/53
S: (1/2-)37-45-60-61

T; N
R: 25-51/53
S: (1/2-)37-45-61

N
R: 50/53
S: 60-61

Xn; N
R: 22-51/53
S: (2-)23-61

T; N
R: 25-50/53
S: (1/2)22-37-45-60-61

T; N
R: 25-50/53
S: (1/2-)24-37-45-60-61

006-038-00-4

sulfallate (ISO)
2-chlorallyl N,N-dimethyldithiocarbamate

E

202-388-9

95-06-7

Carc. Cat. 2; R45
Xn; R22
N; R50-53

T; N
R: 45-22-50/53
S: 53-45-60-61

006-039-00-X tri-allate (ISO)

218-962-7

2303-17-5 Xn; R22-48/22

S-2,3,3-trichlorallyl diisopropylthiocarbamate

R43
N; R50-53

Xn; N
R: 22-43-48/22-50/53
S: (2-)24-37-60-61

006-042-00-6 monuron (ISO)

3-(4-chlorphenyl)-1,1-dimethylurea

205-766-1

150-68-5

Carc. Cat. 3; R40
Xn; R22
N; R50-53

Xn; N
R: 22-40-50/53
S: (2-)36/37-60-61

006-043-00-1 monuron-TCA

(cid:151)

140-41-0 Xi; R36/38

3-(4-chlorophenyl)-1,1-dimethyluronium
trichloroacetate

Carc. Cat. 3; R40
N; R50-53

Xn; N
R: 36/38-40-50/53
S: (2-)36/37-60-61

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Chemical name

Notes
related to
substances

EC No

CAS No

Classification

Labelling

Concentration limits

Notes
related to
preparations

006-045-00-2 methomyl (ISO)

240-815-0

16752-77-5

1-(methylthio)ethylideneamino
N-methylcarbamate

006-046-00-8

bendiocarb (ISO)
2,2-dimethyl-1,3-benzodioxol-4-yl
N-methylcarbamate

006-047-00-3

bufencarb (ISO)
A mixture of 3-(1-methylbutyl)phenyl
N-methylcarbamate and
3-(1-ethylpropyl)phenyl N-methylcarbamate

245-216-8

22781-23-3

(cid:151)

8065-36-9

T+; R28
N; R50-53

T; R23/25
Xn; R21
N; R50-53

T; R24/25
N; R50-53

T+; N
R: 28-50/53
S: (1/2-)22-36/37-45-60-61

T; N
R: 21-23/25-50/53
S: (1/2-)22-36/37-45-60-61

T; N
R: 24/25-50/53
S: (1/2-)28-36/37-45-60-61

006-048-00-9

ethiofencarb (ISO)
2-(ethylthiomethyl)phenyl N-methylcarbamate

249-981-9

29973-13-5 Xn; R22

N; R50-53

006-050-00-X fenuron-TCA

1,1-dimethyl-3-phenyluronium trichloroacetate

(cid:151)

4482-55-7 Xi; R38

N; R50-53

006-053-00-6

isoprocarb (ISO)
2-isopropylphenyl N-methylcarbamate

220-114-6

2631-40-5 Xn; R22

N; R50-53

T+; R28
Xn; R21
N; R50-53

006-054-00-1 mexacarbate (ISO)

206-249-3

315-18-4

3,5-dimethyl-4-dimethylaminophenyl
N-methylcarbamate

006-057-00-8

nitrapyrin (ISO)
2-chloro-6-trichloromethylpyridine

217-682-2

1929-82-4 Xn; R22

N; R51-53

006-060-00-4

oxycarboxin (ISO)
2,3-dihydro-6-methyl-5-(N-phenylcarbamoyl)-
1,4-oxothiine 4,4-dioxide

226-066-2

5259-88-1 Xn; R22
R52-53

006-069-00-3

thiophanate-methyl (ISO)
1,2-di-(3-methoxycarbonyl-
2-thioureido)benzene

006-070-00-9

furmecyclox
N-cyclohexyl-N-methoxy-2,5-dimethyl-
3-furamide

245-740-7

23564-05-8 Muta. Cat. 3; R40

N; R50-53

262-302-0

60568-05-0

Carc. Cat. 3; R40
N; R50-53

Xn; N
R: 22-50/53
S: (2-)60-61

Xi; N
R: 38-50/53
S: (2-)60-61

Xn; N
R: 22-50/53
S: (2-)60-61

T+; N
R: 21-28-50/53
S: (1/2-)36/37-45-60-61

Xn; N
R: 22-51/53
S: (2-)24-61

Xn
R: 22-52/53
S: (2-)61

Xn; N
R: 40-50/53
S: (2-)36/37-60-61

Xn; N
R: 40-50/53
S: (2-)36/37-60-61

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Index No

Chemical name

Notes
related to
substances

EC No

CAS No

Classification

Labelling

Concentration limits

Notes
related to
preparations

006-088-00-7

benfuracarb (ISO)
ethyl N-[2,3-dihydro-2,2-dimethylbenzofuran-
7-yloxycarbonyl(methyl)aminothio]-
N-isopropyl-b-alaninate

007-012-00-5 N,N-dimethylhydrazine
1,1-dimethylhydrazine

007-013-00-0 N,N-dimethylhydrazine
1,2-dimethylhydrazine

009-003-00-1

hydrofluoric acid (cid:133) %

(cid:151)

82560-54-1

T; R23/25
N; R50-53

200-316-0

57-14-7

F; R11
Carc. Cat. 2; R45
T; R23/25
C; R34
N; R51-53

T; N
R: 23/25-50/53
S: (1/2-)36/37-45-60-61

F; T; N
R: 45-11-23/25-34-51/53
S: 53-45-61

(cid:151)

540-73-8

Carc. Cat. 2; R45
T; R23/24/25
N; R51-53

T; N
R: 45-23/24/25-51/53
S: 53-45-61

231-634-8

7664-39-3

T+; R26/27/28
C; R35

T+; C
R: 26/27/28-35
S: (1/2-)7/9-26-36/37-45

E

E

B

C ¯ 25 %: T;
R45-23/24/25
3 % ˜ C < 25 %: T;
R45-20/21/22
0,01 % ˜ C < 3 %: T; R45

C ¯ 7 %: T+; C;
R26/27/28-35
1 % ˜ C < 7 %: T;
R23/24/25-34
0,1 % ˜ C < 1 %: Xn;
R20/21/22-36/37/38

015-039-00-9

azinphos-methyl (ISO)
O,O-dimethyl 4-oxobenzotriazin-3-
ylmethyl phosphorodithioate

015-048-00-8

fenthion (ISO)
O,O-dimethyl-O-(4-methylthio-m-
tolyl)phosphorothioate

015-056-00-1

azinphos-ethyl (ISO)
O,O-diethyl 4-oxobenzotriazin-3-
ylmethylphosphorodithioate

015-140-00-8

triazophos (ISO)
O,O-diethyl-O-1-phenyl-1H,2,4-triazol-
3-ylphosphorothioate

201-676-1

86-50-0

T+; R26/28
T; R24
R43
N; R50-53

T+; N
R: 24-26/28-43-50/53
S: (1/2-)28-36/37-45-60-61

200-231-9

55-38-9 Muta. Cat. 3; R40

220-147-6

2642-71-9

245-986-5

24017-47-8

T; R23-48/25
Xn; R21/22
N; R50-53

T+; R28
T; R24
N; R50-53

T; R23/25
Xn; R21
N; R50-53

T; N
R: 21/22-23-40-48/25-50/53
S: (1/2-)36/37-45-60-61

T+; N
R: 24-28-50/53
S: (1/2-)28-36/37-45-60-61

T; N
R: 21-23/25-50/53
S: (1/2-)36/37-45-60-61

016-013-00-X sulphur dichloride

234-129-0

10545-99-0 R14

C; R34
N; R50

C; N
R: 14-34-50
S: (1/2-)26-36/37/39-45-61

C ¯ 10 %: C; R34
5 % ˜ C < 10 %: Xi;
R36/37/38

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u
r
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e
a
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C
o
m
m
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i
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s

.

.

8
6
2
0
0
0

Index No

Chemical name

Notes
related to
substances

EC No

CAS No

Classification

Labelling

Concentration limits

Notes
related to
preparations

016-014-00-5

sulphur tetrachloride

(cid:151)

13451-08-6 R14

016-023-00-4

dimethyl sulphate

E

201-058-1

77-78-1

C; R34
N; R50

Carc. Cat. 2; R45
Muta. Cat. 3; R40
T+; R26
T; R25
C; R34
R43

016-024-00-X dimexano (ISO)

bis(methoxythiocarbonyl) disulphide

215-993-8

1468-37-7 Xn; R22

N; R50-53

016-071-00-6

trisodium-3-amino-6,13-dichloro-
10-((3-((4-chloro-6-(2-sulfophenylamino)-
1,3,5-triazin-2-yl)amino)propyl)amino]-4,11-
triphenoxydioxazinedisulfonate

410-130-3

136248-03-8 R43

022-001-00-5

titanium tetrachloride

231-441-9

7550-45-0 R14

030-004-00-8

dimethylzinc [1]
diethylzinc [2]

208-884-1 [1]
209-161-3 [2]

544-97-8 [1]
557-20-0 [2]

C; R34

R14
F; R17
C; R34
N; R50-53

C; N
R: 14-34-50
S: (1/2-)26-36/37/39-45-61

C ¯ 10 %: C; R34
5 % ˜ C < 10 %: Xi;
R36/37/38

C ¯ 25 %: T+;
R45-25-26-34-43
10 % ˜ C < 25 %: T+;
R45-22-26-34-43
7 % ˜ C < 10 %: T+;
R45-22-26-36/37/38-43
5 % ˜ C < 7 %: T;
R45-22-23-36/37/38-43
3 % ˜ C < 5 %: T;
R45-22-23-43
1 % ˜ C < 3 %: T;
R45-23-43
0,1 % ˜ C < 1 %: T;
R45-20
0,01 % ˜ C < 0,1 %: T;
R45

T+
R: 45-25-26-34-43
S: 53-45

Xn; N
R: 22-50/53
S: (2-)60-61

Xi
R: 43
S: (2-)22-24-37

C
R: 14-34
S: (1/2-)7/8-26-36/37/39-45

C ¯ 10 %: C; R34
5 % ˜ C < 10 %: Xi;
R36/37/38

F; C; N
R: 14-17-34-50/53
S: (1/2-)16-43-45-60-61

050-002-00-0

cyhexatin (ISO)
hydroxytricyclohexylstannane
tri(cyclohexyl)tin hydroxide

236-049-1

13121-70-5 Xn; R20/21/22

N; R50-53

Xn; N
R: 20/21/22-50/53
S: (2-)13-60-61

.

.

8
6
2
0
0
0

E
N

O

f
f
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c
i
a
l

J
o
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r
n
a
l

o
f

t
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e

E
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e
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9

Index No

Chemical name

Notes
related to
substances

EC No

CAS No

Classification

Labelling

Concentration limits

Notes
related to
preparations

050-012-00-5

tetracyclohexylstannane [1]
chlorotricyclohexylstannane [2]
butyltricyclohexylstannane [3]

215-910-5 [1]
221-437-5 [2]
230-358-5 [3]

1449-55-4 [1]
3091-32-5 [2]
7067-44-9 [3]

Xn; R20/21/22
N; R50-53

Xn; N
R: 20/21/22-50/53
S: (2-)26-28-60-61

C ¯ 1 %: Xn; R20/21/22

1

050-017-00-2

fenbutatin oxide (ISO)
bis(tris(2-methyl-2-phenylpropyl)tin)oxide

236-407-7

13356-08-6

T+; R26
Xi; R36/38
N; R50/53

T+; N
R: 26-36/38-50/53
S: (1/2-)28-36/37-45-60-61

082-009-00-X lead sulfochromate yellow

215-693-7

1344-37-2

C.I. Pigment Yellow 34 [This substance is
identified in the Colour Index by Colour Index
Constitution Number, C.I. 77603.]

082-010-00-5

lead chromate molybdate sulfate red
C.I. Pigment red 104. [This substance is
identified in the Colour Index by Colour Index
Constitution Number, C.I. 77605.]

235-759-9

12656-85-8

601-024-00-X cumene [1]

propylbenzene [2]

202-704-5 [1]
203-132-9 [2]

98-82-8 [1]
103-65-1 [2]

601-032-00-3

benzo[a]pyrene
benzo[def]chrysene

200-028-5

50-32-8

601-034-00-4

benz[e]acephenanthrylene

205-911-9

205-99-2

Carc. Cat. 3; R40
Repr. Cat. 1; R61
Repr. Cat. 3; R62
R33
N; R50-53

Carc. Cat. 3; R40
Repr. Cat. 1; R61
Repr. Cat. 3; R62
R33
N; R50-53

R10
Xn; R65
Xi; R37
N; R51-53

Carc. Cat.2; R45
Muta. Cat. 2; R46
Repr. Cat. 2;
R60-61
N; R50-53

Carc. Cat.2; R45
N; R50-53

602-035-00-2

1,4-dichlorobenzene
p-dichlorobenzene

203-400-5

106-46-7 Xi; R36

N; R50-53

602-054-00-6

3-iodopropene
allyl iodide

209-130-4

556-56-9 R10

C; R34

T; N
R: 61-33-40-50/53-62
S: 53-45-60-61

T; N
R: 61-33-40-50/53-62
S: 53-45-60-61

Xn; N
R: 10-37-51/53-65
S: (2-)24-37-61-62

T; N
R: 45-46-60-61-50/53
S: 53-45-60-61

T; N
R: 45-50/53
S: 53-45-60-61

Xi; N
R: 36-50/53
S: (2-)24/25-46-60-61

C
R: 10-34
S: (1/2-)7-26-45

1

1

4

L

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3
6
/
1
0

E
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O

f
f
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c
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a
l

J
o
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n
a
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o
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t
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e

E
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e
a
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C
o
m
m
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n
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s

.

.

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6
2
0
0
0

Index No

Chemical name

Notes
related to
substances

EC No

CAS No

Classification

Labelling

Concentration limits

Notes
related to
preparations

603-076-00-9

but-2-yne-1,4-diol
2-butyne-1,4-diol

203-788-6

110-65-6

T; R23/25
Xn; R21-48/22
C; R34

T
R: 21-23/25-34-48/22
S: (1/2-)26-36/37/39-45

C ¯ 50 %: T;
R21-23/25-34-48/22
25 % ˜ C < 50 %: T;
R21-23/25-36/38-48/22
10 % ˜ C < 25 %: Xn;
R20/22-48/22
3 % ˜ C < 10 %: Xn;
R20/22

603-091-00-0

exo-1-methyl-4-(1-methylethyl)-7-oxabi-
cyclo[2.2.1]heptan-2-ol

603-093-00-1

exo-(+/-)-1-methyl-4-(1-methylethyl)-
2-[(2-methylphenyl)methoxy]-7-oxabi-
cyclo[2.2.1]heptane

402-470-6

87172-89-2 O; R8

Xn; R22
Xi; R36

402-410-9

87818-31-3 Xn; R20

N; R51-53

603-097-00-3

1,1¡,1¢-nitrilotripropan-2-ol
triisopropanolamine

204-528-4

122-20-3 Xi; R36
R52-53

O; Xn
R: 8-22-36
S: (2-)26

Xn; N
R: 20-51/53
S: (2-)23-61

Xi
R: 36-52/53
S: (2-)26-61

603-117-00-0

propan-2-ol
isopropyl alcohol
isopropanol

604-020-00-6

biphenyl-2-ol
2-hydroxybiphenyl
2-phenylphenol (ISO)

604-021-00-1

2-phenylphenol, sodium salt
sodium 2-biphenylate

604-024-00-8

4,4¡-isobutylethylidenediphenol
(alt.): 2,2-bis(4¡hydroxyphenyl)-4-methylpentane

604-041-00-0

acifluorfen [1]
acifluorfen-sodium [2]
5-[2-chloro-4-(trifluormethyl)phenoxy]-
2-nitrobenzoic acid [1]
sodium-5-[2-chloro-4-(trifluormethyl)
phenoxy]-2-nitrobenzoate [2]

200-661-7

67-63-0

F; R11
Xi; R36
R67

F; Xi
R: 11-36-67
S: (2-)7-16-24/25-26

6

201-993-5

90-43-7 Xi; R36/37/38

N; R50

205-055-6

132-27-4 Xn; R22

Xi; R37/38-41
N; R50

401-720-1

6807-17-6 Repr. Cat. 2; R60

256-634-5 [1]
263-560-7 [2]

50594-66-6 [1]
62476-59-9 [2]

Xi; R36
N; R50-53

Xn; R22
Xi; R38-41
N; R50-53

Xi; N
R: 36/37/38-50
S: (2-)22-61

Xn; N
R: 37/38-41-50
S: (2-)22-26-61

T; N
R: 60-36-50/53
S: 53-45-60-61

Xn; N
R: 22-38-41-50/53
S: (2-)24-39-60-61

.

.

8
6
2
0
0
0

E
N

O

f
f
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c
i
a
l

J
o
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r
n
a
l

o
f

t
h
e

E
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C
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1
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Index No

Chemical name

604-043-00-1 monobenzone

4-hydroxyphenyl benzyl ether
hydroquinone monobenzyl ether

604-044-00-7 mequinol

4-methoxyphenol
hydroquinone monomethyl ether

605-016-00-7

glyoxal (cid:133) %
ethandial (cid:133) %

Notes
related to
substances

EC No

CAS No

Classification

Labelling

Concentration limits

Notes
related to
preparations

203-083-3

103-16-2 Xi; R36

R43

Xi
R: 36-43
S: (2-)24/25-26-37

205-769-8

150-76-5 Xn; R22
Xi; R36
R43

Xn
R: 22-36-43
S: (2-)24/25-26-37/39-46

B

203-474-9

107-22-2 Muta. Cat. 3; R40

Xn; R20
Xi; R36/38
R43

T; R25-48/25
N; R50-53

C ¯ 10 %: Xn;
R20-36/38-40-43
1 % ˜ C < 10 %: Xn;
R40-43

Xn
R: 20-36/38-40-43
S: (2-)36/37

T; N
R: 25-48/25-50/53
S: (1/2-)37-45-60-61

Xn; N
R: 22-36/38-50/53
S: (2-)26-60-61

606-016-00-X pindone (ISO)

2-pivaloylindan-1,3-dione

201-462-8

83-26-1

606-018-00-0

dichlone (ISO)
2,3-dichloro-1,4-naphthoquinone

204-210-5

117-80-6 Xn; R22

Xi; R36/38
N; R50-53

606-019-00-6

chlordecone (ISO)
perchloropentacyclo[5,3,0,02,6,03,9,04,8]
decan-5-one
decachloropentacyclo[5,2,1,02,6,03,9,05,8]
decan-4-one

606-034-00-8 metribuzin (ISO)

4-amino-6-tert-butyl-3-methylthio-1,2,4-
triazin-5(4H)-one
4-amino-4,5-dihydro-6-(1,1-dimethylethyl)-
3-methylthio-1,2,4-triazin-5-one

205-601-3

143-50-0

Carc. Cat. 3; R40
T; R24/25
N; R50-53

T; N
R: 24/25-40-50/53
S: (1/2-)22-36/37-45-60-61

244-209-7

21087-64-9 Xn; R22

N; R50-53

Xn; N
R: 22-50/53
S: (2-)60-61

606-035-00-3

chloridazon (ISO)
5-amino-4-chloro-2-phenylpyridazine-3-(2H)-one
pyrazon

216-920-2

1698-60-8 R43

N; R50-53

606-036-00-9

quinomethionate
chinomethionat (ISO)
6-methyl-1,3-dithiolo(4,5-b)quinoxalin-2-one

219-455-3

2439-01-2 Repr. Cat. 3; R62
Xn;
R20/21/22-48/22
Xi; R36
R43
N; R50-53

Xi; N
R: 43-50/53
S: (2-)24-37-60-61

Xn; N
R: 20/21/22-36-43-48/22-50/
53-62
S: (2-)24-37-60-61

606-037-00-4

triadimefon (ISO)
1-(4-chlorphenoxy)-3,3-dimethyl-
1-(1,2,4-triazol-1-yl)butanone

256-103-8

43121-43-3 Xn; R22

N; R51-53

Xn; N
R: 22-51/53
S: (2-)61

L

1
3
6
/
1
2

E
N

O

f
f
i
c
i
a
l

J
o
u
r
n
a
l

o
f

t
h
e

E
u
r
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p
e
a
n

C
o
m
m
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s

.

.

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6
2
0
0
0

Index No

Chemical name

Notes
related to
substances

EC No

CAS No

Classification

Labelling

Concentration limits

Notes
related to
preparations

606-044-00-2

2,4,6-trimethylbenzophenone

403-150-9

607-043-00-X dicamba (ISO)

217-635-6

2,5-dichloro-6-methoxybenzoic acid
3,6-dichloro-2-methoxybenzoic acid

954-16-5 Xn; R22
Xi; R36
N; R50-53

1918-00-9 Xn; R22
Xi; R41
R52-53

607-057-00-6

coumachlor (ISO)
3-[1-(4-chlorphenyl)-3-oxobutyl)-4-hydroxy-
coumarin

607-058-00-1

607-079-00-6

cuomafuryl (ISO)
fumarin
(RS)-3-(1-(2-furyl)-3-oxobutyl)-
4-hydroxycoumarin
4-hydroxy-3-[3-oxo-1-(2-furyl)butyl]coumarin

kelevan (ISO)
ethyl-5-(perchloro-5-hydroxypentacyclo
(5,3,0,02,6,03,9,04,8)decan-5-yl)-4-oxopentanoate
ethyl-5-(1,2,3,5,6,7,8,9,10,10-decachloro-4-
hydroxypentacyclo(5,2,1,02,6,03,9,05,8)dec-4-
yl)-4-oxovalerate

607-097-00-4

benzene-1,2,4-tricarboxylic acid 1,2-anhydride
trimellitic anhydride

201-378-1

81-82-3 Xn; R48/22

204-195-5

117-52-2

R52-53

T; R25-48/25
R52-53

(cid:151)

4234-79-1

T; R24
Xn; R22
N; R51-53

T; N
R: 22-24-51/53
S: (1/2-)36/37-45-61

607-143-00-3

valeric acid

203-677-2

109-52-4

209-008-0

552-30-7 Xi; R37-41

R42/43

C; R34
R52-53

607-152-00-2

2,3,6-TBA (ISO)
2,3,6-trichlorobenzoic acid

200-026-4

50-31-7 Xn; R22

N; R51-53

607-153-00-8

benazolin (ISO)
4-chloro-2,3-dihydro-2-oxo-1,3-benzothiazol-
3-ylacetic acid

607-156-00-4

chlorfenson (ISO)
4-chlorophenyl-4-chlorobenzenesulfonate

223-297-0

3813-05-6 Xi; R36/38

R52-53

201-270-4

80-33-1 Xn; R22
Xi; R38
N; R50-53

607-158-00-5

sodium salt of chloroacetic acid
sodium chloroacetate

223-498-3

3926-62-3

T; R25
Xi; R38
N; R50

T; N
R: 25-38-50
S: (1/2-)22-37-45-61

Xn; N
R: 22-36-50/53
S: (2-)26-60-61

Xn; N
R: 22-41-52/53
S: (2-)26-61

Xn
R: 48/22-52/53
S: (2-)37-61

T
R: 25-48/25-52/53
S: (1/2-)37-45-61

Xn
R: 37-41-42/43
S: (2-)22-26-36/37/39

C
R: 34-52/53
S: (1/2-)26-36-45-61

Xn; N
R: 22-51/53
S: (2-)61

Xi
R: 36/38-52/53
S: (2-)22-61

Xn; N
R: 22-38-50/53
S: (2-)37-60-61

.

.

8
6
2
0
0
0

E
N

O

f
f
i
c
i
a
l

J
o
u
r
n
a
l

o
f

t
h
e

E
u
r
o
p
e
a
n

C
o
m
m
u
n
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6
/
1
3

Index No

Chemical name

Notes
related to
substances

EC No

CAS No

Classification

Labelling

Concentration limits

Notes
related to
preparations

607-159-00-0

chlorobenzilate (ISO)
ethyl 2,2-di(4-chlorophenyl)-2-hydroxyacetate
ethyl-4,4¡-dichlorobenzilate

208-110-2

510-15-6 Xn; R22

N; R50-53

607-176-00-3 A mixture of: a-3-(3-(2H-benzotriazol-2-yl)-

400-830-7

5-tert-butyl-4-hydroxyphenyl)propionyl-
x-hydroxypoly(oxyethylene);a-3-(3-(2H-
benzotriazol-2-yl)-5-tert-butyl-4-
hydroxyphenyl)propionyl-x-3-(3-(2H-
benzotriazol-2-yl)-5-tert-butyl-
4-hydroxyphenyl)propionyloxypoly
(oxyethylene)

(cid:151) R43

N; R51-53

607-188-00-9

hydrogen sodium
N-carboxylatoethyl-N-octadec-9-enylmaleamate

402-970-4

(cid:151) R43

N; R51-53

Xn; N
R: 22-50/53
S: (2-)60-61

Xi; N
R: 43-51/53
S: (2-)36/37-61

Xi; N
R: 43-51/53
S: (2-)24/37-61

607-209-00-1 A mixture of:

403-030-6

O,O-di(1-methylethyl)trithio-bis-thioformate;
O,O-di(1-methylethyl)tetrathio-bis-thio-
formate;O,O-di(1-methylethyl)pentathio-bis-
thioformate

(cid:151) Xn; R22
R43
N; R50-53

Xn; N
R: 22-43-50/53
S: (2-)36/37-60-61

607-213-00-3

ethyl-3,3-bis[(1,1-dimethylpropyl)peroxy]
butyrate

403-320-2

67567-23-1

E; R2
O; R7
R10
N; R51-53

E; N
R: 2-7-10-51/53
S: (2-)3/7-14-33-36/37/39-61

607-217-00-5

2-ethoxyethyl-2-[4-(2,6-dihydro-
2,6-dioxo-7-phenyl-1,5-dioxaindacen-
3-yl)phenoxy]acetate

403-960-2

(cid:151) R43
R53

607-243-00-7

sodium 3,6-dichloro-o-anisate [1]
3,6-dichloro-o-anisic acid, compound with
2,2¡-iminodiethanol (1:1) [2]
3,6-dichloro-o-anisic acid, compound with
2-aminoethanol (1:1) [3]

217-846-3 [1]
246-590-5 [2]
258-527-9 [3]

1982-69-0 [1]
25059-78-3 [2]
53404-28-7 [3]

R52-53

607-248-00-4

naptalam-sodium
sodium N-naphth-1-ylphthalamate

205-073-4

132-67-2 Xn; R22

Xi
R: 43-53
S: (2-)24-37-61

R: 52/53
S: 61

Xn
R: 22
S: (2)

L

1
3
6
/
1
4

E
N

O

f
f
i
c
i
a
l

J
o
u
r
n
a
l

o
f

t
h
e

E
u
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e
a
n

C
o
m
m
u
n
i
t
i
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s

.

.

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6
2
0
0
0

Index No

Chemical name

Notes
related to
substances

EC No

CAS No

Classification

Labelling

Concentration limits

Notes
related to
preparations

607-249-00-X (1-methyl-1,2-ethanediyl)bis[oxy(methyl-2,1-

256-032-2

42978-66-5 Xi; R36/37/38

ethanediyl)diacrylate
tripropylene glycol diacrylate
TPGDA

607-252-00-6

lambda-cyhalothrin (ISO)
a 1:1 mixture of (S)-a-cyano-3-phenoxybenzyl
(Z)-(1R)-cis-3-(2-chloro-3,3,3-trifluoropropenyl)-
2,2-dimethylcyclopropanecarboxylate and
(R)-a-cyano-3-phenoxybenzyl (Z)-(1S)-cis-
3-(2-chloro-3,3,3-trifluoropropenyl)-
2,2-dimethylcyclopropanecarboxylate

607-255-00-2

fluroxypyr (ISO)
4-amino-3,5-dichloro-6-fluoro-
2-pyridyloxyacetic acid

608-003-00-4

acrylonitrile

415-130-7

91465-08-6

R43
N; R51-53

T+; R26
T; R25
Xn; R21
N; R50-53

(cid:151)

69377-81-7 R52-53

Xi; N
R: 36/37/38-43-51/53
S: (2-)24-37-61

C ¯ 10 %: Xi;
R36/37/38-43
1 % ˜ C < 10 %: Xi; R43

T+; N
R: 21-25-26-50/53
S:
(1/2-)28-36/37/39-38-45-60-61

D
E

203-466-5

107-13-1

R: 52/53
S: 61

F; T; N
R: 45-11-23-/24/25-37/38-41-

43-51/53
S: 9-16-53-45-61

F; R11
Carc. Cat. 2; R45
T; R23/24/25
Xi; R37/38-41
R43
N; R51-53

C ¯ 20 %: T;
R45-23/24/25-37/38-
41-43
10 % ˜ C < 20 %: T;
R45-23/24/25-41-43
5 % ˜ C < 10 %: T;
R45-23/24/25-36-43
1 % ˜ C < 5 %: T;
R45-23/24/25-43
0,2 % ˜ C < 1 %: T;
R45-20/21/22
0,1 % ˜ C < 0,2 %: T; R45

608-016-00-5

1,4-Dicyano-2,3,5,6-tetra-chloro-benzene

401-550-8

1897-41-2 R43

N; R50-53

609-030-00-4

dinoterb (ISO)
2-tert-butyl-4,6-dinitrophenol

609-040-00-9

nitrofen (ISO)
2,4-dichlorophenyl 4-nitrophenyl ether

609-044-00-0

tecnazene (ISO)
1,2,4,5-tetrachloro-3-nitrobenzene

E

E

Xi; N
R: 43-50/53
S: (2-)24-37-60-61

T+; N
R: 61-24-28-44-50/53
S: 53-45-60-61

215-813-8

1420-07-1 Repr. Cat. 2; R61

T+; R28
T; R24
R44
N; R50-53

217-406-0

1836-75-5

Carc. Cat. 2; R45
Repr. Cat. 2; R61
Xn; R22
N; R50-53

T; N
R: 45-61-22-50/53
S: 53-45-60-61

204-178-2

117-18-0 Xn; R22

R43
N; R50-53

Xn; N
R: 22-43-50/53
S: (2-)24-37-60-61

.

.

8
6
2
0
0
0

E
N

O

f
f
i
c
i
a
l

J
o
u
r
n
a
l

o
f

t
h
e

E
u
r
o
p
e
a
n

C
o
m
m
u
n
i
t
i
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s

L

1
3
6
/
1
5

Index No

Chemical name

Notes
related to
substances

EC No

CAS No

Classification

Labelling

Concentration limits

Notes
related to
preparations

611-008-00-4

4-aminoazobenzene
4-phenylazoaniline

200-453-6

60-09-3

Carc. Cat. 2; R45
N; R50-53

T; N
R: 45-50/53
S: 53-45-60-61

611-013-00-1

trilithium 1-hydroxy-7-(3-sulfonatoanilino)-
2-(3-methyl-4-(2-methoxy-4-
(3-sulfonatophenylazo)phenylazo)phenylazo)
naphthalene-3-sulfonate

611-031-00-X 4,4¡-(4-iminocyclohexa-2,5-dienylidenemethyl-
ene)dianiline hydrochloride
C.I. Basic Red 9

612-035-00-4

2-methoxyaniline
o-anisidine

612-042-00-2

benzidine
1,1¡-byphenyl-4,4¡-diamine
4,4¡-diaminobiphenyl
biphenyl-4,4¡-ylenediamine

612-051-00-1

4,4¡-diaminodiphenylmethane
4,4¡-methylenedianiline

612-081-00-5

salts of 4,4¡-bi-o-toluidine
salts of 3,3¡-dimethylbenzidine
salts of o-tolidine

612-099-00-3

4-methyl-m-phenylenediamine
2,4-toluenediamine

E

E

E

A
E

E

403-650-7

117409-78-6

E; R2
N; R51-53

209-321-2

569-61-9

Carc. Cat. 2; R45

E; N
R: 2-51/53
S: (2-)35-61

T
R: 45
S: 53-45

201-963-1

90-04-0

202-199-1

92-87-5

Carc. Cat. 2; R45
Muta. Cat. 3; R40
T; R23/24/25

T
R: 45-23/24/25
S: 53-45

Carc. Cat. 1; R45
Xn; R22
N; R50-53

T; N
R: 45-22-50/53
S: 53-45-60-61

202-974-4

101-77-9

Carc. Cat. 2; R45
Muta. Cat. 3; R40
T; R39/23/24/25Xn;
R48/20/21/22
R43
N; R51-53

T; N
R: 45-39/23/24/25-43-48/20/

21/22-51/53

S: 53-45-61

210-322-5
265-294-7
277-985-0

612-82-8
64969-36-4
74753-18-7

Carc. Cat. 2; R45
Xn; R22
N; R51-53

T; N
R: 45-22-51/53
S: 53-45-61

202-453-1

95-80-7

T; N
R: 45-21-25-36-43-51/53
S: 53-45-61

Carc. Cat. 2; R45
T; R25
Xn; R21
Xi; R36
R43
N; R51-53

612-105-00-4

2-piperazin-1-ylethylamine

205-411-0

140-31-8 Xn, R21/22

C; R34
R43
R52-53

C
R: 21/22-34-43-52/53
S: (1/2-)26-36/37/39-45-61

C ¯ 25 %: T; R45-22
0,01 % ˜ C < 25 %: T; R45

L

1
3
6
/
1
6

E
N

O

f
f
i
c
i
a
l

J
o
u
r
n
a
l

o
f

t
h
e

E
u
r
o
p
e
a
n

C
o
m
m
u
n
i
t
i
e
s

.

.

8
6
2
0
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Index No

Chemical name

612-111-00-7

2-methyl-m-phenylenediamine
2,6-toluenediamine

Notes
related to
substances

EC No

CAS No

Classification

Labelling

Concentration limits

Notes
related to
preparations

.

.

8
6
2
0
0
0

212-513-9

823-40-5 Muta. Cat. 3; R40

612-125-00-3

2-methyl-p-phenylenediamine
2,5-toluenediamine

202-442-1

95-70-5

612-144-00-7

flumetralin (ISO)
N-(2-chloro-6-fluorobenzyl)-N-ethyl-a,a,a-
trifluoro-2,6-dinitro-p-toluidine

(cid:151)

62924-70-3 Xi; R36/38

612-151-00-5

diaminotoluene

E

246-910-3

25376-45-8

613-018-00-4 morfamquat (ISO)

(cid:151)

7411-47-4 Xn; R22

1,1¡-bis(3,5-dimethylmorpholinocarbonyl-
methyl)-4,4¡-bipyridilium ion

Xi; R36/37/38
R52-53

613-031-00-5

symclosene
trichloroisocyanuric acid
trichloro-1,3,5-triazinetrion

613-038-00-3

6-phenyl-1,3,5-triazine-2,4-diyldiamine
6-phenyl-1,3,5-triazine-2,4-diamine
benzoguanamine

613-042-00-5

imazalil (ISO)
1-[2-(allyloxy)-2-(2,4-dichlorophenyl)ethyl]-
1H-imidazole

613-043-00-0

imazalil sulphate (ISO)
1-[2-(allyloxy)ethyl-2-(2,4-dicholorphenyl)]-
1H-imidazolium hydrogen sulphate [1]
(–)-1-[2-(allyloxy)ethyl-2-(2,4-dichlorophenyl)]-
1H-imidazolium hydrogen sulphate [2]

201-782-8

87-90-1 O; R8

Xn; R22
R31
Xi; R36/37
N; R50-53

202-095-6

91-76-9 Xn; R22
R52-53

252-615-0

35554-44-0 Xn; R20/22

261-351-5 [1]
281-291-3 [2]

58594-72-2 [1]
83918-57-4 [2]

N; R41
N; R50-53

Xn; R20/22
Xi; R41
N; R50-53

Xn; R21/22
R43
N; R51-53

T; R25
Xn; R20/21
R43
N; R51-53

R43
N; R50-53

Carc. Cat. 2; R45
T; R25
Xn; R20/21
Xi; R36
R43
N; R51-53

Xn; N
R: 21/22-40-43-51/53
S: (2-)24-36/37-61

T; N
R: 20/21-25-43-51/53
S: (1/2-)24-37-45-61

Xi; N
R: 36/38-43-50/53
S: (2-)36/37-60-61

T; N
R: 45-20/21-25-36-43-51/53
S: 53-45-61

Xn
R: 22-36/37/38-52/53
S: (2-)22-36-61

O; Xn; N
R: 8-22-31-36/37-50/53
S: (2-)8-26-41-60-61

Xn
R: 22-52/53
S: (2-)61

Xn; N
R: 20/22-41-50/53
S: (2-)26-39-60-61

Xn; N
R: 20/22-41-50/53
S: (2-)26-39-60-61

E
N

O

f
f
i
c
i
a
l

J
o
u
r
n
a
l

o
f

t
h
e

E
u
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p
e
a
n

C
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6
/
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7

Index No

Chemical name

Notes
related to
substances

EC No

CAS No

Classification

Labelling

Concentration limits

Notes
related to
preparations

613-066-00-6

terbumeton (ISO)
2-tert-butylamino-4-ethylamino-6-methoxy-
1,3,5-triazin

251-637-8

33693-04-8 Xn; R22

N; R50-53

Xn; N
R: 22-50/53
S: (2-)60-61

613-091-00-2 morfamquat dichloride [1]

morfamquat sulfate [2]

225-062-8 [1]

4636-83-3 [1]
29873-36-7 [2]

Xn; R22
Xi; R36/37/38
R52-53

Xn;
R: 22-36/37/38-52/53
S: (2-)22-36-61

613-098-00-0 N-(n-octyl)-2-pyrrolidinone

403-700-8

2687-94-7

C; R34
N; R51-53

C; N
R: 34-51/53
S: (1/2-)23-26-36/37/39-45-61

613-130-00-3

hexaconazole (ISO)
(RS)-2-(2,4-dichlorophenyl)-1-(1H-1,2,4-
triazol-1-yl)hexan-2-ol

613-131-00-9

pyroquilon (ISO)
1,2,5,6-tetrahydropyrrolo[3,2,1-ij]quinolin-
4-one

(cid:151)

79983-71-4 R43

N; R51-53

(cid:151)

57369-32-1 Xn; R22
R52-53

613-134-00-5 myclobutanil (ISO)

(cid:151)

88671-89-0 Repr. Cat. 3; R63

2-(4-chlorophenyl)-2-(1H-1,2,4-triazol-
1-ylmethyl)hexanenitrile

Xn; R22
Xi; R36
N; R51-53

613-137-00-1 methabenzthiazuron (ISO)

242-505-0

18691-97-9 N; R50-53

1-(1,3-benzothiazol-2-yl)1,3-dimethylurea

613-139-00-2 metsulfuron-methyl

(cid:151)

74223-64-6 N; R50-53

methyl 2-(4-methoxy-6-methyl-1,3,5-triazin-
2-ylcarbamoylsulfamoyl) benzoate

Xi; N
R: 43-51/53
S: (2-)24-37-61

Xn
R: 22-52/53
S: (2-)61

Xn; N
R: 22-36-51/53-63
S: (2-)36/37-46-61

N
R: 50/53
S: 60-61

N
R: 50/53
S: 60-61

614-001-00-4

nicotine (ISO)
3-(N-methyl-2-pyrrolidinyl)pyridine

200-193-3

54-11-5

614-006-00-1

brucine
2,3-dimethoxystrychnine

206-614-7

357-57-3

T+; R27
T; R25
N; R51-53

T+; R26/28
R52-53

T+; N
R: 25-27-51/53
S: (1/2-)36/37-45-61

T+
R: 26/28-52/53
S: (1/2-)13-45-61

L

1
3
6
/
1
8

E
N

O

f
f
i
c
i
a
l

J
o
u
r
n
a
l

o
f

t
h
e

E
u
r
o
p
e
a
n

C
o
m
m
u
n
i
t
i
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s

.

.

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0
0
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Index No

Chemical name

Notes
related to
substances

EC No

CAS No

Classification

Labelling

Concentration limits

Notes
related to
preparations

614-007-00-7

615-006-00-4

brucine sulphate [1]
brucine nitrate [2]
Strychnidin-10-one, 2,3-dimethoxy-,
mono[(R)-1-methylheptyl 1,2-benzenedicar-
boxylate] [3]
Strychnidin-10-one, 2,3-dimethoxy-, compd.
with (S)-mono(1-methylheptyl-1,2-benzene-
dicarboxylate (1:1) [4]

2-methyl-m-phenylene diisocyanate [1]
4-methyl-m-phenylene diisocyanate [2]
m-tolylidene diisocyanate [3]
toluene 2,6-di-isocyanate [1]
toluene 2,4-di-isocyanate [2]
toluene-diisocyanate [3]

225-432-9 [1]
227-317-9 [2]
269-439-5 [3]
269-710-8 [4]

4845-99-2 [1]
5786-97-0 [2]
68239-26-9 [3]
68310-42-9 [4]

T+; R26/28
R52-53

T+
R: 26/28-52/53
S: (1/2-)13-45-61

C

202-039-0 [1]
209-544-5 [2]
247-722-4 [3]

91-08-7 [1]
584-84-9 [2]
26471-62-5 [3]

Carc. Cat. 3; R40
T+; R26
Xi; R36/37/38
R42/43
R52-53

T+
R: 26-36/37/38-40-42/43-52/53
S: (1/2-)23-36/37-45-61

2

C ¯ 20 %: T+;
R26-36/37/38-40-42/43
7 % ˜ C < 20 %: T+;
R26-40-42/43
1 % ˜ C < 7 %: T;
R23-40-42/43
0,1 % ˜ C < 1 %: Xn;
R20-42

616-010-00-9

tosylchloramide sodium

204-854-7

127-65-1 Xn; R22

R31
C; R34
R42

616-034-00-X pyracarbolid (ISO)

246-419-4

24691-76-7 R52-53

3,4-dihydro-6-methyl-2H-pyran-5-carboxanilide

616-035-00-5

cymoxanil
2-cyano-N-[(ethylamino)carbonyl]-
2-(methoxyimino)acetamide

261-043-0

57966-95-7 Xn; R22

R43
N; R50-53

617-004-00-9

1,2,3,4-tetrahydro-1-naphthyl hydroperoxide

212-230-0

771-29-9 O; R7

Xn; R22
C; R34
N; R50-53

617-006-00-X bis(a,a-dimethylbenzyl) peroxide

201-279-3

80-43-3 O; R7

617-008-00-0

dibenzoyl peroxide
benzoyl peroxyde

202-327-6

94-36-0

Xi; R36/38
N; R51-53

E; R2
Xi; R36
R43

C
R: 22-31-34-42
S: (1/2-)7-22-26-36/37/39-45

R: 52/53
S: 61

Xn; N
R: 22-43-50/53
S: (2-)36/37-60-61

O; C; N
R: 7-22-34-50/53
S:
(1/2-)3/7-14-26
(cid:150)36/37/39-45-60-61

O; Xi; N
R: 7-36/38-51/53
S: (2-)3/7-14-36/37/39-61

E; Xi;
R: 2-36-43
S: (2-)3/7-14-36/37/39

650-007-00-3

chlordimeform (ISO)
N2-(4-Chlor-o-tolyl)-N1,N1-dimethylformamidine

228-200-5

6164-98-3

Carc. Cat. 3; R40
Xn; R21/22
N; R50-53

Xn; N
R: 21/22-40-50/53
S: (2-)22-36/37-60-61

C ¯ 25 %: C; R22-34
10 % ˜ C < 25 %: C; R34
5 % ˜ C < 10 %: Xi;
R36/37/38

.

.

8
6
2
0
0
0

E
N

O

f
f
i
c
i
a
l

J
o
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r
n
a
l

o
f

t
h
e

E
u
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o
p
e
a
n

C
o
m
m
u
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/
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9

Index No

Chemical name

Notes
related to
substances

EC No

CAS No

Classification

Labelling

Concentration limits

Notes
related to
preparations

650-008-00-9

drazoxolon (ISO)
4-(2-chlorophenylhydrazone)-3-methyl-
5-isoxazolone

650-009-00-4

chlordimeform hydrochloride
N¡-(4-chloro-o-tolyl)-N,N-
dimethylformamidinemonohydrochloride
N2-(4-chloro-o-tolyl)-N1,N1-
dimethylformamidinehydrochloride

650-033-00-5

esfenvalerate (ISO)
(S)-a-cyano-3-phenoxybenzyl-(S)-
2-(4-chlorophenyl)-3-methylbutyrate

227-197-8

5707-69-7

T; R25
N; R50-53

T; N
R: 25-50/53
S: (1/2-)22-24-36/37-45-60-61

243-269-1

19750-95-9

Carc. Cat. 3; R40
Xn; R22
N; R50-53

Xn; N
R: 22-40-50/53
S: (2-)22-36/37-60-61

(cid:151)

66230-04-4

T; R23/25
R43
N; R50-53

T; N
R: 23/25-43-50/53
S: (1/2-)24-36/37/39-45-60-61

650-041-00-9

triasulfuron (ISO)
1-[2-(2-chloroethoxy)phenylsulfonyl]-
3-(4-methoxy-6-methyl-1,3,5(cid:150)triazin-2-yl)urea

(cid:151)

82097-50-5 N; R50-53

N
R: 50/53
S: 60-61

L

1
3
6
/
2
0

E
N

O

f
f
i
c
i
a
l

J
o
u
r
n
a
l

o
f

t
h
e

E
u
r
o
p
e
a
n

C
o
m
m
u
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s

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ANNEX 1D

Index No

Chemical name

Notes
related to
substances

EC No

CAS No

Classification

Labelling

Concentration limits

Notes
related to
prepara-
tions

006-090-00-8

2-(3-iodoprop-2-yn-1-yloxy)ethyl phenylcarba-
mate

408-010-0

88558-41-2 Xn; R20
Xi; R41
R52-53

Xn
R: 20-41-52/53
S: (2-)22-26-39-61

014-016-00-0 A mixture of:

406-490-6

(cid:151) N; R51-53

1,3-dihex-5-en-1-yl-1,1,3,3-tetramethyldisilox-
ane; 1,3-dihex-n-en-1-yl-1,1,3,3-tetramethyldis-
iloxane

015-164-00-9

calcium-P,P¡-(1-hydroxyethylene)bis
(hydrogenphosphonate)dihydrate

015-165-00-4 A mixture of:

thiobis(4,1-phenylene)-S,S,S¡,S¡-tetraphenyldi-
sulfoniumbishexafluorophosphate;diphenyl
(4-phenylthiophenyl)sulfonium hexafluorophos-
phate

015-166-00-X 3,9-bis(2,6-di-tert-butyl-4-methylphenoxy)-
2,4,8,10-tetraoxa-3,9-diphosphaspiro[5.5]
undecane

400-480-5

36669-85-9 R52-53

404-986-7

(cid:151) Xi; R41

N; R50-53

Xi; N
R: 41-50/53
S: (2-)15-26-39-60-61

410-290-4

80693-00-1 R53

015-167-00-5

3-(hydroxyphenylphosphinyl)propanoic acid

411-200-6

14657-64-8 Xi; R41

601-050-00-1

benzene, C10-13-alkyl derivatives

267-051-0

67774-74-7 N; R50

601-051-00-7

4-phenylbut-1-ene

405-980-7

768-56-9 Xi; R38

N; R51-53

602-083-00-4

diphenyl ether, pentabromo
derivativepentabromodiphenyl ether

251-084-2

32534-81-9 Xn; R48/21/22

R64
N; R50-53

Xn; N
R: 48/21/22-50/53-64
S: (1/2-)36/37-45-60-61

602-084-00-X 1,1-dichloro-1-fluoroethane

404-080-1

1717-00-6 N; R52-53-59

603-128-00-0

2-(phenylmethoxy)naphthalene

405-490-3

613-62-7 R53

N
R: 52/53-59
S: 59-61

R: 53
S: 61

N
R: 51/53
S: 61

R: 52/53
S: 61

R: 53
S: 61

Xi
R: 41
S: (2-)26-39

N
R: 50
S: 61

Xi; N
R: 38-51/53
S: (2-)37-61

.

.

8
6
2
0
0
0

E
N

O

f
f
i
c
i
a
l

J
o
u
r
n
a
l

o
f

t
h
e

E
u
r
o
p
e
a
n

C
o
m
m
u
n
i
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i
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s

L

1
3
6
/
2
1

Index No

Chemical name

Notes
related to
substances

EC No

CAS No

Classification

Labelling

Concentration limits

Notes
related to
prepara-
tions

603-129-00-6

1-tert-butoxypropan-2-ol

406-180-0

57018-52-7 R10

603-130-00-1 A mixture of isomers of:

406-325-8

a-((dimethyl)biphenyl)-x-hydroxypoly
(oxyethylene)

Xi; R41

(cid:151) Xn; R22
R52-53

603-131-00-7 A 3:1 mixture of:

407-290-1

(cid:151) Xi; R41

1-deoxy-1-[methyl-(1-oxododecyl)amino]
-D-glucitol; 1-deoxy-1-[methyl-(1-oxotetrade-
cyl)amino]-D-glucitol

603-132-00-2

2-hydroxymethyl-9-methyl-6-(1-methylethyl)-
1,4-dioxaspiro[4.5]decane

408-200-3

63187-91-7 Xi; R38-41

R52-53

(cid:151) Xn; R22
R52-53

603-133-00-8 A mixture of:

408-240-1

3-[(4-amino-2-chloro-5-nitrophenyl)amino]-
propane-1,2-diol;3,3¡-(2-chloro-5-nitro-
1,4-phenylenediimino)bis(propan-1,2-diol)

603-134-00-3 A mixture of substituted dodecyl and/or

410-450-3

(cid:151) R53

tetradecyl, diphenyl ethers. The substance is
produced by the Friedel Crafts reaction. The
catalyst is removed from the reaction product.
Diphenyl ether is substituted by C1-C10 alkyl
groups. The alkyl groups are bonded randomly
between C1 and C6. Linear C12 and C14,
50/50 used.

603-135-00-9

bis[[2,2¡,2¢-nitrilotris[ethanolato]]-1-N,O]bis
[2-(2-methoxyethoxy)ethoxy]-titanium

410-500-4

(cid:151) Xi; R41

N; R51-53

603-136-00-4

3-((4-(bis(2-hydroxyethyl)amino)-2-nitrophe-
nyl)amino)-1-propanol

410-910-3

104226-19-9 R43

R52-53

603-137-00-X A mixture of:

411-130-6

(cid:151) Xi; R41

1-deoxy-1-[methyl-(1-oxohexadecyl[amino]-
D-glucitol; 1-deoxy-1-[methyl-(1-oxooctade-
cyl)amino]-D-glucitol

603-138-00-5

3-(2,2-dimethyl-3-hydroxypropyl)toluene
alt.): 2,2-dimethyl-3-(3-methylphenyl)propanol

403-140-4

103694-68-4 R52-53

Xi
R: 10-41
S: (2-)26-39

Xn
R: 22-52/53
S: (2-)39-61

Xi
R: 41
S: (2-)26-39

Xi
R: 38-41-52/53
S: (2-)26-37/39-61

Xn
R: 22-52/53
S: (2-)22-36-61

R: 53
S: 61

Xi; N
R: 41-51/53
S: (2-)26-39-61

Xi
R: 43-52/53
S: (2-)24-37-61

Xi
R: 41
S: (2-)26-39

R: 52/53
S: 61

L

1
3
6
/
2
2

E
N

O

f
f
i
c
i
a
l

J
o
u
r
n
a
l

o
f

t
h
e

E
u
r
o
p
e
a
n

C
o
m
m
u
n
i
t
i
e
s

.

.

8
6
2
0
0
0

Index No

Chemical name

Notes
related to
substances

EC No

CAS No

Classification

Labelling

Concentration limits

Notes
related to
prepara-
tions

604-050-00-X 4-chloro-o-cresol

4-chloro-2-methyl phenol

216-381-3

1570-64-5

T; R23
C; R35
N; R50

T; C; N
R: 23-35-50
S: (1/2-)26-36/37/39-45-61

C ¯ 25 %: T; C; R23-35
10 % ˜ C < 25 %: C;
R20-35
5 % ˜ C < 10 %: C;
R20-34
3 % ˜ C < 5 %: Xn;
R20-36/37/38
1 % ˜ C < 3 %: Xi;
R36/37/38

604-051-00-5

3,5-bis((3,5-di-tert-butyl-4-hydroxy)benzyl)-
2,4,6-trimethylphenol

604-052-00-0

2,2¡-methylenebis(6-(2H-benzotriazol-2-yl)-
4-(1,1,3,3-tetramethylbutyl)phenol)

604-053-00-6

2-methyl-4-(1,1-dimethylethyl)-6-(1-methyl-
pentadecyl)-phenol

401-110-5

87113-78-8 R52-53

403-800-1

103597-45-1 R53

410-760-9

157661-93-3 Xi; R38

R43
N; R50-53

(cid:151) R43

R52-53

604-054-00-1 A mixture of:

412-020-0

2-methoxy-4-(tetrahydro-4-methylene-
2H-pyran-2-yl)-phenol;4-(3,6-dihydro-4-methyl-
2H-pyran-2-yl)-2-methoxyphenol

604-055-00-7

2,2¡-((3,5¡,5,5¡-tetramethyl-(1,1¡-biphenyl)
-4,4¡-diyl)-bis(oxymethylene))-bis-oxirane

413-900-7

85954-11-6 Muta.Cat.3; R40

605-027-00-7 A mixture of:

3a,4,5,6,7,7a-hexahydro-4,7-methano-1H-in-
dene-6-carboxaldehyde; 3a,4,5,6,7,7a-hexahy-
dro-4,7-methano-1H-indene-5-carboxaldehyde

410-480-7

(cid:151) R43

N; R51-53

606-051-00-0

4-pentylcyclohexanone

406-670-4

61203-83-6 N; R51-53

606-052-00-6

4-(N,N-dibutylamino)-2-hydroxy
-2¡-carboxybenzophenone

607-272-00-5

fluoroxypyr-meptyl (ISO) [1]
fluroxypyr-butometyl (ISO) [2]
methylheptyl, O-((4-amino-3,5-dichloro-6-
fluoro-2-pyridyloxy) acetate [1]
2-butoxy-1-methylethyl, O-(4-amino-3,5-
dichloro-6-fluoro-2-pyridyloxy) acetate [2]

410-410-5

54574-82-2 R52-53

279-752-9 [1]
(cid:151)

81406-37-3 [1]
154486-27-8 [2]

N; R50-53

R: 52/53
S: 61

R: 53
S: 61

Xi; N
R: 38-43-50/53
S: (2-)24-37-60-61

Xi
R: 43-52/53
S: (2-)24-37-61

Xn
R: 40
S: (2-)22-36-37

Xi; N
R: 43-51/53
S: (2-)24-37-61

N
R: 51/53
S: 61

R: 52/53
S: 61

N
R: 50/53
S: 60-61

.

.

8
6
2
0
0
0

E
N

O

f
f
i
c
i
a
l

J
o
u
r
n
a
l

o
f

t
h
e

E
u
r
o
p
e
a
n

C
o
m
m
u
n
i
t
i
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s

L

1
3
6
/
2
3

Index No

Chemical name

Notes
related to
substances

EC No

CAS No

Classification

Labelling

Concentration limits

Notes
related to
prepara-
tions

607-273-00-0

ammonium-7-(2,6-dimethyl-8-(2,2-dimethylbu-
tyryloxy)-1,2,6,7,8,8a-hexahydro-1-naphthyl)-
3,5-dihydroxyheptanoate

404-520-2

(cid:151) R52-53

607-274-00-6

2-(N-benzyl-N-methylamino)ethyl-3-amino-
2-butenoate

405-350-1

54527-73-0 R43

N; R51-53

607-275-00-1

sodium benzoyloxybenzene-4-sulfonate

405-450-5

66531-87-1 R43

607-276-00-7

bis[(1-methylimidazol)-(2-ethyl-hexanoate)],
zinc complex

405-635-0

(cid:151) Xi; R38-41
N; R50-53

607-277-00-2 A mixture of: 2-(hexylthio)ethylamine

405-720-2

hydrochloride; sodium propionate

607-278-00-8 A mixture of isomers of sodium

405-760-0

phenethylnaphthalenesulfonate; sodium
naphthylethylbenzenesulfonate

607-279-00-3 A mixture of n-octadecylaminodiethyl

405-960-8

bis(hydrogenmaleat); n-octadecylaminodiethyl
hydrogen maleate hydrogenphthalate

607-280-00-9

sodium 4-chloro-1-hydroxybutane-1-sulfonate

406-190-5

(cid:151) Xn; R22
Xi; R41
R43
N; R51-53

(cid:151) Xi; R41
R43
R52-53

(cid:151) R43

N; R51-53

R: 52/53
S: 61

Xi; N
R: 43-51/53
S: (2-)24-37-61

Xi
R: 43
S: (2-)24-37

Xi; N
R: 38-41-50/53
S: (2-)26-37/39-60-61

Xn; N
R: 22-41-43-51/53
S: (2-)24-26-37/39-61

Xi
R: 41-43-52/53
S: (2-)24-26-37/39-61

Xi; N
R: 43-51/53
S: (2-)24-37-61

54322-20-2 Xn; R22
Xi; R36
R43

Xn
R: 22-36-43
S: (2-)22-26-36/37

607-281-00-4 A mixture of branched and linear C7-C9 alkyl

407-000-3

127519-17-9 N; R51-53

3-[3-(2H-benzotriazol-2-yl)-5-(1,1-dimethyl-
ethyl)-4-hydroxyphenyl]propionates

607-282-00-X 2-acetoxymethyl-4-benzyloxybut-1-yl acetate

407-140-5

131266-10-9 R52-53

N
R: 51/53
S: 61

R: 52/53
S: 61

L

1
3
6
/
2
4

E
N

O

f
f
i
c
i
a
l

J
o
u
r
n
a
l

o
f

t
h
e

E
u
r
o
p
e
a
n

C
o
m
m
u
n
i
t
i
e
s

.

.

8
6
2
0
0
0

Index No

Chemical name

Notes
related to
substances

EC No

CAS No

Classification

Labelling

Concentration limits

Notes
related to
prepara-
tions

607-283-00-5

E-ethyl-4-oxo-4-phenylcrotonate

408-040-4

15121-89-8 Xn; R21/22
Xi; R38-41
R43
N; R50-53

Xn; N
R: 21/22-38-41-43-50/53
S: (2-)26-36/37/39-60-61

607-284-00-0 A 9:1 mixture of: sodium

410-040-4

136213-76-8 N; R51-53

3,3¡-(1,4-phenylenebis(carbonylimino-3,1-pro-
panediylimino))bis(10-amino-6,13-dichloro)-
4,11-triphenodioxazinedisulfonate);lithium
3,3¡-(1,4-phenylenebis-(carbonylimino-3,1-
propanediyl-imino))bis(10-amino-6,13-dich-
loro)-4,11-triphenodioxazinedisulfonate

607-285-00-6 A mixture of:

410-065-0

(cid:151) R43

7-(((3-aminophenyl)sulfonyl)amino)-naphtha-
lene-1,3-disulfonic acid;sodium
7-(((3-aminophenyl)sulfonyl)amino)-naphtha-
lene-1,3-disulfonate; potassium7-(((3-aminophe-
nyl)sulfonyl)amino)-naphthalene-1,3-disulfonate

607-286-00-1 A mixture of: sodium/potassium

410-070-8

141880-36-6 R43

7-[[[3-[[4-((2-hydroxy-naphthyl)azo)phe-
nyl]azo]phenyl]sulfonyl]amino]-naphthalene-
1,3-disulfonate

R52-53

607-287-00-7 O¡-methyl O-(1-methyl-2-methacryloyloxy-

410-140-8

(cid:151) R52-53

607-288-00-2

ethyl)-1,2,3,6-tetrahydrophthalate

Tetrasodium (c-(3-(1-(3-(e-6-dichloro-5-cyano-
pyrimidin-f-yl(methyl)amino)propyl)-1,6-dihy-
dro-2-hydroxy-4-methyl-6-oxo-3-pyridylazo)-
4-sulfonatophenylsulfamoyl)phtalocyanine-
a,b,d-trisulfonato(6-))nickelato (II), where a is 1
or 2 or 3 or 4, b is 8 or 9 or 10 or 11, c is 15
or 16 or 17 or 18, d is 22 or 23 or 24 or 25
and where e and f together are 2 and 4 or 4
and 2 respectively.

410-160-7

148732-74-5 Xi; R36

R43
R52-53

N
R: 51/53
S: 61

Xi
R: 43
S: (2-)22-24-37

Xi
R: 43-52/53
S: (2-)22-24-37-61

R: 52/53
S: 61

Xi
R: 36-43-52/53
S: (2-)22-26-36/37-61

607-288-00-8

3-(3-(4-(2,4-bis(1,1-dimethylpropyl)phen-
oxy)butylaminocarbonyl-4-hydroxy-1-naph-
thalenyl)thio)propanoic acid

410-370-9

105488-33-3 R53

R: 53
S: 61

607-290-00-3 A mixture (ratio not known) of: ammonium

410-540-2

1-C14-C18-alkyloxycarbonyl-2-(3-allyloxy-2-hy-
droxypropoxycarbonyl)ethane-1-sulfonate;
ammonium 2-C14-C18-alkyloxycarbonyl-
1-(3-allyloxy-2-hydroxypropoxycar-
bonyl)ethane-1-sulfonate

(cid:151) Xi; R38
R43
N; R50-53

Xi; N
R: 38-43-50/53
S: (2-)24-37-60-61

607-291-00-9

dodecyl-x-(C5/C6-cycloalkyl)alkyl carboxylate

410-630-1

104051-92-5 R53

R: 53
S: 61

.

.

8
6
2
0
0
0

E
N

O

f
f
i
c
i
a
l

J
o
u
r
n
a
l

o
f

t
h
e

E
u
r
o
p
e
a
n

C
o
m
m
u
n
i
t
i
e
s

L

1
3
6
/
2
5

Index No

Chemical name

Notes
related to
substances

EC No

CAS No

Classification

Labelling

Concentration limits

Notes
related to
prepara-
tions

607-292-00-4 A mixture of:

[1-(methoxymethyl)-2-(C12-alkoxy)-
ethoxy]acetic acid;
[1-(methoxymethyl)-2-(C14-alkoxy)-
ethoxy]acetic acid

607-293-00-X A mixture of: N-aminoethylpiperazonium
mono-2,4,6-trimethylnonyldiphenyl ether
di-sulfonate; N-aminoethylpiperazonium
di-2,4,6-trimethylnonyldiphenyl ether
di-sulfonate

410-640-6

(cid:151) Xi; R38-41
N; R50-53

Xi; N
R: 38-41-50/53
S: (2-)26-37/39-60-61

410-650-0

(cid:151) Xi; R41
R43
N; R51-53

Xi; N
R: 41-43-51/53
S: (2-)26-36/37/39-61

607-294-00-5

sodium 2-benzoyloxy-1-hydroxyethane-sulfon-
ate

410-680-4

(cid:151) R43

607-295-00-0 A mixture of: tetrasodium

410-800-5

phosphonoethane-1,2-dicarboxylate; hexaso-
dium phosphonobutane-1,2,3,4-tetracarboxy-
late

(cid:151) R43

N; R51-53

607-296-00-6 A mixture of: pentaerythriol tetraesters with

410-830-9

(cid:151) R53

heptanoic acid and 2-ethylhexanoic acid

607-297-00-1

(E-E)-3,3¡-(1,4-phenylenedimethyl-
idene)bis(2-oxobornane-10-sulfonic acid)

410-960-6

92761-26-7 Xi; R41

607-298-00-7

2-(trimethylammonium)ethoxycarboxybenzene-
4-sulfonate

411-010-3

(cid:151) R43

607-299-00-2 methyl 3-(acetylthio)-2-methyl-propanoate

411-040-7

97101-46-7 Xn; R22

607-300-00-6

trisodium [2-(5-chloro-2,6-difluoropyrimidin-
4-ylamino)-5-(b-sulfamoyl-c,d-sulfonatophthalo-
cyanin-a-yl-K4,N29,N30,N31,N32-sulfony-
lamino)benzoato(5-)]cuprate(II) where a = 1, 2,
3, 4 b = 8, 9, 10, 11 c = 15, 16, 17, 18d =
22, 23, 24, 25

R43
N; R50-53

411-430-7

(cid:151) R43

Xi
R: 43
S: (2-)24-37

Xi; N
R: 43-51/53
S: (2-)24-37-61

R: 53
S: 61

Xi
R: 41
S: (2-)26-39

Xi
R: 43
S: (2-)22-36/37

Xn; N
R: 22-43-50/53
S: (2-)24-37-60-61

Xi
R: 43
S: (2-)22-24-37

607-301-00-1 A mixture of: dodecanoic acid; poly(1-7)lactate

411-860-5

esters of dodecanoic acid

(cid:151) Xi; R38-41
R43
N; R51-53

Xi; N
R: 38-41-43-51/53
S: (2-)24-26-37/39-61

L

1
3
6
/
2
6

E
N

O

f
f
i
c
i
a
l

J
o
u
r
n
a
l

o
f

t
h
e

E
u
r
o
p
e
a
n

C
o
m
m
u
n
i
t
i
e
s

.

.

8
6
2
0
0
0

Index No

Chemical name

Notes
related to
substances

EC No

CAS No

Classification

Labelling

Concentration limits

Notes
related to
prepara-
tions

607-302-00-7 A mixture of: tetradecanoic acid;

411-910-6

poly(1-7)lactate esters of tetradecanoic acid

(cid:151) Xi; R38-41
R43
N; R51-53

Xi; N
R: 38-41-43-51/53
S: (2-)24-26-37/39-61

607-303-00-2

1-cyclopropyl-6,7-difluoro-1,4-dihydro-4-oxo-
quinoline-3-carboxylic acid

413-760-7

93107-30-3 Repr. Cat.3; R62

R52-53

608-023-00-3

4-(4-chlorophenyl)-2-phenyl-2-[(1H-1,2,4-tria-
zol-1-yl)methyl]butanenitrile

406-140-2

114369-43-6 N; R50-53

608-024-00-9

2-(4-(N-butyl-N-phenethylamino)phenyl)ethy-
lene-1,1,2-tricarbonitrile

407-650-8

97460-76-9 R53

608-025-00-4

2-nitro-4,5-bis(benzyloxy)phenylacetonitrile

410-970-0

117568-27-1 R53

Xn
R: 62-52/53
S: (2-)22-36/37-61

N
R: 50/53
S: 60-61

R: 53
S: 61

R: 53
S: 61

609-053-00-X hydrazine-tri-nitromethane

414-850-9

(cid:151) E; R3
O; R8
Carc. Cat. 2; R45
T; R23/25
R43

E; T
R: 45-3-8-23/25-43
S: 53-45

610-010-00-2

2-bromo-1-(2-furyl)-2-nitroethylene

406-110-9

35950-52-8 Xn; R22-48/22

C; R34
R43
N; R50-53

Xi; R41
52-53

611-043-00-5 A 2:1:1 mixture of: trisodium

402-850-1

N(1¡)-N(2):N(1£)-N(2¢)-g-6-[2-amino-4-(or 6)-
hydroxy-(or 4-amino-2-hydroxy)phenylazo]
-6¢-(1-carbaniloyl-2-hydroxyprop-1-enylazo)-
5¡,5£-disulfamoyl-3,3¢-disulfonatobis(naphtha-
lene-2,1¡-azobenzene-1,2¡-diolato-O(1),O(2¡))-
chromate; trisodium N(1¡)-N(2):N(1£)-N(2¢)
-g-6,6¢-bis(1-carbaniloyl-2-hydroxyprop-1-eny-
lazo)-5¡,5£-disulfamoyl-3,3¢-disulfonato-
bis(naphthalene-2,1¡azobenzene-1,2¡-diolato-
O(1),O(2¡))-chromate; trisodium N(1¡)-N(2):N
(1£)-N(2¢)-g-6,6¢-bis[2-amino-4-(or 6)-hydroxy-
(or 4-amino-2-hydroxy)phenylazo]5¡,5£-
disulfamoyl-3,3¢-disulfonatobis(naphthalene-
2,1¡azobenzene-1,2¡-diolato-O(1),O(2¡))-
chromate

C; N
R: 22-34-43-48/22-50/53
S: (1/2-)22-26-36/37/39-45-

60-61

Xi
R: 41-52/53
S: (2-)26-39-61

.

.

8
6
2
0
0
0

E
N

O

f
f
i
c
i
a
l

J
o
u
r
n
a
l

o
f

t
h
e

E
u
r
o
p
e
a
n

C
o
m
m
u
n
i
t
i
e
s

L

1
3
6
/
2
7

Index No

Chemical name

Notes
related to
substances

EC No

CAS No

Classification

Labelling

Concentration limits

Notes
related to
prepara-
tions

403-720-7

117527-94-3 N; R51-53

N
R: 51/53
S: 61

611-044-00(cid:150)0 A mixture of: tert-alkyl(C12-C14)ammonium
bis[1-[(2-hydroxy-5-nitrophenyl)azo]-2-naph-
thalenolato(2-)]-chromate(1-); tert-alkyl
(C12-C14)ammonium bis[1-[(2-hydroxy-4-ni-
trophenyl)azo]-2-naphthalenolato(2-)]-chro-
mate(1-); tert-alkyl(C12-C14)ammonium
bis[1-[[5-(1,1-dimethylpropyl)-2-hydroxy-3-ni-
trophenyl]azo]-2-naphthalenolato(2-)]-chro-
mate(1-); tert-alkyl(C12-C14)ammonium-
[[1-[(2-hydroxy-5-nitrophenyl)azo]-2-naph-
thalenolato(2-)]-[1-[(2-hydroxy-5-nitrophe-
nyl)azo]-2-naphthalenolato(2-)]]-chromate(1-);
tert-alkyl(C12-C14)ammonium-[[1-[[5-(1,1-di-
methylpropyl)-2-hydroxy-3-nitrophenyl]azo]
-2-naphthalenolato(2-)]-[1-[(2-hydroxy-5-nitro-
phenyl)azo]-2-naphthalenolato(2-)]]-chromate
(1-); tert-alkyl(C12-C14)ammonium((1-(4(or
5)-nitro-2-oxidophenylazo)-2-naphtholato)
(1-(3-nitro-2-oxido-5-pentylphenylazo)-2-naph-
tholato))chromate(1-)

611-045-00-6

2-[4-[N-(4-acetoxybutyl)-N-ethyl]amino-
2-methylphenylazo]-3-acetyl-5-nitrothiophene

404-830-8

(cid:151) R53

R: 53
S: 61

611-046-00-1

4,4¡-diamino-2-methylazobenzene

407-590-2

43151-99-1

T; R25
Xn; R48/22
R43
N; R50-53

T; N
R: 25-43-48/22-50/53
S: (1/2-)22-28-36/37-45-60-61

611-047-00-7 A 1:1 mixture of:

407-890-3

111381-11-4 R53

2-[[4-[N-ethyl-N-(2-acetoxyethyl)amino]phe-
nyl]azo]-5,6-dichlorobenzothiazole;2-
[[4-[N-ethyl-N-(2-acetoxyethyl)amino]phe-
nyl]azo]-6,7-dichlorobenzothiazole

611-048-00-2 A (1:1) mixture of:

407-900-6

111381-12-5 R53

2-[[4-[bis(2-acetoxyethyl)amino]phenyl]azo]-
5,6-dichlorobenzothiazole; 2-[[4-[bis(2-acetoxy-
ethyl)amino]phenyl]azo]-6,7-dichlorobenzothia-
zole

R: 53
S: 61

R: 53
S: 61

L

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Index No

Chemical name

Notes
related to
substances

EC No

CAS No

Classification

Labelling

Concentration limits

Notes
related to
prepara-
tions

611-049-00-8

7-[4-(3-diethylaminopropylamino)-6-(3-diethy-
lammoniopropylamino)-1,3,5-triazin-
2-ylamino]-4-hydroxy-3-(4-phenylazopheny-
lazo)-naphthalene-2-sulfonate, acetic acid, lactic
acid (2:1:1)

611-051-00-9

2-(4-(N-ethyl-N-(2-hydroxy)ethyl)amino-
2-methylphenyl)azo-6-methoxy-3-methylbenzo-
thiazolium chloride

408-000-6

118658-98-3 Xn; R48/22

R43
R52-53

Xn
R: 43-48/22-52/53
S: (2-(22-36/37-61

411-110-7

136213-74-6 N; R50-53

611-052-00-4 monosodium aqua-[5-[[2,4-dihydroxy-5-

400-720-9

(cid:151) R52-53

[(2-hydroxy-3,5-dinitrophenyl)azo]phe-
nyl]azo]-2-naphthalensulfonate], iron complex

612-156-00-2 A mixture of: trihexadecylmethylammonium
chloride; dihexadecyldimethylammoniumch-
loride

612-157-00-8

(Z)-1-benzo[b]thien-2-ylethanone oxime
hydrochloride

405-620-9

410-780-8

(cid:151) Xi; R41

N; R50-53

Xi; N
R: 41-50/53
S: (2-)26-39-60-61

(cid:151) Xn; R22-48/22
Xi; R41
R43
N; R51-53

Xn; N
R: 22-41-43-48/22-51/53
S: (2-)22-26-36/37/39-61

612-158-00-3 A mixture of:

410-820-4

(cid:151) R53

N
R: 50/53
S: 60-61

R: 52/53
S: 61

R: 53
S: 61

612-159-00-9

bis(5-dodecyl-2-hydroxybenzaldoximate) copper
(II) C12-alkyl group is branched;
4-dodecylsalicylaldoxime

Reaction products of: trimethylhexamethylene
diamine (a mixture of
2,2,4-trimethyl-1,6-hexanediamine and
2,4,4-trimethyl-1,6-hexanediamine, EINECS
listed), Epoxide 8
(mono[(C10-C16-alkyloxy)methyl]oxirane
derivatives) and p-toluene-sulfonic acid

410-880-1

(cid:151) Xn; R22

C; R34
N; R50-53

C; N
R: 22-34-50/53
S: (1/2-)23-26-36/37/39-45-

60-61

613-149-00-7

2-tert-butyl-5-(4-tert-butylbenzylthio)-4-chloro-
pyridazin-3(2H)-one

405-700-3

96489-71-3

T; R23/25
N; R50-53

T; N
R: 23/25(cid:150)50/53
S: (1/2-)36/37-45-60-61

613-150-00-2

2,2¡-[3,3¡-(piperazine-1,4-diyl)dipropyl]bis
(1H-benzimidazo[2,1-b]benzo[l,m,n][3,8]phen-
anthroline-1,3,6-trione

406-295-6

(cid:151) R53

R: 53
S: 61

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Index No

Chemical name

Notes
related to
substances

EC No

CAS No

Classification

Labelling

Concentration limits

Notes
related to
prepara-
tions

613-151-00-8

1-(3-mesyloxy-5-trityloxymethyl-2-D-threofu-
ryl)thymine

406-360-9

104218-44-2 R53

613-152-00-3

phenyl N-(4,6-dimethoxypyrimidin-2-yl)carba-
mate

406-600-2

89392-03-0 R43

N; R51-53

613-153-00-9

2,3,5-trichloropyridine

407-270-2

16063-70-0 R52-53

613-154-00-4

2-amino-4-chloro-6-methoxypyrimidine

410-050-9

5734-64-5 Xn; R22

613-155-00-X 5-chloro-2,3-difluoropyridine

410-090-7

89402-43-7 R10

Xn; R22
R52-53

613-156-00-5

2-butyl-4-chloro-5-formylimidazole

410-260-0

83857-96-9 R43

N; R51-53

613-157-00-0

2,4-diamino-5-methoxymethylpyrimidine

410-330-0

54236-98-5 Xn; R22-48/22

Xi; R36

613-158-00-6

2,3-dichloro-5-trifluoromethyl-pyridine

410-340-5

69045-84-7 Xn; R20/22

613-159-00-1

4-[2-[4-(1,1-dimethylethyl)phenyl]-ethoxy]qui-
nazoline

410-580-0

120928-09-8

Xi; R41
R43
N; R51-53

T; R25
Xn; R20
N; R50-53

613-160-00-7

(1S)-2-methyl-2,5-diazobicyclo[2.2.1]heptane
dihydrobromide

411-000-9

125224-62-6 R43

615-022-00-1 methyl 3-isocyanatosulfonyl-2-thiophene-car-

410-550-7

79277-18-2

boxylate

E; R2
R14
Xn; R48/22
R42/43

R: 53
S: 61

Xi; N
R: 43-51/53
S: (2-)24-37-61

R: 52/53
S: 61

Xn
R: 22
S: (2-)22

Xn
R: 10-22-52/53
S: (2-)23-36-61

Xi; N
R: 43-51/53
S: (2-)24-37-61

Xn
R: 22-36-48/22
S: (2-)22-26-36

Xn; N
R: 20/22-41-43-51/53
S: (2-)24-26-37/39-61

T; N
R: 20-25-50/53
S: (1/2-)37-45-60-61

Xi
R: 43
S: (2-)24-37

E; Xn
R: 2-14-42/43-48/22
S: (2-)22-30-35-36/37

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Index No

Chemical name

Notes
related to
substances

EC No

CAS No

Classification

Labelling

Concentration limits

Notes
related to
prepara-
tions

615-023-00-7

2-(isocyanatosulfonylmethyl)benzoic acid
methyl ester; (alt.): methyl
2-(isocyanatosulfonylmethyl)benzoate

410-900-9

83056-32-0 R10
R14
Muta. Cat.3; R40
Xn; R20-48/22
Xi; R41
R42

Xn
R: 10-14-20-40-41-42-48/22
S: (2-)23-26-36/37/39

616-044-00-4 N-(3,5-dichloro-4-ethyl-2-hydroxyphenyl)-

402-510-2

(cid:151) N; R51-53

2-(3-pentadecylphenoxy)-butanamide

616-045-00-X 2¡-(4-chloro-3-cyano-5-formyl-2-thienylazo)-

405-190-2

5¡-diethylamino-2-methoxyacetanilide

122371-93-1 R43
R53

616-046-00-5 N-(2-(6-chloro-7-methylpyrazolo(1,5-b)-

406-390-2

(cid:151) N; R50-53

1,2,4-triazol-4-yl)propyl)-2-(2,4-di-tert-pentylp-
henoxy)octanamide

616-047-00-0 A mixture of:

406-640-0

(cid:151) R43

2,2¡,2¢,2£-(ethylenedinitrilotetrakis-N,N-di(C16)
alkylacetamide; 2,2¡,2¢,2£-
ethylenedinitrilotetrakis-N,N-di(C18)alkylaceta-
mide

616-048-00-6

3¡-trifluoromethylisobutyranilide

616-049-00-1

2-(2,4-bis(1,1-dimethylethyl)phenoxy)-
N-(3,5-dichloro-4-ethyl-2-hydroxyphenyl)-hex-
anamide

616-050-00-7 N-[2,5-dichloro-4-(1,1,2,3,3,3-hexafluoropro-
poxy)-phenyl-aminocarbonyl]-2,6-difluoroben-
zamide

406-740-4

1939-27-1 Xn; R48/22
N; R51-53

408-150-2

99141-89-6 R53

410-690-9

103055-07-8 R43

N; R50-53

616-051-00-2 A mixture of: 2,4

411-070-0

(cid:151) R53

-bis(N¡-(4-methylphenyl)-ureido)-toluene; 2,6
-bis(N¡-(4-methylphenyl)-ureido)-toluene

N
R: 51/53
S: 61

Xi
R: 43-53
S: (2-)22-24-37-61

N
R: 50/53
S: 60-61

Xi
R: 43
S: (2-)24-37

Xn; N
R: 48/22-51/53
S: (2-)22-36-61

R: 53
S: 61

Xi; N
R: 43-50/53
S: (2-)24-37-60-61

R: 53
S: 61

617-015-00-9

bis(4-methylbenzoyl)peroxide

407-950-9

895-85-2

E; R2
O; R7
N; R50-53

E; N
R: 2-7-50/53
S: (2-)7-14-36/37/39-47-60-61

650-032-00-X cyproconazole (ISO)

(cid:151)

94361-06-5 Repr. Cat. 3; R63

(2RS,3RS;2RS,3SR)-2-(4-chlorophenyl)-3-
cyclopropyl-1-(1H-1,2,4-triazol-1-
yl)butan-2-ol

Xn; R22
N; R50-53

Xn; N
R: 22-50/53-63
S: (2-)36/37-60-61

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L 136/32

EN

Official Journal of the European Communities

8.6.2000

ANNEX 2

R 66

IT: L’esposizione ripetuta pu(cid:242) provocare secchezza e screpolature della pelle.

(Does not concern the ES version)

(Does not concern the DA version)

(Does not concern the DE version)

(Does not concern the EL version)

(Does not concern the EN version)

(Does not concern the FR version)

(Does not concern the NL version)

(Does not concern the PT version)

(Does not concern the FI version)

(Does not concern the SV version)

8.6.2000

EN

Official Journal of the European Communities

L 136/33

ANNEX 3A

S 23

FR: Ne pas respirer les gaz/fumØes/vapeurs/aØrosols [terme(s) appropriØ(s) (cid:224) indiquer par le fabricant].

(Does not concern the ES version)

(Does not concern the DA version)

(Does not concern the DE version)

(Does not concern the EL version)

(Does not concern the EN version)

(Does not concern the IT version)

(Does not concern the NL version)

(Does not concern the PT version)

(Does not concern the FI version)

(Does not concern the SV version)

S 26

DE: Bei Ber(cid:252)hrung mit den Augen sofort gr(cid:252)ndlich mit Wasser absp(cid:252)len und Arzt konsultieren.

(Does not concern the ES version)

(Does not concern the DA version)

(Does not concern the EL version)

(Does not concern the EN version)

(Does not concern the FR version)

(Does not concern the IT version)

(Does not concern the NL version)

(Does not concern the PT version)

(Does not concern the FI version)

(Does not concern the SV version)

S 56

DE: Diesen Stoff und seinen Beh(cid:228)lter der Problemabfallentsorgung zuf(cid:252)hren.

EN: Dispose of this material and its container to hazardous or special waste collection point.

IT:

Smaltire questo materiale e i relativi contenitori in un punto di raccolta di rifiuti pericolosi o speciali.

(Does not concern the ES version)

(Does not concern the DA version)

(Does not concern the EL version)

(Does not concern the FR version)

(Does not concern the NL version)

(Does not concern the PT version)

(Does not concern the FI version)

(Does not concern the SV version)

L 136/34

EN

Official Journal of the European Communities

8.6.2000

ANNEX 3B

S 27/28

DE: Bei Ber(cid:252)hrung mit der Haut beschmutzte, getr(cid:228)nkte Kleidung sofort ausziehen und Haut sofort mit viel (cid:133)

abwaschen (vom Hersteller anzugeben).

(Does not concern the ES version)

(Does not concern the DA version)

(Does not concern the EL version)

(Does not concern the EN version)

(Does not concern the FR version)

(Does not concern the IT version)

(Does not concern the NL version)

(Does not concern the PT version)

(Does not concern the FI version)

(Does not concern the SV version)

S 29/56

ES: No tirar los residuos por el desag(cid:252)e; elim(cid:237)nese esta sustancia y su recipiente en un punto de recogida pœblica de

residuos especiales o peligrosos.

DE: Nicht in die Kanalisation gelangen lassen; diesen Stoff und seinen Beh(cid:228)lter der Problemabfallentsorgung zuf(cid:252)hren.

EN: Do not empty into drains, dispose of this material and its container to hazardous or special waste collection

point.

IT: Non gettare i residui nelle fognature; smaltire questo materiale e i relativi contenitori in un punto di raccolta di

rifiuti pericolosi o speciali.

NL: Afval niet in de gootsteen werpen; deze stof en de verpakking naar een inzamelpunt voor gevaarlijk of bijzonder

afval brengen.

SV: T(cid:246)m ej i avloppet, l(cid:228)mna detta material och dess beh(cid:229)llare till insamlingsst(cid:228)lle f(cid:246)r farligt avfall.

(Does not concern the DA version)

(Does not concern the EL version)

(Does not concern the FR version)

(Does not concern the PT version)

(Does not concern the FI version)

8.6.2000

EN

Official Journal of the European Communities

L 136/35

ANNEX 4A

(cid:145)B.10. MUTAGENICITY (cid:151) IN VITRO MAMMALIAN CHROMOSOME ABERRATION TEST

1.

METHOD

This method is a replicate of the OECD TG 473, In Vitro Mammalian Chromosome Aberration Test (1997).

1.1.

INTRODUCTION

the in vitro chromosomal aberration test

The purpose of
is to identify agents that cause structural
chromosome aberrations in cultured mammalian cells (1) (2) (3). Structural aberrations may be of two types,
chromome or chromatid. With the majority of chemical mutagens, induced aberrations are of the chromatid
type, but chromosome-type aberrations also occur. An increase in polyploidy may indicate that a chemical
has the potential
this method is not designed to measure
numerical aberrations and is not routinely used for that purpose. Chromosome mutations and related events
are the cause of many human genetic diseases and there is substantial evidence that chromosome mutations
and related events causing alterations in oncogenes and tumour suppressor genes of somatic cells are
involved in cancer induction in humans and experimental animals.

to induce numerical aberrations. However,

The in vitro chromosome aberration test may employ cultures of established cells lines, cell strains or primary
cell cultures. The cells used are selected on the basis of growth ability in culture, stability of the karyotype,
chromosome number, chromosome diversity and spontaneous frequency of chromosome aberrations.

Tests conducted in vitro generally require the use of an exogenous source of metabolic activation. This
metabolic activation system cannot mimic entirely the mammalian in vivo conditions. Care should be taken to
avoid conditions which would lead to positive results which do not reflect intrinsic mutagenicity and may
arise from changes in pH, osmolality or high levels of cytotoxicity (4) (5).

This test is used to screen for possible mammalian mutagens and carcinogens. Many compounds that are
positive in this test are mammalian carcinogens; however, there is not a perfect correlation between this test
and carcinogenicity. Correlation is dependent on chemical class and there is increasing evidence that there are
carcinogens that are not detected by this test because they appear to act through mechanisms other than
direct DNA damage.

See also General Introduction Part B.

1.2.

DEFINITIONS

Chromatid-type aberration: structural chromosome damage expressed as breakage of single chromatids or
breakage and reunion between chromatids.

Chromatid-type aberration: structural chromosome damage expressed as breakage, or breakage and
reunion, of both chromatids at an identical site.

Endoreduplication: a process in which after an S period of DNA replication, the nucleus does not go into
mitosis but starts another S period. The result is chromosomes with 4, 8, 16, (cid:133) chromatids.

Gap: an achromatic lesion smaller than the width of one chromatid, and with minimum misalignment of the
cromatids.

Mitotic index: the ratio of cells in metaphase divided by the total number of cells observed in a population
of cells; an indication of the degree of proliferation of that population.

Numerical aberration: a change in the numer of chromosomes from the normal number characteristic of
the cells utilised.

L 136/36

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Official Journal of the European Communities

8.6.2000

Polyploidy: a multiple of the haploid chromosome number (n) other than the diploid number (i.e. 3n, 4n,
and so on).

Structural aberration: a change in chromosome structure detectable by microscopic examination of the
metaphase stage of cell division, observed as deletions and fragments, intrachanges or interchanges.

1.3.

PRINCIPLE OF THE TEST METHOD

Cell cultures are exposed to the test substance both with and without metabolic activation. At predetermined
intervals after exposure of cell cultures to the test substance, they are treated with a metaphase arresting
substance (e.g. Colcemidﬁ or colchicine), harvested, stained and metaphase cells are analysed microscopically
for the presence of chromosome aberrations.

1.4.

DESCRIPTION OF THE TEST METHOD

1.4.1.

Preparations

1.4.1.1. Cells

A variety of cell
hamster fibroblasts, human or other mammalian peripheral blood lymphocytes).

lines, strains or primary cell cultures, including human cells, may be used (e.g. Chinese

1.4.1.2. Media and culture conditions

Appropriate culture media, and incubation conditions (culture vessels, CO2 concentration, temperature and
lines and strains should be checked
humidity) should be used in maintaining cultures, Established cell
routinely for stability in the modal chromosome number and the absence of mycoplasma contamination and
should not be used if contaminated. The normal cell cycle time for the cells and culture conditions used
should be known.

1.4.1.3.

Preparation of cultures

Established cell lines and strains: cells are propagated from stock cultures, seeded in culture medium at a
density such that the cultures will not reach confluency before the time of harvest, and incubated at 37 (cid:176)C.

Lymphocytes: whole blood treated with an anti-coagulant (e.g. heparin) or separated lymphocytes obtained
from healthy subjects are added to the culture medium containing a mitogen (e.g. phytohaemagglutinin) and
incubated at 37 (cid:176)C.

1.4.1.4. Metabolic activation

Cells should be exposed to the test substance both in the presence and absence of an appropriate metabolic
activation system. The most commonly used system is a cofactor-supplemented post-mitochondrial fraction
(S9) prepared from the livers of rodents treated with enzyme-inducing agents such as Aroclor 1254 (6) (7) (8)
(9), or a mixture of phenobarbitone and b-naphthoflavone (10) (11) (12).

The post-mitochondrial fraction is usually used at concentrations in the range from 1(cid:151)10 % v/v in the final
test medium. The condition of a metabolic activation system may depend upon the class of chemical being
tested. In some cases it may be appropriate to utilise more than one concentration of post-mitochondrial
fraction.

A number of developments, including the construction of genetically engineered cell lines expressing specific
activating enzymes, may provide the potential for endogenous activation. The choice of the cell lines used
should be scientifically justified (e.g. by the relevance of the cytochrome P450 isoenzyme for the metabolism
of the test substance).

8.6.2000

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Official Journal of the European Communities

L 136/37

1.4.1.5. Test substance/Preparation

Solid test substances should be dissolved or suspended in appropriate solvents or vehicles and diluted if
appropriate prior to treatment of the cells. Liquid test substances may be added directly to the test systems
and/or diluted prior to treatment. Fresh preparations of the test substance should be employed unless stability
data demonstrate the acceptability of storage.

1.4.2.

Test conditions

1.4.2.1.

Solvent/vehicle

The solvent/vehicle should not be suspected of chemical reaction with the test substance and should be
compatible with the survival of the cells and the S9 activity. If other than well-known solvent/vehicles are
used, their inclusion should be supported by data indicating their compatibility. It is recommended that
wherever possible, the use of an aqueous solvent/vehicle be considered first. When testing water-unstable
substances, the organic solvents used should be free of water. Water can be removed by adding a molecular
sieve.

1.4.2.2.

Exposure concentrations

Among the criteria to be considered when determining the highest concentration are cytotoxicity, solubility
in the test system and changes in pH or osmolality.

Cytotoxicity should be determined with and without metabolic activation in the main experiment using an
appropriate indication of cell
integrity and growth, such as degree of confluency, viable cell counts, or
mitotic index. It may be useful to determine cytotoxicity and solubility in a preliminary experiment.

At least three analysable concentrations should be used. Where cytotoxicity occurs, these concentrations
should cover a range from the maximum to little or no toxicity; this will usually mean the concentrations
should be separated by no more than a factor between 2 and (cid:211)10. At the time of harvesting, the highest
concentration should show a significant reduction in degree of confluency, cell count or mitotic index (all
greater than 50 %). The mitotic index is only an indirect measure of cytotoxic/cytostatic effects and depends
on the time after treatment. However, the mitotic index is acceptable for suspension cultures in which other
toxicity measurements may be cumbersome and impractical.
Information on cell cycle kinetics, such as
average generation time (AGT), could be used as supplementary information AGT, however, is an overall
average that does not always reveal the existence of delayed subpopulations, and even slight increases in
average generation time can be associated with very substantial delay in the time of optimal yield of
aberrations.

For relatively non-cytotoxic substances, the maximum test concentration should be 5(cid:181)l/ml, 5 mg/ml or
0,01 M, whichever is the lowest.

For relatively insoluble substances that are not toxic at concentrations lower than the insoluble concentration,
the highest dose used should be a concentration above the limit of solubility in the final culture medium at
the end of the treatment period. In some cases (e.g. when toxicity occurs only at higher than the lowest
insoluble concentration), it is advisable to test at more than one concentration with visible precipitation. It
may be useful to assess solubility at the beginning and the end of the treatment, as solubility can change
during the course of exposure in the test system due to presence of cells, S9 serum, etc. Insolubility can be
detected by using the unaided eye. The precipitate should not interfere with the scoring.

1.4.2.3. Negative and positive controls

Concurrent positive and negative (solvent or vehicle) controls, both with and without metabolic activation,
should be included in each experiment. When metabolic activation is used, the positive control chemical
should be the one that requires activation to give a mutagenic response.

Positive controls should employ a known elastogen at exposure levels expected to give a reproducible and
detectable increase over background which demonstrates the sensitivity of the test system.

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Positive control concentrations should be chosen so that the effects are clear but do not immediately reveal
the identity of the coded slides to the reader. Examples of positive control substances include:

Metabolic activation condition

Substance

CAS No

Einecs No

Absence of exogenous metabolic
activation

methyl methanesulphonate

66-27-3

200-625-0

ethyl methanesulphonate

62-50-0

200-536-7

ethyl nitrosourea

759-73-9

212-072-2

mitomycin C

50-07-7

200-008-6

4-nitroquinoline-N-oxide

56-57-5

200-281-1

Presence of exogenous metabolic
activation

benzo[a]pyrene

50-32-8

200-028-5

cyclophosphamide

50-18-0

200-015-4

cyclophosphamide monohydrate

6055-19-2

Other appropriate positive control substances may be used. The use of chemical class-related positive control
chemicals should be considered, when available.

Negative controls, consisting of solvent or vehicle alone in the treatment medium, and treated in the same
way as the treatment cultures, should be included for every harvest time. In addition, untreated controls
should also be used unless there are historical control data demonstrating that no deleterious or mutagenic
effects are induced by the chosen solvent.

1.4.3.

Procedure

1.4.3.1. Treatment with the test substance

Proliferating cells are treated with the test substance in the presence and absence of a metabolic activation
system. Treatment of lymphocytes should commence at about 48 hours after mitogenic stimulation.

1.4.3.2. Duplicate cultures should normally be used at each concentration, and are strongly recommended for
negative/solvent control cultures. Where minimal variation between duplicate cultures can be demonstrated
(13) (14), from historical data, it may be acceptable for single cultures to be used at each concentration.

Gaseous or volatile substances should be tested by appropriate methods, such as in sealed culture vessels (15)
(16).

1.4.3.3. Culture harvest time

In the first experiment, cells should be exposed to the test substance, both with and without metabolic
activation, for 3(cid:151)6 hours, and sampled at a time equivalent to about 1,5 normal cell cycle length after the
beginning of treatment (12). If this protocol gives negative results both with and without activation, an
additional experiment without activation should be done, with continuous treatment until sampling at a time
equivalent to about 1,5 normal cell cycle lengths. Certain chemicals may be more readily detected by
treatment/sampling times longer than 1,5 cycle lengths. Negative results with metabolic activation need to be
conformed on a case-by-case basis. In those cases where confirmation of negative results is not considered
necessary, justification should be provided.

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1.4.3.4. Chromosome preparation

Cell cultures are treated with Colcemidﬁ or colchicine usually for 1(cid:151)3 hours prior to harvesting. Each cell
culture is harvested and processed separately for the preparation of chromosomes. Chromosome preparation
involves hypotonic treatment of the cells, fixation and staining.

1.4.3.5. Analysis

including those of positive and negative controls, should be independently coded before
All slides,
microscopic analysis. Since fixation procedures often result in the breakage of a proportion of metaphase
cells with loss of chromosomes, the cells scored should therefore contain a number of centromeres equal to
the modal number ˚ 2 for all cell
least 200 well-spread metaphases should be scored per
concentration and control, equally divided amongst the duplicates, if applicable. This number can be reduced
when a high number of aberrations is observed.

types. At

Though the purpose of the test is to detect structural chromosome aberrations, it is important to record
polyploidy and endoreduplication when these events are seen.

2.

DATA

2.1.

TREATMENT OF RESULTS

is the cell, and therefore the percentage of cells with structural chromosome
The experimental unit
aberration(s) should be evaluated. Different types of structural chromosome aberrations should be listed with
their numbers and frequencies for experimental and control cultures. Gaps are recorded separately and
reported but generally not included in the total aberration frequency.

Concurrent measures of cytotoxicity for all treated and negative control cultures in the main aberration
experiments should also be recorded.

Individual culture data should be provided. Additionally, all data should be summarised in tabular form.

There is no requirement for verification of a clear positive response. Equivocal results should be clarified by
further testing preferably using modification of experimental conditions. The need to confirm negative results
has been discussed in 1.4.3.3. Modification of study parameters to extend the range of conditions assessed
should be considered in follow-up experiments. Study parameters that might be modified include the
concentration spacing and the metabolic activation conditions.

2.2.

EVALUATION AND INTERPRETATION OF RESULTS

There are several criteria for determining a positive result, such as a concentration-related increase or a
reproducible increase in the number of cells with chromosome aberrations. Biological relevance of the results
should be considered first. Statistical methods may be used as an aid in evaluating the test results (3) (13).
Statistical significance should not be the only determining factor for a positive response.

An increase in the number of polyploid cells may indicate that the test substance has the potential to inhibit
mitotic processes and to induce numerical chromosome aberrations. An increase in the number of cells with
endoreduplicated chromosomes may indicate that the test substance has the potential to inhibit cell cycle
progression (17) (18).

A test substance for which the results do not meet the above criteria is considered non-mutagenic in this
system.

Although most experiments will give clearly positive or negative results,
in rare cases the data set will
preclude making a definite judgement about the activity of the test substance. Results may remain equivocal
or questionable regardless of the number of times the experiment is repeated.

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Positive results from the in vitro chromosome aberration test indicate that the test substance induces structural
chromosome aberrations in cultured mammalian somatic cells. Negative results indicate that, under the test
conditions, the test substance does not induce chromosome aberrations in cultured mammalian somatic cells.

3.

REPORTING

TEST REPORT

The test report must include the following information:

Solvent/vehicle:

(cid:151) justification for choice of vehicle,

(cid:151) solubility and stability of the test substance in solvent/vehicle, if known.

Cells:

(cid:151) type and source of cells,

(cid:151) karyotype features and suitability of the cell type used,

(cid:151) absence of mycoplasma, if applicable,

(cid:151) information on cell cycle length,

(cid:151) sex of blood donors, whole blood or separated lymphocytes, mitogen used,

(cid:151) number of passages, if applicable,

(cid:151) methods for maintenance of cell culture, if applicable,

(cid:151) modal number of chromosomes.

Test conditions:

(cid:151) identity of metaphase arresting substance, its concentration and duration of cell exposure,

(cid:151) rationale for selection of concentrations and number of cultures including, e.g. cytotoxicity data and

solubility limitations, if available,

(cid:151) composition of media, CO2 concentration if applicable,

(cid:151) concentration of test substance,

(cid:151) volume of vehicle and test substance added,

(cid:151) incubation temperature,

(cid:151) incubation time,

(cid:151) duration of treatment,

(cid:151) cell density at seeding, if appropriate,

(cid:151) type and composition of metabolic activation system, including acceptability criteria,

(cid:151) positive and negative controls,

(cid:151) methods of slide preparation,

(cid:151) criteria for scoring aberrations,

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(cid:151) number of metaphases analysed,

(cid:151) methods for the measurements of toxicity,

(cid:151) criteria for considering studies as positive, negative or equivocal,

Results:

(cid:151) signs of toxicity, e.g. degree of confluency, cell cycle data, cell counts, mitotic index,

(cid:151) signs of precipitation,

(cid:151) data on pH and osmolality of the treatment medium, if determined,

(cid:151) definition for aberrations, including gaps,

(cid:151) number of cells with chromosome aberrations and type of chromosome aberrations given separately for

each treated and control culture,

(cid:151) changes in ploidy if seen,

(cid:151) dose-response relationship, where possible,

(cid:151) statistical analyses, if any,

(cid:151) concurrent negative (solvent/vehicle) and positive control data,

(cid:151) historical negative (solvent/vehicle) and positive control data, with ranges, means and standard deviations.

Discussion of results.

Conclusions.

4.

REFERENCES

(1) Evans, H.

J.

(1976), Cytological Methods for Detecting Chemical Mutagens,

in: Chemical mutagens,
(ed) Plenum Press, New York and

Principles and Methods for their Detection, Vol. 4, Hollaender, A.
London, pp. 1(cid:151)29.

(2)

Ishidate, M. Jr. and Sofumi, T.
(1985), The In Vitro Chromosomal Aberration Test Using Chinese
Hamster Lung (CHL) Fibroblast Cells in Culture, in: Progress in Mutation Research, Vol. 5, Ashby, J. et al.,
(eds) Elsevier Science Publishers, Amsterdam-New York-Oxford, pp. 427(cid:151)432.

(3) Galloway, S. M., Armstrong, M. J., Reuben, C., Colman, S., Brown, B., Cannon, C., Bloom, A. D.,
Nakamura, F., Ahmed, M., Duk, S., Rimpo, J., Margolin, G. H., Resnick, M. A., Anderson, G. and Zeiger
E. (1978), Chromosome aberration and sister chromatic exchanges in Chinese hamster ovary cells:
Evaluation of 108 chemicals, Environs, Molec. Mutagen 10 (suppl.10), pp. 1(cid:151)175.

(4) Scott, D., Galloway, S. M., Marshall, R. R., Ishidate, M. Jr., Brusick, D., Ashby, J. and Myhr, B. C. (1991),
Genotoxicity under Extreme Culture Conditions. A report from ICPEMC Task Group 9, Mutation Res.,
257, pp. 147(cid:151)204.

(5) Morita, T., Nagaki, T., Fukuda,

I. and Okumura, K.,

(1992), Clastogenicity of low pH to Various

Cultured Mammalian Cells, Mutation Res., 268, pp. 297(cid:151)305.

(6) Ames, B. N., McCann, J. and Yamasaki, E. (1975), Methods for Detecting Carcinogens and Mutagens
with the Salmonella/Mammalian Microsome Mutagenicity Test, Mutation Res., 31, pp. 347(cid:151)364.

(7) Maron, D. M. and Ames, B. N. (1983), Revised Methods for the Salmonella Mutagenicity Test, Mutation

Res., 113, pp. 173(cid:151)215.

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(8) Natarajan, A. T., Tates, A. D., van Buul, P. P. W., Meijers, M. and de Vogel, N. (1976), Cytogenetic
Induction of
Effects of Mutagen/Carcinogens after Activation in a Mircrosomal System In Vitro, I.
Chromosome Aberrations
and
(DEN)
and Sister Chromatid Exchange by Diethylnitrosamine
Dimethylnitrosamine (DMN) in CHO Cells in the Presence of Rat-Liver Microsomes, Mutation Res., 37,
pp. 83(cid:151)90.

(9) Matsuoka, A., Hayashi, M. and Ishidate, M. Jr. (1979), Chromosomal Aberration Tests on 29 Chemicals

Combined with S9 Mix In Vitro, Mutation Res., 66, pp. 277(cid:151)290.

(10) Elliot, B. M., Combes, R. D., Elcombe, C. R., Gatehouse, D. G., Gibson, G. G., Mackay, J. M. and Wolf,
R. C. (1992), Report of UK Environmental Mutagen Society Working Party. Alternative to Aroclor
1254-induced S9 in In Vitro Genotoxicity Assays, Mutagenesis, 7, pp. 175(cid:151)177.

(11) Matsushima, T., Sawamura, M., Hara, K. and Sugimura, T. (1976), A. Safe Substitute for Polychlorinated
Biphenyls as an Inducer of Metabolic Activation Systems, in: de Serres, F. J. Fouts, J. R. Bend, J. R.
and Philpot, R. M. (eds), In Vitro Metabolic Activation in Mutagenesis Testing, Elsevier, North-Holland,
pp. 85(cid:151)88.

(12) Galloway, S. M., Aardema, M. J., Ishidate, M. Jr., Ivett, J. L., Kirkland, D. J. Morita, T., Mosesso, P.,
Sofumi, T. (1994), Report from Working Group on in In Vitro Tests for Chromosomal Aberrations,
Mutation Res., 312, pp. 241(cid:151)261.

(13) Richardson, C., Williams, D. A., Allen, J.A., Amphlett, G., Chanter, D. O. and Phillips, B.

(1989),
Analysis of Data from In Vitro Cytogenetic Assays, in: Statistical Evaluation of Mutagenicity Test Data,
Kirkland, D. J., (cd) Cambridge University Press, Cambridge, pp. 141(cid:151)154.

(14) Soper, K. A. and Galloway, S. M. (1994), Replicate Flasks are not Necessary for In Vitro Chromosome

Aberration Assays in CHO Cells, Mutation Res., 312, pp. 139(cid:151)149.

(15) Krahn, D. F., Barsky, F. C. and McCooey, K. T. (1982), CHO/HGPRT Mutation Assay: Evaluation of
Gases and Volatile Liquids, in: Tice, R. R., Costa, D. L., Schaich, K. M. (eds), Genotoxic Effects of Airborne
Agents, New York, Plenum, pp. 91(cid:151)103.

(16) Zamora, P. O., Benson, J. M., Li, A. P. and Brooks, A. L. (1983), Evaluation of an Exposure System
Using Cells Grown on Collagen Gels for Detecting Highly Volatile Mutagens in the CHO/HGPRT
Mutation Assay, Environmental Mutagenesis, 5, pp. 795(cid:151)801.

(17) Locke-Huhle, C. (1983), Endoreduplication in Chinese hamster cells during alpha-radiation induced G2

arrest, Mutation Res., 119, pp. 403(cid:151)413.

(18) Huang, Y., Change, C. and Trosko, J. E.

(1983), Aphidicolin-induced endoreduplication in Chinese

hamster cells, Cancer Res., 43, pp. 1362(cid:151)1364.(cid:146)

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(cid:145)B.11. MUTAGENICITY (cid:151) IN VIVO MAMMALIAN BONE MARROW CHROMOSOME ABERRATION TEST

ANNEX 4B

1.

METHOD

This method is a replicate of the OECD TG 475, Mammalian Bone Marrow Chromosome Aberration Test
(1997).

1.1.

INTRODUCTION

The mammalian in vivo chromosome aberration test is used for the detection of structural chromosome
aberrations induced by the test substance to the bone marrow cells of animals, usually rodents (1) (2) (3) (4).
Structural chromosome aberrations may be of
two types, chromosome or chromatid. An increase in
polyploidy may indicate that a chemical has the potential to induce numerical aberrations. With the majority
of chemical mutagens, induced aberrations are of the chromatid-type, but chromosome-type aberrations also
occur. Chromosome mutations and related events are the cause of human genetic diseases and there is
substantial evidence that chromosome mutations and related events causing alterations in oncogenes and
tumour suppresser genes are involved in cancer in humans and experimental systems.

Rodents arc routinely used in this test. Bone marrow is the target tissue in this test, since it is a highly
vascularised tissue, and it contains a population of rapidly cycling cells that can be readily isolated and
processed. Other species and target tissues are not the subject of this method.

This chromosome aberration test is especially relevant to assessing mutagenic hazard in that it allows
consideration of factors of in vivo metabolism, pharmacokinetics and DNA-repair processes although these
may vary among species and among tissues. An in vivo test is also useful for further investigation of a
mutagenic effect detected by an in vitro test.

If there is evidence that the test substance, or a reactive metabolite, will not reach the target tissue, it is not
appropriate to use this test.

See also General introduction Part B.

1.2.

DEFINITIONS

Chromatid-type aberration: structural chromosome damage expressed as breakage of single chromatids or
breakage and reunion between chromatids.

Chromosome-type aberration: structural chromosome damage expressed as breakage, or breakage and
reunion, of both chromatids at an identical site.

Endoreduplication: a process in which after an S period of DNA replication, the nucleus does not go into
mitosis but starts another S period. The result is chromosomes with 4, 8, 16, (cid:133) chromatids.

Gap: an achromatic lesion smaller than the width of one chromatid, and with minimum misalignment of the
chromatid(s).

Numerical aberration: a change in the number of chromosomes from the normal number characteristic of
the cells utilised.

Polyploidy: a multiple of the haploid chromosome number (n) other than the diploid number (i.e. 3n, 4n
and so on).

Structural aberration: a change in chromosome structure detectable by microscopic examination of the
metaphase stage of cell division, observed as deletions and fragments, intrachanges or interchanges.

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1.3.

PRINCIPLE OF THE TEST METHOD

Animals are exposed to the test substance by an appropriate route of exposure and are sacrificed at
appropriate times after treatment. Prior to sacrifice, animals are treated with a metaphase arresting agent
(e.g. colchicine or Colcemidﬁ). Chromosome preparations are then made from the bone marrow cells and
stained, and metaphase cells are analysed for chromosome aberrations.

1.4.

DESCRIPTION OF THE TEST METHOD

1.4.1.

Preparations

1.4.1.1.

Selection of animals species

Rats, mice and Chinese hamsters are commonly used, although any appropriate mammalian species may be
used. Commonly used laboratory strains of young healthy adult animals should be employed. At
the
commencement of the study, the weight variation of animals should be minimal and not exceed – 20 % of the
mean weight of each sex.

1.4.1.2. Housing and feeding conditions

General conditions referred in the General Introduction to Part B are applied although the aim for humidity
should be 50(cid:150)60 %.

1.4.1.3.

Preparation of the animals

Healthy young adult animals are randomly assigned to the control and treatment groups. Cages should be
arranged in such a way that possible effects due to cage placement are minimised. The animals are identified
uniquely. The animals are acclimated to the laboratory conditions for at least five days.

1.4.1.4.

Preparation of doses

Solid test substances should be dissolved or suspended in appropriate solvents or vehicles and diluted, if
appropriate, prior to dosing of the animals. Liquid test substances may be dosed directly or diluted prior to
dosing. Fresh preparations of the test substance should be employed unless stability data demonstrate the
acceptability of storage.

1.4.2.

Test conditions

1.4.2.1.

Solvent/vehicle

The solvent/vehicle should not produce toxic effects at the dose levels used, and should not be suspected of
chemical reaction with the test substance. If other than well-known solvents/vehicles are used, their inclusion
should be supported with data indicating their compatibility. It is recommended that wherever possible, the
use of an aqueous solvent/vehicle should be considered first.

1.4.2.2. Controls

Concurrent positive and negative (solvent/vehicle) controls should be included for each sex in each test.
Except for treatment with the test substance, animals in the control groups should be handled in an identical
manner to the animals in the treated groups.

Positive controls should produce structural aberrations in vivo at exposure levels expected to give a detectable
increase over background. Positive control doses should be chosen so that the effects are clear but do not
immediately reveal the identity of the coded slides to the reader. It is acceptable that the positive control be

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administered by a route different from the test substance and sampled at only a single time. The use of
chemical class related positive control chemicals may be considered, when available. Examples of positive
control substances include:

Substance

ethyl methansulphonate

ethyl nitrosourea

mitomycin C

cyclophosphamide

cyclophosphamide monohydrate

triethylenemelamine

CAS No

62-50-0

759-73-9

50-07-7

50-18-0

6055-19-2

51(cid:150)18(cid:150)3

Einecs No

200-536-7

212-072-2

200-008-6

200-015-4

200-083-5

Negative controls, treated with solvent or vehicle alone, and otherwise treated in the same way as the
treatment groups, should be included for every sampling time, unless acceptable inter-animal variability and
frequencies of cells with chromosome aberrations are available from historical control data. If single sampling
is applied for negative controls, the most appropriate time is the first sampling time. In addition, untreated
controls should also be used unless there are historical or published control data demonstrating that no
deleterious our mutagenic effects are induced by the chosen solvent/vehicle.

1.5.

PROCEDURE

1.5.1.

Number and sex of animals

Each treated and control group include at least five analysable animals per sex. If at the time of the study
there are data available from studies in the same species and using the same route of exposure that
demonstrate that there are no substantial differences in toxicity between sexes, then testing in a single sex
will be sufficient. Where human exposure to chemicals may be sex-specific, as for example with some
pharmaceutical agents, the test should be performed with animals of the appropriate sex.

1.5.2.

Treatment schedule

Test substances are preferably administered as a single treatment. Test substances may also be administered as
a split dose,
i.e. two treatments on the same day separated by no more than a few hours, to facilitate
administering a large volume of material. Other dose regimens should be scientifically justified.

Samples should be taken at two separate times following treatment on one day. For rodents, the first
sampling interval is 1,5 normal cell cycle length (the latter being normally 12(cid:150)18 hours) following treatment.
Since the time required for uptake and metabolism of the test substance as well as its effect on cell
cycle kinetics can affect the optimum time for chromosome aberration detection, a later sample collection
24 hours after the first sample time is recommended. If dose regimens of more than one day are used, one
sampling time at 1,5 normal cell cycle lengths after the final treatment should be used.

Prior to sacrifice, animals are injected intraperitoneally with an appropriate dose of a metaphase arresting
agent (e.g. Colcemidﬁ or colchicine). Animals are sampled at an appropriate interval thereafter. For mice this
interval is approximately 3(cid:150)5 hours; for Chinese hamsters this interval as approximately 4(cid:150)5 hours. Cells are
harvested from the bone marrow and analysed for chromosome aberrations.

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1.5.3.

Dose levels

If a range finding study is performed because there are no suitable data available, it should be performed in
the same laboratory, using the same species, strain, sex, and treatment regimen to be used in the main study
(5). If there is toxicity, three dose levels are used for the first sampling time. These dose levels should cover a
range from the maximum to little or no toxicity. At the later sampling time only the highest dose needs to
be used. The highest dose is defined as the dose-producing signs of toxicity such that higher dose levels,
based on the same dosing regimen, would be expected to produce lethality. Substances with specific
biological activities at low non-toxic doses (such as hormones and mitogens) may be exceptions to the
dose-setting criteria and should be evaluated on a case-by-case basis. The highest dose may also be defined as
a dose that produces some indication of toxicity in the bone marrow (e.g. greater than 50 % reduction in
mitotic index).

1.5.4.

Limit test

If a test at one dose level of at least 2 000 mg/kg body weight using a single treatment, or as two treatments
on the same day, produces no observable toxic effects, and if genotoxicity would not be expected based on
data from structurally related substances, then a fully study using three dose levels may not be considered
necessary. For studies of a longer duration, the limit dose is 2 000 mg/kg/body weight/day for treatment up
to 14 days, and 1 000 mg/kig/body weight/day for treatment longer than 14 days. Expected human exposure
may indicate the need for a higher dose level to be used in the limit test.

1.5.5.

Administration of doses

The test substance is usually administered by gavage using a stomach tube or a suitable intubation cannula,
or by intraperitoneal injection. Other routes of exposure may be acceptable where they can be justified. The
maximum volume of liquid that can be administered by gavage or injection at one time depends on the size
of the test animal. The volume should not exceed 2 ml/100g body weight. The use of volumes higher than
these must be justified. Except for irritating or corrosive substances which will normally reveal exacerbated
effects with higher concentrations, variability in test volume should be minimised by adjusting the
concentration to ensure a constant volume at all dose levels.

1.5.6.

Chromosome preparation

Immediately after sacrifice, bone marrow is obtained, exposed to hypotonic solution and fixed. The cells are
then spread on slides and stained.

1.5.7.

Analysis

The mitotic index should be determined as a measure of cytotoxicity in at least 1 000 cells per animal for all
treated animals (including positive controls) and untreated negative control animals.

At least 100 cells should be analysed for each animal. This number could be reduced when high numbers of
aberrations are observed. All slides, including those of positive and negative controls, should be independently
coded before microscopic analysis. Since slide preparation procedures often result in the breakage of a
proportion of metaphases with loss of chromosomes, the cells scored should therefore contain a number of
centromeres equal to the number 2n – 2.

2.

DATA

2.1.

TREATMENT OF RESULTS

Individual animal data should be presented in tabular form. The experimental unit is the animal. For each
animal the number of cells scored, the number of aberrations per cell and the percentage of cells with
structural chromosome aberration(s)
structural chromosome
aberrations should be listed with their numbers and frequencies for treated and control groups. Gaps are
recorded separately and reported but generally not included in the total aberration frequency. If there is no
evidence for a difference in response between the sexes, the data from both sexes may be combined for
statistical analysis.

should be evaluated. Different

types of

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2.2.

EVALUATION AND INTERPRETATION OF RESULTS

There are several criteria for determining a positive result, such as a dose-related increase in the relative
number of cells with chromosome aberrations or a clear increase in the number of cells with aberrations in a
single dose group at a single sampling time. Biological relevance of the results should be considered first.
Statistical methods may be used as an aid in evaluating the test results (6). Statistical significance should not
be the only determining factor for a positive response. Equivocal results should be clarified by further testing
preferably using a modification of experimental conditions.

An increase in polyploidy may indicate that
to induce numerical
chromosome aberrations. An increase in endoreduplication may indicate that the test substance has the
potential to inhibit cell cycle progression (7) (8).

the test substance has the potential

A test substance for which the results do not meet the above criteria is considered non-mutagenic in this test.

Although most experiments will give clearly positive or negative results,
in rare cases the data set will
preclude making a definite judgement about the activity of the test substance. Results may remain equivocal
or questionable regardless of the number of experiments performed.

Positive results from the in vivo chromosome aberration test indicate that a substance induces chromosome
aberrations in the bone marrow of the species tested. Negative results indicate that, under the test conditions,
the test substance does not induce chromosome aberrations in the bone marrow of the species tested.

The likelihood that the test substance or its metabolites reach the general circulation or specifically the target
tissue (e.g. systemic toxicity) should be discussed.

3.

REPORTING

TEST REPORT

Test test report must include the following information:

Solvent vehicle:

(cid:151) justification for choice of vehicle,

(cid:151) solubility and stability of the test substance in solvent/vehicle, if known.

Test animals:

(cid:151) species/strain used,

(cid:151) number, age and sex of animals,

(cid:151) source, housing conditions, diet, etc.,

(cid:151) individual weight of the animals at the start of the test, including body weight range, means and standard

deviation for each group.

Test conditions:

(cid:151) positive and negative (vehicle/solvent) controls,

(cid:151) data from range-finding study, if conducted,

(cid:151) rationale for close level selection,

(cid:151) details of test substance preparation,

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(cid:151) details of the administration of the test substance,

(cid:151) rationale for route of administration,

(cid:151) methods for verifying that the test substance reached the general circulation or target tissue, if applicable,

(cid:151) conversion from diet/drinking water test substance concentration (ppm) to the actual dose (mg/kg body

weight/day), if applicable,

(cid:151) details of food and water quality,

(cid:151) detailed description of treatment and sampling schedules,

(cid:151) methods for measurements of toxicity,

(cid:151) identity of metaphase arresting substance, its concentration and duration of treatment,

(cid:151) methods of slide preparation,

(cid:151) criteria for scoring aberrations,

(cid:151) number of cells analysed per animal,

(cid:151) criteria for considering studies as positive, negative or equivocal.

Results:

(cid:151) signs of toxicity,

(cid:151) mitotic index,

(cid:151) type and number of aberrations, given separately for each animal,

(cid:151) total number of aberrations per group with means and standard deviations,

(cid:151) number of cells with aberrations per group with means and standard deviations,

(cid:151) changes in ploidy, if seen,

(cid:151) dose-response relationship, where possible,

(cid:151) statistical analyses, if any,

(cid:151) concurrent negative control data,

(cid:151) historical negative control data with ranges, means and standard deviations,

(cid:151) concurrent positive control data.

Discussion of the results.

Conclusions.

4.

REFERENCES

(1)

(2)

Adler, I. D. (1984), Cytogenetic Tests in Mammals, in: Mutagenicity Testing: a Practical Approach, S. Venitt
and J. M. Parry (eds), IRL Press, Oxford, Washington D.C., pp. 275(cid:150)306.

Preston, R. J., Dean, B. J., Galloway, S., Holden, H., McFee, A. F. and Shelby, M. (1987), Mammalian In
Vivo Cytogenetic Assays: Analysis of Chromosome Aberrations in Bone Marrow Cells, Mutation Res.,
189, pp. 157(cid:150)165.

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(3)

(4)

(5)

(6)

(7)

(8)

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Richold, M., Chandley, A., Ashby, J., Gatehouse, D. G., Bootman, J. and Henderson, L. (1990), In vivo
Cytogenetic Assays, in: D. J. Kirkland (ed.), Basic Mutagenicity Tests, UKEMS Recommended Procedures.
UKEMS Sub-Committee on Guidelines for Mutagenicity Testing. Report, Part I revised, Cambridge University
Press, Cambridge, New York, Port Chester, Melbourne, Sydney, pp. 115(cid:150)141.

Tice, R. R., Hayashi, M., MacGregaro, J. T., Anderson, D., Blakey, D. H., Holden, H. E., Kirsch-Volders,
M., Oleson Jr., F. B., Pacchierotti, F., Preston, R. J., Romagna, F., Shimada, H., Sutou, S. and Vannier, B.
(1994), Report from the Working Group on the in Vivo Mammalian Bone Marrow Chromosomal
Aberration Test, Mutation Res., 312, pp. 305(cid:150)312.

Fielder, R. J., Allen, J. A., Boobis, A. R., Botham, P. A., Doe, J., Esdaile, D. J., Gatehouse, D. G.,
Hodson-Walker, G., Morton, D. B., Kirkland, D. J. and Richold, M. (1992), Report of British Toxicology
Society/UK Environmental Mutagen Society Working Group: Dose setting in In Vivo Mutagenicity
Assays, Mutagenesis, 7, pp. 313(cid:150)319.

Lovell, D. P., Anderson, D., Albanese, R., Amphlett, G. E., Clare, G., Ferguson, R., Richold, M.,
Papworth, D. G. and Savage, J. R. K. (1989), Statistical Analysis of In Vivo Cytogenetic Assays, in:
UKEMS Sub-Committee on Guidelines for Mutagenicity Testing, Report Part
III. Statistical Evaluation of
Mutagenicity Test Data, D. J. Kirkland (ed.) Cambridge University Press, Cambridge, pp. 184(cid:150)232.

Locke-Huhle, C. (1983), Endoreduplication in Chinese hamster cells during alpha-radiation-induced G2
arrest, Mutation Res., 119, pp. 403(cid:150)413.

Huang, Y., Change, C. and Trosko, J. E.
hamster cells, Cancer Res., 43, pp. 1362(cid:150)1364.(cid:146)

(1983), Aphidicolin-induced endoreduplication in Chinese

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ANNEX 4C

(cid:145)B.12. MUTAGENICITY (cid:151) IN VIVO MAMMALIAN ERYTHROCYTE MICRONUCLEUS TEST

1.

METHOD

This method is a replicate of the OECD TG 474, Mammalian Erythrocyte Micronucleus Test (1997).

1.1.

INTRODUCTION

The mammalian in vivo micronucleus test is used for the detection of damage induced by the test substance
to the chromosomes or the mitotic apparatus of erythroblasts by analysis of erythrocytes as sampled in bone
marrow and/or peripheral blood cells of animals, usually rodents.

The purpose of the micronucleus test is to identify substances that cause cytogenetic damage which results in
the formation of micronuclei containing lagging chromosome fragments or whole chromosomes.

When a bone marrow erythroblast develops into a polychromatic erythrocyte, the main nucleus is extruded;
any micronucleus that has been formed may remain behind in the otherwise anucleated cytoplasm.
Visualisation of micronuclei is facilitated in these cells because they lack a main nucleus. An increase in the
frequency of micronucleated polychromatic erythrocytes in treated animals is an indication of
induced
chromosome damage.

The bone marrow of rodents is routinely used in this test since polychromatic erythrocytes are produced in
that tissue. The measurement of micronucleated immature (polychromatic) erythrocytes in peripheral blood is
equally acceptable in any species in which the inability of the spleen to remove micronucleated erythrocytes
has been demonstrated, or which has shown an adequate sensitivity to detect agents that cause structural or
numerical chromosome aberrations. Micronuclei can be distinguished by a number of criteria. These include
identification of the presence or absence of a kinetochore or centromeric DNA in the micronuclei. The
frequency of micronucleated immature (polychromatic) erythrocytes is the principal end point. The number
of mature (normochromatic) erythrocytes in the peripheral blood that contain micronuclei among a given
number of mature erythrocytes can also be used as the endpoint of the assay when animals are treated for
four weeks or more.

This mammalian in vivo micronucleus test especially relevant to assessing mutagenic hazard in that it allows
consideration of factors of in vivo metabolism, pharmacokinetics and DNA repair processes although these
may vary among species, among tissues and among genetic endpoints. An in vivo assay is also useful for
further investigation of a mutagenic effect detected by an in vitro system.

If there is evidence that the test substance, or a reactive metabolite, will not reach the target tissue, it is not
appropriate to use this test.

See also General Introduction Part B.

1.2.

DEFINITIONS

Centromere (Kinetochore): region(s) of a chromosome with which spindle fibers are associated during cell
division, allowing orderly movement of daughter chromosomes to the poles of the daughter cells.

Micronuclei: small nuclei, separate from and additional
telophase of mitosis (meiosis) by lagging chromosome fragments or whole chromosomes.

to the main nuclei of cells, produced during

Normochromatic erythrocyte: mature erythrocyte that lacks ribosomes and can be distinguished from
immature, polychromatic erythrocytes by stains selective for ribosomes.

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Polychromatic erythrocyte:
that still
contains ribosomes and therefore can be distinguished from mature, normochromatic erythrocytes by stains
selective for ribosomes.

in an intermediate stage of development,

immature erythrocyte,

1.3.

PRINCIPLE OF THE TEST METHOD

Animals are exposed to the test substance by an appropriate route. If bone marrow is used, the animals are
sacrificed at appropriate times after treatment, the bone marrow extracted, and preparations made and
stained (1) (2) (3) (4) (5) (6) (7). When peripheral blood is used, the blood is collected at appropriate times
after treatment and smear preparations are made and stained (4) (8) (9) (10). For studies with peripheral
blood, as little time as possible should elapse between the last exposure and cell harvest. Preparations are
analysed for the presence of micronuclei.

1.4.

DESCRIPTION OF THE TEST METHOD

1.4.1.

Preparations

1.4.1.1.

Selection of animal species

Mice or rats are recommended if bone marrow is used, although any appropriate mammalian species may be
used. When peripheral blood is used, mice are recommended. However, any appropriate mammalian species
may be used provided it is a species in which the spleen does not remove micronucleated erythrocytes or a
species which has shown an adequate sensitivity to detect agents that cause structural or numerical
chromosome aberrations. Commonly used laboratory strains of young healthy animals should be employed.
At the commencement of the study, the weight variation of animals should be minimal and not exceed
– 20 % of the mean weight of each sex.

1.4.1.2. Housing and feeding conditions

General conditions referred in the General Introduction to Part B are applied although the aim for humidity
should be 50(cid:150)60 %.

1.4.1.3.

Preparation of the animals

Healthy young adult animals are randomly assigned to the control and treatment groups. The animals are
identified uniquely. The animals are acclimated to the laboratory conditions for at least five days. Cages
should be arranged in such a way that possible effects due to cage placement are minimised.

1.4.1.4.

Preparation of doses

Solid test substances should be dissolved or suspended in appropriate solvents or vehicles and diluted, if
appropriate, prior to dosing of the animals. Liquid test substances may be dosed directly or diluted prior to
dosing. Fresh preparations of the test substance should be employed unless stability data demonstrate the
acceptability of storage.

1.4.2.

Test conditions

1.4.2.1.

Solvent/vehicle

The solvent/vehicle should not produce toxic effects at the dose levels used, and should not be suspected of
chemical reaction with the test substance. If other than well-known solvents/vehicles are used, their inclusion
should be supported with reference data indicating their compatibility. It is recommended that wherever
possible, the use of aqueous solvent/vehicle should be considered first.

1.4.2.2. Controls

Concurrent positive and negative (solvent/vehicle) controls should be included for each sex in each test.
Except for treatment with the test substance, animals in the control groups should be handled in an identical
manner to animals of the treatment groups.

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Positive controls should produce micronuclei in vivo at exposure levels expected to give a detectable increase
over background. Positive control doses should be chosen so that the effects are clear but do not immediately
reveal the identity of the coded slides to the reader. It is acceptable that the positive control be administered
by a route different from the test substance and sampled at only a single time. In addition, the use of
chemical class-related positive control chemicals may be considered, when available. Examples of positive
control substances include:

Substance

ethyl methansulphonate

N-ethyl-N-nitrosourea

mitomycin C

cyclophosphamide

cyclophosphamide monohydrate

triethylenemelamine

CAS No

62-50-0

759-73-9

50-07-7

50-18-0

6055-19-2

51(cid:150)18(cid:150)3

Einecs No

200-536-7

212-072-2

200-008-6

200-015-4

200-083-5

Negative controls, treated with solvent or vehicle alone, and otherwise treated in the same way as the
treatment groups should be included for every sampling time, unless acceptable inter-animal variability and
frequencies of cells with micronuclei are demonstrated by historical control data. If single sampling is applied
for negative controls, the most appropriate time is the first sampling time. In addition, untreated controls
should also be used unless there are historical or published control data demonstrating that no deleterious or
mutagenic effects are induced by the chosen solvent/vehicle.

If peripheral blood is used, a pre-treatment sample may also be acceptable as a concurrent negative control,
but only in the short peripheral blood studies (e.g. 1(cid:150)3 treatment(s)) when the resulting data are in the
expected range for the historical control.

1.5.

PROCEDURE

1.5.1.

Number and sex of animals

Each treated and control group must include at least five analysable animals per sex (11). If at the time of the
study there are data available from studies in the same species and using the same route of exposure that
demonstrate that there are no substantial differences between sexes in toxicity, then testing in a single sex
will be sufficient. Where human exposure to chemicals may be sex-specific, as for example with some
pharmaceutical agents, the test should be performed with animals of the appropriate sex.

1.5.2.

Treatment schedule

No standard treatment schedule (i.e. one, two or more treatments at 24-hour intervals) can be recommended.
The samples from extended dose regimens are acceptable as long as a positive effect has been demonstrated
for this study or, for a negative study, as long as toxicity has been demonstrated or the limit dose has been
used, and dosing continued until the time of sampling. Test substances may also be administered as a split
dose, i.e. two treatments on the same day separated by no more than a few hours, to facilitate administering
a large volume of material.

The test may be performed in two ways:

(a) animals are treated with the test substance once. Samples of bone marrow are taken at least twice,
starting not earlier than 24 hours after treatment, but not extending beyond 48 hours after treatment
with appropriate intervals between samples. The use of sampling times earlier than 24 hours after
treatment should be justified. Samples of peripheral blood are taken at least twice, starting not earlier

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than 36 hours after treatment, with appropriate intervals following the first sample, but not extending
beyond 72 hours. When a positive response is recognised at one sampling time, additional sampling is
not required;

(b)

If two or more daily treatments are used (e.g. two or more treatments at 24-hour intervals), samples
should be collected once between 18 and 24 hours following the final treatment for the bone marrow
and once between 36 and 48 hours following the final treatment for the peripheral blood (12).

Other sampling times may be used in addition, when relevant.

1.5.3.

Dose levels

If a range finding study is performed because there are not suitable data available, it should be performed in
the same laboratory, using the same species, strain, sex, and treatment regimen to be used in the main study
(13). If there is toxicity, three dose levels are used for the first sampling time. These dose levels should cover
a range from the maximum to little or no toxicity. At the later sampling time only the highest dose needs to
be used. The highest dose is defined as the dose producing signs of toxicity such that higher dose levels,
based on the same dosing regimen, would be expected to produce lethality. Substances with specific
biological activities at low non-toxic doses (such as hormones and mitogens) may be exceptions to the
dose-setting criteria and should be evaluated on a case-by-case basis. The highest dose may also be defined as
a dose that produces some indication of toxicity in the bone marrow (e.g. a reduction in the proportion of
immature erythrocytes among total erythrocytes in the bone marrow or peripheral blood).

1.5.4.

Limit test

If a test at one dose level of at least 2 000 mg/kg body weight using a single treatment, or as two treatments
on the same day, produces no observable toxic effects, and if genotoxicity would not be expected based upon
data from structurally related substances, then a fully study using three dose levels may not be considered
necessary. For studies of a longer duration, the limit dose is 2 000 mg/kg/body weight/day for treatment up
to 14 days, and 1 000 mg/kg/body weight/day for treatment longer than 14 days. Expected human exposure
may indicate the need for a higher dose level to be used in the limit test.

1.5.5.

Administration of doses

The test substance is usually administered by gavage using a stomach tube or a suitable intubation cannula,
or by intraperitoneal injection. Other routes of exposure may be acceptable where they can be justified. The
maximum volume of liquid that can be administered by gavage or injection at one time depends on the size
of the test animal. The volume should not exceed 2 ml/100g body weight. The use of volumes higher can
these must be justified. Except for irritating or corrosive substances which will normally reveal exacerbated
effects with higher concentrations, variability in test volume should be minimised by adjusting the
concentration to ensure a constant volume at all dose levels.

1.5.6.

Bone marrow/blood preparation

Bone marrow cells are usually obtained from the femurs or tibias immediately following sacrifice. Commonly,
cells are removed from femurs or tibias, prepared and stained using established methods. Peripheral blood is
obtained from the tail vein or other appropriate blood vessel. Blood cells are immediately stained supravitally
(8) (9) (10) or smear preparations are made and then stained. The used of a DNA specific stain (e.g. acridine
orange (14) or Hoechst 33258 plus pyronin-Y (15)) can eliminate some of the artifacts associated with using
a non-DNA-specific stain. This advantage does not preclude the use of conventional stains (e.g., Giemsa).
Additional systems (e.g. cellulose columns to remove nucleated cells (16)) can also be used provided that
these systems have been shown to adequately work for micronucleus preparation in the laboratory.

1.5.7.

Analysis

The proportion of immature among total (immature + mature) erythrocytes is determined for each animal by
counting a total at least 200 erythrocytes for bone marrow and 1 000 erythrocytes for peripheral blood (17).
All slides,
including those of positive and negative controls, should be independently coded before
microscopic analysis. At least 2 000 immature erythrocytes per animal are scored for the incidence of

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micronucleated immature erythrocytes. Additional
information may be obtained by scoring mature
erythrocytes for micronuclei. When analysing slides, the proportion of immature erythrocytes among total
erythrocytes should not be less than 20 % of the control value. When animals are treated continuously for
four weeks or more, at least 2 000 mature erythrocytes per animal can also be scored for the incidence of
micronuclei. Systems for automated analysis (image analysis and flow cytometric analysis of cell suspensions)
are acceptable alternatives to manual evaluation if appropriately justified and validated.

2.

DATA

2.1.

TREATMENT OF RESULTS

Individual animal data should be presented in tabular form. The experimental unit is the animal. The number
of immature erythrocytes scored, the number of micronucleated immature erythrocytes, and the number of
immature among total erythrocytes should be listed separately for each animal analysed. When animals are
treated continuously for four weeks or more, the data on mature erythrocytes should also be given if it is
collected. The proportion of immature among total erythrocytes and, if considered applicable, the percentage
of micronucleated erythrocytes is given for each animal. If there is no evidence for a difference in response
between the sexes, the data from both sexes may be combined for statistical analysis.

2.2.

EVALUATION AND INTERPRETATION OF RESULTS

There are several criteria for determining a positive result, such as a dose-related increase in the number of
micronucleated cells or a clear increase in the number of micronucleated cells in a single dose group at a
single sampling time. Biological relevance of the results should be considered first. Statistical methods may be
used as an aid in evaluating the test results (18) (19). Statistical significance should not be only determining
factor for a positive response. Equivocal results should be clarified by further testing preferably using a
modification of experimental conditions.

A test substance for which the results do not meet the above criteria is considered non-mutagenic in this test.

in rare cases the data set will
Although most experiments will give clearly positive or negative results,
preclude making a definite judgement about the activity of the test substance. Results may remain equivocal
or questionable regardless of the number of times the experiment is repeated.

Positive results in the micronucleus test indicate that the substance induces micronuclei which are the result
of chromosomal damage or damage to the mitotic apparatus in the erythroblasts of the test species. Negative
results indicate that, under the test conditions, the test substance does not produce micronuclei
in the
immature erythrocytes of the test species.

The likelihood that the test substance or its metabolites reach the general circulation or specifically the target
tissue (e.g. systemic toxicity) should be discussed.

3.

REPORTING

TEST REPORT

The test report should include the following information:

Solvent/vehicle:

(cid:151) justification for choice of vehicle,

(cid:151) solubility and stability of the test substance in solvent/vehicle, if known.

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Test animals:

(cid:151) species/strain used,

(cid:151) number, age and sex of animals,

(cid:151) source, housing conditions, diet, etc.,

(cid:151) individual weight of the animals at the start of the test, including body weight range, mean and standard

deviation for each group.

Test conditions:

(cid:151) positive and negative (vehicle/solvent) control data,

(cid:151) data from range-finding study, if conducted,

(cid:151) rationale for dose level selection,

(cid:151) details of test substance preparation,

(cid:151) details of the administration of the test substance,

(cid:151) rationale for route of administration,

(cid:151) methods for verifying that the test substance reached the general circulation or target tissue, if applicable,

(cid:151) conversion from diet/drinking water test substance concentration (ppm) to the actual dose (mg/kg body

weight/day), if applicable,

(cid:151) details of food and water quality,

(cid:151) detailed description of treatment and sampling schedules,

(cid:151) methods of slide preparation,

(cid:151) methods for measurements of toxicity,

(cid:151) criteria for scoring micronucleated immature erythrocytes,

(cid:151) number of cells analysed per animal,

(cid:151) criteria for considering studies as positive, negative or equivocal.

Results:

(cid:151) signs of toxicity,

(cid:151) proportion of immature erythrocytes among total erythrocytes,

(cid:151) number of micronucleated immature erythrocytes, given separately for each animal,

(cid:151) mean – standard deviation of micronucleated immature erythrocytes per group,

(cid:151) dose-response relationship, where possible,

(cid:151) statistical analyses and methods applied,

(cid:151) concurrent and historical negative data,

(cid:151) concurrent positive control data.

Discussion of the results.

Conclusions.

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4.

REFERENCES

(1) Heddle, J. A. (1973), A Rapid In Vivo Test for Chromosomal Damage, Mutation Res., 18, pp. 187(cid:150)190.

(2) Schmid, W. (1975), The Micronucleus Test, Mutation Res., 31, pp. 9(cid:150)15.

(3) Heddle, J. A., Salamone, M. F., Hite, M., Kirkhart, B., Mavournin, K., MacGregor, J. G. and Newell, G. W.
(1983), The Induction of Micronuclei as a Measure of Genotoxicity, Mutation Res. 123, pp. 61(cid:150)118.

(4) Mavournin, K. H., Blakey, D. H., Cimino, M. C., Salamone, M. F. and Heddle, J. A. (1990), The In Vivo
the U.S.

Micronucleus Assay in Mammalian Bone Marrow and Peripheral Blood. A report of
Environmental Protection Agency Gene-Tox Program, Mutation Res., 239, pp. 29(cid:150)80.

(5) MacGregor,

J. T., Schlegel, R., Choy, W. N. and Wehr, C. M.

in Circulating
Erythrocytes: A Rapid Screen for Chromosomal Damage During Routine Toxicity Testing in Mice, in:
Developments in Science and Practice of Toxicology, ed. A. W. Hayes, R. C. Schnell and T. S. Miya. Elsevier,
Amsterdam, pp. 555(cid:150)558.

(1983), Micronuclei

(6) MacGregor, J. T., Heddle, J. A. Hite, M., Margolin, G. H., Ramel, C., Salamone, M. F., Tice, R. R., and
Wild, D. (1987), Guidelines for the Conduct of Micronucleus Assays in Mammalian Bone Marrow
Erythrocytes, Mutation Res., 189, pp. 103(cid:150)112.

(7) MacGregor,

J. T., Wehr, C. M., Henika, P. R. and Shelby, M. E.

(1990), The in vivo Erythrocyte
Micronucleus Test: Measurement at Steady State Increases Assay Efficiency and Permits Integration with
Toxicity Studies, Fund. Appl. Toxicol. 14, pp. 513(cid:150)522.

(8) Hayashi, M., Morita, T., Kodama, Y., Sofumi, T. and Ishidate, M. Jr. (1990), The Micronucleus Assay
with Mouse Peripheral Blood Reticulocytes Using Acridine Orange-Coated Slides, Mutation Res., 245,
pp. 245(cid:150)249.

(9) The Collaborative Study Group for the Micronucleus Test (1992). Micronucleus Test with Mouse
Peripheral Blood Erythrocytes by Acridime Orange Supravital Staining: The Summary Report of the 5th
Collaborative Study by CSGMT/JEMMS, MMS, Mutation Res., 278, pp. 83(cid:150)98.

(10) The Collaborative Study Group for the Micronucleus Test (CSGMT/JEMMS, MMS: The Mammalian
(1995), Protocol
test, Mutagenesis, 10,

the short-term mouse peripheral blood micronucleus

the Environmental Mutagen Society of

Japan)

Mutagenesis Study Group of
recommended for
pp. 53(cid:150)159.

(11) Hayashi, M., Tice, R. R., MacGregor, J. T., Anderson, D., Blackey, D. H., Kirsch-Volders, M., Oleson, Jr.
in Vivo Rodent

F. B., Pacchicrotti, F., Romagna, F., Shimada, H. Sutou, S. and Vannier, B. (1994),
Erythrocyte Micronucleus Assay, Mutation Res., 312, pp. 293(cid:150)304.

(12) Higashikuni, N. and Sutou, S. (1995), An optimal, generalised sampling time of 30 – 6 h after double

dosing in the mouse peripheral micronucleus test, Mutagenesis, 10, pp. 313(cid:150)319.

(13)

Fielder, R. J., Allen, J. A., Boobis, A. R., Botham, P. A., Doe, J., Esdaile, D. J., Gatehouse, D. G.,
Hodson-Walker, G., Morton, D. B., Kirkland, D. J. and Rochold, M. (1992), Report of British Toxicology
Society/UK Environmental Mutagen Society Working Group: Dose Setting in In Vivo Mutagenicity
Assays, Mutagenesis, 7, pp. 313(cid:150)319.

(14) Hayashi, M., Sofumi, T. and Ishidate, M. Jr. (1983), An Application of Acridine Orange Fluorescent

Staining to the Micronucleus Test, Mutation Res., 120, pp. 241(cid:150)247.

(15) MacGregor, J. T., Wehr, C. M. and Langlois, R. G. (1983), A Simple Fluorescent Staining Procedure for
Micronuclei and RNA in Erythrocytes Using Hoechst 33258 and Pyromin Y, Mutation Res., 120,
pp. 269(cid:150)275.

(16) Romagna, F. and Staniforth, C. D. (1989), The automated bone marrow micronucleus test, Mutation

Res., 213, pp. 91(cid:150)104.

(17) Gollapudi, B. and McFadden, L. G.

(1995), Sample size for the estimation of polychromatic to
normochromatic erythrocyte ratio in the bone marrow micronucleus test, Mutation Res., 347,
pp. 97(cid:150)99.

(18) Richold, M., Ashby, J., Bootman, J., Chandley, A., Gatehouse, D. G. and Henderson, L. (1990), In Vivo
in: D. J. Kirkland (ed.), Basic Mutagenicity tests, UKEMS Recommended Procedures,
Cytogenetics Assay,
UKEMS Sub-Committee on Guidelines for Mutagenicity Testing. Report, Part 1, revised, Cambridge University
Press, Cambridge, New York, Port Chester, Melbourne, Sydney, pp. 115(cid:150)141.

(19) Lovell, D. P., Anderson, D., Albanese, R., Amphlett, G. E., Clare, G., Ferguson R., Richold, M.,
Papworth, D. G. and Savage, J. R. K. (1989), Staticical Analysis of In Vivo Cytogenetic Assays, in: D. J.
Kirkland (ed.), Statistical Evaluation of Mutagenicity Test Data. UKEMS Sub-Committee on Guidelines for
Mutagenicity Testing. Report, Part III, Cambridge University Press, Cambridge, New York, Port Chester,
Melbourne, Sydney, pp. 184(cid:150)232.(cid:146)

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ANNEX 4D

(cid:145)B.13/14. MUTAGENICITY (cid:151) REVERSE MUTATION TEST BACTERIA

1.

METHOD

This method is a replicate of the OECD TG 471, Bacterial Reverse Mutation Test (1997).

1.1.

INTRODUCTION

The bacterial reverse mutation test uses amino-acid requiring strains of Salmonella typhimurium and Escherichia
coli to detect point mutations, which involve substitution, addition or deletion of one or a few DNA base
pairs (1) (2) (3). The principle of this bacterial reverse mutation test is that it detects mutations which revert
mutations present in the test strains and restore the functional capability of the bacteria to synthesise an
essential amino acid. The revertant bacteria are detected by their ability to grow in the absence of the
amino-acid required by the parent test strain.

Point mutations are the cause of many human genetic diseases and there is substantial evidence that point
mutations in oncogenes and tumour suppressor genes of somatic cells are involved in tumour formation in
humans and experimental animals. The bacterial reverse mutation test is rapid, inexpensive and relatively easy
to perform. Many of the test strains have several features that make them more sensitive for the detection of
mutations including responsive DNA sequences at the reversion sites, increased cell permeability to large
molecules and elimination of DNA repair systems or enhancement of error-prone DNA repair processes. The
specificity of the test strains can provide some useful information on the types of mutations that are induced
by genotoxic agents. A very large data base of results for a wide variety of structures is available for bacterial
reverse mutation tests and well-established methodologies have been developed for testing chemicals with
different physico-chemical properties, including volatile compounds.

See also General Introduction Part B.

1.2.

DEFINITIONS

A reverse mutation test in either Salmonella typhimurium or Escherichia coli detects mutation in an amino
acid requiring strain (histidine or tryptophan, respectively) to produce a strain independent of an outside
supply of amino acid.

Base pair substitution mutagens are agents that cause a base change in DNA. In a reversion test this
change may occur at the site of the original mutation, or at a second site in the bacterial genome.

Frameshift mutagens are agents that cause the addition or deletion of one or more base pairs in the DNA,
thus changing the reading frame in the RNA.

1.3.

INITIAL CONSIDERATIONS

The bacterial reverse mutation test utilises prokaryotic cells, which differ from mammalian cells in such
factors as uptake, metabolism, chromosome structure and DNA repair processes. Tests conducted in vitro
generally require the use of an exogenous source of metabolic activation. In vitro metabolic activation systems
cannot mimic entirely the mammalian in vivo conditions. The test
therefore does not provide direct
information on the mutagenic and carcinogenic potency of a substance in mammals.

The bacterial reverse mutation test is commonly employed as an initial screen for genoloxic activity and, in
particular,
for point mutation-inducing activity. An extensive data base has demonstrated that many
chemicals that are positive in this test also exhibit mutagenic activity in other tests. There are examples of
mutagenic agents which are not detected by this test; reasons for these shortcomings can be ascribed to the

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specific nature of the endpoint detected, differences in metabolic activation, or differences in bioavailability.
On the other hand, factors which enhance the sensitivity of the bacterial reverse mutation test can lead to an
overestimation of mutagenic activity.

The bacterial reverse mutation test may not be appropriate for the evaluation of certain classes of chemicals,
for example highly bactericidal compounds (e.g. certain antibiotics) and those which are thought (or known)
to interfere specifically with the mammalian cell replication system (e.g. some topoisomerase inhibitors and
some nucleoside analogues). In such cases, mammalian mutation tests may be more appropriate.

Although many compounds that are positive in this test are mammalian carcinogens, the correlation is not
absolute. It is dependent on chemical class and there are carcinogens that are not detected by this test
because they act through other, non-genotoxic, mechanisms or mechanisms absent in bacterial cells.

1.4.

PRINCIPLE OF THE TEST METHOD

Suspensions of bacterial cells are exposed to the test substance in the presence and in the absence of an
exogenous metabolic activation system. In the plate incorporation method, these suspensions are mixed with
an overlay agar and plated immediately onto minimal medium. In the preincubation method, the treatment
mixture is incubated and then mixed with an overlay agar before plating onto minimal medium. For both
techniques, after two or three days of incubation, revertant colonies are counted and compared to the
number of spontaneous revertant colonies on solvent control plates.

Several procedures for performing the bacterial reverse mutation test have been described. Among those
commonly used are the plate incorporation method (1) (2) (3) (4), the preincubation method (2) (3) (5) (6) (7)
(8), the fluctuation method (9) (10), and the suspension method (11). Modifications for the testing of gases or
vapours have been described (12).

The procedures described in the method pertain primarily to the plate incorporation and preincubation
methods. Either of
them is acceptable for conducting experiments both with and without metabolic
activation. Some substances may be detected more efficiently using the preincubation method. These
include short chain aliphatic nitrosamines, divalent metals,
substances belong to chemical classes that
aldehydes, azo-dyes and diazo compounds, pyrollizidine alkaloids, allyl compounds and nitro compounds (3).
It is also recognised that certain classes of mutagens are not always detected using standard procedures such
as the plate incorporation method or preincubation method. These should be regarded as (cid:147)special cases(cid:148) and
it is strongly recommended that alternative procedures should be used for their detection. The following
(cid:147)special cases(cid:148) could be identified (together with examples of procedures that could be used for their
detection): azo-dyes and diazo compounds (3) (5) (6) (13), gases and volatile chemicals (12) (14) (15) (16) and
glycosides (17) (18). A deviation from the standard procedure needs to be scientifically justified.

1.5.

DESCRIPTION OF THE TEST METHOD

1.5.1.

Preparations

1.5.1.1. Bacteria

Fresh cultures of bacteria should be grown up to the late exponential or early stationary phase of growth
(approximately 109 cells per ml). Cultures in late stationary phase should not be used. It is essential that the
cultures used in the experiment contain a high titre of viable bacteria. The titre may be demonstrated either
from historical control data on growth curves, or in each assay through the determination of viable cell
numbers by a plating experiment.

The recommended incubation temperature is 37 (cid:176)C.

At least five strains of bacteria should be used. These should include four strains of S. typhimurium (TA 1535;
TA 1537 or TA97a or TA97; TA98; and TA100) that have been shown to be reliable and reproducibly
responsive between laboratories. These four S.
the primary
reversion site and it is known that they may not detect certain oxidising mutagens, cross-linking agents and
hydrazines. Such substances may be detected by E. coli WP2 strains or S. typhimurium TA102 (19) which have
an AT base pair at the primary reversion site. Therefore the recommended combination of strains is:

typhimurium strains have GC base pairs at

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(cid:151) S. typhimurium TA1535, and

(cid:151) S. typhimurium TA1537 or TA97 or TA97a, and

(cid:151) S. typhimurium TA98, and

(cid:151) S. typhimurium TA100, and

(cid:151) E. coli WP2 uvrA, or E. coli WP2 uvrA (pKM101), or S. typhimurium TA102.

In order to detect cross-linking mutagens it may be preferable to include TA102 or to add a DNA
repair-proficient strain of E. coli (e.g. E. coli WP2 or E. coli WP2 (pKM101)).

Established procedures for stock culture preparation, marker verification and storage should be used. The
for growth should be demonstrated for each frozen stock culture preparation
amino-acid requirement
(histidine for S. typhimurium strains, and tryptophan for E. coli strains). Other phenotypic characteristics
should be similarly checked, namely: the presence or absence of R-factor plasmids where appropriate (i.e.
ampicillin resistance in strains TA98, TA100, and TA97a or TA97, WP2 uvrA and WP2 uvrA (pKM101), and
ampicillin + tetracycline resistance in strain TA 102); the presence of characteristic mutations (i.e. rfa mutation
in S. typhimurium through sensitivity to crystal violet, and uvrA mutation in E. coli or uvrB mutation in S.
typhimurium,
(3). The strains should also yield spontaneous
revertant colony plate counts within the frequency ranges expected from the laboratory’s historical control
data and preferably within the range reported in the literature.

through sensitivity to ultra-violet

light)

(2)

1.5.1.2. Medium

An appropriate minimal agar (e.g. containing Vogel-Bonner minimal medium E and glucose), and an overlay
agar containing histidine and biotin or tryptophan to allow for a few cell divisions, is used (1) (2) (9).

1.5.1.3. Metabolic activation

Bacteria should be exposed to the test substance both in the presence and absence of an appropriate
metabolic activation system. The most commonly used system is a cofactor-supplemented post-mitochondrial
fraction (S9) prepared from the livers of rodents treated with enzyme-inducing agents such as Aroclor 1254
(1) (2) or a combination of phenobarbitone and b-naphthoflavone (18) (20) (21). The post-mitochondrial
fraction is usually used at concentrations in the range from 5 to 30 % v/v in the S9-mix. The choice and
condition of a metabolic activation system may depend upon the class of chemical being tested. In some
cases,
it may be appropriate to utilise more than one concentration of post-mitochondrial fraction. For
azo-dyes and diazo-compounds, using a reductive metabolic activation system may be more appropriate (6)
(13).

1.5.1.4. Test substance/preparation

Solid test substances should be dissolved or suspended in appropriate solvents or vehicles and diluted if
appropriate prior to treatment of the bacteria. Liquid test substances may be added directly to the test
systems and/or diluted prior to treatment. Fresh preparations should be employed unless stability data
demonstrate the acceptability of storage.

The solvent/vehicle should not be suspected of chemical reaction with the test substance and should be
than well-known
compatible with the survival of
solvent/vehicles are used, their inclusion should be supported by data indicating their compatibility. It is
recommended that wherever possible, the use of an aqueous solvent/vehicle be considered first. When testing
water-unstable substances, the organic solvents used should be free of water.

the bacteria and the S9 activity (22).

If other

1.5.2.

Test conditions

1.5.2.1. Test strains (see 1.5.1.1)

1.5.2.2.

Exposure concentration

Amongst the criteria to be taken into consideration when determining the highest amount of the test
substance to be used are the cytotoxicity and the solubility in the final treatment mixture.

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It may be useful to determine toxicity and insolubility in a preliminary experiment. Cytotoxicity may be
detected by a reduction in the number of revertant colonies, a clearing or diminution of the background
lawn, or the degree of survival of treated cultures. The cytotoxicity of a substance may be altered in the
presence of metabolic activation systems. Insolubility should be assessed as precipitation in the final mixture
under the actual test conditions and evident to the unaided eye.

The recommended maximum test concentration for soluble non-cytotoxic substances is 5 mg/plate or
5 (cid:181)l/plate. For non-cytotoxic substances that are not soluble at 5 mg/plate or 5 (cid:181)l/plate, one or more
concentrations tested should be insoluble in the final treatment mixture. Test substances that are cytotoxic
already below 5 mg/plate or 5 (cid:181)l/plate should be tested up to a cytotoxic concentration. The precipitate
should not interfere with the scoring.

At least five different analysable concentrations of the test substance should be used with approximately half
log (i.e. (cid:211)10) intervals between test points for an initial experiment. Smaller intervals may be appropriate
when a concentration-response is being investigated. Testing above the concentration of 5 mg/plate or
5 (cid:181)l/plate may be considered when evaluating substances containing substantial amounts of potentially
mutagenic impurities.

1.5.2.3. Negative and positive controls

Concurrent strain-specific positive and negative (solvent or vehicle) controls, both with and without metabolic
activation, should be included in each assay. Positive control concentrations that demonstrate the effective
performance of each assay should be selected.

For assays employing a metabolic activation system, the positive control reference substance(s) should be
selected on the basis of the type of bacteria strains used.

The following substances are examples of suitable positive controls for assays with metabolic activation:

Substance

9,10-dimethylanthracene

7,12-dimethylbenz[a]anthracene

benzo[a]pyrene

2-aminoanthracene

cyclophosphamide

cyclophosphamide monohydrate

CAS No

781-43-1

57-97-6

50-32-8

613-13-8

50-18-0

6055-19-2

Einecs No

212-308-4

200-359-5

200-028-5

210-330-9

200-015-4

The following substance is a suitable positive control for the reductive metabolic activation method:

Substance

Congo Red

CAS No

573-58-0

Einecs No

209-358-4

2-Aminoanthracene should not be used as
If
2-aminoanthracene is used, each batch of S9 should also be characterised with a mutagen that requires
metabolic activation by microsomal enzymes, e.g., benzo[a]pyrene, dimethylbenzanthracene.

the sole indicator of

the efficacy of

the S9-mix.

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The following substances are examples of strain-specific positive controls for assays performed without
exogenous metabolic activation system:

Substance

CAS No

Einecs No

Strain

sodium azide

26628-22-8

247-852-1

TA 1535 and TA 100

2-nitrofluorene

607-57-8

210-138-5

TA 98

9-aminoacridine

90-45-9

201-995-6

TA 1537, TA 97 and TA 97a

ICR 191

17070-45-0

241-129-4

TA 1537, TA 97 and TA 97a

cumene hydroperoxide

80-15-9

201-254-7

TA 102

mitomycin C

50-07-7

200-008-6

WP2 uvrA and TA 102

N-ethyl-N-nitro-N-nitrosoguanidine

70-25-7

200-730-1

4-nitroquinoline-1-oxide

56-57-5

200-281-1

WP2, WP2uvrA and
WP2uvrA(pKM101)

WP2, WP2uvrA and
WP2uvrA(pKM101)

furylfuramide
(AF2)

3688-53-7

plasmid-containing strains

Other appropriate positive control reference substances may be used. The use of chemical class-related
positive control chemicals should be considered, when available.

Negative controls, consisting of solvent or vehicle alone, without test substance, and otherwise treated in the
same way as the treatment groups, should be included. In addition, untreated controls should also be used
unless there are historical control data demonstrating that no deleterious or mutagenic effects are induced by
the chosen solvent.

1.5.3.

Procedure

For the plate incorporation method (1) (2) (3) (4), without metabolic activation, usually 0,05 ml or 0,1 ml of
the test solutions, 0,1 ml of fresh bacterial culture (containing approximately 108 viable cells) and 0,5 ml of
sterile buffer are mixed with 2,0 ml of overlay agar. For the assay with metabolic activation, usually 0,5 ml of
metabolic activation mixture containing an adequate amount of post-mitochondrial fraction (in the range
from 5 to 30 % v/v in the metabolic activation mixture) are mixed with the overlay agar (2,0 ml), together
with the bacteria and test substance/test solution. The contents of each tube are mixed and poured over the
surface of a minimal agar plate. The overlay agar is allowed to solidify before incubation.

For the preincubation method (2) (3) (5) (6), the test substance/test solution is preincubated with the test
strain (containing approximately 108 viable cells) and sterile buffer or the metabolic activation system (0,5 ml)
usually for 20 minutes or more at 30(cid:150)37 (cid:176)C prior to mixing with the overlay agar and pouring onto the
surface of a minimal agar plate. Usually 0,05 or 0,1 ml of test substance/test solution, 0,1 ml of bacteria and
0,5 ml of S9-mix or sterile buffer are mixed with 2,0 ml of overlay agar. Tubes should be aerated during
pre-incubation by using a shaker.

For an adequate estimate of variation, triplicate plating should be used at each dose level. The use of
duplicate plating is acceptable when scientifically justified. The occasional loss of a plate does not necessarily
invalidate the assay.

Gaseous or volatile substances should be tested by appropriate methods, such as in sealed vessels (12) (14)
(15) (16).

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1.5.4.

Incubation

All plates in a given assay should be incubated at 37 (cid:176)C for 48(cid:150)72 hours. After the incubation period, the
number of revertant colonies per plate is counted.

2.

DATA

2.1.

TREATMENT OF RESULTS

Data should be presented as the number of revertant colonies per plate. The number of revertant colonies on
both negative (solvent control, and untreated control if used) and positive control plates should also be given.
Individual plate counts, the mean number of revertant colonies per plate and the standard deviation should
be presented for the test substance and positive and negative (untreated and/or solvent) controls.

There is no requirement for verification of a clear positive response. Equivocal results should be clarified by
further testing preferably using a modification of experimental conditions. Negative results need to be
confirmed on a case-by-case basis. In those cases where confirmation of negative results is not considered
justification should be provided. Modification of study parameters to extend the range of
necessary,
conditions assessed should be considered in follow-up experiments. Study parameters that might be modified
include the concentration spacing, the method of treatment (plate-incorporation or liquid pre-incubation), and
metabolic activation conditions.

2.2.

EVALUATION AND INTERPRETATION OF RESULTS

There are several criteria for determining a positive result, such as a concentration-related increase over the
range tested and/or a reproducible increase at one or more concentrations in the number of revertant
colonies per plate in at least one strain with or without metabolic activation system (23). Biological relevance
of the results should be considered first. Statistical methods may be used as an aid in evaluating the test
results (24). However, statistical significance should not be the only determining factor for a positive
response.

A test substance for which the results do not meet the above criteria is considered non-mutagenic in this test.

Although most experiments will give clearly positive or negative results,
in rare cases the data set will
preclude making a definite judgement about the activity of the test substance. Results may remain equivocal
or questionable regardless of the number of times the experiment is repeated.

Positive results from the bacterial reverse mutation test indicate that the substance induces point mutations
by base substitutions or frameshifts in the genome of either Salmonella typhimurium and/or Escherichia coli.
Negative results indicate that under the test conditions, the test substance is not mutagenic in the tested
species.

3.

REPORTING

TEST REPORT

The test report must include the following information:

Solvent/vehicle:

(cid:151) justification for choice of solvent/vehicle,

(cid:151) solubility and stability of the test substance in solvent/vehicle, if known.

Strains:

(cid:151) strains used,

(cid:151) number of cells per culture,

(cid:151) strain characteristics.

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Test conditions:

(cid:151) amount of test substance per plate (mg/plate or (cid:181)l/plate) with rationale for selection of dose and number

of plates per concentration,

(cid:151) media used,

(cid:151) type and composition of metabolic activation system, including acceptability criteria,

(cid:151) treatment procedures.

Results:

(cid:151) signs of toxicity,

(cid:151) signs of precipitation,

(cid:151) individual plate counts,

(cid:151) the mean number of revertant colonies per plate and standard deviation,

(cid:151) dose-response relationship, where possible,

(cid:151) statistical analyses, if any,

(cid:151) concurrent negative (solvent/vehicle) and positive control data, with ranges, means and standard

deviations,

(cid:151) historical negative (solvent/vehicle) and positive control data with ranges, means and standard deviations.

Discussion of results.

Conclusions.

4.

REFERENCES

(1) Ames, B. N., McCann, J. and Yamasaki E. (1975), Methods of Detecting Carcinogens and Mutagens with

the Salmonella/Mammalian-Microsome Mutagenicity Test, Mutation Res., 31, pp. 347(cid:150)364.

(2) Maron, D. M. and Ames, B. N. (1983), Revised Methods for the Salmonella Mutagenicity Test, Mutation

Res., 113, pp. 173(cid:150)215.

(3) Gatehouse, D., Haworth, S., Cebula, T., Gocke, E., Kier, L., Matsushima, T., Melcion, C., Nohmi, T.,
Venitt, S. and Zeiger, E. (1994), Recommendations for the Performance of Bacterial Mutation Assays,
Mutation Res., 312, pp. 217(cid:150)233.

(4) Kier, L. D., Brusick D. J., Auletta, A. E., Von Halle, E. S., Brown, M. M., Simmon, V. F., Dunkel, V.,
McCann,
(1986), The Salmonella
typhimurium/Mammalian Microsomal Assay: A Report of the U.S. Environmental Protection Agency
Gene-Tox Program, Mutation Res., 168, pp. 69(cid:150)240.

J., Mortelmans, K., Prival, M., Rao, T. K.

and Ray V.

(5) Yahagi, T., Degawa, M., Seino, Y. Y., Matsushima, T., Nagao, M., Sugimura, T. and Hashimoto, Y.

(1975), Mutagenicity of Carcinogen Azo Dyes and their Derivatives, Cancer Letters 1, pp. 91(cid:150)96.

(6) Matsushima, M., Sugimura, T., Nagao, M., Yahagi, T., Shirai, A. and Sawamura, M. (1980), Factors
in: Short-term Test Systems for Detecting Carcinogens, ed.

Modulating Mutagenicity Microbial Tests,
Norpoth K. H. and Garner, R. C., Springer, Berlin-Heidelberg-New York, pp. 273(cid:150)285.

(7) Gatehouse, D. G., Rowland, I. R., Wilcox, P., Callender, R. D. and Foster, R. (1980), Bacterial Mutation
Assays, in: Basic Mutagenicity Tests: UKEMS Part 1 Revised, ed. D. J. Kirkland, Cambridge University Press,
pp. 13(cid:150)61.

(8) Aeschacher, H. U., Wolleb, U. and Porchet, L.

(1987), Liquid Preincubation Mutagenicity Test for

Foods, J. Food Safety, 8, pp. 167(cid:150)177.

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(9) Green, M. H. L., Muriel, W. J. and Bridges, B. A. (1976), Use of a simplified fluctuation test to detect

low levels of mutagens, Mutation Res., 38, pp. 33(cid:150)42.

(10) Hubbard, S. A., Green, M. H. L., Gatehouse, D. and Bridges, J. W. (1984), The Fluctuation Test in
Bacteria, in: Handbook of Mutagenicity Test Procedures, 2nd Edition, ed. Kilbey, B. J., Legator, M., Nichols,
W. and Ramel, C., Elsevier, Amsterdam-New York-Oxford, pp. 141(cid:150)161.

(11) Thompson, E. D. and Melampy, P. J. (1981), An Examination of the Quantitative Suspension Assay for

Mutagenesis with Strains of Salmonella typhimurium, Environmental Mutagenesis, 3, pp. 453(cid:150)465.

(12) Araki, A., Noguchi, T., Kato, F. and Matsushima, T. (1994), Improved Method for Mutagenicity Testing

of Gaseous Compounds by Using a Gas Sampling Bag, Mutation Res., 307, pp. 335(cid:150)344.

(13) Prival, M. J., Bell, S. J., Mitchell, V. D., Reipert, M. D. and Vaughan, V. L. (1984), Mutagenicity of
Benzidine and Benzidine-Congener Dyes and Selected Monoazo Dyes in a Modified Salmonella Assay,
Mutation Res., 136, pp. 33(cid:150)47.

(14) Zeiger, E., Anderson, B. E., Haworth, S., Lawlor, T. and Mortelmans, K. (1992), Salmonella Mutagenicity

Tests. V. Results from the Testing of 311 Chemicals, Environ. Mol. Mutagen., 19, pp. 2(cid:150)141.

(15) Simmon, V., Kauhanen, K. and Tardiff, R. G. (1977), Mutagenic Activity of Chemicals Identified in
in Progress in Genetic Toxicology, D. Scott, B. Bridges and F., Sobels (eds.) Elsevier,

Drinking Water,
Amsterdam, pp. 249(cid:150)258.

(16) Hughes, T. J., Simmons, D. M., Monteith, I. G. and Claxton, L. D. (1987), Vaporisation Technique to
/Salmonella Assay,

Measure Mutagenic Activity of Volatile Organic Chemicals
Environmental Mutagenesis, 9, pp. 421(cid:150)441.

in the Ames

(17) Matsushima, T., Matsumoto, A., Shirai, M., Sawamura, M., and Sugimura, T. (1979), Mutagenicity of
the Naturally Occurring Carcinogen Cycasin and Synthetic Methylazoxy Methane Conjugates in
Salmonella typhimurium, Cancer Res., 39, pp. 3780(cid:150)3782.

(18) Tamura, G., Gold, C., Ferro-Luzzi, A. and Ames, B. N. (1980), Fecalase: A Model for Activation of
Dietary Glycosides to Mutagens by Intestinal Flora, Proc. Natl. Acad. Sci. USA, 77, pp. 4961(cid:150)4965.

(19) Wilcox, P., Naidoo, A., Wedd, D. J. and Gatehouse, D. G. (1990), Comparison of Salmonella typhimurium

TA 102 with Escherichia coli WP2 Tester strains, Mutagenesis, 5, pp. 285(cid:150)291.

(20) Matsushima, T., Sawamura, M., Hara, K. and Sugimura, T. (1976), A Safe Substitute for Polychlorinated
Biphenyls as an Inducer or Metabolic Activation Systems, in: In vitro Metabolic Activation in Mutagenesis
Testing, eds. F. J. de Serres et al. Elsevier, North Holland, pp. 85(cid:150)88.

(21) Elliot, B. M., Combes, R. D., Elcombe, C. R., Gatehouse, D. G., Gibson, G. G., Mackay, J. M. and Wolf,
R. C. (1992), Alternatives to Aroclor 1254-induced S9 in in vitro Genotoxicity Assays, Mutagenesis, 7,
pp. 175(cid:150)177.

(22) Maron, D., Katzenellenbogen, J. and Ames, B. N. (1981), Compatibility of Organic Solvents with the

Salmonella/Microsome Test, Mutation Res., 88, pp. 343(cid:150)350.

(23) Claxton, L. D., Allen, J., Auletta, A., Mortelmans, K., Nestmann, E. and Zeiger, E. (1987), Guide for the
Salmonella typhimurium/Mammalian Microsome Tests for Bacterial Mutagenicity, Mutation Res., 189, pp.
83(cid:150)91.

(24) Mahon, G. A. T., Green, M. H. L., Middleton, B., Mitchel, I., Robinson, W. D. and Tweats, D. J. (1989),
Analysis of Data from Microbial Colony Assays, in: UKEMS Sub-Committee on Guidelines for Mutagenicity
Testing, Part II. Statistical Evaluation of Mutagenicity Test Data, ed. Kirkland, D. J., Cambridge University
Press, pp. 28(cid:150)65.(cid:146)

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ANNEX 4E

(cid:145)B.17. MUTAGENICITY (cid:151) IN VITRO MAMMALIAN CELL GENE MUTATION TEST

1.

METHOD

This method is a replicate of the OECD TG 476, In Vitro Mammalian Cell Gene Mutation Test (1997).

1.1.

INTRODUCTION

The in vitro mammalian cell gene mutation test can be used to detect gene mutations induced by chemical
substances. Suitable cell lines include L5178Y mouse lymphoma cells, the CHO, CHO-AS52 and V79 lines of
Chinese hamster cells, and TK6 human lymphoblastoid cells (1). In these cell lines the most commonly used
genetic endpoints measure mutation at thymidine kinase (TK) and hypoxanthine-guanine phosphoribosyl
transferase (HPRT), and a transgene of xanthine-guanine phosphoribosyl transferase (XPRT). The TK, HPRT
and XPRT mutation tests detect different spectra of genetic events. The autosomal location of TK and XPRT
may allow the detection of genetic events (e.g.
large deletions) not detected at the HPRT locus on X
chromosomes (2)(3)(4)(5)(6).

In the in vitro mammalian cell gene mutation test, cultures of established cell lines or cell strains can be used.
The cells used are selected on the basis of growth ability in culture and stability of the spontaneous mutation
frequency.

Tests conducted in vitro generally require the use of an exogenous source of metabolic activation. This
metabolic activation system cannot mimic entirely the mammalian in vivo conditions. Care should be taken to
avoid conditions which would lead to results not reflecting intrinsic mutagenicity. Positive results which do
not reflect intrinsic mutagenicity may arise from changes in pH, osmolality or high levels of cytotoxicity (7).

This test is used to screen for possible mammalian mutagens and carcinogens. Many compounds that are
positive in this test are mammalian carcinogens; however, there is not a perfect correlation between this test
and carcinogenicity. Correlation is dependent on chemical class and there is increasing evidence that there are
carcinogens that are not detected by this test because they appear to act through other, non-genotoxic
mechanisms or mechanisms absent in bacterial cells (6).

See also General Introduction Part B:

1.2.

DEFINITIONS

Forward mutation: a gene mutation from the parental type of the mutant form which gives rise to an
alteration or a loss of the enzymatic activity of the function of the encoded protein.

Base pair substitution mutagens: substances which cause substitution of one or several base pairs in the
DNA.

Frameshift mutagens: Substances which cause the addition or deletion of single or multiple base pairs in the
DNA molecule.

Phenotypic expression time: a period during which unaltered gene products are depleted from newly
mutated cells.

Mutant frequency: the number of mutant cells observed divided by the number of viable cells.

increase in cell number over time compared to a control population of cells;
Relative total growth:
calculated as the product of suspension growth relative to the negative control times cloning efficiency
relative to negative control.

Relative suspension growth: increase in cell number over the expression period relative to the negative
control.

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Viability: the cloning efficiency of the treated cells at the time of plating in selective conditions after the
expression period.

Survival: the cloning efficiency of the treated cells when plated at the end of the treatment period; survival is
usually expressed in relation to the survival of the control cell population.

1.3.

PRINICIPLE OF THE TEST METHOD

Cells deficient in thymidine kinase (TK) due to the mutation TK+/ﬂ R TKﬂ/ﬂ are resistant to the cytotoxic
effects of the pyrimidine analogue trifluorothymidine (TFT). Thymidine kinase proficient cells are sensitive to
TFT, which causes the inhibition of cellular metabolism and halts further cell division. Thus mutant cells are
able to proliferate in the presence of TFT, whereas normal cells, which contain thymidine kinase, are not.
Similarly, cells deficient in HPRT or XPRT are selected by resistance to 6-thioguanine (TG) or 8-azaguanine
(AG). The properties of the test substance should be considered carefully if a base analogue or a compound
related to the selective agent is tested in any of the mammalian cell gene mutation tests. For example, any
suspected selective toxicity by the test substance for mutant and non-mutant cells should be investigated.
Thus, performance of the selection system/agent must be confirmed when testing chemicals structurally
related to the selective agent (8).

Cells in suspension or monolayer culture are exposed to the test substance, both with and without metabolic
activation, for a suitable period of time and subcultured to determine cytotoxicity and to allow phenotypic
expression prior to mutant selection (9) (10) (11) (12) (13). Cytotoxicity is usually determined by measuring
the relative cloning efficiency (survival) or relative total growth of the cultures after the treatment period. The
treated cultures are maintained in growth medium for a sufficient period of time, characteristic of each
selected locus and cell type, to allow near-optimal phenotypic expression of induced mutations. Mutant
frequency is determined by seeding known numbers of cells in medium containing the selective agent to
detect mutant cells and in medium without selective agent to determine the cloning efficiency (viability). After
a suitable incubation time, colonies are counted. The mutant frequency is derived from the number of mutant
colonies in selective medium and the number of colonies in non-selective medium.

1.4.

DESCRIPTION OF THE TEST METHOD

1.4.1.

Preparations

1.4.1.1. Cells

A variety of cell types are available for use in this test including subclones of L5171Y, CHO, CHO-AS52, V79
or TK6 cells. Cell types used in this test should have a demonstrated sensitivity to chemical mutagens, a high
cloning efficiency and a stable spontaneous mutant frequency. Cells should be checked for mycoplasma
contamination and should not be used if contaminated.

The test should be designed to have a predetermined sensitivity and power. The number of cells, cultures and
concentrations of test substance used should reflect these defined parameters (14). The minimal number of
viable cells surviving treatment and used at each stage in the test should be based on the spontaneous
mutation frequency. A general guide is to use a cell number which is at least 10 times the inverse of the
spontaneous mutation frequency. However, it is recommended to utilise at least 106 cells. Adequate historical
data on the cell system used should be available to indicate consistent performance of the test.

1.4.1.2. Media and culture conditions

Appropriate culture media, and incubation conditions (culture vessels, temperature, CO2 concentration and
humidity) should be used. Media should be chosen according to the selective systems and cell type used in
the test. It is particularly important that culture conditions should be chosen that ensure optimal growth of
cells during the expression period and colony forming ability of both mutant and non-mutant cells.

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1.4.1.3.

Preparation of cultures

Cells are propagated from stock cultures, seeded in culture medium and incubated at 37 (cid:176)C. Prior to use in
this test, cultures may need to be cleansed of pre-existing mutant cells.

1.4.1.4. Metabolic activation

Cells should be exposed to the test substance both in the presence and absence of an appropriate metabolic
activation system. The most commonly used system is a cofactor-supplemented post-mitochondrial fraction
(S9) prepared from the livers of rodents treated with enzyme-inducing agents such as Aroclor 1254 (15) (16)
(17) (18) or a combination of phenobarbitone and b-naphthoflavone (19) (20).

The post-mitochondrial fraction is usually used at concentrations in the range from 1(cid:151)10 % v/v in the final
test medium. The choice and condition of a metabolic activation system may depend upon the class of
chemical being tested.
In some cases it may be appropriate to utilise more than one concentration of
post-mitochondrial fraction.

A number of developments, including the construction of genetically engineered cell lines expressing specific
activating enzymes, may provide the potential for endogenous activation. The choice of the cell lines used
should be scientifically justified (e.g. by the relevance of the cytochrome P450 isoenzyme for the metabolism
of the test substance).

1.4.1.5. Test substance preparation

Solid test substances should be dissolved or suspended in appropriate solvents or vehicles and diluted if
appropriate prior to treatment of the cells. Liquid test substances may be added directly to the test systems
and/or diluted prior to treatment. Fresh preparations of the test substance should be employed unless stability
data demonstrate the acceptability of storage.

1.4.2.

Test conditions

1.4.2.1.

Solvent/vehicle

The solvent/vehicle should not be suspected of chemical reaction with the test substance and should be
compatible with the survival of the cells and the S9 activity. If other than well-known solvent/vehicles are
used, their inclusion should be supported by data indicating their compatibility. It is recommended that
wherever possible, the use of an aqueous solvent/vehicle be considered first. When testing water-unstable
substances, the organic solvents used should be free of water. Water can be removed by adding a molecular
sieve.

1.4.2.2.

Exposure concentrations

Among the criteria to be considered when determining the highest concentration are cytotoxicity, solubility
in the test system and changes in pH or osmolality.

Cytotoxicity should be determined with and without metabolic activation in the main experiment using an
appropriate indication of cell integrity and growth, such as relative cloning efficiency (survival) or relative
total growth. It may be useful to determine cytotoxicity and solubility in a preliminary experiment.

At last four analysable concentrations should be used. Where there is cytotoxicity, these concentrations
should cover a range from the maximum to little or no toxicity; this will usually mean that the concentration
levels should be separated by no more than a factor between 2 and (cid:211)10. If the maximum concentration is
based on cytotoxicity then it should result in approximately 10(cid:151)20 % (but not less than 10 %) relative
survival
(relative cloning efficiency) or relative total growth. For relatively non-cytotoxic substances, the
maximum test concentration should be 5 mg/ml, 5 (cid:181)l/ml, or 1,01 M, whichever is the lowest.

Relatively insoluble substances should be tested up to or beyond their limit of solubility under culture
conditions. Evidence of insolubility should be determined in the final treatment medium to which cells are
exposed. It may be useful to assess solubility at the beginning and the end of the treatment, as solubility can
change during the course of exposure in the test system due to presence of cells, S9, serum etc. Insolubility
can be detected by using the unaided eye. The precipitate should not interfere with the scoring.

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1.4.2.3. Controls

Concurrent positive and negative (solvent or vehicle) controls, both with and without metabolic activation
should be included in each experiment. When metabolic activation is used, the positive control chemical
should be the one that requires activation to give a mutagenic response.

Examples of positive control substances include:

Metabolic activation condition

Locus

Substance

CAS No

Einecs No

Absence of exogenous
metabolic activation

HPRT

Ethyl methansulphonate

62-50-0

200-536-7

Ethyl nitrosourea

759-73-9

212-072-2

TK (small and
large colonies)

Methyl methansulphonate

66-27-3

200-625-0

XPRT

Ethyl methanesulphonate

62-50-0

200-536-7

Ethyl nitrosourea

759-73-9

212-072-2

Presence of exogenous
metabolic activation

HPRT

3-Methylcholanthrene

56-49-5

200-276-4

N-Nitrosodimethylamine

62-75-9

200-549-8

7,12-Dimethylbenzanthracene

57-97-6

200-359-5

TK (small and
large colonies)

Cyclophosphamide

50-18-0

200-015-4

Cyclophosphamidmonohydrate

6055-19-2

Benzo[a]pyrene

50-32-8

200-028-5

3-Methylcholanthrene

56-49-5

200-276-5

XPRT

N-Nitrosodimethylamine (for
high levels of S-9)

62-75-9

200-549-8

Benzo[a]pyrene

50-32-8

200-028-5

Other appropriate positive control reference substances may be used, e.g. if a laboratory has a historical data
base on 5-bromo 2¡-deoxyuridine (CAS No 59-14-3, Einecs No 200-415-9), this reference substance could be
used as well. The use of chemical class-related positive control chemicals should be considered, when
available.

Negative controls, consisting of solvent or vehicle alone in the treatment medium, and treated in the same
way as the treatment groups, should be included. In addition, untreated controls should also be used unless
there are historical control data demonstrating that no deleterious or mutagenic effects are induced by the
chosen solvent.

1.4.3.

Procedure

1.4.3.1. Treatment with the test substance

Proliferating cells should be exposed to the test substance both with and without metabolic activation.
Exposure should be for a suitable period of time (usually 3(cid:151)6 hours is effective). Exposure time may be
extended over once or more cell cycles.

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Either duplicate or single treated cultures may be used at each concentration tested. When single cultures are
used, the number of concentrations should be increased to ensure an adequate number of cultures for
analysis (e.g. at least eight analysable concentrations). Duplicate negative (solvent) control cultures should be
used.

Gaseous or volatile substances should be tested by appropriate methods, such as in sealed culture vessels (21)
(22).

1.4.3.2. Measurement of survival, viability and mutant frequency

At the end of the exposure period, cells are washed and cultured to determine survival and to allow for
expression of
the mutant phenotype. Measurement of cytotoxicity by determining the relative cloning
efficiency (survival) or relative total growth of the cultures is usually initiated after the treatment period.

Each locus has a defined minimum time requirement to allow near optimal phenotypic expression of newly
induced mutants (HPRT and XPRT require at least 6-8 days, and TK at least two days). Cells are grown in
medium with and without selective agent(s) for determination of numbers of mutants and cloning efficiency,
respectively. The measurement of viability (used to calculate mutant frequency) is initiated at the end of the
expression time by plating in non-selective medium.

If the test substance is positive in the L5178Y TK+/ﬂ test, colony sizing should be performed on at least one
of the test cultures (the highest positive concentration) and on the negative and positive controls. If the test
substance is negative in the L5178Y TK+/ﬂ test, colony sizing should be performed on the negative and
positive controls. In studies using TK6TK+/ﬂ, colony sizing may also be performed.

2.

DATA

2.1.

TREATMENT OF RESULTS

Data should include cytotoxicity and viability determination, colony counts and mutant frequencies for the
treated and control cultures. In the case of a positive response in the L5178Y TK+/ﬂ test, colonies are scored
using the criteria of small and large colonies on at least one concentration of the test substance (highest
positive concentration) and on the negative and positive control. The molecular and cytogenetic nature of
both large and small colony mutants has been explored in detail (23) (24). In the TK+/ﬂ test, colonies are
scored using the criteria of normal growth (large) and slow growth (small) colonies (25). Mutant cells that
have suffered the most extensive genetic damage have prolonged doubling times and thus form small
colonies. This damage typically ranges in scale from the losses of the entire gene to karyotypically visible
chromosome aberrations. The induction of small colony mutants has been associated with chemicals that
induce gross chromosome aberrations (26). Less seriously affected mutant cells grow at rates similar to the
parental cells and form large colonies.

Survival (relative cloning efficiencies) or relative total growth should be given. Mutant frequency should be
expressed as number of mutant cells per number of surviving cells.

Individual culture data should be provided. Additionally, all data should be summarised in tabular form.

There is no requirement for verification of a clear positive response. Equivocal results should be clarified by
further testing preferably using modification of experimental conditions. Negative results need to be
confirmed on a case-by-case basis. In those cases where confirmation of negative results is not considered
necessary,
justification should be provided. Modification of study parameters to extend the range of
conditions assessed should be considered in follow-up experiments for either equivocal or negative results.
Study parameters that might be modified include the concentration spacing and the metabolic activation
conditions.

2.2.

EVALUATION AND INTERPRETATION OF RESULTS

There are several criteria for determining a positive result, such as a concentration-related increase or a
reproducible increase in mutant frequency. Biological relevance of the results should be considered first.
Statistical methods may be used as an aid in evaluating the test results. Statistical significance should not be
the only determining factor for a positive response.

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A test substance for which the results do not meet the above criteria is considered non-mutagenic in this
system.

Although most studies will give clearly positive or negative results, in rare cases the data set will preclude
making a definite judgement about the activity of the test substance. Results may remain equivocal or
questionable regardless of the number of times the experiment is repeated.

Positive results from the in vitro mammalian cell gene mutation test indicate that the test substance induces
gene mutations in the cultured mammalian cells used. A positive concentration-response that is reproducible
is most meaningful. Negative results indicate that, under the test conditions, the test substance does not
induce gene mutations in the cultured mammalian cells used.

3.

REPORTING

TEST REPORT

The test report must include the following information:

Solvent/vehicle:

(cid:151) justification for choice of vehicle/solvent,

(cid:151) solubility and stability of the test substance in solvent/vehicle, if known.

Cells:

(cid:151) type and source of cells,

(cid:151) number of cell cultures,

(cid:151) number of cell passages, if applicable,

(cid:151) methods for maintenance of cell culture, if applicable,

(cid:151) absence of mycoplasma.

Test conditions:

(cid:151) rationale for selection of concentrations and number of cultures including, e.g. cytotoxicity data and

solubility limitations, if available,

(cid:151) composition of media, CO2 concentration,

(cid:151) concentration of test substance,

(cid:151) volume of vehicle and test substance added,

(cid:151) incubation temperature,

(cid:151) incubation time,

(cid:151) duration of treatment,

(cid:151) cell density during treatment,

(cid:151) type and composition of metabolic activation system, including acceptability criteria,

(cid:151) positive and negative controls,

(cid:151) length of expression period (including number of cells seeded, and subcultures and feeding schedules, if

appropriate),

(cid:151) selective agents,

(cid:151) criteria for considering tests as positive, negative or equivocal,

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(cid:151) methods used to enumerate numbers of viable and mutant cells,

(cid:151) definition of colonies of which size and type are considered (including criteria for (cid:147)small(cid:148) and

(cid:147)large(cid:148)colonies, as appropriate).

Results:

(cid:151) signs of toxicity,

(cid:151) signs of precipitation,

(cid:151) data on pH and osmolality during the exposure to the test substance, if determined,

(cid:151) colony size if scored for at least negative and positive controls,

(cid:151) laboratory’s adequacy to detect small colony mutants with the L5178Y TK+/ﬂ system where appropriate,

(cid:151) dose-response relationship, where possible,

(cid:151) statistical analyses, if any,

(cid:151) concurrent negative (solvent/vehicle) and positive control data,

(cid:151) historical negative (solvent/vehicle) and positive control data with ranges, means and standard deviations,

(cid:151) mutant frequency.

Discussion of results.

Conclusions.

4.

REFERENCES

(1) Moore, M.M., DeMarini, D.M., DeSerres, F.J. and Tindal, K.R.

(eds.)

(1987), Banbury Report 28;

Mammalian Cell Mutagenesis, Cold Spring Harbor Laboratory, New York.

(2) Chu, E.H.Y. and Malling H.V. (1968), Mammalian Cell Genetics. II. Chemical Induction of Specific
Locus Mutations in Chinese Hamster Cells In Vitro, Proc. Natl. Acad. Sci. USA, 61, pp. 1306(cid:151)1312.

(3) Liber, H.L. and Thilly, W.G. (1982), Mutation Assay at the Thymidine Kinase Locus in Diploid Human

Lymphoblasts, Mutation Res. 94, pp. 467(cid:151)485.

(4) Moore, M.M., Harington-Brock, K., Doerr, C.L. and Dearfield, K.L.

(1989), Differential Mutant

Quantitation at the Mouse Lymphoma TK and CHO HGPRT Loci, Mutagenesis, 4, pp. 394(cid:151)403.

(5) Aaron, C.S., and Stankowski, Jr. L.F.,

(1989), Comparison of the AS52/XPRT and the CHO/HPRT

Assays: Evaluation of Six Drug Candidates, Mutation Res. 223, pp. 121(cid:151)128.

(6) Aaron, C.S., Bolcsfoldi, G., Glatt, H.R., Moore, M., Nishi, Y., Stankowski,

J. and
Thompson, E. (1994), Mammalian Cell Gene Mutation Assays Working Group Report. Report of the
International Workshop on Standardisation of Genotoxicity Test Procedures. Mutation Res. 312, pp.
235(cid:151)239.

Jr. L.F., Theiss,

(7) Scott, D., Galloway, S. M., Marshall, R. R., Ishidate, M., Brusick, D., Ashby, J. and Myhr, B.C. (1991),
Genotoxicity Under Extreme Culture Conditions. A report from ICPEMC Task Group 9, Mutation Res.
257, pp. 147(cid:151)204.

(8) Clive, D., McCuen, R., Spector, J. F. S., Piper, C. and Mavournin, K. H. (1983), Specific Gene Mutations
in L5178Y Cells in Culture. A Report of the U.S. Environmental Protection Agency Gene-Tox Program,
Mutation Res., 115, pp. 225(cid:151)251.

(9) Li, A. P., Gupta, R. S., Heflich, R. H. and Wasson, J. S. (1988), A Review and Analysis of the Chinese
Hamster Ovary/Hypoxanthine Guanine Phosphoribosyl Transferase System to Determine
the
Mutagenicity of Chemical Agents: A Report of Phase III of the U.S. Environmental Protections Agency
Gene-Tox Program, Mutation Res., 196, pp. 17(cid:151)36.

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(10) Li, A. P., Carver, J. H., Choy, W. N., Hsie, A. W., Gupta, R. S., Loveday, K. S., (cid:211)Neill, J. P., Riddle, J. C.,
Stankowski, L. F. Jr. and Yang, L. L. (1987), A Guide for the Performance of the Chinese Hamster
Ovary Cell/Hypoxanthine-Guanine Phosphoribosyl Transferase Gene Mutation Assay, Mutation Res.,
189, pp. 135(cid:151)141.

(11) Liber, H. L., Yandell, D. W. and Little, J. B. (1989), A Comparison of Mutation Induction at the TK and
HPRT Loci in Human Lymphoblastoid Cells: Quantitative Differences are Due to an Additional Class of
Mutations at the Autosomal TK Locus, Mutation Res., 216, pp. 9(cid:151)17.

(12) Stankowski, L. F. Jr., Tindal,, K. R., and Hsie, A. W. (1986), Quantitative and Molecular Analyses of
Ethyl Methanosulphonate (cid:151) and ICR 191-Induced Molecular Analyses of Ethyl Methanosulphonate (cid:151)
and ICR 191 Induced Mutation in AS52 Cells, Mutation Res., 160, pp. 133(cid:151)147.

(13) Turner, N. T., Batson, A. G. and Clive, D. (1984), Procedure for the L5178Y/TK+/ﬂ (cid:151) TK+/ﬂ Mouse
Lymphoma Cell Mutagenicity Assay, in: Kilbey, B. J. et al (eds.) Handbook of Mutagenicity Test Procedures,
Elsevier Science Publishers, New York, pp. 239(cid:151)268.

(14) Arlett, C. F., Smith, D. M., Clarke, G. M., Green, M. H. L., Cole, J., McGregor, D. B. and Asquith S. C.
(1989), Mammalian Cell Gene Mutation Assays Based upon Colony Formation, in: Statistical Evaluation
of Mutagenicity Test Data, Kirkland, D. J., ed., Cambirdge University Press, pp. 66(cid:151)101.

(15) Abbondandolo, A., Bonatti, S., Corti, G., Fiorio, R., Loprieno, N. and Mazzaccoro, A. (1977), Induction
of 6-Thioguanine-Resistant Mutants in V79 Chinese Hamster Cells by Mouse-Liver Microsome-
Activated Dimethylnitrosamine, Mutation Res. 46, pp. 365(cid:151)373.

(16) Ames, B. N., McCann, J. and Yamasaki, E. (1975), Methods for Detecting Carcinogens and Mutagens
with the Salmonella/Mammalian-Microsome Mutagenicity Test, Mutation Res. 31, pp. 347(cid:151)364.

(17) Clive, D., Johnson, K. O., Spector, J. F. S., Batson A. G. and Brown M. M. M. (1979), Validation and
Characterisation of the L5178Y/TK+/ﬂ (cid:151) Mouse Lymphoma Mutagen Assay System, Mutat. Res. 59, pp.
61(cid:151)108.

(18) Maron, D. M. and Ames, B. N. (1983), Revised Methods for the Salmonella Mutagenicity Test, Mutation

Res. 113, pp. 173(cid:151)215.

(19) Elliott, B. M., Combes, R. D., Elcombe, C. R., Gatehouse, D. G., Gibson, G. G., Mackay, J. M. and Wolf,
R. C. (1992), Alternatives to Aroclor 1254-Induced S9 in: In Vitro Genotoxicity Assays, Mutagenesis 7,
pp. 175(cid:151)177.

(20) Matsushima, T., Sawamura, M., Hara, K. and Sugimura, T. (1976), A Safe Substitute for Polycholrinated
Biphenyls as an Inducer of Metabolic Activation Systems, in: In Vitro Metabolic Activation in Mutagenesis
Testing, de Serres, F. J., Fouts, J. R., Bend, J. R. and Philpot, R. M. (eds), Elsevier, North-Holland, pp.
85(cid:151)88.

(21) Krahn, D. F., Barsky, F. C. and McCooey, K. T. (1982), CHO/HGPRT Mutation Assay: Evaluation of
Gases and Volatile Liquids, in: Ticc, R. R., Costa, D. L., Schaich, K. M. (eds), Genotoxic Effects of Airborne
Agents, New York, Plenum, pp. 91(cid:151)103.

(22) Zamora, P. O., Benson, J. M., Li, A. P. and Brooks, A. L. (1983), Evaluation of an Exposure System
Using Cells Grown on Collagen Gels for Detecting Highly Volatile Mutagens in the CHO/HGPRT
Mutation Assay, Environmental Mutagenesis, 5, pp. 795(cid:151)801.

(23) Applegate, M. L., Moore, M. M., Broder, C. B., Burrell, A. and Hozier, J. C. (1990), Molecular Dissection
of Mutations at the Heterozygous Thymidine Kinase Locus in Mouse Lymphoma Cells, Proc. Natl. Acad.
Sci. USA, 87, pp. 51(cid:151)55.

(24) Moore, M. M., Clive, D. Hozier, J. C., Howard, B. E., Batson, A. G., Turner, N. T. and Sawyer, J. (1985),
Analysis of Trifluoronthymidine, Resistant (TFT+) Mutants of L5178Y/TK+/ﬂ (cid:151) Mouse Lymphoma Cells,
Mutation Res. 151, pp. 161(cid:151)174.

(25) Yandell, D. W., Dryja, T. P. and Little, J. B. (1990), Molecular Genetic Analysis of Recessive Mutations

at a Heterozygous Autosomal Locus in Human Cells, Mutation Res. 229, pp. 89(cid:151)102.

(26) Moore, M. M. and Doerr, C. L. (1990), Comparison of Chromosome Aberration Frequency and Small
Colony TK-Deficient Mutant Frequency in L5178Y/TK+/ﬂ (cid:151) 3.7.2C Mouse Lymphoma Cells,
Mutagenesis, 5, pp. 609(cid:151)614.(cid:146)

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ANNEX 4F

(cid:145)B.23. MAMMALIAN SPERMATOGONIAL CHROMOSOME ABERRATION TEST

1.

METHOD

This method is a replicate of the OECD TG 483, Mammalian Spermatogonial Chromosome Aberration Test
(1997).

1.1.

INTRODUCTION

The purpose of the in vivo mammalian spermatogonial chromosome aberration test is to identify those
substances that cause structural chromosome aberrations in mammalian spermatogonial cells (1) (2) (3) (4)
(5). Structural aberrations may be of two types, chromosome or chromatid. With the majority of chemical
mutagens, induced aberrations are of the chromatid type, but chromosome-type aberrations also occur. This
method is not designed to measure numerical aberrations and is not routinely used for that purpose.
Chromosome mutations and related events are the cause of many human genetic diseases.

This test measures chromosome events in spermatogonial germ cells and is, therefore, expected to be
predictive of induction of inheritable mutations in germ cells.

Rodents are routinely used in this test. This in vivo cytogenetic test detects chromosome aberrations in
spermatogonial mitoses. Other target cells are not the subject of this method.

the first mitotic cell division following
To detect chromatid-type aberrations in spermatogonial cells,
treatment should be examined before these lesions are lost
in subsequent cell divisions. Additional
information from treated spermatogonial stem cells can be obtained by meiotic chromosome anaylsis for
chromosome-type aberrations at diakinesis-metaphase I when the treated cells become spermatocytes.

This in vivo test is designed to investigate whether somatic cell mutagens are also active in germ cells. In
addition, the spermatogonial test is relevant to assessing mutagenicity hazard in that it allows consideration
of factors of in vivo metabolism, pharmacokinetics and DNA repair processes.

A number of generations of spermatogonia are present in the testis with a spectrum of sensitivity to chemical
treatment. Thus, the aberrations detected represent an aggregate response of treated spermatogonial cell
populations, with the more numerous differentiated spermatogonial cells predominating. Depending on their
position within the testis, different generations of spermatogonia may or may not be exposed to the general
circulation, because of the physical and physiological Sertoli cell barrier and the blood-testis barrier.

If there is evidence that the test substance, or a reactive metabolite, will not reach the target tissue, it is not
appropriate to use this test.

See also General Introduction Part B.

1.2.

DEFINITIONS

Chromatid-type aberration: structural chromosome damage expressed as breakage of single chromatids or
breakage and reunion between chromatids.

Chromosome-type aberration: structural chromosome damage expressed as breakage, or breakage and
reunion, of both chromatids at an identical site.

Gap: an achromatic lesion smaller than the width of one chromatid, and with minimum misalignment of the
chromatids.

Numerical aberration: a change in the number of chromosomes from the normal number characteristic of
the animals utilised.

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Polyploidy: a multiple of the haploid chromosome number (n) other than the diploid number (i.e. 3n, 4n
and so on).

Structural aberration: a change in chromosome structure detectable by microscopic examination of the
metaphase stage of cell division, observed as deletions, intrachanges or interchanges.

1.3.

PRINCIPLE OF THE TEST METHOD

Animals are exposed to the test substance by an appropriate route of exposure and are sacrificed at
appropriate times after treatment. Prior to sacrifice, animals are treated with a metaphase-arresting substance
(e.g. colchicine or Colcemidﬁ). Chromosome preparations are then made from germ cells and stained, and
metaphase cells are analysed for chromosome aberrations.

1.4.

DESCRIPTION OF THE TEST METHOD

1.4.1.

Preparations

1.4.1.1.

Selection of animal species

Male Chinese hamsters and mice are commonly used. However, males of other appropriate mammalian
species may be used. Commonly used laboratory strains of young healthy adult animals should be employed.
At the commencement of the study the weight variation of animals should be minimal and not exceed –
20 % of the mean weight.

1.4.1.2. Housing and feeding conditions

General conditions referred in the General Introduction to Part B are applied although the aim for humidity
should be 50(cid:151)60 %.

1.4.1.3.

Preparation of the animals

Healthy young adult males are randomly assigned to the control and treatment groups. Cages should be
arranged in such a way that possible effects due to cage placement are minimised. The animals are identified
uniquely. The animals are acclimated to the laboratory conditions for at least five days prior to the start of
the study.

1.4.1.4.

Preparation of doses

Solid test substance should be dissolved or suspended in appropriate solvents or vehicles and diluted,
if
appropriate, prior to dosing of the animals. Liquid test substances may be dosed directly or diluted prior to
dosing. Fresh preparations of the test substance should be employed unless stability data demonstrate the
acceptability of storage.

1.4.2.

Test conditions

1.4.2.1.

Solvent/vehicle

The solvent/vehicle should not produce toxic effects at the dose levels used and should not be suspected of
chemical reaction with the test substance. If other than well-known solvents/vehicles are used, their inclusion
should be supported by data indicating their compatibility. It is recommended that wherever possible, the use
of an aqueous solvent/vehicle should be considered first.

1.4.2.2. Controls

Concurrent positive and negative (solvent/vehicle) controls should be included in each test. Except
for
treatment with the test substance, animals in the control groups should be handled in an identical manner to
animals in the treated groups.

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Positive controls should produce structural chromosome aberrations in vivo in spermatogonial cells when
administered at exposure levels expected to give a detectable increase over background.

Positive control doses should be chosen so that the effects are clear but do not immediately reveal the
identity of the coded slides to the reader. It is acceptable that the positive control be administered by a route
In addition, the use of chemical
different from the test substance and sampled at only a single time.
class-related positive control chemicals may be considered, when available. Examples of positive control
substances include:

Substance

cyclophosphamide

CAS No

50-18-0

cyclophosphamide monohydrate

6055-19-2

cyclohexylamine

mitomycin C

monomeric acrylamide

triethylenemelamine

108-91-8

50-07-7

79-06-1

51-18-3

Einecs No

200-015-4

203-629-0

200-008-6

201-173-7

200-083-5

Negative controls, treated with solvent or vehicle alone, and otherwise treated in the same way as the
treatment groups, should be included for every sampling time, unless acceptable inter-animal variability and
frequency of cells with chromosome aberrations are demonstrated by historical control data. In addition,
untreated controls should also be used unless there are historical or published control data demonstrating
that no deleterious or mutagenic effects are induced by the chosen solvent/vehicle.

1.5.

PROCEDURE

1.5.1.

Number of animals

Each treated and control group must include at least five analysable males.

1.5.2.

Treatment schedule

Test substances are preferably administered once or twice (i.e. as a single treatment or as two treatments).
Test substances may also be administered as a split dose, i.e. two treatments on the same day separated by no
more than a few hours, to facilitate administering a large volume of material. Other dose regimens should be
scientifically justified.

In the highest dose group two sampling times after treatment are used. Since cell cycle kinetics can be
influenced by the test substance, one early and one late sampling time are used around 24 and 48 hours after
treatment. For doses other than the highest dose, a sampling time of 24 hours or 1,5 cell cycle length after
treatment should be taken, unless another sampling time is known to be more appropriate for detection of
effects (6).

In addition, other sampling times may be used. For example in the case of chemicals which may induce
chromosome lagging, or may exert S-independent effects, earlier sampling times may be appropriate (1).

The appropriateness of a repeated treatment schedule needs to be identified on a case-by-case basis. Following
a repeated treatment schedule the animals should then be sacrificed 24 hours (1,5 cell cycle length) after the
last treatment. Additional sampling times may be used where appropriate.

Prior to sacrifice, animals are injected intraperitoneally with an appropriate dose of a metaphase arresting
substance (e.g. Colcemidﬁ or colchocine). Animals are sampled at an appropriate interval thereafter. For mice
this interval is approximately 3(cid:151)5 hours, for Chinese hamsters this interval is approximately 4(cid:151)5 hours.

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1.5.3.

Dose levels

If a range finding study is performed because there are no suitable data available, it should be performed in
the same laboratory, using the same species, strain and treatment regimen to be used in the main study (7). If
there is toxicity, three dose levels are used for the first sampling time. These dose levels should cover a range
from the maximum to little or no toxicity. At the later sampling time only the highest dose needs to be used.
The highest dose is defined as the dose-producing signs of toxicity such that higher dose levels, based on the
same dosing regimen, would be expected to produce lethality.

Substances with specific biological activities at low non-toxic doses (such as hormones and mitogens) may be
exceptions to the dose-setting criteria and should be evaluated on a case-by-case basis. The highest dose may
also be defined as a dose that produces some indication of toxicity in the spermatogonial cells (e.g. a
reduction in the ratio of spermatogonial mitoses to first and second meiotic metaphases; this reduction
should not exceed 50 %).

1.5.4.

Limit test

If a test at one dose level of at least 2 000 mg/kg body weight/day using a single treatment, or as two
treatments on the same day, produces no observable toxic effects, and if genotoxicity would not be expected
based upon data from structurally related substances, then a full study using three dose levels may not be
considered necessary. Expected human exposure may indicate the need for a higher dose level to be used in
the limit test.

1.5.5.

Administration of doses

The test substance is usually administered by gavage using a stomach tube or a suitable intubation cannula,
or by intraperitoneal injection. Other routes of exposure may be acceptable where they can be justified. The
maximum volume of liquid that can be administered by gavage or injection at one time depends on the size
of the test animal. The volume should not exceed 2 ml/100g body weight. The use of volumes higher than
these must be justified. Except for irritating or corrosive substances, which will normally reveal exacerbated
effects with higher concentrations, variability in test volume should be minimised by adjusting the
concentration to ensure a constant volume at all dose levels.

1.5.6.

Chromosome preparation

Immediately after sacrifice, cell suspensions are obtained from one or both testes, exposed to hypotonic
solution and fixed. The cells are then spread on slides and stained.

1.5.7.

Analysis

For each animal at least 100 well-spread metaphase should be analysed (i.e. a minimum of 500 metaphases
per group). This number could be reduced when high numbers of aberrations are observed. All slides,
including those of positive and negative controls, should be independently coded before microscopic analysis.
in the breakage of a proportion of metaphases with loss of
Since fixation procedures often result
chromosomes, the cells scored should contain a number of centromeres equal to the number 2n – 2.

2.

DATA

2.1.

TREATMENT OF RESULTS

Individual animal data should be presented in a tabular form. The experimental unit is the animal. For each
individual animal
the number of cells with structural chromosome aberrations and the number of
chromosome aberrations per cell should be evaluated. Different types of structural chromosome aberrations
should be listed with their numbers and frequencies for treated and control groups. Gaps are recorded
separately and reported but generally not included in the total aberration frequency.

If mitosis as well as meiosis is observed, the ratio of spermatogonial mitoses to first and second meiotic
metaphases should be determined as a measure of cytotoxicity for all treated and negative control animals in
a total sample of 100 dividing cells per animal to establish a possible cytotoxic effect. If only mitosis is
observed, the mitosis index should be determined in at least 1 000 cells for each animal.

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2.2.

EVALUATION AND INTERPRETATION OF RESULTS

There are several criteria for determining a positive result, such as a dose-related increase in the relative
number of cells with chromosome aberrations or a clear increase in the number of cells with aberrations in a
single dose at a single sampling time. Biological relevance of the results should be considered first. Statistical
methods may be used as an aid in evaluating the test results (8). Statistical significance should not be the only
determining factor for a positive response. Equivocal results should be clarified by further testing preferably
using a modification of experimental conditions.

A test substance for which the results do not meet the above criteria is considered non-mutagenic in this test.

Although most experiments will give clearly positive or negative results,
in rare cases the data set will
preclude making a definite judgement about the activity of the test substance. Results may remain equivocal
or questionable regardless of the number of times the experiment is repeated.

Positive results from the in vivo spermatogonial chromosome aberration test indicate that the test substance
induces structural chromosome aberrations in the germ cells of the species tested. Negative results indicate
that, under the test conditions, the test substance does not induce chromosome aberrations in the germ cells
of the species tested.

The likelihood that the test substance or its metabolites reach the target tissue should be discussed.

3.

REPORTING

TEST REPORT

The test report must include the following information:

Solvent/vehicle:

(cid:151) justification for choice of vehicle,

(cid:151) solubility and stability of the test substance in solvent/vehicle, if known.

Test animals:

(cid:151) species/strain used,

(cid:151) number and age of animals,

(cid:151) source, housing conditions, diet, etc.,

(cid:151) individual weight of the animals at the start of the test, including body weight range, mean and standard

deviation for each group.

Test conditions:

(cid:151) data from range finding study, if concluded,

(cid:151) rationale for dose level selection,

(cid:151) rationale for route of administration,

(cid:151) details of test substance preparation,

(cid:151) details of the administration of the test substance,

(cid:151) rationale for sacrifice times,

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(cid:151) conversion from diet/drinking water test substance concentration (ppm) to the actual dose (mg/kg body

weight/day), if applicable,

(cid:151) details of food and water quality,

(cid:151) detailed description of treatment and sampling schedules,

(cid:151) methods for measurement of toxicity,

(cid:151) identity of metaphase arresting substance, its concentration and duration of treatment,

(cid:151) methods of slide preparation,

(cid:151) criteria for scoring aberrations,

(cid:151) number of cells analysed per animal,

(cid:151) criteria for considering studies as positive, negative or equivocal.

Results:

(cid:151) signs of toxicity,

(cid:151) mitotic index,

(cid:151) ratio of spermatogonial mitoses cells to first and second meiotic metaphases,

(cid:151) type and number of aberrations, given separately for each animal,

(cid:151) total number of aberrations per group,

(cid:151) number of cells with aberrations per group,

(cid:151) dose-response relationship, if possible,

(cid:151) statistical analyses, if any,

(cid:151) concurrent negative control data,

(cid:151) historical negative control data with ranges, means and standard deviations,

(cid:151) concurrent positive control data,

(cid:151) changes in ploidy, if seen.

Discussion of results.

Conclusions.

4.

REFERENCES

(1) Adler, I. D., (1986), Clastogenic Potential in Mouse Spermatogonia of Chemical Mutagens Related to their
Cell-Cycle Specifications, in: Genetic Toxicology of Environmental Chemicals, Part B: Genetic Effects and Applied
Mutagenesis, Ramel, C., Lambert, B. and Magnusson, J. (eds) Liss, New York, pp. 477-484.

(2) Adler, I. D., (1984), Cytogenetic tests in Mammals, in: Mutagenicity Testing: a Practical Approach, (ed.) S.

Venitt and J. M. Parry, IRL Press, Oxford, Washington DC, pp. 275-306.

(3) Evans, E. P., Breckon, G. and Ford, C. E. (1964), An Air-drying Method for Meiotic Preparations from

Mammalian Testes, Cytogenetics and Cell Genetics, 3, pp. 289-294.

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(4) Richold, M., Ashby, J., Chandley, A., Gatehouse, D. G. and Henderson, L. (1990), In Vivo Cytogenetic
J. Kirkland (ed.), Basic Mutagenicity Tests, UKEMS Recommended Procedures. UKEMS
I revised, Cambridge University Press,

Assays,
Subcommittee on Guidelines for Mutagenicity Testing. Report. Part
Cambridge, New York, Port Chester, Melbourne, Sydney, pp. 115-141.

in: D.

(5) Yamamoto, K. and Kikuchi, Y. (1978), A New Method for Preparation of Mammalian Spermatogonial

Chromosomes, Mutation Res., 52, pp. 207-209.

(6) Adler, I. D., Shelby M. D., Bootman, J., Favor, J., Generoso, W., Pacchierotti, F., Shibuya, T. and Tanaka
N. (1994), International Workshop on Standardisation of Genotoxicity Test Procedures. Summary Report
of the Working Group on Mammalian Germ Cell Tests, Mutation Res., 312, pp. 313-318.

(7) Fielder, R.

J., Allen,

J. A., Boobis, A. R., Botham, P. A., Doe,

J., Gatehouse, D. G.,
Hodson-Walker, G., Morton, D. B., Kirkland, D. J. and Richold, M. (1992), Report of British Toxicology
Society/UK Environmental Mutagen Society Working Group: Dose setting in In Vivo Mutagenicity Assays,
Mutagenesis, 7, pp. 313-319.

J., Esdaile, D.

(8) Lovell, D. P., Anderson, D., Albanese, R., Amphlett, G. E., Clarc, G., Ferguson, R., Richold, M., Papworth,
D. G. and Savage J. R. K. (1989), Statistical Analysis of In Vivo Cytogenetic Assays, in: D. J. Kirkland (ed.),
Statistical Evaluation of Mutagenicity Test Data. UKEMS Subcommittee on Guidelines for Mutagenicity Testing,
report, Part III. Cambridge University Press, Cambridge, New York, Port Chester, Melbourne, Sydney, pp.
184-232.(cid:146)

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(cid:145)B.39. UNSCHEDULED DNA SYNTHESIS (UDS) TEST WITH MAMMALIAN LIVER CELLS IN VIVO

ANNEX 4G

1.

METHOD

This method is a replicate of the OECD TG 486, Unscheduled DNA Synthesis (UDS) Test with Mammalian
Liver Cells In Vivo (1997).

1.1.

INTRODUCTION

The purpose of the unscheduled DNA synthesis (UDS) test with mammalian liver cells in vivo is to identify
test substances that induce DNA repair in liver cells of treated animals (1), (2), (3), (4).

This in vivo test provides a method for investigating genotoxic effects of chemicals in the liver. The end point
measured is indicative of DNA damage and subsequent repair in liver cells. The liver is usually the major site
of metabolism of absorbed compounds. It is thus an appropriate site to measure DNA damage in vivo.

If there is evidence that the test substance will not reach the target tissue, it is not appropriate to use this test.

The end point of unscheduled DNA synthesis (UDS) is measured by determining the uptake of labelled
nucleosides in cells that are not undergoing scheduled (S-phase) DNA synthesis. The most widely used
technique is the determination of the uptake of tritium-labelled thymidine (3H-TdR) by autoradiography. Rat
livers are preferably used for in vivo UDS tests. Tissues other than the livers may be used, but are not the
subject of this method.

The detection of a UDS response is dependent on the number of DNA bases excised and replaced at the site
of the damage. Therefore, the UDS test is particularly valuable to detect substance-induced (cid:147)longpatch repair(cid:148)
(20 to 30 bases). In contrast (cid:147)shortpatch repair(cid:148) (one to three bases) is detected with much lower sensitivity.
Furthermore, mutagenic events may result because of non-repair, misrepair or misreplication of DNA lesions.
The extent of the UDS response gives no indication of the fidelity of the repair process. In addition, it is
possible that a mutagen reacts with DNA but the DNA damage is not repaired via an excision repair process.
The lack of specific information on mutagenic activity provided by the UDS test is compensated for by the
potential sensitivity of this end point because it is measured in the whole genome.

See also General Introduction Part B.

1.2.

DEFINITIONS

Cells in repair: a net nuclear grain (NNG) higher than a preset value, to be justified at the laboratory
conducting the test.

Net nuclear grains (NNG): quantitative measure for UDS activity of cells in autoradiographic UDS tests,
calculated by subtracting the average number of cytoplasmic grains in nucleus-equivalent cytoplasmic areas
(CG) from the number of nuclear grains (NG): NNG = NG ﬂ CG. NNG counts are calculated for individual
cells and then pooled for cells in a culture, in parallel cultures, etc.

Unscheduled DNA synthesis (UDS): DNA repair synthesis after excision and removal of a stretch of DNA
containing a region of damage induced by chemical substances or physical agents.

1.3.

PRINCIPLE OF THE TEST METHOD

The UDS test with mammalian liver cells in vivo indicates DNA repair synthesis after excision and removal of
a stretch of DNA containing a region of damage induced by chemical substances or physical agents. The test
is usually based on the incorporation of 3H-TdR into the DNA of liver cells which have a low frequency of
cells in the S-phase of the cell cycle. The uptake of 3H-TdR is usually determined by autoradiography, since
this technique is not as susceptible to interference from S-phase cells as, for example,
liquid scintillation
counting.

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1.4.

DESCRIPTION OF THE METHOD

1.4.1.

Preparations

1.4.1.1.

Selections of animal species

Rats are commonly used, although any appropriate mammalian species may be used. Commonly used
laboratory strains of young healthy adult animals should be employed. At the commencement of the study
the weight variation of animals should be minimal and not exceed – 20 % of the mean weight for each sex.

1.4.1.2. Housing and feeding conditions

General conditions referred in the General Introduction to Part B are applied although the aim for humidity
should be 50 to 60 %.

1.4.1.3.

Preparation of the animals

Healthy young adult animals are randomly assigned to the control and treatment groups. Cages should be
arranged in such a way that possible effects due to cage placement are minimised. The animals are identified
uniquely and kept
the study to allow for
in their cages for at
acclimatisation to the laboratory conditions.

five days prior to the start of

least

1.4.1.4. Test substance/preparation

Solid test substances should be dissolved or suspended in appropriate solvents or vehicles and diluted, if
appropriate, prior to dosing of the animals. Liquid test substances may be dosed directly or diluted prior to
dosing. Fresh preparations of the test substance should be employed unless stability data demonstrate the
acceptability of storage.

1.4.2.

Test conditions

1.4.2.1.

Solvent/vehicle

The solvent/vehicle should not produce toxic effects at the dose levels used, and should not be suspected of
chemical reaction with the test substance. If other than well-known solvents/vehicles are used, their inclusion
should be supported with data indicating their compatibility. It is recommended that wherever possible, the
use of an aqueous solvent/vehicle should be considered first.

1.4.2.2. Controls

Concurrent positive and negative controls (solvent/vehicle) should be included in each independently
performed part of the experiment. Except for treatment with the test substance, animals in the control group
should be handled in an identical manner to the animals in the treated groups.

Positive controls should be substances known to produce UDS when administered at exposure levels expected
to give a detectable increase over background. Positive controls needing metabolic activation should be used
at doses eliciting a moderate response (4). The doses may be chosen so that the effects are clear but do not
immediately reveal the identity of the coded slides to the reader. Examples of positive control substances
include:

Sampling times

Substance

CAS-No

Einecs No

Early sampling times (2 to 4 hours) N-nitrosodimethylamine

62-75-9

200-249-8

Late sampling times (12 to 16
hours)

N-2-fluorenylacetamide (2-AAF)

53-96-3

200-188-6

Other appropriate positive control substances may be used. It is acceptable that the positive control should
be administered by a route different from the test substance.

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1.5.

PROCEDURE

1.5.1.

Number and sex of animals

An adequate number of animals should be used to take account of natural biological variation in test
response. The number of animal should be at least three analysable animals per group. Where a significant
historical database has been accumulated, only one or two animals are required for the concurrent negative
and positive control groups.

If at the time of the study there are data available from studies in the same species and using the same route
of exposure that demonstrate that there are no substantial differences in toxicity between sexes, then testing
in a single sex, preferably males, will be sufficient. Where human exposure to chemicals may be sex-specific,
as for example with some pharmaceutical agents,
the
appropriate sex.

the test should be performed with animals of

1.5.2.

Treatment schedule

Test substances are generally administered as a single treatment.

1.5.3.

Dose levels

Normally, at least two dose levels are used. The highest dose is defined as the dose producing signs of
toxicity such that higher dose levels, based on the same dosing regimen, would be expected to produce
lethality. In general, the lower dose should be 50 % to 25 % of the high dose.

Substances with specific biological activities at low non-toxic doses (such as hormones and mitogens) may be
exceptions to the dose-setting criteria and should be evaluated on a case-by-case basis. If a range finding
study is performed because there are no suitable data available,
it should be performed in the same
laboratory, using the same species, strain, sex, and treatment regimen to be used in the main study.

The highest dose may also be defined as a dose that produces some indication of toxicity in the liver (e.g.
pyknotic nuclei).

1.5.4.

Limit test

If a test at one dose level of at least 2 000 mg/kg body weight, applied in a single treatment, or in two
treatments on the same day, produces no observable toxic effects, and if genotoxicity would not be expected,
based upon data from structurally related substances, then a full study may not be necessary. Expected
human exposure may indicate the need for a higher dose level to be used in the limit test.

1.5.5.

Administration of doses

The test substance is usually administered by gavage using a stomach tube or a suitable intubation cannula.
Other routes of exposure may be acceptable where they can be justified. However, the intraperitoneal route is
not recommended as it could expose the liver directly to the test substance rather than via the circulatory
system. The maximum volume of liquid that can be administered by gavage or injection at one time depends
on the size of the test animal. The volume should not exceed 2 ml/100g body weight. The use of volumes
higher than these must be justified. Except for irritating or corrosive substances, which will normally reveal
exacerbated effects with higher concentrations, variability in test volume should be minimised by adjusting
the concentration to ensure a constant volume at all dose levels.

1.5.6.

Preparation of liver cells

Liver cells are prepared from treated animals normally 12 to 16 hours after dosing. An additional earlier
sampling time (normally two to four hours post-treatment) is generally necessary unless there is a clear
positive response at 12 to 16 hours. However, alternative sampling times may be used when justified on the
basis of toxicokinetic data.

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Short-term cultures of mammalian liver cells are usually established by perfusing the liver in situ with
collagenase and allowing freshly dissociated liver cells to attach themselves to a suitable surface. Liver cells
from negative control animals should have a viability (5) of at least 50 %.

1.5.7.

Determination of UDS

Freshly isolated mammalian liver cells are incubated usually with medium containing 3H-TdR for an
appropriate length of time, e.g. three to eight hours. At the end of the incubation period, medium should be
removed from the cells, which may then be incubated with medium containing excess unlabelled thymidine
to diminish unincorporated radioactivity ((cid:147)cold chase(cid:148)). The cells are then rinsed, fixed and dried. For more
prolonged incubation times, cold chase may not be necessary. Slides are dipped in autoradiographic
emulsion, exposed in the dark (e.g. refrigerated for 7 to 14 days), developed, stained, and exposed silver
grains are counted. Two to three slides are prepared from each animal.

1.5.8.

Analysis

The slide preparations should contain sufficient cells of normal morphology to permit a meaningful
assessment of UDS. Preparations are examined microscopically for signs of overt cytoxicity (e.g. pyknosis,
reduced levels of radiolabelling).

Slides should be coded before grain counting. Normally 100 cells are scored from each animal from at least
two slides; the scoring of less than 100 cells/animals should be justified. Grain counts are not scored for
S-phase nuclei, but the proportion of S-phase cells may be recorded.

The amount of 3H-TdR incorporation in the nuclei and the cytoplasm of morphologically normal cells, as
evidenced by the deposition of silver grains, should be determined by suitable methods.

2.

DATA

2.1.

TREATMENT OF RESULTS

Individual slide and animal data should be provided. Additionally, all data should be summarised in tabular
form. Net nuclear grain (NNG) counts should be calculated for each cell, for each animal and for each dose
and time by subtracting CG counts from NG counts. If (cid:147)cells in repair(cid:148) are counted, the criteria for defining
(cid:147)cells in repair(cid:148) should be justified and based on historical or concurrent negative control data. Numerical
results may be evaluated by statistical methods. If used, statistical tests should be selected and justified prior
to conducting the study.

2.2.

EVALUATION AND INTERPRETATION OF RESULTS

Examples of criteria for positive/negative responses include:

positive

(i)

NNG values above a preset threshold which is justified on the basis of laboratory historical
data;

or

(ii) NNG values significantly greater than concurrent control;

negative

(i)

NNG values within/below historical control threshold;

or

(ii) NNG values not significantly greater than concurrent control.

The biological relevance of data should be considered: i.e. parameters such as inter-animal variation, dose
response relationship and cytotoxicity should be taken into account. Statistical methods may be used as an
aid in evaluating the test results. However, statistical significance should not be the only determining factor
for a positive response.

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Although most experiments will give clearly positive or negative results,
in rare cases the data set will
preclude making a definite judgement about the activity of the test substance. Results may remain equivocal
or questionable regardless of the number of times the experiment is repeated.

A positive result from the UDS test with mammalian liver cells in vivo indicates that a test substance induces
DNA damage in mammalian liver cells in vivo that can be repaired by unscheduled DNA synthesis in vitro. A
negative result indicates that, under the test conditions, the test substance does not induce DNA damage that
is detectable by this test.

The likelihood that the test substance reaches the general circulation or specifically the target tissue (e.g.
systemic toxicity) should be discussed.

3.

REPORTING

TEST REPORT

The test report must include the following information.

Solvent vehicle:

(cid:151) justification for choice of vehicle,

(cid:151) solubility and stability of the test substance in solvent/vehicle, if known.

Test animals:

(cid:151) species/strain used,

(cid:151) number, age and sex of animals,

(cid:151) source, housing conditions, diet, etc.,

(cid:151) individual weight of the animals at the start of the test, including body weight range, mean and standard

deviation for each group.

Test conditions:

(cid:151) positive and negative vehicle/solvent controls,

(cid:151) data from range-finding study, if conducted,

(cid:151) rationale for dose level selection,

(cid:151) details of test substance preparation,

(cid:151) details of the administration of the test substance,

(cid:151) rationale for route of administration,

(cid:151) methods for verifying that test agent reached the general circulation or target tissue, if applicable,

(cid:151) conversion from diet/drinking water test substance concentration (ppm) to the actual dose (mg/kg body

weight/day), if applicable,

(cid:151) details of food and water quality,

(cid:151) detailed description of treatment and sampling schedules,

(cid:151) methods for measurement of toxicity,

(cid:151) method of liver cell preparation and culture,

(cid:151) autoradiographic technique used,

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(cid:151) number of slides prepared and numbers of cells scored,

(cid:151) evaluation criteria,

(cid:151) criteria for considering studies as positive, negative or equivocal.

Results:

(cid:151) individual slide, animal and group mean values for nuclear grains, cytoplasmic grains, and net nuclear

grains,

(cid:151) dose-response relationship, if available,

(cid:151) statistical evaluation if any,

(cid:151) signs of toxicity,

(cid:151) concurrent negative (solvent/vehicle) and positive control data,

(cid:151) historical negative (solvent/vehicle) and positive control data with range, means and standard deviations,

(cid:151) number of (cid:147)cells in repair(cid:148) if determined,

(cid:151) number of S-phase cells if determined,

(cid:151) viability of the cells.

Discussion of results

Conclusions

4.

REFERENCES

(1) Ashby, J. Lefevre, P. A., Burlinson, B. and Penman, M. G. (1985), An Assessment of the In Vivo Rat.

Hepatocyte DNA Repair Assay, Mutation Res., 156, pp. 1(cid:151)18.

(2) Butterworth, B. E., Ashby, J., Bermudez, E., Casciano, D., Mirsalis, J., Probst, G. and Williams, G. (1987),
A Protocol and Guide for the In Vivo Rat Hepatocyte DNA Repair Assay, Mutation Res., 189, pp.
123(cid:151)133.

(3) Kennelly, J. C., Waters, R., Ashby, J., Lefevre, P. A., Burlinson, B., Benford, D. J., Dean, S. W. and
Mitchell, I. de G. (1993), In Vivo Rat Liver UDS Assay, in: Kirkland D. J. and Fox M., (eds), Supplementary
Mutagenicity Tests: UKEM Recommended Procedures. UKEMS Subcommittee on Guidelines for Mutagenicity
revised, Cambridge University Press, Cambridge, New York, Port Chester,
Testing. Report. Part
Melbourne, Sydney, pp. 52(cid:151)77.

II

(4) Madle, S., Dean, S. W., Andrac, U., Brambilla. G., Burlinson, B., Doolittle, D. J., Furihata, C., Hertner, T.,
McQueen, C. A. and Mori, H. (1993), Recommendations for the Performance of UDS Tests In Vitro and
In Vivo, Mutation Res., 312, pp. 263(cid:151)285.

(5)

Fautz, R., Hussain, B., Efstathiou, E. and Hechenberger-Freudl. C. (1993), Assessment of the Relation
Between the Initial Viability and the Attachment of Freshly Isolated Rat Hepatocytes Used for the In
Vivo/In Vitro DNA Repair Assay (UDS), Mutation Res., 291, pp. 21(cid:151)27.

(6) Mirsalis, J. C., Tyson, C. K. and Butterworth, B. E. (1982), Detection of Genotoxic Carcinogens in the In

Vivo/In Vitro Hepalocyte DNA Repair Assay, Environ. Mutagen, 4, pp. 553(cid:151)562.(cid:146)

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ANNEX 5

SV:

3.2.3.

Farligt

R65 Farligt: kan ge lungskador vid f(cid:246)rt(cid:228)ring.

Flytande (cid:228)mnen och beredningar som p(cid:229) grund av sin l(cid:229)ga viskositet utg(cid:246)r en fara f(cid:246)r m(cid:228)nniska vid aspiration

a)

F(cid:246)r (cid:228)mnen och beredningar som inneh(cid:229)ller alifatiska, alicykliska och aromatiska kolv(cid:228)ten i en total kon-
centration av 10 % eller mer och

(cid:151) har en fl(cid:246)destid mindre (cid:228)n 30 sekunder, uppm(cid:228)tt med en 3 mm utloppsb(cid:228)gare enligt ISO 2431, eller

(cid:151) har en kinematisk viskositet l(cid:228)gre (cid:228)n 7 (cid:215) 10ﬂ6 m2/s vid 40 (cid:176)C, uppm(cid:228)tt med en kalibrerad kapill(cid:228)rvis-

kosimeter av glas , enligt ISO 3104 och ISO 3105, eller

(cid:151) har en kinematisk viskositet l(cid:228)gre (cid:228)n 7 (cid:215) 10ﬂ6 m2/s vid 40 (cid:176)C, best(cid:228)md fr(cid:229)n rotationsviskosimetri enligt

ISO 3219.

˜mnen och beredningar, som uppfyller dessa kriterier, beh(cid:246)ver dock inte klassificeras om de har en
genomsnitlig ytsp(cid:228)nning h(cid:246)gre (cid:228)n 33 mN/m vid 25 (cid:176)C, uppm(cid:228)tt med du No(cid:252)ytensiometer eller enligt de
testmetoder som finns beskrivna i bilaga V del A.5.

b)

F(cid:246)r (cid:228)mnen och beredningar, baserat p(cid:229) praktiska erfarenheter fr(cid:229)n m(cid:228)nniska.

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FI:

3.2.6.1 Ihon tulehtuminen

Seuraava vaaraa osoittava lauseka m(cid:228)(cid:228)r(cid:228)ytyy alla esitelt(cid:228)vien perusteitten mukaan:

R38 ˜rsytt(cid:228)(cid:228) ihoa

(cid:151) Aineet ja valmisteet aiheuttavat ihon merkitt(cid:228)v(cid:228)n tulehtumisen enint(cid:228)(cid:228)n nelj(cid:228)n tunnin altistuksessa m(cid:228)(cid:228)-

ritettyn(cid:228) kanilla liitteess(cid:228) V mainitulla iho(cid:228)rsytstestill(cid:228). Tulehdus kest(cid:228)(cid:228) v(cid:228)hint(cid:228)(cid:228)n 24 tuntia.

Ihon tulehdus on merkitt(cid:228)v(cid:228)(cid:228), jos:

a)

b)

punoituksen ja ruvenmuodostuksen tai turvotuksen voimakkuutta kuvaavien lukuarvojen keskiarvo
laskettuna kaikista koe-el(cid:228)imist(cid:228) on v(cid:228)hint(cid:228)(cid:228)n 2;

tai kun liitteess(cid:228) V tarkoitettua testi(cid:228) on t(cid:228)ydennetty k(cid:228)ytt(cid:228)m(cid:228)ll(cid:228) kolmea koe-el(cid:228)int(cid:228), v(cid:228)hint(cid:228)(cid:228)n kahden
koe-el(cid:228)imen ihon punoituksen ja ruvenmuodostuksen tai
turvotuksen voimakkuutta kuvaavien
lukuarvojen keskiarvo on, jokaiselle koe-el(cid:228)imelle laskettuna erikseen, v(cid:228)hint(cid:228)(cid:228)n 2.

Kummassakin tapauksessa keskiarvojen lasekmiseen on k(cid:228)ytett(cid:228)v(cid:228) kaikkia niit(cid:228) lukuarvoja, jotka saadaan
arvioitaessa vaikutusta 24 tunnin, 48 tunnin ja 72 tunnin v(cid:228)lein.

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Tulehdusta pidet(cid:228)(cid:228)n my(cid:246)s merkitt(cid:228)v(cid:228)n(cid:228),
jos ihon tulehtuminen jatkuu ainakin kahdella koe-el(cid:228)imell(cid:228)
havainnointiajan p(cid:228)(cid:228)ttymiseen asti. Erityiset vaikutukset kuten esimerkiksi hyperplasia, hilseileminen, v(cid:228)rin
muutokset, halkeamat, ruvet ja karvojenl(cid:228)ht(cid:246) on otettava huomioon.

T(cid:228)h(cid:228)n liittyvi(cid:228) tietoja voidaan saada my(cid:246)s el(cid:228)imill(cid:228) teht(cid:228)vist(cid:228) ei-akuuttisista altistuskokeista (katso lause-
ketta R48 koskevat huomautukset jaksossa 2.d). Vaikutuksia pidet(cid:228)(cid:228)n merkitt(cid:228)vin(cid:228), jos ne vastaavat edell(cid:228)
kuvattuja vaikutuksia.

(cid:151) Aineet ja valmisteet, jotka aiheuttavat ihmisill(cid:228) merkitt(cid:228)v(cid:228)(cid:228) ihotulehdusta, kun kosketus on ollut v(cid:228)lit(cid:246)n,

jatkuva tai toistuva.

(cid:151) Orgaaniset peroksidit, paitsi jos on olemassa n(cid:228)ytt(cid:246)(cid:228) siit(cid:228), ett(cid:228) t(cid:228)llaista vaikutusta ei ole.

Tuntoharha ((cid:146)paresthesia(cid:146)):

Pyretroiditorjunta-aineen ihokosketuksen aiheuttamaa tuntoharhaa ihmisess(cid:228) ei pidet(cid:228) (cid:228)rsytysvaikutuksena, joka
oikeuttaisi luokituksen Xi; R38. S-lauseketta S24 on kuitenkin sovellettava aineisiin, joilla on t(cid:228)llainen vaikutus.

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In point 6.2 (Safety phrases for substances and preparations):

DE:

S 28 Bei Ber(cid:252)hrung mit der Haut sofort mit viel (cid:133) abwaschen (vom Hersteller anzugeben)

(cid:151) Anwendungsbereich:

(cid:151) sehr giftige, giftige oder (cid:228)tzende Stoffe und Zubereitungen;

(cid:151) Verwendung:

(cid:151) obligatorisch f(cid:252)r sehr giftige Stoffe und Zubereitungen;

(cid:151) empfohlen f(cid:252)r sonstige obengenannte Stoffe und Zubereitungen, inbesondere, wenn Wasser nicht die geeignete

Sp(cid:252)lfl(cid:252)ssigkeit ist;

(cid:151) empfohlen f(cid:252)r (cid:228)tzende Stoffe und Zubereitungen, die an die allgemeine (cid:214)ffentlichkeit abgegeben werden.

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FI:

S 29 Ei saa tyhjent(cid:228)(cid:228) viem(cid:228)riin

(cid:151) Soveltamisala:

(cid:151) eritt(cid:228)in helposti syttyv(cid:228)t tai helposti syttyv(cid:228)t veteen sekoittumattomat nesteet,

(cid:151) eritt(cid:228)in myrkylliset tai myrkylliset aineet ja valmisteet,

(cid:151) ymp(cid:228)rist(cid:246)lle vaaralliset aineet.

(cid:151) K(cid:228)yt(cid:246)n perusteet:

(cid:151) pakollinen yleisess(cid:228) kulutuksessa todenn(cid:228)koisesti k(cid:228)ytett(cid:228)ville ymp(cid:228)rist(cid:246)lle vaarallisille ja tunnuksella N luokitel-

luille aineille, jollei kyseess(cid:228) ole aineen tarkoitettu k(cid:228)ytt(cid:246),

(cid:151) suositeltava yleisess(cid:228) kulutuksessa todenn(cid:228)koisesti k(cid:228)ytett(cid:228)ville muille edell(cid:228) mainituille aineille tai valmisteille,

jollei kyseess(cid:228) ole kemikaalin tarkoitettu k(cid:228)ytt(cid:246).

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ANNEX 6

(cid:145)ANNEX IX

PART A

Provisions relating to child-proof fastenings

In addition to the provisions in Article 22(1)(e) of this Directive, containers of whatever capacity containing substances
presenting an aspiration hazard (Xn; R65) and classified and labelled according to paragraph 3.2.3 of Annex VI to this
Directive, with the exception of substances placed on the market in the form of aerosols or in a container fitted with a
sealed spray attachment, shall be fitted with child-proof fastenings.

1. Reclosable packages

Child-proof fastenings used on reclosable packages shall comply with ISO standard 8317 (1 July 1989 edition)
relating to (cid:147)Child-resistant packages (cid:151) Requirements and methods of testing for reclosable packages(cid:148) adopted by
the International Standard Organisation (ISO).

2. Non-reclosable packages

Child-proof fastenings used on non-reclosable packages shall comply with CEN standard EN 862 (March 1997
edition)
for
non-reclosable packages for non-pharmaceutical products(cid:148) adopted by the European Committee for Standardisation
(CEN).

relating to (cid:147)Packaging (cid:151) Child-resistant packaging (cid:151) Requirements and testing procedures

3. Notes

1.

Evidence of conformity with the above standards may be certified only by laboratories which conform with
European Standards Series EN 45 000.

2.

S p e c i f i c c a s e s

If it seems obvious that packaging is sufficiently safe for children because they cannot get access to the
contents without the help of a tool, the test does not need to be performed.

In all other cases and when there are sufficient grounds for doubting the security of the closure for a child, the
national authority may ask the person responsible for putting the product on the market to give it a certificate
from a laboratory, described in 3.1, stating that either:

(cid:151) the type of closure is such that it is not necessary to test to the ISO and CEN standards referred to above,

or

(cid:151) the closure has been tested and has been found to conform with the standards referred to above.

PART B

Provisions relating to tactile warning devices

The technical specifications for tactile warning devices shall conform with EN ISO standard 11683 (1997 edition)
relating to (cid:147)Packaging (cid:151) Tactile warnings of danger (cid:151) Requirements(cid:148).(cid:146)